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10th Congress of International Society of Systemic Auto-Inflammatory Diseases (ISSAID)

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    Oral presentations

    Molecular determinants of innate immunity

    O01 New MVK mutant mouse avatars of mevalonate kinase deficiency mimic the underlying defect in protein prenylation in patients

    Marcia A. Munoz1, Oliver P. Skinner1, Julie Jurczyluk1, Kristen Perry1, Robert Brink2, David Zahra2, Rob J. Arts3, Anna Simon3, Michael J. Rogers1
    1Bone Biology; 2Immunology, Garvan Institute of Medical Research, Sydney, Australia; 3Medical Centre, Radboud University, Nijmegen, Netherlands
    Correspondence: Marcia A. Munoz

    Introduction: Mevalonate Kinase Deficiency (MKD) is a periodic fever syndrome characterised by recurrent bouts of high fever and systemic inflammation. MKD is caused by recessive, hypomorphic mutations in the mevalonate kinase gene (MVK) encoding a key enzyme in the mevalonate pathway. This pathway is responsible for cholesterol synthesis and the production of isoprenoid lipid tags required for post-translational prenylation of proteins. It is believed that inflammation in MKD is triggered by shortage of isoprenoid lipids and defective prenylation of small GTPases. We have found that protein prenylation is indeed compromised in PBMCs from patients and this defect distinguishes MKD from other periodic fever syndromes. However, the link between protein prenylation and inflammation in MKD is still far from understood, largely due to the lack of suitable genetic mouse models.

    Objectives: To generate new Mvkmutant mouse models of MKD that mimic the human disease.

    Methods: CRISPR/Cas9 gene editing was used to generate different heterozygous mouse lines with hypomorphic mutations in exon 11 of theMvkgene: a V377I substitution (the most frequent mutation in MKD), and 8, 13 or 91 base pair deletions. These lines were then crossed to generate homozygous MvkV377I/V377Ior MvkV377I/deIcompound heterozygous mice. To assess the effect of the mutations on the mevalonate pathway, we measured the accumulation of unprenylated Rab GTPases and Rap1A using an in vitroprenylation assay and western blotting. For quantification of inflammatory cytokines in serum, as well as in culture supernatants from LPS-stimulated PBMCs and bone marrow macrophages, we used ELISA and multiplex cytokine bead arrays.

    Results: Homozygous mice carrying complete loss of function deletion mutations were not viable and, as expected, wildtype and heterozygous Mvkmutant mice had normal protein prenylation. However, in a similar pattern to patient PBMCs, MvkV377I/deIimmune cells from blood, spleen and bone marrow had a dramatic accumulation of unprenylated Rab and Rap1A GTPases, with a much milder defect in MvkV377I/V377Icells. This is consistent with reportedly less severe clinical disease associated with the homozygous V377I mutation. Furthermore, MvkV377I/deI mice had slightly elevated levels of inflammatory serum cytokines, including IL-6 and G-CSF, and cultures of PBMCs and bone marrow macrophages responded more robustly to LPS stimulation than cells from control mice. Interestingly, like patient-derived cell lines, the prenylation defect was dramatically enhanced in bone marrow cells from Mvkmutant mice after briefly culturing at higher temperature (39-40oC). Importantly, addition of the missing isoprenoid lipid geranylgeraniol could rescue the prenylation defect in primary cell cultures in vitroand in peritoneal macrophages in vivo.

    Conclusion: To our knowledge, we have generated the first genetic mouse avatars of MKD. These mice, like MKD patients, have defective protein prenylation and an exaggerated inflammatory response. Furthermore, as with patient-derived cell lines, Mvkmutant mouse cells are temperature-sensitive, suggesting that elevations in body temperature (e.g. caused by mild infections) could quickly precipitate devastating defects in protein prenylation that lead to systemic inflammatory flares. These mice are exciting new tools to study the pathophysiology of MKD and can be used to develop new therapeutic approaches, such as supplementation with isoprenoid lipids.

    Disclosure of Interest

    None Declared

    O02 Generation and analysis of mice carrying a novel heterozygous missense mutation of a proteasome subunit, PSMB9, in patients with autoinflammation and immunodeficiency

    Hiroaki Hemmi1, Nobuo Kanazawa2, Noriko Kinjo3, Satoru Hamada3, Hidenori Ohnishi4, Tsunehiro Mizushima5, Akira Kinoshita6, Koh-Ichiro Yoshiura6, Tsuneyasu Kaisho1
    1Department of Immunology, Wakayama Medical University Institute of Advanced Medicine; 2Department of Dermatology, Wakayama Medical University, Wakayama; 3Department of Child Health and Welfare (Pediatrics), University of the Ryukyus Graduate School of Medicine, Nishihara; 4Department of Pediatrics, Gifu University Graduate School of Medicine, Gifu; 5Picobiology Institute, University of Hyogo Graduate School of Life Science, Kamigori; 6Department of Human Genetics, Nagasaki University Atomic Bomb Disease Institute, Nagasaki, Japan
    Correspondence: Tsuneyasu Kaisho

    Introduction: The proteasome is a large protein complex involved in degradation of unnecessary or useless proteins. Homozygous, compound heterozygous or digenic mutations of proteasome subunits cause autoinflammatory diseases, termed proteasome-associated autoinflammatory syndromes (PRAAS). De novo, heterozygous missense mutation in the proteasome subunit PSMB9 (encodes β1i) gene (hereafter, PSMB9 X mutation), was commonly found in two unrelated patients showing PRAAS-like but distinct manifestations, on which two other posters are presented in this meeting. The PSMB9 X mutation is novel and causes a substitution of an amino acid conserved among multiple species.

    Objectives: It is unclear whether and how the PSMB9 X mutation contributes to the manifestations of the patients. In order to clarify this issue, we have generated and analyzed mutant mice carrying the mutation.

    Methods: The mice carrying the Psmb9 X mutation were generated with CRISPR/Cas9 technology. Homozygous Psmb9 X mutant mice died within 6 months old. Therefore, the heterozygous Psmb9 X mutant mice were mainly analyzed. Not only biochemical but also immunological analyses including histological and flow cytometry analyses were performed.

    Results: Heterozygous Psmb9 X mutant mice appeared healthy at glance. The β1i subunit becomes mature after processing by the proteasome, but this maturation process was impaired in splenocytes of the heterozygous Psmb9 X mutant mice. In the mutant mice, thymus was small and the cortico-medullary junction was unclear. All thymocytes such as CD4+, CD8+, double positive and double negative cells were decreased. In the spleen, B cells as well as CD4+ and CD8+ T cells were decreased. Furthermore, serum levels of immunoglobulins, including IgM, IgGs and IgA, were severely decreased. Dendritic cells (DCs) and all DC subsets were also decreased, although the conventional DC1 subset, defined as CD8α+CD11b- cells, was most severely decreased. Meanwhile, CD11b+ cells consisting mainly of neutrophils and monocytes were increased in the bone marrow. These phenotype of the heterozygous Psmb9 X mutant mice were not identical to, but overlapping significantly with the manifestations of the two patients.

    Conclusion: We here generated mutant mice carrying a novel heterozygous missense mutation in the proteasome subunit Psmb9 gene, which was identified in two unrelated PRAAS-like patients. Multiple defects in both innate and adaptive immune cells were observed in the heterozygous Psmb9 X mutant mice and some, although not all, defects were also observed in the two patients. These results indicate that the heterozygous Psmb9 X mutation can be the cause of the PRAAS-like phenotypes in the two patients. The findings that the mutation causes not only autoinflammation but also combined immunodeficiency prompt us to propose a novel category of autoinflammatory diseases distinct from PRAAS as “proteasome-associated autoinflammation and immunodeficiency disease (PRAID)”. The mutant mice are unique and quite useful for clarifying how the proteasome dysfunction leads to various manifestations of PRAID.

    Disclosure of Interest

    None Declared

    Mechanisms of inflammasome activation

    O03 Cofilin-1 is an essential redox sensor for NLRP3 inflammasome activation

    Wonyong Lee, Yong Hwan Park, Daniel L. Kastner, Jae Jin Chae
    NHGRI, Bethesda, United States
    Correspondence: Wonyong Lee

    Introduction: NLRP3 has a pivotal role in nucleating the inflammasome, a cytoplasmic multiprotein complex that mediates the maturation of the proinflammatory cytokine interleukin-1β (IL-1β) by activating caspase-1. Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases, the cryopyrin-associated periodic syndromes (CAPS). The generation of reactive oxygen species (ROS) is one of the major NLRP3 inflammasome activating factors. However, the molecular basis of the relationship between change of cellular redox state and NLRP3 inflammasome activation has not been elucidated.

    Methods: We utilized mouse bone marrow-derived macrophage (BMDM) to analyze interaction of cofilin-1 and NLRP3 by co-immunoprecipitation (co-IP). Mouse BMDMs were used to ectopically express wild-type (WT) or mutant cofilin-1 proteins, and to transfect siRNA for knockdown assay. Cofilin-1 knock-in (KI) mice (C39A or C39S) were generated by microinjection of sgRNA and Cas9 ribonucleoprotein (RNP) complex.

    Results: To identify an ROS-mediated regulator for NLRP3 inflammasome activation, the immune complexes precipitated by NLRP3 specific antibody from BMDMs of WT or NLRP3-KO mice were analyzed by mass spectrometry. We found cofilin-1, the actin severing protein, as a negative regulator for the NLRP3 inflammasome. Cofilin-1 interacted with the nucleotide-binding domain (NBD) of NLRP3 and dissociated from NLRP3 when the cells were stimulated with known NLRP3 inflammasome activators, such as ATP or nigericin. The NLRP3 inflammasome activators generate ROS that leads to cofilin-1 oxidation, which is intramolecular disulfide bond formation between two cysteine residues at amino acids 39 and 80. This oxidation induces conformational change of cofilin-1 and dissociation from NLRP3, which results in the activation of the NLRP3 inflammasome. Indeed, the assembly of NLRP3 inflammasome components is impaired and the IL-1β release was significantly suppressed in BMDMs ectopically expressing oxidation-resistant mutant cofilin-1 (C39A or C80A). In addition, knockdown of cofilin-1 in LPS-primed BMDMs induced NLRP3 inflammasome activation without activator treatment. We also observed that the interaction of cofilin-1 with the CAPS-associated mutant NLRP3 proteins was substantially diminished relative to WT NLRP3, which resulted in constitutive activation of the NLRP3 inflammasome. To examine the role of cofilin as a redox sensor for NLRP3 inflammasome activation in vivo, we have generated KI mice expressing oxidation-resistant mutant cofilin-1 (C39A or C39S). Unexpectedly, the IL-1β release from the BMDMs of the KI mice was higher than WT BMDMs when the cells were stimulated with ATP after LPS priming. Consistently, IL-1β levels in the serum of KI mice after injection of lipopolysaccharide (LPS) intraperitoneally was significantly higher than WT mice. This inconsistent result may be due to the low level of mutant cofilin-1 in KI mice. Indeed, similarly to the result of cofilin-1 knockdown, LPS-primed KI BMDMs release substantial IL-1β without activators.

    Conclusion: Taken together, these findings suggest that cofilin-1 is a key component in regulating the NLRP3 inflammasome in response to ROS. In addition, our data suggest cofilin-1 as a potential therapeutic target for the inflammatory conditions involving the NLRP3 inflammasome, including gout, type 2 diabetes mellitus, atherosclerosis, and Alzheimer’s disease.

    Disclosure of Interest

    None Declared

    O04 Autoinflammatory mutation in NLRC4 reveals an LRR-LRR oligomerization interface

    Fiona Moghaddas1,2,3, Ping Zeng4, Yuxia Zhang5, Heike Schuetzle6, Sebastian Brenner6, Sigrun Hofmann6, Reinhard Berner6, Yuanbo Zhao5,7, Bingtai Lu5, Xiaoyun Chen5, Li Zhang5, Suyun Cheng4, Stefan Winkler6, Kai Lehmberg8, Scott W. Canna9, Peter E. Czabotar10,11, Ian P. Wicks2,11,12, Dominic De Nardo2,11, Christian Hendrich6,13,14, Huasong Zeng4, Seth L. Masters2,11
    1Clinical Immunology and Allergy, The Royal Melbourne Hospital, Melbourne; 2Inflammation Division, The Walter and Eliza Hall Institute of Medical Research; 3Department of Medical Biology, The University of Melbourne, Parkville, Australia; 4Department of Rheumatology; 5Immunology Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou, China; 6Department of Pediatrics, University Hospital and Faculty of Medicine Carl Gustav Carus, Dresden, Germany; 7Department of Chemical Biology, Guizhou Medical University, Guiyang, China; 8Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg Eppendorf, Hamburg, Germany; 9Pediatric Rheumatology/RK Mellon Institute, Children’s Hospital of Pittsburgh ofUPMC, Pittsburgh, United States; 10Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville; 11Department of Medical Biology, The University of Melbourne, Melbourne; 12Rheumatology Department, The Royal Melbourne Hospital, Parkville, Australia; 13Department of Women’s & Children’s Health, nstitute of Translational Medicine, University of Liverpool; 14Department of Pediatrics Rheumatology, Alder Hey Children's NHS Foundation Trust Hospital, Liverpool, United Kingdom
    Correspondence: Fiona Moghaddas

    Introduction: Monogenic autoinflammatory disorders are characterised by dysregulation of the innate immune system. A significant number of this broadening group of disorders are caused by gain-of-function mutations in inflammasome forming proteins, such as NLRC4. A number of mutations in NLRC4 have been described, leading to a spectrum of NLRC4-associated autoinflammatory disorders (NLRC4-AID).

    Objectives: We studied two patients with early onset macrophage activation syndrome caused by the same de novo mutation in NLRC4 (c.G1965C, p.W655C). Unlike other mutations in NLRC4 described to date, p.W655 is located within the leucine rich repeat (LRR) domain. For this reason, we investigated mechanisms by which this mutation contributes to the pathogenesis of autoinflammatory disease.

    Methods: Next generation and Sanger sequencing techniques were used for genetic analysis. ELISA was performed to quantify serum cytokine levels. In vitro, inflammasome complex formation was quantified using flow cytometric analysis of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks.Monocyte-like cell lines were generated by genetic deletion of NLRC4 from THP-1 cells using CRISPR/Cas9 techniques followed by lentiviral transduction of wild type (WT) or mutant NLRC4 cDNA. Cell death and release of IL-1b/IL-18 were quantified using flow cytometry and ELISA respectively.

    Results: Both reported patients succumbed to macrophage activation syndrome early in life, associated with increased IL-18 serum levels. The NLRC4 mutation identified, c.G1965C/p.W655C, caused increased ASC speck formation in vitro. In THP-1 cells, introduction of c.G1965C/p.W655C NLRC4 resulted in increased cell death, IL-1b and IL-18 production. The enhanced response was independent of NLRP3 and caspase-8. ASC contributed to p.W655C NLRC4 mediated cytokine release, but not cell death. p.W655 is located at the interface between adjacent LRR domains in the oligomeric inflammasome structure. Mutation of p.W655 activates the NLRC4 inflammasome complex by engaging with two interfaces on the opposing LRR domain. One key set of residues (p.D1010, p.D1011, p.L1012 and p.I1015) participates in LRR-LRR oligomerization when it is triggered by NLRC4-AID mutations or T3SS effector (PrgI) stimulation of the NLRC4 inflammasome complex.

    Conclusion: This is the first report of a mutation in the LRR domain of NLRC4 causing NLRC4-AID. c.G1965C/p.W655C NLRC4 increases inflammasome activation, leading to constitutive IL-18 production and increased IL-1b release upon priming, where ASC contributes to the cytokine response, but not to cell death. Data generated from various NLRC4 mutations suggests that the tryptophan at p.W655 does not tolerate substitution, and provides evidence that the LRR-LRR interface has an important, previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.

    Disclosure of Interest

    None Declared

    Multifactorial vs monogenic autoinflammatory diseases

    O05 Canakinumab, on a reduced dose or a prolonged dose interval without concomitant corticosteroids and methotrexate, maintains efficacy in systemic juvenile idiopathic arthritis patients in clinical remission

    Pierre Quartier1, Ekaterina Alexeeva2, Carine Wouters3, Inmaculada Calvo4, Tilmann Kallinich5, Bo Magnusson6, Nico Wulffraat7, Xiaoling Wei8, Alan Slade9, Ken Abrams9, Alberto Martini10
    1AP-HP, Institut des Maladies Génétiques (IMAGINE), and Université Paris-Descartes, Necker-Enfants Malades Hospital, Paris, France; 2National Medical Research Center of Children's Health and Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation, Moscow, Russian Federation; 3Gasthuisberg University Hospital,Leuven, Belgium; 4Hospital Universitario La Fe, Valencia, Spain; 5Charité Berlin Campus Virchow, Berlin, Germany; 6Karolinska University Hospital, Stockholm, Sweden; 7University Medical Center Utrecht, Utrecht, Netherlands; 8China Novartis Institutes for Biomedical Research Co., Ltd, Beijing, China; 9Novartis Pharmaceuticals Corporation, East Hanover, United States; 10Universita di Genova Pediatria II, Genova, Italy
    Correspondence: Ekaterina Alexeeva

    Introduction: Treatment with canakinumab (CAN), a selective, human anti-IL-1β monoclonal antibody, has shown sustained therapeutic effect along with corticosteroid dose reduction/discontinuation in patients with systemic juvenile idiopathic arthritis (SJIA), in a long-term extension study (NCT00891046).1

    Objectives: To evaluate the efficacy and safety results from a study evaluating 2 different canakinumab tapering regimens in SJIA patients who were in clinical remission (NCT02296424).

    Methods: This Phase 3b/4 study had two parts. In Part I, 182 patients with inactive disease from the extension study1 (cohort 1) and CAN-naïve patients (cohort 2) with active disease were administered subcutaneous CAN 4 mg/kg q4w. Per protocol titration off corticosteroids and/or methotrexate was attempted during Part I. Eligible patients (inactive disease for 24 consecutive weeks and being corticosteroid- and methotrexate-free for at least 4 weeks) advanced to Part II. Patients were randomised to either a 3-step CAN dose reduction regimen (2mg/kg/q4w, followed by tapering to 1 mg/kg/q4w and then discontinuation) or dose interval prolongation regimen (4mg/kg q8w, followed by tapering to 4 mg/kg/q12w and then discontinuation); patients advanced to the next tapering step if inactive disease was maintained for 24 weeks. The primary objective was to evaluate if at least 40% of patients were able to maintain inactive disease status for at least 24 consecutive weeks on either 2mg/kg q4w or 4mg/kg q8w.

    Results: In Part II, a total of 75 patients were randomised to a dose reduction (n=38) or dose interval prolongation (n=37) in CAN tapering regimen. The proportion of patients who maintained inactive disease for 24 consecutive weeks exceeded the predefined threshold of 40% for Step 1 of: the reduced CAN dose (71%; 2 mg/kg q4w) and prolonged dose interval (84%; 4 mg/kg q8w) treatment arms. A total of 68% (26/38) and 79% (30/37) of the dose reduction and interval prolongation arms, respectively were successful in Step 2, while only 33% (25/75) of patients successfully discontinued CAN and maintained inactive disease for 24 consecutive weeks. Adverse events (AEs) and serious AEs observed within the 2 treatment cohorts and across Parts I and II were similar without any specific pattern or relationship to patients’ disease status at baseline or treatment regimen. The most frequent AEs were common infections such as nasopharyngitis, upper respiratory tract infection, and pharyngitis followed by SJIA-related events such as rash, pyrexia and arthralgia. Clinical laboratory abnormalities were consistent with expected findings in patients with active SJIA and the known safety profile of CAN.

    Conclusion: SJIA patients who are able to maintain inactive disease status on CAN monotherapy can successfully taper CAN by either reducing the dose or prolonging the dosing interval. However, only a minority of patients successfully discontinued CAN treatment for 24 weeks. The safety profile for both CAN titration regimens was similar and consistent with other CAN SJIA studies. No new safety signals were identified.

    Reference

    1. Brunner et al. Arthritis Rheumatol.2016; 68 (S10).

    Disclosure of Interest

    P. Quartier Consultant for: Abbvie, Lilly, Novimmune, Novartis and SOBI,Speaker Bureau of: AbbVie, Lilly, Novartis and SOBI, E. Alexeeva Grant / Research Support from: Roche, Abbott, Pfizer, Bristol-Myers Squibb, Centocor, Novartis, C. Wouters Consultant for: GSK, Roche, Pfizer , I. Calvo: None Declared, T. Kallinich Speaker Bureau of: Novartis, B. Magnusson: None Declared, N. Wulffraat Consultant for: Novartis, X. Wei Employee of: Novartis, A. Slade Conflict with: Novartis Pharmaceuticals CorporationShareholder of: Novartis Pharmaceuticals Corporation,Employee of: Novartis Pharmaceuticals Corporation, K. Abrams Conflict with: Novartis Pharmaceuticals CorporationShareholder of: Novartis Pharmaceuticals Corporation,Employee of: Novartis Pharmaceuticals Corporation, A. Martini: None Declared

    O06 IL-18:CXCL9 ratio as a predictor of treatment response in patients with systemic juvenile idiopathic arthritis treated with canakinumab

    Tanja Hinze1, Christoph Kessel1, Claas Hinze1, Julia Seibert2, Hermann Gram2, Dirk Foell1
    1Department of Pediatric Rheumatology and Immunology, Muenster University Hospital, Muenster, Germany; 2Novartis Pharma, Basel, Switzerland
    Correspondence: Tanja Hinze

    Introduction: Canakinumab, a monoclonal anti-interleukin (IL)-1β antibody, is highly effective for treating patients with systemic juvenile idiopathic arthritis (SJIA) but biomarkers predicting treatment response are desirable.

    Objectives: The objective of this study was to analyze the association of various serum biomarkers with treatment outcomes.

    Methods: Serum samples from 54 patients treated with canakinumab in an open-label long-term extension study were studied by Luminex at different time points during the study, including days 1 (baseline), 3, 15 and weeks 4, 8, 24, 48. Treatment outcomes included modified pediatric American College of Rheumatology (pACR) 30/50/70/90/100 responses within 15 days of treatment, clinically inactive disease (CID) according to the Wallace criteria within 15 days of treatment and sustained complete response, defined as pACR100or CID within 15 days of treatment plus no disease flare or macrophage activation syndrome (MAS) during the study. Data were analysed using non-parametric testing and receiver operating characteristic (ROC) analysis.

    Results: Within 15 days of treatment with canakinumab, 79%/77%/68%/49%/34% of the patients reached a modified pACR 30/50/70/90/100 response and 34% CID. Within a median follow-up of 23 months, 12 of 54 (22%) patients had a sustained complete response and 5 (9%) had developed MAS. Biomarkers did not correlate significantly with age, duration of disease or active joint count. Most biomarkers were elevated when compared to healthy controls at baseline and some rapidly decreased within 15 days of therapy (IL-1RA, IL-6, IL-18 and S100A12). A pattern was apparent when comparing responders and non-responders; responders had higher IL-18 and IFN-γ levels and lower CXCL9 levels at baseline, most emphasized by the IL-18:CXCL9 and the IFN-γ:CXCL9 ratios (Table 1). As determined via ROC analyses, these ratios had good accuracy in predicting treatment responses relating to pACR30/50/70/90/100/CID responses at day 15 and sustained complete response (area under the curve [AUC] for IL-18:CXCL9 ratio 0.79/0.75/0.67/0.72/0.77/0.71 and 0.80, respectively; and for the IFN-γ:CXCL9 ratio 0.79/0.76/0.71/0.75/0.77/0.76 and 0.79). Higher baseline CXCL9 levels predicted future MAS during the course of the study (ROC analysis AUC = 0.77).

    Conclusion: Several serum biomarkers were markedly elevated in patients with SJIA at baseline. A dysregulation of the IL-18-IFN-γ-CXCL9 axis is present in patients with SJIA, confirming findings from other investigators. However, our findings indicate that patients with a lower response to IL-18 and IFN-γ, as measured by CXCL9, an IFN-γ-induced chemokine, may have a better clinical response to canakinumab treatment. These findings will have to be confirmed in other cohorts.

    Disclosure of Interest

    T. Hinze Grant / Research Support from: Study was funded by Novartis Pharma , C. Kessel Grant / Research Support from: Study was funded by Novartis Pharma, C. Hinze Grant / Research Support from: Study was funded by Novartis Pharma, J. Seibert Employee of: Novartis Pharma, H. Gram Employee of: Novartis Pharma, D. Foell Grant / Research Support from: Study was funded by Novartis Pharma

    Table 1 (abstract O06). Median (range) IFN-gamma: CXCL9 and IL-18:CXCL9 ratios at baseline

    Update of autoinflammatory diseases

    O07 The NIH cohort study of DADA2 patients: novel insights into pathophysiology and treatment with TNF inhibitors

    Qing Zhou1,2, Natalie Deuitch1, Dan Yang3, Natalia Sampaio Moura1, Xiaomin Yu4, Oskar Schnappauf1, Patrycja Hoffmann1, Deborah Stone1, Amanda Ombrello1, Manfred Boehm3, Daniel Kastner1, Ivona Aksentijevich1
    1NHGRI/NIH, Bethesda, United States; 2Zhejiang University, Hang Zhou, China; 3NHLBI/NIH; 4NIAID/NIH, Bethesda, United States
    Correspondence: Qing Zhou

    Introduction: Deficiency of Adenosine Deaminase 2 (DADA2) is a recessively inherited disorder caused by a loss of functional ADA2 protein. A broad spectrum of features, including systemic inflammation, cutaneous, neurologic, musculoskeletal, and immunological manifestations have now been associated with DADA2. Given the highly polymorphic nature of the ADA2 and the complex clinical presentations of DADA2, large cohort studies are critical to advancing our knowledge of ADA2’s role in disease manifestation and pathogenesis.

    Objectives: To expand on the genetics of patients with DADA2 and explore the pathophysiology and the underlying mechanisms of TNF inhibitor response in these patients.

    Methods: We performed Sanger sequencing of the ADA2 gene (previously known as CECR1) and measured ADA2 enzyme activity in serum samples of patients and family members. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry and cell differentiation experiments to define an inflammatory signature in DADA2 patients and studied their response to TNF inhibitor treatment.

    Results: We have identified 60 patients with DADA2 and detected 10 pathogenic variants, which had not previously been associated with DADA2 – p.R9W, p.R34W, p.W204C, p.E244A, p.D329N, p.L351Q, p.A357T, p.P425A, p.P435A, p.W501*.Patients tested for ADA2 enzymatic activity had significantly decreased protein activity. Symptoms were highly variable among patients, even in cases with identical genotypes.

    We identified distinct inflammatory signatures. Patients had significantly higher subsets of CD14+ inflammatory monocytes than healthy controls. Phosphorylation of STAT1 was upregulated in patients CD4+ cells and monocytes following stimulation. Additionally, we observed increased gene expression of IP-10 in patients’ monocytes and macrophages. Furthermore, we observed strong NF-κB signaling in patients. Intracellular cytokine staining and qPCR for IL-1β, IL-6, and TNF cytokines were elevated in patients’ monocytes.

    After TNF inhibitor treatment, IFN and NF-κB inflammatory signatures were normalized. Analyses of post-treatment skin, lung and brain biopsies showed minimal perivascular TNF and resolution of inflammatory myeloid cell infiltrates. Immunostaining of skin biopsies revealed intact blood vessels with normal endothelial layers after treatment. Together, the data provides evidence that TNF inhibition both reduces systemic inflammation and improves endothelial integrity in the small vessels.

    ADA2 deficiency affects the differentiation of monocytes to anti-inflammatory M2 macrophages. Treatment with TNF-inhibitors rescued the impairment of M2 differentiation as demonstrated by improved cell morphology and a higher number of M2 macrophages.

    Conclusion: We report 10 novel pathogenic variants in ADA2, which is valuable for future DADA2 molecular diagnostic. Most important, we showed the cellular mechanism underlying effective treatment with anti-TNF therapies. DADA2 vasculitis is strongly related to the presence of activated myeloid cells and is reversible.

    Disclosure of Interest

    None Declared

    O08 Recommendation on colchicine dosing and definition of colchicine resistance/intolerance in the management of FMF

    Seza Ozen1, Erdal Sag1, Eldad Ben-Chetrit2, Marco Gattorno3, Ahmet Gul4, Philip Hashkes5, Isabelle Kone-Paut6, Helen Lachmann7, Elena Tsitsamis8, Marinka Twilt9, Fabrizio de Benedetti10, Jasmin B. Kuemmerle-Deschner11
    1Pediatric Rheumatology, Hacettepe University, Ankara, Turkey; 2Rheumatology Unit, Hadassah-Hebrew University Hospital, Jerusalem, Israel; 3Clincal Pediatrics and Rheumatology, Gaslini Institute, Genova, Italy; 4Department of Internal Medicine, Division of Rheumatology, Istanbul University, Istanbul, Turkey; 5Pediatric Rheumatology Unit, Department of Pediatrics, Shaare Zedek Medical Center, Jerusalem, Israel; 6Service de rhumatologie pédiatrique, CHU de Bicêtre, Le Kremlin-Bicêtre, France; 7Royal Free Campus, National Amyloidosis Centre, London, United Kingdom; 8First Department of Pediatrics, Aghia Sophia Childrens Hospital, University of Athens Medical School, Athens, Greece; 9Rheumatology, Department of Pediatrics, Alberta Children’s Hospital, University of Calgary, Calgary, Canada; 10Division of Rheumatology, Ospedale Pediatrico Bambino Gesù, Rome, Italy; 11Department of Pediatrics, Division of Pediatric Rheumatology, University Hospital Tuebingen, Tuebingen, Germany
    Correspondence: Jasmin B. Kuemmerle-Deschner

    Introduction: FMF is the most common monogenic autoinflammatory disease and colchicine is the drug of choice for the treatment. However, about 5-10% of FMF patients do not respond to colchicine even when they are fully compliant. Anti-IL1 treatments are used for the patients who are resistant to colchicine. Treatment with IL-1 inhibitors have been shown to be effective in clinical trials and in several case series.

    Objectives: The objective of this report is to produce evidence-based recommendations to define “colchicine resistance“, as well as compliance and intolerance, to guide rheumatologists and other health professionals in the treatment and follow-up of patients with colchicine-resistant FMF.

    Methods: A consensus meeting with 12 experts followed a systemic literature review and Delphi questionnaire. The expert committee consisted of adult and pediatric rheumatologists with expertise in FMF. Parameters for colchicine resistance/intolerance/compliance derived from the literature were evaluated by a pre-meeting online questionnaire. All parameters were discussed with a nominal group technique during the meeting. Recommendations were accepted if more than 80% agreement was reached. If agreement was below 80% a second round of discussion was held.

    Results: The systematic literature review yielded 264 articles. Of these, 38 were selected for expert review. After the literature review, Delphi survey, and round table discussion, recommendations that reached consensus levels were:

    Colchicine is the drug of choice for the treatment of FMF and compliance is a critical issue. For the following statements, it is assumed that the patient is compliant with colchicine.

    When utilizing colchicine to treat FMF, it is recommended to adjust the dose based on disease activity with the maximal dose in children depending on age (and weight).

    The maximum recommended colchicine dose for the treatment of FMF is between 1-3 mg per day depending on age, limited by signs of toxicity and tolerability.

    For a patient receiving the maximum tolerated dose of colchicine, resistance to colchicine is defined as ongoing disease activity (as reflected by either recurrent clinical attacks (average one or more attacks per month over a three-month period), or persistently elevated CRP or SAA in between attacks (depending on which is available locally)), in the absence of any other plausible explanation.

    AA amyloidosis develops as a consequence of persistent inflammation, which may be a manifestation of colchicine resistance.

    Colchicine intolerance, which generally manifests as GI symptoms (such as diarrhea and nausea), is common and can limit the ability to achieve or maintain the effective dose. Dose-limiting toxicity is rare and may include elevated LFT, leukopenia, azoospermia etc.

    Active disease and intolerance to colchicine affect quality of life.

    Various patient reported outcomes to be used to guide FMF disease management were outlined.

    Conclusion: The suggested recommendations are intended to improve patient care in FMF, to make a personalized treatment plan.

    Disclosure of Interest

    S. Ozen Speaker Bureau of: Novartis, SOBI, Pfizer, E. Sag: None Declared, E. Ben-Chetrit: None Declared, M. Gattorno: None Declared, A. Gul: None Declared, P. Hashkes Grant / Research Support from: Novartis,Consultant for: Novartis, I. Kone-Paut: None Declared, H. Lachmann: None Declared, E. Tsitsamis: None Declared, M. Twilt: None Declared, F. de Benedetti: None Declared, J. Kuemmerle-Deschner Grant / Research Support from: Novartis, SOBI,Consultant for: Novartis, SOBI

    Oral communications – clinical

    O09 The clinical spectrum of the deficiency of adenosine deaminase 2 (DADA2) continues to expand

    Karyl Barron1, Amanda Ombrello2, Debra Stone2, Patrycja Hoffmann2, Tina Romeo2, Anne Jones2, Natalia Sampaio Moura2, Oskar Schnappauf2, Ivona Aksentijevich2, Jenna Bergerson1, Ariane Soldatos3, Camilo Toro2, Dan Kastner2
    1NIAID; 2NHGRI; 3NINDS, NIH, Bethesda, United States
    Correspondence: Karyl Barron

    Introduction: The original reports of the deficiency of adenosine deaminase 2 (DADA2) in 2014 emphasized early-onset lacunar strokes, livedoid rash, intermittent fevers and early-onset polyarteritis nodosa. Since then, there have been reports of antibody deficiency, pure red cell aplasia, and cytopenias observed in DADA2 patients.

    Objectives: To document the range of clinical manifestations of DADA2.

    Methods: 46 patients were enrolled in an IRB approved study at the NIH. Sequencing of ADA2, the gene encoding ADA2 (adenosine deaminase 2), was performed on all patients. All underwent extensive clinical, laboratory & radiologic evaluation.

    Results: We evaluated 46 patients with DADA2 (24 F/22 M) including 6 sibling pairs & 2 families with 3 affected individuals. All patients had biallelic germline mutations in ADA2. Serum ADA2 enzyme activity levels were obtained in 32 patients and revealed absent to low levels compared to age-matched controls.

    The 46thpatient, seen 5 years after bone marrow transplantation for presumed GATA2 deficiency, was not included in our summary calculations.

    32 patients (71%) reported a history of recurrent fevers. 6 patients (13%) had diffuse lymphadenopathy.

    Skin involvement was seen in 38 patients (84%) including livedo in 34 (76%), cutaneous polyarteritis nodosa in 27 (60%), and Raynaud’s in 9 (20%).

    22 patients (49%) had a history of at least one stroke.Brain MRI showed evidence of ischemic infarcts in l6/22 (73%), 5 had both ischemic & hemorrhagic strokes (23%) and 1 had a hemorrhagic stroke (5%). There were 55 strokes in the 22 patients, the majority occurring in the brain stem and cerebellum (38%) and deep brain nuclei (36%). The average age at the time of the first stroke was 5.6 years (range 5 months-20 years). Stroke patients had an average of 3 strokes (range of 1-11). 3 patients manifest severe sequelae of hemorrhagic strokes.

    Abdominal ultrasound revealed hepatomegaly in 20 patients (44%) & splenomegaly in 26 (58%). Portal hypertension was observed in 7 patients (16%). Liver biopsies revealed hepatoportal sclerosis in 5 patients and focal nodular regenerative hyperplasia in 2. Abdominal MRA was abnormal in 7/13 patients, revealing arteritis and aneurysms.

    Significant peripheral vasculopathy was seen in 4 patients, one requiring multiple amputations of gangrenous digits. Systemic hypertension was observed in 11 patients (24%).

    Laboratory evaluation revealed hypogammaglobulinemia in 26 patients (58%). Immunoglobulin replacement was required in 10 patients. Lymphocyte phenotyping revealed arrested B cell class switching in 24/34 patients (71%) and decreased memory T cells in 11/34 (32%). Severe hematologic abnormalities, including anemia, leukopenia, lymphopenia and/or thrombocytopenia in 18 patients (40%), with 6 developing pancytopenia and 3 pure red cell aplasia. Immune mediated neutropenia was observed in 12 patients. ESR and C-reactive protein were elevated in 73% & 86%, respectively.

    6 patients underwent bone marrow transplantation with 4 patients successfully engrafted (2 requiring a second transplant), and 2 recently transplanted.

    Conclusion: The spectrum of DADA2 continues to expand to include ischemic and hemorrhagic strokes, cutaneous findings, portal and systemic hypertension, hematologic abnormalities, vascular pathology, immune deficiency and bone marrow failure. As the phenotypic presentation is likely to continue to expand, it is important to investigate any new complaints.

    Disclosure of Interest

    None Declared

    O10 Serum S100A8/A9 (calprotectin) in familial mediterranean fever and carriers of MEFV mutations does not correlate with disease activity

    Ruth Pepper1, Mathew Hutchinson2, Scott R. Henderson3, Sarah K. Todd3, Alan D. Salama3, Philip N. Hawkins4, Dorota Rowczenio4, Helen J. Lachmann4
    1Centre for Nephrology, UCL; 2Rheumatology, University College Hospital; 3Centre for Nephrology; 4National Amyloidosis Centre, UCL Division of Medicine and Royal Free Hospital NHS Foundation Trust, London, United Kingdom
    Correspondence: Ruth Pepper

    Introduction: Familial Mediterranean Fever (FMF) is caused by mutations in MEFV. The protein product pyrin is expressed in monocytes, neutrophils and eosinophils. Acute inflammatory attacks are accompanied by a dramatic hepatic acute phase response. S100A8/A9 is damage associated molecular pattern and a TLR4 ligand expressed in neutrophils, monocytes and early infiltrating macrophages.

    Objectives: We aimed to investigate S100A8/A9 in 39 patients with FMF, 45 healthy carriers and 16 wild type controls.

    Methods: All patients were genotyped. Patients and healthy controls (HC) serum S100A8/A9 levels, cell surface expression on monocytes and neutrophils as well as intracellular peripheral blood mononuclear cells (PBMC) expression were measured by flow cytometry (FACS). CD14 cells were isolated and following overnight incubation with or without LPS, S100A8/A9 was measured in the supernatants by ELISA. Patient and HC monocyte apoptosis was compared.

    Results: Serum levels were measures in 84 samples from 31 patients with homozygous or compound mutations (median 9061ng/ml [range 500-38470], 79 samples from 39 symptomatic patients who were MEFV heterozygotes (median 9394ng/ml [range 1744-38119], 80 samples from 45 individuals with MEFV variants but without clinical features of FMF (median 10939ng/ml [range 2447->40000]. There was no difference in calprotectin concentrations between the different mutations and no correlation with levels of the hepatic acute phase response, CRP or SAA. All the groups described had significantly higher levels than healthy controls (n=16 median 2836ng/ml [range 1058-6175])(p<0.001). Minimal monocyte and neutrophil cell surface expression was detectable. Following LPS stimulation there was significantly more S100A8/A9 detected in the supernatants in patients than healthy control CD14. There was also a trend to an increased intracellular monocyte S100A8/A9 expression.

    Conclusion: Patients with pyrin mutations both with and without clinical disease have greatly elevated serum S100A8/A9 levels without detectable cell surface expression in well-controlled disease with a trend to an increased monocyte intracellular expression. Upon monocyte stimulation with LPS, increasedS100A8/A9 is secreted. The exact mechanism by which these patients, especially those with mutations but no clinical disease, demonstrate sustained elevated serum S100A8/A9 remains to be elucidated but does not appear to result in a significant clinical sequelae.

    Disclosure of Interest

    None Declared

    O11 PAPA syndrome: novelties from the Eurofever registry

    Roberta Caorsi, Daniela Marotto, Antonella Insalaco, Angelo Marzano, Joost Frenkel, Graciela Espada, Immaculada Calvo Penades, Marijia Jelusic, Maria Cristina Maggio, Joost Swart, Esther Hoppenreijs, Ozgur Kasapcopur, Fabrizio De Benedetti, Marco Gattorno, The Pediatric Rheumatology International Trial Organization (PRINTO) and the Eurofever Project
    Center for Autoinflammatory Diseases and Immunodeficiency, Istituto G. Gaslini, Genova, Italy
    Correspondence: Roberta Caorsi

    Introduction: PAPA syndrome is a very rare autoinflammatory condition. Few data are nowadays available about the clinical characteristic, the response to treatment and the outcome of this disease.

    Objectives:To analyse the data of the PAPA patients enrolled to the Eurofever registry.

    Methods: the data analysed in the study were extracted from the Eurofever registry, which is hosted in the PRINTO website (www.printo.it). The patients were included in the study in the presence of mutations in the PSTPIP1 gene or, in genetically negative patients, in the presence of at least two of the following clinical manifestation: recurrent pyogenic arthritis, pyoderma gangrenousm or skin abscess with negative cultural tests. Demographic data, clinical manifestations and response to treatment were analysed.

    Results: In may 2018 baseline and clinical information were available of near 4000 patients in the Eurofever registry. Of the 36 patients classified as PAPA syndrome, 2 were excluded from the study. 34 PAPA patients, from 11 different centers, were analysed: the genotype was confirmatory in 29 patients, while in 5 was not available. 10 patients were of the same family, in 4 cases one parent was affected (2 included in the registry), while in other 8 patients the family history was negative. At the time of enrolment, 15 patients were in the paediatric age, while 19 were adults. The mutations detected in the PSTPIP1 gene were E250Q (13 pts), E250K (5 pts), A230T (3 pts), G258A (3 pts), E277D (2 pts), E257G (1 pt), G940A (1pts) and R365W (1 pts).

    The disease course was recurrent in 24 patients, while the other 10 presented a chronic disease course with periodic recrudesces. Joint and skin involvement were present at disease onset in 24 and 9 patients respectively. In other 12 patients skin involvement appeared over time. 20 out of the 34 patients presented clinical manifestations not typical of PAPA syndrome (psoriasis, uveitis, osteolytic bone lesions, chronic renal failure, muscular abscesses, gastrointestinal symptoms anaemia and hepatosplenomegaly).

    10 patients were treated with NSAID with partial and poor response in 6 and 4 patients respectively, while steroids caused a complete or partial control of disease manifestations in 6 and 10 patients respectively. Five patients were treated with methotrexate with partial response. Etanercept was used in 6 patient with complete response in 2 and partial in 4, adalimumab in 4 patients (1 partial and 1 complete responders, 2 failure) and anakinra in 9 patients (3 partial and 6 complete responders). 2 patients were treated with Canakinumab with complete response.

    Conclusion: This study enlightens the phenotypic variability of PAPA syndrome. The unusual clinical manifestations and the lack of the clinical triad of the disease may be responsible for the under recognition of this disease. Between biologic drugs, IL-1 inhibitors were more effective in the analysed cohort of patients.

    Disclosure of Interest

    None Declared

    O12 New classification criteria for recurrent autoinflammatory diseases applied to an independent cohort: experience from the JIR cohort database

    Glory Dingulu1, Sophie Georgin-Lavialle2, Isabelle Koné-Paut3, Pascal Pillet4, Anne Pagnier5, Etienne Merlin5, Daniela Kaiser6, Alexandre Belot7, Michael Hofer8, Véronique Hentgen9
    1Centre Hospitalier Versailles, Le Chesnay; 2Service de Médecine Interne-CEREMAIA, Centre Hospitalier Universitaire Tenon, Paris; 3Service de Rhumatologie Pédiatrique-CEREMAIA, Centre Hospitalier Universitaire Le Kremlin Bicêtre, Le Kremlin Bicêtre; 4Service d’Accueil des Urgences Pédiatriques, Centre Hospitalier Universitaire Pellegrin, Bordeaux; 5Service de Pédiatrie Générale, Centre Hospitalier Universitaire Clermont Ferrand, Clermont Ferrand, France; 6Service de Pédiatrie Générale, Centre Hospitalier Cantonal Luzern, Luzern, Switzerland; 7Service de Néphrologie-Rhumatologie Pédiatrique, Centre Hospitalier Universitaire Mère-Enfant, Bron, France; 8Service de Rhumatologie Pédiatrique, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 9Service de Pédiatrie Générale-CEREMAIA, Centre Hospitalier Versailles, Le Chesnay, France
    Correspondence: Glory Dingulu

    Introduction: New classification criteria for the inherited periodic fever syndromes Cryopyrin Associated Periodic Syndrom (CAPS), Tumour Necrosis Factor Receptor Associated Periodic Syndrom (TRAPS), Familial Mediterranean Fever (FMF), Mevalonate Kinase Deficiency (MKD), have recently been developed during a Consensus Conference held in Genoa in March 2017.

    Objectives: The aim of our study was to compare these new classification criteria for monogenic recurrent fever syndromes with the diagnoses of clinicians in a real-life setting.For this purpose we used the JIRcohort database, an international platform gathering data of patients with pediatric inflammatory disease.

    Methods: Patients with recurrent fever syndrome and complete clinical and genetic data were enrolled to the study from the auto-inflammatory module of the JIRcohort database. Patients genotype were characterized with HRF pathogenicity classification. A score from 0 to 2, 0 (no mutation), 1 (non-confirmatory genotype) and 2 (confirmatory genotype) was attributed to each gene screened in one patient. The new Genoa classification criteria were applied to all the patients and then compared to the diagnosis of the treating physician. The treating physician diagnosis was considered as standard reference. If the treating physician hesitated between two or more diagnoses, patients were redefined as Syndrome of Unexplained Recurrent Fever (SURF). SURF and PFAPA patients were pooled together. Finally, for each criteria sensitivity and specificity were determined before an analytical study, describing true positive, false positive and false positive patients.

    Results: 455 patients were included:10 CAPS, 11 MKD, 27 TRAPS, 122 FMF and 285 SURF/PFAPA patients.

    CAPS classification criteria showed a 60 % sensitivity and 98% specificity. 6 patients were true positive patients with confirmatory and non-confirmatory NLRP3 genotype. 4 were false negative patients with non-confirmatory genotype or no mutation in NLRP3. 8 were false positive with no mutation in NLRP3.

    TRAPS classification criteria showed a 100 % sensitivity and 98% specificity. 22 were true positive patients with confirmatory and non-confirmatory TNFRSF1A genotype. 5 patients were false positive with non-confirmatory genotype or no mutation in TNFRSF1A.

    FMF classification criteria showed a 96 % sensitivity and 88% specificity. 117 patients were true positive with confirmatory, non-confirmatory and non mutated MEFV genotype. 5 were false negative with non-confirmatory and non mutated genotype. 37 werefalse positive patients with patients with non-confirmatory genotype or non mutated genotype or no MEFV screening.

    MKD classification criteria showed a 64 % sensitivity and 66% specificity. 7 patients were true positive patients with confirmatory MVK genotype.4 were false negative patientswith non-confirmatory genotype or non mutated MVK genotype. 148 were false positive patients with non mutated MVK genotype.

    Conclusion: This work is the first to study Genoa criteria, in real-life setting, in a cohort of patients seen with recurrent fever. Genoa criteria showed tremendous performance for patients with confirmatory genotype and helped classifying patients with non-confirmatory genotype.

    Genoa classification criteria were less effective when patients did not display at least one genetic variant. Implementation of biological criteria in MKD might improve MKD criteria performance.

    The major limit of our study is the lack of a proper gold standard when genotype is not confirmatory. Nevertheless our study shows that the new classification criteria are of a high risk of misclassification in patients displaying a recurrent fever syndrome without genetic test.

    Disclosure of Interest

    None Declared

    O13 Impaired platelet functions in patients treated with colchicine

    Özlem Çimen1, Selcan Demir2, Erdal Sağ2, Armağan Keskin1, Yelda Bilginer2, Şule Ünal Cangül3, Seza Özen2
    1Department of Pediatrics; 2Department of Pediatric Rheumatology; 3Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey
    Correspondence: Selcan Demir

    Introduction: Colchicine has been used in the treatment of Familial Mediterranean Fever (FMF) since 1972. Apart from the inhibiting mitosis in all cells, colchicine has an anti-inflammatory effect by inhibiting activation and migration of neutrophils. Colchicine is a safe drug at recommended doses, but it can cause rare side effects including hematological findings such as lymphopenia, thrombocytopenia and neutropenia.

    Objectives: In this study we aimed to define the adverse effect of colchicine on platelet function and its clinical relevance.

    Methods: A total of 220 FMF patients between June 2016-2017, followed at Hacettepe University Pediatric Rheumatology Department and were on colchicine treatment for at least one year, were included to the study.

    Results: Among the selected 220 FMF patients, 100 of them (54% female) described hematological symptoms when questioned in detail. The mean age of these patients was 11.74 ± 4.86 years. The mean cumulative colchicine exposure was 5.7±3.8 years.

    The most common referral symptom was frequent epistaxis (79%) followed by easy bruising (69%), and menstrual disorder including prolonged or heavy menstrual bleeding (21.8% among female patients). Among these 100 patients, 36 of them had prolonged bleeding time and impaired platelet aggregation test. Patients who had abnormal platelet function tests (the group with abnormal bleeding time) were receiving higher colchicine doses (median 0.05 vs 0.03 mg/kg/day; p:0.001) compared to the patients who had normal platelet function tests (bleeding time normal group) However there were no significant difference in terms of cumulative colchicine exposure (median 6.5 vs 4.5 years; p:0.07) and total platelet counts (median 288500 vs 279000/mm3; p:0.61). Patients with abnormal platelet function tests (bleeding time abnormal group) also had more epistaxis (47% vs 7%; p<0.001), bruising (51% vs 3%; p<0.001) and dysmenorrhea (among female patients 100% vs 26%; p<0.001) (Figure 1 and 2). Colchicine was not reduced in these patients and no life-threatening event was observed.

    Conclusion: In our study, we have shown prolonged bleeding time for the first time in the literature. Colchicine may cause microtubule inhibition in platelets as well as in other cells and impair platelet function. Further prospective studies are needed to clarify the significance of this side effect.

    Disclosure of Interest

    None Declared

    O14 Autoinflammatory disorders in patients with myelodysplastic syndrome: the role of distinctive karyotypes and somatic mutations

    Mark Kacar1, Abdulla Watad2, Nicola Bragazzi3, Qiao Zhou2, Catherine Cargo4, Dennis McGonagle2, Sinisa Savic2
    1Department of Clinical Immunology and Allergy, St James University Hospital; 2LIRMM, University of Leeds, Leeds, United Kingdom; 3Department of Health Sciences, University of Genoa, Genoa, Italy; 4Department of Haematology, St James University Hospital, Leeds, United Kingdom
    Correspondence: Mark Kacar

    Introduction: A higher prevalence of autoimmune and autoinflammatory complications has been reported in patients with myelodysplastic syndrome (MDS). The exact cause of this remains to be elucidated.

    Objectives: To determine the correlation between patients’ demographic, clinical and molecular (karyotype, somatic genetic mutation status) features of MDS with specific autoinflammatory complications and long-term outcome.

    Methods: This was a retrospective study of 140 MDS patients referred to the Haematological Malignancy Diagnostic Service (HMDS) in Leeds, UK, between 2012-2018. As part of their diagnostic workup, all patients had karyotype assessment and targeted genetic sequencing performed. Patients’ medical records were examined to collect demographic and clinical information, and to identify patients with autoinflammatory complications. Patients were classified as having ‘non-specific autoinflammatory features’ if CRP was found to be elevated (>10.0 mg/L) on 5 or more separate occasions and this elevation could not be explained by infection, malignancy or autoimmunity. Chi-squared test, Student t-test, analysis of variance (ANOVA), univariate and multivariate logistic regression analyses were performed.

    Results: The average age was 77.08±11 years (median 79 years), with a male (n=91, 65.0%) preponderance. The 72 patients who had non-specific autoinflammatory features (51%) tended to be younger (75.15±11.23 years versus 79.15±11.92, p=0.0395), and more frequently had arthritis (n=25, 34.7%, versus n=12, 17.6%, p=0.0225), arthralgia (n=32, 44.4%, versus n=18, 26.5%, p=0.0271), skin rash (n=22, 30.6%, versus n=10, 14.7%, p=0.0261), and pleuritis (16, 22.2%, versus n=3, 4.4%, p=0.0022). 26.4% of MDS patients had a well-defined diagnosis of autoinflammatory disorder, with neutrophilic dermatosis and polymyalgia rheumatic occurring most commonly. Mutations affecting the transcription factor pathway (NPM1, RUNX1, BCOR, WTI, TP53, MYD88) (OR 3.15 [95%CI 1.04-9.56], p=0.0426) and deletion of chromosome 5 (OR 3.37 [95%CI 1.01-11.22], p=0.0479) were associated with autoinflammatory complications in general. Stratifying the patients into a “well-defined” and “non-specific” autoinflammatory disease group showed that deletion of chromosome 7 was associated with well-defined conditions, whilst deletion of chromosome 5 was linked with a non-specific autoinflammatory status. Furthermore, a higher rate of acute leukaemia transformation was reported in MDS patients with autoinflammatory status (n=25, 34.7%, versus n=8, 11.8%, p=0.0002).

    Conclusion: Autoinflammatory conditions were found to be more prevalent than expected in patients with MDS and were linked to a worse prognosis. Transcription factor pathway gene mutations and an abnormal karyotype were also associated with autoinflammation. Autoinflammatory features were associated with malignant transformation, hinting at the possibility that treatment of the autoinflammation might play a role in preventing disease progression. Further studies are required to replicate our findings and study the effect of anti-inflammatory therapy on disease progression.

    Disclosure of Interest

    None Declared

    Table 1 (abstract O14). See text for description

    O15 A novel MEFV mutation associated with an autosomal dominant FMF complicated by AA amyloidosis in a large British kindred

    Dorota Rowczenio, Taryn Youngstein, Hadija Trojer, Charalampia Papadopoulou, Tamer Rezk, Philip Hawkins, Helen Lachmann
    National Amyloidosis Centre, UCL, London, United Kingdom
    Correspondence: Dorota Rowczenio

    Introduction: Hereditary systemic autoinflammatory diseases (SAIDs) are rare genetic disorders characterised by recurrent, spontaneously resolving episodes of fever and systemic inflammation that predominantly involves serosal surfaces. AA amyloidosis is the most serious complication of SAIDs and is associated with proteinuric renal dysfunction that progresses to end stage renal failure and premature death.

    Objectives: To find a genetic cause in a large British family with a dominantly inherited autoinflammatory syndrome complicated by AA amyloidosis.

    Methods: Initially the Next Generation Sequencing (NGS) targeting 20 autoinflammatory genes was performed in the index patient and his sister, both of whom have been diagnosed with AA amyloidosis. There was a clear autosomal dominant inheritance affecting three generations. Upon finding a genetic cause the DNA obtained from other affected family members were analysed by Sanger sequencing.

    Results: The index case was diagnosed with sJIA aged 7 and developed end stage renal failure in his early twenties. He had haemodialysis for four years and underwent renal transplantation at the age of 32. He was referred to the National Amyloidosis Centre (NAC) with a suspicion of AA amyloid deposits in the transplanted kidneys. He reported suffering with fever accompanied by severe abdominal and chest pain, arthritis, erythema and night sweats from early life. Interestingly having been started on colchicine for post-transplant gout he felt significantly better. Subsequently his sister developed nephrotic syndrome due to AA amyloidosis. She describes similar symptoms throughout most of her life. Their parents were of white British origin from a non-consanguineous kindred. Their father had died aged 52 years from complications immediately following a cadaveric renal transplantation. The post-mortem examination of his renal biopsy revealed deposition of AA amyloid fibrils. The index case grandmother also suffered with cyclical episodic abdominal cramping and had a history of osteoarthritis. The index case’s two paternal cousins and two of their children described similar symptoms.

    NGS revealed a single MEFV allele to be affected in both the index case and his sister, resulting in the p.P373L variant in exon 3. Subsequently this variant was confirmed by Sanger sequencing in all living affected members. The mutant allele was not identified in the unaffected cases. All symptomatic individuals were treated with colchicine which suppressed their FMF related inflammation. We sequenced SAA1 gene, as homozygosity for the SAA1.1 allele is a known susceptibility factor for AA amyloidosis, but all cases were heterozygous.

    Conclusion: In the Northern Caucasian population FMF is extremely rare in comparison to the Mediterranean region. Typically FMF is an autosomal recessive disorder, nonetheless very rare cases of dominantly inherited disease have previously been reported, namely caused by the deletion of methionine residue at position 694 identified in British patients and three substitutions affecting threonine 577: p.T577N, p.T577S and p.T577A found in British, Turkish and Dutch patients respectively.

    Here we report a novel MEFV variant p.P373L causing dominant FMF in three generations of a large British family and in three cases this was complicated by AA amyloidosis indicating a severe pathogenicity of this variant.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Oral communications – Immunology

    O16 PYRIN inflammasome dysregulation in FMF patients: implication for a fast diagnostic test

    Thomas Henry, Flora Magnotti, Alexandre Belot, Yvan Jamilloux
    Inserm U1111, CNRS UMR5308, Univ. Lyon, ENS Lyon, CIRI, Lyon, France
    Correspondence: Thomas Henry

    Introduction: Familial Mediterranean Fever (FMF) is usually associated with bi-allelic mutations in the MEFV gene. MEFV encodes Pyrin, an inflammasome sensor leading to IL-1β release and a fast cell death termed pyroptosis. The relationship between MEFV mutations and Pyrin inflammasome regulations is still poorly understood. Furthermore, due to the large number of MEFV variants and a substantial proportions of FMF patients presenting mutations in a single MEFV allele, the genetic confirmation of the FMF diagnosis is often unconclusive.

    Objectives: The objective of the study were:

    1. 1.

      to understand at the molecular levels the Pyrin inflammasome dysregulation in monocytes from FMF patients

    2. 2.

      to assess whether monitoring Pyrin inflammasome activation could lead to a novel functional diagnostic test for FMF

    Methods: Monocytes from healthy donors (HD) or FMF patients were isolated and treated with a kinase inhibitor targeting the PKC superfamily, which includes PKN1/2 known to regulate Pyrin inflammasome activation. IL-1β release andcell death kinetics were monitored.

    In parallel, a human monocyte cell line was engineered to modelize FMF and HD monocytes and assess the role of Pyrin phosphorylation and inflammasome components in cell death and cytokine release.

    Results: Dephosphorylation of Pyrin was sufficient to trigger Pyrin inflammasome activation in monocytes from FMF patients while no inflammasome activation was observed in monocytes from healthy controls.

    Using a human monocyte cell line, we demonstrated that dephosphorylation of Pyrin was similar in cells expressing wild-type or mutated MEFV but that a mutated MEFV was necessary and sufficient to progress to active inflammasome upon PKC superfamily inhibition.

    Finally, thanks to a cohort of FMF patients, we demonstrate that FMF patients can be efficiently and specifically diagnosed based on the response of their monocytes to PKC superfamily inhibitors.

    Conclusion: Our results demonstrate that Pyrin dephosphorylation is sufficient to trigger inflammasome activation in monocytes from FMF patients but not from healthy donors. This indicates that in healthy donors, the progression to an active Pyrin inflammasome requires two independently-controlled steps and that the second mechanism of control is deficient in monocytes from FMF patients.

    Our study also demonstrates that monitoring inflammasome activation upon PKC superfamily inhibition in monocytes can discriminate FMF patients from patients with other inflammatory conditions opening the way to a fast functional test to diagnose FMF.

    Disclosure of Interest

    None Declared

    O17 Unraveling the molecular pathogenesis of proteasome-associated autoinflammatory syndromes

    Frédéric Ebstein1, Anja Brehm2, Sébastien Küry3, Thomas Besnard3, Bertrand Isidor3, Stéphane Bézieau3, Pawel Stankiewicz4, Elke Krüger1
    1Institut für Medizinische Biochemie und Molekularbiologie (IMBM), Universitätsmedizin Greifswald, Greifswald; 2Institut für Biochemie, Charité Universitätsmedizin Berlin, Berlin, Germany; 3Service de Génétique Médicale, CHU de Nantes, Nantes, France; 4Dept of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Correspondence: Frédéric Ebstein

    Introduction: In most eukaryotic cells, the degradation of abnormal and/or regulatory proteins is ensured by large multi-subunit ATP-dependent proteases known as proteasomes which consist in two distinct sub-complexes, a 20S core particle and a 19S regulatory particle. Since the early 2010s, an increasing number of loss-of-function mutations have been identified in genes encoding proteasome subunits including PSMB8, PSMB9, PSMB4, PSMA3 and PSMD12 and/or the proteasome maturation protein POMP. Fascinatingly, depending on the subunit affected, such genomic alterations result in the development of two seemingly distinct phenotypes, namely: (i) systemic autoinflammation or (ii) cognitive impairment. Herein, mutations of the 20S core particle subunits (i.e. PSMB8, PSMB9, PSMB4 and PSMA3) and/or POMP are typically associated with a group of autoinflammatory syndromes sharing the same constellation of clinical signs and frequently referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) and/or proteasome-associated autoinflammatory syndromes (PRAAS). By contrast, genetic disruption of the subunits of the 19S regulatory particle (i.e. PSMD12) leads to a syndromic form of intellectual disability (ID).

    Objectives: Our aim in this study is to determine whether syndromic ID disorders caused by variants in genes encoding 19S proteasome subunits share similarities with CANDLE/PRAAS in their etiology and/or pathogenesis particularly regarding inflammation.

    Methods: Peripheral blood mononuclear cells (PBMC) from subjects carrying 19S proteasome loss-of-function mutations diagnosed with syndromic ID as well as related control were collected and subjected to RNA isolation and protein extraction prior to quantitative PCR and western-blot analysis, respectively.

    Results: Like CANDLE/PRAAS, syndromic ID disorders caused by genomic alterations in 19S proteasome genes were associated with perturbed proteasome function, as evidenced by increased levels of intracellular ubiquitin-protein conjugates. Most importantly, our data show that PBMC from patients with syndromic ID exhibit high transcription levels of various IFN-induced genes (ISG) including CXCL10, CXCL9, IFI44 and IFI44L. Interestingly, the strength of the IFN signatures in these patients correlated with the magnitude of the unfolded protein responses (UPR) initiated by proteasome dysfunction, suggesting a cause-and-effect relationship between endoplasmic reticulum (ER) stress and inflammation.

    Conclusion: In this work, we provide evidence on the association of 19S proteasome dysfunction with the generation of autoinflammation in subjects diagnosed with syndromic ID disorders. From these data, we expect to convey a more integrated picture of the pathophysiology of syndromic ID and identify new therapeutic targets for the treatment of cognitive impairment.

    Disclosure of Interest

    None Declared

    O18 A novel knock-in mouse model of CAPS that develops amyloidosis: therapeutic efficacy of proton pump inhibitors

    Arinna Bertoni1, Sonia Carta2, Chiara Baldovini3, Federica Penco1, Enrica Balza2, Silvia Borghini4, Marco Di Duca4, Emanuela Ognio5, Paolo Nozza3, Francesca Schena1, Patrizia Castellani2, Claudia Pastorino1, Carola Perrone1, Laura Obici6, Alberto Martini7, Isabella Ceccherini4, Marco Gattorno1, Anna Rubartelli2, Sabrina Chiesa1
    1Centro Malattie Autoinfiammatorie ed Immunodeficienze, IRCCS Istituto G. Gaslini; 2Unità di Biologia Cellulare, IRCCS Ospedale Policlinico San Martino; 3Anatomia Patologica; 4Genetica Medica, IRCCS Istituto G. Gaslini; 5S.S Animal Facility, IRCCS Ospedale Policlinico San Martino, Genova; 6Centro per lo Studio e la Cura delle Amiloidosi Sistemiche, Fondazione IRCCS Policlinico San Matteo, Pavia; 7Direzione Scientifica, IRCCS Istituto G. Gaslini, Genova, Italy
    Correspondence: Arinna Bertoni

    Introduction: Cryopyrin associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NLRP3 gene that cause uncontrolled IL-1β secretion. CINCA syndrome is the most severe CAPS disease characterized by central nervous system disabilities with a long-term risk of secondary amyloidosis.

    Proton pump inhibitors (PPIs), commonly used as inhibitors of gastric acidproduction, also display anti-inflammatory properties, making them promising drugs in sepsis and in inflammatory disorders.

    Objectives: To develop a novel NLRP3 knock-in (KI) mouse model of CAPSto evaluate amyloid deposition and to test alternative therapeutic approaches.

    Methods: We generated KI mice by engineering N475K mutation associated with CAPS phenotype into mouse Nlrp3 gene. KI and Wild Type (WT) mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. Cytokines secretion from bone marrow derived dendritic cells (BMDCs) and peritoneal macrophages (PMs) was evaluated by ELISA. Hystological analysis of all organs was evaluated by hematoxylin and eosin staining. Amyloid deposition was quantified through Congo Red staining.

    Results: Mutant NLRP3 KI mice displayed features that recapitulates the immunological and clinical phenotype of CAPS. These mice had systemic inflammation, with high levels of serum pro-inflammatory cytokines compared to WT controls. Hystological analysis revealed the presence of acute and chronic inflammatory cell infiltrates and amyloid deposits in spleen, liver and kidneys. As in CAPS monocytes , BMDCs and PM from KI mice showed a strong increase in IL-1β, IL-18, and IL-1α secretion and decreased levels in interleukin-1 receptor antagonist (IL-1Ra), the naturally occurring IL-1b inhibitor.

    PPIs treatment of KI mice showed a clear clinical impact with improvement of inflammatory conditions and regression of amyloid deposits. Remarkably, BMDCs and PMs from PPI-treated mice presentedreduced secretion of pro-inflammatory cytokines and re-established the levels of IL-1RA.

    Conclusion: NLRP3 KI mice display a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness associated with lack oftoxicity indicates that PPIs could represent relevant adjuvants to the anti-IL-1 drugs in IL-1 driven diseases.

    Disclosure of Interest

    None Declared

    O19 T cell defects in patients with ARPC1B germline mutations account for combined immunodeficiency

    Immacolata Brigida1, Matteo Zoccolillo1,4, Maria Pia Cicalese1,2,3, Laurène Pfajfer5-9, Federica Barzaghi2,4, Serena Scala1, Carmen Oleaga-Quintas10,11, Jesus A. Alvarez12, Lucia Sereni1, Stefania Giannelli1, Claudia Sartirana1, Francesca Dionisio1, Luca Pavesi13, Marta Benavides-Nieto14,15, Luca Basso-Ricci1, Paola Capasso1, Benedetta Mazzi16, Jeremie Rosain10,11,28, Nufar Marcus17, Yu Nee Lee18, Raz Somech18, Massimo Degano19, Giuseppe Raiola20, Roberta Caorsi21, Paolo Picco21, Marcela Moncada Velez12, Joelle Khourieh11,12, Andrés Augusto Arias12,29, Aziz Bousfiha22, Thomas Issekutz23, Andrew Issekutz23, Bertrand Boisson11,12,24, Kerry Dobbs25, Anna Villa1,26, Angelo Lombardo1,3, Benedicte Neven14, Despina Moshous14,15, Jean-Laurent Casanova11,12,14,24,27, José Luis Franco12, Luigi D Notarangelo25, Cristina Scielzo13, Stefano Volpi21,30, Loïc Dupré5-9, Jacinta Bustamante11,12,24,28, Marco Gattorno21,31‡, and Alessandro Aiuti1,2,3‡
    1San Raffaele Telethon Institute for Gene Therapy, SR-TIGET; 2Pediatric Immunohematology, San Raffaele Scientific Institute, Milan; 3Vita-Salute San Raffaele University, Milan, Italy; 4Department of Systems Medicine, Tor Vergata University, Rome, Italy; 5INSERM, UMR1043, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; 6CNRS, UMR5282, Toulouse, France; 7Université Toulouse III Paul-Sabatier, Toulouse, France; 8Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria; 9CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 10Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, 75015 Paris, France, EU; 11Paris Descartes University, Imagine Institute, 75015 Paris, France, EU; 12Group of Primary Immunodeficiencies, Department of Microbiology & Parasitology, School of Medicine, University of Antioquia UdeA, Medellin, Colombia; 13Division of Experimental Oncology, Unit of B-cell Neoplasia, San Raffaele Scientific Institute, Milan; 14Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France, EU; 15Genome Dynamics in the Immune System, Université Paris Descartes – Sorbonne Paris; 16Immunogenetics Laboratory, HLA & Chimerism, Dept. of Immunohematology & Blood Transfusion, IRCCS Ospedale San Raffaele, Milano, Italy; 17Kipper Institute for Allergy and Immunology, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel, affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 18Pediatric Department A and the Immunology Services, “Edmond and Lily Safra” Children's Hospital, Jeffrey Modell Foundation Center, Sheba Medical Center, Tel Hashomer affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 19Division of Immunology, Transplantation, and Infectious Diseases, Biocrystallography Unit. San Raffaele Scientific Institute, Milan; 20Unità Operativa di Pediatria, Azienda Ospedaliera “Pugliese-Ciaccio” di Catanzaro; 21U.O. Clinica Pediatrica e Reumatologia, Istituto Giannina Gaslini, Genova, Italy; 22Clinical Immunology Unit, Department of Pediatrics, King Hassan II University, Ibn-Rochd Hospital, Casablanca, Morocco; 23Department of Pediatrics & Department of Microbiology-Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; 24St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA; 25Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, USA; 26Istituto di Ricerca Genetica e Biomedica (IRGB), Consiglio Nazionale delle Ricerche (CNR), Milan Unit, Milan, Italy; 27Howard Hughes Medical Institute, NY, USA; 28Center for the Study of Primary Immunodeficiencies, Assistance Publique-Hôpitaux de Paris AP-HP, Necker Hospital for Sick Children, Paris, France, EU; 29School of Microbiology, University of Antioquia UdeA, Medellin, Colombia; 30Università degli Studi di Genova, Genova, Italy; 31UOSD Centro Malattie e Autoinfiammatorie e Immunodeficienze, Istituto Giannina Gaslini, Genova, Italy
    Correspondence: Alessandro Aiuti

    Introduction: ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex, involved in actin branching from an existing filament. Germline mutations in ARPC1B have been recently described in six unrelated patients, with clinical features of combined immunodeficiency, whose neutrophils and platelets but not T lymphocytes were studied.

    Objectives: We hypothesized that ARPC1B-deficiency may also lead to cytoskeleton and functional defects in T cells

    Methods: Next-generation sequencing in 6 patients; flow cytometry, proliferation and migration, confocal and electron microscopy) to characterize defects in T cells; lentiviral-mediated gene transfer for genetic correction of ARPC1B.

    Results: We identified bi-allelic mutations in 6 unrelated patients with severe infections, autoimmune manifestations and platelet defects showing altered protein structure, and absent/low expression of the ARPC1B protein. Confocal microscopy showed altered expression of ARPC1B with actin. T cells displayed impaired TCR-mediated proliferation and SDF1-α directed migration. Gene transfer of ARPC1B in patient’s T cells using a lentiviral vector restored ARPC1B expression, leading to improved T-cell proliferation in vitro. In 2 patients normal ARPC1B levels in a fraction of lymphocytes were associated with in vivo somatic reversion and improved T cell migration in vitro. In one of the patient somatic revertant was present only in memory CD8+ T-cells, which showed improved in vitro T-cell migration.

    Conclusion: Inherited ARPC1B deficiency alters T cell cytoskeletal dynamics and functions, contributing to the clinical features of combined immunodeficiency.

    Disclosure of Interest

    None Declared

    O20 B cell defect in ADA2 deficiency patients

    Francesca Schena1, Federica Penco1, Stefano Volpi1,2, Claudia Pastorino1, Roberta Caorsi1, Arinna Bertoni1, Francesca Kalli3, Daniela Fenoglio3,4,5, Annalisa Salis6, Ignazia Prigione1, Paola Bocca1, Francesca Antonini7, Antonella Insalaco8, Alice Grossi9, Gianluca Damonte6, Isabella Ceccherini9, Gilberto Filaci3,4,5, Alberto Martini1,2, Elisabetta Traggiai10, Marco Gattorno1
    1UOSD Centro Malattie Autoinfiammatorie e Immunodeficienze and Clinica Pediatrica e Reumatologia, IRCCS Istituto Giannina Gaslini; 2Università degli studi di Genova; 3Center of Excellence for Biomedical Research; 4Department of Internal Medicine, Clinical Immunology Unit, Università di Genova; 5Ospedale Policlinico San Martino; 6Department of Experimental Medicine and Center of Excellence for Biomedical Research, Università di Genova; 7Core Facilities Flow-Cytometry and Cell imaging Lab, Istituto Giannina Gaslini, Genova; 8Division of Rheumatology, Department of Pediatric Medicine, IRCCS, Bambino Gesù Children’s Hospital, Roma; 9Medical Genetics, IRCCS Istituto Giannina Gaslini, Genova, Italy; 10Novartis Institutes for Biomedical Research, Basel, Switzerland
    Correspondence: Francesca Schena

    Introduction: Adenosine Deaminase 2 Deficiency (DADA2) is an autoinflammatory disease characterized by systemic vasculopathy, strokes and mild immunodeficiency, mainly affecting B cell compartment. The defect is due to a loss of function mutation of ADA2 gene, coding for Adenosine Deaminase 2, a protein which regulates the catabolism of extracellular adenosine.

    Objectives: Hypogammaglobulinemia and recurrent infections are associated to DADA2. We therefore investigated phenotype and in vitro B and T cell responses in DADA2 patients to address if ADA2 mutation affects B and T cell function and in particular we focused on B cell-T cell interaction.

    Methods: 14 patients carrying loss of function mutations in ADA2 were examined. They showed clinical history with livedo reticularis, fever, vasculitis and neurological symptoms. We analyzed peripheral B and T cell phenotype by flow cytometry, in vitro B-cell proliferation and differentiation to Immunoglobulin secreting cells in response to TLR9 agonist and T cell help. Moreover B cells isolated from DADA2 patients or HD have been cultured in co-culture with CD4+ T cells and in vitro B cell proliferation has been evaluated by CFSE dilution, whereas B cell differentiation and immunoglobulin secretion in response to TLR9 agonist and T cell help have been evaluated by ELISA and ELISPOT assay. Simultaneously cytokine production from Tfh cells has been analyzed.

    Results: Flow-cytometric analysis of DADA2 peripheral blood showed a significant reduction of switch memory B cells and an increased frequency of CD21low B cells. Regarding T cell compartment memory CD4+ and CD8+ T cells are significantly reduced; interestingly we identified an expansion in frequency of circulating Tfh cells.

    Then we investigated a role of ADA2 in B cells: we found that ADA2 is expressed in all B cell subsets, secreted but its enzymatic activity is strongly impaired in DADA2. We show that Naïve B cells from DADA2 patients are impaired in their proliferation, suggesting an intrinsic defect due to the mutation.

    We also addressed the interaction between B and helper T cells; DADA2 CD4+ T cells showed an impairment in the IL21 production and a downregulation of CD40L, suggesting a functional defect of Tfh cells; then we found that proliferation and differentiation of patients’ B cells were not sustained from patients’ CD4+ T cells.

    Conclusion: Our findings suggest that ADA2 mutations could lead to an intrinsic defect in B cell function and to a reduced T cell dependent B cell response.

    Disclosure of Interest

    None Declared

    O21 Periodic Fever, Aphthous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome and obstructive sleep apnea are distinct inflammatory disorders of oropharyngeal lymphoid tissue

    Kalpana Manthiram1, Silvia Preite2, Fatma Dedeoglu3, Maranda Lawton3, Pamela Mudd4, Hemalatha Srinivasalu4, Greg Licameli3, Kathryn Edwards5, Pamela Schwartzberg2, Daniel Kastner1
    1National Human Genome Research Institute; 2National Institute of Allergy and Infectious Diseases, NIH, Bethesda; 3Boston Children’s Hospital, Harvard Medical School, Boston; 4Children’s National Medical Center, George Washington University School of Medicine, Washington; 5Vanderbilt University School of Medicine, Nashville, United States
    Correspondence: Kalpana Manthiram

    Introduction: Tonsillectomy is one of the most common surgical procedures in children and is performed typically for recurrent tonsillitis or obstructive sleep apnea (OSA), and less frequently for periodic fever, aphthous stomatitis pharyngitis, cervical adenitis (PFAPA) syndrome. The pathogenesis of OSA and PFAPA is unknown.

    Objectives: In order to understand the pathogenesis of these two disorders, we studied the immunologic profile of tonsils removed from children with PFAPA and OSA in comparision with control tonsils.

    Methods: After getting informed consent, we obtained tonsils from children undergoing tonsillectomy for (1) PFAPA (N=12) and (2) OSA (N=12), and from (3) children undergoing tonsillectomy during oropharyngeal anatomic correction surgery like pharyngeal flap preparation (“controls”, N=9).Mononuclear cells from the tonsils were separated by standard methods and cells were analyzed by flow cytometry and gene expression with Nanostring. Cytokine production by isolated tonsillar mononuclear cells was measured by flow cytometry following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation for 4 hours ex vivo.

    Results: Tonsils from patients with PFAPA, which were removed during asymptomatic intervals, had evidence of germinal center suppression with significantly fewer T follicular helper cells, more T follicular regulatory cells, fewer plasma cells and reduced IgG class switching compared to tonsils from either OSA patients or controls. Gene expression analysis revealed downregulation of pro-inflammatory genes in tonsillar myeloid cells from patients with PFAPA. However, upon stimulation with PMA and ionomycin, CD4+ T cells from tonsils of patients with PFAPA produced more IFNγ and IL-17 than that of controls, but less than those of patients with OSA.

    On the other hand, tonsils from patients with OSA, had significantly larger germinal center areas by histology compared to those from patients with PFAPA and controls. Moreover, in tonsils from OSA patients, we found higher IFNγ production by CD4+ T, CD8+ T and NK cells, and greater IL-17 production by CD4+ T cells and NK cells upon stimulation with PMA and ionomycin when compared with tonsils from PFAPA patients and controls.

    Conclusion: We show that tonsils from both patients with PFAPA and OSA exhibit evidence of immune dysregulation. During asymptomatic periods, PFAPA patients display myeloid cell and germinal center suppression in the tonsils, suggesting a compensatory response to inflammatory flares. However, upon stimulation, CD4+ T cells produce high levels of pro-inflammatory cytokines. We hypothesize that alternating periods of heightened immune activation and suppression lead to the periodicity of PFAPA. In comparison, tonsils from patients with OSA display relatively constant germinal center hypertrophy likely due to unchecked chronic T cell activation. Further studies are necessary to understand the genetic and environmental risk factors for T cell activation in both of these disorders and why PFAPA patients may have cycling of inflammatory periods in the tonsils while OSA patients have chronic tonsillar inflammation.

    Disclosure of Interest

    None Declared

    O22 PFAPA syndrome: NK cells infiltrating tonsils support the crucial role of innate immunity in the pathogenesis

    Sabrina Chiesa1, Roberta Caorsi2, Francesca Bellora3, Mariella Della Chiesa3, Ilaria Ingrosso1, Federica Penco1, ArinnaBertoni1, Claudia Pastorino1, Ignazia Prigione1, Alessia Omenetti2, Martina Finetti2, Silvia Borghini4, Angela Sementa5, Roberto D’Agostino6, LuciaSemino6, Alberto Martini7, Cristina Bottino3, Marco Gattorno1
    1Centro Malattie Autoinfiammatorie e Immunodeficienze; 2Clinica Pediatrica e Reumatologica, IIRCCS G. Gaslini; 3Medicina Sperimentale, University of Genoa; 4Laboratorio di Genetica Molecolare; 5Anatomia Patologica; 6UO Otorinolaringoiatria, IIRCCS G. Gaslini; 7Clinica Pediatrica e Reumatologica, University of Genoa, Genova, Italy
    Correspondence: Sabrina Chiesa

    Introduction: Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is a more frequent cause of recurrent fever in children. The exact etiology of this pediatric disorder is still unknown. Elevated serum levels of IL-1β, IL-18, IL-6, and IFNγ suggest innate immunity dysregulation as the key pathomechanism of PFAPA attacks. Palatine tonsils are sites where innate immunity leads to the onset of adaptive immunity, mediated by B and T lymphocytes. Natural killer (NK) cells, the most important effectors of the innate lymphoid cells (ILCs), play a fundamental role in innate immune responses.

    Objectives: Tonsillectomy is one of the therapeutic options for PFAPA patients. We tested whether specific infiltrating inflammatory cells in pediatric tonsils contribute to PFAPA pathogenesis.

    Methods: Tonsils were collected from 2 groups of pediatric patients undergoing tonsillectomy: PFAPA patients (n=32) and children with bacterial tonsillitis (control group, CG) (n=25). Phenotypic analysis of subpopulations of tonsil cell suspensions and tissues was performedby flow cytometry and immunohistochemistry.

    Results: During the asymptomatic phase of disease the number of monocytes did not differ between the PFAPA and control tonsils. We observed a considerable recruitment of NK cells in tonsils of PFAPA patients respect to CG. In particular, we detected a significant expansion of both CD56bright CD16- and CD56dim CD16+ NK cell subsets in PFAPA samples compared to CG. A fine characterization of activating and inhibitory NK receptors suggested a crucial role of CD56dim CD16+ cell subset in PFAPA disease. Specially, activating receptors, as natural cytotoxicity receptors (NCRs) and NKG2D, were higher in NK cells of PFAPA patients than in NK cells from CG. Remarkably, CD56+ NK cells from PFAPA tonsils werecytotoxic and contained much more perforine and granzyme than those from CG tonsils. NK cells from PFAPA tonsils exhibited increased IFN-g production, compared to NK from CG. Accordingly, plasmacytoid dendritic cells, the main source of type I interferon cytokines, were significantly increased in PFAPA tonsils. We detected a higher number of naïve and a significantly lower percentage of effector memory CD4+ and CD8+ T cells in PFAPA tonsils compared to CG. Additionally, PFAPA tonsils presented a significant decrease of both functional follicular helper T cells (CXCR5+ICOS+) and T regulatory cells (CD39+Foxp3+). Finally, CD19+CD38+ B cellswere reduced in this cohort of PFAPA tonsils.

    Conclusion: These results suggest aninvolvement of NK cells in the pathogenesis of PFAPA and support the crucial role ofinnate immunity in the disease. Nonetheless, the abundant and activated CD56+NK cells might shape adaptive immunity that is impaired in the tonsils of PFAPA patients.

    Disclosure of Interest

    None Declared

    Novel AID pathways and genes

    O23 Identification of rare coding variants in IL-1-related pathways in patients with adult-onset still’s disease

    Giulio Cavalli1,2, Rosanne Van Deuren2, Peer Arts2, Marloes Steerhower2, Paolo Sfriso3, Paola Galozzi3, Serena Colafrancesco4, Roberta Priori4, Luca Cantarini5, Stefano Rodolfi1, Elena Baldissera1, Frank van der Veerdonk2, Lorenzo Dagna1, Alexander Hoischen2, Charles A. Dinarello2,6
    1Unit of Immunology, Rheumatology, Allergy and Rare Diseases (UniRAR), Vita-Salute San Raffaele University, Milan, Italy; 2Department of Medicine, Radboud University Medical Centre, Nijmegen, Netherlands; 3Rheumatology, University of Padua, Padua; 4Rheumatology, Sapienza University, Rome; 5Rheumatology, University of Siena, Siena; 6Department of Medicine, University of Colorado Denver, Aurora, CO, Italy
    Correspondence: Giulio Cavalli

    Introduction: Adult-onset Still's disease (AOSD) is a rare autoinflammatory disease characterized by fever, arthritis, and multi-organ involvement. Inflammation in AOSD is mediated by interleukin (IL)-1β, as confirmed by the dramatic clinical efficacy of selective blockers of this cytokine. The genetic predisposition to this rampant IL-1-driven inflammation remains nevertheless elusive. Previous studies failed to identify associations between polymorphisms in the genes encoding IL-1 and AOSD, thus pointing at more complex genetic mechanisms. This ‘missing heritability’ cannot be adequately investigated with traditional techniques for genetic partitioning, such as GWAS, which only assess common variants and polymorphisms. Studies focusing on highly penetrant rare variants or different types of mutations (i.e. small copy-number variations; insertions/deletions) are warranted.

    Objectives: We hypothesized that genetically determined changes in IL-1-related pathways resulting in excessive IL-1β activity lead to the development of autoinflammation in AOSD. Scope of this study was to unravel the combined mutational variation of a network of IL-1-related receptors, pathways, counter-regulators, and cellular processes possibly involved in the pathogenesis of AOSD and IL-1-mediated inflammation in general.

    Methods: We collected clinical, demographic, and genetic data from a large cohort of 76 AOSD patients and developed an innovative platform based on molecular inversion probes (MIP) technology for performing highly multiplexed targeted-resequencing. This allows efficient sequencing of the coding sequence of 48 genes related to the IL-1-pathway, and allows studying rare and common variants in one assay. We have also screened 500 healthy controls, and 1000s of samples with other disorders using the same assay.

    Results: We identified rare and unique (i.e. private variants) in the IL1 pathway in several individuals with AOSD. Whether any these are involved in a strong predisposition to AOSD is currently followed-up. Rare genetic variants have been identified in six IL-1-pathway ‘clusters’:

    1. 1.

      Deregulated activation of the inflammasome and release of IL‑1β and IL-18.

    2. 2.

      IL-1 family receptors and intracellular signaling mediators.

    3. 3.

      Other pro-inflammatory cytokines and receptors.

    4. 4.

      Regulatory molecules, including IL-1Ra or IL-37.

    5. 5.

      Cellular processes regulating production of IL-1 and IL-18 (i.e. autophagy).

    6. 6.

      Production of ROS, which function as markers of cellular damage and trigger inflammation.

    Conclusion: Unraveling the genetic bases of inflammation in AOSD deepens our understanding of the human innate immunome. Of note, this study platform may serve for the genetic analysis of other IL-1-mediated conditions, including gout and other autoinflammatory diseases, whose genetic predisposition remains elusive. Equally important, the identification of pathways amenable to targeting with small molecules or biologics may translate into remarkable clinical implications.

    Disclosure of Interest

    None Declared

    O24 Trisomy 8-associated autoinflammatory disease (TRIAD) is characterized by increased monocyte activation

    Kalpana Manthiram1, Alina Dulau-Florea2, Deborah Bruns3, Amanda Ombrello1, Karyl Barron4, Tina Romeo1, Anne Jones1, Karin Weiss1, Sandro Perazzio5, Isabelle Kone-Paut6, Nora Al-Mutiari7, Troy Torgerson5, Daniel Kastner1
    1National Human Genome Research Institute; 2Department of Laboratory Medicine, NIH, Bethesda; 3Department of Counseling, Quantitative Methods, and Special Education, Southern Illinois University Carbondale, Carbondale; 4National Institute of Allergy and Infectious Diseases, NIH, Bethesda; 5Department of Pediatrics, University of Washington School of Medicine, Seattle, United States; 6Bicêtre University Hospital, APHP, CEREMAIA, University of Paris Sud, Paris, France; 7Department of Pediatrics, Sabah Hospital, Kuwait City, Kuwait
    Correspondence: Kalpana Manthiram

    Introduction: Many adults with acquired trisomy 8 and myelodysplasia have been reported to have Behçet’s-like inflammatory disease. A few patients with constitutional trisomy 8 mosaicism and ulcerative disease have also been reported. The spectrum of phenotypic abnormalities, inflammatory profiles, and treatment responses of patients with constitutional trisomy 8 are not well-characterized.

    Objectives: We analyzed the clinical and immunologic features of a cohort of patients with trisomy 8 mosaicism in order to understand the pathogenesis of the disorder.

    Methods: Whole blood gene expression was analyzed with the Nanostring Human Immunology panel. Copy number of two genes on the p and q arms of chromosome 8 were determined by digital droplet PCR in sorted CD3+ T cells, CD19+ B cells, and CD14+ monocytes from peripheral blood to determine the percentage of mosaicism in different cell populations. Platelet electron microscopy was performed in a clinical laboratory.

    Results: Eleven patients with trisomy 8 mosaicism ranging in age from 5 to 37 years were recruited. Eight-two percent had recurrent fever, 82% had oral ulcerations, and 72% had severe oral ulcerations larger than 1 cm or lasting more than one week. Five patients reported genital, esophageal, or colonic ulcers. Nine patients had cognitive or motor delay. Two patients had symptoms of a bleeding diathesis with subdural hematoma, petechiae, and/or menorrhagia with normal platelet counts; the platelets of these patients had fewer dense granules by electron microscopy. Patients had elevated peripheral absolute monocyte count (average 970 cells/uL), but none had chronic myelomonocytic leukemia, acute myeloid leukemia or myelodysplasia. Whole blood gene expression revealed upregulation of IL-1-, TLR- and NF-ĸB-related genes. CD14+ monocytes had a significantly higher percentage of cells with trisomy 8 compared to CD3+ T cells and CD19+ B cells (chromosome 8 copy number was 2.8 in CD14+ cells vs. 2.5 in CD19+ [p=0.01] and 2.3 in CD3+ cells [p =0.001]). Four out of 5 patients had partial improvement on daily colchicine, 3 out of 6 reported improvement with intermittent or daily anakinra, 2 out of 3 had improvement on TNFα inhibitors (etanercept or infliximab), and one patient improved following hematopoietic stem cell transplant.

    Conclusion: We report the spectrum of clinical and immunologic manifestations in patients with trisomy 8 mosaicism, a disease we name trisomy 8-associated autoinflammatory disease (TRIAD). Patients with TRIAD present with (1) recurrent fever and/or severe mucosal ulcerations, (2) bleeding diatheses, and (3) developmental delay. Patient monocytes are elevated in number and have higher percentage of trisomy 8, indicating that an extra copy of chromosome 8 may confer a survival advantage preferentially to monocytes. In addition, activation pathways in monocytes are upregulated, and patients have symptom improvement in response to blockade of TNFα and IL-1, prominent cytokines associated with myeloid cell activation. Like monocytes, megakaryocytes and platelets develop from a common myeloid progenitor suggesting that cell development in the myeloid lineage may be affected. Further studies are underway to better characterize the inflammatory and survival pathways in myeloid cells with trisomy 8.

    Disclosure of Interest

    None Declared

    Deficiencies in key regulatory signals

    O25 A combined immunodeficiency with severe infections, inflammation and allergy caused by ARPC1B deficiency

    Stefano Volpi1,2, Maria Pia Cicalese3, Paul Tuijnenburg4,5, Anton T. J. Tool6, Eloy Cuadrado7, Hamid Ahanchian8, Raed Alzyoud9, Zeynep C. Akdemir10, Federica Barzaghi11, Alexander Blank12, Bertrand Boisson13, Cristina Bottino14, Roberta Caorsi15, PaoloPicco15, Jean-Laurent Casanova13,16, Sabrina Chiesa17, Ivan Kingyue Chinn18, Gregor Dückers19, Anselm Enders20, Hans Christian Erichsen21, LisaR. Forbes18, Tomasz Gambin22,23, Marco Gattorno24, Ehsan G. Karimiani25, Silvia Giliani26, Michael S. Gold27, Marwan Abu-Halaweh28, Eva-Maria Jacobsen12, Machiel H. Jansen29,30, Jovanka R. King27, Ronald M. Laxer31, James R. Lupski22,32, Emily Mace18, Stefania Marcenaro33, Reza Maroofian34, Alexander B. Meijer35, Tim Niehues19, Luigi D. Notarangelo36, Jordan Orange18, Ulrich Pannicke37, Chris Pearson38, Patrick J. Quinn27, Ansgar Schulz12, Filiz Seeborg18, Asbjørg Stray-Pedersen39, Hasan Tawamie40, Ester M. M. van Leeuwen30, Alessandro Aiuti11, Rae Yeung31,41, Klaus Schwarz37,42, Taco W. Kuijpers29,43
    1Clinica Pediatrica e Reumatologia, Centro per le malattie Autoinfiammatorie e Immunodeficienze, Istituto Giannina Gaslini; 2DINOGMI, Università degli Studi di Genova, Genova; 3Pediatric Immunohematology, San Raffaele Hospital and San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan, Italy; 4Department of Pediatric Immunology, Rheumatology and Infectious diseases, Emma Children’s Hospital, Amsterdam UMC, University of Amsterdam; 5Department of Experimental Immunology, Amsterdam Infection & Immunity Institute; 6Department of Blood Cell Research; 7Department of Immunopathology, Sanquin Research and Landsteiner Laboratory AMC, University of Amsterdam, Amsterdam, Netherlands; 8Department of Allergy and immunology, School of medicine, Mashhad university of Medical Sciences, Mashhad, Iran, Islamic Republic Of; 9Immunology, Allergy and Rheumatology section- Bone Marrow Transplantation for Primary Immunodeficiency Disorders, Queen Rania Children's Hospital, Amman, Jordan; 10Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Huston, United States; 11Pediatric Immunohematology, San Raffaele Hospital and San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Milan, Italy; 12Department of Pediatrics, University Medical Center Ulm, Ulm, Germany; 13St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, United States; 14Department of Experimental Medicine (DIMES), University of Genoa; 15Centro per le Malattie Infiammatorie e Immunodeficienze, Clinica Pediatrica e Reumatologia, Istituto Giannina Gaslini, Genova, Italy; 16Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, APHP, Paris, France; 17Clinica Pediatrica e Reumatologia, Centro per le malattie Autoinfiammatorie e Immunodeficienze Istituto Giannina Gaslini, Genova, Italy; 18Department of Pediatrics, Section of Allergy, Immunology, and Rheumatology & Center for Human Immunobiology, Texas Children's Hospital, Houston, United States; 19Center for Child and Adolescent Medicine, Helios-Clinic, Krefeld, Germany; 20Department of Immunology and Infectious Disease, John Curtin School of Medical Research and Centre for Personalised Immunology, Australian National University, Canberra, Australia; 21Section of Paediatric Medicine and Transplantation, Division of Paediatric and Adolescent Medicine, Oslo University Hospital, oslo, Norway; 22Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; 23Institute of computer science, Warsaw University of Technology, Warsaw, Poland; 24. Clinica Pediatrica e Reumatologia, Centro per le malattie Autoinfiammatorie e Immunodeficienze Istituto Giannina Gaslini, Genova, Italy; 25Molecular and Clinical Sciences Institute, St. George’s, University of London, Cranmer Terrace, London, United Kingdom; 26Medical Genetics Unit and “A. Nocivelli” Institute for Molecular Medicine, Spedali Civili Hospital, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; 27Discipline of Pediatrics, School of Medicine, University of Adelaide and Department of Allergy and Clinical Immunology, Women's and Children's Health Network, Adelaide, Australia; 28Department of Biotechnology and Genetics Engineering in Philadelphia University, Jordan, United States; 29Emma Children’s Hospital, Amsterdam UMC, University of Amsterdam, Department of Pediatric Immunology, Rheumatology and Infectious diseases; 30Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Amsterdam Infection & Immunity Institute, Amsterdam, Netherlands; 31Division of Rheumatology, Department of Paediatrics and Department of Medicine, University of Toronto, The Hospital for Sick Children, Toronto, Canada; 32Department of Pediatrics, Baylor College of Medicine, Houston, United States; 33Istituto Giannina Gaslini, Genova, Italy; 34Medical Research, RILD Welcome Wolfson Centre, Exeter Medical School, Royal Devon and Exeter NHS Foundation Trust, Exeter and Genetics and Molecular Cell Sciences Research Centre, St George's University of London, London, United Kingdom; 35Department of Plasma proteins, Sanquin Research and Landsteiner Laboratory AMC, University of Amsterdam, Amsterdam, Netherlands; 36Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, United States; 37Institute for Transfusion Medicine, University Ulm, Ulm, Germany; 38Department of General Medicine, Women's and Children's Health Network, Adelaide, Australia; 39Norwegian National Unit for Newborn Screening, Division of Pediatric and Adolescent Medicine, Oslo University Hospital, Oslo, Norway; 40The Institute of Human genetics of Leipzig, Leipzig, Germany; 41Departments of Paediatrics, Immunology, Institute of Medical Science, University of Toronto, Cell Biology Program, The Hospital for Sick Children, Toronto, Canada; 42Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Service Baden-Wuerttemberg – Hessen, Ulm, Germany; 43Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory AMC, University of Amsterdam, Amsterdam, Netherlands
    Correspondence: Stefano Volpi

    Introduction: Genetic defects in regulatory proteins of the cytoskeleton are known to cause different syndromes, mostly dominated by hematologic and immune phenotypes. Recently has been reported a novel syndrome of combined immunodeficiency, allergy and autoinflammation caused by mutations in the ARPC1B gene, one of the seven subunits of the ARP2/3 complex, which regulates actin polymerization.

    Methods: We report the natural history, clinical manifestations, genetics, and immunohematological findings in 14 patients from 12 families with ARPC1B deficiency.

    Results: Although consanguinity was not revealed by clinical history in five families, all cases carried homozygous mutations in ARPC1B. The mutations resulted in undetectable or reduced protein expression. Early-onset gastrointestinal bleeding and skin rash were common findings (7/14 and 9/14, respectively), whereas bacterial (12/14) and viral (10/14) infections (including warts, molluscum and CMV infections), vasculitis (9/14) and growth failure (10/14) became prominent features later in life. The majority of children developed severe allergy in the presence of extensive eczema (8/14) and a universal increase in IgA and IgE levels. Moderate thrombocytopenia was present in the majority of patients while overt bleeding tendency was absent after infancy. Immunophenotyping showed a B-cell lymphocytosis and abnormalities in T- and NK-lymphocyte subsets. In vitro T- and B-lymphocyte proliferation and in vivo response to vaccination were normal. Most noticeable was the strongly reduced regulatory T-cell function in those patients tested.

    Conclusion: In conclusion, our cohort delineates the spectrum of clinical, hematological and immunological manifestations of subjects with ARPC1B deficiency. The disease appears progressive in most cases and challenging to manage clinically with prophylactic measures and immunosuppression alone.

    Disclosure of Interest

    None Declared

    O26 ARPC1B-deficiency causes defective cell migration, loss of actin polymerization and hyperresponsiveness in zebrafish and patient-derived cells

    Gabriella Leung1,2, Aleixo M. Muise2,3
    1Cell Biology; 2Gastroenterology, Hepatology and Nutrition; 3Inflammatory Bowel Disease Centre, Hospital for Sick Children, Toronto, ON, Canada
    Correspondence: Gabriella Leung

    Introduction: ARPC1B-deficiency is a rare paediatric genetic disorder identified in 2017 with autoinflammatory components. Primary symptoms include cutaneous vasculitis, increased susceptibility to infection, microthrombocytopenia, and colitis. ARPC1B is a component of the Arp2/3 complex which regulates branched actin polymerization. Its expression is limited to the haematopoietic compartment, however its function in macrophages and B cells has not been characterised.

    Objectives: To determine the effect of ARPC1B-deficiency on macrophage migration and B cell activation.

    Methods: Mutant Arpc1b-deficient zebrafish were generated by targeting CRISPR-Cas9 to exon 4 of arpc1b in AB zebrafish embryos, and crossing F1 mutants to mpeg1-GFP+ fish. Four days post-fertilization, tails were cut transversely just distal to the notochord, and GFP+ cells imaged over an 8 hr period. Cells were tracked individually and migration behaviour quantified using Volocity. EBV-transformed lymphoblasts (LCLs) were derived from three previously characterised ARPC1B patients. Patient 1 has a null mutation (c.387_388insCT, L90fs). Patients 2 and 3 are brothers with two SNPs (c.434C>T, A105V; c832G>A, A238T). LCLs were also derived from the parents of Patient 2 and 3, heterozygous for both mutations. Cell phenotype was assessed by immunoblotting, immunofluorescence, and flow cytometry. Calcium flux was analysed by flow cytometry using ratiometric dye Fura Red-AM and stimulating LCLs with anti-IgG. The VCA domain of WASP binds and activates the Arp2/3 complex and was used to stimulate pyrene-actin polymerization activity. Protein interaction between ARPC1B vs. ARPC1A to VCA and WASP were also tested by immunoprecipitation.

    Results: ARPC1B-deficient zebrafish were significantly smaller, and monocytes migrated slower compared to WT fish in response to injury. In the patient LCLs, loss of ARPC1B protein, compensatory upregulation of isoform ARPC1A, and loss of total F-actin were confirmed. Patient 1 LCLs were unusually adherent in the absence of stimulation, suggesting constitutive activation. To measure activation, LCLs were stimulated with anti-IgG to measure calcium flux; Patient 1 had a statistically significant higher calcium flux peak compared to control. To address whether Arp2/3 function was compromised, LCL lysates were stimulated with (activating domain) VCA and actin assembly was measured. Although both ARPC1B and ARPC1A isoforms were found to interact with VCA and full-length WASP in immunoprecipitation experiments, VCA-induced actin polymerization in cell lysates was completely abolished in Patients 1-3. Patient 1 was treated with an allogeneic HSCT in January 2018.

    Conclusion: Loss of ARPC1B leads to impaired cell migration, loss of Arp2/3 function, and hyperactivation in B cells, which together likely contribute to the dysfunctional immune phenotype. HSCT may represent a curative option for patients with severe ARPC1B-deficiency.

    Disclosure of Interest

    None Declared

    Nucleic acid sensing and interferon

    O27 Heterozygous mutations in COPA are associated with enhanced type I interferon signalling

    Marie-Louise Frémond1, Alice Lepelley1, Carolina Uggenti2, Maria José Martin-Niclos1, Marine Depp2, Vincent Bondet3, Darragh Duffy3, Gillian I. Rice4, Mary Brennan5, Caroline Thumerelle6, Siham Boulisfane6, Marie Legendre7, Serge Amselem7, Thierry Molina8, Nadia Nathan9, Yanick J. Crow2
    1Laboratory of Neurogenetics and Neuroinflammation, Imagine Institute, Paris, France; 2Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; 3Immunobiology of Dendritic Cells, Institut Pasteur, Paris, France; 4Division of Evolution and Genomic Sciences, Manchester Academic Health Science Centre, Manchester; 5Department of Paediatric Rheumatology, Royal Hospital for Sick Children, Edinburgh, United Kingdom; 6Pediatrics Department, CHRU de Lille, Lille; 7Genetic Department and Inserm UMR S933, Trousseau Hospital-APHP and Sorbonne Université; 8Pathology Department, Necker Hospital-APHP; 9Inserm UMR S933 and Pediatric Pulmonology department and Reference Centre for Rare Lung Diseases, RespiRare, Trousseau Hospital-APHP and Sorbonne Université, Paris, France
    Correspondence: Marie-Louise Frémond

    Introduction: Heterozygous mutations in COPA, encoding coatomer protein subunit alpha, cause an autosomal dominant inflammatory syndrome associating lung, joint and renal disease, showing some overlap with STING-associated vasculopathy with onset in infancy (SAVI). Mutations were originally described to cause endoplasmic reticulum (ER) stress and priming of a T helper 17 response. More recently, increased transcription of interferon (IFN)-stimulated genes (ISGs) was reported in blood circulating cells of affected individuals. However, the precise pathophysiology of this disease remains unclear.

    Objectives: To better decipher the mechanism of COPA syndrome.

    Methods: We studied 8 patients from 3 unrelated families, each segregating a heterozygous mutation in COPA. We assessed type I IFN status by IFNa ultra-sensitive digital quantification in plasma, STAT1 phosphorylation and RNA expression of ISGs in whole blood from patients. In vitro assays also were performed in HEK293T and THP-1 cells to study IFN signalling in the context of COPA mutations.

    Results: We observed commonalities in the lung pathology between COPA and SAVI, as well as an IFN signature, raised levels of IFNa protein in the serum and phosphorylation of STAT1 in patient T cells. In a cellular model of HEK293T, phosphorylation of IRF3 and increased ISG expression were observed in cells co-transfected with wild type STING and mutant COPA plasmids. In THP-1 cells, short hairpin RNA knockdown of COPA induced IFN signalling that was abrogated in the absence of STING.

    Conclusion: Our data suggest that mutations in COPA lead to constitutive activation of type I IFN signalling through STING. Based on these results, one patient has been treated with the JAK1/2 inhibitor ruxolitinib for the last 12 months. How COPA interacts with ER-resident STING remains to be investigated.

    References

    Watkin et al, Nat Genet 2015;47:654-60.

    Volpi et al, Clin Immunol 2018;187:33-36.

    Disclosure of Interest

    None Declared

    O28 Sting-associated vasculopathy in mice requires adaptive immunity but not cGAS or type I interferon

    Hella Luksch1, Angela Rösen-Wolff1, Alexander Gerbaulet2, W. Alexander Stinson3, Brock G. Bennion3, Gowri Kalugotla4, Wei Qian3, Catherine A. Miner4, Jonathan Miner5
    1Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden; 2Department of Immunology, Faculty of Medicine,TU Dresden, Dresden, Germany; 3Department of Pathology and Immunology; 4Department of Medicine, Washington University School of Medicine; 5Departments of Pathology and Immunology, Medicine and Molecular Microbiology, Washington University School of Medicine, Saint Louis, St. Louis, United States
    Correspondence: Angela Rösen-Wolff

    Introduction: It is assumed that monogenic interferonopathies are mediated by type I interferon (IFN) inducing autoinflammation. For instance, it has been shown that a gain-of-function mutation in STING (STING N153S) up-regulates type I IFN-stimulated genes (ISGs) and results inperivascular inflammatory lung disease in mice. The corresponding mutation in humans also causes lung disease. It is thought that signaling via the cGAS-STING pathway and subsequent activation of type I IFN, IFN regulatory factors (IRF) 3/7, and ISGs are involved.

    Objectives: We decided to characterize the role of cGAS, IRF3/7, the type I IFN receptor (IFNAR1), and adaptive immunity in the spontaneous inflammatory lung disease in STING N153S mice.

    Methods: Hence, we crossed STING N153S mice to animals lacking cGAS, IRF3, IRF7, IFNAR1, adaptive immunity, alph/beta T cells, and mature B cells. As read out we evaluated the mice for development of spontaneous inflammatory lung disease.In addition we generated bone marrow chimeric mice and examined severity of the lung disease and survival of the transplanted animals for 322 days.

    Results: We found that spontaneous inflammatory lung disease in STING N153S mice developed independently of cGAS, IRF3, IRF7, and type I IFN signaling. Bone marrow transplantation experiments revealed that certain aspects of STING N153S-associated disease are intrinsic to the hematopoietic system. In additon, we discovered that Rag1-/- STING N153S mice have histologically normal lungs without perivascular infiltrates.Tcr beta-/- STING N153S animals developed a mild lung phenotype.

    Conclusion: Spontaneous inflammatory lung disease in STING N153S mice develops independently of cGAS and type I IFN signaling.STING N153S depends on adaptive immunity to induce inflammatory lung disease in mice.

    Disclosure of Interest

    None Declared

    New frontiers in the treatment of SAID

    O29 Preclinical studies of gene therapy for deficiency of adenosine deaminase type 2 (DADA2)

    Ying Hong, Marina S. Casimir, Barbara Jensen, Benjamin Houghton, Adrian Thrasher, Paul Brogan, Despina Eleftheriou
    Infection, immunity and inflammation, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
    Correspondence: Ying Hong

    Introduction: Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive autoinflammatory disease and vasculitis, caused by loss-of-function mutations in ADA2. Treatment with anti-TNF-α is effective for the autoinflammation and vasculitis of DADA2, but may add to the burden of immunosuppression, is expensive, and is required for life. This treatment may also not effectively treat bone marrow failure or the associated immunodeficiency. Since haematopoietic stem cell transplantation (HSCT) may be curative, but is toxic, we are currently exploring gene therapy for DADA2.

    Objectives: (i) develop specific self-inactivating (SIN) lentivirus vectors encoding ADA2 cDNA; (ii) explore the efficacy of gene transfer using this vector in monocyte derived macrophages (MDM) from DADA2 patients; and in an ADA2 knockout (KO) monocyte cell-line (THP-1) model generated using CRISPR/CAS9.

    Methods: We generated an ADA2-SIN lentivirus under the control of the elongation-1a-factor (EFS) promoter, that we then used to transduce ADA2 KO THP-1/ wild type (WT) control THP-1 cells; or patient and healthy control-derived MDM, at different multiplicity of infection, and examined the following:

    1. (i)

      ADA2 protein expression.

    2. (ii)

      ADA2 enzyme activity

    3. (iii)

      M1/M2 macrophage immunophenotype. MDM and THP-1 differentiated macrophages were incubated with IL-4, IL-10 and IL-13 to obtain M2 polarized macrophages or with IFN-γ/LPS to induce polarization to M1. Immunophenotyping was assessed in transduced or non-transduced M1 and M2 polarised macrophages using qPCR, flow cytometry, and ELISA-cytokine quantification in culture supernatants.

    4. (iv)

      macrophage induced endothelial activation. Transduced or non-transduced MDM from DADA2 patients and healthy controls were also cultured with endothelial cells, and CD62E expression (marker of endothelial activation) was examined with flow cytometry

    Results: In the THP-1 KO cell line we observed:

    1. (i)

      full restoration of ADA2 protein expression and ADA2 enzyme activity in transduced ADA2 KO THP-1 cells compared to non-transduced cells;

    2. (ii)

      amelioration of M1 proinflammatory molecule gene expression: polarized M1 cells derived from transduced ADA2 KO THP-1 cells exhibited a similar gene expression profile for proinflammatory cytokines (TNF-α, CXCL-10, STAT-1, and IL-1β) to that observed in M1 derived from WT control THP-cells; in comparison, non-transduced ADA2 KO THP-1 cells exhibited significant up-regulation of gene expression for these proinflammatory cytokines (p<0.0001).

    Using DADA2 patient derived cells (n=3) we also established:

    1. (iii)

      full restoration of ADA2 protein expression and enzyme activity: transduction of DADA2 MDM led to full restoration of ADA2 protein expression and enzyme activity to levels observed in healthy controls (p=0.0001);

    2. (iv)

      amelioration of M1 proinflammatory gene expression in patient cells: transduced DADA2 MDM were restored to levels seen in healthy control MDM cells for proinflammatory cytokine gene expression (p=0.0001); cytokine production at protein level (p=0.01); and reduced M2 apoptosis (p=0.0001).

    3. (v)

      prevention of endothelial activation: transduction of DADA2 MDM significantly prevented endothelial activation in co-culture experiments, compared with non-transduced DADA2 MDM, that continued to induce significant endothelial activation (p=0.0002).

    Conclusion: We have used a gene therapeutic approach to successfully demonstrate rescue of the immunophenotype of DADA2 using a THP-1 ADA2 KO cell line model; and primary cells from DADA2 patients, thus providing proof of principle that gene therapy might work in patients with DADA2. Next steps now include: 1. gene correction in CD34 cells from DADA2 patients; and 2. safety studies in animal models.

    Disclosure of Interest

    None Declared

    O30 Experience with and management of HLH-like toxicities following chimeric antigen receptor T-cell therapy for treatment of relapsed/refractory pre-B ALL

    Amanda K. Ombrello1, Bonnie Yates2, Haneen Shalabi2, Terry J. Fry3, Nirali N. Shah2
    1NHGRI; 2NCI, NIH, Bethesda; 3Children's Hospital of Colorado, Denver, United States
    Correspondence: Amanda K. Ombrello

    Introduction: Chimeric antigen receptor T-cell (CAR-T) therapy is a highly effective form of adoptive cell immunotherapy combining antigen specific targeting capabilities with T-cell based cytotoxicity. Particularly effective against B-cell antigens (CD19/CD22), CAR-T cell activation leads to a systemic inflammatory response called cytokine release syndrome (CRS) that can further evolve into hemophagocytic histiocytosis (HLH) symptomatology.

    Objectives: To evaluate the presentation, incidence and management of HLH toxicities in children/young adults with relapsed/refractory pre-B acute lymphoblastic leukemia (pre-B ALL) treated on a phase I study of anti-CD22 CAR-T cell therapy (Clinicaltrials.gov NCT02315612).

    Methods: Using modified diagnostic criteria to define CAR-T cell related HLH, it was established in those with ferritin of > 100,000 ng/mL and at least 1 of the following: > grade 3* AST/ALT elevation or hyperbilirubinemia; > grade 3* oliguria or increase in serum creatinine; > grade 3* pulmonary edema; or hemophagocytosis in the bone marrow. Serial inflammatory markers (ferritin, CRP) and serum cytokines were prospectively monitored from CAR-T cell infusion through day +28 (+/-4) and retrospectively analyzed comparing peak values in those who did/did not develop HLH. Treatments included supportive care, glucocorticoids +/- anakinra.

    *Grading as per Common Terminology Criteria for Adverse Events, v4.03

    Results: In 52 subjects, 46 experienced CRS, of whom 37 (80.4%) achieved complete remission and 18 (39.1%) developed HLH. Median ferritin in those with/without HLH was 206740 vs. 22758 (ng/mL) (Table 1). Clinical manifestations included: > grade 3* creatinine (n=2); > grade 3* AST/ALT elevation or hyperbilirubinemia (n=23, including 6 without HLH), > grade 3* pulmonary edema (n=5, including 2 without HLH); and hemophagocytosis in the bone marrow (n=9). Cytokine profiling demonstrated significantly higher levels of IFN-y, IL-1B, IL-6, IL-10, TNFa and MIP-1a (Table 1). Limited paired samples of sIL-2R showed statistically significant increase from baseline (median level 1254) to HLH presentation (median 9310), with 6/9 having substantial increase (peak 123700). All had resolution of HLH symptoms, except 1 who died from gram-negative rod sepsis complications prior to resolution. Three had asymptomatic lab abnormalities that self-resolved (median 14 days) without intervention. Anakinra monotherapy (median 5 mg/kg/day) was used in 3 and the remainder (n=3) received anakinra + steroids. Both regimens resolved HLH. There was no statistically significant difference in underlying leukemia burden or efficacy following CAR-T cell therapy in those who did/did not develop HLH. Notably, use of anakinra +/- steroids did not diminish therapeutic efficacy of CAR-T cells.

    Conclusion: Ferritin and cytokine profiling revealed HLH patients had a different inflammatory response independent of disease burden. Anakinra +/- steroids was effective and did not impede CAR-T cell expansion. Further analysis to identify HLH-predictive parameters and optimizing interventions to treat/prevent these complications are ongoing.

    Disclosure of Interest

    None Declared

    Table 1 (abstract O30). See text for description

    Novel targets and therapies in autoinflammation

    O31 Novel NLRP3 targeted therapy in caps

    Laela M. Booshehri1, Matthew McGeough1, Ben Keer1, Milos Lazic2, Christopher McBride2, Davide Povero2, James Veal2, Gretchen Bain2, Hal M. Hoffman1
    1Pediatrics, University of California San Diego, La Jolla; 2Jecure Therapeutics, San Diego, United States
    Correspondence: Hal M. Hoffman

    Introduction: Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease characterized by a hyperactive inflammasome leading to the overproduction of interleukin-1b (IL-1b). Assembly of the NLRP3 inflammasome is central to the CAPS disease process resulting in subsequent activation and prolific release of inflammatory cytokines, which further propagate inflammation and disease. Current therapies for CAPS patients directly target IL-1b or IL-1 receptor to mitigate excess inflammation caused by NLRP3 activation. While there are a number of new compounds in pre-clinical development that target NLRP3 directly, there is little data on the efficacy of these compounds in CAPS versus healthy controls.

    Objectives: To study the ex vivo efficacy of a novel NLRP3 selective small-molecule inhibitor compound 1 (C1) in monocytes from CAPS patients with multiple Nlrp3 mutations as compared to healthy controls and in bone marrow derived dendritic cells from murine Nlrp3 mutant CAPS models as compared to wild type mice, with the goal of identifying an effective NLRP3 specific inhibitor for CAPS patients.

    Methods: Peripheral blood mononuclear cells from 7 CAPS patients with 6 different NLRP3 mutations and 4 healthy donor controls were isolated by gradient centrifugation, and monocytes were allowed to adhere for 4 hours prior to treatment and stimulation. Samples were obtained under an approved Institutional Review Board protocol for human subjects. Bone marrow was isolated from MWS Nlrp3A350V/+ CreT, FCAS Nlrp3L351P/+ CreT, and NOMID Nlrp3D301N/+ CreT conditional knock-in mice and cells were cultured with GMCSF for 1 week and treated with tamoxifen 1 day prior to drug treatment to induce expression of the mutation. Cells were treated with C1 at varying concentrations prior to stimulation with LPS (mutants) or LPS and ATP (controls), and supernatants were collected after overnight incubation for analysis of IL-1b by ELISA. C1 was also orally administered to MWS Nlrp3A350V/+ CreT mice prior to in vivo tamoxifen administration to determine in vivo efficacy and pharmacodynamics. Whole body and spleen weights were measured and blood was obtained for complete blood counts in treated and untreated mice. Animal studies were performed in accordance with the University of California San Diego and IACUC policies and procedures.

    Results: Robust inhibition of IL-1b release from C1 treated cells was shown with comparable activity across multiple human NLRP3 mutations and murine Nlrp3 mutant models. Compound efficacy and pharmacodynamics were also shown to be similar between CAPS mutant cells and cells from respective healthy donors or wild-type controls. Daily oral administration of C1 was well tolerated and demonstrated efficacy in preventing weight loss, splenomegaly, and neutrophilia in MWS Nlrp3A350V/+ CreT mice.

    Conclusion: The novel NLRP3 inhibitor C1 was shown to significantly reduce LPS induced IL-1b release in cells from CAPS patients and murine Nlrp3 mutant models indicating direct and effective inhibition of mutant NLRP3. C1 and other emerging NLRP3 inhibitors present an additional avenue for future CAPS patient therapy by directly targeting the inflammasome. Similar efficacy of C1 on both normal and mutant NLRP3 likewise indicate potential applications in other inflammatory diseases beyond CAPS.

    Disclosure of Interest

    None Declared

    O32 Preclinical efficacy of NLRP3 small molecule inflammasome inhibitors: implications for future treatment of autoinflammatory syndromes

    Angela Abad-Perez1, Stefan Frischbutter1,2, Niklas A. Mahnke1, Jens v. Kries3, Marc Nazaré4, Marcus Maurer1, Jörg Scheffel1,2, Karoline Krause1,2
    1Department of Dermatology, Venereology and Allergology; 2Autoinflammation Reference Center Charité (ARC2), Charité Universitätsmedizin Berlin; 3Screening Unit Cell Biology and High Content Screen; 4Medicinal Chemistry, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
    Correspondence: Angela Abad-Perez

    Introduction: Systemic autoinflammatory diseases (SAIDs) are characterized by abnormally increased inflammation affecting different organs. They are mediated predominantly by the cells and molecules of the innate immune system. Inflammasome activation represents the critical pathogenic mechanism shared by most SAIDs. Current treatment strategies are limited to downstream cytokine blockade. Specific inflammasome inhibitors are not available so far.

    Objectives: To address this unmet medical need, we performed a high content screening of more than 60.000 small molecules from the compound collection of the …Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)”, which includes the ChemBioNet and LOPAC®1280 libraries as well as donations of academic chemists, FDA approved drugs (Selleck library) and a natural product collection from AnalytiCon Discovery.

    Methods: For the primary screen, we used a fluorescent murine inflammasome reporter cell line to detect ASC speck formation, a marker of inflammasome activation. Compounds were selected based on their inhibitory capacity on ASC speck formation, as observed by automated fluorescence microscopy, and IL-1ß release after activation with canonical NLPR3-inflammasome inducers ATP and nigericin. The 10 most potent and druggable hit compounds were tested in peripheral blood mononuclear cells (PBMCs) obtained from venous blood of patients with Schnitzler syndrome (N=8), familial Mediterranean fever, FMF (N=8) and from unmatched healthy donors (N=18). In vitro effect on cellular IL-1ß release was measured by ELISA.

    Results: Selected compounds proved to efficiently inhibit the secretion of IL-1ß in PBMCs from both patients and healthy controls in a dose dependent manner, validating their inhibitory capacities in human and murine cellular assays. Among these compounds were known anti-inflammatory drugs such as auranofin and a VEGFR2 tyrosine kinase inhibitor. The median inhibitory capacity upon stimulation with lipopolysaccharide (LPS) and ATP at 10 μM ranged between 50% to 80% with IC50s in the low μM region. A similar inhibitory profile could be observed for the previously reported inflammasome inhibitor MCC950, which was included in our assays as a reference substance. Moreover, compounds had similar efficacy in inflammasome inhibition PBMCs obtained from patients and healthy controls.

    Conclusion: Based on our results in murine and human cells in vitro, small molecule inflammasome inhibitors my complement current treatment options for SAIDs in the future.

    Disclosure of Interest

    None Declared

    Oral communications – new diseases

    O33 Biallelic loss of function mutations in sharpin cause autoinflammation

    Hirotsugu Oda1, David Beck1, Kalpana Manthiram1, Hye Sun Kuehn1, Natalia Sampaio Moura1, Rao Anand2, Mariana Kaplan1, Douglas Kuhns3, Wanxia Li Tsai1, Hiroyuki Yoshitomi4, Junya Toguchida4, Gustavo Gutierrez-Cruz1, Jeremy Davis1, Massimo Gadina1, Jennifer Stoddard1, Kazuhiro Iwai4, Sergio Rosenzweig1, Luigi Notarangelo1, Daniel L. Kastner1, Ivona Aksentijevich1
    1NIH, Bethesda, United States; 2Manipal Hospital, Bangalore, India; 3NIH, Frederick, United States; 4Kyoto University, Kyoto, Japan
    Correspondence: Hirotsugu Oda

    Introduction: The linear ubiquitination chain assembly complex (LUBAC) consists of HOIP, HOIL1 and SHARPIN and mediates linear ubiquitination. LUBAC is essential for NF-κB signaling and thus proper innate and adaptive immunity. Patients with HOIP and HOIL1 deficiencies have been reported to have immunodeficiency, autoinflammation and amylopectinosis. Although mice deficient in Sharpin have severe TNF-dependent skin inflammation due to enhanced apoptosis and necroptosis of the keratinocytes, the role of SHARPIN in human diseases is unknown.

    Objectives: We aimed to investigate a 14 year-old boy from a consanguineous family in India with polyarthritis, parotitis, hepatosplenomegaly and colitis associated with anorectal fistula, but without skin manifestations or any history of severe infections. The patient’s symptoms dramatically improved on anti-TNF therapy.

    Methods: We performed whole exome sequencing to identify the genetic cause of the patient's phenotypes.

    Results: We identified a homozygous frameshift mutation in SHARPIN in our proband (c.220dupC). Patient derived dermal fibroblasts have no detectable SHARPIN protein with markedly reduced HOIP, and HOIL1 suggesting destabilization of the LUBAC complex. These cells also displayed impaired canonical NF-κB activity, as exemplified by induction of IκBα phosphorylation and nuclear translocation of p65. However, in contrast to HOIP and HOIL1 deficiencies, the patient’s monocytes did not show hyperresponsiveness to IL-1β stimulation.SHARPIN deficient patient fibroblasts demonstrated enhanced apoptosis induced by FAS stimulation as compared to control cells, which parallels the enhanced apoptosis observed in the Sharpin deficient mice. We knocked out SHARPIN in a human immortalized osteoblast cell line (hFOB1.19) and interestingly, despite attenuated NF-κB activity, these cells secrete higher amounts of IL-6 more rapidly after IL-1β stimulation than control cells.

    Conclusion: We identified the first case of human SHARPIN deficiency in a patient with autoinflammation. Molecular consequences of the SHARPIN deficiency are currently being investigated in comparison to other LUBAC deficiencies.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    O34 A loss-of-function mutation in USP43, a deubiquitinase gene, is linked to an interferon-mediated autoinflammatory disorder with proteasome defects

    Hongying Wang1, Qing Zhou1, Anna Kozlova2, Vasili Burlakov2, Daniel Kastner1, Ivona Aksentijevich1, Anna Shcherbina2
    1Inflammatory Disease Section, National Human Genome Research Institute (NHGRI) / NIH, Bethesda, United States; 2National Research and Practical Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation
    Correspondence: Hongying Wang

    Introduction: Deubiquitinase enzymes (DUBs) function in the removal of poly-ubiquitin chains from substrate proteins to regulate their activity and degradation by the ubiquitin-proteasome system (UPS). Deficiency of DUB activity may lead to excessive immune signaling as has been observed in patients with haploinsufficiency of A20 (HA20) and OTULIN deficiency. Through whole exome sequencing analysis (WES), we identified a novel homozygous missense mutation (c. 2509G>A; p. E837K) in a deubiquitinase encoding gene USP43, in a Russian patient of Tatar ancestry. The patient presented with early-onset recurrent fevers, rash, subcutaneous skin nodules, lipodystrophy, and prominent arthritis, and this phenotype was suggestive of the CANDLE syndrome.

    Objectives: USP43 is a poorly characterized ubiquitin specific protease (USP).We aimed to study the disease-causing mechanism underlying this novel mutation, E837K, as well as the biological function and targets of USP43.

    Methods: We performed WES in the patient’s family and RNA sequencing in whole blood samples. We generated a fibroblast cell line and EBV-transformed B cells from the patient’s primary cells. We used USP43 knockdown and CRISPR/Cas9 knockout in 293T cells to study the effects of the USP43 depletion. A series of USP43 truncated mutants were generated to study the effect of protein domains on its DUB activity. Immunoprecipitation and immunoblot, luciferase assays, serum and plasma cytokine profiling, immunofluorescence, real-time PCR, and flow cytometry were used to investigate abnormalities in patient-derived cells.

    Results: We found that the novel USP43 mutation leads to an upregulation in interferon signaling and causes an impairment in the proteasome-mediated protein degradation pathway. The patient’s EBV-transformed B cells had increased phospho-STAT levels in response to interferon stimulation and spontaneously produced a significantly higher level of IL-6. This cellular phenotype was rescued by transfection with wild-type USP43, which suggests that this mutation is loss-of-function.The patient’s primary cells showed decreased proteasome activity and excessive accumulation of K48-ubiquinated proteins following stimulation with a proteasome inhibitor MG132. Similarly, transient knockout of USP43 in 293T cells led to increased levels of ubiquitinated proteins. Reintroducing wildtype USP43 to EBV-transformed patient B cells markedly decreased the expression of ubiquitinated proteins. These data suggest that the mutant USP43/E837K protein loses the ability to remove K48-ubiquitin chains from target proteins. Overexpression of a series of truncated USP43 mutants with K-48 Ub chains in 293T cells confirmed that the C-terminal domain is required for the DUB function of USP43. In addition, USP43 mutants possessing the E837K mutation lost the ability to clear accumulated K48-Ub chains. Interferon-stimulated patient’s EBV cells and fibroblasts showed decreased protein levels of PSMB8.PSMB8 encodes the catalytic subunit of the immunoproteasome. Co-transfection of USP43/E837K mutant with PSMB8 in 293T cells reduced the expression of PSMB8 precursor protein compared to cells transfected with USP43 wild type.

    Conclusion: Our data suggest that the loss-of-function mutation in USP43 decreases immunoproteasome activity and causes an upregulation in type I interferon signaling, similar to what is observed in patients with CANDLE. Treatment with a JAK inhibitor has been very effective in controlling the disease activity in this patient. To our knowledge, this is the first report of a human disease caused by mutation in USP43.

    Disclosure of Interest

    None Declared

    O35 A novel autoinflammatory disease characterized by neonatal-onset cytopenia with autoinflammation, rash, and hemophagocytosis (NOCARH) due to aberrant CDC42 function

    Michael T. Lam1,2,3, Simona Coppola4, Oliver H. Krumbach5, Giusi Prencipe6, Antonella Insalaco6, Cristina Cifaldi7,8, Immacolata Brigida9, Serena Scala9, Marcello Niceta10, Andrea Ciolfi10, Alexandre F. Carisey1,2, Mohammad Akbarzadeh5, Andrea Finocchi7,8, Franco Locatelli11, Caterina Cancrini7,8, Alessandro Aiuti9,12,13, Mohammad R. Ahmadian5, Jordan S. Orange2, Fabrizio De Benedetti6, Marco Tartaglia10
    1Department of Pediatrics, Baylor College of Medicine, Houston; 2Department of Pediatrics, Columbia University, Irving Medical Center, New York; 3Translational Biology and Molecular Medicine Graduate Program and Medical Scientist Training Program, Baylor College of Medicine, Houston, United States; 4National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy; 5Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, Düsseldorf, Germany; 6Division of Rheumatology; 7Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, IRCCS; 8Department of Systems Medicine, University of Rome Tor Vergata, Rome; 9San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan; 10Genetics and Rare Diseases Research Division; 11Department of Pediatric Hematology and Oncology, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome; 12Pediatric Immunohematology, San Raffaele Scientific Institute; 13Vita Salute, San Raffaele University, Milan, Italy
    Correspondence: Antonella Insalaco

    Introduction: Despite continuous advances in the identification of novel causative genes, several patients with a clinical autoinflammatory phenotype remain unclassifiable.

    Objectives: to describe a novel hematological and autoinflammatory disorder in three unrelated patients caused by a de novo missense mutation of CDC42

    Methods: Whole exome sequencing was used to identify the novel variant. The functional impact of altered CDC42 function on hematopoiesis and inflammation was assessed through patient peripheral blood and bone marrow analyses, protein behavior and immune and non-immune cell functioning through in vitro biochemical and functional assays and in vivo C. elegans modeling.

    Results: Patients shared the same de novo missense mutation of CDC42 (NM_001791, Chr1:22417990, c.556C>T, p.R186C).Disease features included neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and hemophagocytosis (collectively termed NOCARH syndrome) (Table). An altered hematopoietic compartment (prevalence of early differentiation elements and substantially decreased clonogenic progenitors) was demonstrated. Complementary assays documented the unique consequences of this mutation on CDC42 localization and function, and its disruptive effect on cell behavior and developmental processes, possibly linked to actin dysregulation. Increased secretion of IL-1β, and particularly of IL-18, was observed via ex vivo spontaneous release from unstimulated bone marrow mononuclear cells and by high levels in bone marrow supernatants and plasma. IFNγ was alsoincreased and correlated to CXCL9 levels which were strictly related to ferritin levels. Treatment with anakinra and emapalumab, a monoclonal antibody to IFNγ, was identified as critical in the survival of one patient, who underwent successful hematopoietic stem cell transplantation.

    Conclusion: The p.R186C amino acid substitution in CDC42 underlies a novel, unique syndrome where CDC42 functional dysregulation has pleiotropic effects, causing hematopoietic disturbance, hyperinflammation, and immune impairment. Early recognition and control of HLH, through neutralization of IFNγ, followed by hematopoietic stem cell transplantion, appear to be crucial to survival.

    Disclosure of Interest

    None Declared

    Table 1 (abstract O35). See text for description

    O36 PSMB10, The last immunoproteasome gene missing for PRAAS (Proteasome-Associated Autoinflammatory Syndrome)

    Guillaume Sarrabay1,2, Déborah Méchin1,2, Aicha Salhi3, Guilaine Boursier1, Cécile Rittore1, Yanick Crow4, Gillian Rice5, Tu-Ahn Tran2,6,7, Renaud Cezar7, Darragh Duffy8, Vincent Bondet8, Lakhtar Boudehane9, Sylvie Grandemange1,2, Florence Apparailly2, Isabelle Touitou1,2
    1Department of Medical Genetics, Rare diseases and Personalized medicine, Rare and Autoinflammatory diseases unit; 2IRMB, INSERM, CHU Montpellier, Univ Montpellier, Montpellier, France; 3Dermatology department, Alger medicine University, Alger, Algeria; 4Laboratory of Neurogenetics and Neuroinflammation, Institut Imagine, Paris Descartes University, Paris, France; 5Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom; 6Paediatrics department, University Hospital Nimes; 7Immunology department, CHU Nîmes, Univ Montpellier, Nîmes; 8ICD Unit, Inserm U1223, Institut Pasteur, Paris, France; 9Paediatrician office, Liberal, Sétif, Algeria
    Correspondence: Guillaume Sarrabay

    Introduction: PRAAS defines a clinical spectrum encompassing JMP (joint contractures, muscle atrophy, microcytic anemia and panniculitis-induced childhood-onset lipodystrophy syndrome), NNS (Nakajo-Nishimura syndrome) and CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature). PRAAS is caused by autosomal recessive, autosomal dominant, or digenic mutations in several genes encoding for either constitutive proteasome subunits (PSMB4, PSMA3), immunoproteasome subunits (PSMB8, PSMB9) or chaperone protein (POMP). We describe here a 3-year-old female patient with clinical features evocative of PRAAS in whom no mutations have been found using our 62 gene panel sequencing that includes known PRAAS genes.

    Objectives: The aim of the study was to identify the molecular cause responsible for the PRAAS phenotype in this patient.

    Methods: We performed a trio-based whole exome sequencing (WES) in the patient and her parents. Functional assays were conducted to confirm the pathogenic effect of the mutated candidate gene. They included: enzymatic protease activity in the patient’s peripheral blood monocyte cells (PBMC) and in transfected HEK293T cells, interferon (IFN) signature and IFNα dosage, multiplexed cytokines measurement from patient’s serum, and protein maturation assays by WB (western-blot) analyses in transfected wild-type and mutant HEK293T cells.

    Results: The patient is a 3-year-old female patient of Algerian descent, born to related parents. She developed a cutaneous rash on the 7th day of life and became febrile at the age of one year. The rash was polymorphic, annular shaped and predominantly periorbital She failed to thrive and has long-lasting hepatosplenomegaly. She has an emaciated face, a distinctive nose, and long and gracile fingers. She had elevated acute phase reactants, microcytic anemia and hypertriglyceridemia. She exhibited partial response to steroid and methotrexate treatment and relapsed when the doses were lowered. WES revealed a homozygous missense mutation in the candidate gene PSMB10, located in the N-terminal part of protein which is cleaved in the mature form. This variant is absent from the GnomAD cohort, and predicted to be pathogenic according to in silico bioinformatic tools. The patient had a positive interferon signature and elevated IFNα protein in the serum on the one occasion tested. Cytokines multiplexed measurement showed raised IL-6, TNFα, MIG and MCP-3 in the patient’s serum whereas IL-1β level was similar to healthy pediatric controls. WB assays in HEK293T cells showed defective cleavage of the mutant protein upon IFNγ induction compared to the wild-type protein. Patient’s PBMC and mutant HEK293T cells showed an alteration in trypsin-like proteasome activity.

    Conclusion: We report here a patient with clinical and biological criteria consistent with PRAAS, with a homozygous PSMB10 mutation. This is the third and last immunoproteasome subunit involved in this disease, and this new gene responsible for PRAAS expands the number of genes involved in this spectrum.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    O37 WNT6 mutation causes an early onset granulomatosus intestinal disease with recurrent hemophagocytic lymphohistiocytosis (HLH)

    Claudia Bracaglia1, Daniela Knafelz2, Fiammetta Bracci2, Antonella Insalaco1, Giulia Marucci1, Manuela Pardeo1, Giusi Prencipe1, Ivan Caiello1, Antonia Pascarella1, Marcello Niceta3, Francesca Pantaleoni3, Andrea Ciolfi3, Bronislava Papadatou2, Marco Tartaglia3, Giuliano Torre2, Fabrizio De Benedetti1
    1Division of Rheumatology; 2Hepatology, Gastroenterology and Nutrition Unit; 3Genetics and Rare Diseases Research Division, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy
    Correspondence: Claudia Bracaglia

    Introduction: Use of NGS in patients with unclassifiable disease lies a possible approach to the identification of novel disease causing genes.

    Objectives: We report a patient with an early onset inflammatory bowel disease with granulomatous lesions and recurrent HLH episodes carrying a missense mutation in the WNT6 gene.

    Methods: A trio based Whole Exome Sequencing (WES) approach was used. Cytokine levels were measured by multiplex assay and by specific ELISAs.

    Results: Ten years old Caucasian boy affected by early onset pan-colitis from 9 months of age. Since the disease onset the patient is on glucocorticoid treatment with amino acidic enteral nutrition and oligo antigenic diet. Because of recurrent disease relapses at any attempt of glucocorticoid withdrawal, azathioprine and cyclosporine treatments were also added. At 2 years of age he received total colectomy with ileostomy. Because of insufficient disease control, treatment with a TNF-inhibitor (infliximab) was started with apparent improvement of intestinal symptoms.However, persistent granulomatous inflammatory disease of the distal portion of the ileus-rectal anastomosis persisted. Moreover, the patient presented recurrent HLH episodes that required high dose of glucocorticoid and cyclosporine-A treatment. Except one HLH episode related to a varicella zoster infection, the other HLH events were most likely triggered by his underlying inflammatory condition. During the HLH episodes levels of IL-18 were moderately elevated (10.880 pg/ml) the IFN-gamma induced chemokine CXCL9 was markedly high (21.871 pg/mL) and remained markedly elevated also during clinical and laboratory HLH remission (3.121 pg/ml and 9.929 pg/ml respectively). Considering the early disease onset, primary immunodeficiency and early intestinal bowel disease onset were genetically ruled out as well as chronic granulomatosis diseases through extensive NGS panels. WES revealed carriage of a private (MAF: 1/125568, TOPMED), predicted pathogenic (CADD: 31), homozygous variant of WNT6 (c.793G>C; p.(Asp265His); NM_006522.3). The patient is now partially controlled on low dose of oral glucocorticoid (0.1 mg/kg), cyclosporine-A (5mg/kg) and antimicrobic treatment.

    Conclusion: WNT signalling has been primarily described as a regulatory pathway in ontogeny and homeostatic processes. Schaale at al. demonstrated that WNT6 is expressed in granulomatous lesions in the lung of Mycobacterium tuberculosis–infected mice. Moreover, they found that the transcription factor c-Myc is significantly induced in murine macrophages by WNT6. This identifies WNT6 as a novel factor driving macrophage polarization toward an M2-like phenotype, suggesting a role for WNT6 in macrophage differentiation. Our case suggests defective function of WNT6 might be involved in the development of a granulomatous disease. WNT6 role in macrophage differentiation and polarization might also be important in the activation of the IFN-gamma pathway and in recurrent HLH episodes.

    Reference

    K. Schaale et al. Wnt6 Is Expressed in Granulomatous Lesions of Mycobacterium tuberculosis–Infected Mice and Is Involved in Macrophage Differentiation and Proliferation. J Immunol 2013; 191:5182-5195.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    C. Bracaglia: None Declared, D. Knafelz: None Declared, F. Bracci: None Declared, A. Insalaco: None Declared, G. Marucci: None Declared, M. Pardeo: None Declared, G. Prencipe: None Declared, I. Caiello: None Declared, A. Pascarella: None Declared, M. Niceta: None Declared, F. Pantaleoni: None Declared, A. Ciolfi: None Declared, B. Papadatou: None Declared, M. Tartaglia: None Declared, G. Torre: None Declared, F. De Benedetti Grant / Research Support from: Novartis, Novimmune, Hoffmann- La Roche, SOBI, AbbVie, Pfizer

    O38 NFIL3 mutations alter immune homeostasis and sensitize for arthritis pathology

    Stephanie Humblet-Baron1, Susan Schlenner2, Emanuela Pasciuto2, Vasiliki Lagou1, Oliver Burton2, Teresa Prezzemolo1, Steffie Junius1, Carlos Roca1, Cyril Seillet3, Cynthia Louis3, James Dooley1, Kylie Luong3, Erika Van Nieuwenhove1, Ian P Wicks3, Gabrielle Belz3, Adrian Liston1, Carine Wouters4
    1Immunology and Microbiology, Center for Brain and disease research, KU Leuven-VIB; 2Immunology and Microbiology, Center for Brain and disease research, KU Leuven - VIB, LEUVEN, Belgium; 3Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; 4KU Leuven and University Hospitals Leuven, LEUVEN, Belgium
    Correspondence: Stephanie Humblet-Baron

    Introduction: Juvenile idiopathic arthritis (JIA) is the most common of the childhood rheumatic diseases. JIA is characterized as juvenile-onset persistent arthritis with no defined cause. A high degree of clinical heterogeneity is observed within the JIA group of diseases, thought to reflect a diversity in genetic and environmental factors and mechanistic drivers. JIA shows similarities to adult autoimmune diseases,but also has similarities to autoinflammatory diseases, such as genetic associations to innate inflammatory pathways and response to IL-1β blockade. The recent success in identifying monogenic causes of autoinflammatory diseases suggests that monogenic causes may also underlie a subset of JIA patients.

    Objectives: NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans.

    Methods: Here we characterized a kindred of two monozygotic twin girls with juvenile idiopathic arthritis at the genetic and immunological level, using whole exome sequencing, single cell sequencing and flow cytometry. Parallel studies were performed in a mouse model.

    Results: The patients inherited a novel p.M170I in NFIL3 from each of the parents. The mutant form of NFIL3 demonstrated reduced stability in vitro. The potential contribution of this mutation to arthritis susceptibility was demonstrated through a pre-clinical model, where Nfil3-deficient mice upregulated IL-1β production, with more severe arthritis symptoms upon disease induction. Single cell sequencing of patient blood quantified the transcriptional dysfunctions present across the peripheral immune system, converging on IL-1β as a pivotal cytokine.

    Conclusion: NFIL3 mutation can sensitize for arthritis development, in mice and humans, and rewires the innate immune system for IL-1β over-production.

    Disclosure of Interest

    None Declared

    O39 EROS/CYBC1 mutations: a novel cause of chronic granulomatous disease and more

    David C. Thomas1, Louis M. Charbonnier2, Andrea Schejtman3, Hasan Aldhekri4, Eve Coomber5, Elizabeth Dufficy6, Anne Beenken1, James Lee1, Simon Clare7, Anneliese Speak7, Adrian Thrasher8, Giorgia Santilli8, Hamoud Al-Mousa9, Fowzan Alkuraya10, Talal Chatila11, Kenneth Smith1
    1Department of Medicine, University of Cambridge, Cambridge, United Kingdom; 2Paediatrics, Harvard, Boston, United States; 3University College London, London, United Kingdom; 4Department of Paediatrics,King Faisal Specialist Hospital and Research Center , Riyadh, Saudi Arabia; 5Wellcome Trust Sanger Institute, Cambridge; 6Medicine, University of Cambridge, Camridge; 7WTSI, Cambridge; 8Institute of Child Health, UCL, London, United Kingdom; 9Paediatrics; 10Genetics, KFSH, Riyadh, Saudi Arabia; 11Paediatrics, Harvard University, Boston, United States
    Correspondence: David C. Thomas

    Introduction: The multi-subunit phagocyte nicotinamide adenine dinucle- otide phosphate oxidase generates reactive oxygen species and is crucial for host defence. Deficiencies in individual subunits (gp91phox, p22phox, p47phox, p67phox, and p40phox) cause chronic granulomatous disease (CGD), but some patients with CGD do not have mutations in these genes. We recently found that Eros, a hitherto undescribed protein, is essential for the generation of reactive oxygen species because it is necessary for protein (but not mRNA) expression of the gp91phox-p22phox heterodimer, which is almost absent in Eros-deficient mice. Eros-/- animals succumb quickly following infection with Salmonella typhimurium or Listeria monocytogenes. Eros is highly conserved and has a human orthologue CYBC1 (alias C17ORF62), hereafter referred to asCYBC1 gene and essential for reactive oxygen species (EROS) protein.

    Objectives: We asked:

    1. 1.

      Whether the gene fulfilled the same function in humans.

    2. 2.

      Whether mutaions in juman EROS/CYBC1/C17ORF62 could cause a human disease

    Methods: We performed CRISPR-mediated deletion of CYBC1/EROS in PLB-985 cells and identified 2 clones with 8 bp and 1 bp deletions, respectively. Neither clone expressed detectable EROS protein. We also identified a patient with a homozygous EROS/CYBC1/C17ORF62 who was subsequently diagnosed with chronic granulomatous disease secondary to this mutation.

    Results: We show that the function of CYBC1/EROS is conserved in human cells. Knockout of EROS in cell lines or primary human ips derived macrophages results in abswnt gp91phox-p22phox expression and aboloishges the phagocyte respiratory burst.We also describe a case of CGD secondary to a homozygous CYBC1/EROSmutation that abolishes EROS protein expression. This work demonstrates the fundamental importance of CYBC1/EROSin human immunity and describes a novel, 6thcause of CGD.

    However, EROS also regulates the expression of other proteins. Eros-/- macrophages also express very low levels of P2X7, a ligand gated ion channel that functions as a danger receptor by binding extracellular ATP released from damaged or dying cells. and driving activation of the NLRP3 inflammasome. We show that Eros co-immunoprecipiates with P2X7 and that P2X7 driven calcium flux and inflammasome activation are markedly abnormal in Eros-/- cells. Eros also affects T cell biology, underlining key roles beyond NADPH oxidase activation.

    Conclusion: This work demonstrates the fundamental importance of CYBC1/EROSin human immunity and describes a novel, 6thcause of CGD as well as highlighting role of EROS that are independent of the geneartion of reactive oxygen species.

    Disclosure of Interest

    None Declared

    O40 Cold-induced urticarial autoinflammatory syndrome related to factor XII activation

    Jörg Scheffel1, Niklas Mahnke1, Zonne Hofman2, Steven de Maat2, Jim Wu1, Hanna Bonnekoh1, Reuben Pengelly3, Sarah Ennis3, John Holloway3, Martin Church1, Marcus Maurer1, Coen Maas2, Karoline Krause1
    1Charite - Universitaetsmedizin Berlin, Berlin, Germany; 2University Medical Center Utrecht, Utrecht, Netherlands; 3University of Southampton, Southampton, United Kingdom
    Correspondence: Karoline Krause

    Introduction: Early onset cold-induced urticarial rash with systemic inflammatory symptoms are hallmarks of hereditary autoinflammatory diseases caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). However, in many cases genetic tests are negative, suggesting the existence of unrecognized genetic variants.

    Methods: We studied eight members of a four-generation family, four of whom were affected. Genetic analysis involved exome sequencing followed by targeted Sanger sequencing. Functional analyses included immunoblotting, mononuclear cell stimulation and immunohistochemistry. We generated recombinant protein variants and assessed cytokines and proteins in plasma and skin.

    Results: Affected patients had cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identified a novel deleterious variant in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Occurrence of the mutation segregated with disease status. Immunoblotting for FXII exhibited a distinct 50kDa band that was also present in recombinant W268R-mutated proteins suggesting unusual fragmentation and spontaneous activation of FXII. Furthermore, we observed contact system activation with reduced plasma prekallikrein and profound cleavage of high molecular weight kininogen, representing bradykinin production. Skin and blood neutrophils were found to be a prominent source of FXII. Interleukin-1ß (IL-1ß) was upregulated in lesional skin and in mononuclear cells of healthy donors exposed to recombinant proteins. In accordance with these findings, treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduced disease activity in patients.

    Conclusion: We identified a novel autoinflammatory syndrome characterized by a substitution in the F12 gene resulting in activation of the contact system and cytokine-mediated inflammation.

    Disclosure of Interest

    None Declared

    Poster presentations – Monday 1 April

    Guided poster tour 1A

    PT1A01 Multi-omics analysis of ADA2 deficiency in Japanese cohort

    Hiroshi Nihira1, Kazushi Izawa1, Takahiro Yasumi1, Moeko Ito2, Sachiko Iwaki-Egawa2, Yoji Sasahara3, Hirokazu Kanegane4, Tadateru Yasu5, Tomohiro Kubota6, Syuji Takei6, Dai Keino7, Etsuro Nanishi8, Hidetoshi Takada9, Shoichi Ohga8, Syunsuke Kajikawa10, Makio Takahashi11, Naoko Nakano12, Osamu Ohara13, Toshio Heike14, Junko Takita1, Ryuta Nishikomori1
    1Pediatrics, Kyoto University, Kyoto; 2Life Sciences, Hokkaido University of Science, Sapporo; 3Pediatrics, Tohoku University, Sendai; 4Pediatrics, Tokyo Medical and Dental University, Tokyo; 5Pediatrics, Nagasaki Medical Center, Omura; 6Pediatrics, Kagoshima University, Kagoshima; 7Pediatrics, St. Marianna University, Kawasaki; 8Pediatrics, Kyusyu University, Fukuoka; 9Pediatrics, Tsukuba University, Tsukuba; 10Neurology, Kyoto University, Kyoto; 11Neurology, Osaka Red Cross Hospital, Osaka; 12Pediatrics, Ehime University, Toon; 13Applied Genomics, Kazusa DNA Research Institute, Kisarazu; 14Pediatrics, Hyogo Prefectural Amagasaki General Medical Center, Amagasaki, Japan
    Correspondence: Hiroshi Nihira

    Introduction: Adenosine deaminase type 2 deficiency (DADA2) is caused by recessive loss-of-function variants in ADA2. Most of DADA2 patients reveal systemic vasculopathy consistent with polyarteritis nodosa and large phenotypic variability has been reported [1, 2, 3]. However, pathogenesis of DADA2 remains unclear.

    Objectives: The objective of this study is to reveal clinical and genetic characteristics of Japanese DADA2 patients, and to gain insight into the pathogenesis of DADA2 by multi-omics analysis.

    Methods: We performed the genetic analysis of the ADA2 gene and measured ADA2 activity of the patients from 2016 to 2018 in Japan. Multi-omics analysis had been done in 4 out of 8 DADA2 patients and 4 healthy donors using their peripheral blood mononuclear cells (PBMCs). The samples were taken before and after introduction of anti-TNFα agents (meaning acute and remission phase) in the patients.

    Results: We found 8 DADA2 patients. In this cohort, central neurological manifestations were present in 5 (63%), including asymptomatic small lacunar infarction. Seven subjects (88%) had livedo racemose, but there was no digital ulcer or necrosis. Low levels of IgG and IgM were revealed in 3 (37.5%) and 5 (62.5%) patients respectively, but there was no recurrent infectious episode in this case series. There were two (25.0%) who revealed pure red cell aplasia (PRCA); one revealed only anemia without any inflammation, the other revealed anemia transiently and recovered from it spontaneously but he revealed chronic inflammation afterwards. All 8 patients received anti-TNFα agent and all except one with CsA-dependent PRCA were well controlled.

    We identified 6 previously described and 4 novel variants in ADA2, which included two that we reported before [3]. Overexpression of ADA2 variant constructs in HEK 293 cells showed that some variants had comparable protein expression levels to wild-type in cell lysate but most of them were not secreted and all the variants had low or absent ADA2 enzyme activities.

    In multi-omics analysis, differentially expressed (DE) genes were analyzed at the mRNA and protein levels. We found 64 and 58 genes that were differentially expressed in acute phase vs control and remission phase vs control in common at the transcriptome and proteome levels respectively. Gene ontology analysis of these datasets revealed constitutive up-regulation of type 1 and type 2 interferon pathway. Some genes were common to both datasets.

    Conclusion: We have found 8 DADA2 patients in Japan and identified some novel disease-causing variants. Using multi-omics analysis, we also have found differentially expressed genes in DADA2 patients. Some genes were consistently up-regulated even in the remission period. This may provide further insights into the pathogenesis of DADA2.

    References

    [1] Zhou Q., et al. N Engl J Med, 2014.

    [2] Navon Elkan P., et al. N Engl J Med, 2014.

    [3] Meyts I., Aksentijevich I. J Clin Immunol, 2018.

    [4] Nihira H., et al. Scand J Rheumatol, 2017.

    Disclosure of Interest

    None Declared

    PT1A02 The clinical and immunological profiles of haploinsufficiency of A20 in Japan

    Hidenori Ohnishi1, Tomonori Kadowaki1, Norio Kawamoto1, Tomohiro Hori1, Kenichi Nishimura2, Chie Kobayashi3, Tomonari Shigemura4, Shohei Ogata5, Yuzaburo Inoue6, Tomoki Kawai7, Eitaro Hiejima7, Kazushi Izawa7, Tadashi Matsubayashi8, Kazuaki Matsumoto9, Masatoshi Takagi9, Kohsuke Imai9, Ryuta Nishikomori7, Shuichi Ito2, Toshio Heike7, Osamu Ohara10, Tomohiro Morio11, Hirokazu Kanegane12, Toshiyuki Fukao1
    1Pediatrics, Gifu University Graduate School of Medicine, Gifu; 2Pediatrics, Yokohama City University, Kanagawa; 3Child Health, Faculty of Medicine, University of Tsukuba, Ibaraki; 4Pediatrics, Shinshu University School of Medicine, Matsumoto; 5Pediatrics, Kitasato University Hospital, Kanagawa; 6Allergy and Rheumatology, Chiba Children’s Hospital, Chiba; 7Pediatrics, Kyoto University Hospital, Kyoto; 8Pediatrics, Seirei Hamamatsu General Hospital, Shizuoka; 9Community Pediatrics, Perinatal and Maternal Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo; 10Applied Genomics, Kazusa DNA Research Institute, Chiba; 11Pediatrics and Developmental Biology; 12Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
    Correspondence: Hidenori Ohnishi

    Introduction: A20, encoded by the TNFAIP3gene, is a negative regulator of the tumor necrosis factor (TNF)-nuclear factor (NF)-κB signaling pathway. Recently, the haploinsufficiency of A20 (HA20) caused by heterozygous mutations in the TNFAIP3gene was identified to cause early onset autoinflammatory disease resembling Behçet’s disease.

    Objectives: In this study, we performed a multicenter survey investigating HA20 patients found in Japan and characterized immunological profile.

    Methods: We summarized the detailed clinical manifestations, genetic analyses and several immunological parameters of Japanese patients with HA20. Serum cytokine levels and the production levels of IL-1β and TNF-α from the peripheral blood mononuclear cells (PBMCs) were measured using ELISA. Multicolor flowcytometry analysis was performed. To detect A20 protein expression, immunoblot analysis was performed for PHA blast derived from the patients and A20 transfected HEK293T cells. The NF-κB reporter gene activity was analyzed using the dual-luciferase reporter assay system.

    Results: A total 32 patients from 10 independent families were enrolled in this study. Age of onset was 0 to 20 years. Three mutations in the TNFAIP3gene were previously reported; however, seven were novel. All these mutations were evaluated to be functionally pathogenic by several in vitroassays. The production levels of proinflammatory cytokines such as TNF-α and IL-1β from PBMCs were increased. In the detailed analysis of lymphocyte subsets including T, B and NK cells, regulatory T cells (Treg) were increased in all analyzed patients. The increase of double-negative T (DNT) and T helper 17 cell (Th17) cells were observed in 7 and 3 out of 18 analyzed patients, respectively. In addition, follicular helper T cells (Tfh) were significantly increased especially in younger patients. Memory B cells were decreased in most of analyzed patients. Intriguingly, in the complications of HA20 patients, 9 out of 32 of them had not only autoinflammatory phenotypes but also several autoimmune disorders including psoriatic arthritis, Hashimoto’s thyroiditisand autoimmune lymphoproliferative syndromewere observed. The immune dysregulation derived from the defect of A20 may cause the increase of the autoimmune related T cell subsets and the secondary Treg expansion as well as the phonotype of the previously reported A20 knockout mice.

    Conclusion: Our study revealed unexpected variation in phenotypes of HA20 including autoimmunity. In the analysis of lymphocyte subsets for HA20 patients, the characteristic findings such as the increase of Treg and the decrease of memory B cells were observed. The increase of DNT, Th17 and Tfh cells may be involved in onset of several autoimmune disorders.

    Disclosure of Interest

    None Declared

    PT1A03 Use of SIGLEC1/CD169 as a biomarker for monogenic interferonopathies

    Banu Orak1,2, Axel Panzer3, Manuela Theophil3, Elke Krüger4, Frédéric Ebstein4, Barbara Zieba4, Nadine Unterwalder5, Christian Meisel5, Tilmann Kallinich2
    1Center for chronically sick children, Charité University Medicine Berlin; 2Department of Pediatrics, Division of Pneumology, Immunology with intensive Medicine, Charité University Medicine Berlin; 3Pediatric Neurology, DRK Klinikum Berlin-Westend, Berlin; 4Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald; 5Department of Immunology, Labor Berlin GmbH, Berlin, Germany
    Correspondence: Banu Orak

    Introduction: Monogenic Interferonopathies represent a rare group of inflammatory diseases with difficulties in early diagnosis. Expression of SIGLEC1, also known as CD169, on monocytes is the second highest interferon stimulated gene (ISG) in systemic lupus erythematodes (SLE).A correlation of SIGLEC1 expression with ISG in SLE is well established. Furthermore, SIGLEC1 seems to estimate disease activity more accurately than anti-dsDNA antibodies.

    Objectives: To show the relevance of SIGLEC1 as a diagnostic marker for detection of Interferonopathies.

    Methods: Eight patients with genetically confirmed monogenic Interferonopathies were included. Clinical data, classical inflammatory markers and blood count were obtained by patients file. SIGLEC1 expression was measured by flow cytometry with a highly standardized quantitative assaywith a reference range in healthy controls less than 2500 SIGLEC1 molecules/monocyte.In order to quantify the antigen expression by every single cell QuantiBRITE™ PE tubes were applied. Additionally, transcriptional level of SIGLEC1, IFI44L, IFI27, ISG15 and RSAD2 as type I Interferon stimulated genes were assessed by real-time PCR.

    Results: All patients showed homozygous mutations. Three patients displayed TREX-1, two patients IFIH-1, two patients SAMDH1 and one patient RNASE2HB mutations. Mean age of patients was 12 years (min. 6 months, max. 49 years, SD+/- 17 years). Six of eight patients showed neurological symptoms consistent with Aicardi-Goutières-Syndrome like neurological development retardation and microcephaly. Five patients showed abnormalities on brain MRI, like periventricular calcifications or corpus callosum thinning. Two patients (homozygous for IFIH-1 mutation) were diagnosed with Singleton-Merten-Syndrome presenting abnormal ossification of extremities and dental anomalies.One patient with homozygous TREX1 mutation presented with postnatal glaucoma, microcephaly, developed sensorimotor polyneuropathia and suffered from recurrent fever with persistent chilblain lesions.

    All eight patients (100%) showed elevated results for SIGLEC1 expression (mean molcules/monocyte +/- SD: 10272 +/- 3746) without having high levels of standard inflammatory markers. In six patients elevated SIGLEC1 expression showed dysregulation of the type 1 interferon pathway prior to genetic testing. In three patients with unclear disease phenotype measuring expression of SIGLEC1 contributed to establish the right diagnosis. On transcriptional level SIGLEC1 and the other ISGs were also elevated in comparison to healthy controls.

    Conclusion: In all patients with monogenic Interferonopathies like Aicardi-Goutières-Syndrome high expression of SIGLEC1 was observed, either before diagnosis was established or during disease course.

    Therefore, SIGLEC1 qualifies as an easy accessible and cheap diagnostic marker with short turnaround time to screen patients with suspected Interferonopathy.

    Disclosure of Interest

    None Declared

    PT1A04 Screening of patients with idiopathic polyarteritis nodosa, granulomatosis with polyangiitis, and microscopic polyangiitis for deficiency of adenosine deaminase 2

    Oskar Schnappauf1, Monique Stoffels2, Ivona Aksentijevich1, Amanda Ombrello1, Natalia Sampaio Moura1, Karyl Barron1, Daniel Kastner1, Peter Grayson3, Peter Merkel4, on behalf of Vasculitis Clinical Research Consortium
    1National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH); 2National Human Genome Research Institute (NHGRI), National Institutes of Health; 3National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda; 4Division of Rheumatology and the Department of Biostatistics, Epidemiology, and Informatics (DBEI), University of Pennsylvania, Philadelphia, United States
    Correspondence: Oskar Schnappauf

    Introduction: Deficiency of adenosine deaminase 2 (DADA2) is the first described monogenic vasculitis. Patients usually present in childhood, but age of onset, disease severity, and organ involvement of DADA2-associated vasculitis is highly variable. Clinical manifestations of DADA2 overlap with the typical features of necrotizing vasculopathies such as polyarteritis nodosa (PAN), granulomatosis with polyangiitis (GPA), and microscopic polyangiitis (MPA).

    Objectives: This study aimed to test the prevalence of DADA2 in patients with presumed idiopathic PAN, GPA or MPA.

    Methods: Patients (n=117) with idiopathic PAN, all of whom tested negative for hepatitis B virus infection, and patients (n=1107) with GPA or MPA were screened for mutations in ADA2. To further assess the pathogenicity of identified variants on a functional level, ADA2 activity was determined on available serum samples of patients with PAN.

    Results: Nine of 118 patients with PAN (7.6%) were identified as having rare missense variants in ADA2 with a minor allele frequency of < 0.005. Four patients (3.4%) were homozygous or compound heterozygous for variants in ADA2. Of the seven distinct variants present in these four patients, G47A, G47W, R169Q, E328K, F355L, and G383S had previously been reported as causative for DADA2. The remaining variant, P106S, is a rare variant predicted to be damaging to protein function by in silico algorithms. Five additional patients were carriers for the monoallelic variants R34W, T65M, M309I, V349I, and Y453C. R34W and Y453C were reported in DADA2 before, while the three remaining variants are of unknown clinical significance. None of the patients with GPA or MPA were biallelic for rare missense variants in the ADA2 gene but 32 individuals (2.9%) were carriers for monoallelic rare missense variants.

    Serum samples of patients with PAN were available on the individuals with the G383S/G383S and E328K/F355L genotypes and showed markedly reduced ADA2 enzyme activity, comparable to levels seen in patients with DADA2. ADA2 activity of three of the four available serum samples on monoallelic carriers was not reduced, confirming the non-pathogenicity of T65M, M309I, and V349I. The serum sample on the individual carrying the pathogenic variant Y453C showed ADA2 activity in the range of carriers. ADA2 enzyme activity testing of the remaining serum samples revealed one additional individual with strongly reduced ADA2 activity levels as well as five individuals with enzymatic activity in the range of carriers. Sanger sequencing of the ADA2 gene in these individuals did not identify any pathogenic variants and indicates the presence of cryptic mutations undetectable by conventional sequencing. In summary, for five out of 118 patients with PAN the diagnosis of DADA2 can be applied.

    Conclusion: This is the first study to report biallelic pathogenic variants in ADA2 in patients with adult-onset, idiopathic PAN, and demonstrates that DADA2 specifically accounts for a subset of patients with idiopathic PAN but not GPA or MPA. Given the potential efficacy of TNF-inhibitors in DADA2, that anti-TNF treatment is not the conventional therapy in PAN, and the consequences for other family members, these findings suggest that ADA2 testing and/or ADA2 activity testing should be considered in patients with HBV-negative idiopathic PAN, especially in patients with an early onset of this potentially life-threatening disease.

    Disclosure of Interest

    None Declared

    PT1A05 Diagnosis and long term management of type I interferonopathies in a pediatric rheumatology center

    Stefano Volpi1,2, Elettra Santori3, Margherita Ricci1, Paolo Picco1, Alessandra Tesser4, Gillian I. Rice5, Roberta Caorsi1, Alice Grossi6, Isabella Ceccherini6, Alberto Martini2, Yanick J. Crow7,8, Alberto Magnasco9, Alberto Tommasini4, Fabio Candotti3, Marco Gattorno1
    1Centro per le Malattie Infiammatorie e Immunodeficienze, Clinica Pediatrica e Reumatologia, Istituto Giannina Gaslini; 2DINOGMI, Università degli Studi di Genova, Genova, Italy; 3Allergy and Immunology, Lausanne University Hospital, Lausanne, Switzerland; 4IRCCS Buro Garofalo, Trieste, Italy; 5Genetic Medicine, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom; 6UOC Genetica Medica e UOSD Genetica e Genomica delle Malattie Rare, Istituto Giannina Gaslini, Genova, Italy; 7Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; 8Institute Imagine, University Paris Decartes, Paris, France; 9Nephrology, Dialysis, Transplantation Unit, Istituto Giannina Gaslini, Genova, Italy
    Correspondence: Stefano Volpi

    Introduction: in recent years several genetic diseases linked to pathologic activation of type 1 interferon (IFN) pathway have been described

    Objectives: To identify type I interferonopathies in a cohort of children with early-onset rheumatic disease and to report the long-term efficacy and side effect of pathway-specific treatment with a Janus Kinase (JAK) inhibitor

    Methods: Patients were selected based on the presence of any of the following: i) early onset (infancy or before puberty) inflammatory SLE-like symptoms; ii) vasculopathy (skin ulcers, chilblains, strokes) iii) panniculitis with or without lipodystrophy iv) persistent or recurrent systemic inflammation with or without lung involvement v) polyarthritis with lung involvement. Type 1 IFN activation was assessed by quantitative PCR, measuring the expression of 6 type 1 interferon-related genes (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) in peripheral blood. In selected patients, molecular analysis was performed using Sanger sequencing, a NGS panel of 41 inflammatory-related genes or whole exome sequencing. Off label therapy with the JAK inhibitor Ruxolitinib was considered in patients with a definitive genetic diagnosis and incomplete disease control by standard treatment

    Results: We screened 324 patients evaluated in the rheumatology unit of our Institute. 132 out of 324 patients had a positive type 1 IFN signature. Based on the clinical presentation and the result of the IFN signature we further analyzed a subset of patients for an underline genetic defect. In 6 patients we identified pathogenic mutations affecting TMEM173 (p.V155M in one patient and p.R281Q in a second patient), DNASE2 (p.D121V), DNASE1L3 (c.289_290delAC in 2 patients) and COPA (p.R233H) genes were identified. Therapy with Ruxolitinib was started in 4 patients and allowed to partially control disease manifestations and reduce or stop steroids, however, we observed the following limitation: the patient with DNASE1L3 mutations and a severe kidney involvement at therapy onset, despite the control of his systemic symptoms progressed to kidney failure; one patient with SAVI and a severe lung involvement experienced severe recurrent viral lung infections requiring intensive care and extracorporeal membrane oxygenation (ECMO) in one episode; the patient with DNASE2 mutationsexperienced a Herpes Zoster infection controlled with antiviral therapy and decreasing Ruxolitinib dosage.

    Conclusion: Patients with type 1 interferonopathies might present with symptoms overlapping pediatric rheumatic diseases. The combination of type 1 interferon pathway activation assessment and NGS is an effective strategy for the diagnosis of this heterogeneous class of diseases and can guide the choice for targeted therapies. JAK inhibitors represent a therapeutic resource in controlling these difficult-to-treat patients, however further studies are needed to assess their efficacy and long-term safety

    Disclosure of Interest

    None Declared

    PT1A06 Results of international Delphi survey for the diagnosis, investigation and management of deficiency of ADA2

    Taryn A.-B. Youngstein1, Eugene P. Chambers2, on behalf of DADA2 Foundation, Helen J. Lachmann1, DADA2 Delphi Study Participants
    1National Amyloidosis Centre, UCL Division of Medicine, London, United Kingdom; 2Vanderbilt University Medical Centre, Vanderbilt University, Nashville, United States
    Correspondence: Taryn A.-B. Youngstein

    Introduction: DADA2 is phenotypically heterogenous in its presentation, even within families with the same mutation, and the absence of a murine ortholog has placed emphasis on the study of known cases of the disease.

    Objectives: We conducted an E-Delphi study to explore consensus on the diagnosis, investigations and management of those with suspected disease.

    Methods: A Delphi method over two rounds of consensus building was used. Invitation email to participate in the study was sent to all published authors on DADA2 and physicians and scientists known to the DADA2 Foundation, a patient advocacy group supporting research into DADA2.Consensus was defined as > 80% agreement.

    Results: 69 respondents contributed to Round 1 and 53 respondents in Round 2, 75% of respondents were paediatricians, the rest adult physicians, a nurse specialist and basic scientists.

    Consensus was reached in 1. Suggestive diagnostic features; Livedoid Rash, Stroke, Fever, Digital Gangrene, 2. The combined use of ADA2 gene sequencing and ADA2 enzyme activity levels to confirm diagnosis. 3. Use of anti-TNF therapy in acute and chronic presentations, 4. Indefinite use of anti-TNF until more information is available, and 5. The avoidance of anticoagulation.

    A wide variety in practice was identified in the use of baseline and follow up investigations including imaging.

    Conclusion: There is consensus that rash, stroke, fever, digital gangrene are highly suggestive of DADA2 but there may be a separate haematological phenotype.Gene sequencing and ADA2 activity levels in combination are the diagnostic gold standard but not generally available.There is not yet consensus on the baseline screening investigations, such as neuroimaging, for these cases. Follow-up investigations are currently guided by clinical course and practice remains highly variable.

    92% of treating physicians believe that anti-TNF therapy is the correct treatment approach currently, and its use should be indefinite until more information is available.

    The authors recommend the creation of a detailed international case registry and a standardised series of baseline and follow-up investigations during this accelerated learning phase about this recently described disease.

    Disclosure of Interest

    None Declared

    PT1A07 Somatic mutations in the NLRP3-inflammasome gene in late adulthood-onset chronic urticaria

    Eman Assrawi1, Camille Louvrier1, Fawwaz Awad1, Clemence Lepelletier2, JD Bouaziz2, William Piterboth1, Florence Moinet3, Philippe Moguelet4, Claire Jumeau1, Laetitia Cobret1, Elma El-Khouri1, Philippe Duquesnoy1, Marie Legendre1, Sophie Georgin-Lavialle5, Gilles Grateau5, Sonia Athina Karabina1, Serge Amselem1, Irina Giurgea1
    1Sorbonne Université, inserm UMRS 993; 2Hôpital Saint-Louis , Service de Dermatologie, Paris; 3Centre Hospitalier Universitaire de Martinique, Service de médecine interne, Martinique; 4Hôpital Tenon , Anatomie et cytologie pathologiques; 5Hôpital Tenon, Service de médecine interne, Paris, France
    Correspondence: Eman Assrawi

    Introduction: Chronic urticaria is a common dermatological disorder and one of the most prominent symptoms of Cryopyrin-Associated Periodic Syndrome (CAPS), a systemic autoinflammatory condition. Besides urticaria, CAPS cardinal symptoms are fever, arthralgia and deafness; however, absence of pathognomonic symptoms makes this diagnosis challenging. NLRP3, the disease-causing gene, encodes the cryopyrin, which upon activation initiates NLRP3 inflammasome assembly and proinflammatory cytokine secretion. Familial cases of CAPS are due to heterozygous germ-line NLRP3 mutations; however, sporadic cases, more often identified in children, are related to de novo or somatic NLRP3 mutations.

    Objectives: We aimed to establish the etiological diagnosis of two elderly unrelated patients presenting with idiopathic chronic urticaria for two decades.

    Methods: Molecular study of patients’ DNA was performed using a NGS panel targeting genes involved in autoinflammatory disorders. Functional analyses of the identified NLRP3 variants were performed using two cellular models, HEK293T cells (stably expressing ASC-GFP and pro-caspase1-FLAG) to study ASC speck formation, and THP1 cells to assess IL1β secretion.

    Results:

    In two sporadic unrelated patients, we identified two mosaic NLRP3 mutations: a novel in-frame deletion (c.926_934del, p.Gly309_Phe311del) and a recurrent CAPS mutation (c.1705G>A, p.Glu569Lys). In whole blood DNA, the mosaicism level was of 17.2% in the first patient and of 11% in the second one. The patients, who are about 70 years old, presented for two decades with late onset chronic idiopathic urticaria, occasionally associated with fever, arthralgia and myalgia. Deafness was diagnosed at the age of 50 years in the first patient, and of 70 years, after the identification of a NLRP3 mutation, in the second one.

    To assess the pathogenicity of the identified variants, we studied the activation of NLRP3 inflammasome after transient expression of NLRP3 wild-type (WT) or of NLRP3 carrying either the p.Gly309_Phe311del or the p.Glu569Lys mutation. Both mutations were found to significantly increase ASC speck formation andIL1β secretion as compared to NLRP3-WT.

    The diagnosis of CAPS was therefore established on the bases of these molecular and functional data. Accordingly, complete remission was achieved with anti-interleukin 1 receptor antagonists in both patients.

    Finally, we studied the mosaicism level in several cell types from both patients and showed a wide distribution profile of the mutant alleles, suggesting that, in both cases, the mutational event occurred early during embryogenesis.

    Conclusion: In late adulthood-onset chronic urticaria, the search for autoinflammatory markers and for somatic NLRP3 mutations may have important diagnostic and therapeutic issues. Importantly, despite the onset of the disease after 50 years old, NLRP3 mutations are not restricted to myelomonocytic cells.

    Disclosure of Interest

    None Declared

    PT1A08 Idiopathic recurrent pericarditis: clinical findings and treatment approach

    Camilla Celani1, Silvia Federici1, Anna Tulone1, Brigitte Bader Meunier2, Virginia Messia1, Manuela Pardeo1, Claudia Bracaglia1, Pierre Quartier Dit Maire2, Fabrizio De Benedetti1, Antonella Insalaco1
    1Division of Rheumatology, IRCCS, Ospedale Pediatrico Bambino Gesù, Rome, Italy; 2Unité d’Immunologie-Hématologie et Rhumatologie pédiatrique, Hôpital Necker-Enfants, Paris, France
    Correspondence: Camilla Celani

    Introduction: Recurrent pericarditis affects 15-30% of patients with acute pericarditis. The etiology is poorly understood, with about 80% being idiopathic. Several treatment options are available for recurrences, including NSAIDs, colchicine, glucocorticoides and IL-1 inhibitors (i.e. Anakinra). Standardized guidelines for the management of these patients are still lacking

    Objectives: To analyze clinical findings and treatment approach in a cohort of pediatric patients with recurrent pericarditis

    Methods: Patients with at least two episodes of idiopathic pericarditis, followed at two Pediatric Rheumatology centers between 2006 and 2018, were included

    Results: A total of 42 patients (18 males ) were included. Mean age at disease onset was 11.8 years (range 4-17). Chest pain and fever were the presenting symptoms in all patients. In 47% pleural effusion was detected. Laboratory tests showed increased white blood cell count (mean 14.509/mm3), C-reactive protein (mean 18.01 mg/dl) and erythrocyte sedimentation rate (mean 39 mm/h) in all patients. The first episode was variably treated: 18/42 (43%) received NSAIDs alone, 5/42 (11.9%), colchicine alone or associated to NSAIDs and 3/42 patients (7%) received antibiotics alone. 16/42 (38%), not responsive to NSAIDs or colchicine, received glucocorticoides. Patients who received glucocorticoids at the first episode relapsed earlier (median time of 2.1 months range 10 days-5 months), than patients treated with NSAIDs ( 6.6 months range 10 days -24 months) or with colchicine (5 months range 10 days-5 months) (p<0.05). In our study, initial treatment of the first episode did not affect the number of subsequent flares. To evaluate treatment strategy at relapses, we divided our study population in two groups: Group 1 (20 pts) in which recurrence was treated with NSAIDs, colchicine or glucocorticoid (alone or combined); group 2 (22 patients) in which anakinra was started. Among patients belonging group 2, 9 received anakinra at first relapse, 7 at the second, 2 at the third and 2 at the fourth. Anakinra treatment was followed by a prompt resolution of symptoms and inflammatory signs within 2 days. During daily treatment with full dose anakinra, no relapses were reported over a median of 13.3 months (range 5-24 months). In 13 out of 22 patients, anakinra was gradually tapered reducing the days of administration during the week. Four of these patients relapsed. The mean time from the start of anakinra to tapering was 17±4 months (range 14-23 months) in the 4 patients who experienced a relapse versus 14±4 months (range 7-21 months) in patients who did not flare, with no statistical difference. Among the 22 patients belonging to group 2 anakinra was finally discontinued in 11 after a mean time of 23.4 months (range 12-36). Among these, 8 relapsed after anakinra withdrawal (including 2 of the 4 patients already relapsed during tapering). Only 3 patients didn’t present any relapse (up to 20.3 months of follow-up). All patients who relapsed responded quickly to the reintroduction of anakinra

    Conclusion: Our study confirms the lack of a standardized treatment approach in patients with recurrent pericarditis. Patients treated with glucocorticoid at first episode relapse before than those treated with other drugs. Anakinra is an effective treatment; however, tapering/discontinuation of the drug lead to relapses in several cases. Further experience on larger population is needed to define the best treatment duration and approach to withdrawal of IL-1 inhibitor

    Disclosure of Interest

    None Declared

    Guided poster tour 1B

    PT1B01 Monocytes proteomic profile of patients with different autoinflammatory diseases: a new approach to characterize these diseases

    Federica Penco1, Andrea Petretto2, Chiara Lavarello2, Ilaria Gueli3, Arinna Bertoni1, Alessia Omenetti3, Claudia Pastorino1, Marco Gattorno1
    1Centro Malattie Autoinfiammatorie ed Immunodeficienze; 2Laboratorio Core Facilities - Proteomica e Metabolomica Clinica; 3Clinica Pediatrica e Reumatologica, Istituto Giannina Gaslini, Genova, Italy
    Correspondence: Federica Penco

    Introduction: Autoinflammatory diseases are a group of inherited diseases characterized by early onset and systemic inflammation, often manifesting with unexplained fevers.These pathologies are usually caused by mutations in genes involved in the regulation of innate immune response with a consequent inflammatory phenotype. The most common genetically defined periodic fevers are Familial Mediterranean Fever (FMF), Cryopyrin-associated periodic syndromes (CAPS), TNF receptor-associated periodic syndrome (TRAPS) and mevalonate kinase deficiency/hyperimmunoglobulin D syndrome (MKD/HIDS). Some patients show clinical features similar to autoinflammatory diseases but no genetic mutation has been found.

    Objectives: Our aim is to evaluate the differences in the expression of proteins or pathway in monocytes, and plasma metabolites in patients with autoinflammatory diseases compared with healthy subjects to clusterize and better understand the mechanisms underlying different genetically defined disorders and try to characterize the genetically undefined pathologies.

    Methods: Monocytes, purified from peripheral blood and incubated for 4 hours with or without LPS, were collected from 5 patients for each pathology (FMF, CAPS, TRAPS and MKD) and healthy donors. The samples have been processed by iST protocol. Each digested sample was analyzed by high-resolution liquid chromatography and tandem mass spectrometry (LC-MS/MS) based on Orbitrap technology. The quantification strategy is a label-free approach (LFQ) available in MaxQuant suite.

    Results: Here we identified a median of about 5000 proteins from the monocyte samples of each 4000 is quantified by LFQ approach. PCA analysis and Person’s correlation show good reproducibility of data and a good separation between the different groups. The data were then submitted to an appropriate statistic. The T-Tests highlighted the differentially expressed proteins and through the use of Cytoscape with the ClueGo app we obtained the differently regulated pathways in the different conditions. It has also been constructed, starting from significative proteins, a network, related to disease using the information of String Disease db. This was done to highlight the proteins associated with the disease as well as to reveal new possible biomarkers. We observed that the expression of proteins is differently enriched according to the different conditions. For each autoinflammatory disease, a list of significantly modulated proteins was obtained: some of which are already known to be related to the disorders, while others have not yet been described. In FMF, MEFV, RhoA and some related proteins were significantly up-regulated together with genes linked to the interferon pathway. In TRAPS relevant proteins turn up related to the maintenance of Golgi and cellular trafficking. The bioinformatics analysis allows us to better understand the functional interaction between these monocytes proteins and map which are involved in the diseases. Proteins thus analyzed were then contextualized in dominant pathways for each pathology through Cytoescape network analysis.

    Conclusion: Here, we addressed how a high-resolution proteomics approach could be used to better understand the biology of autoinflammatory diseases. The characterization of a broad spectrum of proteins and their interaction network will allow us to identify new biomarkers for the different pathologies and better comprehend and recognize the genetically undefined disorders.

    Disclosure of Interest

    None Declared

    PT1B02 Prulipotent stem cell derived myeloid cell lines for dissecting the mechanism of autoinflammation

    Megumu Saito
    Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Introduction: Autoinflammatory disorders (AIDs) is defined as an disorders associated with dysregulation of innate immune systems. In typical case, patients with AIDs show various inflammatory symptoms, such as periodic fever, skin rash and sterile serositis. Human induced pluripotent stem cell (iPSC) models of AIDs are supposed to be feasible and useful, because 1) most of typical AIDs are monogenic, 2) responsible cells are usually innate immune cells which can be robustly differentiated from PSCs, and 3) improvement of therapeutic or diagnostic approach is still needed in many AIDs. We therefore focused on establishing PSC models of AIDs, and applied these models to identification of genetic and biological background of patients’ pathophysiology.

    Results: We established iPSCs from patients with AIDs such as CINCA/NOMID, Nakajo-Nishimura syndrome (NNS), and Blau syndrome (BS). We also corrected the disease-causing mutations by genome editing technology in selected clones. iPSCs were then differentiated into hematopoietic cells, especially into monocytic lineage cells. To stably obtain functional monocytic cells, monocytic progenitor cells derived from iPSCs were immortalized. The immortalized monocytic cell lines (MLs) were further differentiated into mature macrophages and then used for functional assays. The monocytic cells from AID-iPSCs showed increased secretion of proinflammatory cytokines such as IL-6, TNF and IL-1β. In case of CINCA/NOMID, iPSCs established from a NLRP3-mutation negative patient were useful for the identification of somatic NLRC4 mutation of the patient. The AID-iPSCs were also used for dissecting underlying disease mechanism, and suitable for high-throughput phenotypic screening.

    Conclusion: We applied iPSC technology for studying autonflammatory disorders. When combined with other novel technologies such as next generation sequencing and genome editing, iPSC-based phenotypic dissection was useful for diagnosis of the patients and understanding the disease mechanism.

    Disclosure of Interest

    None Declared

    PT1B03 Loss of protein prenylation in human monocytes promotes the formation of an NLRP3-dependent inflammasome in a model of mevalonate kinase deficiency

    Oliver Skinner1, Julie Jurczyluk1,Paul Baker2, Seth Masters2, Avril Robertson3, Kate Schroder4, Sam Mehr5, Marcia Munoz1, Michael Rogers1
    1Bone Biology Division, Garvan Institute of Medical Research, Darlinghurst, Sydney; 2Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Melbourne; 3School of Chemistry and Molecular Biosciences; 4Institute of Molecular Bioscience, University of Queensland, Brisbane; 5Dept of Allergy/Immunology, Royal Children's Hospital, Melbourne, Australia
    Correspondence: Oliver Skinner

    Introduction: Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in an enzyme of the mevalonate pathway, leading to loss of isoprenoid lipids necessary for protein prenylation. Defective prenylation in MKD is thought to trigger inflammasome activation, IL-1β release and recurrent episodes of systemic inflammation. However, how loss of prenylation causes inflammasome activation remains controversial. Recent studies have suggested that lack of Rho or K-Ras prenylation leads to assembly of the Pyrin inflammasome in macrophages, whereas others have shown that disruption of the mevalonate pathway in monocytes triggers IL-1β release via NLRP3 inflammasome activation.

    Objectives: To determine which type of inflammasome is activated in human monocytes upon loss of protein prenylation in MKD.

    Methods: THP-1 human monocytic cells were treated for 24 hours with 5μM simvastatin (SIM) to pharmacologically block the mevalonate pathway, followed by stimulation with LPS or Pam3CSK4 (TLR4 and TLR2 agonists, respectively) to induce an inflammatory response. IL-1β and IL-18 release was quantified by ELISA whilst caspase-1 enzyme activity, ASC immunostaining and permeability to propidium iodide were used as measures of inflammasome formation and pyroptosis. To assess the contribution of Pyrin or NLRP3, we used inducible CRISPR/Cas9 knockout THP-1 cells, as well as the small molecule inhibitor of NLRP3, MCC950. Finally, inflammasome activation was examined in peripheral blood mononuclear cells (PBMCs) from an MKD patient (bearing V377I/H20N mutations in MVK) and heterozygous parents.

    Results: SIM treatment of THP-1 cells caused a clear accumulation of unprenylated Rab and Rap1A proteins, without affecting cell viability. Furthermore, LPS or Pam3CSK4 stimulation of SIM-treated cells resulted in a 4-fold increase in IL-1β and IL-18 release and significantly higher ASC-containing speck formation, caspase-1 activity and pyroptosis. Importantly, all these effects were reversed to normal levels by restoring protein prenylation using geranylgeraniol (the missing lipid metabolite necessary for protein prenylation). In support of a predominant role for NLRP3, MCC950 completely inhibited the stimulatory effect of SIM on LPS-induced ASC speck formation, caspase-1 activation, IL-1β release and pyroptosis. All of these were also abolished by knocking out NLRP3, whereas deletion of Pyrin had no effect. Similarly, LPS stimulation of MKD patient-derived PBMCs resulted in much higher IL-1β release compared to heterozygous parents, which was completely blocked by NLRP3 inhibition with MCC950.

    Conclusion: We clearly demonstrate that lack of prenylation in human monocytic cells, using statin treatment (a pharmacologic model of MKD) or using authentic cells from an MKD patient, leads to enhanced formation of an NLRP3-dependent, Pyrin-independent, inflammasome upon TLR2/4 stimulation. In contrast to reports that lack of prenylation activates the Pyrin inflammasome in macrophages, these findings indicate a prominent additional role for NLRP3 in the pathogenesis of MKD and demonstrate that targeting the Pyrin inflammasome in isolation may not be sufficient to resolve all the pathology associated with MKD. Rather, approaches to overcome the metabolic defect in the mevalonate pathway and restore normal protein prenylation could be more effective at preventing broader inflammasome activation.

    Disclosure of Interest

    None Declared

    PT1B04 Performance of targeted NGS for routine diagnosis of autoinflammatory diseases

    Guilaine Boursier1, Cécile Rittore1, Déborah Méchin2, Muriel Gutierrez1, Florian Milhavet1, Guillaume Sarrabay2, Isabelle Touitou2
    1Department of Medical Genetics, Rare diseases and personalized medicine, Rare and autoinflammatory diseases unit, CHU Montpellier, Univ Montpellier; 2IRMB, INSERM, Univ Montpellier, Department of Medical Genetics, Rare diseases and personalized medicine, Rare and autoinflammatory diseases unit, CHU Montpellier, Montpellier, France
    Correspondence: Guilaine Boursier

    Introduction: Monogenic systemic autoinflammatory diseases (SAIDs) are characterized by mutations in genes coding for proteins involved in innate immunity. Since the discovery of the first gene MEFV (OMIM 608107) responsible for familial Mediterranean fever, more than 30 new conditions have been identified, notably through high-throughput sequencing approaches. Currently, next generation sequencing (NGS) allows the simultaneous investigation of multiple genes at a manageable cost. We present here our 4 years of experience of targeted NGS as a routine diagnostic for SAIDs.

    Objectives: To evaluate the performance of a panel of 49 genes targeting well-defined autoinflammatory diseases and clinical concordance after retro-phenotyping.

    Methods: DNAs from 577 patients clinically suspected for SAIDs (age 3 months to 79 years) were sequenced by NGS between September 2014 and December 2018. The libraries were prepared using Nextera (Illumina) or SureSelect (Agilent) Target Enrichment Capture custom kits. Sequencing reactions were performed on MiSeq or NextSeq500 equipment (Illumina). The mutations were classified according to a 5-class scale provided by the Infevers database or according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Epidemiological data, clinical symptoms and biological markers were collected on a form provided with all genetic diagnosis requests.

    Results: Almost a half of patients (261/577) had at least one mutation. Mutations that were probably pathogenic (class 4) or clearly pathogenic (class 5) accounted for one third of the mutations and were detected in only 30/49 genes. We identified 87% (100/115) missense variants and 11% (13/115) truncating mutations including whole gene deletions. The genes in which we identified most of the pathogenic mutations were MEFV, MVK, PSMB8, ADA2, NLRP3 and RNASEH2B then NOD2, PSTPIP1 and SLC29A3. The most recurrent pathogenic variants were MVK:p.(Val377Ile), MEFV:p.(Met694Val) and RNASEH2B:p.(Ala177Thr). The yield of conclusive genetic diagnosis (one class 4 or 5 mutation including mosaicism in a dominant condition or two non-allelic mutations in a recessive condition) using this NGS strategy was 8% over this 4-year period. We observed a rather good, though incomplete clinical concordance in patients with a genetic diagnosis.

    Conclusion: The simultaneous investigation of multiple genes using targeted NGS is a successful routine diagnostic for SAIDs and is of particular interest for the detection of low-level mosaic mutations and copy number variations. However, our targeted NGS approach has resulted in genetic confirmation in a relatively small proportion of patients. One of the reasons may be related to the stricter definition we have used compared to previous reports. On the other hand, SAID genes or molecular mechanisms that are still unknown are likely to be found. Finally, a better clinical filter for ordering genetic tests through a consultation with experts could be encouraged.

    Disclosure of Interest

    None Declared

    PT1B05 Late-onset TRAPS with low-grade mosaicism in TNFRSF1A

    Barend P. Kant1, Marco J. Koudijs1, Ruben van‘t Slot2, Joyce van Kuik3, Lisanne M. Sikkema1, Joost Frenkel4, Anna Simon5, Mariëlle E. van Gijn1
    1Department of Genetics; 2Center for Molecular Medicine; 3Department of Pathology; 4Department of Pediatrics, University Medical Center Utrecht, Utrecht; 5Department of Internal Medicine, Radboudumc Expertisecenter for Immunodeficiency and Autoinflammation, Radboud University Medical Center, Nijmegen, Netherlands
    Correspondence: Barend P. Kant

    Introduction: The diagnosis of patients with systemic autoinflammatory diseases (SAID) is difficult which can result in delayed treatment and irreversible organ damage. In several patients, low grade mosaicism of an autosomal dominant form of SAID has been detected. In NLRP3, even mosaic mutations with allele frequency <5% in whole blood can result in severe disease. In recent years, high-grade mosaic mutations have been detected in the TNFRSF1A gene in patients with late-onset TRAPS. With a new screening assay, we investigate whether low-grade mosaic mutations in TNFRSF1A might contribute to autoinflammatory disease.

    Objectives: To examine the presence of low-grade mosaicism in a patient with clinically suspected late-onset TRAPS.

    Methods: DNA was extracted from whole blood, FACS sorted hematopoietic cells and non hematopoietic cells. Mosaic mutation screening was performed using a single molecule molecular inversion probe (smMIP) assay. Positive results were confirmed with droplet digital (dd)PCR technology.

    Results: Our patient is a 70 year old male with a history of episodes of fever with migratory erythematous rash and myalgia since his 30s. Also pleuritis, cervical lymphadenopathy and eye involvement were recorded and there was a single episode with abdominal pain and vomiting. On average, the episodes lasted for 2 weeks and occurred every 5 weeks. Family history was negative. Complete remission of the symptoms was achieved after starting treatment with anakinra.

    We previously screened the TNFRSF1A gene by Sanger sequencing and next generation sequencing (NGS) with negative results. With our smMIP assay, we detected a c.269C>A p.(Thr90Asn) likely pathogenic missense mutation with 1,3% allele frequency in whole blood. This result was confirmed by ddPCR. The mutation was also present in sorted granulocytes, monocytes, T and B cells, but not in non-hematopoietic cells. This distribution is different from late-onset CAPS patients who were found to have myeloid restricted mosaic mutations in NLRP3.

    Conclusion: We report the first low-grade mosaic variant in TNFRSF1A. Based on clinical symptoms, favorable response to anakinra and genetic findings, we diagnosed our patient with mosaic TRAPS. Our data suggest that even mutations present in a very small number of cells can cause systemic autoinflammatory disease.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    PT1B06 Heterozygous TNFAIP3 mutation as the cause of an interferon-mediated neuroinflammatory disorder

    Ciara M. Mulhern1, Ying Hong1, Ebun Omoyinmi1, Dara McCreary1, Marina Casimir1, Cheryl Hemingway2, Felice D’Arco2, Paul Brogan1, Despina Eleftheriou1
    1Infection, Inflammation and Rheumatology, Great Ormond Street Institute of Child Health; 2Neurology Department, Great Ormond Street Hospital for Children, NHS Foundation Trust, London, United Kingdom
    Correspondence: Ciara M. Mulhern

    Introduction: Heterozygous loss-of-function mutations in TNFAIP3 lead to haploinsufficiency of A20 (HA20) commonly manifesting with severe orogenital ulceration and ocular inflammation. Central nervous system (CNS) inflammation has been observed in patients with haploinsufficiency of HA20 but CNS involvement as the sole clinical manifestation of heterozygous TNFAIP3 mutation in humans has never been described however. Herein, we report on the case of an 8 year old female of non-consanguineous Pakistani-Indian descent who presented with left sided focal seizures and hemiparesis, acute uveitis and congitive decline. Magnetic resonance imaging (MRI) of her brain revealed contrast-enhancing T2 hypointense intracranial mass lesions, in the grey matter of the paracentral lobule and the thalamus on the left with surrounding oedema. Brain biopsy revealed necrotising granulomatous inflammation. Brain CT revealed intracerebral calcification. Extensive screening of infectious or malignant causes was negative.She failed to respond to multiple anti-inflammatory treatments but had an excellent radiological and clinical response to an oral JAK-STAT inhibitor suggesting this was likely an interferon (IFN) mediated inflammatory disease.

    Objectives: To use next generation sequencing to identify the genetic cause of the progressive neuroinflammatory disorder in this case and characterise the mechanisms underpinning neuroinflammation

    Methods: Whole exome sequencing (WES; illumina MiSeq) was carried out in all family members. Expression of phosphorylated- p65, IRF3, STAT1 and STAT3 in both patient and healthy control derived peripheral blood mononuclear cells was assessed by flow cytometry. Meso Scale Discovery (MSD) assays was used to quantify cytokines in patient serum. qPCR assays measured the expression levels of IFN stimulated gene expression. Co-immunoprecipitation analysis assessed the binding capacity of the mutated protein to Tank-binding kinase 1 (TBK1)

    Results: WES revealed a heterozygous missense p.T647P mutation in TNFAIP3 as the cause of the progressive neuroinflammatory disorder in this case. Patient-derived cells exhibited enhanced phosphorylation of the p65 transcription factor, and increased expression of NF-ƙB mediated proinflammatory cytokines. The mutated p.T647P A20 protein failed to control interferon-regulatory-factor-3 (IRF3) activation and interferon (IFN)-dependent gene transcription, in comparison to healthy control cells. We also show that the mutant A20 protein cannot efficiently bind to TBK1, in order to turn off IRF3 activation leading to an enhanced IFN signature in the patient. We believe that failure to regulate IFN-mediated immune responses, was the driver of the CNS inflammation observed. Furthermore, treatment with an oral JAK 1/2 inhibitor resulted in marked clinical improvement, complete radiological resolution of neuroinflammation, and normalisation of IFN-stimulated gene expression in whole blood. IFN mediated immune responses were also impaired in an additional disease control case of HA20 in a 4 year old heterozygote for the p.N98Tfs25 TNFAIP3 variant.

    Conclusion: We describe for the first time heterozygous p.T647P missense mutation in TNFAIP3 as the cause ofprogressive neuroinflammation. The p.T647P mutated A20 proteinfailed to control IRF3 activation and IFN dependent transcription. Treatment with an oral JAK 1/2 inhibitor was highly effective. Our report now adds TNFAIP3 mediated neuroinflammation to the ever-expanding group of monogenic interferonopathies with propensity to CNS involvement.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    PT1B07 Systemic evaluation of genetic and biochemical testing for deficiency of ADA2 (DADA2) from the NIH patient cohort

    Natalia Sampaio Moura, Oskar Schnappauf, Natalie Deuitch, Qing Zhou, Daniel Kastner, Ivona Aksentijevich
    Inflammatory Disease Section, National Institutes of Health, Bethesda, United States
    Correspondence: Natalia Sampaio Moura

    Introduction: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disorder caused by loss-of-function mutations in the ADA2 gene, which encodes the adenosine deaminase 2 (ADA2) enzyme. Patients with DADA2 can present with many different manifestations including early-onset lacunar stroke, recurrent fever, hepatosplenomegaly, livedo reticularis, immune dysregulation, and hematopoietic abnormalities. They also exhibit absent or significantly reduced ADA2 enzyme activity. Despite these conserved features, some individuals display discordance between clinical characteristics and genetic or enzyme activity testing.

    Objectives: The goal of our retrospective study is to evaluate sensitivity of diagnostic testing for DADA2 in our CLIA-certified laboratory at the National Institutes of Health (NIH). We compared the diagnostic efficacy of the different types of assays, such as Sanger sequencing, ADA2 enzymatic analysis and multiplex ligation-dependent probe amplification (MLPA).

    Methods: We reviewed all ADA2 Sanger sequencing results of individuals tested between 2014 and 2018, including confirmatory testing in referral patients, and determined the diagnostic yield of this method in patients we suspected to have DADA2 based on clinical phenotype. We then analyzed if subsequent ADA2 enzyme assay and/or MLPA further increased our diagnostic yield.

    Results: Out of 190 individuals with clinical characteristics suggestive of DADA2, 56 patients tested positive for biallelic mutations (29%). Eleven tested as carriers for a monoallelic pathogenic mutation (6%), and 123 (65%) were mutation-negative based on conventional Sanger sequencing. ADA2 enzyme activity assay was performed on 45 patients who had available serum samples. The assay corroborated the DADA2 diagnosis on 30 patients and identified 7 individuals with monoallelic mutations (detected via Sanger) from three different families as patients due to low enzyme activity. MLPA successfully identified a second pathogenic copy number variation (CNV) in all patients who displayed low ADA2 activity. Addition of ADA2 protein assay and MLPA increased our DADA2 diagnostic rate to 33%. Thus, we observed a 13.8% (4/29) relative increase in our diagnostic yield due to the incorporation of these testing modalities.

    Conclusion: Our results suggest that the serum ADA2 enzyme assay should be considered first tier testing for patients with suspected DADA2, given that pathogenic mutations in ADA2 are highly heterogeneous and not always detectable by Sanger sequencing alone. Sanger sequencing in combination with MLPA remain indispensable assays to support and confirm enzymatic testing when indeterminate or unclear, and to identify causal variants. Detection of mutations at the DNA level is important for genetic counseling. Targeted next-generation testing is another method able to identify a diverse scope of pathogenic mutations and has the potential to increase the diagnostic yield of genetic testing.

    Disclosure of Interest

    None Declared

    Monogenic autoinflammatory diseases (clinical)

    P1001 Is there any difference between M694V heterozygote and non-exon 10 mutations on symptoms onset and response to colchicine treatment?

    Hatice Adiguzel Dundar1, Serkan Turkucar1, Ceyhun Acari1, Ozge Altug Gucenmez2, Balahan Makay2, Erbil Unsal1
    1Department of Pediatrics, Pediatric Rheumatology Unit, Dokuz Eylul University Faculty of Medicine; 2Pediatric Rheumatology Unit, Dr. Behcet Uz Childrens’ Hospital, Izmir, Turkey
    Correspondence: Hatice Adiguzel Dundar

    Introduction: Familial Mediterranean fever (FMF) is the most common inherited autoinflammatory syndrome throughout the world. It is caused by mutations of the MEFV gene encoding a protein called pyrin. The most frequent genotype-phenotype correlation is in a certain part of exon 10, especially M694V mutation. There are also a group of patients with non-exon 10 mutations, who have a similar clinical spectrum of the disease.

    Objectives: We aim to investigate the genotype-phenotype differences between M694V heterozygote mutations and non-exon 10 mutations.

    Methods: Data charts of children (n=431) with FMF from Dokuz Eylul University childrens’ hospital and Dr.B.Uz childrens’ hospital were reviewed. Patients were divided into two groups with regard to having M694V heterozygote or non-exon 10 mutations. Genotype-phenotype features and response to treatment were compared.

    Results: There were M694V heterozygote mutations in 128 (29.7%) patients and non-exon 10 mutations in 303 (70.3%) patients. The follow-up period was 54.5 (33-105) months. There was no difference between the age of symptoms onset, the age of diagnosis, and the diagnosis delay time. The family history in patients with M694V heterozygote mutation was statistically positive compared to non-exon 10 mutation group (p:0.000). The symptoms of joint involvement as arthritis were significantly higher in the M694V heterozygotegroup (p:0.026). Additionally, biological agent need due to colchicine unresponsiveness was statistically higher in M694V heterozygote group than group with non-exon 10 mutation (p:0.004) (Table 1).

    Conclusion: There is a significant difference between children with M694V and non-exon 10 mutations, even when the M694V mutation is present in one allele only. Family history with FMF, musculoskeletal symptoms, and unresponsiveness to colchicine are main parameters.

    Disclosure of Interest

    None Declared

    P1002 A case report of aicardi goutieres syndrome type 5 in two siblings mimicking juvenile idiopathic arthritis

    Buthaina Al Adba1, Hajar Dauleh2
    1Paediatric Rheumatology; 2Paediatric, Sidra Medicine, Doha, Qatar
    Correspondence: Buthaina Al Adba

    Introduction: Aicardi-Goutières syndrome (AGS) is a genetically determined encephalopathy characterized by calcification of the basal ganglia and white matter, demyelination, and raised levels of lymphocytes and IFNα in the cerebrospinal fluid [1]. Neurological dysfunction becomes clinically apparent in infancy and manifests as progressive microcephaly, spasticity, dystonia, and psychomotor retardation. Expression of interferon-regulated genes (IGS) in peripheral blood is also upregulated, which is sustained over time [2]. The genes mutated in AGS have been defined to encode proteins implicated in the metabolism of nucleic acids TREX1, the RNASEH2 complex and SAMHD1,which may all function as cellular nucleases [3].

    Objectives: Arthritis and progressive arthropathy with distal joint contractureshave been reported in SAMHD1 mutation along with neurological symptoms [4]. We are describing two siblings with SAMHD1 mutation who only presented with early onset polyarthritis and no systemic or CNS manifestations.

    Methods: Chart review of clinical data includes IFN signature and molecular analysis.

    Results: Two brothers of consanguineous parents with no significant family history of auto inflammatory or autoimmune disease, were born healthy, at term with normal growth parameters.Patient one is a six yearold boy who presented at age of four with one-year history of joint pain and morning stiffness. He had active arthritis in his both wrists, knees and elbows. Lab testing revealed normal blood work ESR, CRP, and negative ANA and RF. Interestingly HLAB27 was present.A diagnosis of JIA was made and he was started on naproxen, and had intra-articular in jectionsarticular injections twice. His arthritisHis arthritis was difficult to control and Etanercept andsubcutaneous Methotrexate were started. His arthritis improved significantly,with residual mild disease atboth wrist joints.His brother, patient two is a 3 year old boy who presented similarly at age of 18 months with morning stiffness and abnormal gait for one-month duration. He had active arthritis in both knees, hips and wrists. He had similar lab findings and positive HLAB27.He was treated with naproxen, a short course of oral steroid and Methotrexate. He responded faster than his brother, but continue to have mild arthritis atboth wrists. Due to difficult to treat, early arthritis in two consanguineous brothers, wesent genetic testing with whole exome sequencing (WES) to look for other causes of their arthritis.Genetic testing for both siblings showed Homozygous mutation at the SAMHD1 gene for the P.Arg 290His (CGT>CAT):c.869G >A.Both parents were heterozygous for the same mutation. The R290H variant in the SAMHD1 gene has been reported previously in association with AGS in an affected individual with multiple clinical features including CNS symptoms and arthritis [5].Peripheral blood analysis of type I interferon-related biomarkers demonstrated that both siblings have an interferon signature characteristic of Aicardi-Goutieres syndrome (Fig 1). Both brothers continued to have normal development with no CNS symptoms or rash and their eye exam showed no glaucoma, which was previously reported in AGS with the SAMHD1 mutation. Despite being asymptomatic from CNS disease we are considering brain MRI to look for inflammatory cerebral vasculopathy that has been reported previously [6].

    Conclusion: We are reporting a spectrum of AGS with a SAMHD1 mutation that mimicks difficult to treat early onset polyarticular arthritis without any CNS or skin manifestations. This illustrates the need to consider mutation analysis of SAMHD1 in similar presentations as CNS and skin disease may evolve later. Finally, even though there has been partial response to treatments,we may consider changing therapy to Janus Kinase inhibitors to achieve complete remission of arthritis.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1003 Phenotypic and genotypic characteristics and damage accrual of monogenic autoinflammatory diseases other than familial Mediterranean fever from the pediatric rheumatology Arab group (PRAG)

    Sulaiman Al-Mayouf1, Abdulaziz Almutairi1, Safia Albrawi2, Abdulatif AlEnazi3, Basil Fatallah4, Abdulallh Alsonbul1, Mohammed Abu-shukair5, Raed Alzyoud5, Adel Alwahadneh5, Mabruka Zlenti6, Ebtisam Kawaja6, Khloud Khawaja7, Zakia Almusawi8, Wafa Madan8, Muna AlMutairi9, Nora Almuatiri10 and Pediatric Rheumatology Arab Group (PRAG)
    1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; 2Royal Hospital, Muscat, Oman; 3King Fahad Medical City, Riyadh, Saudi Arabia; 4AlJalial Children Hospital, Dubai, United Arab Emirates; 5Queen Rania Children Hospital, Amman, Jordan; 6Tripoli Children Hospital, Tripoli, Libya; 7Mafraq Hospital, Abu Dhabi, United Arab Emirates; 8Salmaniya Hospital, Bahrain, Bahrain; 9Al Adan Hospital; 10AlSabah Hospital, Kuwait, Kuwait
    Correspondence: Sulaiman Al-Mayouf

    Introduction: Monogenic autoinflammatory diseases (AIDs) are a group of rare hereditary recurrent multisystem inflammatory diseases.The available published data from Arab countries about monogenic AIDs other tahn Familial Mediterranean fever (FMF) is very limited.

    Objectives: To report the phenotype, genotype and response to treatment in Arab children with monogenic AIDs other than FMF, with focus on accrual damage.

    Methods: We retrospectively reviewed patients with clinical and/ or genetically proven monogenic AIDs other than FMF seen between 1990 and 2018 at 10 rheumatology clinics from seven Arab countries. Data were collected at the last follow-up visit comprising history of consanguinity, age at onset and diagnosis, follow-up duration, clinical and laboratory findings, as well as the damage accrual and death related to monogenic AIDs.

    Results: Seventy (46 female) patients with monogenic AIDs were analyzed. Consanguinity rate among the enrolled patients was 74.6% and a family history of AIDs was present in 60%. The mean age at disease onset was 3.2±2 years and mean duration of follow-up was 7.5±4 years. The initial diagnosis was inaccurate in 47% and the mean diagnosis delay was 4±3 years. The most frequent monogenic AIDs were LACC1 associated monogenic disorders (monogenic JIA and monogenic Crohn’s) (23) followed by cryopyrin-associated periodic syndrome (CAPS) (12), tumor necrosis factor receptor associated periodic syndrome (TRAPS) (12), hyperimmunoglobulinemia D syndrome (HIDS) (9), Majeed’s syndrome (6) and eight patients with other monogenic AIDs. Musculoskeletal involvement was the main feature in LACC1 associated monogenic disorders while fever and mucocutaneous and gastrointestinal involvement were the most prevalent features in the other monogenic AIDs. Genetic testing was performed in 67 patients, 69% had genetically confirmed disease. Patients with mutation c.T850C p.C284R in exon 4 of LACC1 had severe arthritic changes. Three CAPS patients with NLRP3 mutation had cognitive impairment and one with significant hearing and ocular damage. Two HIDS patients had homozygous p.V3771 mutation and other two patients with p.V3771/compound heterozygous MEFV: p.E148Q/p.P369S/p.R408G. Three different LPIN2 mutations were recorded for Majeed’s syndrome. Overall, growth failure was the most frequent (36%), followed by cognitive impairment (13%). There were three deaths due to infection.

    Conclusion: The number of genetically confirmed patients with monogenic AIDs other than FMF are not uncommon among Arab children probably due to a high consanguinity rate. Diagnostic delay and high damage accrual emphasize the need for more awareness and early referral to specialized centers.

    Disclosure of Interest

    None Declared

    P1004 Auto-inflammatory diseases: a retrospective single center study in Saudi Arabia

    Abdulrahman A. Alrasheed1, Ashwag Al Harthi2, Banan Al Rewaithi2, Wafa Suwairi2, Jubran Al Qanatish2, Fayhan Al Roqi2
    1King Abdullah Specialized Children Hospital; 2Pediatric, King Abdullah Specialized Children Hospital, Riyadh, Saudi Arabia
    Correspondence: Abdulrahman A. Alrasheed

    Introduction: The auto-inflammatory conditions represent an emerging and over-expanding series of diseases unified by a dysregulation of innate immunity. The current increase in the prevalence of chronic inflammatory diseases makes this subject of interest. Early recognition and treatment with immunomodulator agents have the potential to improve the outcomes and the quality of life.

    Objectives: The purpose of this review is to describe the clinical features, investigations, therapeutic modalities and outcome of auto-inflammatory syndromes seen at King Abdullah Specialized Children’s Hospital, Riyadh, Saudi Arabia.

    Methods: We carried out a descriptive retrospective analysis of pediatric patients with auto-inflammatory diseases who were seen and managed at a single tertiary center, in Riyadh, Saudi Arabia, between 2004 and 2018. Multiple measures were investigated including time of disease onset, clinical features, investigations, treatment, and outcome.

    Results: 67 patients (30 females and 37 males) with auto-inflammatory diseases were identified and formed the basis of the study (Table 1).

    Autoinflammatory diseases include Systemic Juvenile Idiopathic Arthritis (sJIA) 46%, Chronic Recurrent Multifocal Osteomyelitis (CRMO) 15%, Familial Mediterranean fever (FMF) 7%, Hyperimmunoglobulin D syndrome (HIDS) 6%, Periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) 6%, Neonatal onset multisystemic inflammatory disease (NOMID) 3%, STING-associated vasculopathy with onset in infancy (SAVI) 3%, ), Aicardi-Goutières syndrome (AGS) 1%, Deficiency of Adenosine Deaminase 2 (DADA2) 5%, Deficiency of interleukin thirty-six receptor antagonist (DITRA) 1%, Blau syndrome 1% and Uncharacterized Auto-inflammatory Disease (UAD) 3%.

    The mean age at initial presentation was 58.2 months. The median Erythrocyte Sedimentation Rate (ESR) and C - Reactive protein (CRP) were 72 mm/hr and 86 mg/dl, respectively. Treatment modalities that have been used include Disease-modifying antirheumatic drugs (DMARDs) and/ or biologic therapy including Anti TNF alpha, Anti IL-1 or Anti IL-6 and others (Table1).

    Conclusion: We believe this cohort provides the common features of auto-inflammatory diseases in Saudi Children and the outcome after frequently used target therapies. Early recognition and treatment of auto-inflammatory diseases with immunomodulator agents have the great potential to alleviate the debilitating symptoms and to improve the quality of life.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1004). See text for description

    P1005 Clinical and genetic characteristics of myalgia in Armenian children with familial Mediterranean fever

    Gayane G. Amaryan1, Tamara F. Sarkisian2, Nune G. Mkrtchyan3, Marina M. Papazyan4, Artashes E. Tadevosyan5
    1National Pediatric Centre for Familial Mediterranean Fever of “Arabkir” Medical Centre - Institute of Child and Adolescent Health, Department of Pediatrics Yerevan State Medical University; 2Centre of Medical Genetics and Primary Health Care, Depatment of Medical Genetics Yerevan State Medical University; 3National Pediatric Centre for Familial Mediterranean Fever of “Arabkir” Medical Centre - Institute of Child and Adolescent Health, Department of Pediatrics Yerevan State Medical University; 4NationalPediatric Centre for Familial Mediterranean Fever, “Arabkir” Medical Centre - Institute of Child and Adolescent Health; 5Department of Public Health and Health Care Organization , Yerevan State Medical University, Yerevan, Armenia
    Correspondence: Gayane G. Amaryan

    Introduction: Familial Mediterranean Fever (FMF) as an ethnic disease is wide-spread in Armenia. In most cases FMF manifests in childhood. Myalgia is an essential feature of musculoskeletal symptoms (MSM) of FMF, which can be presented as acute muscle attacks - spontaneous myalgia (SM) and exercise-induced myalgia (EIM), as well as prolonged protracted febrile myalgia (PFM). Awareness of these symptoms are important for early diagnosis and differential diagnosis of FMF.

    Objectives: to study the frequency of different types of myalgia in Armenian children with FMF and their correlation with MEFV genotype and disease severity.

    Methods: A group of 715 children with FMF was observed at the National Pediatric Centre for FMF (438 boys, 277 girls, mean age 8.64±0.17). The diagnosis of FMF was based on the Tel-Hashomer criteria and detection of 12 MEFV mutations common for Armenians using Viennalab Diagnostics molecular-genetic assay. For statistical analysis standard statistical Epi-Info 2000 Program was performed. For comparison of two nominal variables in table “two by two” Yaet’s corrected for continuity chi-square test was used, significance level p<0.05

    Results: Myalgia was observed in 37.5% (268 out of 715) of FMF patients and manifested mainly as SM and /or EIM in 34.7% (248 patients), as well as PFM in 2.7% (20 patients).

    SM and EIM manifested as transient pains of the legs without fever, disappearing after resting or taking NSAIDs. The frequency of development of SM and EIM varied from 43.6% to 31.2% depending on the genotype and was relatively common in M694V and V726A heterozygotes compared to homozygotes(χ2= 3.41; p> 0.05). Risk of development of SM and EIM was associated with severity of FMF (χ2= 23.20, p <0.0001) and was higher in severe and moderate course of FMF compared to mild 5.6 times (χ2= 18.28; P <0.0001) and 1.9 times (χ2= 3.77; P <0.05) respectively.

    We observed prolonged PFM in 2.7% of FMF patients with more severe phenotypes, which were characterized by 4 times higher risk of early onset (χ2= 5.94; p = 0.015) (mean age 3 years), as well as late diagnosis of FMF (9.42 ± 0.72) and the delayed start of colchicine therapy . This cohort of patients had also high frequency of severe FMF attacks with generalized febrile muscle pain during 1-3 weeks, prevalence of acute recurrent arthritis, erysipelas-like erythema. PFM manifestation usually was after 5-6 year of FMF onset. Because of PFM is considered also as vasculitis, steroid therapy was started along with colchicine. Development of PFM was associated with severe M694V-homozygous genotype (χ2 = 8.27; p <0.02) and was observed at 4.6% of M694V-homozygous patients.

    Conclusion: The frequency of different types of myalgia in Armenian children with FMF is high (37.5%), especially acute muscle attacks - SM and/or EIM (34.7%). PFM is diagnosed in 2.7% patients.

    The development of SM and EIM is associated with the severity of FMF and does not depend on the MEFV genotype. The risk of development of PFM, as a part of prolonged MSM, is associated with both: the MEFV genotype (mostly M694V- homozygous) and the severity of the disease.

    This allows to consider PFM as a marker of severe course of FMF and early disease onset and the M694V homozygous genotype as a risk factor for the development of PFM.

    MEFV mutation genetic screening is recommended for Armenian paediatric patients with different types of myalgia, especially with PFM, for early diagnosis of FMF, treatment and prevention of complications.

    Disclosure of Interest

    None Declared

    P1006 The impact of aging on familial Mediterrinean fever patients

    Okan Aydin1, Serdal Ugurlu1, Bugra Egeli2, Ece Soykut2, Deniz Demir2, Huri Ozdogan1
    1Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty; 2University of Istanbul - Cerrahpasa, Istanbul, Turkey
    Correspondence: Okan Aydin

    Introduction: Familial Mediterranean Fever (FMF) is a monogenic autoinflammatory disorder with innate immune activation with an onset before age 20 in approximately 90% of the patients. There is scarce data on the effect of aging on FMF patients over 40 years of age

    Objectives: This study aims to collect data on FMF patients who have survived over 40 years of age. Here we report our preliminary data on disease course and treatment status and comorbidities of our patients with FMF.

    Methods: Among the FMF patients who have been followed in our FMF outpatient clinic with a pool of approximately 5000 patients, those who have aged 40 and over are being included to the study. As by today 180 patients are considered for evaluation. The files of patients were reviewed and a standard questionnaire was used to interview the patients. Here we report the results of 100 of these patients (56%) who were contacted for this purpose. These patients were questioned on their demographic characteristics, comorbid conditions, colchicine treatment details, and attack information. In order to see the trend of the change in the parameters assessed , the patients were divided into two groups based on their present age(Group 1: 40-50 years, Group 2: ≥50 years).

    Results: A total of 100 (78 F, 22M) patients were evaluated. There were 61(46F, 15M) patients aged between 40-50 years and 39 (32F, 7M) over 50. The demographic characteristics and clinical features of these patients are given in Table 1.Besides 3, all patients were still on colchicine regularly. Ninety-six percent of the patients declared overall benefit from colchicine therapy, however 38% experienced a side effect related to this treatment.Over 88% of the patients reported decrease in severity and frequency of FMF attacks.The mean daily colchicine dose was lower in the age 50 and over group (1.7±0,77 mg versus 1,35 ±0,38 mg). There were no patients with AA amyloidosis in neither age group. The mean duration from the last attack increased from 15.3 ±19.7 months to 35.6± 52 months in the older patients. One or more additional disease was present in 75% of this patient group.Among the comorbidities hypertension was the most frequent, diagnosed in 25% of the patients, followed by hypothyroidism (16%), diabetes mellitus (10%) and cardiac disease (5% ). Sixty-five of the patients were recieving other medications in addition to colchicine.

    Conclusion: According to our preliminary data the majority of the patients continue to take colchicine after age of 40. However the frequency of FMF attacks as well as daily colchicine dose decrease as the patients get older.With well designed trials stopping colchicine treatment may be considered in a subgroup of patients after 50 years of age. Approximately ¾ of the FMF population over 40 years of age has a comorbidity that neccecitates additional medications which underlines the need for special attention.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1006). Clinical course and co-morbidities in two age groups over 40 years

    P1007 Comorbidities in familial Mediterranean fever

    Ummusen Kaya Akca1, Banu Balci Peynircioglu2, Zehra S. Arici1, Edibe Avci2, Zulfiye Y. Akkaya Ulum2, Engin Yilmaz2, Yelda Bilginer1, Seza Ozen1
    1Pediatric Rheumatology; 2Medical Biology, Hacettepe University, Ankara, Turkey
    Correspondence: Banu Balci Peynircioglu

    Introduction: Familial Mediterranean Fever (FMF) is a periodic fever syndrome, characterized by recurrent episodes of fever and serosal inflammation accompanied with high acute phase reactants.The analysis of possible comorbidities is important to understand the impact of these conditions on clinical care and whether they share a common etiological pathway.

    Objectives: We aimed to evaluate the comorbidities associated with FMF patients in a large genetically diagnosed cohort.

    Methods: We retrospectively evaluated the medical records of FMF patients who were followed up at Department of Pediatric Rheumatology in Hacettepe University between 2000 and 2015. This study was approved by the Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki. The diagnosis of FMF was made according to Tel Hashomer diagnosis criteria for patients who applied prior to April 2009 and to the Turkish FMF pediatric diagnosis criteria after April 2009. The FMF patients who had homozygous or compound heterozygous mutations were included in the study. Comorbidities associated with FMF were divided into three groups; associated with increased inflammation, associated with FMF and incidental.

    Results: A total of 1999 patients were enrolled in the study. Of all 1999 FMF patients, 636 were children (31.8 %), 1029 were males (51.4%), with a mean age of 31.60±16.01 years. The mean follow up time was 4.50±3.99 years (median:3.84 range from 0.21-29.4 years). 880 of 1999 (44%) FMF patients had homozygous MEFV gene mutation, the most common mutation was M694V homozygous. The remaining were compound heterozygous. 656 patients (32.8%) had one or more than one comorbidity associated with FMF. Ankylosing spondylitis was the most common comorbidity associated with increased inflammation while the most common comorbidity in FMF related comorbidities was renal amyloidosis. The frequency of ankylosing spondylitis, henoch schonlein purpura, juvenile idiopathic arthritis, polyarteritis nodosa (PAN), multiple sclerosis (MS) and Behçet’s disease were increased in patients with FMF when compared to those in the literature. Systemic lupus erythematosus was observed less frequently in the patients with FMF than in the population. While the increase in the frequency of MS was 3.3 times, the frequency of PAN was increased 110 times.

    Conclusion: This study shows that FMF is a hereditary disease associated with significant comorbidity. We also confirm that inflamatory and rheumatic diseases are more common in FMF.

    Disclosure of Interest

    None Declared

    P1008 Bone metabolism in systemic autoinflammatory diseases (SAIDS): a case- control study from Padova cohort

    Sara Bindoli1, Giulio Franceschet2, Paola Galozzi1, Martina Zaninotto3, Valentina Camozzi2, Paolo Sfriso1
    1Rheumatology Unit, Department of Medicine; 2Endocrinology Unit, Department of Medicine; 3Department of Laboratory Medicine, University of Padova, Padova, Italy
    Correspondence: Sara Bindoli

    Introduction: Systemic autoinflammatory diseases (SAIDs) represent a group of disorders characterized by recurrent fever attacks, polyserositis, skin, musculo-skeletal and articular manifestations. A dysregulation of the innate immune response in a genetic predisposed host leads to the development of SAIDs. In our study we included patients affected by Familial Mediterranean Fever (FMF), TNF Receptor Associated Periodic Syndrome (TRAPS) and Hyper-IgD Syndrome (HIDS). It is assessed that chronic inflammation, perpetuated by pro-inflammatory cytokines, may exert a role on bone metabolism leading, in the long run, at the onset of osteoporosis (OP). However, how OP may occur in the context of autoinflammatory diseases remains partially unknown.

    Objectives: The aims of our study are focused on the assessment of bone metabolism in patients affected by SAIDs compared to healthy subjects and on the relationship between bone turnover markers (Receptor activator of nuclear factor kappa-Βligand, RANKL and osteoprotegerin, OPG) and serum inflammation markers (serum amyloid A, SAA).

    Methods: 40 adults patients referring to the Rheumatology Unit of Padova University Hospital affected by FMF, TRAPS and HIDS and 40 healthy subjects were recruited between March and June 2018. Fasting blood samples were collected in order to determinate calcium (Ca), phosphorus (P), magnesium (Mg), 24-h urine calcium, 24-h urine phosphorus, albumin, parathyroid hormone (PTH), Vitamin D, creatinine, serum amyloid A (SAA), c-terminal telopeptide of type I collagen (CTX), bone alkaline phosphatase (b-ALP). Moreover, serum OPG and RANK-L were determined by a commercially available ELISA kit (Pantec, Turin, Italy). Femur and lumbar dual-energy X-ray absorptiometry (DXA) was performed with the QDR Bone Densitometer Discovery (Hologic Inc.,Waltham, MA). Trabecular Bone Score (TBS) was calculated on DXA lumbar images, using iNsight Software (version 1.8.0.0; Medimaps, Merignac, France). The statistical analysis was performed using Mann-Whitney U Test.

    Results: We did not observe a statistically significant difference between bone mineral density (BMD) and TBS of patients compared to controls (p=0.5037 and p=0.8031). Also, the values of phospho-calcic metabolism samples were not statistically different between patients and controls. As expected, SAA levels were significantly higher in patients (p= 0.0144), and interestingly also OPG levels were significantly higher if compared to the healthy subjects (p= 0.0018). For b-ALP, CTX and RANK-L no differences were observed between the two groups (p=0.1466, p=0.8861, and p= 0.7890 respectively).

    Conclusion: Patients of our cohort affected by FMF, TRAPS and HIDS do not present an increased risk of OP compared to the healthy controls. Indeed, TBS and BMD are similar between the two groups highlighting a preserved bone quality in our patients. Interestingly, OPG levels are higher compared to controls and this could suggest a protective role and a bone re-balancing activity in a context of inflammation. Finally, a regulatory effect of serum amyloid A on bone homeostasis should be taken into account.

    Disclosure of Interest

    None Declared

    P1009 Two cases of hyperzincaemia/hypercalprotectinaemia show novel phenotypic properties

    Anikó Szabó1, Péter Blazsó1, Viktória Sümegi2, Tibor Kalmár1, Zoltán Maróti1, Csaba Bereczki1
    1Department of Paediatrics; 2Department of Rheumatology and Immunology, University of Szeged, Szeged, Hungary
    Correspondence: Péter Blazsó

    Introduction: Hyperzincaemia/hypercalprotectinaemia (Hz/Hc) is a recently discovered autoinflammatory disorder showing monogenic, autosomal dominant inheritance pattern. Until now the p.E250K and p.E257K missense mutations of PSTPIP1 gene have been found in the background. These result in the extreme overproduction of the pro-inflammatory cytokine IL-1β leading to the striking elevation of serum calprotectin and zinc through positive feedback loops. Uncontrolled inflammation elicited by the continuous activation of these danger signals end up in the classical constellation of systemic and cutaneous inflammation, hepatosplenomegaly, arthritis, pancytopenia, and failure to thrive.

    Objectives: We aimed to characterize the clinical phenotypes accompanied the mutations of PSTPIP1 gene found in two unrelated Hungarian male patients. These data were compared to the clinical presentations of the already published cases and novel symptoms were demonstrated.

    Methods: Clinical cases of both boys were investigated thoroughly. In order to clarify the diagnosis targeted exome sequencing panel (Illumina TruSight One) was applied in each case to pre-screen for possible mutations. Nucleotide variants in the probands and in their parents were confirmed by Sanger sequencing.

    Results: A p.E250K mutation was detected in one patient and a p.E257K in the other. Both variants were de novo and heterozygous. p.E250K in the 2,5-year-old boy was associated with additional recurrent non-infectious diarrhea, macrocephaly and pericardial fluid on the top of the classical signs. p.E257K in the 7-year-old boy was joined with haemangiomatosis, mental retardation, autism and overweight besides the already known manifestations of Hz/Hc.

    Conclusion: Atypical and potentially misleading clinical findings in Hz/Hc underline the importance of genetic testing in order to get to the proper diagnosis. The presented cases might extend our knowledge of the phenotypic traits observable in Hz/Hc.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1010 Identification of novel NLRC4 and IL2RA variants in a family cohort with juvenile-onset arthritis and rash

    Jessica L. Bloom1, Megan L. Curran1, Scott Canna2, Harold Hoffman3, Elena Hsieh4
    1Department of Pediatrics, University of Colorado, Aurora; 2Departments of Pediatrics and Immunology, University of Pittsburgh, Pittsburgh; 3Departments of Pediatrics and Medicine, University of California San Diego, San Diego; 4Departments of Pediatrics and Immunology and Microbiology, University of Colorado, Aurora, United States
    Correspondence: Jessica L. Bloom

    Introduction: Identification of genetic etiologies of autoinflammatory syndromes can inform targeted therapy to improve outcomes.

    Objectives: We aimed to identify a genetic etiology for an underlying autoinflammatory syndrome in a 3-year-old boy presenting with failure to thrive, rash, and polyarthritis since infancy without significant fevers. The patient’s mother and maternal aunt also had similar symptoms since infancy.

    Methods: We obtained a history, exam, routine laboratory evaluation, chromosomal microarray, immune phenotyping and functional assays, and genetic sequencing of familial autoinflammatory syndromes via INVITAE.

    Results: The patient’s first examination showed small and large joint polyarthritis and maculopapular rash. Laboratory results included anemia, positive ANA, negative RF and anti-CCP, mildly raised ferritin, and very elevated platelet level, LDH, ESR, CRP and IgG. He had recurrent diarrhea and tested positive for Campylobacter. INVITAE’s autoinflammatory panel showed two heterozygous variants of unknown significance (VUS): NLRC4, exon 4, c.741_742insAlu (p.Leu247fs) and IL2RA, exon 2, c.76G>C (p.Asp26His).

    Genetic testing revealed the same two variants in the patient’s mother and aunt, while unaffected relatives had one or the other (see Table 1). The patient and mother are HLA-B27+ and have the same 2.8 Mb duplication from 3q28 to 3q29 on chromosomal microarray including IL1RAP. Signal transducer and activator of transcription phosphorylation (pSTAT5) studies showed increased baseline pSTAT5 induction without IL-2 stimulation in patient compared to control.

    The patient’s arthritis improved partially with naproxen and steroid joint injections. Given genetic results, subcutaneous anakinra was initiated at 10 mg/kg daily with significant improvement in arthritis, rash, and fatigue. Labs after one month showed resolved anemia, normal inflammatory markers, and high IL-18 (7,824 pg/mL, normal 89-540). He switched to 4.29 mg/kg of canakinumab monthly. After one month, he had mildly active arthritis, occasional fatigue, and stable labs apart from an increase of IL-18 to 15,329 pg/mL. The mother, who is poorly controlled off therapy, has elevated IL-18 (7,176 pg/mL)

    Conclusion: We present a family cohort with juvenile-onset arthritis and rash, found to have elevated IL-18, VUS in both NLRC4 and IL2RA, and a chromosomal duplication consistent with a heritable autoinflammatory syndrome. While it appears that both variants are required for symptomatology, his presentation is most consistent with an unrecognized gain of function NLRC4 mutation despite lack of recurrent fevers or enterocolitis. Genetic evaluation led to targeted therapy and improved outcomes, with additional testing underway to further personalize therapy.

    The patient's family consented for publication.

    References

    1. Canna SW, et al. An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nature Genetics. 2014;46(10):1140-6.

    2. Romberg N, et al. Mutation of NLRC4 causes a syndrome of enterocolitis and autoinflammation. Nature Genetics. 2014;46(10):1135-9.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1010). INVITAE Panel Results

    P1011 Discontinuation of colchicine therapy in children with familial Mediterranean fever

    Yonatan Butbul1, Rawan Silman1, Shafe Fahoum2, Yackov Berkun3
    1Department of Pediatrics B, , Rappaport Children's Hospital, Rambam Medical Center, Haifa; 2Department of Pediatrics B, Rappaport Children’s Hospital, Rambam Medical Center, Haifa; 3Department of Pediatrics and FMF Clinic, Hadassah-Hebrew University Medical Center, Mount Scopus, Jerusalem, Israel
    Correspondence: Yonatan Butbul

    Introduction: Clinical phenotype of FMF exists in some carriers of MEFV mutation. These patients tend to have a mild disease. Prolonged colchicine free remission was reported in a small group of FMF patients.

    Objectives: To describe and characterize a group of children with FMF in whom colchicine was discontinued.

    Methods: The study cohort consisted of all children with FMF followed at 2 referral centers in Israel in whom colchicine was discontinued following prolonged attack free period.

    Clinical presentation, mutations in MEFV gene and disease outcome of patients who successfully ceased colchicine therapy were compared with patients with relapse of FMF attacks.

    We performed a retrospective study in two referral centers in Israel of 43patients with FMF with 1 or non-mutated MEFV allele who ceased colchicine therapy following prolonged attack free period. The phenotype of the patients was investigated in detail, and the MEFV mutations, laboratory findings, clinical picture and outcome of 30 (70%) patients that successfully ceased colchicine therapy were compared to 13 (30%) patients whofailed.

    Results: 47 patients (55% males), mean age 6±3.2 years at the diagnosis, were enrolled in the study, of them 4patients were excluded due to poor follow up. Fever (93%), abdominal pain (79%), arthralgia (19%) and arthritis (12%) were the most common symptom at attack.The average period free of attacks before enrolment was 11.3±9.2 months. The average follow-up after ceasing colchicine was 5.1± 2.9 years. Thirteenpatients (30.2%) had anattack during follow up with most common symptomsof fever (92%) and abdominal pain (77%) and colchicine therapy was restarted within 10.1 months (1.1-36.4months). There were no differences between the groups of patients that were able to stop colchicine and the group that needed to renew therapy in demographic, genetic and most clinical parameters, including the age (13.4±3.9 vs 11.9±3.7p-0.26), level ofSAA at enrolment (4±3.6 vs 3.3±2.4p-0.7) and time of last attackprior to enrolment (12.6±9.6 vs 8.6±8.2 monthsp-0.08). Myalgia and arthritis were more common among children that required to renew therapy compared to the group that didn’t (31% vs 6.7% p-0.058 and 31% vs 3% p-0.024 respectively).

    Conclusion: Cessation ofcolchicine therapy following prolonged remission in selected group of patients who are not homozygous for MEFV mutation could be considered. Patients with arthritis or arthralgia are more likely to have an attack after ceasing colchicine therapy.

    Disclosure of Interest

    None Declared

    P1012 Long-term follow up of a mevalonate deficiency kinase patients cohort

    Inmaculada Calvo Penades, Berta Lopez Montesinos, M. Isabel Gonzalez Fernandez, Miguel Marti Masanet, Elena Fernandez De La Puebla
    Pediatric Rheumatology Unit, HUIP la Fe, Valencia, Spain
    Correspondence: Inmaculada Calvo Penades

    Introduction: The syndrome mevalonate kinase deficiency (MKD) is part of the syndromes of periodic fever. With typical clinical manifestations and good response to treatment with IL-1 blockade, but the long-term manifestations are little known

    Objectives: To assess the clinical features, treatment and evolution of 11 patients diagnosed with Mevalonate Kinase Deficiency (MKD).

    Methods: Baseline demographic and clinical characteristics of the patients were considered, including age at symptoms onset, age at diagnosis, time to follow up, clinical features, laboratory data, results of the MVK gene sequencing study, administered treatments and evolution.

    Results: Overall, 11 patients. Gender: M/W 7:4. Onset age: 0.8 m (0-15 years). Age at diagnosis: 9.6 years (4m-15 years), time of follow up: 9.8 years (4m-11 years). Clinical features: Fever 93% of the patients, adenopathy 93%, abdominal pain 66%, oral aphtha 66%, diarrhea 60%, arthritis 60%, amygdalitis 60%, other symptoms: skin rash 34%, headache, 34%, hepatomegaly 27%, splenomegaly 20% and serositis 10%. Three patients presented atypical manifestations: intestinal obstruction (6 episods), intestinal invagination (4 episodis), orquitis (3 episodis) and chylothorax. All patients showed IgD levels > 100 U/mL (100-1500). MVK mutations: 6 patients homozygotes (2: V250I, 4: V377I) and 5 patients double heterozygotes: 4(I268T, V377I), 1(N205D, R388X). The genetic study of the whole family was performed in 7 families. Corticoid treatment used for 100% of the patients. Anakinra in 45% (parcial-complete response), Canakinumab 82% (complete response), Etanercept 9% (no response) and adalimumab 9% (hydradenitis). Long-term manifestations: 2 patients with cutaneous abscess and 1 patient with hydradenitis suppurativa.

    Conclusion: In this cohort the high response to canakinumab treatment is shown and late clinical manifestations are described, to our knowledge, for the first time in literature.

    Disclosure of Interest

    None Declared

    P1013 Novel assay to diagnose and monitor cryopyrin associated periodic syndromes (CAPS)

    Fortunata Carbone1,2, Luca Cantarini3, Teresa Micillo4, Maria Alessio5, Alma Nunzia Olivieri6, Maria Francesca Gicchino7, Antonella Insalaco8, Maria Cristina Maggio9, Orso Maria Lucherini3, Roberto Scarpioni10, Matteo Piga11, Maria Maddalena Angioni11, Laura Obici12, Antonella Simpatico3, Pietro Leccese13, Rita Consolini14, Raffaele Manna15, Paolo Sfriso16, Sara Bindoli16, Paola Galozzi16, Ida Orlando3, Sabrina Chiesa17, Marco Gattorno17, Giuseppe Matarese18,19
    1Istituto per l'Endocrinologia e l'Oncologia Sperimentale-Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli; 2Unità di Neuroimmunologia, IRCCS Fondazione Santa Lucia, Roma; 3Research Centre of Systemic Autoinflammatory Diseases, Behçet's Disease Clinic and Rheumatology-Ophthalmology Collaborative Uveitis Centre, Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena; 4Dipartimento di Biologia, Università di Napoli “Federico II”; 5Department of Translational Medical Sciences, Section of Pediatrics, Federico II University; 6Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica; 7Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università degli Studi della Campania “Luigi Vanvitelli”, Napoli; 8Department of Pediatric Medicine, Division of Rheumatology, Bambino Gesù Children's Hospital, Roma; 9Ospedale dei Bambini “Di Cristina” , Palermo; 10Ospedale AUSL “Guglielmo da Saliceto” , Piacenza; 11Reumatologia, Policlinico Universitario, Cagliari; 12Centro per lo Studio e la Cura delle Amiloidosi, Fondazione IRRCS, Policlinico San Matteo, Pavia; 13Rheumatology Institute of Lucania (IReL), Rheumatology Department of Lucania, “San Carlo” Hospital of Potenza and “Madonna delle Grazie” Hospital of Matera, Potenza; 14Laboratory of Immunology, Division of Pediatrics, Department of Clinical and Experimental Medicine, University of Pisa, Pisa; 15Centro delle febbri periodiche e malattie rare, Policlinico Gemelli, Università Cattolica Roma, Roma; 16Unità di Reumatologia, DIMED, Policlinico Universitario, Padova; 17Dipartimento di Scienze Pediatriche Generali e Specialistiche, IRCCS Istituto Giannina Gaslini, Genova; 18Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II; 19Istituto per l'Endocrinologia e l'Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy
    Correspondence: Fortunata Carbone

    Introduction: Cryopyrin associated periodic syndromes (CAPS) are rare autoinflammatory disorders associated with dominantly gain-of-function mutations in the NLRP3 gene that result in overactivation of the inflammasome, increased secretion of interleukin (IL)-1beta and IL-18, and systemic inflammation. It has been reported that oligomeric particles of the adaptor ASC (apoptosis-associated Speck-like protein with a caspase-recruitment domain) are released together with IL-1beta and active caspase-1 subunits after activation of the inflammosome complex and that patients with CAPS show an increased serum concentration of ASC+ particles.

    Objectives: The diagnosis of CAPS is a critical factor due to both the lack of specific laboratory results and the sharing of similar clinical manifestations with other autoinflammatory diseases, our aim is to develop a simple assay to evaluate the levels of ASC particles in the serum of CAPS patients to provide novel biomarkers facilitating early disease diagnosis and able to monitor treatment responses.

    Methods: We developed an ELISA for the quantification of ASC particles in serum and plasma of normal and pathological subjects. We analysed samples from CAPS patients and from patients with autoimmune disorders (Multiple Sclerosis (MS), Type 1 Diabetes (T1D) and juvenile idiopathic arthritis), to confirm that ASC presence in the serum is not due to other chronic inflammatory processes characterizing autoimmunity. In addition, we also evaluated the concentration of ASC in the sera of TNF receptor–associated periodic syndrome (TRAPS) patients to reinforce the concept of specificity of this biomarker in CAPS patients and not in individuals suffering from others inflammatory disorders.

    Results: We observed that untreated CAPS patients are characterized by the presence of a significant higher amount of ASC particles when compared with healthy controls (HS) and with patients suffering from MS and T1D. This tendency was also evident in patients with arthritis and TRAPS even if the difference was not statistically significant due to the small number of samples. In addition there is a tendency through a reduction of ASC levels in CAPS patients after pharmacological treatment, which require future investigations.

    Conclusion: These data suggest that ELISA quantitation of ASC protein could represent a novel and additional strategy for the diagnosis and monitoring of CAPS.

    Disclosure of Interest

    F. Carbone: None Declared, L. Cantarini: None Declared, T. Micillo: None Declared, M. Alessio: None Declared, A. N. Olivieri: None Declared, M. F. Gicchino: None Declared, A. Insalaco: None Declared, M. C. Maggio: None Declared, O. M. Lucherini: None Declared, R. Scarpioni: None Declared, M. Piga: None Declared, M. M. Angioni: None Declared, L. Obici: None Declared, A. Simpatico: None Declared, P. Leccese: None Declared, R. Consolini: None Declared, R. Manna: None Declared, P. Sfriso: None Declared, S. Bindoli: None Declared, P. Galozzi: None Declared, I. Orlando: None Declared, S. Chiesa: None Declared, M. Gattorno: None Declared, G. Matarese Grant / Research Support from: Giuseppe Matarese reports research grants from Merck-Serono, Biogen Idec, Novartis and IBSA.

    P1014 An Italian family with FCAS

    Maria Carrabba1, Marina Zarantonello2, Isabella Ceccherini3, Giovanna Fabio1
    1Internal Medicine - Rare Diseases Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 2Department of Clinical Sciences and Community Health , Università degli Studi, Milan; 3UOC Genetica Medica, UOS Diagnostica Molecolare e Malattie Ereditarie, Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Maria Carrabba

    Introduction: The prevalence of Cryopyrin-Associated Periodic Syndrome (CAPS) is estimated at about one per million. The Familial Cold Autoinflammatory Syndrome (FCAS) presents in about 95% of the patients by 6 months with cold-induced, urticaria-like rash, fever, and arthralgia. FCAS causes lifelong debilitating effects that restrict patients’ daily activities. Diagnostic delay related to lack of knowledge in Autoinflammatory Diseases is still an important problem.

    Objectives: To report an Italian family with FCAS successfully treated with anti-IL1.

    Methods: Three subjects, members of the same family, were screened by an experienced doctor. Clinical and laboratory variables, Brain-CT scan, X-ray, audiometry and lung function tests were performed. A targeted review of clinical feature for Autoinflammatory diseases and standardized questioning for CAPS-associated symptoms was conduct. Genetic testing for the NLRP3 mutation was performed.

    Results: A 51 years old woman presented with a long history of maculopapular rash after cold exposure starting in early childhood associated with low-grade fever, higher during childhood. During cold months, she needed daily FANS because of fever, and topical steroids for skin rash, prescribed by dermatologists. The patient was in chronic therapy with local steroids because of recurrent ulcerative keratitis. Some corneal scars are present as outcomes. She experienced ocular manifestation on exposure to cold and summertime in contact with air conditioning. She has a 18-years daughter and a 15-years son with a long history of maculopapular rash after cold exposure starting in childhood associated with low-grade fever, fatigue, headache and arthralgia (more severe in the son) lasting less than 24hours. The two children had the same ocular manifestations of the mother and underwent local steroids therapy 4-5 times per year. They all carried the A439V mutation on NPLR3 gene exon 3. Patients were vaccinated (Pneumococcus, HBV, HAV) before starting treatment for anti-IL1 (canakinumab, a fully human anti-IL-1β monoclonal antibody, at a dose of 150 mg subcutaneous every 8 weeks). The Auto-Inflammatory Disease Activity Index (AIDAI, the simplified items scored 0–1 (CAPS range 0–155)) and the Autoinflammatory Disease Damage Index (ADDI) scores were assessed before every treatment. Before treatment, the AIDAI score calculated during a winter and summer month was respectively 59 and 5 for the mother, 30 and 3 for the daughter and 40 and 3 for the son. After 12-months of treatment, the AIDAI score is zero for the mother and the daughter as well as the ADDI score. During treatment mother experienced a urinary tract infection and the daughter two distinct infectious episodes: one of pharyngitis and one of diarrhoea. The son delayed his treatment because of a severe chronic sinusitis that needed surgical treatment. He just received his first dose last month.

    Conclusion: The NLRP3 mutation A439V was first entered into the Infevers database in 2002 by Hal Hoffman. It is one of the most common mutations in multiplex families with CAPS and it is considered a CAPS disease-causing mutation, although the overall clinical phenotype is rather mild. Most of the patients reported with FCAS carrying A439V have ocular manifestations (conjunctivitis, keratitis, uveitis) that causes lifelong debilitating effects that restrict patients’ daily activities, as well as rash or low-grade fever or myalgia/arthralgia.FCAS-associated ocular manifestations need treatment with anti-IL1, which typically results in dramatic improvement in clinical and laboratory measures of inflammation, and is well tolerated. Unfortunately, ophthalmologists, dermatologist as well as other specialists do not recognise FCAS and do not appropriately treat this disease nor send patients to Autoinflammatory Diseases experienced Centres.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1015 Scores assessment for clinical management of familial Mediterranean fever

    Maria Carrabba1, Marina Zarantonello2, Giovanna Fabio1
    1Internal Medicine - Rare Diseases Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; 2Department of Clinical Sciences and Community Health, Università degli Studi, Milan, Italy
    Correspondence: Maria Carrabba

    Introduction: Clinical spectrum of Familial Mediterranean fever (FMF) is heterogeneous, ranging between minimal activity of few affected sites and excellent response to colchicine and large number of frequent, intolerable, treatment-resistant attacks. Frequent and severe FMF attacks may result in serious complications. Severity scoring systems for FMF have been developed and validated in adults (1998 Pras et al., 2005 Mor et al.) to objectively quantify the disease severity for both therapeutic and prognostic purposes. In 2016 the ISSF score has been developed with a consensus-driven methodology by paediatricians and internists, with expertise in this disease, and validated in a large database comprising both children and adults with FMF. Recently, the ADDI score, which was developed based on consensus building, purposes to measure the chronic damage by Autoinflammatory Diseases.

    Objectives: This study aims to assess the performance of the existing severity scores (Mor 2005 and ISSF 2016) for FMF in predicting the therapeutic outcome (colchicine dosage and residual attacks); and to assess the relation between the above scores and the ADDI score.

    Methods: All the patients with FMF in charge since 2003 have been enrolled. Severity scores have been calculated at the diagnosis and ADDI has been calculated at the latest visit. ROC curves and statistical analysis have been performed.

    Results: Forty-five consecutive patients affected by FMF (follow-up 1-41 years) with a median age of 39 years (range 9-89) have been evaluated. Ten patients were diagnosed for FMF in childhood and 35 in adulthood, with a median of diagnostic delay of 12 years (range 1-45). Eight patients are homozygous carries of one mutation on MEFV gene exon 10, twelve patients are heterozygous carriers of two mutations on MEFV gene exon 10, and twenty patients are heterozygous carriers of one mutation on MEFV gene exon 10.

    All patients except four are under therapy with colchicine. Nine patients need more than 1mg per day of colchicine. Ten patients reported one or two mild FMF attacks during the year before the last visit. According to the severity score of Mor et al., 15 patients were stratified as mild disease, 20 as intermediate and 10 as severe. According to the ISSF score, 37 patients had a mild disease, eight intermediate and no one severe. Sixteen patients have a positive ADDI score (range 0-4). The ROC analysis shows that the score of Mor performed better than the ISSF (AUC 0.903vs0.661) to identify patients needing more than 1mg of colchicine per day. The ISSF has a good performance (AUC 0.694) to identify patients referring one or two mild attacks in the year before the last visit. The ROC curves analysis shows that both the Mor (AUC 0.677) and the ISSF (AUC 0.657) scores can identify patients with chronic damage (positive ADDI score). ROC analysis of Mor, ISSF and ADDI scores for FMF genetic pattern, showed that only ADDI has a good performance (AUC 0.811) to discriminate the FMF homozygous patients.

    Conclusion: The score of Mor et al. can be useful for prediction of FMF patients needing higher colchicine dosage. The ISSF score seems to predict better patients with one or two mild attacks per year. The most of homozygous FMF patients have a positive ADDI score despite therapy.

    Disclosure of Interest

    None Declared

    P1016 Single center experience with 402 familial Mediterranean fever patients

    Ozlem Ozdemir Isik, Senem Tekeoglu, Duygu Temiz Karadag, Ayten Yazici, Ayse Cefle
    Department of Internal Medicine Division of Rheumatology, Kocaeli University Faculty of Medicine, Kocaeli, Turkey
    Correspondence: Ayse Cefle

    Introduction: Familial Mediterranean Fever (FMF) is an autosomal recessive genetic disorder that causes recurrent episodes of fever, poliserositis, arthritis, skin eruptions.

    Objectives: In this study, we aim to present clinical and demographic features of FMF patients followed in our clinic.

    Methods: The clinical, demographic, genetic features and management of 402 FMF patients (fulfilling Tel-Hashomer Diagnostic Criteria) were analyzed.

    Results: The mean age was 36,8±11,2 (10-71), the mean diagnosis age was 28±11,9 (3-66) years, and mean duration of disease was 189±125 months. Mean duration between disease onset and treatment was 93.6±104 months. 43% (174) of the patients had positive family history for FMF, 7% (29) had consanguineous marriage in family. 24% of the patients had appendectomy. Fever and abdominal pain both were initial symptoms in 72% of the patients, while 7% of them had chest pain, 4% had only fever, 15% had arthritis, 1% had erysipelas like erythema (ELE) as the first symptom of FMF.

    Fever was observed 76% of the patients, abdominal pain in 86%, ELE in 13%, chest pain in 21%, and arthritis in 32%. The frequency of monoarthritis was 21%, oligoarthritis 10,5% and polyarthritis was identified 0,5% of the patients. Ankle arthritis was the most frequent(20%) one among the patients who had monoartritis. 16% of the patients had inflammatory back pain, while 11% of the patients were identified with sacroiliitis. 8 patients (2%) suffered from chronic kidney disease and 2 of them were on dialysis programme. Amyloidosis (diagnosed with biopsy) was identified among 14 patients (3,5%).

    At least one mutation of MEFV gene was identified in 78% of the patients. No mutation could be identified in 8%. MEFV gene analyses was not performed in 14% of the patients. The most frequent mutation was M694V mutation and its allel frequency was found to be 54%. For V726A, M680I, E148Q, R761H and A744S allels, mutation frequency were in order of 11%, 7%, 7%, 2%, 1%. The frequency of compound heterozygositywas 38% and the most common genotype wasM694V/R202Q (11%).

    There was a significant relationship between M694V mutation and arthritis, ELE, amyloidosis and sacroiliitis (p<0.001). Amyloidosis was more frequent in patients with M694 homozygous mutation. Mean age of disease onset was lower in patient who had M694V homozygous mutation than M694V heterozygous mutation (p<0.001).

    Conclusion: The most common mutation in our patients is M694V mutation and it is significantly associated with arthritis, ELE, sacroiliitis, and amyloidosis. Tight control and regular treatment are important in FMF patients to protect from amyloidosis.

    Disclosure of Interest

    None Declared

    P1017 Testicular ischemia in deficiency of adenosine deaminase 2 (DADA2)

    Katherine Clarke, Cathy Campbell, Ebun Omoyinmi, Ying Hong, Muthana Al Obaidi, Neil Sebire, Paul Brogan
    Paediatric Rheumatology, Great Ormond Street Hospital, London, United Kingdom
    Correspondence: Katherine Clarke

    Introduction: Deficiency of adenosine deaminase 2 (DADA 2) is a rare autosomal recessive autoinflammatory condition. Recognised features include vasculitis predominantly affecting medium sized vessels, livedoid skin rash, central and peripheral nervous system involvement, variable degrees of immunodeficiency, and marrow failure amongst other clinical presentations. We present the case of a six year old male with DADA 2 who presented with acute testicular ischaemia secondary to vasculitis, the first such description in DADA2.

    Objectives: We wish to highlight the ever-expanding phenotype of DADA2, and to emphasise the ongoing controversies regarding management of this lifelong genetic disease. Improvements in our understanding of the pathogenesis will ultimately lead to better biomarkers, better treatments, and ultimately even cure using gene therapy based on the favourable clinical outcomes of patients undergoing allogeneic HSCT thus far.

    Methods: A six year old male presented acute right-sided testicular pain. His history included transient infantile neutropenia, resolved hepatosplenomegaly, and longstanding livedo racemosa, leading to screening and confirmation of DADA2 caused by homozygous c.139G>C(p.G47R) mutation of ADA2. As his only clinical feature was that of mild livedo racemosa with normal laboratory parameters at diagnosis, he was being actively monitored prior to starting any treatment. At a routine clinic follow-up a 24 hour history of testicular pain was noted on systems review; he was afebrile, and his only physical signs were that of moderate livedo racemosa, and tenderness of the right testicle. Laboratory parameters revealed C-reactive protein 8mg/L (reference range [RR]<20mg/L); erythrocyte sedimentation rate 28 mm/hr (RR<10); and serum amyloid A 5mg/L (RR<10). Ultrasound-scan of the scrotum revealed significantly reduced perfusion of the right testes, without torsion; and surgical scrotal exploration confirmed testicular ischaemia without torsion. Histology demonstrated ischaemic seminiferous tubules with intervening haemorrhage and acute inflammatory cells, consistent with vasculitis of the testis as the cause. He was treated with high dose intravenous methyl-prednisolone followed by a weaning course of oral prednisolone, and subcutaneous adalimumab (anti-tumour necrosis factor alpha, anti-TNFα). Repeat ultrasound-scan 3 weeks later revealed good testicular perfusion, with a small area of focal infarction. At last follow-up (11 months post-event) he remained asymptomatic, on treatment with adalimumab.

    Results: Figure 1: Livedo racemosa noted on both lower limbs.

    Figure 2: Doppler study of both testes (transverse views) showing globally reduced (but not absent) perfusion in the right testes compared to the left.

    Figure 3: Photomicrograph of testicular biopsy showing patchy areas of tubular necrosis with loss of cellular and nuclear detail and nuclear ‘smudging’ (Right side) with more normal, viable tubules (Left side); H&E original magnification x100

    Figure 4: Acute phase markers before, during, and after acute testicular infarction in DADA2. Acute phase responses were completely normal prior to testicular infarction. C-reactive protein (CRP) and serum amyloid A (SAA) remained within the normal range throughout the episode and follow-up. Erythrocyte sedimentation rate (ESR) was normal, but was transiently modestly elevated on the day of tissue infarction (arrowed), remaining normal at follow-up.

    Conclusion: The phenotype of DADA2 continues to expand, and we add testicular infarction to the features of DADA2. CRP and SAA cannot be relied on as reliable biomarkers to predict tissue ischaemia and hence who to target for anti-TNFα therapy in DADA2, since these remained steadfastly normal before, during, and after testicular infarction in this case

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1018 A novel duplication in the X-linked inhibitor of apoptosis protein gene leading to recurrent hemophagocytic lymphohistiocytosis that is responsive to interleukin-1 blockade

    Dilan Dissanayake1, Rebecca Marsh2, Ahmed Naqvi3, Michael Jordan2, Rae Yeung1, Ronald Laxer1
    1Paediatric Rheumatology, The Hospital for Sick Children, Toronto, Canada; 2Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital, Cincinnati, United States; 3Paediatric Haematology/Oncology, The Hospital for Sick Children, Toronto, Canada
    Correspondence: Dilan Dissanayake

    Introduction: We present here a 9 year-old male with recurrent episodes of hemophagocytic lymphohistiocytosis (HLH), characterized by fever, cytopenias, transaminitis, hyperferritinemia, hypofibrinogenemia, and elevated levels of soluble CD25 and soluble CD163, in whom we investigated for an underlying genetic defect.

    Methods: Peripheral blood samples were obtained from the patient and his family members for analysis using next generation sequencing by gene panels commonly associated with HLH and recurrent fever syndromes, followed by whole exome and whole genome sequencing.The patient’s blood was also assessed using an enzyme-linked immunosorbent assay for serum interleukin (IL)-18. Intracellular X-linked Inhibitor of Apoptosis Protein (XIAP) levels were measured by flow cytometric analysis of peripheral blood mononuclear cells (PBMC).The function of XIAP was assessed by a previously established assay that measures intracellular cytokine staining for tumor necrosis factor alpha (TNFa) production following stimulation of PBMC by muramyl dipeptide (MDP), the ligand for the Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2) receptor.

    Results: The HLH and recurrent fever syndrome gene panels, as well as whole exome sequencing were initially unable to identify a contributory variant in this patient.However, we strongly suspected a primary form of HLH after having measured persistently elevated levels of serum IL-18 to 2524 pg/ml in between episodes of HLH.Furthermore, we discovered a family history of recurrent fevers in the maternal grandfather, which was suspicious for an X-linked condition, such as XIAP deficiency. While awaiting results of whole genome sequencing, we performed flow cytometry for XIAP, which demonstrated reduced protein levels in the patient’s T-lymphocyte, B-lymphocyte and natural killer cell populations.Subsequent results from whole genome sequencing demonstrated a large duplication spanning three exons within the XIAP gene, which was present in the patient, his asymptomatic mother, and his maternal grandfather. In addition to decreased protein expression, MDP stimulation of the patient’s PBMC confirmed a significant functional defect in XIAP function downstream of the NOD2 receptor.While the above testing was being completed, the patient was started on anakinra and successfully weaned off steroids, with no further recurrences of HLH to date.

    Conclusion: This case describes the identification of a previously undescribed large duplication within the XIAP gene, which was initially not identified on gene panels and whole exome sequencing.This genetic variant is associated with reduced expression of XIAP in peripheral blood mononuclear cells, as well as decreased function downstream of the NOD2 receptor.Interestingly, while patients with XIAP deficiency are typically treated with hematopoietic stem cell transplantation, this patient has been successfully maintained on anakinra, suggesting an IL-1 dependent link between XIAP and recurrent HLH. We thereby illustrate the utility of combining genetic and molecular tools for the identification of contributors to unexplained autoinflammatory presentations, and present IL-1 blockade as a potential safe and effective alternative for some patients with XIAP deficiency.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1019 10 year prognosis of patients diagnosed with familial Mediterranean fever

    Serdal Ugurlu1, Bugra H. Egeli2, Asli E. Soykut2, Bilgesu Ergezen2
    1Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty; 2University of Istanbul - Cerrahpasa, Istanbul, Turkey
    Correspondence: Bugra H. Egeli

    Introduction: In Familial Mediterranean Fever (FMF), other than amyloidosis factors affecting mortality are being debated. In our previous study, we did not observe any atherosclerotic plaque formation in carotid or femoral artery. We thought that the risk of atherosclerosis did not increase in patients diagnosed with FMF.

    Objectives: The aim of this study was to assess the 10 year prognosis and comorbidity of patients diagnosed with FMF who have been treated in our rheumatology clinic.

    Methods: The sample group is a subset of 2009 study. In 2009, the patients who already had myocardial infarction or cancer diagnosis were excluded. The patients were interviewed with polar questions of whether they were diagnosed with acute myocardial infarction (AMI), cerebrovascular events, cancer, diabetes, and hypertension.

    Results: We studied 71 patients (37 males, 34 females; mean age: 49.66±6.91) with FMF, and 59 patients (24 males, 35 females) in healthy control (HC) group. The gender and age difference between two groups was not found significant.

    During 10 year follow-up, 8% of FMF patients had either a cardiovascular or cerebrovascular event comparing to 5% in HC (p>0.05). 3% of FMF patients had a cancer diagnosis comparing to 3% in HC (p>0.05). Even though diabetes mellitus diagnosis rate was higher in FMF patients (15% to 10%), results were still not significant (p>0.05). Hypertension diagnosis was 5% higher in FMF group (p<0.05)

    Conclusion: Even though there was a significant increase in hypertension, increased diabetes, cancer, and AMI/Stroke ratio was not found significant when compared to the HCs. Therefore, any cardiovascular and malignancy related comorbidities are not associated with FMF.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1019). Prognostic Factors of FMF patients compared with Healthy Controls

    P1020 In a familial Mediterranean fever prevalent region, are familial Mediterranean fever and Behçet’s disease associated?

    Ozgur Alparslan1, Bugra H. Egeli2, Yeltekin Demirel3, Serdal Ugurlu4
    1Gaziosmanpasa University, Tokat; 2university of Istanbul- Cerrahpasa, Istanbul; 3Sivas Cumhuriyet University, Sivas; 4Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul- Cerrahpasa, Istanbul, Turkey
    Correspondence: Bugra H. Egeli

    Introduction: The co-existence of Familial Mediterranean Fever (FMF) and Behçet’s Disease (BD) has been questioned. There have been a variety of claims on a common pathogenesis.

    Objectives: We intended to report the prevalence of Familial Mediterranean Fever (FMF) and Behçet’s disease (BD) and comorbidity ratio of these two diseases in Sivas, Turkey, a city where FMF is known to be very high.

    Methods: Seventy-two primary schools in the center of Sivas participated in the study. A total of 14881 randomized sample children from 6th, 7th, and 8th grades, and also 985 of them with their parents (n: 978) were interviewed. During these interviews, the family tree up to second degree relatives was drawn. The presence of a diagnosis of FMF or BD was questioned. The ones who have a diagnosis were confirmed by contacting the medical centers. The ones who were suspected of a disease were further investigated at Sivas Cumhuriyet University Medical Faculty, Family Medicine Outpatient unit. For each disease a disease related history, physical examination, eye examination and pathergy test for BD were performed when needed.

    Results: 985 students, 978 mothers, 953 fathers and 1876 relatives (4792 in total) were included in the study. Only 30 (0.6%) of the sample was diagnosed with FMF, and 3 (%0.06) was diagnosed with BD. One of them had concomitant FMF diagnosis.

    Conclusion: The prevalence of FMF in Sivas is higher than Turkey’s prevalence; however, BD prevalence was found very low. According to these findings, it is not easy to conclude that these two diseases share a similar background of pathogenesis.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1020). FMF symptoms within the last year

    P1021 Does testing for SAA is more beneficial than CRP for the follow-up of patients with FMF?

    Oguzhan Selvi, Serdal Ugurlu, Bilgesu Ergezen, Huri Ozdogan
    Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul - Cerrahpasa, Istanbul, Turkey
    Correspondence: Bilgesu Ergezen

    Introduction: In order to follow subclinical inflammation and adjust the therapy for an optimal disease control, clinicians seek for readily accessible, affordable and reproducible markers. C reactive protein (CRP) is widely used for this purpose. Some suggest that CRP measures are not conclusive in all cases, especially in initial stages of inflammation. It is suggested that Serum Amyloid A (SAA) may be more reliable and sensitive in predicting an ongoing inflammation.

    Objectives: It is aimed to evaluate if SAA and CRP is correlated in M694V homozygous FMF cases and if one is superior to the other with regards to early sensitivity.

    Methods: In order to evaluate and to compare the sensitivity of SAA and CRP, 234 measurements from 40 FMF patients with M694V homozygous mutation were obtained during a mean follow-up of 5 months.For the analysis, the folds of normal CRP and SAA values were used for correlation.Serum levels of the given markers were measured with nephelometric kits (normal CRP levels <5 mg/L and SAA levels <6,8 mg/L).

    Results: All patients were on prophylactic colchicine. Among 40 patients 1 patient was being treated with tocilizumab, 2 patients with adalimumab, 19 patients with anti-IL-1 regimens. There were a total of 234 measurements of CRP and SAA from 40 patients.A similar significant correlation was found when we tested only the values obtained during 202 attack-free occasions (r = 0,863, p < 0,01). Both acute phase reactants were increased in 169 measurements, while in 15 CRP was high but SAA was normal and in 40 SAA was high however CRP was within normal limits. 13 patients has amyloidosis. A number of 86 CRP and SRR measures, these two parameters were correlated in patients with FMF Amyloidosis (r = 0,818, p < 0,01). The mean increase in CRP of the population was 2,82 ± 4,52 fold, whereas mean increase in SAA was 8,47 ± 15,15 fold of the normal.

    Conclusion: According to these results, serial testing of SAA does not provide any additional advantages over CRP. Follow-up with CRP measures particularly in patients with amyloidosis might be adequate when it is considered that SAA is not superior to CRP. Readily accessible and affordable bio-marker CRP seems to be sufficient for follow-up of patients with FMF.

    Disclosure of Interest

    None Declared

    P1022 The pregnancy outcomes in FMF patients who are exposed to IL-1 blockade with anakinra

    Bilgesu Ergezen, Serdal Ugurlu, Huri Ozdogan
    Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul - Cerrahpasa, Istanbul, Turkey
    Correspondence: Bilgesu Ergezen

    Introduction: Some colchicine resistant and/or intolerant FMF cases as well as some patients who experience severe FMF episodes during pregnancy may require alternative therapies during their pregnancies. An Anti-IL-1 agent, Anakinra is being used for this purpose although is still under investigation of researchers.

    Objectives: To assess the safety of anakinra in pregnant FMF patients and its effect on fetal and maternal outcomes.

    Methods: Thirteen patients who were exposed to anakinra during their pregnancies were monitored closely for disease activity, side effects, fetal USG and pregnancy outcome.

    Results: A total of 13 FMF cases followed in our clinic were exposed to Anakinra during the course of their pregnancies due to severe protracted febrile myalgia in 4, thrombocytopenia in 1 and amyloidosis in 1 and severe attacks during pregnancy in 6. One patient had a single injection of anakinra due to pericarditis at 2nd GW and had a spontaneous abortus at 4th GW. Among 13 patients, 2 are still pregnant and both at 8th GW, one started Anakinra at 5th GW, while the other concieved on Anakinra. One of our patients was reported previously by Lachman et al1. We represent detailed data of 9 pregnancies which we have conclusive data regarding the whole pregnancy as well as the follow up period after birth. The relation of the use of the drug and pregnancy are represented in Table 1.

    Conclusion: Anakinra seems to be a very effective alternative in the treatment of protracted febrile myalgia non-responsive to colchicine and corticosteroid therapy, even in pregnant FMF patients who have active disease despite colchicine or intolerant and also can be given transiently during pregnancy and successfully stopped after delivery

    Reference

    1) Lachman HJ, et al. Anti IL-1 therapies and pregnancy outcome. Pediatric Rheumatology 2013, 11 (Suppl 1):A269

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1022). See text for description

    P1023 Clinical picture of 7 PAPA patients followed in a single pediatric rheumatologic center

    Silvia Federici1, Camilla Celani1, Virginia Messia1, Giulia Marucci1, Christoph Kessel2, Fabrizio De Benedetti1, Antonella Insalaco1
    1Division of Rheumatology, IRCCS, Ospedale Pediatrico Bambino Gesu’, Rome, Italy; 2Department of pediatric Rheumathology & immunology, University Children’s Hospital, Muenster, Germany
    Correspondence: Silvia Federici

    Introduction: Pyogenic sterile arthritis, pyoderma and acne (PAPA) syndrome is an autosomal dominant inflammatory disorder caused by mutations in the PSTPIP1 gene primarily affecting joints and skin. The E250K mutation cause the hyperzincaemia/hypercalprotectinemia syndrome termed PSTPIP1-associated-related proteinemia inflammatory (PAMI) syndrome in which a bone marrow involvement is reported

    Objectives: To describe the clinical presentation of 7 PAPA patients followed at a single pediatric rheumatology center

    Methods: For each patient clinical and laboratory data were collected from medical charts. PSTPIP1 was sequenced through Sanger Sequencing or targeted resequencing using a customized panel and analyzed with the NextSeq® platform (Illumina)

    Results: We describe 7 patients from 4 unrelated families with the E250K mutation in a mother and 2 siblings, the A230T variant in a father and his son and the R405C and D266N respectively in the last 2 unrelated patients. Disease onset occurred within the 7thyear of life in all patients. Patients 3 and 4 (table) presented since the 1st year of life recurrent episodes of fever without any cutaneous or articular symptoms. In both patients inflammatory markers were elevated during fever episodes but persistently negative during well-being not requiring any therapy. The variants described in these patients were not previously reported. However their pathogenic role is supported by the detection of markedly high serum calprotectin levels (>10.000 microg/ml). The predominant feature of patients 1 and 2 was articular involvement with recurrent episodes of arthritis associated to acne. Patient 1 was initially treated with prednisone with good clinical response but relapse of arthritis at discontinuation followed bythe development of a sterile muscle abscess. An anti-TNF drug was started in both patients with complete clinical response. Patient 5 reported severe acne and psoriasis, and recurrent episodes of sterile arthritis. She presented a persistent elevation of acute phase reactants with severe anemia and leukopenia not resolving after splenectomy. His son (pts 6) presented with recurrent episodes of sterile arthritis, hepato-splenomegaly, anemia and neutropenia. Zinc and calprotectin serum levels resulted respectively 728 micromol/l and 2600 microg/ml. IL-1 inhibition determined a complete normalization of inflammatory parameters with no effects on anemia and neutropenia. In patient 6 zinchemia decreased to almost normal value after 4 months of therapy. Patient 7 presented at the age of 4 years a sterile lymphnode abscess. She also presented with splenomegaly and neutropenia with persistent elevation of acute phase reactants. Anakinra was proposed but not administered for poor compliance

    Conclusion: The clinical picture of patients carrying PSTPIP1 mutation may be heterogeneous. In our cohort TNF-inhibitors were successfully used in PAPA patients preventing new arthritis episodes and resolving cutaneous manifestation where present. In 2 patients the clinical picture was mild not requiring continuous treatment. One PAMI patient had a good response to IL-1 inhibition, which however, had no effect on hematological manifestations

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1023). See text for description

    P1024 Clinical presentation, genetic analysis and IFN-score in patients with undefined interferonopathies

    Silvia Federici1, Gianmarco Moneta1, Chiara Passarelli1, Claudia Bracaglia1, Luana Raffaele1, Fabrizio De Benedetti2, Antonella Insalaco1
    1Division of Rheumatology; 2Ospedale Pediatrico Bambino Gesù, Rome, Italy
    Correspondence: Silvia Federici

    Introduction: A group of genetic disorders with a disturbance of the homeostatic control of IFN-mediated immune responses, have been identified (type I interferonopathies). An increased expression of type I IFN regulated genes, IFN signature (IS), is reported

    Objectives: To evaluate the correlation between clinical presentation, genetic analysis and IFN-score in 10 pts with undefined interpheronopathies

    Methods: Patients with suspected interferonopathy based on the presence of typical clinical manifestations (neurological, muco-cutaneous symptoms), laboratory parameters (complement deficiency, low platelet count, presence of autoimmunity), instrumental abnormalities (cerebral calcification), were screened for the IFN-score. Defined IFN-mediated diseases were excluded. Patients with IFN-score above 10 underwent genetic screening by running a panel of 24 genes involved in interferonopathies

    Results: 10 pts followed in a pediatric rheumatology center were included. 7/10 presented with recurrent fever (table). Pts 2, 3 and 7 displayed neurological manifestation, respectively epilepsy, epilepsy and mental retardation and progressive hemiplegia. To note epilepsy in pts 2 might be due to a bilateral intraventricular hemorrhage presented at birth. In pts 1,4 gastrointestinal manifestation resembling inflammatory bowel diseases were described while pts 5,7 and 10 suffered from recurrent abdominal pain, diarrhea and patient 10 from hypertransaminasemia. Half of the patients complained arthromyalgia; arthritis developed in pts 2. Cutaneous involvement presented in pts 1,3,6 respectively with widespread panniculitis of trunk and limbs, aspecific vasculitis and Schonlein Henoch purpura (HSP). Other cutaneous manifestation were urticarial rash (pts2) and an erythematous, desquamative confluent eczema (pts 4). Autoimmunity was detected in 2/10 pts. Pts 4 8 had an immunological defect with recurrent infections. The genetic analysis resulted negative in pts 1 and 7 and is ongoing in patients 5,6 and 8. Patients 2,3,4,9 and 10 carried one mutation in at least one IFN correlated gene not confirming the diagnosis. All patients presented an increased IS ranging from 14 to 172.

    Conclusion: An elevated IFN-score represent a useful instrument in clinical practice to classify patients with suspected interferonopaties. It may be an important tool to select pts to be genetically screened with a defined panel of interferonopathies correlated genes. In pts in which the genetic analysis results negative, the presence of a positive IFN-score, may guide clinicians in the management of these patients and may support therapeutic decisions

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1024). See text for description

    P1025 Lesson from eurofever registry after the first ten years of enrollment

    Martina Finetti, Ilaria Gueli, Joost Frenkel, Seza Ozen, HelenLachmann, Fabrizio De Benedetti, Isabelle Koné-Paut, Carine Wouters, Paul Brogan, Hermann Girschick, Benedicte Neven, Alberto Martini, Nicola Ruperto, Marco Gattorno, on behalf of the Paediatric Rheumatology International Trials Organisation (PRINTO) and the Eurofever Project
    UOSD Centro Malattie Autoinfiammatorie e Immunodeficienze, on the behalf of the Paediatric Rheumatology International Trials Organisation (PRINTO) and the Eurofever Project, IRCCS Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Martina Finetti

    Introduction: In 2008 the Paediatric Rheumatology European Society (PReS) promoted an International Project for the study of Autoinflammatory Diseases (AIDs) named Eurofever, whose main purpose is to create a web-based registry for the collection of information in AIDs patients.

    Objectives: To assess the impact of the Eurofever Registry on scientific community with particular interest in the geographical coverage, diagnostic delay, access to treatment and pubblications.

    Methods: The data analyzed in the study were extracted from the Eurofever registry, which is hosted in the PRINTO website.

    Results: Up to date 4175 patients have been enrolled from 62 countries (3843 of them with complete demographic data). Most of patients (72%) are resident in Western Europe, 8% in Central-Eastern Europe, 11% in Southern-Eastern Mediterranean, 2% in South America and 7% in other countries. Compared to the first Eurofever report (Toplak et al, 2012) we have observed an increase of enrolled patients from 1388 to 2651 in Western Europe, from 106 to 313 in Central-Eastern Europe, from 294 to 406 in Southern-Eastern Mediterranean. The median onset age is 4 years (range 1 month – 75 years), the median diagnosis age is 8 years (range 1 month – 78 years). The median diagnostic delay observed in 2012 was 7.3 years (range 0.3–76), from patients enrolled after 2012 it was 1.9 years (range 0-57). Comparing the mean diagnostic delay from 1980 to 2018, we have observed an encouraging constant reduction of period between AIDs onset e diagnosis (from a mean diagnostic delay value of 20 years for patients born before 1980, to a mean value of 1 year for patients born after 2011). Complete information on access to treatment were available in 2430 patients. DMARDs were used in 1031 (42%), biologics in 396 (16%) patients. According to the number of enrolled patients, biologics were used in 361/1782 (20%) of Western europeans, 17/342 (5%) of Central-Eastern europeans, 6/259 (2%) of Southern-Eastern Mediterranean patients. Regarding Eurofever impact on Scientific Community, during this first 10 years the Registry provided 12 papers with more than 800 citations. Detailed analysis of clinical features collected in Eurofever database allowed to perform studies with large cohort of patients, to purpose new classification criteria (Federici et al, 2015), to validate damage and activity score (Piram et al, 2014 and N Ter Haar et al, 2017) and to evaluated genotype/phenotype correlation (Papa et al, 2017).

    Conclusion: In the last years we have observed an encouraging increase of involved Countries, with a greater number of patients coming from geographic area poorly represented in the first epidemiologic study of Toplak et al. Eurofever data analysis has confirmed an improvement of diagnostic ability during the last years, with a significant reduction of mean diagnostic delay. Long-term studies will help understand the efficacy and safety of different treatments used in these rare conditions.

    Disclosure of Interest

    M. Finetti: None Declared, I. Gueli: None Declared, J. Frenkel: None Declared, S. Ozen: None Declared, H. Lachmann: None Declared, F. De Benedetti: None Declared, I. Koné-Paut: None Declared, C. Wouters: None Declared, P. Brogan: None Declared, H. Girschick: None Declared, B. Neven: None Declared, A. Martini Grant / Research Support from: The Gaslini Hospital, which is the public Hospital where AM worked till 31/dec/2018as a full time public employee, has received contributions from the following industries:Abbvie, Ablynx, Aim Group, Amgen, Astrazeneca, Biogen, BMS, Boehringer, Celgene, Emd Serono, GSK, Janssen, Novartis, Pfizer, R-Pharm. This money has been reinvested for the research activities of the hospital in a fully independent manner without any commitment with third parties., N. Ruperto Grant / Research Support from: The Gaslini Hospital, where NR works as full-time public employee, has received contributions (> 10.000 USD each) from the following industries in the last 3 years: BMS, Eli-Lilly, GlaxoSmithKline, F Hoffmann-La Roche, Janssen, Novartis, Pfizer, Sobi. This funding has been reinvested for the research activities of the hospital in a fully independent manner, without any commitment with third parties.,Speaker Bureau of: NR has received honoraria for consultancies or speaker bureaus (< 10.000 USD each) from the following pharmaceutical companies in the past 3 years: Ablynx, AbbVie, Astrazeneca-Medimmune, Biogen, Boehringer, Bristol Myers and Squibb, Eli-Lilly, EMD Serono, Glaxo Smith and Kline, Hoffmann-La Roche, Janssen, Merck, Novartis, Pfizer, R-Pharma, SanofiServier, Sinergie, Sobi and Takeda., M. Gattorno Grant / Research Support from: MG has received unrestricted grants from Sobi and Novartis

    P1026 Clinical study of Japanese patients with fever of unknown origin: investigation of mutations in 22 genes related to autoinflammatory diseases

    Kyoko Fujimoto1, Yukiko Hidaka1, Yumi Yoshida1, Makiko Hayashi1, Takuma Koga1, Shinjiro Kaieda1, Satoshi Yamasaki2, Tomoaki Hoshino1, Hiroaki Ida1
    1Division of Respirology, Neurology and Rheumatology, Department of Medicine, Kurume University School of Medicine; 2Center for Rheumatology, Kurume University Medical Center, Kurume-shi, Fukuoka, Japan
    Correspondence: Kyoko Fujimoto

    Introduction: Autoinflammatory diseases cause systemic inflammation mainly by innate immune abnormality. It is important to have a diagnosis of autoinflammatory diseases in patients with fever of unknown origin. Examining gene mutations is valuable for the diagnosis of autoinflammatory diseases; however, the frequency of genetic mutations in patients with fever of unknown origin is not reported in Japan.

    Objectives: We comprehensively analyzed genetic mutations related to autoinflammatory diseases and clinical features in patients with fever of unknown origin.

    Methods: We analyzed mutations of 22 genes related to autoinflammatory diseases as follows: TNFRSF1A, MEFV, NLRP3, MVK, NOD2, IL1RN, NLRP12, PSTPIP1, PSMB8, PSMB9, PSMA3, PSMB4, POMP, NLRC4, PLCG2, HMOX1, CECR1, COPA, TNFAIP3, FAM105B, RNF31, RBCK1,in 84 patients with fever of unknown origin who introduced to our hospital from May 2017 to June 2018. Genetic analysis was performed by the next generation sequencer. Furthermore, we investigated precise clinical features in 53 cases.

    Results: Fifteen genes were identified as having novel or rare variants. The most frequent variants were determined in NLRP12 (8 cases, 6 sites) and NLRP3 (8 cases, 4 sites). In addition, missense mutations including genetic polymorphisms were observed in MEFV(66.7%). In 53 cases in which clinical symptomatic investigations were possible, 40 cases (75.5%) had definite periodic fever. Among cases with periodic fever, joint symptoms were observed in 52.5%, and abdominal symptoms were in 35.0%. Colchicine treatment was effective in 37.5%. Sixty-five percent of patients with periodic fever had MEFVmutations, including genetic polymorphisms. Almost half of them had novel or rare mutations in autoinflammatory diseases related genes other than MEFV. The most frequent variants were determined in NLRP3.

    Conclusion: Novel or rare variants in NLRP12and NLRP3were noted in patients with fever of unknown origin. Sixty-five percent of patients with periodic fever had MEFVmutations including genetic polymorphism.

    Disclosure of Interest

    None Declared

    P1027 A case of adenosine deaminase 2 deficiency (DADA2) with an uncommon clinical presentation and response to IV IG

    Francesca Garbarino1, Roberta Caorsi2, Stefano Volpi2, Alice Grossi3, Isabella Ceccherini3, Marco Gattorno2
    1Università Degli Studi Di Genova; 2Clinica Pediatrica Reumatologica, UOSD Malattie Autoinfiammatorie-Immunodeficienze; 3UOC Genetica Medica e UOSD Genetica e Genomica delle Malattie Rare, Ist. Giannina Gaslini, Genova, Italy
    Correspondence: Francesca Garbarino

    Introduction: DADA2 is an autoinflammatory disease with autosomal recessive inheritance characterized by a heterogeneous clinical phenotype ranging from multisystemic inflammation (fever, polyarteritis nodosa, cerebral stroke, livedo reticularis, gastro-intestinal involvement, peripheral neuropathy etc.) to immune-dysregulation and immunodeficiency (lymphoproliferation, recurrent infections, cytopenia etc.).

    Objectives: To extend the clinical spectrum of DADA2 reporting a case of isolated nonspecific systemic inflammatory syndrome associated with slight signs of immune-dysregulation in a patient with a novel ADA2 mutation.

    Methods: In a patient with nonspecific inflammatory phenotype associated to susceptibility to viral infections, Next Generation Sequencing (NGS) panel was performed; mutations detected were confirmed by direct sequencing (Sanger analysis). ADA2 enzymatic activity was analyzed in monocyte isolated from the patient and incubated with adenosine and an ADA1 inhibitor.

    Results: The girl, adopted and of Asian origin, began to suffer from nonspecific systemic inflammatory symptoms (high persistent fever and arthralgias) at the age of 6 years. In past history recurrent respiratory infections and impaired immunological response to viruses (CMV related hepatitis, measles after vaccination) were reported. After few months the patient developed clinical and laboratory findings of HLH (Hemophagocytic Lympho-Histocytosis), confirmed on bone marrow samples; treatment with intravenous (IV) high dose (HD) steroids was started, with prompt response. During steroids tapering fever and systemic inflammation reappeared; anti-IL1 treatment (anakinra) was not effective. Immunologic assessment demonstrated mild hypogammaglobulinemia and moderate NK deficiency on lymphocyte subsets. HD IV Immunoglobulins (IG) (2 g/kg every month) allowed to achieve a complete control of the clinical picture; the frequency of administration was progressively reduced to every 4 months due to persistent wellbeing. At the age of 9, after switching IG to the substitutive dosage, the patient experienced an Herpes Zoster virus reactivation (requiring prolonged antiviral treatment), followed by the reappearance of the inflammatory phenotype complicated by HLH with neurological involvement (irritability and lethargy), responsive to HD steroids and IG. A later cerebral MRI evidenced a small gliotic area in left Centrum Ovale. After steroids suspension, monthly HD IV IG administrations maintained clinical remission. Further immunological studies confirmed a reduction of NK cells with normal function. Hereditary HLH, Autoimmune Lympho-Proliferative Syndrome (ALPS) and main primary immunodeficiencies were ruled out. Given the clinical picture, a large NGS diagnostic panel (courtesy by K. Botzug, Vienna) for autoinflammatory diseases and immunodeficiencies was performed revealing the homozygous LEU141PRO ADA2 mutation, confirmed by Sanger analysis. Being this mutation novel, an ADA2 enzymatic activity test was performed revealing a complete loss of ADA2 activity. The parents refused anti-TNF treatment and the patient is still on monthly HD IG with a complete wellbeing after 3 years of follow-up.

    Conclusion: The current report enlarges the clinical spectrum associated with DADA2 to a persistent unspecific inflammatory syndrome, complicated by HLH. This case further emphasizes the possibility that NGS could unravel unusual phenotypes of already known inflammatory syndromes. Even if further reports are required, the response to high doses IG observed in the present case it is of interest. Even if anti-TNF is the treatment of choice HD IV IG could be a possible treatment in DADA2, especially during the acute phase.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1028 Non-amyloid kidney diseases and autoinflammatory diseases: report of 20 cases and literature review

    Charlotte Borocco1,2, Isabelle Kone-Paut1,2, Alexandre Belot3, Marine Desjonqueres3, Alexandre Karras4, Corinne Miceli-Richard5, Bruno Moulin6, Tim Ulinski7, Jean-Jacques Boffa8, David Buob9, Gilles Grateau10,11, Sophie Georgin-Lavialle10,11
    1Pediatric Rheumatology, Bicetre University Hospital; 2CeReMAIA, Le Kremlin-Bicêtre; 3Pediatric Rheumatology, Lyon University Hospital, Bron; 4Department of Nephrology, Georges Pompidou European Hospital; 5Department of Rheumatology, Cochin University Hospital, Paris; 6Department of Nephrology, Civil Hospital, Strasbourg; 7Pediatric Nephrology, Trousseau University Hospital; 8Department of Nephrology; 9Department of Pathology; 10Department of Internal Medicine, Tenon University Hospital; 11CeReMAIA, Paris, France
    Correspondence: Sophie Georgin-Lavialle

    Introduction: Autoinflammatory diseases (AID) are associated with abnormalities of innate immunity. Patients display recurrent fever and various signs including digestive disorders, cutaneous rashes and joint features. One of the most severe complication of AID is inflammatory amyloidosis (AA) including renal involvement, that can lead to kidney failure. However, other nephropathies exist and can be misdiagnosed.

    Methods: This study was a retrospective, multicentric French study carried out by medical societies and national reference centers of AID. All patients with non-amyloid kidney diseases (NAKD) occurring at pediatric or adult age with a genetically proven or not AID were included.

    Results: Twenty patients were included: 13 patients with familial Mediterranean fever (FMF), 3 with mevalonate kinase deficiency (MKD), one with TNF-receptor associated periodic syndrome (TRAPS) and 3 with unclassified AID (uAID). Among the 13 FMF patients, 8 displayed homozygous M694V MEFV mutation, the mean age at the occurrence of nephrological symptoms was 28.6 years old. Renal diagnosis were: Henoch-Schönlein purpura (HSP) nephritis (n=3), minimal change disease (n=3), IgA nephropathy (n=2), diabetic nephropathy (n=2), nephroangiosclerosis (n=2) and idiopathic nephrotic syndrome (n=1). Renal treatments were represented by angiotensin-converting enzyme inhibitors (ACEIs) (n=6), then colchicine (n=4; dose adjustment or reinstatement of treatment), corticosteroids (n=4), antihypertensive medication (n=4), dialysis (n=2), levamisol (n=1), kidney transplantation (n=1) and anakinra (n=1). After treatment, 5 patients were in renal remission, 2 patients had a transient proteinuria, 5 patients a persistent proteinuria, 2 patients had a renal insufficiency, and 1 patient was in remission after kidney transplantation.

    Among the 3 MKD patients, the mean age at onset of nephrological symptoms was 17.7 years. Renal biopsies found: pauci immune extra capillary glomerulonephritis (n=1), HSP nephritis (n=1) and segmental focal hyalinosis lesions associated with nephrosclerosis (n=1). One patient received a kidney transplant with a relapse after it, one was in pre-transplant status, one was in remission under treatment. They all were treated with anti IL1 agent.

    The TRAPS patient displayed rapidly progressive glomerulonephritis and was treated with steroids, etanercept and then anti IL1 agent.

    The 3 uAID patients displayed: C3 depositional glomerulonephritis (n=1) an IgA nephropathy (n=2) at a median age of 36 years old.

    In our literature review including our cases, we found 124 cases of FMF with NAKD biopsy proven, 4 MKD patients with NAKD, only 1 TRAPS patient and no cryopyrin associated periodic syndrome.

    Conclusion: Non-amyloid nephropathies can occur in AID; the most frequent ones being IgA nephropathy and HSP nephropathy. Kidney biopsy is necessary in case of nephrological signs to optimize the treatment and to limit the evolution towards kidney failure.

    Disclosure of Interest

    None Declared

    P1029 The transition from pediatrics to adulthood in auto-inflammatory diseases: comparison of 2 transition strategies among 72 patients

    Sophie Georgin-Lavialle1, Pierre Quartier2, Brigitte Bader-Meunier2, Katia Stankovic Stojanovic3, Virginie Avellino3, Samuel Deshayes3, Isabelle Melki4, Alexandre Belot5, Isabelle Kone-Paut6, Gilles Grateau7, Véronique Hentgen8 on behalf of transition group project, CEREMAIA, FAI2R and Transition Working Group, National Reference Center for Autoinflammatory Diseases and AA Amyloidosis
    1Internal Medicine, Tenon hospital, AP-HP, Paris; 2Pediatric Rheumatology, AP-HP, Necker Hospital; 3Internal Medicine, Tenon Hospital; 4Pediatric Rheumatology, AP-HP, PARIS; 5Pediatric Rheumatology, HCL, Hopital Mère Enfant, LYON; 6Pediatric Rheumatology, AP-HP, Kremlin-Bicêtre hospital, Kremlin-Bicêtre; 7Internal Medicine, AP-HP, Tenon hospital, PARIS; 8General pediatry, CH Versailles André mignot, Versailles, France
    Correspondence: Sophie Georgin-Lavialle

    Introduction: Transition is the period during which the young patient will move from the pediatric team to the adult team for the follow-up of his or her chronic disease. It requires perfect coordination between the two medical teams but also the active involvement of the young person and his or her family. A specific transition program increases the chances of good health and well-being and reduces mortality in this age group. We experienced two different transition strategies from two different pediatric wards to a single adult ward specialized in auto-inflammatory diseases (AIDs) and compare the efficacy of the 2 strategies.

    Objectives:To compare the effectiveness of the transition process in the two strategies, evaluated by the lost of sight patients avec a median 5 years of follow up.

    Methods: Children from 2 pediatric centers were addressed for transition in a single adult ward. The first strategy concerned 53 patients that were to prepare since the age of 14 years old and at the age of 18, they were presented for transition thru a joint consultation with the pediatrician in the adult clinics. The second strategy concerned 19 patients who came directly from pediatrics without the pediatrician for an adult medical consultation; the pediatrician explained bye mail and/or phone the case and sent the patient with his medical file.

    Results: Seventy-two young people (29 girls, 43 boys) passed the 10-year transition step between 2007 and 2017. The AIDs encountered were: familial Mediterranean fever (n=46), PFAPA syndrome, mevalonate kinase deficiency (n=6 of each), Still's disease (n=4), TRAPS syndrome (n=3) or another rare MAI (NRLP12 mutation, CAPS, or other rare) (1 to 2 cases each time). A total of 81% young patients came regularly (about once a year). Only 3 patients are currently out of sight (4%); none had a clearly identified single gene MAI requiring very regular follow-up. The young people interviewed are generally satisfied.

    Conclusion: No difference was observed in the transition efficacy process when comparing the two transition strategies (joint consultation or not). Joint consultation is experienced as necessary, but our results show that this strategy is not mandatory for a successful transition. When asked, young people are eager to have written documentation about their illness and to have the telephone number and e-mail address of the doctor to contact in case of problems.

    These positive results of the effectiveness of a transition program require close collaboration between the pediatrician and the adult physician regardless of the transition strategy chosen. The adult doctor must invest in taking care of young people who are very connected and in case of problems expect a quick response by email most often. Indeed, more than a joint consultation, the involvement of the adult physician seems essential to the successful transition from pediatrics to adult service.

    Disclosure of Interest

    None Declared

    P1030 The longitudinal eurofever project: an update on enrollment

    Ilaria Gueli, Martina Finetti, Fabrizio De Benedetti, Jordi Anton Lopez, Maria Alessio, Joost Frenkel, Luca Cantarini, Romina Gallizzi, Judith Sanchez Manubens, Marco Cattalini, Efimia Papadopoulou-Alataki, Rolando Cimaz, Donato Rigante, Alma Nunzia Olivieri, Pavla Dolezalova, Alberto Martini, Nicola Ruperto, Marco Gattorno, on behalf of the Paediatric Rheumatology International Trials Organisation (PRINTO) and the Eurofever Project
    UOSD Centro Malattie Autoinfiammatorie e Immunodeficienze, on the behalf of the Paediatric Rheumatology International Trials Organisation (PRINTO) and the Eurofever Project, IRCCS Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Ilaria Gueli

    Introduction: In 2008 the Paediatric Rheumatology European Society (PReS) promoted an International Project for the study of Autoinflammatory Diseases (AIDs) named Eurofever, whose main purpose is to create a web-based registry for the collection of information in AIDs patients

    Objectives: To implement the Registry with the new recently described AIDs and to increase the collection of longitudinal data

    Methods: The data were extracted from the Eurofever registry, which is hosted in the PRINTO website (http://www. printo.it). From February 2015 we started the longitudinal collection of follow-up data with particular focus on treatment, modification of the clinical picture, onset of complication/adverse events. We have enrolled patients included in the registry up to 28 September 2018.

    Results: Up to date 4175 patients have been enrolled (3843 of them with complete demographic information, 1903 M and 1940 F) from 62 countries. For 3356 (87%) patients also clinical data from onset to diagnosis, collected during the first visit performed at referred pediatric or adult center, are available. For each disease the number of enrolled patients is: FMF 1086 pts (951 with complete clinical data); TRAPS 273 pts (256 complete); CAPS 298 pts (279 complete); MKD 205 pts (190 complete); Blau’s disease 49 pts (26 complete); PAPA 42 pts (41 complete); NLRP-12 mediated periodic fever 13 pts (11 complete); DADA2 14 pts (9 complete); DIRA 3 pts (all complete); SAVI 3 pts (all complete); CANDLE 1 pt (complete) and Majeed 4 pts (all complete). Among multifactorial autoinflammatory diseases: PFAPA 676 pts (551 complete); CNO 581 pts (540 complete); Behcet 214 pts (186 complete), undefined periodic fever 368 pts (292 complete) and Schnitzler syndrome 13 pts (all complete). The median onset age is 4 years (range 1 month – 75 years), the median diagnosis age is 8 years (range 1 month – 78 years). Most of patients (3509, 91%) presented disease onset during pediatric age (<16 years), 334 (9%) during adult age (81 FMF, 31 CAPS, 53 TRAPS, 40 CRMO, 12 Schnitzler syndrome and 90 unknown fever). 405 of 3509 (12%) patients with pediatric onset received diagnosis during adult age. The median diagnostic delay is 5 years; diseases with longer diagnostic delay are: NLRP12 (24 years, range 4-76), CAPS (15 years, range 0-77), PAPA (14 years, range 2-57), TRAPS (12 years, range 0-77). 396 patients have been treated with at least one biologic drug, 1031 with DMARDs, 427 with systemic steroid and 686 with others drugs. The most frequent diseases treated with biologic drugs are: CAPS (38%), multifactorial diseases (22%), TRAPS (14%), MKD (11%), rare monogenic (8%: 1 CANDLE, 2 DIRA, 2 NLRP12, 3 Majeed, 8 DADA2 and 14 PAPA), and FMF (7%). Since February 2015, longitudinal visits have been inserted for 477 (12%) patients, with detailed data on treatment and safety.

    Conclusion: The enrollment in Eurofever Registry is still ongoing. The analysis of data will improve our knowledge both on the natural history of the single disease and on the efficacy/safety of treatment commonly used in the clinical practice.

    Disclosure of Interest

    I. Gueli: None Declared, M. Finetti: None Declared, F. De Benedetti: None Declared, J. Anton Lopez: None Declared, M. Alessio: None Declared, J. Frenkel: None Declared, L. Cantarini: None Declared, R. Gallizzi: None Declared, J. Sanchez Manubens: None Declared, M. Cattalini: None Declared, E. Papadopoulou-Alataki: None Declared, R. Cimaz: None Declared, D. Rigante: None Declared, A. N. Olivieri: None Declared, P. Dolezalova: None Declared, A. Martini Grant / Research Support from: The Gaslini Hospital, which is the public Hospital where AM worked till 31/dec/2018as a full time public employee, has received contributions from the following industries:Abbvie, Ablynx, Aim Group, Amgen, Astrazeneca, Biogen, BMS, Boehringer, Celgene, Emd Serono, GSK, Janssen, Novartis, Pfizer, R-Pharm. This money has been reinvested for the research activities of the hospital in a fully independent manner without any commitment with third parties., N. Ruperto Grant / Research Support from: The Gaslini Hospital, where NR works as full-time public employee, has received contributions (> 10.000 USD each) from the following industries in the last 3 years: BMS, Eli-Lilly, GlaxoSmithKline, F Hoffmann-La Roche, Janssen, Novartis, Pfizer, Sobi. This funding has been reinvested for the research activities of the hospital in a fully independent manner, without any commitment with third parties.,Speaker Bureau of: NR has received honoraria for consultancies or speaker bureaus (< 10.000 USD each) from the following pharmaceutical companies in the past 3 years: Ablynx, AbbVie, Astrazeneca-Medimmune, Biogen, Boehringer, Bristol Myers and Squibb, Eli-Lilly, EMD Serono, Glaxo Smith and Kline, Hoffmann-La Roche, Janssen, Merck, Novartis, Pfizer, R-Pharma, SanofiServier, Sinergie, Sobi and Takeda., M. Gattorno Grant / Research Support from: MG has received unrestricted grants from Sobi and Novartis

    P1031 Digital brachial index testing as a noninvasive tool in diagnosing peripheral vascular disease in DADA2

    Patrycja M. Hoffmann1, Deborah L. Stone1, Karyl Barron2, Cornelia Cudrici3, Alessandra Brofferio3, Anne Jones1, Tina Romeo1, Ivona Aksentijevich1, Daniel L. Kastner1, Amanda K. Ombrello1
    1NHGRI; 2NIAID; 3NHLBI, National Institutes of Health, Bethesda, MD, United States
    Correspondence: Patrycja M. Hoffmann

    Introduction: Deficiency of adenosine deaminase 2 (DADA2), is an autosomal recessive disease caused by biallelic loss-of-function mutations in the ADA2 gene. Deficiency of ADA2 is the first molecularly described monogenic vasculitis syndrome. In some cases, DADA2-associated peripheral vascular disease (PVD) can be severe enough to require amputation.

    Objectives: To examine the results of upper and lower digital brachial index (DBI) and to compare upper and lower DBI tests to corresponding upper and/or lower extremity MRA tests in patients with DADA2 who have clinical features of PVD.

    Methods: Three out of 45 patients with DADA2 seen at the NIH were found to have moderate to severe PVD. Complete histories were obtained and physical exams were performed. Upper and/or lower extremity DBI tests and MRA exams were performed.

    Results: 28 yo male with DADA2 who had triphasic Raynaud’s syndrome and pustular-like lesions, bone resorption, and necrotic tissue of various digits, three of which eventually required partial amputation. DBI of upper and lower extremities showed decreased waveform and pressure in 1st-5th left upper digits, 1st, 3rd-5th right upper digits, 2nd-5th left lower digits and 2nd-5th right lower digits. MRA findings showed a single patent common artery, supplying blood to 2nd-3rd left upper digits, adequate blood flow in right upper 2nd digit and little to no blood flow in right 1st, 3rd-5th digits. Right and left lower 1st digit blood flow was patent. There were numerous collaterals to all lower extremity digits. Thus DBI and angiography were in agreement. DBI results demonstrated a lack of waveform or pressure in digits that showed poor flow on angiography. Based on this data, DBI can be considered as an alternative to MRA if cost or renal insufficiency is a concern.

    26 yo female with DADA2 with polyarteritis nodosum and intermittent bilateral 1st toe swelling and pain. Right and left lower DBI showed no waveform or pressure in 1st-5th digits. MRA of lower extremity was limited to medium vessel arteries which were patent bilaterally. Small vessels of the feet were not well visualized because of MRA limitations so digital blood flow could not be analyzed or compared to abnormal DBI results.

    39 yo female with DADA2 with multiple chronic ankle ulcerations and Raynoud’s syndrome. Bilateral lower DBI waveform and pressure was normal in the 1st digits bilaterally but there was decreased pressure and waveform in the 2nd-5th digits bilaterally. MRA of lower extremity was limited to medium vessel arteries which were patent bilaterally. Small vessels were not well visualized because of MRA limitations so digital blood flow could not be analyzed or compared to abnormal DBI results.

    Conclusion: The complexity of DADA2 increases the need for expanding diagnostic tests, some of which can be long and invasive. In the NIH cohort of 45 patients, 3 were found to have clinical findings of PVD. The upper and lower extremity DBI test helped diagnose patients with moderate to severe PVD. In our 2nd and 3rd patient, the MRA exam was limited to medium vessel blood flow testing and therefore blood flow in the small vessels of the feet were not well visualized.Based on the noninvasive less expensive DBI diagnostic testing, patients with DADA2 can be screened effectivey and in a timely manner and avoid exposure to contrast, especially in the setting of renal deficiency. The additional close monitoring can help expedite diagnosis and treatment to possibly avoid future amputation as was the case in Patient 1.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1032 The expanding clinical and laboratory spectrum of PAPA syndrome: the NIH cohort

    Patrycja Hoffmann1, Amanda K. Ombrello1, Deborah L. Stone1, Karyl Barron2, Anne Jones1, Tina Romeo1, Michele Nehrebecky1, Jae Chae1, Ivona Aksentijevich1, Daniel L. Kastner1
    1NHGRI; 2NIAID, National Institutes of Health, Bethesda, MD, United States
    Correspondence: Patrycja Hoffmann

    Introduction: The dominantly inherited PAPA syndrome is caused by mutations in PSTPIP1. It is one of the least understood of the known monogenic autoinflammatory diseases, both from a pathogenic and treatment perspective. Symptoms include arthritis, cystic acne, and pyoderma gangrenosum. Therapy includes corticosteroids, interleukin-1 receptor antagonists, and tumor necrosis factor inhibitors.

    Objectives: To review noninfectious epiglottis and sterile osteomyelitis, rare complications not previously published in PAPA syndrome, and to compare LDH and aldolase levels in patients with PAPA syndrome vs patients with mutation-negative PAPA-like phenotype.

    Methods: The NIH cohort of 20 patients with PAPA syndrome and 10 patients with PAPA-like phenotype were reviewed. Comprehensive evaluation was performed. MRI, CBC with differential, LDH, aldolase, CK, CRP and ESR were examined. Patients’ labs were drawn during times of flare and no flare. CK was drawn to ensure there was no inflammatory muscle involvement.

    Results: One patient with PAPA syndrome developed severe sore throat. Clinical and radiographic evaluation showed piriform sinus swelling consistent with supraglottitis. CRP and ESR were elevated. WBC was normal. Treatment was started with IV clindamycin and ceftriaxone; doxycycline was added later. Severe sore throat continued and repeat radiographic findings showed ongoing swelling. Clindamycin was stopped.Methylprednisolone and a scheduled dose of golimumab were initiated with significant symptomatic improvement and normalization of radiographic findings, CRP and ESR.

    A second patient with PAPA syndrome developed severe right wrist pain and swelling. MRI revealed distal right radial epiphyseal marrow abnormalities consistent with osteomyelitis without synovitis. ESR and CRP were elevated. WBC was normal. The patient was treated with clindamycin without symptomatic benefit or improvement in MRI findings. Clindamycin was discontinued. Methylprednisolone and anakinra were initiated which resulted in decreased pain and improvement in marrow inflammation on MRI. CRP and ESR improved.

    Of the 20 patients with PAPA syndrome, LDH, aldolase, and CK levels were drawn. LDH was elevated in 19/20 patients. Aldolase was elevated in 19/19 patients. CK was elevated in 5/20 patients. Of the 10 patients with PAPA-like phenotype, LDH was elevated in 1/10 patients. Aldolase was elevated in 2/10. CK was elevated in 1/10. MRIs done on two patients with PAPA syndrome did not show muscle inflammation during a flare.

    Upon further analysis of LDH isoenzymes I-V tested in 12/20 patients with PAPA syndrome, 12/12 had elevated LDH isoenzyme V, a skeletal muscle isoenzyme.

    Conclusion: Our findings indicate an expanding clinical spectrum in PAPA syndrome that includes aseptic supraglottitis and non-bacterial osteomyelitis. Improvement in clinical symptoms, MRI findings, and acute phase reactants as well as a normal WBC on methylprednisolone support an inflammatory rather than infectious process.

    We do not have a clear understanding as to the relevance of elevation in LDH and aldolase in patients with PAPA syndrome and overall normal LDH and aldolase in the PAPA-like phenotype during periods of flare and no flare. LDH isoenzyme V and aldolase are markers of skeletal muscle involvement. In looking at muscle MRIs and obtaining CK levels, we did not see any muscle inflammation. Therefore, in addition to pursuing further testing to rule out muscle damage, additional etiologies for elevation of LDH and aldolase should be considered.

    Disclosure of Interest

    None Declared

    P1033 Pseudodominant inheritance of Behçet-like autoinflammatory disease associated with TNFAIP3 (A20) and MEFV mutations in a Turkish family with familial Mediterranean fever

    Nobuyuki Horita1, Ahmet Gul2, Ivona Aksentijevich1, Daniel Kastner1, Elaine Remmers1
    1NIH, Bethesda, United States; 2Istanbul University, Istanbul, Turkey
    Correspondence: Nobuyuki Horita

    Introduction: Familial Mediterranean Fever (FMF) is an auto-inflammatory disorder that causes recurrent fevers and painful serosal inflammation in the abdomen and pleura as well as synovial inflammation in the joints. Missense M694V, V726A, M694I, M680I and other mutations in exon 10 of the MEFV gene are well known to cause FMF, usually with a recessive mode of inheritance. The MEFV gene encodes pyrin, a key component of the pyrin inflammasome. FMF-associated mutations in pyrin cause dysregulated activation of the inflammasome, leading to activation of caspase, which converts pro-interleukin-1 (IL-1) beta to its active cleaved form.

    Objectives: We obtained a Turkish kindred with multiple cases with Familial Mediterranean Fever (FMF) and Behçet's disease (BD)-like manifestations. The index case and her two daughters, all with Behçet-like disease, were previously found to have a TNFAIP3 frameshift mutation. The high frequency of affecteds could be consistent with a dominantly inherited inflammatory disease in this family, although other individuals had clinical features consistent with recessively inherited FMF. We sequenced DNA from members of this family to determine whether the TNFAIP3 frameshift mutation and MEFV variants could explain this autoinflammatory disease pedigree.

    Methods: Patients were clinically diagnosed to have FMF or BD. Sanger sequence targeting TNFAIP3 exon 5 and MEFV exon 10 was carried out.

    Results: A maternal uncle of the index case and the mother of the index case had compound heterozygous FMF-associated MEFV exon 10 mutations, M680I and R761H. Two daughters of the maternal uncle also had compound heterozygous FMF-associated MEFV mutations, M680I and V726A. The index case and her two daughters had a TNFAIP3 exon 5 frameshift mutation (TNFAIP3 c.799delG, p.Pro268Leufs*19), which is consistent with their HA-20 diagnosis, and also carried a single allele (heterozygous) of the MEFV R761H mutation.

    Conclusion: Segregation of BD-like manifestations in a single Turkish family with multiple FMF patients could be explained by co-inheritance of a heterozygous exon 10 MEFV variant and one TNFAIP3 mutation, and contribution of MEFV variants on the BD-like manifestations of HA20 requires further studies. 

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1034 Systematic review of biological treatment of deficiency of interleukin-36 receptor antagonist (DITRA) in children and adolescents

    Toni Hospach1, Fabian Glowatzki1, Friederike Blankenburg1, Dennis Conzelmann1, Christian Stirnkorb1, Sandra Müllerschön2, Peter von den Driesch2, Lisa Koehler3, Meino Rohlfs3, Christoph Klein3, Fabian Hauck3
    1Olgahospital Stuttgart; 2Klinikum Stuttgart, Stuttgart; 3Dr. von Haunersches Kinderspital, Universität München, München, Germany
    Correspondence: Toni Hospach

    Introduction: Deficiency of interleukin-36 receptor antagonist (DITRA) is a life threatening autoinflammatory disease caused by autosomal recessive mutations of the IL36RN gene leading to recurrent episodes of generalized pustular psoriasis with systemic inflammation and fever. For this disease no standardized treatment guidelines do exist.

    Objectives: To systematically review and analyze the data of biologically treated pediatric DITRA patients.

    Methods: For systematic research we made a “pubmed” research using the term “Deficiency of interleukin-36 receptor antagonist”, “Deficiency of interleukin-36 antagonist”, “IL36RN mutation” and “DITRA” with age restriction to 18 years.

    Results: Our literature research revealed 13 pediatric patients with DITRA and biolocial treatment. Ten patients were homozygous including six with the p.Leu27Pro, three with the p.Arg10 Argfs* and one with the p.Thr123Met mutation and four were compound heterozygous. We add an unreported DITRA patient with a compound heterozygous IL36RN p.Pro76Leu/pSer113Leu mutation. In total 29 flares in 14 patients were treated with biological agents- targeting IL-1/R, IL-17, IL-12/23 and TNF-α. Complete response was achieved in 15 (52%), partial in 4 (14 %), and no response in 10 (34 %) of the flares. Response rates were heterogeneous among the different agents. While complete/partial/no response with inhibition of TNF-alpha could be achieved in 6 (46%)/3 (23%)/4 (31%), the inhibition of IL-17 and of IL-12/23 led in each 4 flares to a 100 % complete response. IL-1/R inhibition led to complete/partial response in each 1 (13 %) and was not effective in 6 (75%) flares. Of note, the unreported patient was successfully treated with weekly dosed adalimumab.

    Conclusion: DITRA is a rare disease that has to be considered in patients with generalized pustular psoriasiswith systemic inflammation and fever. It can be effectively treated with specific biological inhibition of TNF-alpha, IL-12/23 and IL- 17, while anti-IL-1/R treatment seems less effective. Weekly dosed adalimumab appears to be a novel treatment option for pediatric patients. Further reports and studies of biological treated pediatric DITRA patients are warranted for evaluation of optimal treatment.

    Disclosure of Interest

    None Declared

    P1035 Familial Mediterranean fever in Slovakia – clinical and genetic characteristics of Slovak cohort

    Milos Jesenak1,2,3, Lenka Kapustova1, Katarina Hrubiskova4, Tomas Dallos5, Peter Banovcin1
    1Centre for Periodic Fever Syndromes, Department of Pediatrics; 2Centre for Periodic Fever Syndromes, Department of Pulmonology and Phthisiology, Jessenius Faculty of Medicine, Comenius University in Bratislava; 3Department of Clinical Immunology and Allergology, University Teaching Hospital in Martin, Martin; 4Centre for Periodic Fever Syndrome, 5th Department of Internal Medicine, Comenius University in Bratislava, Faculty of Medicine, University Teaching Hospital; 5Department of Pediatrics, National Institute of Children’s Diseases, Comenius University in Bratislava, Faculty of Medicine, Bratislava, Slovakia
    Correspondence: Milos Jesenak

    Introduction: Familial Mediterranean fever (FMF) is considered to be a rare disease in the region of Central Europe (CE). True prevalence is still not known and awareness of FMF is very low.

    Objectives: Since the data about FMF prevalence are missing, we aimed to find all the FMF cases from the whole area of Slovakia. Then we aimed to analyse the clinical and genetic characteristics of Slovakian FMF patients’ cohort.

    Methods: We performed the questionnaire based survey in outpatient clinics for clinical immunology and rheumatology and hospital departments in Slovakia. We collected all the genetically-confirmed FMF patients and invite them for clinical examination in the National Centre for Periodic Fever Syndromes in Martin. They underwent complex clinical and laboratory examination and detailed history analysis.

    Results: All together, we detected 53 FMF patients (males 23, 42%), aged 33.64±17.33 years (males 28.93±17.42 years, females 35.39±17.34 years). The age of first symptoms was 12.90±13.37 years and the age of FMF confirmation was 31.30±18.18 years, so the average diagnostic delay was almost 19 years. All the patients had recurrent abdominal pain (100%) which was accompanied by recurrent fever in 50 pts (94.3%). Other reported symptoms associated with FMF were: fatigue (77.4%), arthralgia/arthritis (66.0%), chest pain (56.6%), cervical lymphadenopathy (32.1%), tonsillitis (28.3%), headache (26.4%) and skin rash during flares (15.1%). Pleuritis was confirmed in 18.9%, pericarditis in 11.3% and ascited in 22.6%. 3 patients suffered from renal amyloidosis. 3 pts (5.7%) were homozygotes for pathogenic mutation, 10 (18.9%) compound heterozygotes, 28 (52.8%) with pseudo-AD inheritance and 12 (22.6%) patients carried mutations of unknown or possible benign origin. 79% pts were treated by colchicine (mean dose was 1.03±0.51 mg/day), 10 with anakinra (3 regular, 7 on demand) and 9 by canakinumab. 4 patients were intolerant to colchicine. Isolated elevation of serum amyloid A without concomitant elevation of C-reactive protein between the flares before the initiation of the treatment was observed in 34% pts. In 5 of them (9.4%), the IgD elevation was found. In 7 patients, the origin from FMF endemic regions was confirmed.

    Conclusion: This the first complex report about the epidemiology of FMF in Central European Country with presumed low incidence. We confirmed much higher prevalence as was expected. The majority of our patients have pseudo-AD form of FMF. All the available therapeutic options were applied. It is necessary to raise the awareness of FMF and to shorten the diagnostic delay.

    Disclosure of Interest

    None Declared

    P1036 Diagnostic criteria for proteasome-associated autoinflammatory syndromes (PRAASS) including Nakajo-Nishimura syndrome, JMP syndrome and CANDLE syndrome

    Nobuo Kanazawa1, Hiroaki Ida2, Noriko Kinjo3, Tomoaki Ishikawa4, Ryuta Nishikomori5
    1Department of Dermatology, Wakayama Medical University, Wakayama; 2Department of Medicine, Division of Respirology, Neurology, and Rheumatology, Kurume University School of Medicine, Kurume; 3Department of Pediatrics, University of the Ryukyus Graduate School of Medicine, Nishihara; 4Department of Pediatrics, Nara Medical University, Kashihara; 5Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
    Correspondence: Nobuo Kanazawa

    Introduction: Nakajo-Nishimura syndrome (NNS) was described originally as “secondary osteoperiostosis with pernio” in Japanese by Nakajo in 1939 and by Nishimura in 1950, and then reported in English as “a syndrome with nodular erythema, elongated and thickened fingers, and emaciation” in 1985 and as “hereditary lipo-muscular atrophy with joint contracture, skin eruptions and hyper-gamma-globulinemia” in 1993. It was in 2011 that similar syndromes have first been reported from outside Japan, which included “joint contractures, muscular atrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP)” syndrome and “chronic atypical neutrophilic dermatitis with lipodystrophy and elevated temperature (CANDLE)” syndrome. As PSMB8 mutations have commonly been identified in NNS and these two syndromes, they are now collectively called as “proteasome-associated autoinflammatory syndromes (PRAASs)”. Recently, their responsible genes are expanding to other proteasomal genes and, on the other hand, a similar but distinct proteasome-associated entity “proteasome-associated autoinflammation and immunodeficiency disease (PRAID)” has been proposed. In Japan, NNS is now registered as an officially-recognized intractable disease diagnosed by officially-approved criteria.

    Objectives: To define the diagnostic criteria of PRAASs.

    Methods: So far reported 30 NNS, 3 JMP and 21 CANDLE/PRAAS cases are reviewed and the temporal diagnostic criteria of NNS are verified for these cases.

    Results: Among 8 points of 1) autosomal recessive inheritance (parental consanguinity or familial occurrence), 2) pernio-like purplish rash in hands and feet (appearing in winter since infancy), 3) haunting nodular erythema with infiltration and induration (sometimes circumscribed), 4) repetitive spiking fever (periodic, not necessarily), 5) Long clubbed fingers and toes with joint contractures, 6) progressive partial lipomuscular atrophy and emaciation (marked in the upper part of body), 7) hepatosplenomegaly, and 8) basal ganglia calcification, more than 5 are required for temporal clinical diagnosis of NNS. 80% of NNS, 100% of JMP and 67% of CANDLE/PRAAS cases meet these criteria, while most of NNS cases reported before 1990 and CANDLE/PRAAS cases without any description on pernio-like rash do not meet the criteria. If the point 7) is changed to hepatomegaly and 2 points of 9) microcytic anemia and 10) hyper-gamma-globulinemia are added and more than 6 of the final 10 points are required for the diagnosis, positivity of the new criteria reaches 93% of NNS, 100% of JMP and 76% of CANDLE/PRAAS cases. PRAID cases do not meet these criteria. Genetically, 11 cases of NNS, 3 cases of JMP and 11 cases of CANDLE/PRAAS cases have homozygous or compound heterozygous PSMB8 mutations, while other digenic (PSMB8 + PSMA3, PSMB8 + PSMB4, PSMB9 + PSMB4), compound heterozygous PSMB4 or heterozygous POMP mutations are observed in 8 CANDLE/PRAAS patients. PRAID cases have distinct heterozygous POMP or PSMB9 mutations.

    Conclusion: We propose a new diagnostic criteria for PRAASs: clinically, at least 6 points are required among above-mentioned 10 points, and genetically, “definite” when disease-associated proteasomal gene mutation(s) are identified and “probable” even if such mutation(s) are not identified, but when other diseases are differentiated.

    Disclosure of Interest

    None Declared

    P1037 Short term follow-up results of children with familial Mediterranean fever after cessation of colchicine: is it possible to quit?

    Ayşe Tanatar, Hafize Emine Sönmez, Şerife Gül Karadağ, Mustafa Çakan, Nuray Aktay Ayaz
    Pediatric Rheumatology, Health Sciences University Kanuni Sultan Süleyman Training and Research Hospital, Istanbul, Turkey
    Correspondence: Şerife Gül Karadağ

    Introduction: To define the characteristics of children with familial Mediterranean fever (FMF) whose colchicine treatment was discontinued and then to compare these features of the patients whose colchicine was restarted with the ones not restarted.

    Methods: Sixty-four out of 1786 children with FMF whom colchicine was stopped by the physician or patients/parents own decision were enrolled. These patients were grouped into two as: group 1; children whose colchicine was re-started and group 2; children whose colchicine was not re-started. The demographic, clinical and genetic data were collected and compared between group1 and group 2.

    Results: Colchicine was stopped in 59.4% (38/64) by the physician and 40.6% (26/64) of them had stopped colchicine by patients/parents will. Colchicine was ceased at a median of 10.6 (2.120.5) years of age, and attack- and inflammation-free periods of 18.2 (6-148) months. The median follow-up of 64 patients after colchicine cessation was 37.4 (6.4-154.7) months. It was re-started in seventeen patients due to attacks (n=11) or elevated acute phase reactants (n=6), while remaining 47 patients did not require colchicine. The age at cessation of the colchicine was lower (p= 0.04) and duration of colchicine treatment until its cessation was shorter (p= 0.007) in group 1 than group 2.

    Conclusion: Even though the results of our study are not satisfactory enough to endorse the hypothesis that colchicine may be discontinued by close follow-up; older age and long duration of colchicine treatment before cessation may be two important features that should be considered in the future studies

    Disclosure of Interest

    None Declared

    P1038 Periodic fever syndromes: a year follow-up of a tertiary pediatric rheumatology outpatient clinic in Turkey

    Nuray Aktay Ayaz, Şerife G. Karadağ, Hafize Emine Sönmez, Ayşe Tanatar
    Pediatric Rheumatology, Health Sciences University Kanuni Sultan Süleyman Training and Research Hospital, Istanbul, Turkey
    Correspondence: Şerife G. Karadağ

    Introduction: To define the frequency of patients who were suspected to have periodic fever syndromes (PFS) and their final diagnosis.

    Methods: We prospectively evaluated the patients who were initially referred to our department with suspicion of PFS in a year period. These findings cover only ten months results as a preliminary study.

    Results: A total of 2317 new patients (1142 male/1175 female) were seen. Among them, 724 patients were referred to evaluate for the presence of PFS. Finally, 553 patients were classified as having PFS. Of those, 444 patients were diagnosed with familial Mediterranean fever, 43 with periodic fever with aphthous stomatitis, pharyngitis, and adenitis, 2 with cryopyrin-associated periodic fever syndromes, and 1 with hyper-immunoglobulin D syndrome. Genetic analyses are still in progress in the remaining 63 patients. Most common MEFV variant was M694V in our patients. The rest of the patients who were suspected to have PFS were diagnosed as follows: gastrointestinal disorders (n= 161), infections (n=6), dysmenorrhea (n=2), immunodeficiency (n=1).

    Conclusion: Diagnosing PFS requires a careful evaluation. As our study shows nearly one third of patients referred to our center were not accepted as PFS.A detailed evaluation of the patient’s signs and symptoms and also taking the recurrency of these features into account will help to exclude unnecessary referrals to pediatric rheumatology units. Although recommendations are present for rheumatologists, generating some clear-cut algorithms about PFS may provide a practical approach for general pediatricians while they are evaluating and referring these patients.

    Disclosure of Interest

    None Declared

    P1039 Assessing French liberal pediatricians awareness and referral for reccurent fever syndromes: the fireville survey

    Valerian Koskas1, Remy Assathiany2, Sylvie Hubinois3, Corinne Levy4, Marc Koskas5, Isabelle Koné-Paut6
    1Pediatric Rheumatology, APHP, University of Paris Sud, Le Kremlin Bicêtre; 2Pediatrics, Liberal Exercise, Issy les Moulineaux; 3AFPA (Association Française de Pédiatrie Ambulatoire), Saint Germain en Laye; 4Association Clinique et Thérapeutique Infantile du Val de Marne; 5Liberal Pediatrician, Saint Maur; 6Pediatric rheumatology, APHP, Bicetre Hospital, Le Kremlin Bicêtre, France
    Correspondence: Valerian Koskas

    Introduction: Patients with recurrent fevers almost always refer initially to their family (liberal) paediatrician, their general physician and/or the emergency hospital departments. If in most cases, benign recurrent viral infections are in causes, it may underlie rare well–defined disorders such as systemic auto inflammatory diseases (SAID). At present few is known on the degree of awareness of liberal paediatricians on SAID and nothing on how they deal with these patients in terms of further investigations, treatment and referral. FIREVILLE study tries to better understand why patients with SAID undergo complex medical journey before appropriate referral in our country.

    Objectives: To survey, liberal paediatricians on theirs knowledge and practices regarding children with recurrent fevers

    Methods: We took the facilities of the AFPA (French Association of Ambulatory Paediatrics) to email 1248 active liberal paediatricians between June and September 2018, to answer a Survey-Monkey type questionnaire. The survey, included 36 questions divided into 3 parts; i.e.Socio-demographic characteristics of paediatricians and their knowledge on recurrent fever syndromes (RFS), clinical case analysis, then more general evaluation of knowledge on and analysis of city-hospital networks.

    Results:

    360 paediatricians (28.8%) answered the survey after 4 email reminders. 77% were women aged 44 to 60 years with a completion rate of 79%. 22% had part time of hospital practice. The top 3 regions were: Paris ile de France: 24.9%, Auvergne-Rhône-Alpes: 18.3% and Provence-Alpes-Côte d'Azur f 9,9%. 69% of paediatricians considered their knowledge on HRF, and 83% on SAID. PFAPA was the best known (46%), on the basis of the frequency (92%) and the regularity (89%) of fever. Only 66% considered the duration of fever as an added diagnostic value. The clinical case was a PFAPA chart. 92% of paediatricians required whole blood cell count and 91.8% a CRP. 70% of paediatricians ruled out radiologic evaluations. They were 80.6% to suspect PFAPA and 14% to suspect FMF. 57% asked second opinion, in a paediatric rheumatology unit (46%) and in general paediatrics (35%). Youngest paediatricians chose more significantly a paediatric rheumatology unit (p = 0.009).Only 44% of responders knew a SAID referral centre in their area. Finally, 90% were interested in joined city-hospital care.

    Conclusion: This is the first study surveying liberal paediatricians on their knowledge and practices with a child with supposed HRF. In spite of their thought insufficient knowledge, their answers were accurate in most cases, however the questionnaire revealed insufficient knowledge of the dedicated resources and network for SAID.

    Disclosure of Interest

    None Declared

    P1040 Crimean Tatars is new target nationality for the familial Mediterranean fever

    Olga Zhogova1, Natalya Lagunova1, Sergey Ivanovskiy1, Evgeny Suspitsin2,3, Mikhail Kostik2
    1V.I. Vernadskiy, Crimean Federal University, Simferopol; 2Saint-Petersburg State Pediatric Medical University; 3N.N. Petrov Institute of Oncology, Saint-Petersburg, Russian Federation
    Correspondence: Mikhail Kostik

    Introduction: Familial Mediterranean Fever (FMF) is a monogenic autoinflammatory disease with high prevalence in some nationalities, such as Jew, Armenians, Turkish, Arabians and other nationalities with Mediterranean origin. Before 2016 there were no data about FMF distribution in the Crimea region, but the first 15 new cases of FMF were diagnosed in the last 2 years.

    Objectives: The aim of our study is the evaluation of the distribution of FMF in the Crimea region.

    Methods: Our cohort consists of 13 children and 2 adults, among them were 2 parents and 6 kids from 1 family. All belong to Crimea Tatar nationality. This nationality is close to Turkish. The diagnosis of FMF was based on the Tel-Hashomer criteria and later was confirmed by MEFV gene sequence.

    Results: 10 children with FMF have M694V heterozygous mutation, 3 have M694V homozygous mutations, and 2 adults (parents) have M694V homo (father) and heterozygous (mother) mutations. Ten children and 2 adults are treated with colchicin. 2 kids and 1 adult (all M694V/M694V) received canakinumab due to inefficacy and 1 child (M694V/N) due to intolerance of increased doses. Clinical characteristic of the studied population are in the table.

    Conclusion: Crimean Tatars is a new nationality with an increased prevalence of FMF with typical high penetrance mutations. Further epidemiological studies required about MEFV alleles distribution in the healthy population and in the FMF patients.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1040). See text for description

    P1041 New variant in the IL1RN-gene associated with late onset and atypical presentation of DIRA – follow-up

    Jasmin B. Kuemmerle-Deschner1, Kerstin Reicherter2, Susanne Schlipf3, Sandra Hansmann1, Anton Hospach4, Ilias Tsiflikas5, Xiao Liu6, Susanne Benseler7, Alexander Weber6
    1Department of Pediatrics, Division of Pediatric Rheumatology, University Hospital Tuebingen; 2CEGAT, Tuebingen; 3Kinderarztpraxis Dr. Lakner, Schwäbisch Gmünd; 4Zentrum für Pädiatrische Rheumatologie, Klinikum Stuttgart, Olgahospital, Stuttgart; 5Pediatric Radiology, Department of Radiology, University Hospital Tuebingen; 6Department of Immunology, University of Tuebingen, Tuebingen, Germany; 7Rheumatology, Department of Pediatrics, University of Calgary, Calgary, Canada
    Correspondence: Jasmin B. Kuemmerle-Deschner

    Introduction: Deficiency of the Interleukin-1 receptor antagonist (DIRA) is an autoinflammatory disease characterized by severe systemic inflammation with bone and skin involvement present in the first days of life. Here we report on diagnosis, treatment and follow up of a novel variant in the IL1RN-gene associated with late onset and atypical phenotype of DIRA.

    Objectives: Here we report on diagnosis, treatment and follow up of a novel variant in the IL1RN-gene associated with late onset and atypical phenotype of DIRA.

    Methods: A 3 year-old boy presented with recurrent monthly episodes of fever and fatigue, associated with lymphadenopathy, pericarditis, pleuritis, pancreatitis, and arthritis involving sacroiliac, hip, knee and ankle joints in the absence of any skin involvement. Symptoms had started at age one and had progressed over time to life-threatening episodes requiring intensive care therapy. Throughout, inflammatory parameters including ESR, CRP, SAA, S100A8/9, leukocytes and platelet counts were highly elevated. Treatment with colchicine and steroids improved symptoms but did not prevent flares. Typical immune deficiencies were ruled out; genetic testing for FMF, CAPS, TRAPS, HIDS and DITRA did not reveal variants in the associated genes.

    Results: Whole exome sequencing detected a novel homozygous stop variant c.62C>G; p.Ser21* in the ILRNgene (NM_173842.2). Mother, father and brother were heterozygous for the same variant. In addition, three variants of unknown significance were identified in the patient's PCGF5, CPA1and SPTA1genes. Functional studies revealed only marginal secretion of IL-1RA in the patient’s unstimulated leukocytes and after stimulation with IL-1βand LPS, confirming the disease-causing nature of the variant.

    IL-1 inhibition with anakinra at 2 mg/kg/d was started and resulted in complete resolution of clinical symptoms and signs of inflammation on MRI, in normal inflammatory markers, and dramatically improved energy levels. Intolerance to daily subcutaneous injections prompted a switch to canakinumab at 4 mg/kg/4 weeks. However, as per the patient’s and mother’s assessment of disease activity, canakinumab was inferior to anakinra. After four months a flare occurred, prompting re-start of anakinra and resulting in sustained and complete resolution of symptoms in an observation period of 14 months.

    Conclusion: This is the first report of the novel c.62C>G; p.Ser21* variant in the IL1RN-gene primarily causing severe serositis in a homozygous carrier, while heterozygous family members were completely symptom-free. Skin disease, one of the most prominent features in other patients with DIRA was not observed in this patient, while IL-1 inhibition was likewise effective.

    The different phenotype in the patient reported here, may be due to the selective loss of secreted IL1RN. Unlike one report of successful canakinumab treatment of one patient with late onset of DIRA [1], our patient did not respond favourably to canakinumab, but treatment with anakinra lead to sustained remission.

    1 Ulusoy et al. J Med Case Reports 2015; 9:145

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    J. Kuemmerle-Deschner Grant / Research Support from: Novartis, SOBI,Consultant for: Novartis, SOBI, K. Reicherter Employee of: CEGAT, S. Schlipf: None Declared, S. Hansmann: None Declared, A. Hospach Consultant for: Novartis, Chugai-Roche, SOBI, I. Tsiflikas: None Declared, X. Liu: None Declared, S. Benseler Consultant for: Novartis, SOBI, abbvie, A. Weber: None Declared

    P1042 Efficacy of anti-IL-1 treatment in familial Mediterranean fever: a single-center experience

    Tuba Kurt, Halide O. Basaran, Fatma Aydın, Nermin Uncu, Banu Celikel Acar
    Pediatric Rheumatology, Ankara, Child Health Hematology and Oncology Education and Research Hospital, Ankara, Turkey
    Correspondence: Tuba Kurt

    Introduction: In 5%–10% of patients with familial Mediterranean fever (FMF), colchicine is not effective in preventing inflammatory attacks. Furthermore, another 5%–10% of patients are intolerant to effective doses of colchicine and experience serious side effects. In recent years, it has been shown that interleukin-1 (IL-1) plays a central role in the pathogenesis of FMF.Several reports have pointed out the efficacy of IL-1 blockade in colchicine resistant FMF.

    Objectives: To review the patients followed with FMF who received anti-IL-1 treatment, in terms of outcome and side effects

    Methods: 18 FMF patients who were treated with anti-IL-1 treatment were retrospectively reviewed with regard to indication, effect on disease activity and acute phase response, adverse events and AIDAI score and patient global assessment. Colchicine resistance was defined as at least one attack per month for three consecutive months and elevated erythrocyte sedimentation rate or C-reactive protein in-between attacks despite taking adequate dose of colchicine.

    Results: There were 18 patients with FMF (9 M/ 9 F) who were treated with Anakinra and Canakinumab for various indications (colchicine resistant recurrent febrile attacks in 16, colchicine related side effects in 1, subclinic inflamation in 1). 11 patients were treated with Anakinra while were 15 patients with Canakinumab. All patients except 5 had homozygous or compound heterozygous exon ten mutations. The mean age of onset of anti-IL-1 treatment was 15± 2 (11-18 years) years. The mean duration of the disease was 11.1 ± 4.34 years. All patients were taking adequate dose of colchicine for their age before treatment with a median dosage of 0,03 ±0,012 mg/kg/day before anti-IL-1 treatment (0.03–0.06 mg/kg/day). 11 patients were treated with anakinra with a median duration of 29,7 months (8- 60 months), but 6 of them switched to canakinumab because of noncompliance and side affects (2 headache, 1 urticerial rash), all responded.After the initiation of Anakinra treatment 6 patients became attack-free, 2 patients reported more than 50% decrease, and 3 patients no change in the frequency of the attacks.

    15 patients were treated with Canakinumab but 2 (%13,3) of them swiched to anakinra because had increase in frequency of the attacks. All of patients complete respondend and any of them were no side effects.

    A significant decrease was observed in the mean CRP (from 8.5± 8.7 mg/dLto 0.68 ± 0.75 mg/dL), WBC (from 10335 ± 3206 mm³ to 7047 mm³± 2108mm³) and ESR levels (from 44.35 ± 18.7 mm/h to 9.5 ± 7.6 mm/h), respectively (referance range for CRP: 0–0.3mg/dL).

    AIDAI score decreased from 25±17,5 to 0,33±1 and mean pyscian’s global assessment 7,4±1,5to 1,25±1,1 respectively, under anti-IL-1 treatment treatment.

    As for the adverse events, 1 patients (%9) had allergic reactions under Anakinra treatment (severe disseminated rash in 1 patient ) and 2 patients (%18,1) had headache which necessitated termination of treatment in 3 patients. There were no adverse events in the remaining all patients during the course of treatment.

    Conclusion: Based on the results of our study, anti-IL-1 therapy seems to be a safe and effective alternative for patients with FMF who do not respond to or cannot tolerate colchicine, however approximately one fourth of the patients stop anakinra for insufficient response and injection site reaction. The treatment should be modified and decided for each patient on an individual basis.

    Disclosure of Interest

    None Declared

    P1043 Erysipelas-like erythema as the primary gripe of FMF

    Omer Kuru1, Ahmet Ki̇vanc Cengi̇z2
    1Physical Medicine and Rehabilitation, Division of Rheumatology, University of Heath Sciences, Istanbul Okmeydani Research and Training Hospital, Istanbul; 2Physical Medicine and Rehabilitation-Rheumatology, 19 Mayis University Faculty of Medicine, Samsun, Turkey
    Correspondence: Omer Kuru

    Introduction: Familial Mediterranean Fever (FMF) is a monogenic autoinflammatory disease characterized by recurrent polyserositis attacks, mainly involving peritoneum, pleura and synovium. Erysipelas-like erythema (ELE) is an aseptic neutrophilic dermatose defined as well-demarcated, warm, tender, erythematous and infiltrated plaques which resolves spontaneously in 2-3 days. ELE is a pathognomic cutaneous manifestation of FMF. The reported frequency of ELE is 5-30 % in different populations. It typically developes on the extensor surface of lower extremities especially on dorsum of ankles and feet. It is usually unilateral and associated with more severe FMF clinical phenotype and M694V homozygosity.

    Objectives: Here we would like to present a 12 year old girl admitted to the emergency service with an erythematous, painful rash at the dorsum of her foot. This was her fourth admission in approximately 9 monthstime with a similar skin lesion at dorsum of the same foot. In her medical records previous diagnosis for the described lesion were insect bite, contact dermatitis and cellulitis. Oral and topical antibiotics, topical steroids were prescribed in her previous visits. She told that regardless of the medication used the lesions resolved spontaneously in 3-4 days time. She had also noticed that the lesions usully occured after her volleyball matches. Exercise and long time standing usually has caused pain in her legs and if she insists on the activity despite this pain, the lesions occured. She had no prominant abdominal or pleural pain. She didn’t have arthritis but had episodes of fever lasting 36-48 hours accompanying the skin lesions. Her family history was unremarkable except FMF in one of her cousins. Her laboratory tests revealed leukocytosis, elevated crp and fibrinogen levels. She did not have proteinuria.

    Methods: Case report

    Results: The recurrence of the typical lesion, spontaneous recovery in 3-4 days time, accompanying fever and positive family history reminds the diagnosis of FMF. Genetic analysis was performed and compound heterozygous genotype (M694V/V726A) was detected. Colchicine was prescribed. She is 14 years old now and did not have any typical serositis attack yet. Under colchicine treatment the ELE attacks did not disappear completely but their frequency and severity decreased prominently and fever did not accompany her ELE attacks any more.

    Conclusion: Spontaneous recovery, recurrence of the attacks, accompanying fever and elevated acute phase reactants during the attacks points out FMF especially in inhabitants of the Mediterranean basin. Occurence of ELE as the first manifestation of FMF is rare but this possibility must also be kept in mind.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1044 Analysis of new referrals to a specialist UK adult autoinflammatory disease service

    Serdal Ugurlu1, Philip N. Hawkins2, Charalampia Papadopoulou2, Tamer Rezk2, Dorota Rowczenio2, Helen J. Lachmann2
    1Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul - Cerrahpasa, Istanbul, Turkey; 2National Amyloidosis Centre, Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, Royal Free Campus, UCL, London, United Kingdom
    Correspondence: Helen J. Lachmann

    Introduction: Diagnosis of the SAIDs requires a high index of suspicion and previous series has suggested that there are often long diagnostic delays, particularly in TRAPS and MKD.

    Objectives: To look at the case mixed referred to a single adult clinic in London specialising in assessment of potential SAIDS over the course of the year of 2017.

    Methods: All new referrals were accepted for clinical assessment. At the first visit patients had a full history and examination, genetic testing – varying from single gene to a 20 gene panel depending on clinical features, and laboratory testing including fortnightly blood draws for serial analysis of the hepatic acute phase response proteins, CRP and SAA over a 3 month period.

    Patients with a non suggestive history, non contributory genetic testing and no evidence of inflammation accompanying symptoms were felt not to have SAIDS and referred back to their local hospitals for further management. Other cases were diagnosed based on full clinical assessment, other investigations – for example ferritin in AOSD, genetic testing results, serial monitoring of CRP and SAA and therapeutic trials, for example colchicine in presume FMF and anti IL-1 therapies in CAPS and Schnitzler’s syndrome.

    Results: 273 new patients were referred. Median age at referral was 37.4 years, the oldest patient was 84.3 years old and 59% were female. 174 (64%) were of northern European ancestry, 68 (25%) were eastern Mediterranean, west Asian or southern European ancestry, 19 (7%) were of south or east Asian ethnicity and 4% were of African or Afro-Caribbean ancestry. 76% of referrals were from hospital specialities. The referral source was: rheumatology 38%, general practitioner 24%, dermatology 8%, immunology 8%, gastroenterology 6%, infectious diseases 3%, clinical genetics 3%, nephrology 2%, haematology 2%, gynaecology 2%, emergency department 1%, respiratory 1%, other 2%.

    After work up 135 (49.5%) were felt not to have a SAID as the cause of their symptoms. Of the remaining 138 patients who did have evidence of a SAID the diagnoses made were: FMF 33%, uncharacterised SAID 26%, CAPS 9%, AOSD 8%, recurrent idiopathic pericarditis 6%, Schnitzler’s Syndrome 5%, TRAPS 4%, variant PFAPA 4%, DADA2 1%, MKD 1%, CRMO 1%, Behcets 1%, Cattleman’s disease 1%.

    The median interval between reported symptom onset and diagnosis were as follows: 16 yrs for FMF, 28.1 yrs for CAPS, 5.0 years for recurrent idiopathic pericarditis, 4.5 yrs for Schnitzler’s Syndrome, 5.7 yrs for TRAPS, 20.5 yrs for variant PFAPA,12.5 yrs for DADA2, 17 yrs for MKD and 2 years for CRMO.

    Conclusion: This series suggests that recognition and diagnosis of the SAIDS remains a challenge. More than 1/3 of referrals were from rheumatology, referrals from primary care were almost exclusively from patients with a known family history of one the inherited syndromes. The wide variety of referring specialities reflects the diverse nature of SAIDS and the importance of almost all specialities considering the possibility of SAIDS. Only just over 50% referrals had evidence of diseases falling within the recognised SAID spectrum and 26% of these have currently uncharacterised disease with non diagnostic genetic testing. Of those in whom a diagnosis could be made there are significant diagnostic delays fortunately despite late initiation of treatment no patients had evidence of systemic AA amyloidosis.

    Disclosure of Interest

    None Declared

    P1045 Application of autoinflammatory disease damage index (ADDI) to other autoinflammatory diseases in a tertiary referral hospital

    Mireia Lopez, Estefania Moreno
    Pediatric Rheumatology, Hospital Vall d’Hebron, Barcelona, Spain
    Correspondence: Mireia Lopez

    Introduction: Autoinflammatory diseases (AIDs) cause systemic inflammation that can be chronic and result in damage to multiple organs. Recently, the autoinflammatory disease damage index (ADDI) has been developed and validated to the four most common monogenic AIDS, cryopyrin-associated periodic syndrome (CAPS), familial Mediterranean Fever (FMF), mevalonate kinase deficiency (MKD) and tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS). ADDI could be useful in other AIDs different than the four reported.

    Objectives: The aim of the study is to asses the application of ADDI to patients with AIDs followed in our hospital with the four most common monogenic diseases and other AIDs, determine the reliability and point out encountered comments about the scoring.

    Methods: All patients with AIDs followed in Transitional Care unit or Pediatric Rheumatology specialized in AIDs consult from Hospital Universitari Vall d’Hebron were identified. A cross- sectional, descriptive study was performed applying ADDI by two pediatric rheumatologists (EM, ML). Laboratory test including C-reactive protein (CRP) mg/dl, amyloid protein (AP) mg/L, erythrocyte sedimentation rate (ESR) mm/h and protein/creatinine rate (mg/g Cr) were performed at the moment ADDI was applied. Variables related with disease duration, current treatment and accumulated corticosteroids treatment was assessed. The continuous data are presented as mean and standard deviation (SD). Categorical variables are presented by percentages.

    Results: A total of 41 patients with AIDs were included, of whom 61% were female, with a median age of 20 years (SD 11.9) at inclusion. Disease duration has a mean of 11 years (SD 8.2). AIDs included were 11 patients with FMF (26.8%), TRAPS n=4 (9.8%), MKDn=3 (7.3%), CAPS n= 2 (4,9%), Blau syndrome n= 7 (17.1%), SAVI syndrome n=3 (7.3%), CRMO n=4 (9.8%), PFAPA n=2 (4.9%), APLAID n=1 (2.4%), Stickler syndrome n=1 (2.4%), and 3 unknown AIDs with genetic test negative n=3 (7.3%). Current treatment is variable among patients, 6 (15.8%) are taking disease-modifying antirheumatic drugs (DMARDs), 9 (23.7%) Colchicine, 8 (21.1%) Anakinra, 13 anti-TNF therapy (34.2%), 1 (2.6%) Ruxolitinib and 1 (2.6%) Abatacept.Just 6 patients were receiving corticoids with mean prednisone dose of 7.5 mg/day.

    ADDI mean score was 2.3 (SD 2.2) for all patients. Regarding the eight different items evaluated, musculoskeletal domain was the highest punctuated with a mean of 1.02 points, followed by ocular domain with 0.42 points. Laboratory test results were mean ESR 27.2 mm/h (SD 26.7), CRP 0.7 mg/dl (SD 1.3), AP 13.9 mg/L (SD 18.6). Proteinuria was present in 2 patients with mean 286.5 mg/g (SD 246.1). EM and ML applied ADDI in 5-10 minutes average.

    Conclusion: ADDI is a feasible index suitable to measure damage in a single patient. Despite it was performed to the four most common AIDs it could be applied to other diseases. In our cohort mean ADDI was low with musculoskeletal item as the highest punctuated. This result could be explained by the correct control of the disease with the current treatment.Laboratory tests also support this finding. Despite good response to medication, it is difficult to reverse bone deformities or joint restriction. Nevertheless, some organ systems are not represented like respiratory, cardiovascular or cutaneous damage, important in some syndromes included in our cohort. Knowing the difficulties of conducting an unified index for all diseases, ADDI must be applied in longitudinal cohorts.

    Disclosure of Interest

    None Declared

    P1046 Vasculitis in autoinflammatory diseases in a tertiary hospital

    Rosa M. Alcobendas, Sara M. Loza, Pablo F. Fraga, Clara U. Gascon, Catarina Fervenza, Agustin Remesal
    Pediatric Rheumatology, University Hospital La Paz, Madrid, Spain
    Correspondence: Sara M. Loza

    Introduction: Autoinflammatory diseases with vasculitis and interpheronopaties have been recently described and may present with variable clinical signs. Such entities are considered rare diseases and potentially life-threatening. Clinicians should be aware of the existence of autoinflammatory vasculitis to diagnose and treat them correctly.

    Objectives: To describe the initial manifestations, laboratory findings and therapeutic approaches of patients diagnosed with autoinflammatory vasculitis during the last 5 years in a pediatric rheumatology unit of a tertiary hospital.

    Methods: Retrospective chart review. Inclusion criteria were: children under 18 years diagnosedwith monogenic autoinflammatory vasculitis in a tertiary hospital.

    Results: During the study period, 5 patients were identified (2 boys and 3 girls). The main clinical manifestations were severe cutaneous lesions (5/5), intermittent fever (4/5) and neurological involvement (4/5) (see table 1).The only patient without neurologic manifestations had relatives with psychomotor retardation and sensorineural symptoms of unknown etiology. All patients were diagnosed of with another rheumatology condition,with no response to conventional treatment.

    Conclusion: Autoinflammatory vasculitis are extremely rare entities but potentially fatal. The early age of onset, the intense skin involvement as well as the presence of affected relatives may be suggestive data. Clinical suspicion is important in order to establish an early and adequate treatment.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1046). Clinical characteristics of the patients

    P1047 Diagnosis and treatment of periodic fevers: a single centre experience

    Peter McNaughton1,2, Jane Peake1,3, Ben Whitehead1,3, Su Han Lum2, Kahn Preece4
    1Queensland Children's Hospital, Brisbane, Australia; 2Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom; 3University of Queensland, Brisbane; 4John Hunter Hospital, Newcastle, Australia
    Correspondence: Peter McNaughton

    Introduction: Diagnosis of periodic fever syndromes is difficult due to atypical presentations and overlap in inflammatory symptoms. Treatment of suspected periodic fevers varies widely due to the lack of established clinical guidelines.The utility of genetic testing in identifying monogenic periodic fever syndromes is also unclear due to high frequency of variants of uncertain significance, somatic mutations and heterozygous mutations in genes associated with autosomal recessive conditions.

    Objectives: This study aims to evaluate the diagnosis, treatment and use of genetic testing of patients diagnosed with periodic fever syndromes at a single tertiary paediatric hospital.

    Methods: We retrospectively reviewed the clinical history of patients diagnosed with periodic fever syndromes at Queensland Children’s Hospital, Brisbane, Australia between November 2014 and June 2018.

    Results: 43 patients were diagnosed based on their clinical presentation with periodic fever syndromes.10 patients were diagnosed with PFAPA, 9 with TRAPS, 6 with CAPS, 4 with MKD and 14 unspecified.Median age of onset of symptoms was 24m (range: birth-96m) and median age of diagnosis was 60m (9m-180m).Median time to diagnosis from onset of symptoms was 24m (0-149m).

    Multiple medications were used in 15 patients.The medications used varied widely (prednisolone (22), anakinra (9), etanercept (5), tofacitinib (2), tocilizumab (2), cimetidine (2)).

    Genetic testing of between 1-26 genes was performed in 26 patients (60%).1-3 genes were tested in 13 patients, targeted panels in 10 patients, SNP only in 1 patient.Genetic variants were identified in 9 patients (34% of those tested) however only 2 of these variants were clearly pathogenic (7.7% of those tested).

    Clinical diagnosis and the Eurofever classification criteria were in agreement for patients diagnosed with CAPS (p=0.046) and TRAPS (p=0.025) but not for patients diagnosed with MKD (p=0.47).10 of the patients where clinical diagnosis and Eurofever classification criteria were in agreement had 2 diagnoses positive on the classification score.Two patients diagnosed with CAPS were exclusively positive for CAPS on the classification score and none of the patients diagnosed with TRAPS were exclusively positive for TRAPS.6 of the 7 TRAPS patients had a positive eurofever score for at least one PFS diagnosis.

    Conclusion: As reported in previous studies there was a significant delay between onset of symptoms and diagnosis.This reflects an ongoing need to raise awareness of these conditions with primary care providers. The large number of patients treated with multiple medications and the broad range of medications used reflects the lack of established treatment protocols and varied response to treatments in this group of patients.

    The clinical diagnosis and diagnostic score showed agreement for CAPS and TRAPs however many patients had a diagnostic score positive for more than one diagnosis meaning that a combination of clinical score and clinical judgement is required to make a diagnosis.

    Consistent with previous studies many patients with heterozygous mutations in genes associated with periodic fevers were identified.The significance of these variants is not clear and the diagnostic yield from genetic testing in this cohort was low. Further improvements in availability of next generation sequencing and molecular understanding of autoinflammatory conditions will hopefully improve this yield and allow more confident diagnoses and targeted therapies.

    Disclosure of Interest

    None Declared

    P1048 Multifocal osteomyelitis revealing a PSTPIP1- associated myeloid-related proteinemia inflammatory (PAMI) syndrome: case report and review of the literature

    Manel Mejbri, Katerina Theodoropoulou, Michael Hofer
    Femme-Mère-Enfant, Centre Hospitalier Universitaire Vaudois CHUV, Lausanne, Switzerland
    Correspondence: Manel Mejbri

    Introduction: PAMI syndrome is a recently described condition, previously known as Hyperzincemia/Hypercalprotectinemia(Hz/Hc) syndrome. It is a very rare auto-inflammatory disorder characterized by a chronic systemic inflammation, cutaneousand osteo-articular manifestations, hepatosplenomegaly, anemia and neutropenia. Increased blood levels of MRP 8/14(S100A8/A9 or calprotectin) and zinc distinguish this condition. Specific pathogenic mutations in PSTPIP1 gene (p.E250K andp.E257K) were identified as the genetic cause of this condition.

    Objectives: Case report and review of the literature of PAMI syndrome

    Methods: We report a case of 13 months age female referred to our unity for recurrent episodes of osteoarthritis.Physical examination showed hepatosplenomegaly. Blood work revealed a systemic inflammation, a microcytic anemia and neutropenia. A complete workup for metabolic disorders, oncologic processes and uncommon infections was negative.Because of history of recurrent osteoarthritis, a whole-body MRI was performed and confirmed a multifocal osteomyelitis.

    Whole exome sequencing identified the missense p.E250K in the PSTPIP1 gene.

    A literature search on PAMI syndrome was performed until the15 October 2018. Pubmed was screened using a combination of the following terms: Hyperzincemia, Hypercalprotectinemia, E250K mutation, PSTPIP1 mutation, PAPA with E250K mutation.

    Results: We identified 20 cases of PAMI syndrome in the literature. PAMI syndrome is an early onset inflammatory diseasewith a median age of 2.4 years. Clinical manifestations include Osteo-articular manifestations (80%), skin lesions (71%), splenomegaly (89%), hepatomegaly (68%), lymphadenopathy (42%), growth failure (58%) and hemorragic diasthesis withrecurrent epistaxis and/or haematoma tendency in 5 patients. All cases had relevant abnormalities in hematologic parameters:mild to severe neutropenia and anemia (100%). Thrombocytopenia (42%). Systemic inflammation was confirmed in 94% using the monitoring of CRP, ESR or SAA. Zinc and MRP 8/14 blood concentrations were markedly elevated in all tested patients. Genetic analyses of PSTPIP1 gene revealed the two specific identified mutations (p.E250K and p.E257K) in all patients. Response to the treatment was variable with no consistently effective therapy. Most common therapeutic optionswere AINS, Corticosteroids (n=9), Anakinra (n=9), Anti-TNF (n=6) and Cyclosporine A (n=4).

    Conclusion: PAMI syndrome is a rare auto inflammatory condition which should be considered in patients with undefined systemic inflammation and neutropenia, even without skin or osteo-articular manifestations. Zinc and serum MRP 14/8measurement may be helpful tools for the diagnostic orientation in these cases.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Monogenic autoinflammatory diseases (basic science)

    P1049 Investigation of inflammasome components in the process of cell migration in FMF patients

    Tayfun H. Akbaba1, Z. Yeliz Akkaya-Ulum1, Selcan Demir2, Zeynep Tavukcuoglu1, Seza Ozen2, Banu Balci-Peynircioglu1
    1Medical Biology; 2Pediatric Nephrology and Rheumatology, Hacettepe University, Ankara, Turkey
    Correspondence: Tayfun H. Akbaba

    Introduction: Autoinflammatory diseases, periodic fever syndromes, are a group of diseases that develop recurrent inflammatory response in the absence of infection. Familial Mediterranean fever, which is the most common disease among autoinflammatory diseases, is caused by mutant pyrin production due to mutations in MEFV gene. Pyrin protein is thought to be involved in inflammatory pathways by means of protein-protein interactions. There are studies supporting the role of pyrin as a proinflammatory regulator in the literature and involved in pathways associated with cell migration.

    Objectives: The aim of this study was to investigate the effect of pyrin inflammasome on cell migration in FMF patients, in CAPS patient as disease control and healthy controls.

    Methods: Within the scope of the thesis; pyrin inflammasome was examined in mononuclear cells isolated from the blood samples of 11 FMF patients homozygous for M694V mutation, 1 CAPS patient with somatic mosaicism and 7 healthy individuals in the process of inflammatory cell migration. Lipopolysaccharide was used to induce the activation of inflammasome in the cells, while arachidonic acid was used for the inhibition of inflammasome. IL-1 beta secretion was analyzed by western blot as an indicator of inflammasome activation, and transwell filter assay was performed in order to examine the differences in cell migration.

    Results: We demonstrated that the inhibition of inflammasome in the cells of FMF patients compared to the controls was less under the same application conditions. Following inflammasome activation and inhibition, filter experiments were performed and fetal bovine serum was used to induce inflammatory cell migration. In both inflammasome activation and suppression conditions; we observed a statistically significant increase in the ratio of cell migration of the mononuclear cells of the FMF patients compared to CAPS patient and control indivuals.

    Conclusion: These findings support the idea of increased cell migration ratio in patients with FMF due to the more active pyrin inflammasome seen in patients. Although the involvement of other inflammasome components remains to be defined, this study has made significant contributions to illuminate the role of pyrin protein in inflammatory cell migration through the structure of inflammasome.

    Keywords: FMF, pyrin inflammasome, cell migration.

    This thesis was supported by Hacettepe University Scientific Research CoordinationUnit (Project Number: TYL-2018-17354)

    Disclosure of Interest

    None Declared

    P1050 Cell migration defect in hyperimmunoglobulin D syndrome

    Tayfun H. Akbaba1, Selcan Demir2, Z. Yeliz Akkaya-Ulum1, Seza Ozen2, Banu Balci-Peynircioglu1
    1Medical Biology; 2Pediatric Nephrology and Rheumatology, Hacettepe University, Ankara, Turkey
    Correspondence: Tayfun H. Akbaba

    Introduction: Hyper-IgD syndrome or Mevalonate Kinase Deficiency(HIDS/MKD, MIM #260920) is a ultra rare, autosomal recessive hereditary autoinflammatory syndrome caused by compound heterozygous or homozygous mutations in the mevalonate kinase(MVK) gene. It is characterized by recurrent febrile episodes with abdominal pain, lymphadenopathy and an increased serum immunoglobulin D (IgD) level.Anti-IL-1 and anti-TNF treatment are applied to alleviate the symptoms of the disease and to reduce the number of attacks.

    Objectives: In this study, we aimed to analyze cell migration, an important process of inflammation,in HIDS patients and compare with FMF patients and controls in inflamasome activated and inhibited conditions.

    Methods: Peripheral blood mononuclear cell isolation from HIDS patients, FMF patients and controls was performed. LPS was used to induce inflammasome in isolated cells while arachidonic acid was used for inhibition of inflammasome.After activation and inhibition of inflammasome, transwell filter experiments were performed with isolated cells from patients. As a result of the experiment, the migrating cells were stained with calcein-AM and analyzed by Image J programme.

    Results: We did not observe cell migration in HIDS patients’ mononucleer cells under normal conditions, after LPS stimulation. We observed increased cell migration in FMF patients and normal rate in controls as a positive control of these experiments.Considering that 2 HIDS and 1 FMF patients involved in these experiments were receiving anti-IL-1 treatment, it was thought that the drug used by the patients had no effect in cell migration pattern that we observed.

    Conclusion: Our preliminary data is the first study identified cell migration defect seen in HIDS. Due to the mutant protein produced as a result of MVK gene mutations, the deficiency in the function of the mevolanate kinase enzyme observed in HIDS leads to the depletion of geranylgeranyl pyrophosphate, a key substrate for protein prenylation.Considering the close relationship between protein prenylation and cytoskeleton elements related with cell migration, a possible decrease in prenylation may be a potential explanation of cell migration defect seen in HIDS patients.

    Keywords: HIDS, cell migration defect, prenylation

    This project was supported by Hacettepe University Scientific Research Coordination Unit (Project Number: TYL-2018-17354)

    Disclosure of Interest

    None Declared

    P1051 Possible regulatory effects of miRNAs in the pathogenesis of systemic auto inflammatory diseases, from the perspective of familial Mediterranean fever

    Z. Yeliz Akkaya-Ulum1, Zeynep Tavukcuoglu1, Ezgi Deniz Batu-Akal2, Tayfun Hilmi Akbaba1, Hafize Emine Sonmez2, Banu Balci-Peynircioglu1, Seza Ozen2
    1Medical Biology; 2Pediatric Nephrology and Rheumatology, Hacettepe University, Ankara, Turkey
    Correspondence: Tayfun Hilmi Akbaba

    Introduction: Systemic Auto Inflammatory Diseases (SAID) is a group of rare and hereditary periodic fever syndromes with recurrent inflammatory involvement developing in the absence of infection. Among same SAID patients, phenotypic heterogeneity is common, and modifier mechanisms such as epigenetic factors may be considered as one of the reason of these variations. MicroRNAs (miRNAs), a type of epigenetic mechanisms, are regulated in most of the biological processes like inflammation.

    Objectives: This study aimed to explore the potential effect of miRNAs in the auto inflammation mechanism seen in SAID from the perspective of Familial Mediterranean fever (FMF) which is the most common auto inflammatory disorder.

    Methods: The expression levels of miRNAs were analyzed by miRNA array, performed on whole blood RNA samples from healthy controls, homozygous FMF patients with severe phenotype, homozygous FMF patients with mild phenotype, carriers who displayed the disease phenotype, healthy carriers and other rare SAIDs, in pediatric term. The raw data was analyzed by Multi Experiment Viewer (MeV) and TAC programs. Then we performed pathway analyses using DAVID v6.8. and Panther analysistools. Candidate miRNAs shown to be related with inflammatory pathways by bioinformatics analysis, are further studied functionally for their possible effect on expression levels of inflammatory genes, caspase I activation and cell migration (transwell and wound healing experiments) in SW982 (synovial fibroblast) cell lines.

    Results: The four patient groups were compared in between and miR-30e-3p, miR-374b-5p, miR-329-3p, miR-29c-3p, miR-25-5p were found to be significantly down regulated in the patient groups. The expression levels of these miRNAs were validated with qRT-PCR. All these miRNAs were found to be known regulators in TGF-beta and Toll-like receptor signaling pathway, apoptosis and actin cytoskeleton regulation by bioinformatics. After pre-miR transfection of miR-30e-3p, miR-374b-5p, miR-29c-3p; expression levels of inflammatory genes (IL-1β, IL-18, TNF-α, TGF-β) were decreased, caspase I activation and cell migration ratio were significantly decreased (p<0.05).

    Conclusion: Functional results of this study showed that these miRNAs may possibly have an anti-inflammatory effect and can regulate the expression of genes found in inflammatory pathways. The investigation of potential target genes of these miRNAs by bioinformatics tools and 3’ UTR luciferase assay is underway. The results of this study will be informative for understanding and investigating the possible effect of miRNAs in other auto inflammatory diseases.

    This project has been funded by E-RARE-3 project (INSAID, grant 003037603) and The Technical and Scientific Research Council of Turkey (TUBITAK), Grant number: TUBITAK 1001-SBAG 315S096.

    Keywords: SAID, FMF, epigenetics, miRNA, inflammation.

    Disclosure of Interest

    None Declared

    P1052 MIR-197 regulates inflammation in monocytes and synovial fibroblasts by targeting IL1R1

    Yeliz Z. Akkaya Ulum1, Zeynep Tavukcuoglu1, Tayfun Hilmi Akbaba1, Engin Yilmaz2, Banu Balci-Peynircioglu1
    1Medical Biology, Hacettepe University, Ankara; 2Medical Biology, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey
    Correspondence: Yeliz Z. Akkaya Ulum

    Introduction: Familial Mediterranean Fever (FMF); is an autosomal recessively inherited autoinflammatory disease. FMF is caused by the mutations in the Mediterranean Fever (MEFV) gene which encodes the pyrin protein. In many studies, pyrin has been implicated in important cellular processes like apoptosis, cytoskeleton dynamics, signal transduction, and inflammation. Recent studies have shown that epigenetic control mechanisms, particularly non-coding RNAs, may play a role in the pathogenesis of autoinflammation. microRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating host genome expression at the post-transcriptional level. Dysregulated miRNA expression patterns have been documented in a broad range of diseases including cancer, inflammatory, and autoimmune diseases.

    Objectives: Phenotypic heterogeneity seen in FMF disease indicated that FMF is not a simple monogenic disease. Therefore it has been suggested that epigenetic factors can be one of the reason for the variations.

    Methods: Previously we identified miR-20a-5p and miR-197-3p as significantly differentially expressed among healthy controls (-/-) and patients (M694V/M694V). The validation of differentially expressed miRNAs was done by quantitative reverse transcription polymerase chain reaction (qRT-PCR). miRNA target genes were determined in miRWalk, the database on predicted and validated miRNA targets. Then pathway analysis was performed in DAVID. For functional assays, SW982 (synovial fibroblast) and THP-1 (monocyte) cell lines were cultured and transfected with miR-197 mimic and scramble control. After transfection, cytokines expression levels (MEFV, IL-1b, IL-18, TNF-α, TGF-β), caspase I activity, apoptosis and cell migration rate were determined. Migration was analyzed in two ways by Transwell migration chamber and wound healing assays. For target gene studies, 3’UTR lusiferase activity assay was done.

    Results: qRT-PCR analysis confirmed the results of the miRNA profiling for 2 miRNAs. From two of them; miR-20a showed induction and the other one; miR-197 showed reduction in the homozygote group compared to controls (Akkaya-Ulum et al., 2017).

    For functional studies, after pre-miR-197 transfection, the expression levels of cytokines were decreased, cells showed less migration and caspase 1 activity. There was no effect in apoptosis. These results showed that miR-197 could have an anti-inflammatory effect by causing less migration and cytokine secretion. miR-197 was found to bind to the interleukin-1beta (IL-1β) receptor, type I (IL1R1), which is one of the key molecules of the inflammatory pathways.

    Conclusion: These findings provide evidence that miR-197 may play role in FMF pathogenesis. Therefore, this study may contribute to understand the inflammatory process seen in FMF disease, as well as to development of the new drug targets and biomarkers in addition to the existing colchicine and similar treatments.

    As miRNA-based therapeutics are promising approaches for treating autoinflammatory diseases, we are planning to continue to study on potential therapeutic usage of miR-197 in mouse model of FMF disease.

    This study was supported by The Scientific and Technological Research Council of Turkey TUBITAK 1001-SBAG Project Number: 214S106 and Hacettepe University Scientific Research Projects Coordination Unit, Thesis Support, PhD Project Number: TDK-2017-16253 and BAP Comprehensive Project Number: 013D05101005 and Turkey Rheumatology Association.

    Keywords: Familial Mediterranean Fever, inflammation, microRNA, miR-197, IL1R1.

    Reference

    Akkaya-Ulum YZ, Balci-Peynircioglu B, Karadag O, Eroglu FK, Kalyoncu U, Kiraz S, et al. Alteration of the microRNA expression profile in familial Mediterranean fever patients. Clin Exp Rheumatol. 2017 Nov-Dec;35 Suppl 108(6):90-94.

    Disclosure of Interest

    None Declared

    P1053 Comparison of FMF patients with age of onset before 20 versus 40 years and over

    Okan Aydin, Serdal Ugurlu, Huri Ozdogan
    Division of Rheumatology, Department of Internal Medicine, Cerrahpasa Medical Faculty, University of Istanbul - Cerrahpasa, Istanbul, Turkey
    Correspondence: Okan Aydin

    Introduction: Familial Mediterranean fever (FMF) isa disease withan onset before 20 years of age in 90% of the patients.However late onset FMF defined as age of onset over 40 years is being recognised more frequently.

    Objectives: To better define patients with FMF who had their first attack before age 40 and compare them with early onset patient group in Turkish population

    Methods: The files of 2180 FMF patients followed in a single center between 2008-2017 who have fulfilled Tel-Hashomer criteria, were reviewed with regard to age of onset 40 years and over (index patients, Group 1).For control purposesfiles before and after the index patients were browsed and

    first patients with an onset before age 20 years (Group 2) were included. The demographic, clinical and geneticcharacteristics are compared between these 2 subgroups.

    Results: Patients with an onset after 40 years consisted 2.7% of our FMF population. 50 of the 59 patients with an onset 40 yearsor over were re-evaluated and compared with early onset group consisting of 100 patients (Table 1).The delay in diagnosis, and disease durationwere significantly longer and number of patients with M694V homozygosity and M694V allele frequencywere significantly more frequent among group 2. In general, phenotypes of both onset groups were similar, the only significant differences being the frequency of fever and myositis which were less common amonggroup 1. Also response to colchicine was more pronouncedingroup 1. One other interesting observation was the low incidence ofamyloidosis in a group with such a significant delay in diagnosis and thus treatment.

    Conclusion: FMF should be included among the differential diagnosis of patients over 40 years of age with recurrent autoinflammatory manifestations. Less than 3% of FMF patients experience their first attacks after 40 years of age. The frequency of M694V is significantly less in the late onset group, pointing out a milder disease.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1053). Demographic, clinical and genetic features of the study groups

    P1054 What history of appendectomy will tell us about the course of familial Mediterranean fever in adulthood?

    Erdal Bodakçi1, Nazife S. A. Bilge1, Nuh Ataş2, Berkan Armağan3, Hasan Satış2, Alper Sarı3, Hakan Babaoğlu2, Gözde K. Yardımcı3, Reyhan B. Salman2, Levent Kılıç3, Mehmet A. Öztürk2, Berna Göker2, Seminur Haznedaroğlu2, Umut Kalyoncu3, Abdurrahman Tufan2, Timuçin Kaşifoğlu1
    1Deparment of Internal Medicine, Division of Rheumatology, Eskişehir Osmangazi University Faculty of Medicine, Eskişehir; 2Deparment of Internal Medicine, Division of Rheumatology, Gazi University Faculty of Medicine; 3Deparment of Internal Medicine, Division of Rheumatology, Hacettepe University Faculty of Medicine, Ankara, Turkey
    Correspondence: Erdal Bodakçi

    Introduction: Peritonitis attacks of FMF usually requires emergency medical admissions and it’s hard to distinguish a typical attack from surgical causes of acute abdomen. Therefore, unrevealing abdominal surgery history, particularly appendectomy, is very common in patients with FMF. However, history of appendectomy might also give some clues about the disease course of FMF.

    Objectives: to determine whether history of appendectomy is associated with disease course of FMF in adulthood.

    Methods: All patients recruited from FMF in Central Anatolia (FiCA) cohort, comprising 970 (mean age 35.3±12 years, 61.5%female) adult subjects. All patients fulfilled Tel Hashomer criteria. Demographic data, FMF disease characteristics, co-morbid conditions, past medical history including surgeries, disease complications were meticulously questioned and laboratory features and genotype data (if available) were recruited from patient files. Disease severity and FMF associated damage were assessed with International Severity Scoring System (ISSF) for FMF and Autoinflammatory Disease Damage Index (ADDI), respectively.

    Results: Appendectomy history was evident in 240 (24.7%) subjects.Peritonitis (4.4±6.7 vs 2.9±4.3 attacks/per year, p<0.001) and pleuritis (3.9±5.2 vs 2.8±4.4 attacks/per year, p=0.03) attacks were more frequent in appendectomy performed (AP) group than appendix intact (AI) group. However, there were no difference between AP and AI groups for the attack frequencies of musculoskeletal and skin components. Considering all types of attacks, AP group had more attacks (6.4±8.2 vs 4.3±6.6 attacks, p<0.001), despite they had used more higher doses of colchicine (1.43±0.6 mg/day vs 1.27±0.5 mg/day, p=0.002). ISSF scores were also higher in AP group 3.13±1.68 vs 2.73±1.48, p=0.001). Colchicine nonresponse were more prevalent in AP group as well (15.1% vs 6.7%, pearson X2=20.2, p<0.001). However, ADDI damage scores were similar between AP and AI groups.

    Conclusion: Appendectomy history is common in FMF patients and associated with frequent serositis attacks in adulthood. These patients require more colchicine doses with a lower response rate. Hence, history of appendectomy would be a worthy clue for the management of FMF patients.

    Disclosure of Interest

    None Declared

    P1055 Complement endorse the pathogenesis in autoinflammation

    Juergen Brunner1, Wilfried Posch2, Doris Wilflingseder2
    1Pediatrics; 2Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria
    Correspondence: Juergen Brunner

    Introduction: The complement system represents a major part of the innate immune system, consisting of more than 30 different proteins in plasma and on cell surfaces and can be activated through three different pathways.Inflammasomes are also part of the innate immune system.A group of disorders in inflammasomes have been associated with autoinflammatory diseases (AIDs). Familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological, cutaneous and articular syndrome/neonatal onset multisystem inflammatory disease (CINCA/NOMID) were originally described as three distinct diseases. After the identification of their common genetic origin, i.e. mutations in the NLRP3 gene on chromosome 1q44, they are perceived as a continuum of one disease entity and labelled cryopyrin-associated periodic syndromes (CAPS).

    Objectives: Aim of this preliminary study in a patient with MWS was to find a correlation between the complement system and a disorder of autoinflammation.

    Methods: PBMCs (peripheral blood mononuclear cells) were isolated from blood of a healthy donor and of an individual suffering from MWS by density gradient centrifugation using a Ficoll Paque Premium (GE Healthcare). After washing, PBMCs were incubated with anti-human CD14 Magnetic Beads (BD) to obtain CD14+ monocytes. These were stimulated by addition of cytokines (IL-4 and GM-CSF) for five days to generate immature moDCs (iDCs), which were used for cytokine ELISAs and flow cytometric analyses. IL-6 and IL-1β cytokine ELISAs were performed according to the manufacturer (Biolegend) following stimulation of cells using either LPS or differentially complement opsonized HIV-1. Phenotypical characterization of pathogen-exposed DCs was performed by analyzing characteristic surface markers (CD11c, DC-SIGN, CD86) by multi-color flow cytometry.

    Results: IL-1β production of iDCs is higher in the patients cells than in the cells of the healthy donor. However, the most significant difference was shown in complement opsonized iDCs. DC-SIGN is higher expressed in complement opsonized iDCs in patient cells compared to cells of a healthy donor (37,12% v28,64%). DC-SIGN is also higher expressed in the iDCs of the MWS patient after stimulation with LPS.

    Conclusion: The complement system may play an important role in the development of an proinflammatory milieu in patients with disorders of autoinflammation. The phenomenon shown in a patient with MWS has to be reproduced in more MWS patients a well as in patients with other disorder of autoinflammation.

    Disclosure of Interest

    None Declared

    P1056 Generation of ADA2 genetic knockout in a myeloid cell line using CRISPR/CAS9 genome editing: an in vitro cell line model to study DADA2

    Marina S. Casimir, Ying Hong, Paul Brogan, Despina Eleftheriou
    Infection, Inflammation and Rheumatology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
    Correspondence: Marina S. Casimir

    Introduction: Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive genetic disease causing systemic inflammation and vasculitis resembling polyarteritis nodosa, caused by loss of function mutations in the ADA2 gene. We are curently exploring an alternative treatment strategy of curing DADA2 using gene therapy. An important first step for this line of work was to develop a cell line model, with abrogation of ADA2 protein and enzyme expression and to determine whether the immunophenotype and characteristics of this cell line mimic the phenotype of primary cells derived from DADA2 patients.

    Objectives: To develop an ADA2 knockout (KO) monocyte cell line (THP-1) derived using Clustered Regularly-Interspersed Small Palindromic Repeats (CRISPR)/CAS9 and to confirm that this KO has comparable immunophenotype to monocytes from DADA2 patients (reduced ADA2 expression; M1/M2 skewing and pro-inflammatory cytokine production).

    Methods: A plasmid delivered CRISPR/CAS9 system was used to generate the ADA2 KO THP-1 cell line. ADA2 enzyme activity was assessed using a modified commercially available assay and ADA2 protein expression using immunoblotting. Cells were treated with PMA to induce differentiation into macrophages before polarisation into M1 through LPS/ INF-γ stimulation; and M2 polarisation through IL-4, IL-10 and IL-13 stimulation. Immunophenotyping was assessed using gene expression analysis by qPCR. We performed similar experiments in monocyte-derived macrophages (MDM) from 4 DADA2 patients using the same protocol as for the cell line. Cytokine production was evaluated using MSD electrochemiluminescence.

    Results: We first confirmed effective knockout of ADA2 at the genetic and protein level and a complete loss of ADA2 enzyme activity in culture supernatants when compared to scramble control THP-1 cells (p<0.01).M1 polarised ADA2 KO THP1 cells exhibited increased activation of pro inflammatory markers such as TNF-α (p<0.001), CXCL-10 (p<0.001), STAT-1 (p<0.01) and IL-1β (p<0.001) when compared to control THP-1 cells. The M1/M2 ratio was reversed in the ADA2 KO THP-1 cell line compared to the wild type THP-1 cells. In MDM cells from DADA2 patients, we observed similar induction of the pro-inflammatory pathway as indicated by increased TNF-α and IL-1β gene expression. Release of TNF-α in culture supernatants from M1 polarised cells was also enhanced for both the ADA2 KO THP-1 cell line and in cells derived from DADA2 patients.

    Conclusion: We have generated an effective ADA2 genetic KO myeloid cell line using a CRISPR/Cas9 system and confirmed that these cells have a complete loss of ADA2 enzyme activity and a comparable immunophenotype to monocytes from DADA2 patients. This in vitro system can now be used to examine the possibility to reverse the defects associated with DADA2 using gene therapy strategies.

    Disclosure of Interest

    None Declared

    P1057 Promising blood test for diagnosis and treatment options in patients with suspected chronic auto-inflammatory syndromes

    Anne-Laure N. Chetaille1, Nathalie Pagé2, Marie-Pier Longchamps2, Louis Bessette2, Laetitia Michou2, Paul Fortin2, Philippe Tessier2, Martin Pelletier2
    1Rhumatologie Adulte et Pédiatrique; 2CHU de Québec-Université Laval, Québec, Canada
    Correspondence: Anne-Laure N. Chetaille

    Introduction: Diagnosis and treatments of Auto-Inflammatory Syndromes (AIS) patients are challenging as clinical symptoms are non-specific especially compared with Systemic Autoimmune Rheumatic Diseases (SARD) and detection rate of genetic mutations in patients with high suspicion for AIS is low. Even if a mutation is found, genotypes don’t correlate with phenotypes neither with the response to treatments. As a consequence, patients can be misdiagnosed, receive inappropriate treatment, leading to severe complications and substantial socio-economic costs.

    Objectives: The cytokines abnormally secreted by each individual are unknown. We hypothesized that the secretion of cytokines by peripheral blood mononuclear cells (PBMC) could be an indicator of the disease and could guide the treatment to the most suitable anti-cytokine. Quantification of the secretome of leukocytes may guide diagnosis and treatment of AIS patients.

    Methods: Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls, suspected AIS patients, and rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients from the CHU de Québec SARD Biobank Repository Database (SBRD). PBMCs were stimulated with well-known immune activators to trigger inflammasome activation, and cytokine levels were analyzed in the plasma and the supernatants by multiplex assays.

    Results: Plasma cytokine levels were similar and did not allow to discriminate between suspected AIS patients, SARD patients or healthy volunteers. PBMCs, on the contrary, had a distinct profile of cytokine secretion between groups. PBMCs from AIS patients spontaneously secreted more IL-1a, IL‑12 and IL-18 than cells from SARD patients or healthy control subjects, as well as IFNg in response to NLRP3, AIM2 and pyrin inflammasome activation. PBMCs from some suspected AIS patients secreted excessive IL-1a or IL-1b in the absence of the antagonist IL‑1RA, suggesting blockers of IL-1 could be used as biotherapeutic approach. PBMCs from other suspected AIS patients secreted high levels of IFNg, IL-12 and IL‑18, pointing to the usefulness of treatments such as Janus kinase small molecule inhibitors, and may be the anti-IL‑12/IL-23 p40 antagonist and/or the novel anti-IL-18 in clinical development.

    Conclusion: This study demonstrates that analysis of leukocytes’ secretome is reliably more sensitive than serum to reveal cytokine signatures and to predict biologic treatment options in patients with suspected chronic AIS. Quantification of leukocytes’ secretome allows personalized medicine by guiding diagnosis and treatment options of AIS patients.

    Disclosure of Interest

    None Declared

    P1058 Specific dysbiosis associated with familial Mediterranean fever complicated or not with AA amyloidosis

    Samuel Deshayes1, Soraya Fellahi2, Jean-Philippe Bastard2, Jean-Marie Launay3, Jacques Callebert3, Thibault Fraisse4, David Buob5, Jean-Jacques Boffa6, Irina Giurgea7, Charlotte Dupont8, Sarah Jegou9, Marjolène Straube9, Alexandre Karras10, Achille Aouba1, Gilles Grateau4, Harry Sokol11, Sophie Georgin-Lavialle4 and AA Amyloidosis Study Group
    1Internal Medicine, CHU Côte de Nacre, Caen; 2Biochemistry, Hôpital Tenon; 3Biochemistry, Hôpital Lariboisière; 4Internal Medicine; 5Anatomopathology; 6Nephrology, Hôpital Tenon; 7Medical Genetics, Hôpital Trousseau; 8Reproduction Biology, Hôpital Tenon; 9AP-HP Laboratoire des Biomolécules (LBM), UPMC Université Paris 06; 10Nephrology, Hôpital Européen Georges Pompidou; 11Gastroenterology, Hôpital Saint-Antoine, Paris, France
    Correspondence: Samuel Deshayes

    Introduction: Familial Mediterranean fever (FMF) can be complicated by inflammatory (AA) amyloidosis, but the reason why only some patients will develop amyloidosis is not completely understood.

    Objectives: To assess gut microbiota composition and inflammatory markers in FMF patients complicated or not by AA amyloidosis.

    Methods: We included 34 FMF patients without AA amyloidosis, 7 FMF patients with AA amyloidosis, 19 patients with AA amyloidosis from another origin, and 26 controls. The gut microbiota was studied by 16S ribosomal ribonucleic acid gene sequencing on an Illumina MiSeq platform. Associations between bacterial taxa and clinical phenotype were evaluated using the multivariate association with linear models (MaAsLin) statistical method. Blood dosages of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were carried out by enzyme-linked immunosorbent assay.

    Results: Compared to healthy subjects, significant decreases in α-diversity were noted in FMF patients without amyloidosis and in patients with non-FMF-related AA amyloidosis. β-diversity analysis also showed significant differences between healthy controls and two same groups. After multivariate association testing with the MaAsLin statistical method to control for the effects of potential confounding factors (such as age, gender, body mass index and treatments), several operational taxonomic units belonging to the Clostridiales order were associated with FMF. Moreover, 2 operational taxonomic units belonging to the Clostridiales order were overrepresented in FMF-related AA amyloidosis compared to FMF patients without AA amyloidosis.

    All studied groups had higher blood levels of IL-1β, IL-6 and tumor necrosis factor-α than controls.

    Conclusion: FMF was associated with a gut microbiota dysbiosis characterized by a decreased α-diversity and a significant alteration in composition. Although these results are descriptive, they suggest that the gut microbiota might be involved in the clinical expression of FMF. In FMF patients, amyloidosis was independently associated with a specific alteration in the microbiota composition, suggesting that the gut microbiota may play a role in AA amyloidosis pathogenesis. These data need to be further consolidated in mechanistic and interventional studies.

    Disclosure of Interest

    None Declared

    P1059 Proinflammatory cytokines induced by PBMCS from a patient with a NLRP1 variant showing severe corneal intraepithelial dyskeratosis

    Troels Herlin1, Sofie E. Jørgensen2, Christian Høst1, Mette Christiansen3, Dorte A. Larsen4, Trine H. Mogensen5
    1Pediatrics, Aarhus University Hospital; 2Biomedicine, Aarhus University; 3Clinical Immunology; 4Ophthalmology; 5Infectious diseases, Aarhus University Hospital, Aarhus, Denmark
    Correspondence: Troels Herlin

    Introduction: Corneal intraepithelial dyskeratosis is an extremely rare autosomal dominant inherited disorder with the classical form known as hereditary benign intraepithelial dyskeratosis (HBID). Variants of the nucleotide-binding leucine-rich repeat containing purine domain 1, NLRP1gene, have recently been associated with autoinflammatory disorders with arthritis, vitiligo and dyskeratosis, including HBID.

    Objectives: In a boy with severe HBID, with a variant p.A59P in the autoinhibitory PYD domain of NLRP1, to examine the level of NF-kB activation and proinflammatory cytokines in stimulated PBMCs.

    Methods: \5-year-old boy with severe inflammation of the gingival mucosa with premature loss of several teeth, painful corneal and conjunctival inflammation of both eyes leading to marked photophobia, corneal opacification and loss of vision. He had mild eczema, but no dyskeratosis of the skin. He is the only child of non-consanguineous Danish parents. Grandfather’s brother and father’s grandfather both had hyperkeratotic dermatosis and swollen gingival mucosa but no eye inflammation.

    NLRP1Sanger sequencing was performed. Peripheral blood mononuclear cells (PBMCs) from patient and 3 healthy controls were stimulated with NF-kB inducers (TNFa and LPS or left untreated. Phosphorylated IkBa, as a measure of NF-kB activation, was measured by Luminex technology. PBMCs were stimulated with different ligands (TNFa and LPS and muramyl dipeptide (MDP) or left untreated for 16 hrs. Proinflammatory cytokines (IL-1b, IL-6, IL-18, and TNFa) were measured in the supernatants using Mesoscale U-plex multiplex assays.

    Results: Sanger sequencing identified a heterozygous variant (c.175G>C, p.A59P) in the autoinhibitory PYD domain of NLRP1. The variant has a high CADD score of 24.1 predicting a high likelihood of deleteriousness and is not present in the gnomAD database. The sequencing revealed other variants (p.L155H, p.V1063M and p.M1188V) with high minor allele frequencies, most likely benign. Family analysis showed that A59P and L155H were carried by the father and the grandfather’s brother but none by the mother.

    Patient PBMCs demonstrated significantly increased levels of Ser32/36 phosphorylated IkBa in response to TNFa and LPS stimulation compared to controls, indicating increased NF-kB activation. In response to especially MDP, but also TNFa stimulation, patient PBMCs expressed extremely high levels of IL-1b and IL-6 and increased IL-18 levels in unstimulated PBMCs compared to controls. Clinically and biochemically the patient responded well to monthly treatment with subcutaneous canakinumab (5 mg/kg) with rapid improvement of the gingivitis, and ophthalmologically administration of diluted anakinra (2.5% solution) as 1 eye-droplet three times per day had a dramatic effect on ocular pain and inflammation.

    Conclusion: We here report A59P NLRP1 as a novel defect causing inflammasome hyperactivation/autoinflammatory disease and a clinical picture including severe inflammation of the gingival mucosa and keratitis in a 5-year-old boy with a family history suggesting HBID. Patient PBMCs exhibited increased NF-kB activation and elevated levels of IL-1 and IL-6 in response to proinflammatory stimuli. Thus, our results enabled us to select relevant targeted therapy deploying IL-1binhibition with systemic canakinumab and topical anakinra.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1060 Gain-of-function mutation of NOD2 impairs its ligand specific immune responses in BLAU syndrome patient-derived IPS cells

    Naotomo Kambe1, Nhung T. M. Ly1, Megumu K. Saito2, Hiroyuki Okamoto1
    1Dermatology, Kansai Medical University, Hirakata, Osaka; 2Clinical Application, CiRA, Kyoto University, Kyoto, Japan
    Correspondence: Naotomo Kambe

    Introduction: NOD2 is crucial for innate immune response and mainly expressed in hematopoietic lineage cells, especially in monocytic cells. On the recognition of its ligand, muramyl dipeptide (MDP), NOD2 leads to activation of NF-κB pathway, causing upregulation of pro-inflammatory cytokines.

    Objectives: Mutations of NOD2 have been associated with Blau syndrome, but the details regarding mechanisms associated mutant NOD2 leads to granuloma formation are still unclear. By using iPS cells derived from the patients, we tried to reveal the molecular mechanism of Blau syndrome.

    Methods: The iPS cells with R334W mutation in NOD2 have been established from a Blau patient and we corrected the mutation of the iPS cells into wild type (WT) by using a CRISPR-Cas9 system. These isogenic iPS cells were differentiated into monocytic cell lineages, then transfected with the lentiviral vectors and maintained in the StemPro-34 medium with M-CSF and GM-CSF.

    Results: IFNγ induced the same upregulation of NOD2 in iPS-derived monocytes with WT and those with R334W. Without MDP stimulation, pro-inflammatory cytokine production was only found in those with mutant NOD2, suggesting that Blau-associated NOD2 is gain-of-function mutation. However, after stimulating with MDP, the R334W mutant cells showed NF-κB pathway activation and cytokine secretions less than WT cells even with or without IFNγ treatment. On the other hand, both the cell groups showed the comparative immune response to TNFα and LPS treatment, unrelated to NOD2 mutation.

    Conclusion: The response to MDP by mutant NOD2 was selectively impaired in Blau syndrome, that may incompletely lead to neutrophilic inflammation but compensatively induce granuloma formation in host defense.

    Disclosure of Interest

    None Declared

    P1061 The role of DNA methylation for disease severity in patients with heterozygous mutations in the Mediterranean fever gene MEFV

    Julie Krainer1, Walter Pulverer1, Dirk Foell2, Seza Özen3, Andreas Weinhäusel1
    1AIT - Austrian Institute Of Technology, Vienna, Austria; 2Pediatric Rheumatology & Immunology, University Children's Hospital, Muenster, Germany; 3Department of Pediatric Rheumatology, Hacettepe University, Ankara, Turkey
    Correspondence: Julie Krainer

    Introduction: Familial Mediterranean Fever (FMF) is the most common hereditary SAID, with a high prevalence in patients of eastern Mediterranean decent. It is known as an autosomal dominant disease with mutations in the MEFV gene that encodes for Pyrin, an important innate immunity regulator. However, some heterozygous individuals also show an FMF phenotype, which leads to the assumption that other modifying factors lead to a manifestation of the phenotype1. In recent years, DNA methylation has demonstrated suitable as biomarker and its potential for disease diagnosis by several studies. DNA methylation plays an important epigenetic regulatory effect on gene expression, and aberrances can result in pathological disease states.

    Objectives: The main goal of this study was to evaluate the DNA methylation in patients carrying heterozygous mutations in the MEFV gene but show different phenotypes. We hypothesis that alterations in DNA methylation can add important and valuable information about the disease etiopathogenesis.

    Methods: The study included 32 patients, 12 of the patients show a typical FMF phenotype and SNP analysis showed a heterozygous mutation, 9 patients showed similar heterozygous mutations but lack FMF characteristics and are healthy. We also included 12 control samples without any mutation in the MEFV gene.

    We performed a genome wide DNA methylation analysis using Illumina’s EPIC BeadArray, interrogating over 850.000 CpG sites at single C resolution covering the first exon of >84% of all Genes2. The results were processed and normalized using the R package Champ3 and group comparisons were performed with limma4.

    Results: In total four different group comparisons were calculated including Heterozygous Healthy vs. Heterozygous Disease, Heterozygous Healthy vs. Control, Heterozygous Disease vs. Control and Heterozygous (Disease + Healthy) vs. Control. Each comparison revealed over 30000 significant cpgs (p<0.05) where between 28 and 75 cpgs showed at least 15% differences in mean methylation between the groups. Eighty of the significant cpgs were present in every group comparison covering 62 unique genes. During hierarchical clustering the three clusters separated visibly, where two Heterozygous Healthy patients cluster within the control samples. A group comparison between the two Heterozygous groups revealed 71 differentially methylated sites (p<0.05, difference ≥ 15%), able to separate the two groups.

    Conclusion: During our experiments we were able to detect differentially methylated sites separating Heterozygous Healthy and Heterozygous Disease patients. We hypothesize that the identified CpG sites can contribute to provide information to the etiopathogenesis of FMF in general.

    REFERENCES

    [1] Sönmez et al.. Familial Mediterranean fever. Current perspectives. In: Journal of Inflammation Research 9; 2016. S. 13–20. DOI: 10.2147/JIR.S91352.

    [2] Pidsley et al. “Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling.” Genome Biology 17 (1), S. 208; 2016. DOI: 10.1186/s13059-016-1066-1.

    [3] Morris et al. “Champ: 450k chip analysis methylation pipeline.” Bioinformatics, 30(3), 428-30; 2014. DOI: 10.1093/bioinformatics/btt684.

    [4] Ritchie ME et al. “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research, 43(7), e47; 2015. DOI: 10.1093/nar/gkv007

    Disclosure of Interest

    None Declared

    P1062 Differential kinetic of actin polymerization in neutrophil from patients with familial Mediterranean fever

    David Poghosyan1, Anush Martirosyan1, Nune Mkrtchyan2,3, Sona Margaryan1, Susanna Ghonyan1, Gayane Amaryan2,3, Gayane Manukyan1
    1Laboratory of Molecular and Cellular Immunology, Institute of Molecular Biology; 2Arabkir Medical Centre, Institute of Child and Adolescent Health, National Pediatric Centre for Familial Mediterranean Fever; 3Yerevan State Medical University, Yerevan, Armenia
    Correspondence: Gayane Manukyan

    Introduction: Seemingly unprovoked trafficking of neutrophils to the serosal/synovial membranes is a key event in acute inflammatory attacks of familial Mediterranean fever (FMF). Migratory events behind abnormal trafficking of the cells remain largely unknown.

    Objectives: The aim of the present study is to analyze kinetic of actin polymerization and migration rate of neutrophils from colchicine naïve and colchicine receiving FMF patients.

    Methods: FMF patients in acute attack (colchicine naïve A-FMF), FMF patients in remission period (receiving colchicine, R-FMF), and healthy controls (HD) were enrolled in the study. We have measured the amounts of F-actin in untreated and pre-treated with colchicine neutrophils induced by fMLP at 5s, 15s, 30s, 60s, 120s, and 180s time points in vitro. Actin polymerization was investigated by flow cytometry using FITC-Phalloidin. Neutrophils migration to fMLP was assessed using Transwell cell migration assay.

    Results: Neutrophils from A-FMF displayed higher migration rate compared to R-FMF and HD (P< 0.05). R-FMF patients, who were receiving colchicine, displayed the lowest actin polymerization activity in neutrophils at all-time points. The cells from FMF patients in A-FMF stimulated with fMLP displayed a maximal F-actin polymerization earlier (at 5s) than in R-FMF (at 30s) and HD (at 15s) (P< 0.05). In opposite, plateau levels of F-actin content were reached earlier in HD neutrophils (after 1 min), while plateau levels in A-FMF and R-FMF cells were reached later (after 2 min). Colchicine-pretreated cells in all studied groups reached maximal F-actin polymerization later than in untreated with colchicine cells, namely at 15s in A-FMF and 30s in R-FMF and HD groups.

    Conclusion: We have demonstrated actin dysfunctions in neutrophils from FMF patients which might cause defects in neutrophil functioning. The results suggest that colchicine may affect actin polymerization in the cells due to unknown mechanism.

    Disclosure of Interest

    None Declared

    P1063 CARD15 mutations in an Indian cohort of Blau syndrome

    Amit Rawat1, Deepti Suri1, Rajni Kumrah1, Sagar Bhattad2, Sandesh Guleria1, Vignesh Pandiarajan1, Ankur Jindal1, Anju Gupta1, Isabelle Ceccherini3, Marco Gattarno4, Surjit Singh1
    1Pediatrics, Postgraduate Institute of Medical Education and Research, Chandigarh; 2Pediatric Immunology and Rheumatology, Aster CMI Hospital, Bengaluru, India; 3Genetica Medica; 4U.O.C. Pediatria II - Reumatologia, Istituto Giannina Gaslini, Genoa, Italy, Genoa, Italy
    Correspondence: Amit Rawat

    Introduction: Blau syndrome is an autosomal dominant disorder resulting from genetic variants in the CARD15 gene previously known as NOD2. It is characterized by a clinical triad of arthritis, uveitis and skin rash. Classic features also include skin lesion with non-caseating granulomas, boggy synovitis or tenosynovitis and camptodactyly. CARD15 gene comprises eleven exons encoding for a 1044 amino acid protein. The protein has N-terminal caspase recruitment domains (CARDs) linked to a nucleotide binding domain (NBD) and C- terminal leucine-rich repeats (LRRs).

    Objectives: The objective of the study was to determine the underlying genetic variants in our cohort of patients with Blau syndrome and to establish a genotype-phenotype correlation if any.

    Methods: Genetic variants in CARD15 were determined in 11 of our patients with Blau syndrome. Targeted next- generation sequencing was employed to detect these variants in six patients at the Istituto Giannina Gaslini, Genoa, Italy. In rest of the 5 patients, conventional Sanger sequencing was used. Since most of the variants have been detected in Exon 4, the exon4 was amplified by polymerase chain reaction. Four pairs of specific primers described in Resource of Asian Primary Immunodeficiency Diseases Database were used for amplification of the Exon 4 and the PCR amplifications generated were sequenced using conventional Sanger sequencing.

    Results: Single nucleotide substitutions resulting in missense variants were detected in all of the eleven patients. Nine of the eleven patients had a recurrent, previously reported missense variant in Exon 4 which encodes for the nucleotide binding domain (NBD). This variant resulting from the substitution of Thymine for cytosine at c.1000; c.1000C>T causes the replacement of Arginine with Tryptophan at codon 344; p.R344W. This variant has been reported in the Infevers database. Three of the nine patients with this variant were related, an affected mother with her son and daughter. Rest of the six were from unrelated families. However, six of the nine patients were from the north Indian state of Haryana, and one was from the adjoining northern state of Punjab. The remaining two patients were from the eastern part of India. Two patients had another missense variant in the Exon 4; p.G299D with replacement of Glycine with Aspartic acid at codon 299. This variant has not been reported in the Infevers database or the Human Gene Mutation Database (HGMD), but it has been reported in the Exome Aggregation Consortium (ExAC).

    Conclusion: The detection of a similar previously reported missense variant in 9 of the 11 patients might denote a founder mutation especially given the fact that seven of the nine patients with the p.R344W variant were from the same geographical locale in North India. This recurrent variant also provides an opportunity for development of an amplification-refractory mutation system for its detection without resorting to sequencing.

    Disclosure of Interest

    None Declared

    P1064 Differential activation of the Pyrin inflammasome in monocytes and macrophages predicts the pathological significance of MEFV variants in familial Mediterranean fever (FMF) patients

    Takeshi Shiba1, Takayuki Tanaka1, Hiroaki Ida2, Misa Watanabe3, Haruna Nakaseko4, Mitsujiro Osawa5, Hirofumi Shibata1, Kazushi Izawa1, Takahiro Yasumi1, Yuri Kawasaki5, Megumu K. Saito5, Junko Takita1, Toshio Heike6, Ryuta Nishikomori1
    1Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto; 2Department of Respiratory, Neurology, and Rheumatology, Kurume University School of Medicine, Fukuoka; 3Department of Pediatrics, Toho University School of Medicine, Tokyo; 4Department of Infection and Immunology, Aichi Children’s Health and Medical Center, Aichi; 5Department for Clinical Application, Center for iPS cell Research and Application, Kyoto University, Kyoto; 6Department of Pediatrics, Hyogo Prefectural Amagasaki General Medical Center, Hyogo, Japan
    Correspondence: Takeshi Shiba

    Introduction: While numerous MEFV sequence variants have been reported, the impact of these variants on pyrin inflammasome functions remains unknown.

    Objectives: To determine the effect of MEFV variants on IL-1β production by monocytes and monocyte-derived macrophages from Familial Mediterranean Fever (FMF) patients and to extend this approach to the functional evaluation of rare MEFV variants.

    Methods: Freshly isolated monocytes and monocyte-derived macrophages from patients with typical FMF and healthy donors were analyzed for IL-1β secretion in response to the pyrin inflammasome activator Clostridium difficile toxin A (TcdA). Induced pluripotent stem cell (iPSC) clones derived from FMF patients and healthy donors were differentiated into macrophages and analyzed for pyrin inflammasome activation. Rare MEFV variants were expressed in iPSC-derived macrophages.

    Results: IL-1β secretion by FMF monocytes in response to TcdA was comparable to that of healthy donors, and colchicine inhibited IL-1β secretion from healthy donors, but not from FMF monocytes. FMF macrophages secreted significantly higher levels of IL-1β than healthy donor macrophages, and their IL-1β secretion could be inhibited with colchicine. The characteristics observed in monocyte-derived macrophages were recapitulated in iPSC-derived macrophages. Finally, iPSC-derived macrophages transgenically expressing the N679H variant were functionally similar to those expressing the pathogenic MEFV variant M694I, while cells expressing the T577N variant had a similar phenotype as control cells.

    Conclusion: While FMF monocytes carrying the typical MEFV mutations were distinct from healthy monocytes in their unresponsiveness to colchicine, FMF macrophages showed hyperactivation of the pyrin inflammasome and were sensitive to colchicine inhibition. Our novel approach may be useful for identifying MEFV variants that cause the typical FMF phenotype according to their capacity to induce pyrin inflammasome activation.

    Disclosure of Interest

    None Declared

    P1065 Cross-talk between type I IFN pathway and TLR sensing in DNase2 mutated fibroblasts

    Alessandra Tesser1, Elisa Piscianz2, Valentina Boz2, Giulia Piperno3, Federica Benvenuti3, Alberto Tommasini1
    1IRCCS Burlo Garofolo, Trieste, Italy; 2University of Trieste, Italy; 3ICGEB, Trieste, Italy
    Correspondence: Alessandra Tesser

    Introduction: Cytoplasmic sensing of nucleic acids via cGAS-TBK1 pathway leads to type I interferon (IFN) production. DNase2 deficiency impairs DNA digestion in phagosomes resulting in cGAS-dependent IFN hyper-production (1). However, other sensors like TLRs can stimulate this pathway, as recently shown in animal models (2).

    Objectives: To investigate the interaction between cGAS and TLRs stimulation in DNAse2-deficient fibroblasts.

    Methods: Fibroblasts from a subject with DNase2 deficiency and from healthy controls were treated with different Multiplicity Of Infection (MOI) of E. coli and concentration of bacterial LPS, 2’3’-cGAMP and Poly(I:C) for 1 hour. After stimulation, phosphorilated-TBK1 was measured by flow-cytometry with intracellular staining.

    Results: Fibroblasts with DNase2 deficiency responded to an overload of bacteria with a greater activation of the IFN pathway compared to healthy controls. DNase2-mutated cells showed an enhanced IFN response even after stimulation with pure E. coli derived-LPS (Tab. 1). Moreover, the combined stimulation of the cGAS-STING pathway and on TLRs (cGAMP + LPS, cGAMP + Poly(I:C)) resulted in a synergic increase of TBK1 phosphorylation compared with any stimulus alone (Tab. 1).

    Conclusion: The possibility that the dysregulated IFN pathway in DNase2-deficient fibroblasts may sensitise cells to an increased response to LPS is supported by others preliminary data (not shown) proving that intact STING is necessary for LPS-induced IFN production. Indeed, there are evidences that STING can be activated not only by 2’3’-cGAMP, but also by other molecules collecting the signals from distinct TLRs. Considering these results, we can assume that a dysfunctional IFN pathway could display a “permissive” role towards other stimuli acting on STING-TBK1 signaling. Ongoing experiments are being carried out to clarify the convergence of different TLRs stimuli on the cGAS-TBK1 axis.

    1. Rodero MP, et al. Type I interferon-mediated autoinflammation due to DNase II deficiency. Nat Commun. 2017 Dec 19;8(1):2176.

    2. Manfredo VS, et al. Translocation of a gut pathobiont drives autoimmunity in mice and humans. Science. 2018 Mar 09:Vol. 359, Issue 6380, pp. 1156-1161.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1065). TBK1 phosphorylation assessed by flow-cytometry after infection with different MOI (20, 120, 400) of E. coli, single stimulation with LPS (0.5 ug/ml), cGAMP (20 ug/ml) and Poly(I:C) (20 ug/ml), and combined stimulation of cGAS/TBK1 and TLRs pathways (cGAMP + LPS; cGAMP + Poly(I:C); Poly(I:C) + LPS)

    P1066 Auto-inflammatory diseases (SAIDS) in Western Switzerland: a descriptive study through the JIRcohorte platform

    Lorenzo Tosetti, Manel Mejbri, Aikaterini Theodoropoulou, Michael Hofer
    Pediatric Immunology Allergology Rheumatology Unit, University Hospital Lausanne and University Hospital Geneva, Lausanne and Geneva, Switzerland
    Correspondence: Lorenzo Tosetti

    Introduction: SAIDs are a large and heterogeneous group of inflammatory conditions, including monogenetic and multifactorial diseases, associated with a dysregulation of innate immune system. Early diagnosis and treatment of these conditions are essential to prevent serious complications, in particular the development of amyloidosis. Biological treatments blocking the Interleukins (IL-) 1 and 6 can lead to rapid remission and are expected to improve long-term outcome in these patients. Five of them received an indication to be treated by these medications in Switzerland; therefore, we are interested to evaluate the number of patients who may receive these treatments.

    Objectives: To describe and estimate the prevalence of FMF, MKD, TRAPS, CAPS and SoJIA in Switzerland.

    Methods: This is a monocentric, prospective and descriptive cohort study, through the JIRcohorte platform. Patients with juvenile-onset sAIDs attending the pediatric rheumatology unit of Western Switzerland in the University Hospitals of Lausanne and Geneva were enrolled at the study until August 2018. AIDs included diagnosis of Systemic onset juvenile idiopathic arthritis (SoJIA), Familial Mediterranean fever (FMF), Cryopyrin-associated periodic syndromes (CAPS), Mevalonate kinase deficiency (MKD) and Tumor necrosis factor receptor-associated periodic syndrome (TRAPS). A projection for Switzerland was made using the population data extracted from the Swiss Federal Statistical Office in 2016.

    Results: A total of 123 patients were enrolled, including 67 % females. The median age at last visit was 12.3 years[TA1] . Patients were distributed as follows: 61 patients with SoJIA, 35 with FMF, 15 with CAPS, 8 with MKD and 4 patients with TRAPS. Biologic agents (anti IL-1 or anti IL-6) were used for treatment in 55 % of our patients. The prevalence in our population is 1.4 per 100.000 for sAIDS and 1.5 per 100.000 for SoJIA. The total number of patients with sAIDs is estimated at 139 for monogenic fevers and 131 for SoJIA in Switzerland.

    Conclusion: AIDs are rare but not negligible conditions in Switzerland. A national study through the JIRcohorte database is ongoing aiming to evaluate the number of pediatric and adult patients with sAIDs in Switzerland, in order to study clinical presentation, treatments and long term complications.

    Disclosure of Interest

    None Declared

    P1067 Familial Mediterranean fever associated infertility and underlying factors

    Nuh Atas1, Berkan Armagan2, Erdal Bodakci3, Timucin Kasifoglu3, Hasan Satis1, Alper Sari2, Nazife S. Y. Bilge3, Hakan Babaoglu1, Gozde K. Yardimci2, Reyhan B. Salman1, Levent Kilic2, Mehmet A. Ozturk1, Berna Goker1, Seminur Haznedaroglu1, Umut Kalyoncu2, Abdurrahman Tufan1
    1Department of Internal Medicine, Division of Rheumatology, Gazi University Faculty of Medicine; 2Department of Internal Medicine, Division of Rheumatology, Hacettepe University Faculty of Medicine, Ankara; 3Department of Internal Medicine, Division of Rheumatology, Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey
    Correspondence: Abdurrahman Tufan

    Introduction: Familial Mediterranean Fever (FMF) is characterized by recurrent attacks of fever, serositis and arthritis, but some patients may experience long-term complications of disease such as infertility/subfertility.The published data about FMF associated infertility is still limited.

    Objectives: The aim of this study is to investigate the frequency and to determine potential factors for FMF associated infertility /subfertility.

    Methods: All patients recruited from FMF in Central Anatolia (FiCA) cohort, currently comprising 970 adult subjects. All patients fulfilled Tel Hashomer criteria and all were using colchicine for at least 1 year. Demographic data, FMF disease characteristics and genotype data (if available), disease complications, autoinflammatory damage index (ADDI), laboratory parameters and treatment features were recorded. For this study data on 582 patients (mean age 41±10.6, 65.8% female) who had been willing to have children were used.

    Results: Proportions of FMF manifestations were fever 82.3%, peritonitis 90.5%, pleuritis 48.1%, arthritis 41.1% and skin rash 24.7%. MEFV mutations were available in 454 subjects and 79.5% of subjects were harboring M694V mutation (44.5% homozygous for M694V). Among all, 53 (9.1%) patients were colchicine resistant (crFMF). Infertility was present in 64 patients (14.6 % of females and 4 % of males). Multivariate analysis showed female sex (odds ratio, 8.19; 95% confidence interval [CI95%] 2.69-24.97; p<0.05), FMF disease onset <20 years (odds ratio, 9.9; [CI95% 2.06-47.82]; p<0.05), damage accrual (ADDI score) (odds ratio, 2.02; [CI95% 1.65-2.50]; p<0.05) and colchicine nonresponse (odds ratio, 2.07; 95% confidence interval, [CI95% 1.47-2.93]; P<0.05) are the independent predictors of infertility.

    Conclusion: Damage accrual (ADDI), FMF disease onset <20 years, colchicine nonresponse and female sex were found to be the independent predictors of infertility. The value of effective therapeutic interventions must be determined to treat infertility in these patients.

    Disclosure of Interest

    None Declared

    P1068 MEFV-mutant induced pluripotent stem-cells have reduced anti-inflammatory properties

    Katharina Kessel1,2, Christoph Kessel1, Nadine Ludwig3, Toni Weinhage1, Dirk Foell1, Helmut Wittkowski1
    1Pediatric Rheumatology and Immunology, University Children’s Hospital; 2Department of Nuclear Medicine; 3Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Muenster, Germany
    Correspondence: Helmut Wittkowski

    Introduction: Familial Mediterranean Fever (FMF) is a prototypic autoinflammatory disorder associated with MEFV pyrin-encoding gene mutations, characterized by unprovoked episodes of inflammation. On a molecular level, pyrin inflammasome activation with consecutive increased IL-1beta maturation is an important mechanism of inflammation in FMF.

    Objectives: Experiments with human MEFV-mutated cells rely on FMF patient material, but patients are often already treated with an anti-inflammatory therapy, i. e. Colchicine. In order to systematically study functional consequences of MEFV pyrin-encoding gene mutations on a therapy-naïve background we aimed to generate human induced pluripotent stem cells (hiPSCs) from M694V homo- and heterozygous FMF-patients as well as healthy controls and differentiated those to functional human monocytes.

    Methods: HiPSCs were generated from MEFV-mutant patients with clinical FMF and from healthy controls by reprogramming of peripheral blood-derived (PB) CD34+ HSCs. After confirmation of MEFV genotypes, HiPSCs were differentiated to monocytes using an embryoid body (EB)-based differentiation protocol. Terminally differentiated cells were phenotyped regarding extra- and intracellular marker expression as well as cell morphology and were subjected to different functional assays. Cells were stimulated with LPS, ATP and S100A12 and cytokine secretion into culture supernatants was quantified by multiplexed bead array assay. For confirmation of observed phenotypes, ex vivo purified primary monocytes from FMF-patients were likewise stimulated and analysed.

    Results: Terminally differentiated hiPSC-derived monocytes from both FMF patients and HCs were larger in cell area (mm2) compared to primary human monocytes. On the level of 25F9, CD16a and CD68 expression hiPSC derived monocytes ranged between that detected on primary cells and in vitro differentiated primary monocyte derived macrophages. All cells expressed identical levels of CD14. Similar to our observations on primary monocytes from FMF patients and controls both CD14 and CD16 expression was lowest on p.M694V homozygous hiPSC derived monocytes. On the contrary intracellular S100A9 and A12 expression in both primary as well as hiPSC derived monocytes increased with p.M694V gene dose. Correspondingly, p.M694V homozygous hiPSC-derived monocytes revealed increased S100A8 expression at base line and already spontaneous protein release in stimulation experiments. In contrast, Il1rn expression in p.M694V mutant hiPSC-derived monocytes was low compared to HCs. When induced by IFNa, Il1rn expression was reciprocal according to p.M694V gene dose. In similar lines, p.M694V mutant hiPSC-derived monocytes revealed strongly decreased IL-10 expression on both gene and protein level compared to healthy control cells.

    Conclusion: We successfully generated mature monocytes from hiPSC derived from p.M694V hetero- and homozygous FMF-patients as well as healthy individuals. Apart from S100 proteins p.M694V mutant cells did not reveal a pronounced overexpression of inflammatory cytokines but rather demonstrated an intrinsic lack of anti-inflammatory counter-regulation on the level of IL-1Ra and IL-10 expression.

    Disclosure of Interest

    None Declared

    Monogenic autoinflammatory diseases (genetics)

    P1069 Three novel cases of late-onset cryopyrin-associated periodic syndromes due to somatic NLRP3 mosaicism

    Anna Mensa-Vilaró1, María Teresa Bosque2, Enrique Gómez de la Fuente3, Natalia Palmou4, Luis Martín-Penagos5, Concha Delgado2, Susana Plaza1, Rocío Lara1, María Carmen Antón1, Helios Martinez-Banaclocha6, Juan J. Martinez-García6, Jordi Yagüe1,7,8, Miguel Ángel González-Gay4, Pablo Pelegrin6, Juan I. Arostegui1,7,8
    1Immunology, Hospital Clinic, Barcelona; 2Rheumatology, Hospital Universitario Lozano Blesa, Zaragoza; 3Dermatology, Hospital Universitario Fundación Alcorcón, Alcorcón; 4Rheumatology; 5Nephrology, Hospital Universitario Marqués de Valdecilla, Santander; 6Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Murcia; 7Institut d’investigacions Biomèdiques August Pi i Sunyer; 8Universitat de Barcelona, Barcelona, Spain
    Correspondence: Juan I. Arostegui

    Introduction: Monoallelic gain-of-function NLRP3 mutations are the genetic cause of the dominantly-inherited cryopyrin-associated periodic syndromes (CAPS), which represent the prototypical early-onset, interleukin-1b-mediated disease (1). Post-zygotic NLRP3 mutations leading to somatic mosaicism have been shown as the disease-causing mechanism in a moderate, but increasing number of patients (2-3). Recent reports have also shown that somatic NLRP3 mosaicism may appear later in life, leading to forms of the disease that started during adulthood (4-7).

    Objectives: To describe the clinical, analytical and genetic features as well as the outcome of administered treatments in three unrelated Spanish patients with late-onset CAPS carrying somatic NLRP3 mosaicism.

    Methods: Clinical and analytical data, and outcome of treatments were collected from patients’ medical charts. Genetic studies were performed using both Sanger and amplicon-based deep sequencing.

    Results: The following table summarizes the clinical data of the three enrolled patients.

    All patients displayed increased counts of white blood cells, neutrophils and platelets, as well as increased plasma levels of acute-phase reactants.

    Genetic studies revealed in each patient a post-zygotic NLRP3 variant: p.Gln306His variant in patient 1 (minor allele frequency [MAF]: 5.1%), p.Ala352Thr variant in patient 2 (MAF: 18.7%) and p.Gln636Glu in patient 3 (MAF: 18.4%). All these NLRP3 variants were classified as pathogenic and gain-of-function variants on the basis of their absence among healthy controls and in public databases, the damaging predictions by using different bioinformatics analyses and additional ex vivo evidences of inflammasome hyperactivation.

    All three patients are being treated with anti-IL-1 drugs since several years, resulting in a successful clinical control of the disease, normalization of acute phase reactants and hematological parameters, and improvement of renal function in patient 1 who suffered from AA amyloidosis.

    Conclusion: The results here shown add additional evidences of the relevant role of somatic NLRP3 mosaicism as the disease-causing mechanism in patients with late onset, but otherwise typical CAPS. Due to the serious consequences that these data could have with regard to their treatments, this genetic mechanism should be seriously considered in the design of genetic analyses to be done in candidate patients.

    References

    1Nat Immunol 2017; 18: 832-842

    2Arthritis Rheum 2011; 63: 3625-3632

    3Ann Rheum Dis 2015; 74: 603-610

    4J Allergy Clin Immunol 2015; 135: 561-564

    5Arthritis Rheumatol 2015; 67: 2428-2436

    6Arthritis Rheumatol 2016; 68: 3035-3041

    7Front Immunol 2017; 8: 1410.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1069). See text for description

    P1070 The NLRP3 p.A441V mutation in cryopyrin-associated periodic syndrome pathogenesis: functional consequences, phenotype-genotype correlations and evidence for a founder effect

    Eman Assrawi1, Fawwaz Awad2, Claire Jumeau1, Sylvie Odent3, Veronique Despert4, G. Cam5, Aleth Perdriger6, Camille Louvrier1, Laetitia Cobret1, Bruno Copin1, Phillipe Duquesnoy1, William Piterboth1, Claire Le Jeunne7, Sophie Georgin-Lavialle8, Gilles Grateau8, Marie Legendre1, Irina Giurgea1, Sonia Athina Karabina1, Serge Amselem1
    1Sorbonne Université, Inserm UMR_S933; 2Sorbonne Université,Inserm UMR_S933, Paris; 3Centre Hospitalier Universitaire de Rennes, Service de Génétique; 4Centre Hospitalier Universitaire de Rennes, Département de Médecine de L’enfant et de L’adolescent, Rennes; 5Centre Hospitalier de Saint-Malo, Service de Néphrologie, Saint-Malo; 6Centre Hospitalier Universitaire de Rennes, Service de Rhumatologie, Rennes; 7Hôpital Cochin; 8Hôpital Tenon , Service de Médecine Interne, Paris, France
    Correspondence: Eman Assrawi

    Introduction: Cryopyrin‑associated periodic syndromes (CAPS) are a group of rare monogenic autoinflammatory diseases caused by heterozygous missense gain-of-function mutations in NLRP3 gene, coding for the NLRP3.Upon activation, NLRP3 initiates the formation of a multiprotein complex called inflammasome, which regulates proinflammatory cytokine secretion.

    Objectives: To determine the molecular and cellular basis of autoinflammatory syndromes in two unrelated families with two clinically overlapping CAPS phenotypes; a multigenerational French family with Muckle-Wells syndrome and in a patient originating from Portugal with familial cold autoinflammatory syndrome.

    Methods: Sanger sequencing of NLRP3 exon 3 was performed in all accessible patients. Microsatellites analysis was used to test the intra-familial segregation of the identified variant and to look for a founder effect. Functional analyses included the study of (i) ASC speck formation in HEK293T cells (stably expressing ASC-GFP and pro-caspase1-FLAG) transiently expressing the wild-type or mutated NLRP3 protein, (ii) IL1β secretion from transfected THP1 cells, and (iii) inflammasome-related gene expression and cytokine secretion from monocytes isolated from patients in crisis (probands from the two families), related patients out of crisis, and from controls.

    Results: The same heterozygous mutation (c.1322C>T, p.A441V) in NLRP3 exon 3, segregating with the disease within the first family, was identified in the two families which were shown to share the same mutation-associated NLRP3 haplotype. HEK293T cells transfected with the pNLRP3-A441V construct showed a significantly higher percentage of ASC specks, a common readout of inflammasome activation, as compared to cells transfected with pNLRP3-WT. Transfection of THP1 cells with pNLRP3-A441V led to significantly higher levels of secreted IL1β, a hallmark of inflammasome activation, as compared to cells transfected with pNLRP3-WT. Monocyte inflammasome-related gene expression and cytokine secretion profile was similar in patients out of crisis and in the healthy controls. However, the expression of the inflammasome-related genes in the two probands was different from that of patients without crisis and healthy controls. In addition, this expression pattern was found to be differentially regulated between the two probands, correlating with their phenotypic status.

    Conclusion: These molecular and cellular findings, which indicate a founder effect in these two families, clearly demonstrate the pathogenicity of the p.A441V missense mutation in CAPS and point to the interest of studying patients’ primary cells to assess disease activity.

    Disclosure of Interest

    None Declared

    P1071 The analysis of IL-36RA structural dynamics improves pathogenicity predictions for IL36RN variants observed in generalised pustular psoriasis

    Camilla Davan-Wetton, Niina Karoliina Hassi, Joseph Chifung Ng, Franca Fraternali, Francesca Capon
    King's College London, London, United Kingdom
    Correspondence: Francesca Capon

    Introduction: Generalised pustular psoriasis (GPP) is a potentially life-threatening autoinflammatory condition. While GPP is associated with mutations of the interleukin-36 receptor antagonist (IL-36Ra encoded by IL36RN), commonly used pathogenicity predictors cannot fully differentiate IL36RN disease alleles from polymorphisms.

    Objectives: The aim of this study was to systematically assess the impact of IL-36Ra mutations on the protein three-dimensional structure, in order to identify the computational approaches that best predict in-vitro stability and variant pathogenicity.

    Methods: The IL-36Ra 3D structure was derived by homology modelling. The effects of mutations were assessed using the mCSM and RAPSODY programs. Experimental validation was undertaken by western blot analysis of mutagenized constructs.

    Results: The results of the mCSM analysis partially correlated with those obtained by western blot, particularly for well-characterised mutations like p.Leu27Pro and p.Ser113Leu. An improved correlation with the western blot data was achieved using RAPSODY, which incorporates information on protein structural dynamics. Of note, this programme also out-performed sequence- based predictors such as CADD.

    Conclusion: Computational methods that take into account the dynamic features of the IL-36Ra protein structure show the strongest correlation with experimental data. Extending their implementation to other IL36RN variants (e.g. those that affect the interaction between IL-36Ra and its receptor) will improve our understanding of GPP mutations and enable the development of more accurate pathogenicity predictors to help disease diagnosis.

    Disclosure of Interest

    C. Davan-Wetton: None Declared, N. K. Hassi: None Declared, J. C. Ng: None Declared, F. Fraternali: None Declared, F. Capon Grant / Research Support from: Boheringer Ingelheim, Consultant for: AnaptysBio

    P1072 Spondyloenchondrodysplasia and systemic lupus erythematosus: case report of two sibling

    Bulent Kara1, Ayse Cefle2, Ozgur Kasapcopur3, Ayfer Sakarya Gunes1
    1Department of Pediatrics Division of Child Neurology; 2Department of Internal Medicine Division of Rheumatology, Kocaeli University Faculty of Medicine, Kocaeli; 3Department of Pediatric Rheumatology, Istanbul University Cerrahpasa Medical School, Istanbul, Turkey
    Correspondence: Ayse Cefle

    Introduction: Interferons (IFNs) are signalling proteins that are synthesised and released by immune host cells in response to the presence of the several pathogens. Interferonopathies comprise an expanding group of monogenic diseases characterised by disturbance of the homeostatic control of IFN-mediated immun responses. Although differing in the degree of phenotypic expression and severity, the clinical presentation of the diseases shows a considerable degree of overlap, reflecting their common pathogenetic mechanisms.

    Objectives: Spondyloenchondrodysplasia with immune dysregulation (SPENCDI) is an immuno-osseous dysplasia combining the typical metaphyseal and vertebral bone lesions of SPENCDI and neurologic involvement.

    Methods: 19 years old a male patient presented with arthralgia and rash in 2014. On physical examination short stature and erythematous skin rash were noted. Laboratory studies revealedproteinuria (740 mg/d), lymphopenia (1150/mm3), elevated ESR (51 mm/h), CRP (15 mg/l, N<5) and low C3 level. The C4 level was normal. ANA was homogenously positive, while the anti ds-DNA antibody was also positive. Anticardiolipin IgG and IgM and the lupus anticoagulant were negative. HBsAg, HCV and HIV were negative. IgG, IgA and IgM levels were normal. Skin bipsy revealed perivascular dematitis, while a kidney biopsy showed class II lupus nephritis. The patient was given methyl prednisolone, hydroxycholoroquine and azathioprine. On follow up, the proteinuriadecreased. However, he developed attacks of seizuresand ataxia. The neurologic examination, EEG and EMG, were normal. Cranial tomography revealed calcification of lentiform nucleus, corona radiata, frontal lobe white matter and dentate nuclei. In 2015, he presented with abdominal paine, vomiting and nausea. Abdominal CT revealed edema and inflammation of jejenum and dudenum.The steroid dose was increased. After a few months, the symptoms recurred. The patient was thought to have gastrointestinalinvolvement due to systemic lupus erythematosus (SLE) and rituximab was added to the treatment. Thereafter the patient did not have anysymptoms related toacute abdomen.

    Results: A direct raiographic examination of the spine revealed platyspondyly. The parents were consanguineous. His sister was under follow up due to a diagnosis of juvenil onset SLE.Intracranial calcifications, spastic paraparesis, short stature, systemic lupus erythematosus and platyspondyly suggested a diagnosis of SPENCD. Next generation sequencing of the ACP5 gene showed that the patient and his sisters were homozygous for the c.155A C/p.K52T variant. Both parents were heterozygous forthis variant.

    Conclusion: SPENCDI is a recessive genetic disease caused by homozygous or compound heterozygous mutation in the ACP5 gene on chromosome 19p13. Immune dysregulation ranges from autoimmunity to immunodeficiency. Neurologic and autoimmune manifestations have been observed in different combinations. Skeletal, nneurologic and immune phenotypes were observed between members of the same family.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1073 ADA2 deficiency without ADA2 mutations explained by a structural homozygous variation in 22Q11.1

    Alice Grossi1, Francesca Garbarino2, Roberta Caorsi3, Roberto Cusano4, Marta Rusmini1, Federica Penco3, Francesca Schena3, Rosa Anna Podda5, Paolo Uva4, Isabella Ceccherini1, Marco Gattorno3
    1UOC Genetica Medica, IRCCS Istituto Giannina Gaslini; 2Università degli Studi di Genova; 3Clinica Pediatrica Reumatologia e UOSD Centro Malattie Autoinfiammatorie-Immunodeficienze, IRCCS Istituto Giannina Gaslini, Genova; 4Centre for Advanced Studies Research and Development in Sardinia (CRS4), Science and Technology Park Polaris, Pula; 5Clinica Pediatrica,Talassemie e Malattie Rare, Ospedale Brotzu e Università degli studi di Cagliari, Cagliari, Cagliari, Italy
    Correspondence: Alice Grossi

    Introduction: Adenosine Deaminase 2 deficiency (DADA2) is an autosomal recessive autoinflammatory disease caused by loss of function mutations in the ADA2 gene, located on chromosome 22q11.1. The clinical spectrum of the disease is heterogeneous, ranging from multisystemic inflammation with vascular and multiorgan involvement to immunodeficiency and immunedysregulation.1 A small proportion of patients, despite consistent phenotype and lack of ADA2 enzymatic activity, has an incomplete or negative genotype.

    Objectives: To define the genetics underlying DADA2 in a 9 years old girl with a complete clinical phenotype and deficient enzymatic activity but without any mutation in the coding region of the ADA2 gene.

    Methods: Whole Genome Sequencing (WGS) was performed in the proband by TruSeq Nano DNA Library Prep kit (Illumina) and HiSeq 3000 Instrument (Illumina) with 150bp paired-end reads. Structural variants were identified with Manta.

    Results: At the age of 3 months, the patient began to suffer from intermittent episodes of fever and livedo reticularis. At 1-year old inflammatory symptoms became persistent and the patient developed hypertension and a severe dilatative myocarditis, responsive to high doses of steroids. Anakinra was not effective, hence steroid therapy could not be suspended for years. Hypogammaglobulinemia was also present, together with recurrent upper airways infections. At the age of 6 years, following the withdrawal of steroids, the patient presented two episodes of ischemic and hemorrhagic stroke. Anti-TNF treatment (etanercept) was then started, allowing a complete control of clinical manifestations, as well as the normalization of Ig levels, without the need of any steroid treatment. The patient has been on anti-TNF as monotherapy for 4 years and is still in complete clinical remission. Whole Genome Sequencing (WGS) has revealed a homozygous tandem duplication of the genomic region encompassing exons 3 and 4 (involving 12895 bases), generated by two breakpoints in intron 2 and intron 4 respectively. This has led to a gene transcript postulated to contain 1967 instead of 1536 nucleotides, leading to a frameshift starting from codon V252 followed by a premature stop codon after 11 aminoacid residues (p.V252Gfs11*). PCR reactions properly designed to amplify the junction fragments from both the genomic DNA and the cDNA confirmed such findings.

    Conclusion: Chromosome 22q11, where the ADA2 gene lies, is regarded as an unstable region subjected to copy number variations and other structural genomic alterations, whose occurrence is mediated by low copy repeat sequences or segmental duplications, found in association with several different Syndromes.2 The structural variation identified in the present patient represents the first demonstration of non-allelic homologous recombination (NAHR) occurred in DADA2, different from causative bi-allelic loss-of-function point mutations, typically found in the coding portion of the ADA2 gene. The identification of ADA2 enzymatic impairment in patients with clinical phenotypes consistent with DADA2 should lead to extensive molecular approaches, especially in the absence of any mutation at standard DNA sequencing.

    References

    1. Moens L., Hershfield M., Arts K., Aksentijevich I., Meyts I. “Human adenosine deaminase 2 deficiency: a multi-faceted inborn error of immunity” Immune Rev 2019; 287(1):62-72

    2. Vergés L, Vidal F, Geán E, Alemany-Schmidt A, Oliver-Bonet M, Blanco J. “An exploratory study of predisposing genetic factors for DiGeorge/velocardiofacial syndrome” Sci Rep 2017;7:40031

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1074 Evaluation of the role of SAA1, SAA2 and SAA4 genes in AA amyloidosis

    Claire Jumeau1, Camille Louvrier1, Eman Assrawi1, Fawaz Awad1, Laetitia Cobret1, Sophie Georgin-Lavialle1,2, Cécile Cazorla3, Philippe Duquesnoy1, Jean-Simon Rech3, William Piterboth1, Florence Dastot-Le Moal1, Bruno Copin1, David Buob4, Gilles Grateau1,5, Jean-François Bernaudin1,6, Marie Legendre1, Sonia-Athina Karabina1, Irina Giurgea1, Serge Amselem1
    1Sorbonne Universite, INSERM UMR_S933, Hôpital Trousseau; 2Hôpital Tenon, Service de Médecine Interne, Paris; 3Service de Médecine Interne et Infectiologie, Nouméa; 4Hôpital Tenon, Service d'Anatomie et Cytologie Pathologiques; 5Hôpital Tenon, Service de Médecine Interne, Paris; 6Hôpital Avicenne, Service de Pneumologie, Bobigny, France
    Correspondence: Claire Jumeau

    Introduction: AA amyloidosis is a rare inflammatory disease, mostly seen as a long term complication of chronic inflammation (ie obesity, gout). It is characterized by amyloid A (AA) deposits inside or outside the cells, affecting organ function. AA proteins originate from the cleavage of serum amyloid A proteins (SAA1, SAA2, SAA4) encoded by the corresponding genes. No molecular etiology for AA amyloidosis has been identified so far; however, a specific genotype (SAA1.1/SAA1.1) at the SAA1 locus has been associated with AA amyloidosis in familial Mediterranean fever (FMF) patients of Armenian origin (OR=7). Another genotype at the SAA1 locus (SAA1.3/SAA1.3) was associated with AA amyloidosis in Japanese patients with rheumatoid arthritis.

    Objectives: gout.

    This work aimed at identifying candidate genes in primary AA amyloidosis or secondary to obesity or gout.

    Methods: SAA1, SAA2 and SAA4 genes were screened by Sanger sequencing in all patients and the genotype at the SAA1 locus was determined in 35 independent patients: 18 presented with primary AA amyloidosis, 10 with AA amyloidosis in the context of obesity and 7 with AA amyloidosis in the context of gout.

    Results: We initially searched for rare variants in the 3 SAA genes in the different groups of patients. Only one rare heterozygous variant in SAA2 resulting in a missense variation p.(Ala79Val) was identified in one patient with primary AA amyloidosis.

    The characterization of the genotypes at the SAA1 locus showed a higher frequency (89 and 80%) of the SAA1.1/SAA1.1 genotype in the groups of patients with primary amyloidosis and amyloidosis secondary in the context of obesity, significantly different from its theoretical frequency in healthy controls based on gnomAD database (22%) (Fisher test: p=1,3 .10-4 and p=0.023 respectively). However in the group of patients with AA amyloidosis in the context of gout, 71% presented the SAA1.3/SAA1.3 genotype.

    Conclusion: The variation in SAA2 affects evolutionarily conserved amino-acid. This result paves the way for functional studies on AA fibril formation in order to assess its pathogenicity. The predominance of the SAA1.1/SAA1.1 genotype among patients with primary AA amyloidosis and with amyloidosis in the context of obesity suggests that this genotype could be a risk factor for AA amyloidosis.

    Disclosure of Interest

    None Declared

    P1075 An integrated data sharing and analysis application for systemic autoinflammatory diseases

    Barend P. Kant1, K J. van der Velde2, Mariska K. Slofstra2, Joost Frenkel3, Andreas Weinhäusel4, Isabelle Touitou5, Dirk Föll6, JuanI. Aróstegui7, Seza Ozen8, Marco Gattorno9, Mariëlle E. van Gijn1, Morris A. Swertz2 and the INSAID Consortium
    1Department of Genetics, University Medical Center Utrecht, Utrecht; 2Genomics Coordination Center and Department of Genetics, University of Groningen and University Medical Center Groningen, Groningen; 3Department of Pediatrics, University Medical Center Utrecht, Utrecht, Netherlands; 4Department of Health and Environment, Austrian Institute of Technology, Vienna, Austria; 5Department of Medical Genetics, Rare Diseases and Personalised Medicine, Inserm, Montpellier, France; 6Department of Pediatric Rheumatology and Immunology, University of Münster, Münster, Germany; 7Department of Immunology, Hospital Clínic of Barcelona, Barcelona, Spain; 8Department of Pediatrics, Hacettepe University, Ankara, Turkey; 9Department of Pediatric Rheumatology, IRCCS G. Gaslini Institute, Genoa, Italy
    Correspondence: Barend P. Kant

    Introduction: Systemic autoinflammatory diseases (SAID) are a group of monogenic and multifactorial conditions caused by deregulation of mechanisms controlling the innate immune response. The diagnosis of patients with SAID is difficult, which can result in delayed treatment and irreversible organ damage. Patients benefit from fast molecular diagnosis, but for most patients with a clinical picture strongly resembling autoinflammatory disease, molecular analysis will not provide diagnostic confirmation. These patients are classified as undefined SAID (uSAID).

    Objectives: The INSAID consortium aims to improve the performance of genetic diagnosis by speeding up identification of new conditions, biomarkers and causal DNA variants (in new genes). For this purpose, a central and easy to use bioinformatics platform has been set up, facilitating sharing of clinical, genetics, epigenetics, immunomics and proteomics data among the consortium partners and community-driven data analysis and interpretation.

    Methods: Clinical data were retrieved from the Eurofever registry. Other data were produced by the consortium partners. A MOLGENIS database was set up for data sharing and analysis. MOLGENIS is a user friendly open-source web-application created to collect, manage, analyze, visualize and share large and complex biomedical datasets.

    Results: The INSAID database is being used by the INSAID consortium. All data types are being captured using patient identifier codes as keys for data integration and analysis. New patients can be included and future data and data types can be uploaded and integrated. Consortium progress can be monitored and discussed using the database. The database’s interface is easy to operate and cluster analyses and statistical tests can be written and shared within the database. This enables all consortium partners with or without data analysis skills to query, sort, filter, analyze and visualize the available multi-omics data.

    Conclusion: The INSAID database is set up to facilitate data sharing and to perform multi-omics analysis. This will enable diagnosing patients with currently undefined SAID, discovery of pathways involved and further elucidating disease pathogenesis, ultimately contributing to development of new or improved treatment.

    Disclosure of Interest

    None Declared

    P1076 Comparative analysis ofreported 54 HA20 cases and 520 Japanese Behcet’s disease patients

    Yohei Kirino, Naomi Tsuchida, Yutaro Soejima, Hideaki Nakajima
    Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School of Medicine, Yokohama, Japan
    Correspondence: Yohei Kirino

    Introduction: Phenotype of haploinsufficiency A20 (HA 20) and Behcet’s disease (BD) are reported to be similar. However, the distinction between HA 20 and BD is unknown.

    Objectives: To clarify specific features of HA20 compared to BD.

    Methods: The clinical features of 54 cases of HA 20 cases were examined from the literature including our 4 cases and compared with 520 Japanese BD patients followed at our facility. All BD patients met the 1987 revised diagnostic criteria of the Japanese Bebcet's Disease Eesearch Committee. The research protocol was approved by the facility review committee of Yokohama City University School of Medicine. Statistical analysis was performed using SPSS version 22 (IBM Japan, Tokyo, Japan). Category variables were analyzed using Chi-square test or Cochran-Armitage test. Continuous variables were examined using Student's t test. A p value less than 0.05 was considered to be statistically significant.

    Results: significantly high recurrent fever (74.0%) and gastrointestinal involvement, and less eye involvement than BD. Among gastrointestinal involvement and HA20 patients, 22 (81.8 percent) 18 showed gastrointestinal ulcers including 2 cases with typical deep iliocecal ulcer seen in BD. The positive rate of HLA-B51 was lower (27.3 %) than that of BD (47.8 %), but since the majority of HA 20 has been reported by European populations whose HLA-B51 positive rate is lower than Japanese, it is uncertain whether HLA-B51 affects on HA20 phenotype or not.

    Conclusion: Comparison of HA 20 and BD cohort revealed several important features of HA 20: early onset, familial onset, recurrent fever, gastrointestinal involvement, and less eye involvement. Patients with familial recurrent fever with early onset with BD symptoms, especially gastrointestinal disorders, should consider TNFAIP3 gene screening.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1076). See text for description

    P1077 Novel missense mutation of TNFAIP3 gene in a family with A20 haploinsufficiency

    Charlotte Borocco1,2, Guillaume Sarrabay3,4, Guilaine Boursier3,4, Solene Artru5, Helene Palluy1, Isabelle Touitou3,4, Isabelle Kone-Paut1,2
    1Pediatric Rheumatology, Bicetre University Hospital; 2CeReMAIA, Le Kremlin-Bicêtre; 3Laboratory of Rare and Autoinflammatory Genetic Diseases, Montpellier University Hospital; 4CeReMAIA, Montpellier; 5Department of Pediatric Gastroenterology, Hepatology and Nutrition, Necker Enfants Malades University Hospital, Paris, France
    Correspondence: Isabelle Kone-Paut

    Introduction: A20 haploinsufficiency syndrome is a newly described autoinflammatory disease which involves TNFAIP3 gene. The heterozygous mutations of this gene lead to an impaired negative regulation of the NF-κB pathway. The phenotype is broad and still in process of characterization but encompasses mainly a Behçet phenotype with an unusual gastro intestinal involvement. Patients can encompass an autoinflammatory and an autoimmunity phenotype.

    Objectives: We report a French family with 4 affected members and a new mutation.

    Methods: Four patients over 3 generations were assessed. All the clinical and laboratory data were collected retrospectively. The genetical data were performed by new generation sequencing and Sanger sequencing.

    Results: We identified in all the 4 patients a novel heterozygous missense mutation c.[464C>T] p. Thr155Met in the ovarian tumor domain of the TNFAIP3 gene. This is the second missense mutation described. This mutation seems to have a variable penetration with a carrier patient presenting only psoriasis. The symptoms presented in this Caucasian family were: oral ulcers (n=3), recurrent fever (n=2), arthralgia (n=2), rectorrhagia (n=2), genital ulcers (n=1), abdominal pain (n=2), Gougerot-Sjögren syndrome (n=1), primary biliary cholangitis (n=1), purpuric rash (n=1), Asperger syndrome (n=1) and psoriasis (n=1). For the laboratory abnormalities, one patient presented a transient rheumatoid factor, one had permanent anti-nuclear antibodies associated to anti-SSA and anti-centromere antibodies and one had double negative T lymphocytes increased. One patient was treated by colchicine with a partial effect on oral ulcers.

    Conclusion: A20 haploinsufficiency syndrome is a new cause of autoinflammatory and autoimmunity disease. As an autosomal dominant heritance disease, the penetration can be variable. We confirm the possibility of a second missense mutation in TNFAIP3. Futher funtional studies will be required to confirm the mecanism of pathogenecity in this family.

    Disclosure of Interest

    None Declared

    P1078 Development and clinical application of a targeted next generation sequencing gene panel for monogenic autoinflammatory diseases of the CNS

    Dara McCreary1, Ebun O. Omoyinmi1, Ying Hong1, Ciara Mulhern1, Charalampia Papadopoulou2, Muthana Al Obaidi2, Kshitij Mankad3, Kimberly Gilmour4, Cheryl Hemingway5, Paul Brogan6, Despina Eleftheriou1,7
    1Infection, Immunity and Inflammation, University College London; 2Paediatric Rheumatology Department, GOSH; 3Paediatric Neuroradiology, UCL Great Ormond Street Institute of Child Health; 4Paediatric Immunology Department; 5Paediatric Neurology Department, GOSH; 6Infection, Immunity and Inflammation, UCL GOSH; 7ARUK centre for adolescent rheumatology, University College London, London, United Kingdom
    Correspondence: Dara McCreary

    Introduction: Monogenic autoinflammatory diseases (AID) are severe lifelong systemic inflammatory disorders with dysregulated innate immunity, causing significant morbidity, mortality, and economic burden. Often these diseases present with isolated central nervous system (CNS) involvement and mimic other non-inflammatory CNS disorders. This significantly hampers the clinical management of these patients, who are subjected to expensive and often invasive diagnostic patient pathways. Securing a molecular diagnosis is of major importance in these cases for treatment, prognosis, and genetic counselling. Routine genetic screening is time-consuming, costly, and lacks sensitivity since only common disease harbouring exons of a minority of the known genes causing autoinflammation of the CNS are currently tested using Sanger sequencing. Next-generation sequencing (NGS) offers the ability to rapidly and cost-effectively screen all exons of a gene panel containing hundreds of genes. This approach has not yet been routinely introduced in the UK for these conditions.

    Objectives: To develop and evaluate the performance of a NGS gene panel for AID with predominant CNS involvement.

    Methods: The Agilent SureDesign tool was used to design an NGS panel targeting 256 genes, grouped into the following broad clinical phenotypes: AID; monogenic vasculitis/vasculopathy; interferonopathies; haemophagocytic lymphohistiocytosis (HLH); immunodeficiencies; metabolic diseases and other inherited white matter diseases. The targeted region includes coding exons, conserved non-coding exons, upstream promoter regions, and splice sites. Captured and indexed libraries (QXT Target Enrichment System) were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in paired-end mode. Positive controls for panel validation comprised 16 DNA samples from patients with confirmed mutations. We then applied the panel to test 60 prospective samples with suspected but unconfirmed monogenic autoinflammation of the CNS. Read alignment, variant calling, and annotation were performed using Agilent SureCall v3.0 software.

    Results: In the validation stage, our targeted panel detected all known mutations in the 16 positive control samples. The panel was effective at detecting different types of variants, including rare and common single nucleotide variants (SNVs), insertion/deletions, splice-junction variants, upstream promoter region variants, and somatic mosaicism. Prospective testing of the panel in 60 patients revealed pathogenic variants (class 4 or 5) in 23 of 60 patients giving a detection rate of 38%.A definitive molecular diagnosis was established in 11/60 patients (18%). These included patients with isolated CNS involvement in the context of primary HLH (5/11), monogenic interferonopathies (2/11), AID (2/11), metabolic conditions (1/11) and immunodeficiencies (1/11).

    Conclusion: This study demonstrates the clinical utility of a comprehensive NGS-based targeted gene panel which was both highly sensitive and specific in detecting different sequence variants of clinical significance. We are currently integrating this panel into a routine diagnostic laboratory testing strategy for monogenic autoinflammatory diseases of the CNS.

    Disclosure of Interest

    None Declared

    P1079 Next generation sequencing analysis of an NLRP3 mutation negative CAPS cohort

    Sonia Melo Gomes1, Ebun Omoyinmi1, Juan Arostegui2, Ying Hong1, Nigel Klein1, Paul Brogan1
    1Infection, Inflammation and Rheumatology, UCL GOS Institute of Child Health, London, United Kingdom; 2Immunology -CBD, Hospital Clínic, Barcelona, Spain
    Correspondence: Sonia Melo Gomes

    Introduction:

    Cryopyrin Associated Periodic Syndromes (CAPS) are caused by autosomal dominant gain of function mutations in the NLRP3 gene. However, a subset of these patients with typical clinical features and good response to anti-IL-1 treatment, have no mutation in NLRP3 detected by conventional Sanger DNA sequencing. Somatic mosaicism may account for between 19 to 69% of these, from previous reports. Genetic heterogeneity has also been implicated in patients with CAPS-like phenotypes, with the identification of pathogenic mutations in the NLRP12, PLCG2 and NLRC4 genes (Familial Cold Autoinflammatory Syndrome subsets 2, 3 and 4, respectively).

    Objectives:

    To explore the genetic cause(s) in a paediatric cohort of mutation negative CAPS patients using Next Generation Sequencing (NGS) techniques, by assessing:

    1. 1.

      the rate of somatic NLRP3 mosaicism, and

    2. 2.

      possible contribution of other/novel genes

    Methods:

    Patients from a single paediatric centre who met the CAPS diagnostic criteria and had no NLRP3 mutations identified by partial (exons 3, 4 and 6) or total Sanger sequencing, were identified.

    The NGS approach used in this study was Amplicon-based Deep Sequencing (ADS) for identification of NLRP3 mosaicism; and Whole Exome Sequencing (WES) for exploration genetic heterogeneity (i.e. other causative mutations). ADS was performed on an Ion Torrent platform using the Ion Torrent PGM HiQ sequencing kit. Resulting sequences were analysed using the Amplicon Variant Analyzer software. WES was performed using the Nextera Exome Capture sequencing kit and Hiseq sequencing platforms. Exome data was analysed in the Galaxy web-based suit. The wANNOVER web-based software was used for variant annotation.

    Results: A total of 8 patients who met the CAPS clinical diagnostic criteria and had no mutation identified by conventional Sanger sequencing were included in this study. ASD identified the presence of a somatic mutation in 3 of these patients (37.5%), with an allelic frequency varying from 3.1 to 14.5%.

    The remaining patients were studied by WES. In Patient 4 a novel NLRP3 mutation was identified in exon 5, which had been previously missed due to a restricted Sanger approach.Patient 5 had a novel NOD2 mutation, thus redefining the diagnosis as Blau syndrome. WES findings in the other patients are summarized in the Table.

    Conclusion:

    In this study, somatic NLRP3 mutations were identified in 3/8 (37.5%) of our cohort, and 3/8 had variants in 3 other genes, potentially of relevance to the phenotype. In particular, the identification of a NOD2 mutation and subsequent reclassification of that patient as Blau syndrome, illustrates the limitations of current CAPS diagnostic criteria as well as potential clinicaloverlap between different autoinflammatory syndromes, reiterating the use of a wider NGS approach.At present it is unclear how the variants found in patients 6 and 7 could modulate the phenotype, therefore in 3 patients a clear genetic cause is yet to be found.

    In conclusion, this study suggests that NGS approaches are superior to conventional sequencing for the molecular diagnosis of CAPS and CAPS like phenotypes since they can detect somatic mosaicism or alternative molecular diagnoses mimicking CAPS.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1079). See text for description

    P1080 A recessive mutation in NLRC4 (A160T) causes autoinflammation and immunedysregulation, and predisposes to ulcerative colitis in heterozygous carriers

    Leonardo O. Mendonca1,2, Annemarie Steiner3, Alessandra Pontillo4, Isabella Ceccherini5, Francesco Caroli5, Alice Grossi5, Jonas Moecking6, Fiona Moghaddas6,7,8, Marco Gattorno9, Seth Masters3
    1International Center for Autoinflammatory Diseases and Primary Immunodeficienceis, Istituto Giannina Gaslini; 2Immunogenetics Laboratory, Biomedical Science Institute, University of São Paulo, Sao Paulo, Brazil; 3Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; 4Immunogenetics Laboratory, Biomedical Science Institute, University of São Paulo, São Paulo, Brazil; 5UOC Medical Genetics, Istituto Giannina Gaslini, Genoa, Italy; 6Inflammation Division, The Walter and Eliza Hall Institute for Medical Research; 7Department of Medical Biology, The University of Melbourne; 8Department of Clinical Immunology and Allergy, The Royal Melbourne Hospital, Parkville, Australia; 9International Center for Autoinflammatory Diseases and Primary Immunodeficienceis, Istituto Giannina Gaslini, São Paulo, Brazil
    Correspondence: Leonardo O. Mendonca

    Introduction: NLRC4 associated autoinflammatory disease (NLRC4-AID) was recently described due to autosomal dominant mutations. The clinical spectrum ranges from macrophage activation syndrome with severe enterocolitis to CAPS-like disease. As opposed to mutations that trigger other inflammasomes, which result in IL-1b driven disease, IL-18 seems to be the cytokine responsible for the clinical manifestations of NLRC4-AID.

    Objectives: Report the phenotype of a patient carrying a novel, homozygous mutation in NLRC4 (A160T). Describe in vitro evaluation that supports the pathogenicity of NLRC4 A160T in terms of inflammasome activation and IL-18 production. Provide evidence for a genetic association between heterozygous A160T and ulcerative colitis.

    Methods: Clinical data were acquired from patients records. DNA was extracted and sequenced using standard procedures. Monocytes were isolated by adherence from peripheral blood using Ficoll-Paque gradient for IL-1, IL-6 and IL-18 measured by ELISA. Flow cytometry for quantification of ASC speck formation by TOFIE was examined with overexpressed WT or A160T NLRC4 in HEK 293T cells. THP-1 monocytes were reconstituted with WT and A160T NLRC4, then priming was performed with Pam3CSK4 at a final concentration of 100 ng/ml for 3 hours to evaluate cell death and Il-18 secretion. Genome wide assocaition of NLRC4(A160T) with ulcerative colitis was interrogated using data from the IBD exomes portal.

    Results:Case Report- The adult patient complained since 6 months of life of recurrent episodes of systemic inflammation characterized by recurrent low grade fever, chills, oral ulcer, uveitis, arthralgia and abdominal pain followed by diarrhea with mucus and variable skin rash Low levels of IgG and CD19 were a persistent finding. High doses of corticosteroids were effective in controlling disease and anti-IL1 partially controlled symptoms. Serum cytokine levels during the use of anti-IL1 evidenced increased levels of IL-18 (285 pg/mL) compared to healthy control (median - 100 pg/mL) and undetectable level of IL-1. Genetic analysis- Target gene panel found an homozygous mutation in NLRC4 gene (NM_021209), exon 4, c.478G>A (p.Ala160Thr). Familial genetic segregation was done using standard Sanger Sequencing and the father, mother and a healthy brother are heterozygous for the mutation. The estimated allele frequency is of 0,09% and the frequency for the homozygous status is 0%.In vitro studies- Increased ASC speck formation, and IL-1b/IL-18 secretion was observed when NLRC4 (A160T) was overexpressed in human cell lines. Dominant mutations in NLRC4 (eg S171F) result in stronger inflammasome activation than A160T, which is recessively inherited in these two cases. Patient monocytes evidenced high levels of IL-1b and IL-18 secretion in response to ATP, LPS and Flagellin.GWAS association- The NLRC4(A160T) allele is significantly enriched in ulcerative colitis compared to healthy controls, OR 2.546 (95% 1.778-3.644), p=0.01305.

    Conclusion: Homozygous NLRC4(A160T) causes a monogenic disease characterised by autoinflammation and immunedysregulation. This allele is pathogenic in vitro, however with reduced strength compared to dominant mutations in NLRC4. Carriers with NLRC4(A160T) are at increased risk of developing ulcerative colitis.

    Disclosure of Interest

    None Declared

    P1081 The Australian Autoinflammatory Diseases Registry (AADRY): a streamlined approach to the genetic and immunological evaluation of monogenic autoinflammatory diseases

    Fiona Moghaddas1,2,3, Jenni Harris2, Dunja Vekic4, Harapas Cassandra2, Dale J. Calleja2, Navid Adib5, Jonathan Akikusa6, Roger Allen6, Dan Andrews7, Vanessa Bryant3,8, Geoffrey Cains4, Jeffrey Chaitow9, Britt Christensen10, David Coman11, Matthew Cook12, Angela Cox6,13, Jo A. Douglass1, Peter Gowdie6,13, Laine Hosking14, Matthew F. Hunter15, Paul Kubler16, Senq J. Lee17, Jane Munro6, Samar Ojaimi18, William Renton6, Davinder Singh-Grewal9,19, Charlotte Slade1,3,8, Joanne Smart14, Georgina Tiller6, Ben Whitehead20, Jane Woods4, Philip Wu21, Sam Mehr14, Ian P. Wicks2,3,22, Seth L. Masters2,3 and AADRY
    1Clinical Immunology and Allergy, The Royal Melbourne Hospital; 2Inflammation Division, WEHI; 3Department of Medical Biology, The University of Melbourne; 4Dermatology Department, Liverpool Hospital; 5Queensland Rheumatology Services, Arthur House, Red Hill; 6Rheumatology Department, The Royal Children's Hospital; 7The John Curtin School of Medical Research, Australian National University, Canberra; 8Immunology Division, WEHI; 9Rheumatology Department, The Children’s Hospital at Westmead; 10Gastroenterology Department, The Royal Melbourne Hospital; 11Metabolic Medicine, Queensland Children’s Hospital, Brisbane; 12Centre for Personalised Immunology; 13Paediatric Rheumatology Department, Monash Children’s Hospital; 14Paediatric Immunology Department, The Royal Children’s Hospital; 15Monash Genetics, Monash Health; 16Department of Rheumatology, Royal Brisbane and Women's Hospital; 17Department of Rheumatology, Perth Children's Hospital; 18Department of Infection and Immunity, Monash Children’s Hospital; 19Rheumatology Department, The Sydney Children’s Hospital; 20Rheumatology Department, Queensland Children’s Hospital, Brisbane; 21Australian Phenomics Facility, Australian National University, Canberra; 22Rheumatology Department, The Royal Melbourne Hospital, Australia
    Correspondence: Fiona Moghaddas

    Introduction: Since the original publication in 1971 outlining two cases of FMF complicated by pulmonary amyloidosis, there have been only two studies looking at the status of monogenic autoinflammatory disorders (AIDs) in Australia. Clinicians caring for patients with an AID without a pathogenic mutation in known disease-causing genes do not have a streamlined approach to further genetic evaluation.

    Objectives: The Australian Autoinflammatory Diseases Registry (AADRY) aims to provide clinicians with access to a research team to further evaluate patients without an established genetic diagnosis as well as to address limitations in the current knowledge of AIDs in Australia.

    Methods: Patients with suspected AIDs were recruited as trios. Whole exome sequencing (WES) was performed on whole blood or saliva using the Hiseq 4000 platform and Illumina Sureselect XT V5 library. A variant list was generated using a pipeline, variant caller and filtering strategy previously described. Initial filtering of the variants proceeded by excluding synonymous and common variants, and intronic variants more than 50 base pairs from an intron-exon junction. Sequence data quality was assessed by viewing the BAM file on the Integrated genomics viewer. Variant classification was based on ACMG consensus guidelines.

    Results: From September 2015 to June 2018, 34 index cases underwent WES. Initial curation analysis looked at autoinflammatory genes as per IUIS and ISSAID. A total of 77 variants in 34 patients were filtered with no pathogenic or likely pathogenic variants detected. The majority (82%) of variants were VUS. Nineteen trios underwent WES. Seventy-one de novo variants (mean 4 per index case), 77 AR variants (mean 4) and 253 potential CH variants (mean 13) were curated. Of the 5 families recruited with more than one symptomatic member, a likely pathogenic variant segregating with disease was found in one family. The pedigree demonstrated multiple generations symptomatic of various degrees of pustular acne and hidradenitis suppurtiva. A novel truncating variant in NCSTN (c.1485C>T) encoding nicastrin p.Glu454X segregated with disease.

    Conclusion: AADRY has curated the WES of 34 index cases, including 19 trios. A likely pathogenic variant was discovered in one family with a highly penetrant dominantly inherited dermatological phenotype. No clear genetic diagnosis has been made in the remaining index cases, however a number of candidate novel variants are being evaluated. Further work is now underway to gather data on patients with genetically defined AIDs so that the retrospective cohort of patients with an established diagnosis is captured.

    Disclosure of Interest

    None Declared

    P1082 Genotypic diversity observed within a large cohort of Armenian patients with late-onset familial Mediterranean fever

    Gernot Kriegshäuser1,2, Hasmik Hayrapetyan3,4, Stepan Atoyan3,4, Stefan Nemeth5, Christian Oberkanins5, Tamara Sarkisian3,4
    1Institute of Clinical Chemistry and Laboratory Medicine, General Hospital, Steyr; 2Clinical Institute of Medical and Laboratory Diagnostics, Medical University, Graz, Austria; 3Center of Medical Genetics and Primary Health Care; 4Department of Medical Genetics, Yerevan State Medical University, Yerevan, Armenia; 5ViennaLab Diagnostics, Vienna, Austria
    Correspondence: Christian Oberkanins

    Introduction: Familial Mediterranean fever (FMF) as an autoinflammatory disease, results from mutations in the MEFV gene mainly with an autosomal recessive mode of inheritance. The age of onset of FMF varies, with about 60% and 90% of patients experiencing their first attack before the age of 10 and 20 years, respectively. Hence, FMF with the first attack occurring at the age of ≥ 40 years (i.e. late-onset FMF) is rare and only a few small studies have addressed this disease subset.

    Objectives: This work aimed at investigating the molecular genetic characteristics of Armenian patients diagnosed with late-onset FMF.

    Methods: Genomic DNA isolated from 354 Armenian late-onset FMF patients were analysed for the 12 most common MEFV mutations plus SAA1 isoforms 1.1, 1.3 and 1.5 using multiplex PCR and reverse-hybridisation. Mutational spectra and resulting genotypes were then matched against the clinico-demographic profiles collected for these patients.

    Results: Of all 354 patients, 194 (54.80%) were female and 160 (45.20%) were male. The following genotypes were significantly associated with the late-onset variant: M680I/E148Q (P=0.004), M694V/E148Q (P<0.001) and V726A/V726A (P=0.001). Of note, 12/354 (3.39%) patients were found to be homozygous for the M694V mutation.

    Conclusion: Our data suggest that late-onset FMF is more prevalent in women and is of greater genetic diversity than previously reported. Further studies including late-onset FMF patients homozygous for MEFV mutation M694V are ongoing and may lead to the identification of novel disease-modifying mechanisms.

    Disclosure of Interest

    None Declared

    P1083 Genetic polymorphism of familial Mediterranean fever in Georgia - a snapshot

    Karaman Pagava, Helen Phagava, Davit Tatoshvili
    Tbilisi State Medical University, Tbilisi, Georgia
    Correspondence: Karaman Pagava

    Introduction: There are very few publications about FMF and MEFV mutations in Georgia.

    Objectives: Determination of the MEFV mutations spectrum and their frequency in Georgia.

    Methods: Review of the appropriate literature. 14 sources on the situation in Georgia published from 1981 until now, were analyzed. 8 of them included genetic data. In summary MEFV mutation was investigated in 202 clinically healthy neonates in Tbilisi and 180 patients (children and adolescents) from different regions of Georgia.

    Results: The carrier rate of MEFV mutations in neonates was remarkable: 31/202; 15.3%, most frequently- E148Q (15/31), M680I (5/31), M694V (4/31). This could be explained by high proportion of people of Armenian ethnicity and high frequency of interethnic marriages. In patients the main mutations were: M694V (65.6%), V726A (23.3%), M6801 (20.6%). Overwhelming majority of patients had Armenian roots. This factor explained the similarity of MEFV mutations in both countries. Some difference between data in clinically healthy newborns and patients was found. Maybe the cause is the extent to which a particular mutation is expressed. Additional research in this direction is needed. Recently Georgian Society on Familial Mediterranean Fever, Autoinflammatory and Autoimmune Diseases has been established. Hopefully it will help to create more comprehensive registry of patients with these diseases and obtain new data about their genetic and clinical polymorphism.

    Conclusion: The up-to-date information regarding MEFV genes in Georgia is presented. The awareness of FMF and the availability of appropriate testing should be promoted further in Georgia.

    Disclosure of Interest

    None Declared

    P1084 Exploratory study of MYD88 L265P and clonal haematopoiesis in 30 patients with Schnitzler syndrome, an acquired systemic autoinflammatory disorder

    Shelly Pathak1, Dorota Rowczenio2, Roger Owen3, Gina D. Doody4, Darren Newton4, Claire Taylor4, Catherine Cargo3, Philip Hawkins2, Karoline Krause5, Helen Lachmann2, Sinisa Savic1
    1Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds; 2National Amyloidosis Centre, University College London, London; 3Department of Haematology, St James’s University Hospital; 4Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom; 5Department of Dermatology and Allergy, Universitätsmedizin Berlin, Berlin, Germany
    Correspondence: Shelly Pathak

    Introduction: Schnitzler syndrome (SchS) is rare, acquired, IL-1B mediated, autoinflammatory disorder, for which the pathogenesis is unknown.

    Objectives:

    1. (1)

      Explore the concept of ‘inflamm-ageing’ by looking for evidence of clonal haematopoesis, as this phenomenon has been linked with pro-inflammatory changes of the immune system.

    2. (2)

      Investigate the prevalence of the MYD88 L265P mutation in SchS patients, given that 30% of SchS patients go onto develop WM.

    3. (3)

      To perform deep sequencing of NLRP3 region in an additional 11 SchS patients

    Methods: Informed consent was provided by all subjects, in accordance with the Declaration of Helsinki.

    1. (1)

      Exon-specific regions of 28 genes associated with MDS were amplified using the Fluidigm custom-made 48x48 access array for Illumina library preparation. The sequencing was performed on an Illumina MiSeq using an in-house analysis pipeline. Low level variants were confirmed by repeat sequencing. For the female patients, inactivation of the X chromosome (XCI) was assessed by determination of the androgen receptor (AR) locus methylation status using the HUMARA assay. The XCI ratios are reported as a percentage:<80% is considered random X-inactivation, and percentages >80% are deemed as skewed.

    2. (2)

      The Allele Specific Oligonucleotide-PCR (ASO-PCR) technique was used to identify patients harbouring the mutant ‘C’ allele at chromosomal location 3:38182641, leading to the downstream MYD88 Leu265Pro mutation.This PCR based assay discriminates between the WT and mutated alleles by using 3 sets of primers, followed by direct amplification producing 2 different sized products (400bp – WT; 250bp – mutated).

    Results: Somatic mutations associated with CH were identified in 3/30 SchS patients: two had different variants predicted as variants of unknown significance (VUS) in the TET-2 gene and one had a pathogenic mutation in the STAG2 gene. Though the latter may be a clonal hematopoiesis of indeterminate potential (CHIP) mutation. Of the aCAPS cases one patient had two pathogenic variants in TET-2. As additional evidence of CH, we utilised the HUMARA assay to identify X-allelic skewing in female patients, identifying solely one patient in each cohort as ‘XCI-skewed’. MYD88 L265P variant was detected in peripheral blood (PB) DNA of 9/30 SchS patients.Further deep sequencing of the NLRP3 gene in 11 SchS patients failed to identify somatic variants.

    Conclusion: The rate of CH in SchS and aCAPS is similar to what would be expected for this age group. Therefore, CH does not appear to be a common abnormality underpinning these conditions. The L265P MYD88 variant was not universally found in all SchS patients. However, having not investigated bone marrow-derived DNA we cannot categorically exclude the presence of the MYD88 variant in mutation negative cases. Lastly, including the 21 patients we have reported previously (1), we have now excluded somatic NLRP3 mutations in 32 SchS patients. A molecular mechanism common to all SchS patients remains elusive. It is possible that this could be a heterogeneous disorder, however given the uniform clinical characteristics, including responsiveness to IL-1 inhibition, we favour the likelihood of a common aetiology, yet to be discovered.

    Disclosure of Interest

    None Declared

    P1085 E148Q mutation in family with familial Mediterranean fever

    Tamara F. Sarkisian, Hasmik Hayrapetyan, Anna Yeghiazaryan, Ruzanna Karakhanyan
    Center of Medical Genetics and Primary Health Care, Yerevan, Armenia
    Correspondence: Tamara F. Sarkisian

    Introduction: Familial Mediterranean Fever (FMF) is a chronic autosomal recessive autoinflammatory disease with very high incidence and prevalence in Armenians. First information about recurrent inflammatory syndrome or polyserositis in Armenians was reported in ancient manuscripts of XII Century. It has been confirmed that MEFV (Mediterranean Fever) gene mutations play the main role in occurrence of severity of this disorder. MEFV gene encodes pyrin, the most important regulator of immunity processes. Mutated pyrin is responsible for inflammasome formation and increased inflammatory response. It was reported in “Infever Database” (2015) that more than 310 sequence variants of MEFV gene are revealed in 10 exons of Mediterranean Fever gene. The carrier rate of MEFV gene mutations is extremely high (about one in three) in Armenians. Distribution and spectrum of MEFV gene mutations are responsible for mild and severe forms of recurrent febrile and pain attacks, periodic episodes of fever, peritonitis, pleuritic, erysipelas-like skin lesions, arthralgia. Although FMF is autosomal recessive condition, but in more than 19% of our cohort of FMF patients have been shown to exhibit the same symptoms with heterozygous genotypes. In some heterozygous cases we detected atypical clinical forms of FMF.

    Objectives: In some FMF patients “mild” MEFV mutations, such as P369S, A744S and E148Q, are responsible for the determination of severity of attacks in FMF, along with environmental or possible other genetic factors associated with inflammatory attacks. We have identified E148Q mutation in exon 2 of MEFV gene in three individuals of three generations of one family with confirmed clinical diagnosis of FMF.

    Methods: They were genotyped using Viennalab Reverse Hybridization Strip Assay (Austria). Genetic and clinical data of more than 36000 individuals, including FMF patients and their family members, revealed statistically significance of phenotypes and distribution ofMEFV mutations covering more than 98% of the cases.

    Results: In this family report we present the three FMF patients, including two males and one female of three generations. In the first generation 50 y.o. female has typical clinical FMF with periodic episodes for 1-2 days duration of fever, abdominalgia, thoracalgia, arthralgia, hepato- and splenomegaly. Disease manifested in age of 16 years old. Genetic investigation identified heterozygous E148Q mutation of exon 2. Treatment with Colchicine decreased the intensity of attacks to one-two episodes annually. The son of this patient also inherited the same heterozygous E148Q mutation.The disease onset with first attacks was observed when the boy was in age of 7 years old. The recurrent fever attacks of abdominal pain, myalgia had occurred for 5-6 days. Colchicine treatment decreased the frequency of attacks to 2-4 annually. His son also inherited the same E148Q heterozygous genotype from his grandmother and father. The boy was born in 2018. The disease manifested in 8 months with fever attacks.

    Conclusion: Overall E148Q mutation rate among healthy individuals in our population is 3.4%. In heterozygous carriers of E148Q mutation our study shows an association with typical clinical symptoms of FMF in 3% of patients, and with atypical form - in 6% of FMF patients. Exon 2 mutations (E148Q etc) is correlated with milder disease with almost no arthritis and no amyloidosis. This rare heterozygous genotype resulted in genotype-phenotype associations.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1086 Familial Mediterranean fever: clinical significance of E148Q and P369S mutations

    Tamara F. Sarkisian, Hasmik Hayrapetyan, Stepan Atoyan, Anna Yeghiazaryan, Gohar Shahsuvaryan
    Center of Medical Genetics and Primary Health Care, Yerevan, Armenia
    Correspondence: Tamara F. Sarkisian

    Introduction: FMF represents a significant health care problem in Armenia, because of high prevalence of the carriers of MEFV gene mutations (1:3) among population. Clinical-genetic investigations of 50-90 new FMF cases are performed weekly. Personalized treatment is based on MEFV genotypes and aimed to correct the effect of colchicine therapy.

    Objectives: Disease severity varies from mild, moderate, severe depending on the spectrum of MEFV mutations. We present the evaluation of phenotypic features of FMF patients with E148Q (exon 2) and P369S (exon 3) mutations, suggested that E148Q and P369S are the mildest mutations, and some reports have questioned their association with FMF.

    Methods: Over 36000 individuals, including FMF patients and healthy cohort, have been referred for clinical observation and molecular-genetic detection of MEFV and SAA1 genes mutations. Reverse-hybridisation assay is performing for genetic testing using the Viennalab StripAssay (Austria). Phenotypic, genotypic, and relevant information for patients have been stored in a large database.

    Results: the frequency of carriers of MEFV mutations among Armenians is extremely high (about 20%: 1 in 3 individuals) with FMF prevalence of up to 3%. With the exception of M694I all other common mutations were present in healthy individuals with different frequencies. E148Q and P369S mutations are more frequent for healthy individuals in comparison to affected FMF patients, suggesting their lower penetrance compared to the others, but could still contribute to the overall FMF symptoms. Colchicine treatment is required.

    Among 30 individuals with E148Q/E148Q homozygous genotype 9 have typical FMF, and 3 – non-typical symptoms. Abdominal pain was seen in 77% of the patients, fever in 92.3%, pleuritis in 30.7%, arthralgia in 61.5%. Compound heterozygotes and complex cases had a higher frequency of abdominal pain, fever, arthralgia, arthritis, myalgia, and chest pain than subjects who were homozygous for E148Q, but none of these symptoms reached statistical significance. None of the mentioned patients had developed renal amyloidosis, but two with E148Q/E148Q had a family history of amyloidosis and one had rapidly progressive glomerulonephritis secondary to vasculitis, which progressed to chronic renal failure. Among heterozygous carriers of E148Q mutation 333 had typical FMF, 1336 were without any symptom.

    Homozygous genotype for P369S mutation was detected in only one healthy person. Number of healthy P369S carriers was 159, and in 41 diagnosis of FMF was confirmed.

    In cohort of healthy controls, we detected 10 genotypes with P369S complex alleles, 7 without clinical picture of FMF, 3 manifested mild disease, suggesting that P369S might ameliorate the phenotypic effect of exon 10 mutations. Among E148Q/P369S compound-heterozygous 115 were healthy carriers, 34 are typical and 12 non-typical FMF.

    In one family we confirmed FMF in 2 generations of E148Q/P369S compound-heterozygotes: two sisters with their children.

    Atypical FMF phenotypes was found in: 3 homozygotes, 90 heterozygotes with E148Q mutation, 10 heterozygotes with P369S mutation.

    Conclusion: Genetic counselling, including modern clinical-genetic approach is recommended for disease risk estimation in families with history of FMF, assessment of efficiency of colchicine treatment, prognosis of development of complications. Using a population-based study of healthy persons and FMF patients, we provide evidence that these alleles are a disease-causing mutations and not are a benign polymorphisms among Armenians. We suggest that in some cases other factors along with MEFV genotype, such as environmental or possibly other genetic factors play role in the determination of the severity of the inflammatory attacks in FMF.

    Disclosure of Interest

    None Declared

    P1087 Severe prognosis of late onset CAPS associated with age related clonal hematopoiesis

    Yael Shinar1, Rinat Cohen1, Victoria Marcu1, Itamar Goldstein1, Meital Nagar1, Gleb Slobodin2, Ilan Ben-Zvi1, Michael Rozenbaum2
    1Sheba Medical Center ,Ramat Gan; 2Bnai Zion Medical Center , Haifa, Israel
    Correspondence: Yael Shinar

    Introduction: Sporadic cases with adult Cryopyrin Associated Periodic Syndrome (CAPS) are associated with a somatic mutation in the NLRP3 gene, often restricted to the myeloid lineage.

    Objectives: We investigatedif mutations in myeloprliferative genes that are known to drive age related clonal heatopoiesis (ARCH) associated with the development of CAPS in a late onset case.

    Methods: We sequenced 26autoinflammatory and 52 myeloproliferative genes in peripheral bloodDNA ofa 56 year oldpatient with late onsetCAPS, by the Next Generation Sequencing (NGS) method. The pathogenic mutations were screened in DNA derived from FACS-sorted CD3-positive T cells, CD14-positivemonocytes and granulocytes, and from single cells.

    Results: A somatic NLRP3 mutation, NM_001243133.1:c.1709A>Gp.Tyr570Cys, was present in DNA purified from granulocytes or monocytes (20% NLRP3 gene reads) but was hardly detected in the patient's DNA from T cells (1%). Two pathogenic mutations linked with myeloproliferative disorders were detected: A known frame shiftmutation in the Tet methylcytosine dioxygenase 2 gene (TET2, in 31% ofthegene reads), and a known amino acid substitution in the Serine/arginine-rich splicing factor 2 (SRSF2, in 43% of the SRSF2 reads). Both had the same lineage pattern asthe NLRP3 mutation, yet a higher somatic mosaicism rate than the NLRP3 mutation in peripheral blood cells (PBC). Co-occurence of the NLRP3 mutation with one or two loss-of-function TET2 allele(s) was demonstrated in several single myeloid cells. There was no evidence of hematologic malignancy in the bone marrow or in a CT scan of the patient. Rather, the patient developed macrophage activating syndrome (MAS) that was also the cause of death after 8 years of disease.

    Conclusion: Our results suggest the need to evaluate sporadic late onset CAPS cases for clonality of myeloproliferative genes, with prospect of dynamic and severe disease evolution, including MAS. In the case of TET2deficiency mutations a proinflammatory effect through the NLRP3 inflammasome may be suggested based on mice models. Clonal autoinflammatory diseases may constitute a distinct entity within the autoinflammatory diseases, characterized by clinical complexity and imposing therapeutic challenges.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    Y. Shinar Grant / Research Support from: Novartis Israel, R. Cohen: None Declared, V. Marcu: None Declared, I. Goldstein: None Declared, M. Nagar: None Declared, G. Slobodin: None Declared, I. Ben-Zvi: None Declared, M. Rozenbaum: None Declared

    P1088 CNV analysis from targeted NGS data: case report on a patient with a large deletion in the NLRP12 gene

    Barbara Bangol, Kathrin Starz, Martin Ziegler, Daniel Becker, Jenny Schiller, Sophie Eilitz, Hanns-Georg Klein
    MVZ Martinsried GmbH, Martinsried, Germany
    Correspondence: Kathrin Starz

    Introduction: Diagnostic CNV (copy number variation) analysis in molecular genetics with exon level resolution is performed mainly by hybridization techniques (e.g. multiplex ligation-dependent probe amplification). Therefore, the examinable genomic region is restricted to commercially available test kits in most cases.

    Objectives: The amount of clinically relevant CNVs located beyond the examinable region of these kits remains elusive. Hence, a bioinformatic pipeline for the detection of CNVs from NGS sequencing data was established (MIDAS pipeline). The analysis is routinely performed for patient samples in parallel to NGS testing for single nucleotide variations and small insertions/deletions by targeted enrichment and sequencing of candidate genes.

    Methods: DNA samples from 115 patients with suspected autoinflammatory disease were subjected to NGS panel diagnostics including the genes ELANE, IL1RN, IL36RN, LPIN2, MEFV, MVK, NLRC4, NLRP3, NLRP12, NOD2, PSMB8, PSTPIP1, TMEM173, TNFRSF1A. With the exception of MEFV, no commercial CNV detection kits are available for these genes. In addition to routine NGS testing, binary alignment mapping files (BAM) were further analyzed via the MIDAS CNV pipeline. Coverage of targeted regions was normalized with control samples. CNV calls were performed using CoNVaDING, ExomeDepth and XHMM. Results were further investigated if CNVs were called from two out of three callers.

    Results: Testing in a two-year-old Polish girl presenting with recurrent fever of unknown origin and an increased susceptibility to infections revealed a heterozygous deletion of at least 321 bp comprising exon 5 of the NLRP12 gene (rsa[GRCh37] 19q13.42(54308439_54308760x1, 54310745x2). The deletion was confirmed by quantitative PCR (qPCR) and was not present in 240 control individuals.

    Conclusion: There are known truncating germline mutations in the NLRP12 gene, but no larger deletions have been described in association with the rare familial cold autoinflammatory syndrome 2 (FCAS2) so far. According to Richards et al. (2015) the variant was classified as likely pathogenic, which supports the clinical diagnosis of FCAS2 in the patient. The case described shows that application of CNV analysis of NGS data may increase the diagnostic sensitivity for the molecular diagnostics of hereditary recurrent fever. It is a cost efficient and convenient method to screen for copy number variations beyond regions which are covered by commercially available CNV detection kits. Since very few patients with FCAS2 have been identified, data on the phenotypic presentation is limited. Interestingly, Kostik et al. (2018) described several patients with clinical manifestations characteristic for primary immunodeficiencies and showing increased susceptibility to infections, similar to the present case.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1089 Population based study of frequency of FMF gene mutation carrying in Armenia

    Artashes Tadevosyan, Tigran Avagyan, Anush Budumyan, Hasmik Hayrapetyan, Gayane Amaryan
    Public Health and Helth Care Organization, Yerevan State Medical University, Yerevan, Armenia
    Correspondence: Artashes Tadevosyan

    Introduction: Familial Mediterranean Fever (FMF) is a hereditary autoinflammatory disorder that is very common among Armenian, Turkish, Jewish populations. However, no study was conducted before among general Armenian population on epidemiology of FMF gene mutations.

    Objectives: To study frequency of carrying of FMF gene mutations among Armenian population.

    Methods: Random sample of 290 unrelated persons residing in Armenia, 179 of whom were female and 111 were male were selected. Blood samples were taken from them and analyzed. Identification of FMF and SAA1 gene mutations for 12 more common types of mutations based on PCR and reverse hybridization using ViennaLab diagnostic test was performed.

    Results: This is the first population based study of carrying of FMF gene mutations among Armenian ethnic population in Armenia. Out of 290 participants 98 persons have at least one mutation, among which 59 were female carriers and 39 - male. That makes up 33.45% of the total participants. Out of 98 carriers 87 had heterozygous mode of inheritance, while 10 had compound-heterozygous and 1 had homozygous mode of inheritance.

    Rate of mutation carriers among female participants was 1 out of 3. In female group 55 had heterozygous mode of inheritance (30.73%) and 4 had compound-heterozygous mode of inheritance (2.23%). Following mutations were found in this group: M694V, M680I, V726A, E148Q, K695R, A744S, R761H, F479L and P369S Highest number of cases of identified in female group was M694V (Table1).

    Rate of mutation carriers among male participants was 1 out of 3. In male group 32 had heterozygous mode of inheritance (28.83%), 6 had compound-heterozygous mode of inheritance (5.4%) and 1 had homozygous mode of inheritance (0.9%). Following mutations were found in this group: V726A, M694V, E148Q, M680I, M694I, A744S and F479L. V726A was identified 17 times in male group (Table1).

    Conclusion: The frequency of carrying the FMF gene mutation 1:3 is highest among ever reported.

    Most common type is heterozygous mode of inheritance, which constituted for 88.8% among all carriers.

    The most common mutation among all carriers is M694V - 32 cases, a little less - is carriage of V726A mutation (30 cases). At the third place - is carriage of Е148Q mutation (23 cases).

    Acknowledgments

    This work was supported by the RA MES State Committee of Science, in the frames of the research project № SCS 15T-3D191.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1089). Number and rate (per hundred) of registered mutations

    P1090 Whole genome linkage and exome sequencing analysis in a CRMO family

    Derya Yavuz1, Selcuk Yuksel2, Eda Tahir Turanli1
    1Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul; 2Department of Pediatric Rheumatology and Pediatric Nephrology, Pamukkale University , Denizli, Turkey
    Correspondence: Eda Tahir Turanli

    Introduction: Chronic recurrent multifocal osteomyelitis (CRMO), is an autoinflammatory disorder with incidence varying between 1 – 4 per million and it is most likely due to defects in TLR4/MAPK leading to in anti- and pro-inflammatory cytokine balance (ORPHA: 324964, PMID:29080202). CRMO occurs in childhood and adolescence, characterized with recurrent inflammation and lesions that are predominantly found in long bones. Reported familial cases and associated inflammatory conditions indicate genetic predisposition in the pathogenesis.

    Objectives: In this study, we examined a consanguineous family with 2 affected and 1 healthy siblings and their healthy parents to identify the possible causative locus or gene.

    Methods: Whole genome SNP genotyping by using Illumina OmniExpress-24 BeadChip that targets ~700,000 SNP markers was performed on all the family members that consists of 2 affected sibs, 1 healthy sister and their healthy parents. Resulting genotyping data was used for multipoint parametric linkage analysis that was performed under recessive model with complete penetrance. One of the affected siblings was screened by whole exome genotyping for assessment of possible disease-causing rare, homozygous variants. Variants obtained after filtration of exome data was furthered verified with the linkage analyses data.

    Results: Linkage analyses revealed 137 homozygous regions out of which 44 are longer than 200 kb. Non-synonymous and homozygous variants gathered from the exome data were further filtrated according to 1000G and ExAC databases (MAF < 0.05), revealing 11 candidate variants in NBPF6, DNAH14, MMADHC, KIAA0232, BODIL1, PCDHGB2, PCDHGA6, ZNF316, PWWP2B, CFAB46 and CARNS1. Also, variants in the genes that are listed in Infevers database were checked which revealed 4 intronic variants in NLRP3, NLRC4, PSMA3 and PSTPIP1 genes. Among these variants, rs12786101 in CARNS1 (c.C1145T) and rs55638726 in NLRC4 (g.32461493_32461494insC) were chosen as candidate variants.

    Conclusion: One of the candidate variants that might be related to CRMO phenotype is located in CARNS1 leading to a missense mutation whereas rs55638726 in NLRC4 is found to be located in the splice site that might lead to aberrant splicing. However, these variants were found in homozygous regions that were shared in all the family members. Identification of disease causing potential of the detected variants are still on-going.

    Disclosure of Interest

    None Declared

    P1091 Detection of a rare variant on PSTPIP1 gene through three generations in a Turkish family with systemic autoinflammatory disease

    Merve Ö. Önen1, Serdal Ugurlu2, Ayse Balamir1, Eda Tahir Turanli1, Huri Ozdogan2
    1Department of Molecular Biology and Genetics, Istanbul Technical University; 2Department of Rheumatology, Istanbul University Cerrahpasa, Istanbul , Turkey
    Correspondence: Eda Tahir Turanli

    Introduction: Various monogenic recurrent fever syndromes share common clinical features. Therefore differential diagnosis of systemic autoinflammatory syndromes (sAIS) is a challenge, especially in familial Mediterranean fever prevalent countries.

    Here we describe a family with Turkish descent, with 3 affected members belonging to 3 successive generations (grandmother, daughter, granddaughter) with self-limited, short episodes of fever, abdominal pain, oral ulcers, pharyngitis, arthralgia/arthritis, calf pain with increased acute phase proteins which respond to colchicine prophylactic treatment. None of the patients carry pathogenic MEFV variants.

    Objectives: To identify a causative gene responsible for the autoinflammatory phenotype transmitted in 3 successive generations, in 3 members of the same family.

    Methods: The index patient, the grandmother, was screened with Fever panel including the coding regions and UTRs of MEFV, MVK, TNFRSF1A, NLRP3, NOD2, CECR1, TMEM173, PSTPIP1, NLRP12, LPIN2, PLCG2, CARD14, SLC29A3, IL10RA, NLRC4. After annotation candidate variants were selected via filtering based on frequency and functionality. Candidate variant (p.Arg228Cys) on PSTPIP1 gene was confirmed by Sanger sequencing in grandmother, daughter, and grandchild. Analysis of the possible effect of the variant on protein and transcription levels of PSTPIP1, MEFV and IL1b were performed through kit based-ELISA and Q-PCR methods with the protein and cDNAs, isolated from PBMCs of our three patients and five healthy controls.

    Results: We detected a rare variant; p.Arg228Cys on PSTPIP1 gene, in the index patient and confirmed the variant in her daughter and grandchild. Mutations in the PSTPIP1 gene is related with PAPA (pyogenic arthritis, pyoderma gangrenosum, acne) syndrome. This rare variant is located on F-BAR domain of PSTPIP1 protein, as other PAPA-related pathogenic variants, which is important for PSTPIP1 forming functional trimeric complex with pyrin protein. This binding results in conversion of pro-IL1b to IL1b.Protein levels of PSTPIP1, MEFV and IL1b of patients (44.78 ± 17.47mg/ml , 66.65 ± 31.81 mg/ml , 201.6 ± 63.54 mg/ml respectively) were higher than healthy controls (18.62 ± 3.741 mg/ml, 21.33 ± 6.738 mg/ml, 150.5 ± 15.22 mg/ml) respectively. However, when we calculate the fold change of transcript levels according to GAPDH endogenous gene, an increase in patients compared to healthy controls was detected only for PSTPIP1 (0.2928 ± 0.1324, 0.2136 ± 0.068).

    Conclusion: The systemic autoinflammatory phenotype inherited through three generations in a Turkish family was related to p.Arg228Cys, a rare variant on PSTPIP1. However the mutations in PSTPIP1 gene are associated with PAPA, besides the autosomal dominant transmission, the phenotype of our patients is not compatible with the classical manifestations of this syndrome. Some of these FMF-like disease phenotypes may be attributed to locus heterogeneity such that similar phenotypes may be caused by mutations in different genes of a specific pathway.

    Disclosure of Interest

    None Declared

    Dermatology and Autoinflammation

    P1092 Long-term treatment with ustekinumab for generalized pustular psoriasis in a child with heterozygous mutation in the IL36RN and TYK2 gene

    Troels Herlin1, Mette Christiansen2, Sofie E. Jørgensen3, Christian Høst1, Mia Glerup1, Jens E. Veirum1, Dorte A. Larsen4, Lars Iversen5, Mia Glerup1, Birgitte Mahler1, Trine H. Mogensen6
    1Pediatrics; 2Clinical Immunology, Aarhus University Hospital; 3Biomedicine, Aarhus University; 4Ophthalmology; 5Dermatology; 6Infectious diseases, Aarhus University Hospital, Aarhus , Denmark
    Correspondence: Troels Herlin

    Introduction: Recessive mutations in the IL36RN gene have been identified as the genetic determinants for early onset generalized pustular psoriasis which in its homozygous form results in deficiency of interleukin-36-receptor antagonist (DITRA). Non-receptor tyrosine-protein kinase Tyk2, member of the JAK family, is encoded by the TYK2 gene, which has been identified as a psoriasis susceptibility locus.

    Objectives: To describe the striking long-term effect of ustekinumab, a monoclonal antibody against the p40 subunit of both IL-12 and IL-23, in a girl with DITRA phenotype with heterozygous mutation in the IL36RN and TYK2 gene recalcitrant for several other treatments.

    Methods: Girl, now 5-year-old, being 1st child of unrelated Danish parents, prenatally diagnosed with tetralogy of Fallot (ToF). Array CGH revealed normal karyotype. Pregnancy and delivery were uneventful. Mother has been treated for hemorrhagic proctitis and psoriasis vulgaris, father is healthy. When 3-months-old she presented with scaly psoriasiform plaques. Shortly after operation for ToF at age 4 months she had a precipitous flare of numerous pustules covering more than 80% of skin area. Skin biopsy was compatible with pustular psoriasis.

    Whole exome sequencing and Sanger sequencing was performed.

    Results: Whole exome sequencing identified a heterozygous variant in the IL36RN gene (c.338C>T, p.S113L) with a high CADD score of 27.8 as well as a heterozygous variant in the TYK2 gene (c.157G>A, p.A53T), CADD score 25.1. IL36RN was Sanger sequenced employing DNA from keratinocyte and melanocyte cultures from skin biopsies; the S113L variant was identified in DNA but not present in mRNA.

    Methotrexate treatment was initiated together with topical corticosteroids without any effect on the cutaneous lesions. On suspicion of phenotypic equivalent to DITRA, anakinra (4 mg/kg/day increased to 8 mg/kg/day) caused some improvement although never complete regression of the pustular elements. After 8 weeks anakinra treatment was shifted to infliximab 6.5 mg/kg/dose given three times with 2 weeks interval and thereafter every 4 weeks. The skin lesions, as well as the general condition, improved markedly within few days, but after 2 months she developed a marked flare of pustular skin lesions. She developed an anaphylactoid reaction towards infliximab infusion and the medication was stopped. Treatment with etanercept did not show any improvement, in fact, she developed worsening of the pustulosis. Ustekinumab (0.9 mg/kg subcutaneously every 9 weeks) was then given which had a marked and sustained effect. The treatment with ustekinumab has been well tolerated and gradually anakinra, methotrexate and topical corticosteroids could be stopped. After 1 year of treatment, ustekinumab was paused to allow vaccination for MMR. However, after 5 months intermission she flared again with numerous pustular lesions and ustekinumab treatment was restarted, again with a striking efficacy. She has now been treated with ustekinumab for more than 48 months, the last 43 months as monotherapy.

    Conclusion: Although only homozygous mutations in the IL36RN gene have been associated with DITRA we believe that in our patient the combined heterozygous variants in the IL36RN and TYK2 gene play an essential role in the development of early onset generalized pustular psoriasis, not unlike DITRA syndrome. In contrast to anakinra and infliximab, which only demonstrated a short-lived and partial effect, ustekinumab monotherapy, which has been well tolerated, has enabled complete long-term remission for more than 4 years.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Non-monogenic SAIDs (clinical)

    P1093 Performance of newly proposed periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome criteria in regions endemic for famillial Mediterranean fever (FMF)

    Amra Adrovic, Mehmet Yildiz, Neslihan Gucuyener, Ipek Ulkersoy, Melisa Kanber, Sezgin Sahin, Oya Koker, Kenan Barut, Ozgur Kasapcopur
    Pediatric Rheumatology, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey
    Correspondence: Amra Adrovic

    Introduction: The periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is an autoinflammatory condition characterized by regularly recurrent episodes of high fever lasting 3 to 6 accompanied by aphthosis, cervical adenitis, and pharyngitis, in the absence of upper respiratory tract infections. Diagnosis of PFAPA could be challenging since it shares common characteristics with other auto-inflammatory conditions, including familial Mediterranean fever (FMF), hyper IgD syndrome etc. A relevant number of patients with monogenic periodic fevers also meet the diagnostic criteria for PFAPA syndrome. Especially in regions endemic for FMF, it would be important to avoid useless genetic testing and to be able to identify PFAPA patients by using clinical classification criteria. Despite the high specificity, widely used Marshall’s criteria have been shown to have low specificity. An international consensus among experts has been established recently, in order to define a set of classification criteria for PFAPA syndrome with a better performance in term of sensitivity and specificity.

    Objectives: We aimed to evaluate the performance of recently proposed PFAPA criteria, in order to assess their utility in regions endemic for FMF.

    Methods: Patients diagnosed with PFAPA syndrome, FMF and juvenile idiopathic arthritis (JIA) were consecutively included in the study. PFAPA diagnosis has been established by Marshall’s criteria. Patients with FMF and JIA were diagnosed according to Turkish pediatric FMF criteria and ILAR criteria, respectively. Two investigators blindly evaluated all of patients for the newly proposed PFAPA criteria.

    Results: A total of 321 PFAPA, 118 FMF and 45 JIA patients with mean age of 7.23±2.9, 14.7±3.09, 13.5±4.6 years, respectively, were included in the study. A 45 % (146/323) of PFAPA syndrome, 50% (59/118) of FMF and 58% (23/45) of JIA patients were female. We found quite high sensitivity (90%) of newly proposed PFAPA criteria: 289 out of 321 (90%) patients followed up as PFAPA syndrome fulfilled newly proposed PFAPA criteria, as well. When applied to patients diagnosed with FMF and JIA, 46 out of 118 (39%) FMF and 10 out of 45 (22%) JIA patients also fulfilled newly proposed PFAPA criteria. Specificity of recently proposed PFAPA criteria was found to be 61% and 77%, among FMF and JIA patients, respectively. Positive predictive value was 86% and 97%, negative predictive value was 69% and 50% for FMF and JIA patients, respectively.

    Conclusion: Recently proposed PFAPA criteria have satisfactory high sensitivity. Specificity of recently proposed criteria is still under expectation in regions endemic for FMF. Multicentric studies with higher patients’ number in different regions are needed in order to provide more relevant data on performance of newly proposed PFAPA criteria.

    Disclosure of Interest

    None Declared

    P1094 Spine involvement in patients with non-bacterial multifocal osteomyelitis

    Dmitry Alekseev1, Irina Nikishina1, Dmitry Zubkov1, Aleksandr Smirnov2, Olga Kostareva1
    1Pediatric Rheumatology; 2Radiology, V.A. Nasonova Research Institute of Rheumatology, Moscow, Russian Federation
    Correspondence: Dmitry Alekseev

    Introduction: Non-bacterial osteomyelitis (NBO) is a rare inflammatory disease of the musculoskeletal system, which is difficult to diagnose and treat. Assumed auto-inflammatory nature of the disorder.

    Objectives: The analysis the features and outcome of axial skeleton (AxS) involvement in patients (pts) who were observed in our department for the last 10 years with multifocal NBO.

    Results: Among the whole group of pts with NBO (n = 37) we identified 14 pts with AxS involvement including the lesions of the vertebral bodies and sacroiliac joints; the majority were girls (n = 10, 71.4%). Age at disease onset was 10.1 years in average (from 1.4 to 13.3, Me 9.5). There are no significant differences in clinical and laboratory features between children of different sex and age. Patients underwent standard rheumatologic examination; in order to examine all localizations of the skeletal lesion, a scintigraphy and/or “whole body” MRI scan was performed; a bone biopsy was performed on 5 pts. Other possible causes of damage were excluded, especially infection and oncology.

    AxS lesions were localized in different parts of the spine: cervical – in 5 pts (35.7%), thoracic - 7 (50.0%), lumbar - 3 (21.4%), sacral - 1 (7.1%). Involvement of 2 spinal regions was detected in 5 pts, 3 regions - in 1. There was a lesion of the adjacent regions: cervical and thoracic - in 3 patients; thoracic and lumbar - in 1; cervical, thoracic and lumbar - in 1. In 4 pts 3 vertebral bodies were affected, in 2 - 4, in 8 - 1, in 1 - 17; lesion of the adjacent vertebrae was revealed in 7 pts (50.0%). MRI revealed inflammatory changes, with the presence of an increase in signal intensity in STIR images. In 6 pnts - destructive damage of the vertebrae, in 4 pts – with a decrease in the height of the vertebrae.Peripheral bone lesions were presented with damage of the bones of the extremities in 6 pts, metaphysis mostly (in 3 - the proximal femur, in 2 - the knee and ankle joints), in 1 patient - the distal radius, in 1 patient – tarsal. 9 pts had chronic arthritis of the limbs joints: in 5 pts oligoarthritis with lesions of the hip and knee joints, in 2 pts with polyarthritis with lesions of the hip, knee, ankle joints. Definite evidences of the sacroiliitis was found in 12 pts (85,7%), in 7 pts (50%) the diagnosis of Juvenile ankylosing spondylitis (JAS) fulfilled to New-York modified criteria were established, including severe variant with total ankylosis of sacroiliac and joints, vertebral bodies and multiple syndesmophytes in - 4 pts. But HLA B27 antigen was present just in 5 among 14 pts. Psoriasis of the palmar-plantar localization - in 1 patient; acnae conglobata - 1. Inflammatory bowel disease, uveitis - not identified. Laboratory parameters of activity were detected in the active period of the disease in 6 pnts: ESR acceleration up to 40 mm/h, increase in CRP - up to 73 mg/l; these patients had manifestations of chronic arthritis.

    Treatment included NSAIDs (all), methotrexate (n=4), sulfasalazine (n=3), bisphosphonates (n=5), TNF inhibitors (n=6). Orthopedic measures: regimen with restriction of movements; during the destruction of the vertebrae, a corset was used (n=3), an operation with the fixation of the vertebrae with metal construction (n=3).

    Conclusion: (1) AxS involvement in pts with NBO is a prognostic unfavorable sign associated with the rapid development of an advanced stage of JAS, including the development of syndesmophytes and ankylosis of intervertebral joints.

    (2) Detection of highly active, according to MRI, sacroiliitis in children requires further examination for multifocal NBO.

    Disclosure of Interest

    None Declared

    P1095 DNASE1L3 variant in hypocomplementemic urticarial vasculitis syndrome identifies a different clinical phenotype

    Marco Ranalli1, Chiara Passarelli2, Virginia Messia3, Manuela Pardeo3, Emanuela Sacco3, Antonella Insalaco3, Marina Vivarelli4, Fabrizio De Benedetti3, Claudia Bracaglia3
    1Pediatric Department, La Sapienza University of Rome; 2Unit of Laboratory of Medical Genetics; 3Division of Rheumatology; 4Division of Nephrology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy
    Correspondence: Claudia Bracaglia

    Introduction: Hypocomplementemic urticarial vasculitis syndrome (HUVS) is a rare disease characterized by persistent urticarial lesions and hypocomplementemia associated with systemic features involving musculoskeletal, pulmonary, renal and gastrointestinal systems. Systemic lupus erythematosus (SLE) develops in >50% of patients with HUVS, although the pathogenesis is unknown.

    Objectives: We describe 6 paediatric patients with HUVS, three of whom carry a homozygous variant of DNASE1L3 and present a peculiar clinical phenotype.

    Methods: A Targeted Resequencing using a panel including genes already known to be mainly associated to Interferonopathies Lupus-like (DNASE1DNASE2DNASE1L3TREX1) on the Illumina NextSeq® platform was performed. All variants identified were confirmed by Sanger sequencing and, when possible, family members were tested to study the segregation of identified variants. We applied in silico studies only to variants with an allelic frequency ≤1%.

    Results: All patients described are Caucasian and 3 of them are female. Two patients presented at onset with extended cutaneous manifestation, joints and abdominal involvement with cholecystitis. They did not develop renal or pulmonary involvement. In contrast, the other four patients presented a more severe disease. All of them developed renal involvement (from microhaematuria up to nephrotic syndrome) with renal biopsy showing mesangial glomerulonephritis in three patients and pauci-immune glomerulonephritis (ANCA negative) in one. Moreover, two of them developed also pulmonary vasculitis (Table 1). A homozygous DNASE1L3 variant (c.290_291delCA) was identified in three of these patients. All of them were treated with glucocorticoid and dapsone at onset. Cyclophosphamide, mycophenolate mofetil and azathioprine were used in patients with renal involvement. None of them developed SLE.

    Conclusion: HUVS is very rare disease in childhood. Approximately 50% of HUVS patients develop SLE. Genetic susceptibility to SLE is recognized and DNASE1L3-related SLE have been reported. Özçakar et al. have described 5 children from two families with HUVS who carry the same variant on DNASE1L3 that we report here (1). Our patients confirm that variant in DNASE1L3 can cause HUVS and support the hypothesis that this variant is responsible of a more severe phenotype with major organ involvement (renal and pulmonary). Patients with HUVS need to be followed very strictly for the risk to develop SLE. Presence of variant in DNASE1L3 can identify patients with more severe disease and high risk to develop major organ involvement. These patients need more aggressive and possibly life-long immunosuppressive treatment.

    Reference

    (1) Ozçakar ZB et al. DNASE1L3 Mutations in Hypocomplementemic Urticarial Vasculitis Syndrome. Arthritis Rheum. 2013 Aug;65(8):2183-9.

    Disclosure of Interest

    M. Ranalli: None Declared, C. Passarelli: None Declared, V. Messia: None Declared, M. Pardeo: None Declared, E. Sacco: None Declared, A. Insalaco: None Declared, M. Vivarelli: None Declared, F. De Benedetti Grant / Research Support from: Novartis, Novimmune, Hoffmann- La Roche, SOBI, AbbVie, Pfizer, C. Bracaglia: None Declared

    Table 1 (abstract P1095). Patients’ clinical characteristics

    P1096 A case of idiopathic recurrent pericarditis treated with anakinra but unresponsive to canakinumab

    Francesca Ricci, Silvia Ghetti, Rossana Razza, Camilla Dallavilla, Giulia Verzura, Alessandra Manerba, Marco Cattalini
    University of Brescia and Spedali Civili di Brescia, Pediatric Clinic, Brescia, Italy
    Correspondence: Marco Cattalini

    Introduction: Recurrent pericarditis complicates up to 30% of acute pericarditis cases. First choice initial therapy are non-steroid anti-inflammatory drugs (NSAIDS) and colchicine. Corticosteroids have been used in case of partial response to first-line therapy, allowing rapid control of inflammation but with a high rate of steroid-dependency. Quite recently Anakinra, a recombinant human interleukin-1 competitive receptor antagonist that blocks both IL-1a and b, have been demonstrated to be a valid option in such cases. When to start Anakinra, and how to taper it in case of remission are still open questions, as if other anti-IL1 treatments may also be an option.

    Objectives: We present herein a case of recurrent pericarditis well controlled by Anakinra, that became Anakinra-depent and relapsed after switching to Canakinumab, a recombinant human interleukin-1beta competitive receptor antagonist.

    Results: Results: in September 2008, S.S., a 14-years old boy (weight 69,8 kg, height 160 cm) was admitted in our Emergency Department complaining of three days of acute chest pain that increased in supine position, fever and dyspnoea. A chest X-ray showed heart enlargement. Past medical history revealed that in the previous eight months, S. presented three similar episodes of chest pain, that were effectively home-treated with NSAIDS. Physical examination showed fair general condition, paleness and friction rubs at the cardiac auscultation. Lab tests revealed elevated CRP (270 mcg/ml) and ESR (79 mm/h). At trans-thoracic echocardiography revealed a diffuse pericardial effusion of 300 cc. Serologic studies didn’t suggest a clue for the aetiology of episode. Treatment with naproxen (250 mg twice a day for 7 days) and prednisone (initial dosage 30 mg/die) lead to rapid control of symptoms, inflammatory markers and US findings within few days. From there on, any attempt to taper steroids, even of few mg, led to rapid relapse of symptoms, inflammatory markers, and pericardial effusion. For this reason colchicine was added to initial treatment (1,5mg once a day) with no improvement and, after 3 months from the onset, anakinra (at the dosage of 100 mg/die) was introduced, with prompt normalization of the clinical picture and US findings. Steroids were withdrawn and in the following nine years numerous attempt were made to taper Anakinra but, every time the interval between injection was extended to 4 days, SS experienced a relapse. Genetic tests for TRAPS and Familial Mediterranean Fever resulted negative. In June 2018, we tried to modify the therapy by substituting anakinra with canakinumab, in order to reduced the number of injections. Unfortunately, 48 hours after the administration of canakinumab 150 mg, the patient experienced a flare with thoracic pain and high inflammatory markers. The flare was managed by steroids (prednisone 40mg a day) and, after two weeks, a second dose of Canakinumab was administered. Even after this up-titration it was not possible to taper steroids. Therefore, after an adequate period of wash-out, Anakinra was reintroduced with rapid and stable disease control and steroid withdrawal.

    Conclusion: our case confirms that Anakinra may be a good option for recurrent pericarditis unresponsive to first line treatment, but drug withdrawal may be difficult. Although limited to this case, it seems that canakinumab is not a valid option. One may speculate that recurrent pericarditis (at least in some cases) is an IL-a – mediated disease

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1097 Efficacy of canakinumab as first-line biologic agent in adult-onset still’s disease

    Giulio Cavalli, Alessandro Tomelleri, Corrado Campochiaro, Elena Baldissera, Giacomo De Luca, Lorenzo Dagna
    Unit of Immunology, Rheumatology, Allergy and Rare Diseases (UniRAR), Vita-Salute San Raffaele University, Milan, Italy
    Correspondence: Giulio Cavalli

    Introduction: Adult-onset Still’s disease (AOSD) is a rare auto-inflammatory condition characterized by fever, arthritis, skin rash, and multi-organ inflammation. The pathogenesis of AOSD is centrally mediated by the pro-inflammatory cytokine interleukin (IL)-1β; anakinra, a recombinant inhibitor of the IL‑1β receptor, has thereby become the cornerstone of biologic therapy for AOSD. More recently, a new agent blocking IL‑1β has become available, that is, the monoclonal antibody canakinumab.

    Objectives: So far, canakinumab has been used in the treatment of AOSD following failure of multiple lines of biologic therapy, including anakinra. Here, we describe the use of canakinumab as first-line biologic agent in 4 patients with AOSD, and report the highly promising results of this treatment approach.

    Methods: Four patients with severe, DMARD-refractory AOSD received canakinumab at the approved dose of 4 mg/kg once every 4 weeks following failure of conventional treatment with corticosteroids and methotrexate. Patient characteristics and response to therapy were recorded.

    Results: In all patients, treatment with canakinumab led to striking and rapid clinical responses, within days of initiation. Fever and skin rash disappeared first and did not recur, followed by progressive and sustained improvement in arthritis. Inflammatory organ involvement also responded to treatment: for example, we observed resolution of pericardial inflammation in two patients, as revealed by cardiac ultrasound; another patient had hepatosplenomegaly, which also reverted to normal upon treatment. A striking reduction in serum pro-inflammatory markers C-reactive protein, erythrocyte sedimentation rate, and ferritin mirrored the efficacy on clinical manifestations. These effects amounted to a significant decrease in the modified Pouchot score for measuring disease severity (average score before canakinumab treatment: 6; after: 1.25; statistical significance of difference evaluated with Mann-Whitney: p=0.02), and allowed for tapering or discontinuation of concomitant therapy: specifically, corticosteroid treatment was completely discontinued in two patients and substantially reduced in two patients, whereas methotrexate was stopped in one patient.

    Conclusion: In this study, first-line biologic therapy of AOSD with canakinumab resulted in rapid and marked efficacy, ultimately leading to full clinical remissions in all patients, and allowing for robust steroid-sparing effects.

    Disclosure of Interest

    None Declared

    P1098 CARRA autoinflammatory disease network development- improving care and fostering research in systemic autoinflammatory diseases (SAIDS)

    Julie Cherian1, Theresa L. Wampler Muskardin2, Marinka Twilt3, Ronald M. Laxer4, Jonathon S. Hausmann5, Cagri Yildirim-Toruner6, Maria J. Gutierrez7, Nadine Saad8, Grant S. Schulert9, Evan J. Propst10, Greg Licameli11, Akaluck Thatayatikom12, Gil Amarilyo13, Liora Harel14, Ian C. Michelow15, Peter F. Wright16, Yuriy Stepanovskiy17, Lakshmi N. Moorthy18, Karen Durrant19, Polly J. Ferguson20, Lori B. Tucker21, Fatma Dedeoglu22, Sivia K. Lapidus23 and CARRA Autoinflammatory Network Development Work Group
    1Pediatrics, Stony Brook Children’s Hospital,Stony Brook, NY; 2Medicine and Pediatrics, NYU School of Medicine, NY, United States; 3Pediatrics, Alberta Children's Hospital, Calgary, AB; 4Pediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 5Pediatrics, Boston Children's Hospital, Beth Israel Deaconess Medical Center, Boston, MA; 6Pediatrics, Nationwide Children's Hospital, Columbus, OH; 7Pediatrics, Johns Hopkins University School of Medicine, Baltimore; 8Pediatrics, Hospital for Special Surgery, NY; 9Pediatrics, Cincinnati Children's Hospital, Cincinnati, OH, United States; 10Otolaryngology, Hospital for Sick Children, Toronto, ON, Canada; 11Otolaryngology, Boston Children's Hospital, Boston, MA; 12Pediatrics, University of Florida, Gainesville, FL, United States; 13Pediatrics, Schneider Hospital, Tel Aviv University; 14Pediatrics, Schneider Hospital,Tel Aviv University , Tel Aviv, Israel; 15Pediatrics, Alpert Medical School of Brown University, Providence, RI; 16Pediatrics, Dartmouth-Hitchcock Medical Center,NH, United States; 17Shupyk National Medical Academy of Postgraduate Education, Kyiv, Ukraine; 18Pediatrics, Rutgers/RWJ Medical School, New Brunswick, NJ; 19Pediatrics, Kaiser Permanente San Francisco Medical Center, San Francisco, CA; 20Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA, United States; 21Pediatrics, BC Children's Hospital, UBC Vancouver, BC, Canada; 22Pediatrics, Boston Children's Hospital, Boston, MA; 23Pediatrics, Joseph M. Sanzari Children's Hospital Hackensack Meridian Health, Hackensack, NJ, United States
    Correspondence: Julie Cherian

    Introduction:

    International registries have significantly enhanced the understanding of the genetics, phenotype, prognosis, and treatment responses in Systemic Autoinflammatory Diseases (SAIDs) in the studied populations, and such understanding would be further enhanced by expanding these efforts to include a genetically heterogeneous Northern American cohort.

    Objectives:

    To explore the value of developing a Childhood Arthritis and Rheumatology Research Alliance (CARRA) Autoinflammatory Disease Network in enhancing quality of care, therapeutic monitoring, and determining outcomes in SAIDs.

    Methods: A team within CARRA composed of pediatric rheumatologists, infectious disease physicians, immunologists, otolaryngologists, geneticists, parents/patients with autoinflammatory disorders, and members of the Autoinflammatory Alliance met in person and via teleconference to discuss the benefits of autoinflammatory disease networks from 2016 to the present. Physicians who were involved in already-established autoinflammatory disease clinics in North America shared the benefits of having such programs. A comprehensive literature search on the topic using keywords such as “EuroFever”, “pharmacosurveillance”, and “autoinflammatory registry” were reviewed. Nominal group technique was employed.

    Results:

    A consensus was reached regarding the benefits, feasibility, and intent of developing a CARRA Autoinflammatory Disease Network that will be instrumental for providing quality care for children affected by SAIDs and their families. The network will provide improvements in areas of clinical care, enhanced research, patient and family access to accurate and useful disease-related education, and facilitation of international collaborations. The group’s literature search highlighted the benefits of this approach in rare diseases in preventing diagnostic delay leading to increased morbidity and mortality, understanding the epigenetics of SAIDs that remain poorly understood, and providing an opportunity for pharmacosurveillance in a cohort of patients potentially exposed to biologics.

    Conclusion:

    The development of a CARRA Autoinflammatory Disease Network will facilitate earlier diagnoses, education, and access to expert as well as multidisciplinary quality care.Additionally, this network will create an infrastructure for future clinical and translational investigations into long-term outcomes in SAIDs, epidemiological studies, comparative effectiveness trials, and implementation of optimum standards of care for SAIDs.Given the genetically diverse populations in North America, an autoinflammatory network would facilitate collaborations with international colleagues to benefit patients worldwide.

    Disclosure of Interest

    None Declared

    P1099 The Helios (Hacettepe University Electronic Research Forms) registry: use of biologic drugs in autoinflammatory diseases

    Selcan Demir1, Ezgi D. Batu1, Fuat Akal2, Erdal Sağ1, Ummusen Kaya Akca1, Elif Arslanoğlu Aydın3, Emil Aliyev3, Kübra Yüksel3, Armağan Keskin3, Yelda Bilginer1, Seza Ozen1
    1Department of Pediatric Rheumatology, Hacettepe University, Medical Faculty; 2Department ofComputer Engineering, Hacettepe University; 3Department of Pediatrics, Hacettepe University, Medical Faculty, Ankara, Turkey
    Correspondence: Selcan Demir

    Introduction: Autoinflammatory diseases (AID) are characterized by a dysregulation of innate immunity leading to uncontrolled inflammation. The treatment in AID is critical to control the disease activity, to prevent complications, and to improve the health-related quality of life. Biologic drugs have revolutionized the treatment and outcomes in AID.

    Objectives:Herein we aim to present the clinical characteristics of children to whom biologic drug therapy was initiated for the management of AID.

    Methods: A web-based registry called the Helios Registry (Hacettepe univErsity eLectronIc research fOrmS) has been formed to evaluate the data of all children on biologic treatment. We have been enrolling patients since August 2018 retrospectively and prospectively. We have analyzed the data about the general characteristics of the patients, treatment, the biologic drug used, and adverse effects. Only the patients with the following diagnoses were included: systemic juvenile idiopathic arthritis (SJIA), familial Mediterranean fever (FMF), cryopyrin associated periodic syndrome (CAPS), and chronic recurrent multifocal osteomyelitis (CRMO).

    Results: Of 60 patients included, 19 had FMF (31.7%), 24 had sJIA (40%), 10 had CAPS,(16.7%), and 7 had CRMO (11.7%). Their median age was 10.7 (2-20) years old and disease duration was 2.8 (0-6) years, at the time of biologic drug initiation. 58.3% were currently on canakinmab, 20% anakinra, 10% tocilizumab, 10% etanercept, and 1.7% adalimumab. 63.3% of our patients had previously used at least one other biologic drug. The rate of glucocorticoid use before biologic treatment was 56.6%. The median duration of glucocorticoid treatment after initiating biologic drugs was 7.4 months.56 (93%) patients achieved remission on biologic therapy. There were 15 patients (25%) who received tuberculosis prophylaxis due to positive tuberculin skin test (diameter≥10 mm) and there was no Quantiferon test positivity. Thirteen adverse events (AE) had been noted. 2 of them were serious events as anaphylaxis due to tocilizumab infusions. The rest of the adverse events were mild thrombocytopenia (n=2), varicella infection (n=1), and local side effects (n=8). The median number of the infections per year was one and there were no death or malignancy.

    Conclusion: The most commonly prescribed biologic drugs were IL-1 inhibitors especially for patients with IL-1-mediated AID (FMF, CAPS, and SJIA). The biologic treatment in AID seems effective and reasonably safe.

    Acknowledgment

    This registry was supported by SOBİ

    Disclosure of Interest

    None Declared

    P1100 Pediatric Behcet’s disease with sinus venous thrombosis: three center experience from Turkey

    Selcan Demir1, Ceyhun Açarı2, Özge Basaran3, Erdal Sağ1, Yelda Bilginer1, Şevket Erbil Ünsal2, Seza Ozen1
    1Department of Pediatric Rheumatology, Hacettepe University Faculty of Medicine, Ankara; 2Department of Pediatric Rheumatology, Dokuz Eylül University Faculty of Medicine, İzmir; 3Department of Pediatric Rheumatology, Ankara Child Health and Disease Hematology Oncology Training and Research Hospital, Ankara, Turkey
    Correspondence: Selcan Demir

    Introduction: Behçet’s disease (BD) is a multisystem vascular-inflammatory disorder including the cutaneous, articular, gastrointestinal, and/or central nervous systems (CNS).Adult BD patients with CNS involvement mostly present with parenchymal form, including meningoencephalitis, headache and pleocytosis of the cerebrospinal fluid while non-parenchymal form is more common among pediatric BD patients and younger age. Non-parenchymal form usually presents with cerebral venous sinus thrombosis (CVST) and intracranial hypertension. Neurological symptoms in children and adolescents can be confused with many other disorders and may be the initial symptom of BD.

    Objectives: To report our experiences of the juvenile Behçet’s Disease patients with cerebral venous sinus thrombosis (CVST) and to review previous studies reporting the clinical characteristics and outcomes of Juvenile Behçet’s Disease with CVS

    Methods: The patients who met Pediatric Behçet's Disease (PEDBD) classification criteria for juvenile Behçet’s Disease from 3 referral centers in Turkey were reviewed retrospectively. Disease activity was assessed by BD current activity form (BDCAF). A systematic review of literature of all published data was conducted.

    Results: The study group consisted of 12 juvenile BD patients with CVST. At the time of CVST diagnosis, the most common symptom was headache (%100), followed by vomiting (25%), blurred vision (16.6-7%), and disturbances in eye movements (16.7%). Six (50 %) patients presented with sinus venous thrombosis as an initial symptom. Transverse sinus was the most frequently affected sinus (9/12, 75%) followed by superior sagittal sinus (8/12, 66.6%) and sigmoid sinus (1/12, 8.3%). The median (minimum-maximum) BDCAF was 6 (5-8). Four children (33.3%) had another venous thrombosis apart from CVST. All patients received pulse methylprednisolone for three consecutive days continued with oral prednisolone. Steroid treatment was tapered and discontinued minimum in six months. Eleven patients received azathioprine concomitant to steroid treatment at the time of CVST. All the patients received anticoagulant therapy concomitantly. Only one patient had relapse. Median (min-max) follow-up period was 4 years (1-10).In the literature review, we identified nine articles, describing 35 pediatric CVST patients associated with BD. Thirty patients achieved remission, while five patients had residual neurologic deficit.

    Conclusion: Further multicenter studies with more patients and prospective follow-up may help us to understand the whole spectrum in these patients.

    Disclosure of Interest

    None Declared

    P1101 PFAPA- the Irish experience in a tertiary autoinflammatory clinic

    Ana-Louise Hawke1, Jayne MacMahon1, Emma Jane MacDermott1, Karina Butler2, Ronan Leahy3, Patrick Gavin2, Orla Killeen1
    1Paediatric Rheumatology; 2Paediatric Infectious Disease; 3Paediatric Immunology, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
    Correspondence: Ana-Louise Hawke

    Introduction: Periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome is the most common autoinflammatory disorder in childhood, with multifactorial, polygenic causes postulated.

    Children usually present before the age of 5 years with periodic fevers and one or more associated symptoms of stomatitis, pharyngitis and adenitis, flares usually resolving in adolescence.

    Treatment options include corticosteroids for flares and colchicine, tonsillectomy and increasingly biological agents to prevent disease flare.

    Objectives: The aim of this study was to appraise the presentation and management of children with PFAPA attending a tertiary Autoinflammatory Clinic since it’s establishment in 2016.

    Methods: A retrospective observational chart review of all children with PFAPA, confirmed or likely, attending Autoinflammatory clinic at Our Lady’s Children’s Hospital, Crumlin Dublin from January 2016.

    Data were collected on basic demographics, age at presentation, route of referral, symptoms and signs and inflammatory markers during disease episodes and non-episodes. Details of genetics analysis if performed, were included. Information was also gathered on any treatment methods used and their effect including oral corticosteroids, colchicine, biological agents and tonsillectomy. Results were analysed using Microsoft Excel.

    Results: 13 children were identified as having PFAPA, including 2 sets of siblings. The median age of disease onset was 16 months, ranging from 4 months to 4 years of age. The route to referral was through Immunology (4 patients), Rheumatology (6 patients) and Infectious disease (3 patients) clinics. All children presented with episodic, recurrent febrile episodes with a median duration of 3-4 days and a range of associated features. (See table 1) 3 children presented with the triad of stomatitis, adenitis and pharyngitis.

    Sixty nine percent of patients had documented raised inflammatory markers during a flare, with 84% having high serum amyloid A levels, the highest documented being 1220mg/l (normal <10). 69% of patients had normal inflammatory markers and 76% normal serum amyloid A documented during disease quiescence.

    Genetic testing in 6 children was negative for other causes of Hereditary Autoinflammatory disorders- TRAPS FMF.

    2 sisters were found to have a benign variant NLRP mutation. Serum IgD levels normal in 9 and raised in 1 patient.

    11 patients were initiated with treatment that included a stat of 3-day trial of oral corticosteroids, all of whom reported some improvement in symptoms, but 2 reporting rebound flares. 11 were treated with Colchicine, 9 of whom reported some benefit. Tonsillectomy was performed in 5 patients, 3 who found benefit. Anakinra was used to treat 2 and 1 patient was commenced on Adalimumab.

    Conclusion: This study gives an over view of the burden of disease imposed by PFAPA on an Irish population. While all the children presented with fevers, not all had the classic triad of aphthous stomatitis, pharyngitis and cervical adenitis.Raised CRP ESR were seen in the majority of patients and 84% had raised amyloid levels during disease flare. The majority of patients had prompt relief of symptoms with an initiation trial of corticosteroid. Colchicine being frequently used with some success to prevent occurrence of flares. Tonsillectomy and biological agents were also used in some resistant/severe cases.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1101). Number of patients

    Systemic-onset JIA and AOSD

    P1102 Hepatitis A virus vaccination in autoinflammatory diseases under canakinumab and tocilizumab treatment

    Kenan Barut1, Amra Adrovic1, Sezgin Sahin1, Mehmet Yıldız1, Oya Koker1, Gamze Yalcin1, Omer Faruk Beser2, Bekir Kocazeybek3, Pelin Yuksel3, Ozgur Kasapcopur1
    1Pediatric Rheumatology, Istanbul University Cerrahpasa, Cerrahpasa Medical School; 2Pediatric Gastroenterology, Okmeydani Education and Training Hospital; 3Microbiology, Istanbul University Cerrahpasa, Cerrahpasa Medical School, Istanbul, Turkey
    Correspondence: Kenan Barut

    Introduction: Autoimmune, autoinflammatory mechanism and drugs used in treatment increase the risk of liver disease in patients with chronic rheumatic diseases. Hepatitis A vaccine is a highly effective vaccine that prevents both the formation and spread of clinical hepatitis. In childhood chronic rheumatic diseases, vaccination is of great importance. The risk of various infections increases with the immunosuppressive effect of both the disease and the drugs.Therefore, vaccination of these diseases is of high importance for the prevention of infectious diseases. Systemic juvenile idiopathic (SJİA) arthritis is a juvenile idiopathic arthritis (JİA) subtype with autoinflammatory pathogenesis. Steroid, methotrexate and anti-interleukin 1 and 6 are used for the SJİA treatment. Anti-interleukin 1 treatments are also used in the treatment of Cryopyrin-associated periodic syndromes (CAPS), which is also characterized with autoinflammatory pathogenesis.Studies on the efficacy and safety of vaccines in autoimmune and autoinflammatory diseases are limited.

    Objectives: The aim of this study was to investigate the efficacy and safety of hepatitis A vaccine in patients with autoinflammatory disease on anti-interleukin 1 and 6 treatment.

    Methods: This study was carried out in Pediatric Rheumatology outpatient clinic and Healthy Child clinic between June 2017 and November 2018. A total of 39 patients with autoinflammatory diseases on anti IL-1 and IL-6 therapy were initially evaluated but 25 of them were excluded due to anti-HAV IgG positivity. At the end, 24 patients withautoinflammatory diseases on anti IL-1, anti IL-6 therapyand 39 healthy participants who were seronegative for hepatitis A received two doses of the hepatitis A vaccine in a 0 and 6 month schedule. Hepatitis A virus (HAV) IgG antibodies were measured before vaccination and one month after last dose of the vaccine. Anti-HAV IgG titer as S/ CO;1.1, IU/L was considered positive and protective.

    Results: Total 24 patients with autoinflammatory condition (13 females, 11 males) and 39 healthy controls( 18 female, 21 male) were included in the study.

    Among patients with diagnosis of autoinflammatory disease, 19 were SJİA and 5 were CAPS patients. The mean age was 14.1±3.7 and 12.2±3.3 years respectively. Canakinumab was used in 15 (62.5%) and tocilizumab in 9 (37.5%) all patients. Among all SJİA patients, 10 (52.6%) were treated with canakinumab and 9(47.4%) were treated with tocilizumab. All patients with CAPS (n: 5) were using canakinumab. Among SJIA cases, 15(75%) were also using methotrexate and 14(70%) prednisolone.Anti-HAV IgG concentrations were measured one month after the last dose of hepatitis A vaccine. There was statistically significant difference between patients with autoinflammatory condition and healthy controls regarding the anti-HAV IgG titer (mean 5.3±1.5 IU/L) versus (10.5±7 IU/L) p<0.05. The rate of anti-HAV IgG seropositivity (cut-off 1.1 IU/L) in autoinflammatory disease (24/24 (100%) was significantly different comparing to healthy controls (33/39, 84.6%) (p=0.04). There was no disease flare of disease nor the adverse event detected in any patients after vaccination.

    Conclusion: Anti-HAV IgG seroconversion was detected in patients with autoinflammatory disease on anti-IL1 and anti - IL6 therapy 1 month after the last dose of hepatitis A vaccine. The response to vaccine did not differ between healthy children and patients with autoinflammatory disease under canakinumab and tocilizumab. In this study hepatitis A vaccine was found to be safe in autoinflammatory diseases with canakinumab and tocilizumab treatment.

    Disclosure of Interest

    None Declared

    P1103 Risk score of macrophage activation syndrome in patients with systemic juvenileidiopathic arthritis

    Simone Carbogno1, Denise Pires Marafon2, Giulia Marucci3, Manuela Pardeo3, Antonella Insalaco3, Virginia Messia3, Emanuela Sacco3, Fabrizio De Benedetti3, Claudia Bracaglia3
    1Pediatric Area, University of Milan; 2Pediatric Unit, Fondazione IRCCS Ca’ Grande Ospedale Maggiore Policlinico, Milan; 3Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy
    Correspondence: Claudia Bracaglia

    Introduction: Macrophage Activation Syndrome (MAS) is a severe, life-threatening, complication of rheumatic diseases in childhood, particularly of systemic Juvenile Idiopathic Arthritis (sJIA), occurring in approximately 25% of the patients with sJIA. The mortality rate of MAS is still significantly high. A score that identify sJIA patients who are at high risk to develop MAS would be useful in clinical practice. There are no parameters available to identify from onset sJIA patients with high risk to develop MAS in their disease course.

    Objectives: To evaluate whether routine laboratory parameters at disease onset may predict the development of MAS in patients with active sJIA. To define a risk score of MAS for sJIA patients using these parameters.

    Methods: Laboratory parameters of disease activity and severity (WBC, N, PLT, Hb, ferritin, AST, ALT, gGT, LDH, TGL, fibrinogen, D-dimer and CRP), were retrospectively evaluated in 86 sJIA patients referred to our Division of Rheumatology from 1998 to 2017 with at least one year of follow-up. Laboratory parameters were evaluated during active sJIA, without MAS, at time of hospitalization (T1) and before treatment for sJIA was started (T2). Patients were divided in two groups: group 1 (patients without history of MAS), group 2 (patients with at least one MAS episode during disease course). To calculate a MAS risk score, laboratory parameters, collected at T2, with a statistical significant difference between the two groups of patients were selected.

    Results: Thirty-three patients, that fulfilled the 2016 classification criteria for MAS [1] at time of sampling, were excluded from the analysis. Therefore, we analysed laboratory parameters of 53 patients with sJIA, 33 of whom without history of MAS (group 1) and 20 who developed at least one episode of MAS during disease course (group 2). Levels of ferritin, AST, LDH, gGT and TGL, collected at T2, were statistically significant higher in patients with a history of MAS compared to those without a history of MAS. For each of these parameters an arbitrary cut-off was defined. In order to define the final score an arbitrary rate was attributed to each parameter. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) were calculated to define the best scoring system. The scoring system with the best sensitivity was chosen (Table 1). A MAS risk score >3 identified 19 out of 20 sJIA patients with a history of MAS and 4 out of 33 sJIA patients without history of MAS.

    Conclusion: In conclusion we developed a MAS risk score based on routine laboratory parameters that are available worldwide, that can help clinicians to identify these patients early in the disease course. A validation in a larger population is ongoing.

    Reference

    [1] Ravelli A et al. 2016 Classification Criteria for Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A European League Against Rheumatism/American College of Rheumatology/Paediatric Rheumatology International Trials Organisation Collaborative Initiative. Ann Rheum Dis. 2016 Mar;75(3):481-9.

    Disclosure of Interest

    S. Carbogno: None Declared, D. Pires Marafon: None Declared, G. Marucci: None Declared, M. Pardeo: None Declared, A. Insalaco: None Declared, V. Messia: None Declared, E. Sacco: None Declared, F. De Benedetti Grant / Research Support from: Novartis, Novimmune, Hoffmann- La Roche, SOBI, AbbVie, Pfizer, C. Bracaglia: None Declared

    Table 1 (abstract P1103). Laboratory parameters and cut-off used to create the MAS risk scorein sJIA patients

    P1104 Adult onset Still’s disease complicated with haemophagocytic syndrome: successfull treatment with IVIG and Tocilizumab

    Ayse Cefle, Fatma Tuncer, Ayten Yazici
    Department of Internal Medicine Division of Rheumatology, Kocaeli University Faculty of Medicine, Kocaeli, Turkey
    Correspondence: Ayse Cefle

    Introduction: Adult onset Still's disease (AOSD) is anacute febrile syndrome observed in young adults. Typically, many organs are affected. Haemophagocytic syndrome is a life-threatening condition with high mortality rate. The rate of occurrence of the condition in AOSD patients is 5-10%.

    Objectives: A 24-year-old female patient with fever, rash, joint pain having started 1.5 months ago was admitted to our hospital. In physical examination; She had hepatosplenomegaly and multiple LAPs in bilateral cervical region as well as tenderness on the left wrist and right ankle. The rash was concomittant with fever. CRP, erythrocyte sedimentation rate, white blood cell and ferritin elevation were determined. Hepatitis markers, HIV, brucella, syphilis, CMV, toxoplasma, EBV were found to be negative. The anti-nuclear antibodies, rheumatoid factor and anti-CCP were negative,

    Methods: In the thorax CT, multiple lymph nodes were observed in the mediastinal (13 mm), bilateral hilar, axillary (15 mm largest) and bilateral lower neck. The patient's PET CT revealed increased metabolic rate in the neck, mediastinum, abdominopelvic region, mildly diffused increased metabolism in the bone marrow, and increased metabolism in the basal sections of both lungs. It was reported that lymphoproliferative disease should be included in the differential diagnosis. Right servical lymph node excisional biopsy was reported as reactive lymphoid hyperplasia. Bone marrow biopsy came as normocellular. Her bronchoscopy was normal. Thereforemalignancy and infection were excluded. Prednizolon 60 mg/d and methotrexate 15 mg/w were started.

    Results: The patient developed pancytopenia after two weeks and exhibited an increase in her triglyceride, D-dimer, LDH and hypofibrinogenemia developed. The patient had no atypical cells in the peripheral smear. IVIG as 2g/kg was added to the treatment with the diagnosis of haemophagocytic syndrome. After IVIG, her fever reduced, leukopenia thrombocytopenia improved, her sedimentation CRP reached normal limits. IVIG was administered 3 times with an interval of four weeks. However, one month after, she was admitted to our clinic with common myalgia, subfebrile fever, sore throat, swelling of the elbow, rash on the back. Leukocytosis with neutrophil dominance, increased levels of ferritin, LDH, eryhtrocyte sedimentation rate and CRP were noted. Her methylprednisolone dose was increased and Tocilizumab treatment (8 mg/kg/4w) was begun. After three months, clinical and laboratory findings were normal.

    Conclusion: Macrophage activation syndrome is a reactive form of haemophagocytic syndrome associated with rheumatic diseases. 71% of macrophage activation syndrome cases occur within the first month of AOSD diagnosis. Fever, cytopenia, hypertriglyceridemia, increased ferritin, LDH and liver enzymes, HSM, LAP,coagulopathy and elevation of fibrin degradation products are observed. The syndrome is characterized with haemophagocytosis in bone marrow and RES. Glucocorticoids, cyclosporine, anakinra, tocilizumab and IVIG are administered.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1105 Drug retention rate and predictive factors of drug survival for interleukin-1 inhibitors in systemic juvenile idiopathic arthritis

    Carla Gaggiano1, Jurgen Sota2, Antonella Insalaco3, Rolando Cimaz4, Maria Alessio5, Marco Cattalini6, Romina Gallizzi7, Maria Cristina Maggio8, Giuseppe Lopalco9, Francesco La Torre10, Claudia Fabiani11, Manuela Pardeo3, Alma Nunzia Olivieri12, Paolo Sfriso13, Carlo Salvarani14, Salvatore Grosso1, Claudia Bracaglia3, Fabrizio De Benedetti3, Donato Rigante15, Luca Cantarini2
    1Clinical Pediatrics, Department of Molecular Medicine and Development; 2Research Center of Systemic Autoinflammatory Diseases and Behçet's Disease Clinic, Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena; 3Division of Rheumatology, Department of Pediatric Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome; 4Rheumatology Unit, Meyer Children's Hospital, University of Florence, Florence; 5Department of Pediatrics, University of Naples Federico II, Naples; 6Pediatric Clinic, University of Brescia, Brescia; 7Department of Pediatrics, Azienda Ospedaliera Universitaria Policlinico “G. Martino”, University of Messina, Messina; 8Universitary Department “Pro.S.A.M.I.”, University of Palermo, Palermo; 9Rheumatology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari; 10Pediatric Rheumatology Section, Pediatric Oncoematology Unit, Vito Fazzi Hospital, Lecce; 11Ophthalmology Unit, Department of Medicine, Surgery and Neuroscience, University of Siena, Siena; 12Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Seconda Università degli Studi di Napoli, Naples; 13Rheumatology Unit, Department of Medicine, University of Padua, Padua; 14Rheumatology Unit, Department of Internal Medicine, Azienda Ospedaliera ASMN, Istituto di Ricovero e Cura a Carattere Scientifico, Reggio Emilia; 15Institute of Pediatrics, Periodic Fever Research Center, Università Cattolica Sacro Cuore, Fondazione Policlinico A. Gemelli, IRCCS, Rome, Italy
    Correspondence: Carla Gaggiano

    Introduction: The advent of biologic agents has revolutionized therapeutic approaches in systemic juvenile idiopatic arthritis (sJIA) as their introduction has been shown to modify disease course and improve overall outcomes, particularly when initiated early. Few studies have reported the drug retention rate (DRR) of biologic drugs in JIA, and none of them has specifically investigated the DRR of interleukin (IL)-1 inhibitors on sJIA.

    Objectives: The primary aim of the study was to examine the overall DRR of IL-1 blockers in sJIA patients. Secondary aims of our study were to: (i) explore the influence of biologic line of treatment, adverse events (AEs), type of anti-IL-1 agent and the concomitant use of conventional disease modifying anti-rheumatic drugs (cDMARDs) on DRR; (ii) find eventual predictive factors associated with events leading to drug discontinuation. The corticosteroid sparing effect and the impact of disease duration and treatment delay on survival constituted ancillary aims.

    Methods: sJIA patients – diagnosed according to the revised International League of Association for Rheumatology (ILAR) criteria – treated with anakinra (ANA) and canakinumab (CAN) were enrolled in 15 Italian tertiary referral centers. Demographic, clinical and therapeutic data collected from medical records were retrospectively collected and statistically analyzed.

    Results: Seventy seven patients were enrolled for a total of 86 treatment courses. The cumulative retention rate of the IL-1 inhibitors at 12-, 24-, 48-, and 60-months of follow-up was 79.9, 59.5, 53.5, and 53.5%, respectively, without any statistically significant differences between ANA and CAN (p = 0.056), and between patients treated in monotherapy compared to the subgroup co-administered with conventional immunosuppressors (p = 0.058). On the contrary, significant differences were found between biologic-naive patients and those previously treated with biologic drugs (p = 0.038) and when distinguishing according to AEs occurrence (p = 0.04). In regression analysis, patients pre-treated with other biologics (HR = 3.357 [CI: 1.341–8.406], p = 0.01) and those experiencing AEs (HR = 2.970 [CI: 1.186–7.435], p = 0.020) were associated with a higher hazard ratio of IL-1 inhibitors withdrawal. The mean treatment delay was significantly higher among patients discontinuing IL-1 inhibitors (p = 0.0002).

    Conclusion: Our findings suggest an excellent overall DRR for both ANA and CAN that might be further augmented by paying attention to AEs and employing these agents as first-line biologics in an early disease phase.

    Disclosure of Interest

    None Declared

    P1106 Clinic-laboratory profile of macrophage activation syndrome in children with systemic juvenile idiopathic arthritis

    Sandesh Guleria1, Johnson Nameirakpam1, Anjani Gummadi1, Anju Gupta1, Amit Rawat1, Prateek Bhatia2, Deepti Suri1, Surjit Singh1
    1Pediatric Allergy and Immunology; 2Pediatric Hemato-Oncology, Postgraduate Instiute of Medical Education and Research, Chandigarh, Chandigarh, India
    Correspondence: Sandesh Guleria

    Introduction: Systemic juvenile idiopathic arthritis (SJIA) is an auto-inflammatory disorder with a propensity to develop macrophage activation syndrome (MAS). MAS is an emergent, fulminant, life-threatening condition which can be fatal if not recognized early.

    Objectives: To study the clinic-laboratory profile of MAS in children with SJIA

    Methods: Case records of children of SJIA who had MAS, diagnosed at the Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, during the period 2017-2018, were reviewed. Findings pertaining to history, clinical examination and laboratory investigations were recorded. Diagnosis of MAS was based on criteria given by Ravelli et al (2016).

    Results: A total of 13 children (1-18 years) with 15 MAS events were included in the study. Two children had 2 episodes each of MAS during this period. Mean age was 6.7+ 4.8 years with male to female ratio of 1.17. Five children (38.5%) had MAS, at or during hospital admission on their first presentation. Common clinical features noted were fever (100%), pallor (100%), hepatomegaly and/or splenomegaly (73.3%), rash (46.7%), lymphadenopathy (46.7%), arthritis (33.3%), irritability (26.7%), mucocutaneous bleeding (20%) and jaundice (13.3%). In laboratory investigations (Table 1), all children had anaemia and elevated C-reactive protein (CRP). Leucopenia and leucocytosis was seen in 2 (13.3%) and 8 (53.5%) children respectively. Four (26.6%) children had thrombocytopenia (< 150 x 109/L), 6 (40%) had platelet count < 181 x 109/L and three children (20%) had thrombocytosis. Hypertriglyceridemia (93.3%), hypofibrinoginemia (73.3%), elevated aspartate aminotransferase (73.3%) and alanine aminotransferase (40%) were other prominent laboratory parameters. All children had high serum ferritin levels with mean of 8960.6 + 13662.12 ng/ml. Nine children had serum ferritin/ESR (ratio) of > 80. Three (20%) children died. Two children, who died, had very high serum ferritin levels (52000 ng/ml and 42845 ng/ml).

    Conclusion: MAS is a life threatening multisystemic complication of SJIA with high mortality rate (20%). Fever, pallor, organomegaly and persistent rash are prominent clinical features of MAS. Leucopenia and thrombocytopenia may not be early features of MAS but low or normal platelets can differentiate MAS from a disease flare in which we almost always get thrombocytosis. Very high serum ferritin, Hypertriglyceridemia, hypofibroginemia and transaminitis are better clue for MAS and serum ferritin levels may be a prognostic marker.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1106). Laboratory parameters in children of SJIA with MAS

    P1107 Serum soluble CD25: an useful biomarker of macrophage activation syndrome in systemic juvenile idiopathic arthritis

    Sandesh Guleria1, Anju Gupta1, Amit Rawat1, Prateek Bhatia2, Avinash Sharma1, Deepti Suri1, Surjit Singh1
    1Department of Pediatrics, Division of Allergy and Immunology; 2Department of Pediatrics, Division of Hemato-oncology, Postgraduate Instiute of Medical Education and Research, Chandigarh, Chandigarh, India
    Correspondence: Sandesh Guleria

    Introduction: Systemic juvenile idiopathic arthritis (SJIA) is an auto-inflammatory disorder secondary to innate immune dysfunction with a propensity to develop macrophage activation syndrome (MAS), a life-threatening condition. sCD25 has been used as a sensitive biomarker for the diagnosis of Hemophagocytic lymphohistiocytosis which has similarities in clinical features and pathogenesis to MAS.

    Objectives: To assay serum soluble CD25 in children with systemic juvenile idiopathic arthritis (SJIA) and to compare levels of sCD25 in children with inactive disease, active disease and those with macrophage activation syndrome (MAS).

    Methods: Thisprospective study was conducted in a tertiary care referral centre in North India from January 2017 to June 2018. All patients fulfilling the International League of Associations for Rheumatology (ILAR) 2001 criteria for SJIA were eligible for enrolment. At enrolment, all patients were examined clinically for signs of disease activity. Appropriate investigations were carried out and sCD25 was analyzed by using commercially available sCD25 / IL-2R ELISA kit.

    Results: A total of 35 children (1-18 years) with 43 events were included in the study. Mean age at enrolment in the study was 7.3+3.59 years with male to female ratio of 2.5. Based on clinical features and investigations, events were categorized into 3 groups; SJIA with inactive disease (15; 34.9%), SJIA with active disease (15; 34.9%) and SJIA with MAS (13; 30.2%). Mean sCD25 levels in the study population were 10,966.02 + 10,854.93 pg/ml. Children with inactive disease, active disease and disease with MAS had mean + SD serum sCD25 levels of 4710.6 + 1817.04 pg/ml, 7604 + 2376.24 pg/ml, and 22062.9 + 14335.97 pg/ml respectively. Although, Mean level of sCD25 in children with active disease was higher than those with inactive disease, but no significant difference could be found. sCD25 levels significantly (p value 0.0001) varied between MAS and other 2 groups. sCD25 cut off level of 10,385 pg/ml was found to have sensitivity of 100% and specificity of 96.7% in differentiating MAS in SJIA from disease flare and inactive disease. There was no correlation of sCD25 with demographic parameters, Total leucocyte count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), platelet counts, urea, creatinine, prothrombin time, activated partial prothrombin time and fibrinogen levels. A correlation of sCD25 was found with levels of haemoglobin (p-value .01), ferritin (p-value .001), aspartate aminotransferase (AST) (p-value .000), alanine aminotransferase (ALT) (p-value .000), alkaline phosphatase (ALP) (p-value .005) and triglycerides (p-value .001). Higher sCD25 levels were found in patients who had low Hb, elevated ferritin, elevated AST, ALT, ALP and serum triglyceride levels.

    Conclusion: sCD25 is a useful biomarker in differentiating MAS in SJIA from disease flare and inactive disease with sensitivity and specificity of 100% and 96.7% respectively at a cut off level of 10,385 pg/ml. Coupling sCD25 with other laboratory parameters may be useful for early diagnosis of MAS in SJIA.

    Disclosure of Interest

    None Declared

    P1108 Patient with CASP1 variant with severe systemic JIA and recurrent MAS treated by dual blockade with anakinra and tocilizumab

    Troels Herlin1, Sofie E. Jørgensen2, Mette Christiansen3, Sofie E. Jørgensen2, Christian Høst1, Sofie E. Jørgensen2, Christian Høst1, Mette Christiansen3, Christian Høst1, Mette Christiansen3, Dorte A. Larsen4, Mia Glerup1, Dorte A. Larsen4, Trine H. Mogensen5, Birgitte Mahler1, Trine H. Mogensen5, Trine H. Mogensen5
    1Pediatrics, Aarhus University Hospital; 2Biomedicine, Aarhus University; 3Clinical Immunology; 4Ophthalmology; 5Infectious diseases, Aarhus University Hospital, Aarhus , Denmark
    Correspondence: Troels Herlin

    Introduction: Macrophage activation syndrome (MAS) is a severe complication of rheumatic disease in childhood, particularly in systemic juvenile idiopathic arthritis (sJIA). An increasing body of evidence suggests a genetic background. Human procaspase-1 variants have been associated with febrile episodes and may contribute to inflammation via binding of receptor interacting protein kinase 2 (RIP2) and thereby activate NF-kB signaling.

    Objectives: To describe the genetic, immunological and therapeutic aspects of recalcitrant sJIA and recurrent MAS in a patient with a variant in the CASP1 gene.

    Methods: A 15-year-old, previously healthy girl presented Oct 2016 with a typical clinical and biochemical picture of systemic JIA.

    Based on recurrent MAS, whole exome sequencing (WES) was performed on DNA extracted from whole blood. Peripheral blood mononuclear cells (PBMCs) from patient and a healthy control were stimulated with NF-kB inducers (TNFa and LPS or left untreated). Phosphorylated IkBa, as a measure of NF-kB activation, was measured by Luminex technology. Proinflammatory cytokines (IL-6, CXCL10, MIP-1a and TNFa) were measured in the supernatants using Mesoscale U-plex multiplex assays.

    Results: WES identified a rare heterozygous variant in the CASP1 gene (c.482G>A, p.R161H, CADD score 32; gnomAD frequency 0.000004) inherited from her healthy father and a rather frequent heterozygousvariant in the UNC13D gene (c.2335G>A, p.V779M, CADD score 15.3; gnomAD frequency 0.003). Patient PBMCs demonstrated increased NF-kB activation. Upon stimulation with TNFa and LPS the PBMCs produced extremely high levels of IL-6 and CXCL10 compared to control, whereas the levels of MIP-1a was comparable to control.

    Initially, she responded well to anakinra 2 mg/kg/day and corticosteroids. Three months later she flared with intense pruritus, arthritis and quotidian fever and was changed to tocilizumab. Shortly after she precipitated with the picture of MAS and ferritin rise to 26,000 μg/L, elevated LDH, high triglyceride and low fibrinogen. Bone marrow was normal, spinal fluid showed mild pleocytosis, 36 leukocytes per μL. Etoposide and dexamethasone treatment were started according to the HLH-2004 protocol. Nevertheless, she developed another two episodes of MAS attacks with ferritin peaks >66,000 μg/L and two CNS attacks with generalized seizures (no cells in spinal fluid, but MRI with widespread bilateral cortical involvement). Despite treatment with canakinumab, dexamethasone, high-dose anakinra (8 mg/kg/day), several 3-day pulses with 1 G i.v. methylprednisolone and IVIG the condition deteriorated with repeated hyperferritinemia, fever, and elevated acute phase reactants.

    Based on the findings of increased expression of IL-6 in stimulated PBMCs i.v. tocilizumab 8mg/kg was added every second week to high-dose anakinra, which had a marked clinical and biochemical response. All blood tests rapidly normalized and gradual tapering to low-dose anakinra and subcutaneous tocilizumab for the next 16 months did not precipitate new flares.

    Conclusion: To the best of our knowledge, this is the first description of a CASP1 variant associated with sJIA and recurrent MAS. We suggest that the Arg161His contributes to the phenotype, since it is located in the catalytic domain of procaspase-1 and therefore might stabilize the ASC Pyroptosome. The observed increase in NF-kB activation, possibly explained by procaspase-1 variants inducing stronger interaction with RIP2 than wild-type procaspase-1, may have contributed to the increased expression of proinflammatory proteins, such as IL-6.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1109 Validation and clinical application of MRP8/14 serum levels as biomarker for the diagnosis of systemic juvenile idiopathic arthritis in fever of unknown origin

    Carolin Pretzer1, María Miranda-Garcia1, Rainer Berendes2, Gerd Horneff3, Jasmin Kuemmerle-Deschner4, Gerd Ganser5, Hans-Iko Huppertz6, Kirsten Minden7, Johannes-Peter Haas8, Annette Jansson9, Michael Borte10, Catharina Schuetz11, Prasad Oommen12, Michael Frosch13, Christoph Kessel1, Bernhard Schlueter14, Annette Richter-Unruh15, Helmut Wittkowski1, Johannes Roth16, Dirk Foell1, Dirk Holzinger17
    1Department of Pediatric Rheumatology and Immunology, University Hospital Children’s Muenster, Muenster; 2St. Marien Children's Hospital, Landshut; 3Asklepios Clinic Sankt Augustin, Sankt Augustin; 4University Children's Hospital Tuebingen,, Tuebingen; 5St. Josef-Stift Sendenhorst Hospital, Sendenhorst; 6Prof.-Hess Children's Hospital and Gesundheit Nord Klinikverbund Bremen, Bremen; 7Charité University Medicine and Epidemiology Unit, German Rheumatism Research Centre, Berlin; 8German Center for Pediatric and Adolescent Rheumatology, Garmisch-Partenkirchen; 9Department of Rheumatology and Immunology, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich; 10Paediatric Rheumatology, Immunology and Infectiology, Hospital St. Georg, Leipzig; 11Department of Pediatrics and Adolescents Medicine, University Hospital Ulm, Ulm; 12Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children's Hospital, Medical Faculty, Heinrich-Heine-University, Duesseldorf; 13German Pediatric Pain Centre, Children's and Adolescents’ Hospital, Datteln; 14Center for Laboratory Medicine, University Hospital Muenster; 15Department of Paediatrics, University of Muenster; 16Institute of Immunology, University Hospital Muenster, Muenster; 17Department of Pediatric Hematology-Oncology, University Hospital Essen, Essen, Germany
    Correspondence: Dirk Holzinger

    Introduction: The differential diagnosis of fever of unknown origin (FUO) is a major challenge especially for differentiation of systemic-onset juvenile idiopathic arthritis (SJIA) and infectious diseases and can be supported by biomarker analysis.

    Objectives: In a pilot study the analysis of MRP8/14 serum levels has been demonstrated as an excellent tool for the diagnosis of SJIA, allowing early differentiation with a specificity of 95%. Based on this, the analysis of MRP8/14 serum levels has been offered to pediatric rheumatologists and we aimed to validate these findings in samples from daily clinical practice and establish the analysis by a commercial enzyme-linked immunosorbent sandwich assay (ELISA) and an on-site lateral flow immunoassay (LFIA).

    Methods: In total, 3,002 patients with inflammation of unknown origin were enrolled within 6 years. A full data set was available from 1,935 cases, including symptoms, laboratory parameters (CRP, ESR, leucocytes) and final diagnosis. These cases were divided into two cohorts: (A) For validation of the MRP8/14 test performance with the enzyme-linked immunosorbent sandwich assay (ELISA) used previously as well as a first testing of a commercial ELISA and an innovative point-of-care lateral flow immunoassay (LFIA); and (B) for validating the findings with the latter assays.

    Results: MRP8/14 serum levels of patients with SJIA (12,100 ± 2,670 ng/ml, mean±SEM) were elevated compared to other diagnoses (including infections, vasculitis and other autoinflammatory diseases; 3,020 ± 510 ng/ml) irrespective of fever and anti-inflammatory treatment (p <0.001) in 942 samples. In the group of untreated patients with fever (n=195) MRP8/14 levels in SJIA (19,930 ± 5,260 ng/ml) were even higher compared to other diagnoses (4,730 ± 1,160 ng/ml). Sensitivity and specificity of MRP8/14 to differentiate SJIA vs. other diseases were 72% and 89% respectively (cut-off 9,160 ng/ml). The MRP8/14 levels correlated closely in the commercial ELISA (sensitivity 74%, specificity 87%) and LFIA (sensitivity 77%, specificity 88%). The performance of the commercial ELISA and the LFIA could be confirmed in an independent cohort (n=162). Overall performance was stable in both cohorts: commercial ELISA (sensitivity 79%, specificity 88%) and LFIA (sensitivity 83%, specificity 87%).

    Conclusion: MRP8/14 serum analyses could be validated as a helpful tool for the diagnosis of SJIA in FUO in daily clinical practice. The results of the experimental assay could be confirmed with a commercial ELISA and LFIA making these tests available for routine use. The LFIA enables a quick diagnostic screening test at the point of care.

    Disclosure of Interest

    None Declared

    P1110 Do juvenile idiopathic arthritis patients with systemic onset in the risk of low anti-vaccine antibody: the preliminary report?

    Natalya Lubimova1, Olga Goleva2, Irina Fridman2, Margarita Dubko3, Vera Masalova3, Ludmila Snegireva3, Eugenia Isupova3, Ekaterina Gaidar3, Andrey Santimov3, Olga Kalashnikova3, Susanna Kharit2,3, Mikhail Kostik3
    1Children’s City Hospital#2 n.a. St. Mary Magdalene; 2Pediatric Research and Clinical Center for Infection Diseases; 3Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation
    Correspondence: Mikhail Kostik

    Introduction: Patients with juvenile idiopathic arthritis with systemic onset (soJIA) may have lower protective levels of anti-vaccine antibodies due to high inflammatory activity, frequent using of the systemic corticosteroids and biologics.

    Objectives: The aim of our study was to evaluate the protective levels of anti-vaccine antibodies in patients with soJIA compare to non-systemic ones.

    Methods: In the present study were included data 7 soJIA and 82 non-systemic JIA patients who received scheduled vaccination before the age of 2 years and before JIA onset against measles, parotitis, hepatitis B, diphtheria and rubella. In all patients, the IgG anti-vaccine antibodies levels were detected with ELISA. In each patient, we evaluate the type of the disease, onset age, duration of JIA, treatment with corticosteroids, methotrexate, and biologics.

    Results: We have not found differences in the anti-vaccine antibody levels in soJIA compare to non-systemic, but median levels of the ant-hepatitis B and anti-diphthreia antibody were 12 and 3 times lower, consequently (table 1). The protective levels of anti-measels antibodies was in 4 (57%) vs 47 (57%). p=0.65; anti-parotitis antibody 5(71%) vs 63 (77%), p=0.37; ant-diphtheria 2 (29%) vs 42 (51%), p=0.23; ant-hepatitis B 2 (29%) vs 46 (56%), p=0.23; anti-rubella antibody 7 (100%) vs 80 (99%), p=0.77 in soJIA compare to non-systemic JIA, relatively. The number and levels of soJIA patients with protective anti-hepatitis B and anti-diphtheria antibody were lower compares to non-systemic JIA patients (data insignificant). There was no correlation with antibody concentration and corticosteroids applying, despite soJIA more frequently received them. We have found negative correlation between levels of ant-measels antibodies and JIA duration (r = -0.36, p=0.001), JIA duration (r=-0.26, p=0.02) and treatment with biologics (r=-0.25, p=0.024). Also was detected negative correlation between anti-parotitits antibody (r=-0.25, p=0.021) and anti-diphtheria antibody levels (r=-0.25, p=0.021) with JIA duration and and anti-diphtheria antibody levels (r=-0.29, p=0.009) with methotrexate using.

    Conclusion: patients with systemic JIA may have the risk of lower anti-vaccine antibodies, which required more close monitoring of levels of these antibodies. Further investigations needed.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1110). The levels of anti-vaccine antibody in soJIA and non-systemic JIA patients

    P1111 Have patients with juvenile idiopathic arthritis patients with systemic onset particular features for monocyclic course?

    Mikhail Kostik, Maria Rumyantseva, Eugenia Isupova, Irina Chikova, Margarita Dubko, Vera Masalova, Ludmila Snegireva, Tatyana Kornishina, Tatyana Likhacheva, Ekaterina Gaidar, Andrey Santimov, Olga Kalashnikova
    Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation
    Correspondence: Mikhail Kostik

    Introduction: Juvenile idiopathic arthritis with systemic onset (soJIA) may have a monocyclic, polycyclic or relapsed course. There were not known soJIA course predictors.

    Objectives: Our study aimed to evaluate initial clinical or laboratory features of the patients with soJIA who had a monocyclic course without biologics.

    Methods: In the present study were included data about 130 soJIA patients. After selection, we identified a subgroup of the soJIA patients (n=22) who successfully achieved remission off-biologic medication and were stable in the remission off-medication at least two years. The second group consisted of the patients who required biologics (n=83). The remained 25 patients excluded due to missing data or who did not meet the selection criteria. We evaluated routine clinical (fever, rash, hepatosplenomegaly, serositis, lymphadenopathy, MAS, joint involvement) and laboratory (WBC, PLT, Hb, ferritin, ALS, AST, LDH, GGTP, ALP, albumin, sodium, triglycerides, ESR, CRP, prothrombin, fibrinogen) soJIA features in the onset of the disease.

    Results: Patient with monocycling course have no any differences except the ferritin level: 275 (133; 698) ng/ml vs 950 (150; 3240) ng/ml, (p=0.04), time to achievement remission 17.7 (8.2; 38.0) months vs 60.2 (36.0; 99.0) months (p=0.00002) and rare elbow involvement 4.6% vs 30.9% (p=0.01). The parameters, associated with possible monocycling course are in the table.

    Conclusion: Patients with soJIA initially have quite similar clinical presentations independently the further clinical course. The prediction of a possible course of soJIA is a difficult problem. Patients with soJIA with the monocyclic course were younger and had less intense laboratory activity. Further investigations required.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1111). The parameters, associated with the possible monocyclic course in soJIA patients

    P1112 Single center experience of biological therapy in patients with systemic juvenile idiopathic arthritis associated with macrophage activation syndrome

    Maria I. Kaleda, Irina P. Nikishina
    Pediatrics, V.A. Nasonova Research Institute of Rheumatology, Moscow, Russian Federation
    Correspondence: Irina P. Nikishina

    Introduction: Macrophage activation syndrome (MAS) is a severe complication of systemic juvenile idiopathic arthritis (sJIA) which associated with high risks of the multiple organ failure and mortality. The investigation of safety of Biologics (B) in patients(pts) with risk of MAS is important in real clinical practice to improve the prognosis.

    Objectives: to analyze the results of all cases of therapy with B in pts with sJIA and history of MAS.

    Methods: the study included all pts of single center with sJIA (in accordance to ILAR classification criteria) who developed the MAS diagnosed according to Classification Criteria for MAS 2016 before or under the treatment with B.

    Results: We observed 102 consecutive pts with sJIA, 29 pts (28%) fulfilled the criteria of MAS (Tab.1).

    A total of 37 episodes of MAS was recorded. The total number of B course in this pts was 51 (tocilizumab – 28, canakinumab – 9, anakinra – 2, rituximab – 5, infliximab – 4, etanercept – 2, abatacept – 1). 24 pts (82.7%) had episodes of MAS before starting of B (16 – at onset of sJIA). 13 episodes of MAS was observed during the B therapy (1 on anakinra, 1 - canakinumab, 11 – tocilizumab), in 5 pts among them - for the first time. 8 episodes developed due to deviation of the treatment’s schedule, 2 – associated with flare of sJIA, 2 – associated with acute respiratory infection, 1 – after surgical treatment for chronic osteomyelitis of the clavicle. First features of MAS were bright rash with itching, lower platelet counts, high triglycerides. Pts who developed MAS while were treated with B had rare episodes of fever, a tendency for serum ferritin and CRP decreasing opposite to pts who developed MAS before B, but had no other differences in clinical or laboratory features. Glucocorticoids (per os+iv), intravenous immunoglobulin and cyclosporin were used to treat MAS. Lethal outcome was in 6.7% of sJIA cases with MAS (1.9% of all sJIA pts). 25 pts continue treatment with B (tocilizumab – 19, canakinumab – 6) after the resolution of the acute manifestation of MAS with high efficacy (more than 70-90% response by ACRpedi). Two pts discontinued treatment for organizational reasons.

    Conclusion: Pts with a history of MAS require an earlier prescription of B due to the greater disease activity. It is necessary to consider the probability of a lower frequency of fever and lower level of ferritin and CRP in case of MAS during B therapy. We didn’t observe association between treatment of B and increased risk of MAS, but risk of MAS increase in case of disorder of protocol of the treatment.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1112). Сlinical and demographic characteristics of patients

    Unusual or unsolved case reports

    P1113 Behect disease in pediatrics; a solitary sign can be enough

    Hanan M. Abd El-Lateef
    Pediatric Rheumatology Immunology department, Ain Shams University, Cairo, Egypt

    Introduction: Involvement of the perineal region including vulvovaginitisin prepubertal girls is a common but quite challenging condition because it encompass a wide variety of conditions with subtle differences especially in the absence of systemic manifestations

    Objectives: Considering autoinflammatory disorders as an important differential diagnosis in cases of perineal ulcers in prepubertal females.

    Methods: An 8 years old prepubertal female patient who presented with recurrent painful non itchy perineal ulcerations that interfered with her normal daily activitiesand copious non foul vaginal discharge of five months duration with failure of local topical therapies and systemic antibiotics. No other symptoms were reported.

    Results: On physical examination,patient was average built for age andvitally stable. Examination of the perineal region revealed evidence of subcentimetric scars , an irregular deep ulcer 16 cm at its widest extension with necrotic base and clean margin involving perianal region and extending to labia majorus. Systemic examination revealed no evidence of arthritis, neurological manifestations, muscloskeletal involvement , other dermatologic or mucosal lesions and no organomegaly . Ophthalmological examination was free. Gynecological consultations excluded sexual abuse. Routine Laboratory evaluation showed a within normal CBC picture , ESR 50 mm/1st hr and CRP 48mg/dl. Immunological profile showed a within normal levels serum immunoglobulins assay , CD markers and negative immune markers (ANA , Anti-DNA , ANCA) . Swabs from both the ulcer base and the vaginal discharge cultures were sterile. The diagnosis of Behçet disease versus Genital Crohn's was raised .Double contrast CT pelviabdomen with barium meal follow through showed skipping lesions of edematous bowel wall highly suspicious of Crohn's disease. Anegative ASCA test and a positive HLA-B51 tip the scales and favour the diagnosis of Behçet disease. The investigations showed no involvement of cardiovascular, renal, pulmonary, urologic, joint or central nervous systems. Patient showed marked clinical improvement on Pulse methylprednisolone steroid therapy (1gm/day) for 5 days followed by full dose oral prednisone (60mg/day) but with a progressive severe relapse of the ulcerations on the early withdrawal of the oral therapy (50 mg/day) . Azathioprine was added to the full dose oral steroid therapy for three months but lesions were still full dose steroid- dependant .The last attack was associated with a large pyoderma gangrenosum-like lesion on the anterior tibialsurface of the left leg that responded markedly on pulse steroid therapy.

    Infliximab (Anti-TNFα ) was added (6mg/kg/dose) at 0, 2 and 6 weeks with cautious gradual withdrawal of oral steroids . Patient showed marked clinical and laboratory improvement and was maintained on 15mg oral prednisone per day with a stable remitting course. Further close follow up is done.

    Conclusion: Behçet disease (BD) is a relapsing inflammatory disorder with multi-system involvement , heterogeneous clinical presentations and variable degrees of vascuilitis.BD shares a considerable overlap with other autoinflammatory disorders including Crohn's disease making diagnosis occasionally challenging . Perineal ulcers is a rare presentation in the pediatric age group and being an isolated clinical presentation confirms that in pediatrics the number of symptoms may be too few to apply any classification of diagnosis to a single patient. The severity of the ulcers , the relapsing nature , failure of azathioprine as asteroid sparing therapy lead to applying TNF-α inhibitors which proved a great efficacy to maintainremission.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1114 Childhood pyoderma gangrenosum: diagnostic challenges and managements of two cases from Libya

    Awatif Abushhaiwia1, Nairuz Abushhiwa2, Mohammed Kamel3, Lailasabei4, Mabruka Zletni5, Hafsa Dawi6
    1Pediatric Rheumatology, Tripoli Children’s Hospital; 2Pathology, Tripoli Faculty of Medicine, Tripoli, Libya; 3Adult Rheumatology, Dr Fakhry Hospital, Alkhobar, Saudi Arabia; 4Community Medicine, Tripoli Faculty of Medicine; 5Pediatric Rheumatology, Tripoli Children’s Hospital; 6Dermatology, Central Tripoli Hospital, Tripoli, Libya
    Correspondence: Awatif Abushhaiwia

    Introduction: Pyoderma gangrenousm (PG) is an idiopathic Ulcerative, non-infective chronic inflammatory skin disorder of unknown etiology.The most common reported underlying diseases in pediatrics are inflammatory bowel disease, followed by hematologic disorders, vasculitis, immune deficiencies and pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome. More than half of the cases occur with no underlying disease. The most frequently treatment should be tailored according to the underlying etiology. It includes systemic steroids, corticosteroid sparing agents such as dapsone, cyclosporine, azathioprine and currently TNF –alpha inhibitors such as etenrecpt, adalimumab and infliximab is a promising treatment of refractory PG. Response to treatment is high with cure rates reaching 90%. A high index suspicion and a through workup are mandatory in the management of pediatrics PG.

    Objectives: To describe the clinical presentation, laboratory testes and challenges with treatment trials in two pediatric cases.

    Methods: clinical information was gathered from the history, which was taken from their parents after an informed consent

    Results: for the first time we report two pediatric cases in Libya who diagnosed with refractory pyoderma gangrenousm with no associated systemic diseases except PAPA which can’t be excluded since no genetic tests available in all public hospitals in Libya, we report PG in two Libyan children of 2 year-old and 5 year-old, they presented with skin lesions beginning as a small pustule that progressed to very painful large ulcer with irregular borders. There were healed ulcers with scaring present on chest, both upper and lower extremities and abdominal wall.Laboratory testes for both cases showed an increase in white blood cell counts mainly neutrophils, anemia, increased platelets count, elevated erythrosedmintation rate, and elevated C-reactive protein.No hematological abnormalities were seen in peripheral blood smears except neutrophilic response, the red cell indices were suggestive of iron deficiency anemia.Cultures for bacteria and fungi from skin lesions, and blood cultures were repeatedly negative. Immunoglobulin assay (IgG, IgM, IgA, IgE) was normal. The antinuclear antibody, cytoplasmic antibody, and antiphospholipid antibodies, tests for hepatitis B, C and HIV were all negative. They didn’t have symptoms suggest ulcerative colitis, their colonoscopy was normal. Histopathology from their skin was consistent with pyoderma gangrenousm.

    Both cases were moderate response to high dose of oral prednisolone 2mg\kg per day complicated, however by growth retardation and cushingnoid habitus and minimal response to corticosteroid sparing agent azathioprine. Anti TNF-alpha inhibitors etenrecpt, which has been available at hospital since March 2018, we used it in case 2 because he was inadequate response to azathioprine.

    Both cases showed briefly remitted on azathioprine with relapsed at least 2-3 episodes per year. The ulcer lesions healed with cribriform scaring within 6-8 weeks in each episode.

    Conclusion: Pyoderma gangrenousm has recently been included within the spectrum of auto- inflammatory diseases, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive, which can be diagnosed by genetic tests, which is unfortunately not available in public hospitals in Libya.We couldn’t either be able to start other biological treatment because of the civil war crisis in Libya.

    Disclosure of Interest

    None Declared

    P1115 A girl with a developmental delay and Lupus-like phenotype associated with both neuroblastoma and an MDA5 gain-of-function mutation (Aicardi-Goutières Syndrome 7, AGS7)

    Stefan Berg1, Dara McCreary2, Gunilla Drake af Hagelsrum3, Nina Björkander3, Despina Eleftheriou2
    1Pediatric Immunology and Rheumatology, The Queen Silvia Children's Hospital, Gothenburg, Sweden; 2UCL GOS Institute of Child Health, London, United Kingdom; 3Pediatric Neurology, The Queen Silvia Children's Hospital, Gothenburg, Sweden
    Correspondence: Stefan Berg

    Introduction: Monogenic autoinflammatory diseases (AID) with central nervous system (CNS) involvement are often difficult to diagnose and patients may be subjected to expensive and often invasive diagnostic patient pathways. Securing a molecular diagnosis is of major importance in these cases for treatment, prognosis, and genetic counselling.

    We describe a 7-year-old girl of non-consanguineous Swedish/Lebanese descent with severe developmental delay. Her psychomotor development was normal until 10 months of age. At the age of 10 months she presented with myoclonus and increased muscletone of both legs. She developed a progressive encephalopathy, behavioural change and sleep disturbance during the following months. Cerebral MRI showed white matter disease with high signal on T2 weighted imaging and atrophy. CSF showed normal proteins but minor lymphocytosis, and slight increase in neurological damage markers NFL and Tau (normal GFA-p). Extensive work-up did not reveal a clear diagnosis. Cerebral CT was not performed. At the age of 20 months a neuroblastoma was detected, leading to a diagnosis of atypical opsoclonus-myoclonus syndrome (OMS) despite the absence of opsoclonus, for which she was treated with cyclophosphamide, steroid pulses, IVIG and rituximab. By this time she demonstrated severe psychomotor delay with no speech and a dyskinetic syndrome.

    At the age of 6 years she developed skin rash and vasculitis-like lesions on her toes, which developed to also involved the face and upper limbs. Her ESR was increased (47 mm/h), but CRP was normal (<1 mg/L). Titres of several autoantibodies were raised (ANA, anti-dsDNA and anti-ribo-p). Lumbar puncture showed slightly increased lymphocytes (7, ref <4 x 106/L), and slightly elevated levels of Tau protein (normal NFL and GFA-p), with no increase of protein and no oligoclonal bands in the face of slight increase of IgM-index. Skin biopsy demonstrated findings compatible with SLE. Treatment with MMF and monthly i.v. corticosteroid pulses were required to control her features, resulting in a marked improvement of skin rash and normalization of inflammatory markers.

    Objectives: To use next generation sequencing to establish a genetic diagnosis in this case.

    Methods: An NGS panel targeting 256 genes was used developed and validated as described elsewhere.

    Results: A pathogenic variant (p.R779H, c.G2336A) was found in the IFIH1gene encoding MDA5 consistent with a diagnosis of an interferonopathy syndrome, Aicardi-Goutières syndrome 7 (AGS7). IFN score is pending and we are planning to start JAK inhibition

    Conclusion: We present a child with severe developmental delay having both a classic neuroblastoma and AGS7. She presented with neurodevelopmental delay at the age of 10 months and was found to have a neuroblastoma at the age of 20 months. She was considered to have an atypical OMS even though she didn’t demonstrate opsoclonus. Her developmental delay was more severe than usually seen in OMS. The findings of lupus-like features years later made us consider another cause for her neurological delay. The genetic finding of a pathogenic variant in IFIH1is fully compatible with a type I interferonopathy (AGS7). The fact that she also had a neuroblastoma may also have contributed to her neurological features, although this is not clear. To our knowledge, there is no connection between AGS and neuroblastoma described so far.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1116 Cold urticaria associated with a novel Phospholipase-Gamma-C2 (PLCƔ2) mutation

    Hanna Bonnekoh1,2, Niklas AMahnke1,2, Jörg Scheffel1,2, Marcus Maurer1,2, Karoline Krause1,2
    1Department of Dermatology and Allergy; 2Autoinflammation Reference Center Charité (ARC2), Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
    Correspondence: Hanna Bonnekoh

    Introduction: Phospholipase-Gamma-C2 (PLCƔ2) is a member of the phospholipase C family and plays a key role in the transmembranous signaling of B-lymphocytes, natural killer cells and mast cells. Mutations in PLCG2 were associated with rare dominantly inherited diseases characterized by a variable clinical phenotype including cold urticaria, neutrophilic dermatitis, immunodeficiency and autoimmune features or autoinflammation (PLAID/APLAID). The known mutations comprise in frame loss mutations of exon 19 and exon 20-22 and a missense mutation (p.Ser707Tyr) within the PLCG2 gene.

    Methods: We studied four members of a three-generation family, three of whom were affected by cold-induced urticarial rash. Genetic analysis of the index patient comprised screening for autoinflammatory periodic fever syndrome genes by Sanger sequencing (NLRP3, NLRP12 and PLCG2) and was followed by segregation analysis of further family members. We assessed clinical history and laboratory results including routine laboratory markers, immune status, autoantibodies, immunoglobulins, inflammatory markers (C-reactive protein, S100A8/9) and performed cold contact provocation testing.

    Results: All three affected family members presented with early onset cold-induced urticarial rash since birth associated with an autosomal-dominant inheritance. The patients reported occurrence of pruritic wheals to cold contact and cold evaporation as well as systemic reactions in response to extensive cold. Direct cold contact provocation testing was negative. Treatment with antihistamines could partially reduce the cold-associated skin-symptoms. Laboratory analysis revealed elevated serum S100A8/9 as well as elevated serum IgE levels and reduced IgM levels. Genetic analysis of the index patient showed a heterozygous variant c.2054+5G>T (located in the intronic splice region 18) in PLCG2 which was confirmed in the two other affected family members.

    Conclusion: We identified a novel mutation variant in PLCG2 associated with a phenotype of cold-induced urticarial rash and immune dysregulation. As the substitution is located in the intronic splice region 18, it may cause aberrant splicing and result in truncated mRNA or mRNA decay. Further studies including the identification of the amino acid sequence as well as functional analysis of the PLCƔ2 protein are necessary to clarify the impact of this novel variant.

    Disclosure of Interest

    None Declared

    P1117 Missense variants in SERPINF1 are associated with a novel auto-inflammatory disease presenting with persistent fever and multifocal neutrophilic periostitis

    Marta Bustaffa1, Marta Rusmini2, Marco Cattalini3, Sara Signa1, Riccardo Papa1, Alice Grossi2, Roberta Caorsi1, Stefano Volpi1, Paolo Picco4, Alessandro Plebani3, Angelo Ravelli4, Nicoletta Zoppi5, Marco Ritelli5, Marina Colombi5, Maja Di Rocco4, Isabella Ceccherini2, Marco Gattorno1
    1International Center for Autoinflammatory Disease and Primary Immunodeficiencies - Clinics of Pediatrics and Rhumatology; 2UOC of Genetics, G. Gaslini Institute, Genova; 3Clinica Pediatrica, Univerity of Brescia, Brescia; 4Clinics of Pediatrics and Rhumatology, G. Gaslini Institute, Genova; 5Division of Biology and Genetics, Department of Molecular and Translational Medicine, Univerity of Brescia, Brescia, Italy
    Correspondence: Marta Bustaffa

    Introduction: Caffey disease is a rare condition of early infancy characterized by hyperostotic periostitis, acute inflammation and swelling of soft tissues. It is self-limiting and usually caused by the c.3040C>T p.Arg836Cys mutation in COL1A1. Besides Caffey disease, COL1A1 mutations are reported in Osteogenesis Imperfecta (OI), with a well-known pathogenic mechanism. On the contrary, a link between the molecular defects of Caffey disease and its inflammatory, self-limiting phenotype is not properly understood. Another gene associated with OI is SERPINF1, whereas no inflammatory phenotypes are described related to SERPINF1 mutations.

    Objectives: To describe a case with many common characteristics with Caffey disease, but with a more severe course, steroid-dependency and no spontaneous regression.

    Methods: The proband is a previously healthy 11-month-old girl born from non-consanguineous parents in a community with high risk of endogamy. She presented with persistent fever, elevation of inflammatory markers along with severe pain, functional limitation and progressive swelling of all four limbs. X-ray and MRI documented an aggressive periostitis involving several long bones. Pain was not controlled despite combined antalgic therapies, requiring i.v. morphine administration. Bone biopsy revealed a neutrophilic inflammatory process consistent with acute suppurative osteomyelitis, although broad-spectrum antibiotics were ineffective and an infectious origin was excluded. 41 NGS panel and genetic analysis of COL1A1 excluded DIRA, MKD, Majeed and Caffey disease. With suspicion of a possible auto-inflammatory condition, Anakinra was started and allowed slight clinical improvement but elevation of acute phase reactants and bone swelling persisted.

    Results: Considering the ineffectiveness of different treatments, high dose steroid therapy was introduced with dramatic improvement, maintained even when oral prednisone was started with slow tapering. Anakinra was confirmed as a possible steroid-sparing agent. Serial X-ray displayed persistent improvement until the age of 2 years, when radiology assessment revealed a subclinical disease progression and recurrence of swelling of the left wrist occurred when steroid discontinuation was tried. In the attempt to stop the subclinical disease progression, steroid therapy was maintained to the minimal effective dose. Anakinra treatment was tapered and finally discontinued without any flare. WES revealed compound heterozygosity for 2 missense variants in SERPINF1 (A131D and P132R), inherited from parents. Immunofluorescence analyses of patient’s dermal fibroblasts revealed disorganized collagen type I, III and V in the extracellular matrix, endorsing a possible pathogenic role of both SERPINF1 variants. Biphosphonate treatment was considered because of osteopenic risk due to prolonged steroid treatment and its potential use in OI. 4 doses of neridronate were administered within 9 months and subsequent X-rays displayed improvement of osteopenia and stability of the dysmorphic feature of left wrist.

    Conclusion: We describe a patient with a clinical picture characterized by fever and multifocal neutrophilic hyperostosis without spontaneous regression, presenting dramatic response to high dose corticosteroid and subsequently to bisphosphonate. We suggest that this is a new variant of Caffey disease caused by SERPINF1 missense variants.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1118 Late-onset SAPHO syndrome revealing chronic myelomonocytic leukemia

    Laura Damian1, Simona Grad2, Felicia Mustatea1, Bianca Balan1, Gheorghe Cobzac3, Calin Bolosiu4, Liliana Bene5, Adrian Trifa6, Delia Dima7, Adriana Albu8
    1Rheumatology, Emergency Clinical County Hospital Cluj; 2Gastroenterology, “Iuliu Hatieganu” University of Medicine Cluj; 3Nuclear Medicine; 4Radiology; 5Immunology, Emergency Clinical County Hospital Cluj; 6Genetics, “Iuliu Hatieganu” University of Medicine Cluj; 7Hematology, “I. Chiricuta” Oncologic Institute; 82nd Internal Medicine , “Iuliu Hatieganu” University of Medicine Cluj, Cluj-Napoca, Romania
    Correspondence: Laura Damian

    Introduction: Chronic multifocal osteitis (CMO) and its adult variant, SAPHO syndrome (an acronym of synovitis, acne, hyperostosis, pustulosis, osteitis) are rare polygenic bone autoinflammatory disorders. However, their radiological features are not pathognomonic, as periostitis, bone sclerosis and cysts may be found in other systemic bone diseases, including myeloid neoplasia. Acquired gain-of- function JAK2 (617F) mutations, frequently found in myeloproliferative neoplasms, are sometimes associated to bone marrow fibrosis. JAK2 inhibitors may improve marrow fibrosis in myelofibrosis. JAK inhibition was also successfully employed in a case of refractory SAPHO syndrome.

    Objectives: To present the difficult case of a patient with the clinical picture of a late-onset, incomplete SAPHO syndrome in whom aJAK2-positive myeloproliferative syndrome was found.

    Methods: A 57-year male patient, with no personal or familial history of acne, psoriasis or spondylarthritis, presented with cervical and anterior chest wall intense bone pain, tibio-tarsal arthritis and left plantar fasciitis.

    Results: The laboratory revealed inflammation (ESR 88 mm/h, CRP 18 mg/dl, leukocytosis – WBC 10500 with 70% polymorphonuclear neutrophilsand 11% monocytes), with normal alkaline phosphatase, plasma protein electrophoresis,PSA, CA 19-9 and CEA neoplastic markers.HLA-B27 was negative, as well as the tests for Chlamydia, Mycoplasma, Ureaplasma, Borrelia and hepatitis B, C and HIV serology. Ferritin was 501 ug/l. The radiographs showed left tibial periostitis, asymmetric periarticular bone demineralization, bone cysts, asymmetric sacroiliitis and a dorsal crush fracture. Bone scintigraphy revealed cervical, dorsal sternoclavicular and tibio-tarsal enhancement, with a” bullhead” appearance considered pathognomonic for SAPHO syndrome. Abdominal ultrasound, chest radiograph and CT were unremarkable. He received NSAIDs, sulfasalazine and methotrexate, supplemented afterwards with corticosteroids, cyclosporine and alendronate, with clinical improvement, but persistence of ESR elevation and monocytosis (up to 26%). The bone marrow biopsy showed all series, hypolobulated megakaryocytes and areas of myelofibrosis. The JAK2 (V617F) mutation was positive (allele burden 1%).BCR-ABL was negative. Cytogenetic analysis of the bone marrow revealed a normal karyotype. No free immunoglobulin chains were found. The final diagnosis was chronic myelomonocytic leukemia.

    Conclusion: A late-onset and incomplete SAPHO clinical picture, in the presence of persistently abnormal blood counts, may prompt a search for hematologic malignancies. Common putative mechanisms of osteitis and bone fibrosis may be involved, making plausible a role of JAK inhibition for osteitis therapy.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    L. Damian Grant / Research Support from: Dr. Damian has a travel grant from Pfizer., S. Grad: None Declared, F. Mustatea: None Declared, B. Balan: None Declared, G. Cobzac: None Declared, C. Bolosiu: None Declared, L. Bene: None Declared, A. Trifa: None Declared, D. Dima: None Declared, A. Albu: None Declared

    P1119 Bloody stools and cryopyrin-associated periodic syndrome (CAPS)-- association or coincidence?

    Mariana Correia Marques1, Jonathan S. Hausmann1,2, Edwin Anderson1, Fatma Dedeoglu1
    1Boston Children's Hospital, 2Beth Israel Deaconess Medical Center, Boston, United States
    Correspondence: Fatma Dedeoglu

    Introduction: NLRP3 plays a role in intestinal homeostasis and has been implicated in the development of inflammatory bowel disease (IBD). Mutations in NLRP3 also cause the autoinflammatory disease cryopyrin-associated periodic syndrome (CAPS). Despite the involvement of NLRP3 in both IBD and CAPS, there are no reports of patients with CAPS who develop colitis and other manifestations of IBD.

    Objectives: We report on 3 patients who initially presented with bloody stools and were later diagnosed with CAPS.

    Results: Patient 1 had a history of recurrent painful hives since she was young, triggered by weather changes, stress, illness, or sun, but not cold. At 10 years of age, she developed recurrent uveitis, which was treated with topical steroids. She also had recurrent arthralgias. In her early 20’s she was diagnosed with Crohn’s disease when she developed abdominal pain and ileitis confirmed by colonoscopy. She was treated with mesalamine as needed during ileitis flares. At around the same time, she developed episodes of painful swelling of hands and feet, in addition to daily episodes of fever, fatigue, hives, nausea, and chills, lasting several hours. She had elevated inflammatory markers. She took hydroxychloroquine with no effect. In her 30’s she was diagnosed with CAPS with mutation A441V on the NLRP3 gene. She was started on canakinumab with resolution of most of her symptoms except for her arthritis and conjunctivitis, for which she remained on methotrexate.

    Patient 2 is the son of patient 1. He had occasional fevers before 1 year of age, but at 1 year of age, he started having episodic hives and fevers, often triggered by weather changes but not cold. He had recurrent episodes of bloody stools and diarrhea, which did not improve after dietary restrictions. At around age 2, he started having episodes of ankle arthritis in addition to conjunctivitis without other signs of ocular inflammation.He had normal growth and development. He did not have a colonoscopy. After his mother was diagnosed with CAPS, he was tested and found to be positive for the same NLRP3 mutation. Anakinra was started on an as-needed basis to be used during episodes with good response.

    Patient 3 developed feeding intolerance at around 6 months of age, which improved during his toddler years but recurred at 4 years of age, with the development of diarrhea, vomiting, abdominal pain, bloody stools, elevated inflammatory markers, and weight loss requiring NG tube feeding. He was seen by gastroenterology, and his Prometheus IBD panel was consistent with Crohn’s disease. He was empirically treated with steroids and sulfasalazine. A colonoscopy performed post-treatment did not confirm a pathologic diagnosis. He then developed recurrent episodes of fever and arthralgias lasting 1-2 days before self-resolving. He also suffered from recurrent headaches and there was a question of developmental delays and/or ADHD. At 9 years of age, whole exome sequencing showed the low-penetrance mutation R490K on the NLRP3 gene. His asymptomatic father was also found to be positive for the same mutation. Patient 3 was then referred to rheumatology. Further history revealed he had had urticarial rashes induced by heat and cold about twice per year. His hearing testing was normal. He was treated with anakinra, followed by canakinumab, with a good response except for rare breakthrough fevers, and for persistence of the gastrointestinal symptoms, including occasional bloody stools.

    Conclusion: Patients with CAPS may present with overlapping IBD symptoms such as bloody stools, and failure to thrive that may mask the usual CAPS features, causing delays in diagnosis.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1120 The challenge of treating pulmonary vasculitis in Behçet’s disease: two pediatric cases and literature review

    Selcan Demir1, Erdal Sağ1, Ümmüşen Kaya Akca1, Tuncay Hazırolan2, Yelda Bilginer1, Seza Ozen1
    1Pediatric Rheumatology; 2Radiology, Hacettepe Medical Faculty, Ankara, Turkey
    Correspondence: Selcan Demir

    Introduction: Behçet’s Disease (BD) is a multisystemic autoinflammatory disease and the most severe complication of BD is pulmonary artery involvement (PAI). Data regarding treatment and outcomes of pediatric patients with PAI is very limited.

    Objectives: Herein, we report two pediatric patients with BD presenting with PAI and treated successfully with aggressive immunosuppressive treatment.

    Results: Case 1

    A 15-year-old boy was admitted to a hospital with abdominal pain and feverAn abdominal Doppler ultrasonography (USG) showed stenosis of vena cava inferior (VCI) with a thrombus. Transthoracic echocardiography (TTE) detected that the thrombus extended from VCI to the right atrium. When he started to have hemoptysis, he was referred to our hospital. TTE showed a mass in RV. The computed chest and abdominal tomography angiography (CTA) showed bilateral aneurysmatic dilatation with thrombi in the pulmonary arteries and thrombosis in vena hepatica. The pathergy test was negative and the HLA B5 was negative. According to the ICBD, the patient had been diagnosed as BD due to genital ulcers and vascular involvement. He was given pulse methylprednisolone 500 mg 3 days along and followed by oral prednisolone 1 mg/ kg/day, intravenous cyclophosphamide at a dose of 500 mg (15 mg/kg) every 3 weeks for a total of 6 cycles, and Interferon-α2a (IFN-α2a) 3 times a week. Within one month, hemoptysis and fever disappeared, and CRP and ESR values normalized.After a three-month treatment, TTE and CTA revealed that thrombi shrank significantly. The dosage of prednisolone was tapered gradually and stopped 2 years later. Immunosuppressive treatment was continued with adalimumab. The patient has been followed in remission for nearly 6 years.

    Case 2

    A fifteen-year-old boy was referred to our hospital for the evaluation of fever for over 4 months and a thrombus in his right ventricle. He had a medical history with cough, fever, intermittent hemoptysis and weight loss (14 kg) for the past 3 months. Physical examination revealed acne-like rashes over face and back, ulcers on the buccal mucosa, a 3/6 systolic ejection murmur at the left upper parasternal area.A CTA confirmed the thrombus in RV and showed bilateral multiple aneurysms along the pulmonary artery and its branches. According to ICBD, the patient was diagnosed with BD due to having aphthous ulcers, pseudofolliculitis, and vascular involvement. Iv methylprednisolone (500 mg/day) for 3 days was followed by oral prednisolone 1 mg/ kg/day, which was subsequently tapered. Iv cyclophosphamide at a dose of 500 mg was also given every 3 weeks for a total of 6 cycles, followed by oral azathioprine (AZA). Concomitant subcutaneous IFN-α2a was given two times per week for 6 months. Within two weeks, cough and fever disappeared, CRP and ESR values normalized. After 1 year the pulmonary artery aneurysm disappeared and cardiac thrombosis resolved and returned nearly normal. We have been following the patient with AZA for four years without recurrence.

    Conclusion: We present two pediatric patients with pulmonary involvement of BD. PAI is a life-threatening condition and should be managed with more aggressive medical therapy.Early diagnosis and aggressive immunosuppressive treatment are very important in PAI. We strengthened our treatment with IFN-α2a. There is no data in the literature regarding the use of IFN-α-2a in PAI treatment along with low dose cyclophosphamide. There were no mortality or recurrences within the 6 and 4 years follow up period. An aggressive immunosuppressive therapy leads to better prognosis in this most dreadful complication of BD.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1121 Late diagnosis of cryopyrin-associated periodic syndrome - without gene confirmation - is there any other explanation?

    Raquel Faria1, António Lamas2, Ana Campar1, Graziela Carvalheiras1
    1Unidade de Imunologia Clínica – CHUPorto; 2Serviço de Medicina - CHUPorto, Porto, Portugal
    Correspondence: Raquel Faria

    Introduction: Cryopyrin-associated periodic syndrome (CAPS) is a rare and heterogenous inherited autoinflammatory disease entity: a spectrum of clinical phenotypes associated with NLRP3 mutations. It is characterized by the presence of raised inflammatory markers and typical manifestations: urticaria-like rash, cold-triggered episodes, sensorineural hearing loss, arthralgia/myalgia, chronic aseptic meningitis and skeletal abnormalities.

    Results: A 64-year-old woman presented psychomotor agitation and a recent diagnosis of left subclavian artery thrombosis with digital ischemia. She had a long history (since adolescence) of acute severe systemic inflammatory response syndrome, early-onset sensorineural hearing loss, seronegative arthritis refractory to NSAIDs, steroids, azathioprine and leflunomide, articular deformities of the wrists, knees and feet, bilateral tibial enchondromas and frontal bossing; recurrent episodes of uveitis (anterior and posterior), resulting in blindness; recurrent headaches associated with aseptic meningitis and negative temporal artery biopsy; and chronic kidney disease, category G4/A3 (KDIGO). There was no history of recurrent fever, cold-triggered episodes or rash and no family history of autoimmune or autoinflammatory disease. Markedly elevated serum amyloid A, ESR (erythrocyte sedimentation rate) and CRP (C-reactive protein). Negative autoimmune panel. Non-nephrotic proteinuria and small kidneys on ultrasound. Cerebrospinal fluid analysis revealed elevated protein and glucose levels, without associated pleocytosis. Imaging, micro and mycobacteriologic studies were negative for infectious foci. Pre-mortem genetic analysis results were only available post-mortem, with negative NLRP3 mutation findings by PCR and Sanger sequencing.

    Conclusion: This case report highlights the challenging diagnosis of an adult patient with severe manifestations and organ failure, that fulfils the clinical picture of an autoinflammatory disease, with longstanding uncontrolled inflammation resulting in irreversible organ damage (neurological, osteoarticular, ocular, among others) and complications (prothrombotic state, e.g.). Despite negative NLRP3 mutations, alternative inflammasome mutations (e.g. NLRP12) or mosaicism (not evaluated in the analysis) could explain it. The pattern of manifestations is highly suggestive of CAPS, fulfilling the diagnosis criteria proposed by Kuemmerle-Deschner et al. (2016), and represents a phenotype of increased severity in the CAPS spectrum in an adult patient. Given the patient’s age and clinical course, one may hypothesize an “attenuated” phenotype, possibly associated with genetic mosaicism. The diagnostic challenge of autoinflammatory diseases complicates early diagnosis and control of inflammation, which are critical to prevent irreversible organ damage, thus becoming of vital importance the recognition of autoimmune disease patterns and phenotypes.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1122 Late onset of tumor necrosis factor receptor associated periodic syndrome (TRAPS) due to somatic mosaicism

    Raquel Faria1, Luís Magalhães2, Daniel Oliveira1, António Marinho1, Ana Campar1
    1Unidade de Imunologia Clínica – CHUPorto; 2Serviço de Medicina - Hospital da Arrábida, Porto, Portugal
    Correspondence: Raquel Faria

    Introduction: Somatic mosaicism has been described as one of the major mechanism for autoinflammatory syndromes with late onset in adults.

    Results: Case description:

    We describe a 57 yo previously healthy woman, that after menopause (previous 9 years) developed recurrent 5-10 days episodes of high fever (39ºC), flutuant periorbital edema, abdominal urticarial rash, arthralgia/arthritis, myalgia, abdominal pain (no vomiting, rarely diarrhea), gengival pain, occasional cervical pain and “feeling flu-ish”. The episodes became more severe and frequent, the flu-like symptoms persisted every day. Fever responded to NSAIDs, not the rest, arthritic flares appear twice a month and she was not compliant to steroids. Blood analysis documented ESR >80mm/1st hour on attacks and 50mm/1st hour between attacks, C reactive protein 170mg/dL on attacks and 70mg/dL between attacks; and serum amyloid A ~90mg/dL between flares. Extensive infectious and autoimmune work lab panel was negative. The Eurofever Classification Criteria scored 74 points to CAPS (cryopirinopathies) and 88 points for TRAPS (Tumor Necrosis Factor Receptor Associated Periodic Syndrome). A PCR and Sanger sequencing detected c.176G>A (p.Cys59Tyr) in TNFRSF1A, in 20% of the whole blood sample in EDTA (compatible with somatic mosaicism) – validated pathogenic mutation. Colchicine 1mg/day had no effect on clinical symptoms or inflammatory markers. All her symptoms remitted and C reactive protein and serum amyloid A normalized wtih anakinra 100mg/day subcutaneous. No other family member (living parents or sibling) had ever had any fever or inflammatory symptoms.

    Conclusion: The suspicion level for monogenic autoinflammatory diseases in adults should be high as the clinical picture might be atypical and of slower progression. Genetic testing should include mosaicism screening mainly in adults and a close collaboration with attending clinical physicians is crucial for the final diagnosis.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1123 Hyperzincaemia and hypercalprotectinemia syndrome: more than just autoinflammation?

    Andrea Uva1, Claudia Bracaglia1, Silvia Federici1, Camilla Celani1, Manuela Pardeo1, Christoph Kessel2, Fabrizio De Benedetti1, Antonella Insalaco1
    1Division of Rheumatology, Ospedale Pediatrico Bambino Gesù, Rome, Italy; 2Department of Pediatric Rheumatology & Immunology, University Children’s Hospital, Muenster, Germany
    Correspondence: Silvia Federici

    Introduction: Hyperzincaemia and hypercalprotectinemia (HandH) syndrome has been described as a new rare entity characterized by recurrent infections, dermatological involvement, increased inflammatory markers, hepatosplenomegaly and anemia. Little is known about its heterogeneous presentation, pathophysiology and treatment.

    Objectives: To describe three cases with HandH syndrome

    Methods: Serum calprotectin (MRP8/14) was measured according to Buehlmann assay (ELISA) and plasmatic zinc by atomic absorption spectrometry

    Results: Three patients were referred to our centre because an history characterized by recurrent episodes of skin rash, severe oral aphtosis and increased level of serum amyloid A (SAA). Patient 1 presented, since the age of ten years, with recurrent episodes of fever and rash; skin biopsy showed a picture consistent with a lymphocytic lichenoid vasculitis resembling erythema multiforme. Patient 2 presented at birth, with hemolitic anemia and thrombocytopenia. At the age of 5 she was admitted to another hospital due to EBV related hemophagocytic limphohystiocytosis (HLH). At the age of 8, she was first seen at our center because of a persistent desquamant erythematous rash with recurrent abdominal pain and recurrent arthritis. Intestinal biopsy showed small intestine inflammation (erosions in the digiunum). Patient 3 presented with recurrent episodes of fever, rash, two episodes of transient hip synovitis and muscoloskeletal pain. A bone scintigraphy was performed resulting normal. Patients 1 and 2 suffered from recurrent infections (pneumonia, otitis, skin abscesses). Immunological studies revealed in patient 2 a reduction of memory B cells and a reduced response to Toll like receptor 9 agonist. Of note, all the patients presented in their medical history at least one episode of vasculitis: patients 1 and 3 suffered from Schonlein-Henoch purpura at the age of 11 and 3 respectivelyand patient 2 had at the age of 2 years an undefined vasculitis (evaluated elsewhere). Laboratory tests showed in all patients elevated inflammatory markers, zinchemia and serum calprotectin (table)

    Conclusion: We report three patients with high serum levels of calprotectinemia and zinch presenting with a clinical phenotype consistent with previously reported cases. The presence of vasculitis in all of the patients suggests that it may represent the first symptom of this condition. Vasculitis could lead to an increase of serum calprotectin already proposed as a marker of vascular impairment. Moreover, considering the immunological defect detected in one of our patient, we speculate that recurrent infections described in this syndrome may underline an immune-dysregulation process in which the role of zinc metabolism needs to be assessed

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P1123). See text for description

    P1124 Inherited deficit of proteoglycan mimicking septic arthritis

    Angelo Florio1, Riccardo Papa1, Roberta Caorsi1, Alessandro Consolaro1, Tuula Rinne2, Roberto Gastaldi3, Angelo Ravelli1, Marco Gattorno1, Paolo Picco1
    1Clinica Pediatrica e Reumatologia , IRCCS Istituto Giannina Gaslini, Genova, Italy; 2Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands; 3Clinica Pediatrica ed Endocrinologia, IRCCS Istituto Giannina Gaslini, Genova, Italy
    Correspondence: Angelo Florio

    Introduction: Aggrecanopathies are a heterogeneous group of skeletal disorders caused by ACAN gene mutations leading to aggrecan dysfunction: this latter is a proteoglycan with a pivotal role in extracellular matrix of the growth-plate cartilage organization. Clinically, aggrecanopathies displayed bone dysplasias, idiopathic short stature and facial dysmorphisms. Herein we report a patient with a prevalent inflammatory articular involvement, e.g. osteochondritis dissecans (OCD) mimicking a septic arthritis.

    Methods: A 14-year-old boy displayed pain and swelling at the right elbow. Echo-scan revealed an effusion in both coronoid and olecranon recess. Acute phase reactants were negative. Since non-steroidal anti-inflammatory drugs were ineffective, the patient was admitted in our Institute, a month later clinical onset.

    On physical examination, acute arthritis at right elbow was present, which appeared painful, warm, not erythematous. Laboratory test showed slight elevated inflammatory markers (C reactive protein 1.7 mg/dl, normal value <0.5). Arthrocentesis of the right elbow was performed: sterile synovial fluid was found. Magnetic resonance images of the right elbow displayed bone fragment detachment from humeral condyle with thickening of the synovium and persistent local effusion: the diagnosis of OCD was pointed out.

    It is worth nothing that patient showed minor dysmorphisms (i.e. dolicocephaly, hypotelorism, arched palate and brachydactyly of the IV finger of both hands). Furthermore parents referred that the child presented a previous episode of OCD when he was 12: the symptoms resolved with non-bearwighting and non-steroidal anti-inflammatory therapy.

    The patient went under regular endocrinologist follow-up for short stature since he was 8. At the age of 10 (height cm 123 – SDS 2.4) Growth Hormone (GH) stimulation tests were performed showing only partial response to insulin tolerance test (GH peak 6.27 ng/mL). Bone age was delayed of 12 months. Human recombinant GH replacement therapy was started without a significant growth-velocity improvement.

    Results: Although our patient came to observation because of suspected elbow septic arthritis, we re-considered the diagnosis: namely, i. recurrent episodes of osteochondritis dissecans; ii.short stature poorly responsive to human recombinant GH treatment, iii. Mild facial, skeletal dysmorphisms led us to hypothesize a form of aggrecanopathy. Molecular analysis of the ACAN gene was performed revealing a novel missense variant c.6970T>C, p.Trp2324Arg. A similar mutation in the G3 domain (c.7249G>A) has been previously described and has been connected to aggrecanopathy.

    Conclusion: Aggrecan, an important sub-unit of proteoglycan in cartilage, is synthetized by ACAN gene. Aggrecan-related bone diseases range from severe spondylo-epi-metaphyseal dysplasia to familial osteochondritis dissecans usually associated with short stature. In our experience, a patient with a novel mutation of ACAN gene seems to cause an atypical form of aggrecanopathy mimicking inflammatory and/or septic arthritis associated with slight short stature and bone dysmorphisms. Intra-familial molecular analysis allowed us to recognize other three subjects (mother and 2 siblings) affected only by brachydactily. Further studies need to confirm the role of this variant of ACAN gene as cause of inflammatory arthropathy.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1125 LRBA deficiency in a patient with chronic arthritis

    Şerife Gül Karadağ, Ayşe Tanatar, Mustafa Çakan, Haifize Emine Sönmez, Figen Çakmak, NurayAktay Ayaz
    Pediatric Rheumatology, Health Sciences University Kanuni Sultan Süleyman Training and Research Hospital, Istanbul, Turkey
    Correspondence: Şerife Gül Karadağ

    Introduction: Lipopolysaccharide-responsive, beige-like anchor protein (LRBA) deficiency causes common variable immunodeficiency (CVID) disorders and autoimmunity. Patients with LRBA deficiency may present with a wide spectrum of clinical manifestations. Herein, we report a patient with LRBA deficiency associated chronic arthritis

    Results: A 20-year-old male patient was admitted to our hospital with swelling in knees and ankles for two months. He was initially diagnosed with Evans syndrome when he was four years old. Subsequently, he was diagnosed with Crohn’s disease due to chronic diarrhea at 18-years old. He also suffered from recurrent infections since infancy. He also was followed by endocrinology department due to hypothyroidism. When he was 20-years old, he was referred to our outpatient clinic due to chronic arthritis. In laboratory work-up, he had elevated acute phase reactants and direct coombs positivity. Romatoid factor, anti-nuclear antibody and HLA-B27 were negative. Initially, he was considered to have chronic arthritis due to Crohn’s disease and sulfasalazine was started. In third months, adalimumab was administered due to the inadequate response. Immunodeficiency due to coexistence of endocrinopathy, autoimmune cytopenia and recurrent infections was suspected. Therefore, expression of LRBA protein was found to be low. Mutation in LRBA gene was evaluated with Sanger sequence. A homozygous mutation in LRBA gene was detected (c.2818dupC;p.Gln940Profs*5).Abatacept (CTLA4 fusion protein) and intravenous immunoglobulin was started. Now, he is in remission.

    Conclusion: The association of LRBA deficiency and autoimmunity has previously described. However, the data about the association LRBA deficiency and arthritis is limited. The new finding of this rare disease might further expand the phenotypic spectrum.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1126 Unusual case of recurrent periodic macrophage activation

    Khulood Khawaja1, Tawfiq T. Hen2
    1Adult and Pediatric Rheumatology, Al-Mafraq Hospital; 2Pediatrics, Shaikh Khalifah Medical City, Abu-Dhabi, United Arab Emirates
    Correspondence: Khulood Khawaja

    Introduction: Macrophage activation syndrome (MAS) is a multisystem inflammatory disorder, can be fatal. Most commonly complicating systemic juvenile idiopathic arthritis (JIA)

    Objectives: To discuss treatment pathway of recurrent MAS in systemic JIA when initial therapy fails.

    Methods: Case presentation of severe and recurrent periodic MAS in a child with systemic JIA

    Results: 12-year-old girl presented with 3 weeks history of fever, lethargy and rash.She was having fever in the evening with erythematous rash over her shoulders, face and trunk.She then developed pain in multiple joints with swelling in both knee joints.She had ultrasound scan of her knees, which confirmed effusions and synovial thickening.There had been no preceding infections or travel. Parents are consanguineous.3 siblings fit and well. No family history of significance.

    Initial examination, temperature 38.3°C, she had acanthosis nigricans in axillary areas and behind her neck.No dysmorphic features.Conjunctivanormal.No hair or nail abnormality.She had faint rash over her face and shoulders.Musculoskeletal examination, pediatric gait, arm, leg and spine examination:She was struggling to hop and walk on her heels.Arms:She hadminimal effusions in her wrists with pain on extreme flexion and extension.Legs:She had effusion in both knees and ankles.Spine:Normal.TMJ:Normal. Examination of other systems unremarkable (later developed mild hepatospleenomegaly)

    Initial bloods:ANA negative, double stranded DNA negative, C3 and C4 negative, ENA negative, rheumatoid factor positive.Sugar okay, thyroid function okay, hepatitis B and C serology negative, Quantiferonnegative, Renal function normal. ESR 115 and CRP 129.

    USS of knees, ankles and wrists confirmed synovitis.CHAQ score 0.5, pain VAS 6, general VAS 7,

    Initially treated as JIA with systemic features and was started on Methotrexate 15mg/m2 weekly, joint injections with steroids of wrists, knees and ankles then oral prednisolone reducing dose. She improved. Rash resolved. No fever but continued to complain of joint pains. She was changed to Anti TNF; etanercept (50mg weekly) and small dose of steroids . She also had inadequate response so was changed to IL-6 blocker; tocilizumab.She was well for 6 months then developed MAS presenting with fever, lethargy and feeling nauseated with raisedferritin of >10,000, cytopenia, hyperglyceremia, hypofibrogenemia and raised triglycerides. Bone marrow aspirate normal. Treatment changed to IL-1 blocker Anakinra 100mg daily. She was well on anakinra for 4 months then presented again with MAS requiring IV methyl prednisolone and IV fluids. Has had monthly attacks for 5 months (one with seizure) then every 2 weeks in the last 2 months. Cyclosporin 100mg twice daily was added to her treatment with no benefit. She also had IVIG 2g/kg with no benefit. She was assessed by Oncology and neurology; no concern of malignancy. HLH genetic testing: PRF1, UNC13D, STX11, STXBP2.,SH2D1A, XIAP (BIRC-4), RAB27A, LYST, ITK, GATA2, AP3B1, BLOC1S6, CD27, SLC7A7 all negative. Brain imaging normal. TRAPS,NLRP3 genetic testing neative. She is currently awaiting the result of whole gene sequencing. In terms of treatment, cyclophosphamide was started 375mg/m2. Had first dose. Planning3 doses every 2 weeks then 4 doses every month.

    Conclusion: Our patient had multiplepreseentations of MAS, first following several months of treatment with tocilizumab then Anakinrathen 5 presentations monthly only responding to systemic steroids then every 2 weeks for the last 4 presentations.

    No clear guidelines to treat MAS once standard therapy unsuccessful. Further work is needed to study the genetics of such patients by collaborative work amongst multiple centres.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1127 AA amyloidosis in recipients of cadaveric derived pituitary growth hormone – potential effect of an amyloid enhancing factor?

    Helen J. Lachmann, Dorota Rowczenio, Janet A. Gilbertson, Hadija Trojer, Islam Noor, Thirusha Lane, Julian D. Gillmore, Ashutosh D. Wechalekar, Philip N. Hawkins
    National Amyloidosis Centre, UCL Division of Medicine and Royal Free Hospital NHS Foundation Trust, London, United Kingdom
    Correspondence: Helen J. Lachmann

    Introduction: The development of Creutzfeldt–Jakob disease (CJD) in individuals who had been treated during childhood with human cadaveric pituitary-derived growth hormone (c-hGH) was first reported in 1985 and resulted in its immediate withdrawal and replacement with recombinant protein. Subsequently amyloid-β protein (Aβ) deposits have been described in individuals with CJD and prior exposure to c-hGH. Recently misfolded Aβ peptide was identified in archived vials of human c-hGH, which induced Aβ pathology in an experimental model (Purro et a Nature 2018) supporting its role in the transmission of CJD.These observations are reminiscent of the concept of amyloid enhancing factor (AEF), in experimentally induced AA amyloidosis, in which administration of tiny amounts ofamyloidotic material is associated with massively accelerated AA amyloid deposition.

    Objectives: To look for evidence of prior exposure to c-hGH in patients with AA amyloidosis.

    Methods: We searched our database of 625 patients with AA amyloidosis seen between 1990 and 2017 using the key words pituitary, craniopharyngioma and growth hormone. Only patients whose records suggested exposure to GH prior to 1985 were included.

    Results: 10 cases were identified; accounting for 1.6% of all AA amyloidosis and 8.4% of cases of AA of unknown aetiology. 8 were female, 8 were north European ethnicity and the median age at histological diagnosis of AA amyloidosis was 47.9 yrs with a median time since commencing c-hGH of 35 yrs. c-hGH was started in the 1960’s in 4 cases, the 1970’s in 4 cases and the 1980’s in 2 cases. The indications for GH were: craniopharyngioma in 4, one case each of resection of ependymona, nasal pharyngeal tumour and encephalocoele, 3 cases of unexplained hypopituatism. Chronic inflammation was assessed by serial blood tests after the diagnosis of AA amyloid; only 1 case had severe sustained inflammation with a median SAA 131 mg/L; 4 had medians SAAs of between 20 and 54 mg/L and the others had only low level inflammation with median SAA below 20mg/L. No clear cause of inflammation was identified in the cohort although 50% were obese and 3 had body mass index above 45 kg/m2. Genetic testing was performed in all – in no cases did this result in a diagnosis of a systemic autoinflammatory disorder. Six European patients were homozygous for SAA1.1 (66.7% compared to a reported frequency of 42.7 - 57.9%in European controls), a known predisposing factor for AA amyloidosis. One patient had a fall in SAA after bariatric surgery and one responded completely to anakinra. The other patients had no specific treatment aimed at supressing their SAA production. Five patients eventually required dialysis – 2 of them had subsequent renal transplants; median survival from diagnosis with amyloid is 132 months and 3 patients have died.

    Conclusion: We report 10 patients with AA amyloidosis of unknown aetiology, who were exposed to c-hGH in childhood. We hypothesise that the c-hGH may have contained material that seeded conversion of healthy low concentrations of SAA into AA amyloid fibrils. It is of interest that 6 of this cohort had an additional risk factor in the form of SAA1.1 homozygosity suggesting that a combination of factors may contribute to amyloid formation in individuals who have low levels of circulating potentially amyloidogenic protein.

    Disclosure of Interest

    None Declared

    P1128 Progressive necrotic ulcerative lesions: challenging diagnosig?

    Angela Mauro1, Francesca Orlando2, Maria Tardi3, RobertoRega3, Martemucci Luigi3, Rita Sottile3
    1Pediatrics, Rheumatology Unit, Department of Pediatrics, “Santobono-Pausilipon” Children Hospital, Naples, Italy; 2 Pediatrics; 3Pediatrics, Rheumatology Unit, Department of Pediatrics, “Santobono-Pausilipon” Children Hospital, Naples, Naples, Italy
    Correspondence: Angela Mauro

    Introduction: Pediatric ulcers are less common compared to adult population. No data about prevalence are available. Differential diagnosis is broad, including autoinflammatory, hematologic, infectious andautoimmune causes. .

    Objectives: We describe a case of progressive necrotic ulcerative lesions in a 7- month caucasian boy

    Methods: A 7-month-old Caucasian boy was admitted to our Department with purpuric and telangiectatic lesions on his right hand and left wrist without fever. After one month these lesions evolved in necrotic ulceration of the skin .He was born at term to unrelated parents after a pregnancy complicated by premature contractions.At the age of 9 months he developed a large necrotic lesion (4x4 cm) on the right foot and other similar smaller lesions on the knees. He had purpuric and telangiectatic lesions on the left foot and the medial face of the left leg. Laboratory assessments were performed to exclude infectious diseases (Mycoplasma, Chlamydia, HBV, HCV, HAV, Treponema Pallidum, Rickettisa conorii, Widal-Wright, BorrelliaBurgdoferi, TORCH, EBV) The inflammatory markers (ERS, SAA, CRP), prothrombotic risk factors (PT, APTT, fibrinogen, protein S, protein C), C3, C4, immunoglobulins, antiphospholipid antibodies, ANA, ANCA, ASCA, anti-Dna, celiac disease antibodies, Quantiferon, hemoglobin electrophoresis, blood culturesand urine analysis were negative or in the normal range.The analysis of CERC1 gene was negative for the main mutaions.It was also tested CD18, blood T-lymphocyte subpopulations. cryoglobulins, interferon signature (negative) and CGH ARRAY, karyotype, and for zinc deficiency (ongoing).Skin lesion cultures were positive forProteus mirabilis andStaphylococcus aureus.Ultrasonography of lower limbs resultednegative except for arteritis of the small vessels on the right foot lesion.

    Echocardiography showed interatrial communications ostium secundum type with slight left-right shunt.Abdomen Ultrasoundwas negativeChest radiography (fatta in faseacuta?):hyperinflated lungs, aincrease interstitial markingof the right and left retrocardiacfields. . Flat diaphragm. Expansive ribs.Brain MRI: negativeElectromyography: slight neurogenic problemsSkin biopsy: microscopic epidermis partly necrobiotic, focal spot parakeratosis and spongiosis with vesicles formation. In the dermis, presence of edema and erythrocyte extravasation in the papillae, small thrombosed vessels, sometimes with fibrinoid necrosis and only focal lymphocytic infiltrated, PASS-Giemsa negative staining, negative Z-Nilsen staining

    Results: Findings consistent with livedoid vasculopathy, cryoglobulinemia and gangrenous ecthyma. For gangrenous ecthymahe started Teicoplanin, Meropenem and Fluconazole with a subsequent improvement of the skin lesions and a decrease of necrotic eschars and lesion re-epithelialization, but after 15 days he presented new purpuric lesions on his legs, right heel, and hands.

    Conclusion: Do you have any input about this patient?

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P1129 An undefined systemic autoinflammatory disease complicated by MAS or an unidentified primary HLH?

    Roberta Naddei1, Francesca Orlando1,2, Carolina Porfito1, Teresa Lastella1, Marinabiondina Amico1, Maria Alessio1
    1Pediatric Rheumatology Unit, Mother and Child Department, University of Naples Federico II; 2Department of Pediatrics, Santobono-Pausilipon Children’s Hospital, Naples, Italy
    Correspondence: Roberta Naddei

    Introduction: Systemic auto-inflammatory diseases (SAIDs) are a rare group of illnesses characterized by unprovoked episodes of fever and systemic inflammation. Many patients receive a diagnosis of undefined SAID because they have clinical features not fitting a specific diagnosis or genetic tests are negative.

    Objectives: To describe clinical presentation of a patient affected by recurrent episodes of fever characteristics resembling hemophagocytic lymphohistiocytosis.

    Methods: Case report.

    Results: The patient (male, age 30 months) referred to our Unit at the age of 3 months. At 1 month, he underwent a surgical repair of ventricular septal defect. At 3 months, he was admitted to a hospital for fever and maculopapular rash, with marked elevation of acute phase reactants (Protein C-reactive, PCR, 108 mg/L). Empiric antibiotic therapy was started, but no evidence of infectious diseases was disclosed.At the tenth day of fever, laboratory examinations found thrombocytosis and increase of ferritin (5034 ng/ml), lactate dehydrogenase (1059 U/l), aspartate and alanine aminotransferase (254 and 124 U/l) and triglycerides (266 mg/dl). In the suspicion of atypical Kawasaki disease, even if no coronary artery aneurysm was detected, treatment with intravenous Immunoglobulin and aspirin was started with disappearance of fever; because of persistent cutaneous rash and ferritin, triglycerides, platelets and PCR high levels, he was transferred to our Unit. On admission, physical examination revealed neck and inguinal enlarged lymph nodes. No organomegaly was detected. Laboratory investigations also revealed increased neutrophils (17830/mm3), normocytic anemia (hemoglobin 7,5 g/dl), hypoalbuminemia and hyponatremia. Fibrinogen levels were normal. Bone marrow aspiration revealed no signs of hemophagocytosis or cellular atypia. Intravenous methylprednisolone pulse therapy (30 mg/kg for three days) was started followed by daily oral prednisone (2 mg/kg/day), with clinical resolution and laboratory improvement. After six-months tapering, steroid therapy was withdrawn. Two months later, the patient newly present fever, rash and increase of PCR and ferritin. Anakinra (2 mg/kg/day) was started with improvement of clinical condition. Nevertheless, because of the persistent elevation of inflammatory markers, anakinra dosage was increased up to 4 mg/kg/day during the follow-up. Besides, the mother of child revealed difficulty to adhere daily subcutaneous therapy. At the age of 2 years, he presented a new episode of fever and rash with high ferritin level (2364 ng/ml); anakinra was stopped and canakinumab (4 mg/kg/4 weeks) was started with initial dramatic clinical and laboratory improvement. Fifteen days after the first injection of canakinumab, the patient newly presented fever, without rash. IL18 was raised (184000 pg/ml). Therapy with prednisone (2 mg/kg/die) was added with fever disappearance. During the follow-up, in therapy with canakinumab and prednisone, the patient never obtained normalization of inflammatory markers and fever presented two or three weeks after canakinumab injection.

    So, during the last episode of fever, canakinumab was stopped and cyclosporine was added to steroid therapy. Since the introduction of cyclosporine, the patient has not presented fever and inflammatory markers have normalized.

    During the follow-up, the patient underwent Next Generation Sequencing for HLH and SAID; no mutation was identified, except for a heterozygous CECR1 variant.

    Conclusion: This patient presents recurrent hyperinflammation episodes, resembling HLH/MAS, with low response to anti-IL1 agents. Genetic tests for HLH and SAID are negative; what are we missing? Should we attempt IL18 blockade?

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Allied Health Professionals

    P1130 Towards European harmonisation of healt care for patients with rare immune disorders: ERN RITA result from the registries survey

    Riccardo Papa1, Andrew Cant2, Christoph Klein3, Mark Little4, Nico M. Wulffraat5, Marco Gattorno1, Nicolino Ruperto1 and on behalf of RITA ERN Council
    1Clinica Pediatrica e Reumatologia, IRCCS Istituto Giannina Gaslini, Genova, Italy; 2Great North Children's Hospital, & Institute for Cellular Medicine, University of Newcastle, Newcastle upon Tyne, United Kingdom; 3Department of Pediatrics, Ludwig-Maximilians-University Munich, Munich, Germany; 4Department of Nephrology and Kidney Transplantation, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland; 5Department of Pediatrics, Section Pediatric Rheumatology, Wilhelmina Children's Hospital, University Medical Centrum Utrecht, Utrecht, Netherlands
    Correspondence: Riccardo Papa

    Introduction: Rare Immunodeficiency, AutoInflammatory and AutoImmune Disease (RITA) network is a European Research Network that brings together the leading centres for rare immune disorders (RID).

    Objectives: To know the state of the art about diseases registries and research networks in Europe regarding patients with RID.

    Methods: On April 2018 an on-line survey was sent to all 126 RITA members, of whom 45 are healthcare providers and 8 patients/family organizations. All data were collected and analysed using Excel program.

    Results: Ninety RITA members (71%) replied. Collectively, 27 members are coordinating 25 registries, 53 members are participating in 38 registries, and 27 members knew the existence of 16 registries without participating. Only two members are not involved in any registries.

    Data about 53 different registries were collected across 14 European countries. Almost 50% of registries collect data on autoimmune disorders, while others are dedicated to primary immunodeficiencies and autoinflammatory diseases (respectively 15 registries, 28%, and 12 registries, 23%). Fifteen registries (28%) enrolled patients with a single specific disorder: in particular, three registries are devoted to systemic lupus erythematosus, two registries to Kawasaki disease or Behcet disease, and single registry to juvenile dermatomyositis, juvenile systemic sclerosis, juvenile idiopathic arthritis (JIA)-related uveitis, systemic JIA, Blau syndrome, sarcoidosis, Guillain-Barre syndrome, and myasthenia gravis.

    More than 55000 patients with RID are enrolled. The majority of registries (36; 68%) enrol only patients from national territories. Among the internationals, six collect data on autoimmune disorders (Pharmachild, BrainWorks, EuroMyositis, EULAR web library, UKIVAS registry and JIR cohort), five on primary immunodeficiencies (ESID, EBMT, SCETIDE, PCID and HLH registry), and three are devoted to autoinflammatory diseases (Eurofever, Infevers, and ImmunAID), despite also ESID registry and JIR cohort collect data on autoinflammatory diseases.

    Data usually collected in these registries are demography, diagnosis, clinical manifestations, laboratory tests and treatment, while genetic and imaging data are less frequently reported (respectively in 38% and 9% of registries). A treatment safety profile is reported in 29 registries (55%). Collectively, fifteen biobanks are counted.

    Conclusion: The survey highlighted the pivotal role of national and international organizations in Europe to collect and organize clinical data on immune diseases, allowing the rapidly growing knowledge on these rare disorders, creating research networks and providing significant numbers of data to support new discoveries in the field. RITA network could improve the coordination of these numerous entities, supporting initiatives of collaboration. As a first attempt, the present survey revealed that the collection of key parameters about patient safety, as well as outcome data and quality of life measures should be improved among the registries of RITA network.

    Disclosure of Interest

    None Declared

    Poster presentations – Tuesday 2 April

    Guided poster tour 2A

    PT2A01 The French pediatric cohort of castleman disease

    Charlotte Borocco1,2, Claire Ballot-Schmit3, Oanez Ackermann4, Nathalie Aladjidi5, Jeremie Delaleu6, Vannina Giacobbi-Milet7, Sarah Jannier8, Eric Jeziorski9, Yves Perel5, François Maurier10, Christophe Piguet11, Eric Oksenhendler12,13, Isabelle Kone-Paut1,2,14, Caroline Galeotti1,2,14
    1Pediatric Rheumatology; 2CEREMAIA, Bicetre Hospital, Le Kremlin-Bicêtre; 3General Pediatrics, Jean Minjoz Hospital, Besançon; 4Pediatric hepatology, Bicetre Hospital, Le Kremlin-Bicêtre; 5Pediatric Oncology and Hematology, Pellegrin Hospital, Bordeaux; 6Internal Medicine, Tenon Hospital, Paris; 7Pediatric Oncology and Hematology, Le Mans Hospital, Le Mans; 8Pediatric Oncology and Hematology, Hautepierre Hospital, Strasbourg; 9General Pediatrics, Arnaud de Villeneuve Hospital, Montpellier; 10Internal Medicine, Metz Private Hospital, Metz; 11Pediatric Oncology and Hematology, Limoges Hospital, Limoges; 12Clinical Immunology, Saint-Louis Hospital; 13National Reference Center for Castleman Disease, Paris; 14National Reference Center for Castleman Disease, Le Kremlin-Bicêtre, France
    Correspondence: Caroline Galeotti

    Introduction: Castleman disease (CD) is a very rare non-malignant lymphoproliferation of undetermined origin. CD diagnosis is difficult and often delayed because of insidious onset, low awareness and clinical heterogeneity. Two major disease phenotypes can be distinguished: unicentric CD (UCD) and multicentric CD (MCD). Diagnosis confirmation is based on histopathological findings on an involved lymph node.

    Objectives: We attempted to survey all cases of paediatric CD identified in France so far, in order to set up a national registry aimed to improve CD early recognition, treatment and follow-up, within the context of a new reference centre (http://www.castleman.fr).

    Methods: In 2016, we e-mailed a questionnaire to members of the French paediatric immunohaematology society, the French paediatric rheumatology society and the French Reference Centre for Castleman Disease to retrospectively collect cases of paediatric CD (first symptoms before age 18 years). Anatomopathological confirmation was mandatory.

    Results: We identified 23 patients (12 girls and 11 boys) diagnosed with UCD (n=17) and MCD (n=6) between 1994 and 2018 in 14 centres.

    The average age at first symptoms was 11.5 years for UCD and 8.3 years for MCD. The mean diagnosis delay was 8.2 months for UCD and 5.2 years for MCD.

    In UCD, the initial symptoms were: isolated lymph nodes (n = 10) or lymph node associated with other symptoms (n = 7), fever was present in 3 patients.

    Patients with MCD presented with fever (n=5), abdominal lymph nodes (n=5), failure to thrive (n=3), hepatomegaly and/or splenomegaly (n=3), arthralgia (n=2), abdominal pain (n=2), fatigue (n=2), facial oedema (n=1), isolated lymphadenopathy (n=1), trunk rash (n=1), vascular hepatopathy with oesophageal varicose veins (n=1), diarrhoea (n=1) or cholestasis (n=1). One patient had a Duchenne muscular dystrophy. No patients had HIV or HHV8 infection. The 6 patients met all the diagnosis criteria proposed by Fajgenbaum et al in 2017 for idiopathic MCD.

    One MCD patient with recurrent fevers, had a heterozygous mutation in MEFV gene. Another UCD patient experienced fever episodes and pericarditis two years after surgery. Genetic tests revealed one heterozygous mutation in IL10RA gene (V406L) and one in IL36RN gene (S113L). Nevertheless, our patient did not have any psoriasis or inflammatory bowel disease.

    Treatment of UCD was mainly surgical resection, steroids, and radiotherapy. Treatments of MCD were tocilizumab, rituximab, anakinra, steroids, chemotherapy and splenectomy.

    Overall survival after an average of 6 years of follow-up, was 100 % for both unicentric and multicentric forms.

    Conclusion: Paediatric CD seems still underdiagnosed with a significant diagnosis delay specially for the MCD but new international criteria will help in future. In UCD, surgery is the main treatment. New drugs are used specially for the MCD, but more studies need to be conduct in children. The global survey is better in children than adults.

    Disclosure of Interest

    None Declared

    PT2A02 Use of whole-body magnetic resonance to identify potential diagnostic clues in children with fever of unknown origin (FUO)

    Sara Signa1,2, Roberta Caorsi1, Giorgio Stagnaro3, Francesca Minoia1, Paolo Picco1, Angelo Ravelli1,2, GM Magnano3, Maria B. Damasio3, Marco Gattorno1
    1Clinics of Pediatrics and Rheumatology, G.Gaslini Institute; 2DINOGMI, University of Genoa; 3UO of Radiology, G.Gaslini Institute, Genoa, Italy
    Correspondence: Sara Signa

    Introduction: Whole-body magnetic resonance imaging (WBMRI) is a fast and accurate method to detect diseases throughout the entire body without exposure to ionizing radiation. Possible emerging applications for this technique include rheumatologic field and evaluation of fever of unknown origin (FUO).

    Objectives: To evaluate the ability of WBMRI to identify significant potential diagnostic clue (PDC) in patients presenting a non specific inflammatory clinical picture.

    Methods: We retrospectively collected cases of pediatric patients followed in a single pediatric rheumatology center who underwent WBMRI between January 2010 and December 2015 for the following indications: i) FUO (temperature greater than 38.3°C for more than three weeks or failure to reach diagnosis after one week of investigations), iii) recurrent fever (febrile episodesseparated by periods of normal temperature), iii) Inflammation of unknown origin, IUO (an illness of at least 3 weeks’ duration, with raised inflammatory markers and fever below 38.3°C).WBMRI studies were acquired with coronal and sagittal planes (slice thickness 5mm) with acquisition of several image sets with automatic direct image realignment after acquisition creating a whole-body scan.Sequences include short τ inversion recovery (STIR) and T1-weighted. All studies have been evaluated twice, the second time according to a predefined checklist, defined by an experienced radiologist, considering systematically single /multifocal bone lesion, bone marrow, joint effusion, soft tissues, adenopathies, parenchymal and vessels looking for PDC. We considered as a Potential Diagnostic Clue each alteration of the examined district that can potentially guide the diagnosis. Each alteration found is a PDC. We retrospectively evaluated patients’ clinical history and final diagnosis and we classified the PDCs identified during both first evaluation and re-evaluation as: Not useful (the identified PDC did not guide the diagnosis and is not coherent with the final diagnosis), consistent (the identified PDC is congruent with the patient’s final diagnosis) or diagnostic (the identification of the considered PDC strongly orient the final diagnosis).

    Results: We collected 102 patients who underwent WBMRI; 24 (23%) of them presenting FUO, 27 (26%) presenting recurrent fever and 51 (51%) presenting persistent low grade fever . The mean age of onset symptoms was 7 years and seven months (range: 2 weeks old- 17 years and 6 months). The mean age of execution of WBMRI was 9 years (range: 5 months old- 19 years and one month). After the whole diagnostic work-out a final diagnosis was achieved in 42 patients (41%).PDCs were identified at the first evaluation in 76/102 cases (74.5%).In 22 cases (21.5%) the identified PDCs were consistent with the diagnosis, whereas in 7 cases (7%) the identified PDCs were considered diagnostic. Globally we can consider that at first evaluation PDCs were somehow contributory to the diagnosis in 29 cases (28.5%; 6 JIA, 7 systemic infections, 5 monogenic inflammatory diseases, 4 ALPS, 2 Goldbloom’s Syndrome,2 Vasculitis,1 eosinophilic fasciitis, 1 hystiocytosis, 1 neuroblastoma). Blind re-evaluation of WBMRI allowed the identification of additional PDCs in 52 patients (12 of them previously negative) . In 10 cases the PDC found after re-evaluation were consistent with the final diagnosis (2 JIA, one infectious disease, one neuroblastoma, 3 ALPS, 1 monogenic inflammatory disease, 1Takayasu arteritis, 1 Goldbloom’s syndrome).

    Conclusion: WBMRI can be a powerful diagnostic tool in patients with FUO. A predefined checklist increase sensitivity of WBMRI in the identification of PDC.

    Disclosure of Interest

    None Declared

    PT2A03 A biomarker profile including S100A12 as diagnostic tool for the differential diagnosis of sJIA-associated MAS vs. primary or secondary HLH

    Christoph Kessel1, Ndate Fall2, Alexei Grom2, Wilco de Jager3, Sebastiaan Vastert4, Raffaele Strippoli5, Claudia Bracaglia6, Erik Sundberg7, Anna Horne8, Stephan Ehl9, Sandra Ammann9, Kai Lehmberg10, Fabrizio De Benedetti6, Helmut Wittkowski1, Katharina Kessel1, KarinBeutel11, Dirk Foell1, Dirk Holzinger12
    1Pediatric Rheumatology and Immunology, University Children’s Hospital, Muenster, Germany; 2Division of Rheumatology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; 3Laboratory of Translational Immunolgy, Utrecht Medical Center; 4Pediatric Rheumatology and Immunology, University Medical Center Utrecht, Utrecht, Netherlands; 5Cellular Biotechnology and Hematology, Sapienza University of Rome; 6UO Reumatologia, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy; 7Paediatric Rheumatology Unit; 8Childhood Cancer Research Unit, Karolinska University Hospital Solna, Stockholm, Sweden; 9Center for Chronic Immunodeficiency, University Hospital Freiburg, Freiburg; 10Pediatric Hematology and Oncology, University Medical Center Hamburg Eppendorf, Hamburg; 11Pediatric Hematology and Oncology, Children’s Hospital, TU Munich, Munich; 12Pediatric Hematology-Oncology, University of Duisburg-Essen, Essen, Germany
    Correspondence: Dirk Holzinger

    Introduction: Macrophage activation syndrome (MAS) is a severe complication of autoimmune and autoinflammatory disease and is strongly associated with systemic juvenile idiopathic arthritis (sJIA).

    Objectives: While the granulocytic protein S100A12 is highly overexpressed in sJIA, and the assessment of S100A12 serum levels helps to distinguish sJIA from other febrile illnesses, the presence of elevated levels of IFNγ, IFNγ-induced chemokines and IL-18 have recently been suggested to play a pivotal role during particularly MAS episodes. Clinically, however, MAS is strikingly similar to hemophagocytic lymphohistiocytosis (HLH) and the initial differentiation between sJIA-associated MAS and primary or secondary HLH can be challenging. In this study we set out to define a serum marker profile to support the initial diagnosis of sJIA-MAS and differentiate this from primary or secondary HLH.

    Methods: A multiplexed bead array panel for simultaneous detection of 53 cytokines and chemokines was built based upon their suggested relevance in MAS or HLH according to respective literature. Serum samples from patients with active primary HLH (pHLH) (n=10), secondary HLH (sHLH) (n=12), clinically active or inactive sJIA-MAS (both n=11) as well as healthy controls (n=10) were subjected to this bead array assay as well as quantification of S100A12 levels by ELISA. Based on the data obtained from this discovery cohort we extracted a subset of analytes, which was validated using commercially available bead array reagents in an independent cohort.

    Results: Following analysis of the discovery cohort, out of 54 serum analytes 14 markers were identified that significantly distinguished sJIA-MAS from HLH. These mostly distinguished between pHLH and MAS, while S100A12, IL-18 as well as the MIG/IL-18-ratio also separated between MAS and sHLH. In ROC-analyses six out of these 14 analytes separated MAS from HLH with AUC > 0.9. S100A12 (AUC=0.99), sFASL (0.98) and IL-18 (0.97) were the top-three performing markers in these analyses. The 14plex panel including S100A12 was validated in an independent cohort resulting in IL-18 (AUC=0.83), S100A12 (0.75) and sFASL (0.7) as the top-three performing markers in separating sJIA-MAS from HLH.

    Conclusion: IL-18, S100A12 and sFASL serum levels are useful to differentiate between sJIA-associated MAS and HLH. This can be a helpful diagnostic tool to discriminate sJIA-associated MAS during early onset of the disease from primary or secondary HLH.

    Disclosure of Interest

    None Declared

    PT2A04 Early treatment with anakinra in systemic juvenile idiopathic arthritis

    Manuela Pardeo, Claudia Bracaglia, Anna Tulone, Antonella Insalaco, Giulia Marucci, Rebecca Nicolai, Virginia Messia, Emanuela Sacco, Fabrizio De Benedetti
    Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy
    Correspondence: Manuela Pardeo

    Introduction: Systemic juvenile idiopathic arthritis (sJIA) accounts for 10-20% of all patients with JIA. The prominent systemic clinical features, the marked elevation of inflammatory markers and the absence of autoantibodies make this disease different from other JIA forms. sJIA should be considered as a polygenic autoinflammatory disease. Interleukin 1 (IL-1) has been shown to be a major mediator of the inflammatory cascade that underlies sJIA. Treatment with anakinra has been reported to be effective in a sizable portion of patients with sJIA.

    Objectives: To assess clinical response rate and disease course in sJIA patients treated with anakinra.To evaluated whether the response to anakinra was related to baseline variables.

    Methods: We reviewed 56 (28 F) consecutive patients with sJIA treated with anakinra for at least 6 months in our institution. The diagnosis of sJIA was established according to the International League of Associations for Rheumatology (ILAR) classification criteria. We analyzed the effect of anakinra on fever, rash, number of active joints, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cells count, platelets count and ferritin levels. Clinically inactive disease (CID) was defined according to Wallace criteria. Clinical and laboratory data were obtained using a standard data collection form.

    Results: The median age at the disease onset was 5.7 (IQR 2.9-10.2) years. The median time from onset to received anakinra was 1.9 (IQR 0.7-9.7) months. At baseline 52/56 (93%) of patients had fever and median number of active joints was 2 (IQR 1-4). After 6 months of treatment 39 patients (69.6%) met criteria for inactive disease. Among 56 patients 17 (30.3%) received anakinra in monotherapy and 39 (69.6%) received anakinra with glucocorticoids. There were no statistically significant differences between the two groups for demographic, clinical and laboratory features.13/17 (76.4%) patients treated with anakinra alone and 26/39 (66.6%) patients treated with anakinra and glucocorticoids met criteria for CID off glucocorticoids at 6 months (p=0.54). Among the 56 patients, 29 (51.7%) received anakinra within 2 months from disease onset. There were no statistically significant differences for demographic, clinical and laboratory features among patients who started anakinra in the first 2 months from disease onset compared to those that started anakinra after 2 months. At 6 months after beginning of anakinra treatment, 27/29 patients (93.1%) who started anakinra within 2 months from disease onset and 12/27 (44.4%) who started anakinra after 2 months from disease onset reached clinical inactive disease off glucocorticoids (p=0.0001). Patients who started anakinra after the first 2 months from disease onset have a significantly higher risk of non-response (OR=8.06, 95% CI: 2.03-32.0).

    Conclusion: According with several observations, anakinra is effective in a significant proportion of patients with sJIA. A possible approach to introduce IL-1 inhibitor, with or without concomitant glucocorticoids, early in the disease course taking advantage of a “window of opportunity” has been suggested. Our observation confirms that earlier treatment with anakinra is associated with a better short-term outcome. Moreover, our results show that beginning of treatment after two months from disease onset is correlated with a high risk of non-response.

    Disclosure of Interest

    M. Pardeo: None Declared, C. Bracaglia: None Declared, A. Tulone: None Declared, A. Insalaco: None Declared, G. Marucci: None Declared, R. Nicolai: None Declared, V. Messia: None Declared, E. Sacco: None Declared, F. De Benedetti Grant / Research Support from: Novartis, Novimmune, Hoffmann- La Roche, SOBI, AbbVie, Pfizer

    PT2A05 Antibodies to collagen V and K-Α 1 tubulin in a patient with systemic juvenile idiopathic arthritis associated lung disease

    Elena Tronconi1, Thalachallour Mohanakumar2, Lara Danziger-Isakov3, Grant Schulert1, Alexei Grom1
    1Division of Rheumatology, Cincinnati Children’s Hospital Medical Center, Cincinnati; 2Norton Thoracic Institute Research Laboratory, St. Joseph’s Hospital and Medical Center, Phoenix; 3Division of Infectious Diseases, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Correspondence: Elena Tronconi

    Introduction: Lung transplantation is still characterized by high morbidity and mortality. Bronchiolitis obliterans syndrome (BOS), the clinical correlate of chronic rejection, highly impacts long-term outcomes. It has been shown that recipients can develop antibodies to self-antigens (sAgs) like collagen V (ColV) and K-α 1 tubulin (Kα1t), and this correlates with the development of BOS. ColV and Kα1t are expressed by small airway epithelial cells. The interstitial remodeling that occurs with transplantation seems to enhance ColV and Kα1t exposure. Systemic juvenile idiopathic arthritis (sJIA) is a form of juvenile arthritis characterized by various degree of arthritis and prominent systemic features. In few cases it can be associated with lung involvement, a life-threatening complication, whose exact pathogenetic mechanism is still poorly understood.

    Objectives: We decided to explore the possible role of antibodies to lung sAgs in a single patient affected by sJIA complicated by several episodes of macrophage activation syndrome (MAS), interstitial lung disease and pulmonary alveolar proteinosis.

    Methods: We retrospectively analyzed serum samples collected during clinical follow-up from August 2015 to February 2018. We considered positive values more than 218 ng/ml for Kα1t and 160 ng/ml for colV according to the study by Hachem et al. The results were matched with clinical symptoms and laboratory parameters as white blood cell counts (WBC), hemoglobin (Hb), platelets (PLT), C reactive protein (CRP) and ferritin. Furthermore, we reviewed the ongoing treatment at the time of sample collection.

    Results: In our patient ColV antibodies fluctuated over time, 7/20 samples were positive. The correlation analysis between ColV and WBC, Hb, PLT, CRP and ferritin did not show statistical significance. However, the review of clinical charts showed that at the time of 6 out of the 7 positive samples there was fever, worsening of respiratory symptom like persistent cough, increase respiratory rate or exertional tachypnea, mild flare of sJIA or MAS. Twelve of the 13 negative samples were collected when the patient was in stable clinical conditions, only 1 sample was collected during an episode of early MAS. The trend of Kα1t antibodies did not match completely with ColV. Considering the previously reported normality range, we did not find any positive result. Therapy was a combination of steroid, cyclosporin, anakinra, canakinumab, interleukin 18-binding protein, intravenous immunoglobulins (IVIG). All the treatments, the dosage of steroid and cyclosporin as well as timing of IVIG were tailored on clinical conditions and laboratory test so it is difficult to define a clear association with ColV antibodies values.

    Conclusion: The pathogenic mechanism of sJIA-associated lung diseases is still poorly understood. This preliminary retrospective study showed a good correlation between antibodies to ColV and clinical disease activity, in particular with MAS and worsening of respiratory symptoms. The lack of correlation with laboratory markers can be explain by the prompt modification of treatment according to the clinical condition of the patient and the retrospective nature of the study. Similar to what happens in BOS after transplantation, we hypothesize that during a flare of sJIA or an episode of MAS the inflammation and cellular damage expose sAgs, normally hidden, to the circulation, worsening and amplifying the immune response. This preliminary evidence of increased pulmonary sAgs in sJIA with lung involvement represents a possible paradigm shift in the pathophysiology of the disease although we need more evidence prospectively collected in a larger population.

    Disclosure of Interest

    None Declared

    PT2A06 Recurrent fever, recurrent pyoderma gangrenosum-like and bacterial infections due to a novel homozygous WDR1 mutation in a child treated with allogeneic hematopoietic stem cell transplantation

    Estibaliz Iglesias1, Monica Roldan2, Alexandru Vlagea3, Manuel Solís-Moruno4, Juan Manuel Mosquera1, Violeta Bitterman1, Joan Calzada-Hernández1, Laia Alsina5, Angela Deyà-Martinez5, Rocío Lara3, Maria Carmen Anton3, Susana Plaza3, Helios Martínez-Banaclocha6, María Trabazo7, Georgina Morón7, Manel Juan3, Jordi Yagüe3, Ferran Casals8, Isabel Badell7, Claudia Fortuny9, Asunción Vicente10, Jordi Anton1, Pablo Pelegrin6, Juan I. Aróstegui3, Anna Mensa-Vilaro3
    1Pediatric Rheumatology; 2Confocal Microscopy Unit, Hospital Sant Joan de Déu, Esplugues; 3Immunology, Hospital Clinic; 4Experimental and Health Sciences, Pompeu Fabra University, Barcelona; 5Clinical Immunology Unit, Hospital Sant Joan de Déu, Esplugues; 6Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Murcia; 7Pediatric BMT Unit, Hospital de Sant Pau; 8Genomics, Pompeu Fabra University, Barcelona; 9Pediatrics; 10Pediatric Dermatology , Hospital Sant Joan de Déu, Esplugues, Spain
    Correspondence: Anna Mensa-Vilaro

    Introduction: Immunodeficiency is increasingly recognized as a relevant feature in certain novel monogenic autoinflammatory diseases including the often fatal deficiency of actin-interacting protein 1 (AIP1) caused by biallelic loss-of-function WDR1 mutations.

    Objectives: To characterize the clinical and immunological features, genetic and molecular mechanisms and treatment outcome, underlying a child combining immunodeficiency and sterile inflammation.

    Methods: Whole-exome sequencing (WES) was performed in the family to identify the genetic defect. Immunological evaluations and functional assays were performed to demonstrate the pathogenicity of the candidate variant.

    Results: The patient is a 10-year-old girl born of consanguineous Pakistani parents. She was followed since the age of 5 for recurrent fever, recurrent pyoderma gangrenosum-like, upper respiratory and ocular bacterial infections, one episode of non-bacterial osteomyelitis, anemia, mild-to-moderate thrombocytopenia, neutrophilia, and increase acute-phase reactants. WES detected the novel, homozygous p.(Lys411Asn) WDR1 variant, classified as likely pathogenic according to ACMG guidelines. Immunological investigations revealed moderate B cell lymphopenia with normal immunoglobulin levels, and normal numbers of T and NK cells. Phalloidin staining of patient’s neutrophils showed increased levels of F-actin compared with healthy subjects. Neutrophil respiratory burst was normal with fMLP stimuli, but repeatedly impaired for phagocytosis of opsonized E. coli. Confocal and electronic microscopy reveals aberrant morphology of neutrophils, with nuclear herniations, low cytoplasmatic granule content, and defects in chemotaxis and chemokinetics. Inflammasome-related experiments with patient’s monocytes showed lower levels of both ASC specks formation and active caspase-1 compared with healthy subjects. Finally, she has undergone HLA-identical unrelated hematopoietic stem cell transplantation (HSCT) and she is clinically well with a follow-up time of 50 days.

    Conclusion: Our results strongly support deficiency of AIP1 as the cause of the disease observed in this patient. Allogeneic HSCT might be a curative option for this severe and rare disease.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Guided poster tour 2B

    PT2B01 Adenosine deaminase 2 deficiency: correction of the immune defects by gene therapy

    Immacolata Brigida1, Matteo Zoccolillo1,2, Raisa Jofra Hernandez1, Lucia Sergi Sergi1, Giulia Milardi1,3, Federica Barzaghi2,4, Luigi Naldini1,3, Maria Pia Cicalese1,3,4, Alessandro Aiuti1,3,4, Alessandra Mortellaro1
    1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan; 2Tor Vergata University, Department of Systems Medicine, Rome; 3Vita-Salute San Raffaele University; 4Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
    Correspondence: Alessandra Mortellaro

    Introduction: Adenosine deaminase 2 deficiency (DADA2) is caused by loss-of-function mutations in the ADA2 gene. Clinical features include vasculitis, ischemic strokes, intracranial hemorrhages, hematological abnormalities and immunodeficiency mainly affecting B cells. Anti-TNF therapy reduced the ischemic events, controlled vasculitis and partly restored the B-cell function. Allogeneic hematopoietic stem progenitor cells (HSPCs) transplantation attempted in some patients restored ADA2 activity and improved the inflammatory and hematological manifestations in transplanted DADA2 patients. However, the morbidity and mortality of the allogeneic HSPC transplantation remained high and unacceptable in less severe cases.

    Objectives: Based on the observation that HSPC transplantation may be curative, we hypothesized that strategies based on genetic correction and engraftment of autologous HSPCs could provide a complete cure for DADA2.

    Methods: CD34+ HSPCs isolated from healthy donors and DADA2 patients were transduced with a lentiviral vector (LV) encoding ADA2. HSPCs engraftment and differentiation were assessed in vitro and in vivo in humanized NSGW41 mice.

    Results: Transduction of CD34+ HSPCs isolated from healthy donors and DADA2 patients with the ADA2-LV efficiently restored ADA2 expression in vitro without significant toxicity. Immunological reconstitution of ADA2-transduced CD34+ HSPCs was successfully achieved in vivo in humanized NSGW41 mice. LV-derived ADA2 expression boosted immune reconstitution of myeloid and B cells in vivo.

    Conclusion: LV-mediated gene transfer restored ADA2 expression and enhanced myeloid and B-cell reconstitution. Future studies will be required to explore the safety and efficacy of HSPC gene therapy approach for DADA2.

    Disclosure of Interest

    None Declared

    PT2B02 Analysis of the efficacy of treatment on 45 patients with deficiency of adenosine deaminase 2

    Amanda K. Ombrello1, Deborah L. Stone1, Karyl Barron2, Patrycja M. Hoffmann1, Cornelia Cudrici3, Anne Jones1, Tina Romeo1, Dimana Dimitrova4, Amit Dotan5, Donna Wall5, Monica Thakar6, Jennifer Kanakry4, Daniel Kastner1
    1NHGRI; 2NIAID; 3NHLBI; 4NCI, NIH, Bethesda, United States; 5Hospital for Sick Children, Toronto, Canada; 6Fred Hutchinson Cancer Research Center, Seattle, United States
    Correspondence: Amanda K. Ombrello

    Introduction: Since its initial description in 2014, the deficiency of adenosine deaminase 2 (DADA2) has undergone massive phenotypic expansion. The implementation of anti-TNF agents has resulted in a significant reduction in occurrence of lacunar strokes but variable effects on the other disease manifestations. Hematopoietic stem cell transplantation (hSCT) is another potential curative treatment but, as numbers of patients transplanted rise, specific DADA2 complications have emerged.

    Objectives: To analyze the efficacy of treatment modalities used in DADA2 patients in a single center, 45 patient cohort.

    Methods: Patients with confirmed biallelic mutations in ADA2 were enrolled and a comprehensive multi-system evaluation was undertaken to establish the extent of disease. From June 2013 forward, all patients with DADA2 were offered treatment with anti-TNF agents. Patients were followed longitudinally on an annual basis. At each visit the patients were followed by a core group of consultants with a battery of laboratory, radiologic, and pathological analyses completed. Patients with significant evidence of bone marrow involvement (pure red cell aplasia [PRCA], immune mediated neutropenia, trilineage bone marrow failure) were referred for hSCT.

    Results: Anti-TNF Analysis: In total, 42/45 were initiated on anti-TNF agents. At the time of analysis 38/45 continued treatment (4 underwent hSCT and 3 declined treatment). 15/38 were on adalimumab, 20/38 were on etanercept, 2/38 were on golimumab and 1 was on infliximab. Prior to anti-TNF initiation, there were 66 cumulative strokes and post-anti-TNF initiation, there were 0 strokes. There was a statistically significant decrease in inflammatory burden (ESR, CRP) post-anti-TNF initiation. Pre-treatment transient elastography was abnormal in 11/34 with 6/11 normalizing post-treatment.

    The inflammatory skin manifestations such as erythema nodosum and vasculitic nodules improved on treatment. There was visible improvement of livedo racemosa but little response to peripheral vascular disease/Raynaud’s phenomenon with 1 patient undergoing 3 partial digit amputations despite treatment. The patient with PRCA remained transfusion dependent. Immune mediated neutropenia was present in 10/38 pre-anti-TNF and persisted in 6/10 post-treatment. Trilineage bone marrow failure developed in 1 patient on anti-TNF.

    hSCT Analysis: At the time of analysis 4/45 had undergone hSCT (2 for immune mediated neutropenia, 1 for combined PRCA/immune mediated neutropenia, 1 for trilineage bone marrow failure).All had been on anti-TNF agents leading up to hSCT and 1 developed trilineage bone marrow failure after 4 years of anti-TNF treatment. All received reduced intensity conditioning with varying regimens. One patient was transplanted successfully and is doing well 1 year post-transplant. Two additional patients with immune mediated neutropenia experienced graft failure due to host CD8+T lymphocyte attack requiring second transplant. Patient 4 remains hospitalized after decreasing chimerism and reemergent neutropenia necessitated donor lymphocyte infusion. Three additional patients await hSCT.

    Conclusion: The inflammatory and neurologic manifestations of disease have a robust response to anti-TNF; hepatic and hematologic manifestations have a variable response and vascular manifestations have little response. hSCT can be curative but delayed engraftment/graft failure is a potential complication in patients with neutropenia.

    Disclosure of Interest

    None Declared

    PT2B03 Long-term retention rate of anakinra in adult onset Still’s disease and predictive factors for treatment response

    Antonio Vitale1, Giulio Cavalli2, Serena Colafrancesco3, Roberta Priori3, Guido Valesini3, Lorenza Maria Argolini4, Elena Baldissera2, Elena Bartoloni5, Daniele Cammelli6, Giovanni Canestrari7, Jurgen Sota1, Elena Cavallaro8, Maria Grazia Massaro9, Piero Ruscitti10, Paola Cipriani10, Ginevra De Marchi8, Salvatore De Vita8, Giacomo Emmi6, Gianfranco Ferraccioli7, Micol Frassi11, Roberto Gerli5, Elisa Gremese7, Florenzo Iannone12, Giovanni Lapadula12, Giuseppe Lopalco12, Raffaele Manna9, Alessandro Mathieu13, Carlomaurizio Montecucco14, Marta Mosca15, Ilaria Piazza16, Matteo Piga13, Irene Pontikaki4, Micol Romano4, Silvia Rossi14, Maurizio Rossini16, Elena Silvestri6, Chiara Stagnaro15, Rosaria Talarico15, Angela Tincani11, Ombretta Viapiana16, Gianfranco Vitiello6, Paola Galozzi17, Paolo Sfriso17, Carla Gaggiano18, Donato Rigante19, Lorenzo Dagna2, Roberto Giacomelli10, Luca Cantarini1 and “Working Group” of Systemic Autoinflammatory Diseases of SIR (Italian Society of Rheumatology)
    1Research Center of Systemic Autoinflammatory Diseases and Behçet’s Disease Clinic Surgery and Neurosciences, Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena; 2Unit of Immunology, Rheumatology, Allergy and Rare Diseases (UnIRAR), IRCCS San Raffaele Scientific Institute, Milan; 3Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Rome; 4Division of Rheumatology, ASST Gaetano Pini, Milan; 5Rheumatology Unit, Department of Medicine, University of Perugia, Perugia; 6Department of Experimental and Clinical Medicine, University of Firenze, Firenze; 7Institute of Rheumatology and Affine Sciences, Division of Rheumatology, Catholic University of the Sacred Heart, Rome; 8Department of Medical and Biological Sciences, Rheumatology Clinic, University of Udine, Udine; 9Periodic Fever Research Center, Institute of Internal Medicine, Catholic University of the Sacred Heart, Fondazione Policlinico A. Gemelli, Rome; 10Department of Biotechnological and Applied Clinical Science, Division of Rheumatology, University of L’Aquila, L'Aquila; 11Rheumatology and Clinical Immunology, Spedali Civili and Department of Clinical and Experimental Sciences, University of Brescia, Brescia; 12Rheumatology Unit,Department of Emergency and Organ Transplantation, University of Bari, Bari; 13Rheumatology Unit, Department of Medical Sciences, University and AOU of Cagliari, Cagliari; 14Department of Rheumatology, IRCCS Policlinico San Matteo Foundation, University of Pavia, Pavia; 15Rheumatology Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa; 16Rheumatology Unit, Department of Medicine, University of Verona, Verona; 17Department of Medicine DIMED, Rheumatology Unit, University of Padua, Padua; 18Clinical Pediatrics, Department of Molecular Medicine and Development, University of Siena, Siena; 19Institute of Pediatrics, Università Cattolica Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli I.R.C.C.S., Rome, Italy
    Correspondence: Antonio Vitale

    Introduction: Anakinra (ANA) is an effective treatment choice in patients with adult onset Still’s disease (AOSD). Variables affecting treatment survival include loss of efficacy or adverse events, but also the decision to discontinue treatment after long-term clinical remission.

    Objectives: Aims of this study were: i) to assess the drug retention rate (DRR) of ANA during a long-term follow-up looking for any difference related to the line of biologic treatment, the concomitant use of conventional disease modifying anti-rheumatic drugs (cDMARDs) and the different type of AOSD (systemic versus chronic articular); ii) to identify predictive factors of lack of efficacy, loss of efficacy and ANA withdrawal owing to long-term remission.

    Methods: AOSD patients classified according with Yamaguchi criteria and treated with ANA were retrospectively enrolled in 18 Italian tertiary Centers. Demographic, laboratory, clinical and therapeutic data related to the start of ANA (baseline), the 3-month assessment and the last follow-up visit while on ANA treatment were retrospectively collected and statistically analyzed.

    Results: One hundred and forty-one AOSD patients (48 males, 93 females) treated with ANA for a mean period of 35.96±36.05 months were enrolled. The overall DRR of ANA was 44.6% and 30.5% at the 60- and 120-month assessments, respectively, with no significant differences between: i) biologic naïve patients and those previously treated with other biologics (log-rank p=0.97); ii) monotherapy and concomitant use of cDMARDs (log-rank p=0.45); iii) systemic and chronic articular types of AOSD (log-rank p=0.67). No variables collected at baseline could predict primary inefficacy, while the number of swollen joints at baseline was significantly associated with secondary inefficacy (p=0.01, OR=1.194, C.I. 1.043-1.367). The typical AOSD skin rash was negatively related with ANA withdrawal owing to long-term remission (p=0.03, OR=0.224, C.I. 0.058-0.863).

    Conclusion: Long-term DRR of ANA has been found excellent and is not affected by different lines of biologic treatment, concomitant use of cDMARDs, or type of AOSD. The risk of losing ANA efficacy increases along with the number of swollen joints at the start of therapy, while the typical skin rash is a negative predictor of ANA withdrawal related to sustained remission.

    Disclosure of Interest

    None Declared

    PT2B04 The anti-inflammatory cytokine interleukin 37 is an endogenous inhibitor of trained immunity

    Giulio Cavalli1,2, Mark Gresnigt3, Travis Nemkov4, Rob Arts2, Angelo D'Alessandro4, Silvia Giugliano5, Elan Eisenmensser4, Lorenzo Dagna1, Leo Joosten2, Mihai Netea2, Charles Dinarello2,6
    1Unit of Immunology, Rheumatology, Allergy and Rare Diseases (UniRAR), Vita-Salute San Raffaele University, Milan, Italy; 2Department of Medicine, Radboud university Medical Centre, Nijmegen, Netherlands; 3Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany; 4Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, United States; 5Mucosal Immunology and Microbiota Unit, Humanitas University, Rozzano, Italy; 6Department of Medicine, University of Colorado Denver, Aurora, CO, United States
    Correspondence: Giulio Cavalli

    Introduction: Trained immunity (TI) is a de-facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as a protective mechanism against infections. TI is characterized by rewiring of functional, epigenetic and metabolic programs of innate immune cells such as monocytes and macrophages, which sustain enhanced production of pro-inflammatory cytokines. Since aberrant activation of TI is implicated in inflammatory diseases, tight regulatory mechanisms are likely in place, but the mechanisms responsible for this modulation remain elusive.

    Objectives: Scope of this study was to evaluated the role of IL-37, an anti-inflammatory cytokine that curbs inflammation as well as modulates metabolic pathways, as an endogenous regulator of trained immunity.

    Methods: The effects of recombinant IL-37 were evaluated in a mouse model of TI induced by the administration of beta-glucan in vivo (survival to a lethal inoculum of infectious agents, production of inflammatory cytokines, recruitment of inflammatory cells at the sites of infection). Subsequently, the effects of IL-37 were evaluated ex vivo on splenic and bone marrow monocytes (production of inflammatory cytokines, metabolomic analysis of the activation status of the main pathways of cellular energy metabolism). Finally, we evaluated the association between IL-37 gene polymorphisms and the induction of TI in monocytes of healthy donorswith in vitro functional studies.

    Results: The exogenous administration of IL-37 abrogates the pro-inflammatory effects of TI, significantly reducing the production of pro-inflammatory cytokines and the survival of experimental animals subjected to a disseminated infection model. The inhibitory effects of IL-37 on TI are also associated with reduced recruitment of neutrophils at sites of inflammation. IL-37 and TI programs have differential and opposite effects on the modulation of cellular energy metabolism of monocytes. In humans, polymorphisms in the IL-37 gene are associated with reduced activation of TI programs and reduced production of inflammatory cytokines by healthy donor monocytes.

    Conclusion: In conclusion, IL-37 emerges as an endogenous regulator of TI, which makes this cytokine a potential therapeutic target in immune-mediated pathologies.

    Disclosure of Interest

    None Declared

    PT2B05 Interleukin 18 gene expression by human monocytes is controlled by type I interferon

    Emely Verweyen1, Dirk Holzinger2, Helmut Wittkowski1, Peter Pickkers3, Dirk Foell1, Christoph Kessel1
    1Pediatric Rheumatology and Immunology, University Children's Hospital, Muenster; 2Child and Adolescent Medicine, University Hospital, Essen, Germany; 3Intensive Care Medicine, Radboud University Medical Center, Nijmegen, Netherlands
    Correspondence: Christoph Kessel

    Introduction: Interleukin (IL) 18 is a member of the IL-1 cytokine family and has a pivotal role in natural killer and T cellular interferon (IFN) γ production but its precise function in inflammatory diseases as well as regulatory mechanisms underlying IL-18 expression still remain poorly understood.

    Objectives: Both IL-1β and IL-18 are particularly associated with the autoinflammatory diseases familial mediterranean fever (FMF) or systemic juvenile idiopathic arthritis (sJIA) and IL-18 is thought to drive autoinflammation or infection associated macrophage activation syndrome (MAS). Apart from gathering evidence for the pathophysiological implications of IL-1β and IL-18, several studies have investigated the genetic control of IL-1β expression, highlighting a role for NF-κB in IL-1β transcription. Albeit there are some studies that investigated IL-18 expression by either murine or human cells, it still remains poorly understood whether and how this is regulated.

    Methods: Cytokine expression in plasma of healthy individuals subjected to lipopolysacharide (LPS) re-challenge experiments was quantified. LPS-stimulated primary human monocytes isolated from healthy donors were analyzed for cytokine expression on gene and protein level over time as well as following various drug or recombinant cytokine treatments.

    Results: In plasma of healthy individuals subjected to LPS re-challenge experiments we initially observed that, in contrast to TNFα, IL-6 or IL-1β, the IL-18 levels were unaffected by LPS-tolerance. Similarly, in endotoxin desensitization experiments on primary human monocytes, IL-18 gene expression was resistent to endotoxin tolerance and appeared to rather benefit from endotoxin re-challenge. When studying time kinetics of cytokine gene expression, we found that IL-1 and IL-18 followed identical secretion but completely divergent gene expression patterns, thus providing an explanation for observations on IL18 expression in LPS re-challenge experiments. When studying triggers for the observed attenuated gene expression kinetics of IL-18, we found that both IL-18 gene and protein expression upon LPS-stimulation of primary human monocytes was not affected by other prominent inflammatory cytokines but was sensitive to interference with JAK/STAT-signaling and could be modulated by type I interferon priming of cells or STAT1 gain-of-function. While by itself type I interferon did not induce IL-18 expression, IFNβ-neutralization during LPS-stimulation blunted IL18 transcription. Further, microtubule-destabilizing drugs, such as colchicine, completely abrogated monocytic IL18 expression, which could be reversed by addition of type I interferons.

    Conclusion: Our data suggest monocytic IL-18 expression to require cooperate toll-like receptor and type I interferon signaling, which has implications for IL-18 expression during viral infections and its overexpression in autoinflammation and MAS.

    Disclosure of Interest

    None Declared

    PT2B06 Glycogen synthase kinase 3B regulates TLR3-mediated antiviral response

    Ryeojin Ko, Soo Young Lee
    Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Womans University, Seoul, Korea, Republic Of
    Correspondence: Ryeojin Ko

    Introduction: Toll-like receptor 3 (TLR3) plays a critical role in the antiviral immune response. The protein tyrosine kinase BTK has been reported to regulate signal pathways for TLR3-mediated antiviral gene expression. However, the regulation of BTK activity in TLR3 signaling remains unclear.

    Objectives: Here we investigate the molecular mechanisms underlying GSK3b regulation of BTK in TLR3-mediated antiviral gene expression.

    Methods: To determine whether GSK3b regulates TLR3-mediated antiviral response, we establish the GSK3b knockdown stable macrophage cell lines.

    Results: We demonstrate that GSK3b positively regulates TLR3 signaling via BTK activation. After poly I:C stimulation, GSK3b interacts with BTK in a time-dependent manner. Furthermore, suppression of GSK3b expression or its kinase activity significantly reduces the poly I:C-induced BTK activation and antiviral gene expression such as type I interferon (IFN) and IFN stimulated gene (ISG).

    Conclusion: Together, our results suggest that GSK3b is critical for antiviral response by regulating TLR3 signaling and is a potential molecular therapeutic target in autoimmune disease.

    Disclosure of Interest

    None Declared

    PT2B07 Anticytokine therapy is successful inprevention and treatment OFBCG-associated inflammatory syndrome (IS) after hematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiencies (SCID)

    Alexandra Laberko1, Irina Shipitsina2, Svetlana Rodigina2, Sergei Blagov2, Daria Yukhacheva1, Elena Deripapa1, Anna Kozlova1, Yulia Rodina1, Larisa Shelikhova2, Dmitrii Balashov2, Anna Shcherbina1
    1Immunology; 2Hematopoietic Stem Cell Transplantation, National Medical Research Center of Hematology, Oncology and Immunology Named After D. Rogachev, Moscow, Moscow, Russian Federation
    Correspondence: Anna Shcherbina

    Introduction: Immune recovery IS is a well-known complication in patients with HIV-driven acquired immunodeficiency syndrome after initiation of antiretroviral therapy.It is caused by cytokine storm due to recovering CD4+ lymphocyte activation in response to persistingpathogens. We observed similar phenomena in patients with SCID after allogenic HSCT.

    Objectives: To report BCG-related IS in a group of SCID patients.

    Methods: 22 SCID patients, vaccinated with BCG, received allogenic haploidentical HSCT with TCRab/CD19 graft depletion in our Center between 2012 and 2018. Seven patients had symptoms of BCGitis before HSCT, at the moment of HSCT BCGitis was controlled by antimycobacterial drugs in all of them.

    Post-transplant immunosuppressive therapy before November 2016 ( 11 patients) included tacrolimus, in 2 cases in combination with short course of methotrexate. From November 2016 11 patients received graft versus host disease and cytokine release syndrome prophylaxis with tocilizumab 8mg/kg days -1 , +14, + 28, abatacept 10mg/kg days -1, +5, +14, +28.All patients have been receiving from 2 to 4 antimycobacterial drugs for BCGitis prophylaxis at HSCT and in posttransplant period until full immune recovery.

    Results: 5\22 patients developed early BCG-related IS (4/11 prior to tocilizumab introduction, and only 1/11 – with tocilizumab). Three of them presented at days +4, +6 and +12 after HSCT with sustained fever, focal BCGitis exacerbation and one additionally developed macrophage activation syndrome (MAS). All received therapy with steroids and IL-1 inhibitors, in one of them IS symptoms were controlled, but eventually all three died shortly after. One patient without signs of BCGitis before HSCT at day +38 developed IS with symptoms of systemic BCG infection and MAS. He was successfully treated with combination of etoposide and IL-6 receptor inhibitors – tocilizumab 10mg/kg. In one patient local BCGitis resolved without treatment. In most cases of early IS significant elevation of TCRgd cell level was observed.

    Six patients (5/6 with tocilizumab prophylaxis ) had immune recovery associated IS 2,5 - 5,5 months after HSCT (M 4,4 months), accompanied by CD3+ lymphocytes increase in peripheral blood. Symptoms included focal BCGitis in all 6 (5 of them had no BCGitis before HSCT), fever in 3, lymphadenopathy in 3, MAS in 1. In four patients monotherapy of anakinra 7-12mg/kg/day was used with full response within 2-4 weeks. They were later switched to canakinumab 5mg/kg every 4 weeks for the next 1-3 months. In one patient IS was controlled by 2 infusions of tocilizumab 10mg/kg every 4 weeks. The patient who developed MAS received anakinra 10mg/kg in combination with steroids with full resolution of symptoms.

    Conclusion: BCG vaccinated SCID patients are at high risk of BCG-related early IS after HSCT. Anticytokine therapy with IL6 receptor inhibitor is an effective option for prevention of early BCG-related IS. IL1 inhibitors lead to full resolution of BCG-related immune recovery IS.Evidence-based protocols for IS prevention and treatment are needed.

    Disclosure of Interest

    None Declared

    PT2B08 Allogeneic haematopoietic stem cell transplantation (HSCT) outcome in immunedysregulation and rheumatologic patients: a single center 19 years experience

    Sara Signa1,2, Andrew Gennery1, Sophie Hambleton1, Terence Flood1, Andrew Cant1, Mario Abinun1, Mary Slatter1
    1Great North Children's Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust and Primary Immunodeficiency Group, Institute of Cellular Medicine, Newcastle upon Tyne University, Newcastle upon Tyne, United Kingdom; 2University of Genoa and Center for Autoinflammatory Diseases and Immunodeficiencies-Clinics of Pediatrics and Rheumatology , G.Gaslini Institute, Genoa, Italy
    Correspondence: Sara Signa

    Introduction: Haematopoietic stem cell transplantation (HSCT) is a potential treatment for severe and conventional therapy-resistant immune dysregulation disorders (autoinflammatory diseases ,complex autoimmune diseases, complex rheumatologic diseases).However, it still remains the ‘last resort therapy”, particularly in pediatric age.

    Objectives: To present the outcome of HSCT in 55 children with immune dysregulation in a single center over a 19 year period of time.

    Methods: We retrospectively collected cases of pediatric patients who underwent allogeneic HSCTat Great North Children’s Hospital, Newcastle upon Tyne, UK between 01/1999 -06/2018 for immune dysregulation, rheumatologic and autoinflammatory disorders.Medical charts were reviewed and the following data were extracted: patient demographics, diagnosis, indication for HSCT, donor-recipient matching, conditioning regimen, and graft composition. Post-transplantation data were collected on occurrence of GVHD, infections, and relapse of initial (or occurrence of new, secondary, post-HSCT) immunedysregulation.

    Results: We identified 55 patients who underwent allogeneic HSCT: 8 presented with a defined Rheumatologic disease (5 children JIA, 3 with systemic lupus erythematosus), 7 patients with CTLA-4 deficiency, 6 with STAT 3 gain-of-function mutation disease,6 with IPEX syndrome, 7 with autoinflammatory disorders, 3 children with ALPS, 5 with autoimmune enteropathy, 2 with STAT1 gain-of-function disease, 7 with complex autoimmuny features, and two each with RAS associated autoimmune lymphoproliferative disorder (RALD) and FADD deficiency. 41 patients presented with a monogenic disease, although only 21 were diagnosed pre- HSCT.Mean age of symptom onset was 3.5 years (range: birth-15.34 years). Mean age at HSCT was 8.5 years (range: 0.25-19.34 years). Patients underwent HSCT from 10/10 or 12/12 HLA matched unrelated donors (n 29), 9/10 HLA mismatched unrelated donor (n 5), matched sibling donor (n 14), 9/10 HLA mismatched related donor (n 3) or haploidentical TCR alpha/beta-CD19+ depleted parental donor (n 4). Most patients received reduced toxicity conditioning with fludarabine 3, treosulfan, alemtuzumab(n 27). Peripheral blood was the stem cell source in most cases (n 32). Most received cyclosporine (CsA) and mycophenolate mofetil (MMF) as GVHD prophylaxis (n 37). One did not engraft, needing a rapid second HSCT procedure. 39 patients showed evidence of viral replication (61%) , sometimes from multiple pathogens. Acute GVHD was usually mild and confined to skin (grade I/II, n=27, 49%).Severe acute GVHD occurred in 6 patients (grade III-IV GVHD -10%; 3 died) and two patients experienced extensive chronic GVHD, both deceased.With a median follow-up of 6 .67years (range, 2 months-18 years), 43 of 55 patients were alive and most are either cured and completely healthy (n=21) or significantly improved but with pre-existing end-organ damage (n=11). 19 patients experienced new autoimmune phenomena post-transplant, mostly involving thyroid or hematologic-related.Among the 12 deceased patients, one died from non-transplant related diabetic ketoacidosis, whereas five patients died of GVHD-related causes, five due to infectious causes and one due to extreme induction toxicity, giving a transplant related mortality (TRM) of 20%. Mean age of death was 11 years (range: 0.67-21 years).

    Conclusion: Allogeneic HSCT can be an effective therapeutic option for severe refractory immune dysregulation in childhood, potentially curative. Appropriate and timely patient selection is fundamental to minimise HSCT-related complications.

    Disclosure of Interest

    S. Signa Grant / Research Support from: Research Support from: EFIS-Immunology Letters (IL) Short-Term Fellowship, A. Gennery: None Declared, S. Hambleton: None Declared, T. Flood: None Declared, A. Cant: None Declared, M. Abinun: None Declared, M. Slatter: None Declared

    PT2B09 Global profiling and molecular characterization of the autoantibody repertoire in APECED

    JohnM. Lindner1, Alexander Krämer1, Xavier Leber1, Valerie Caballero1, Frank Kolbinger1, Michail S. Lionakis2, Steven Holland2, Elisabetta Traggiai1
    1Novartis Institutes for BioMedical Research, Basel, Switzerland; 2National Institutes of Health, Bethesda, United States
    Correspondence: Elisabetta Traggiai

    Introduction: Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, also called APS-1), is a phenotypically pleiotropic disease caused by the breakdown of central T-cell tolerance due to mutation of the autoimmune regulator (AIRE) gene. The presence of autoreactive antibodies (Ab) in APECED has implications for T cell-mediated governance of B-cell tolerance. In particular, soluble factors such as cytokines and antimicrobial peptides are frequent autoantigens in APECED. To fully characterize the humoral response against these and other targets, we tested donor sera for reactivity against more than 3000 secreted proteins using customized protein microarrays.

    Results: Applying stringent evaluation methods, we identified more than 50 reactivities enriched among 26 APECED patients relative to 19 healthy donors. We subsequently validated several targets by ELISA, then screened patient IgG memory B cells for Ab of desired specificities using recently developed high-throughput, multiplexed techniques for culture, screening, and cloning. Of particular relevance is the production of Ab against interleukin (IL)-17, given the relationship between TH17-mediated fungal control and chronic candidiasis as a common clinical feature of APECED. To understand the composition and functionality of these Ab, we cloned 15 antigen-specific IgG molecules from 3 APECED patients, and found clear patient-specific skewing toward IL-17A or IL-17F, with some Ab showing cross-reactivity. Furthermore, several also bound the heterodimeric IL-17AF in addition to their respective homodimers, and one Ab specifically bound only the heterodimer, the latter one a specificity which has not yet been described. All Ab showed extensive somatic hypermutation, had sub-nanomolar affinities, and functionally block the interaction between IL-17 and its receptor.

    Conclusion: These features indicate a clear history of interaction with antigen-specific T helper cells, and suggest that autoantibodies in APECED can disrupt immune control, offering insights into the therapeutic control both of APECED and other human diseases.

    Disclosure of Interest

    None Declared

    Monogenic autoinflammatory diseases (clinical)

    P2001 In vitro NLRP3 inflammasome activation assay assists diagnosis of genetically negative CAPS patients and guides anti-IL1 therapy

    Leonardo O. Mendonca1,2,3, Myrthes T. Barros2, Tereza Robazzi4, Pedro F. Giavina-Bianchi2, Francesco Caroli5, Alice Grossi5, Jorge Kalil2, Fabio F. Morato Castro2, Isabella Ceccherini5, Marco Gattorno6, Alessandra Pontillo3
    1International Center for Autoinflammatory Diseases and Primary Immunodeficienceis, Istituto Giannina Gaslini, Genoa, Italy; 2Autoinflammatory Unit, University of São Paulo, School of Medicine; 3Immunogenetics Laboratory, Biomedical Science Institute, University of São Paulo, Sao Paulo; 4Pediatric Rheumatology Unit, School of Medicine of Bahia, Federal University of Bahia, Salvador, Brazil; 5UOC Medical Genetics; 6Center for Autoinflammatory Diseases and Primary Immunodeficienceis, Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Leonardo O. Mendonca

    Introduction: Constitutive activation of NLRP3 inflammasome and the consequent elevate production of inflammatory cytokine IL-1ß are the pathogenic mechanisms involved in CAPS syndromes. However due to the heterogeneity of clinical presentation and to the missing of genetic proving in 40% of patients, both diagnosis and therapeutic choice are often tricky.

    Cryopirin/NLRP3-associated periodic syndromes (CAPS) is a group of rare autoinflammatory diseases characterized by early onset urticarial rash and periodic fever, positive acute reactants markers (CRP, SAA), arthritis and neurologic involvement, including ocular disorders and progressive deafness. The constitutive activation of NLRP3 inflammasome and the consequent elevate production of inflammatory cytokine IL-1ß are the pathogenic mechanisms involved in CAPS. Gain-of-function mutations in NLRP3 were detected in about 60% of patients. Early diagnosis and quick start of anti-IL-1 treatment are very important to prevent severe complications, however due to the heterogeneity of clinical presentation and to the missing of genetic proving in 40% of patients, both diagnosis and therapeutic choice are often trickThe constitutive activation of NLRP3 inflammasome and the consequent elevate production of inflammatory cytokine IL-1ß are the pathogenic mechanisms involved in a group of rare autoinflammatory diseases grouped as Cryopirin Associated Periodic Syndromes. Gain-of-function mutations in NLRP3 were detected in about 60% of patients. Early diagnosis and quick start of anti-IL-1 treatment are very important to prevent severe complications, however due to the heterogeneity of clinical presentation and to the missing of genetic proving in 40% of patients, both diagnosis and therapeutic choice are often trickyThe constitutive activation of NLRP3 inflammasome and the consequent elevate production of inflammatory cytokine IL-1ß are the pathogenic mechanisms involved in a group of rare autoinflammatory diseases grouped as Cryopirin Associated Periodic Syndromes. Gain-of-function mutations in NLRP3 were detected in about 60% of patients. Early diagnosis and quick start of anti-IL-1 treatment are very important to prevent severe complications, however due to the heterogeneity of clinical presentation and to the missing of genetic proving in 40% of patients, both diagnosis and therapeutic choice are often tricky.

    Objectives:

    Demonstrate the usefulness of an in vitro test for NLRP3 inflammasome activation as supportive method for CAPS diagnosis and therapeutic choice.

    Methods: Five patients (P1-P5) with a suspect of CAPS were included in a pilot study between 2016 and 2018. All patients were genetically sequenced using gene panels for mutations in candidate genes (NLRP3, NLPR12 and NLRC4). Monocytes were stimulated with LPS and ATP. IL-1ß concentration was measured in serum and culture supernatants by ELISA.

    Results: All patients presented clinical features compatible with a CAPS. Candidate genes screening revealed a pathogenic mutation in NLRP12 (exon 3; c.C910T; p. H304Y) in P1 and a variant of uncertain significance (VUS) in NLRP3 (exon 4; c.A2176G; p.S726G) in P2. The other 3 patients were genetically negative. Monocytes isolated from P2, P3 and P4 produced high level of IL-1ß when challenged with LPS, and ATP did not amplify cytokine production, suggesting a defect in NLRP3 inflammasome. P1 and P5 produced normal levels of IL-1ß. According with NLRP3-IA test results, P3 and P4 were successfully treated with anti-IL1. P2 presented also an IgM monoclonal gammopathy, therefore receiving a final diagnosis of Schnitzler Syndrome (SS); colchicine ameliorated his clinical presentation. P5 (genetically negative and NLRP3-IA test negative) was successfully treated with corticosteroids.

    Conclusion: These results demonstrate the convenience of the NLRP3-IA test in the examination of suggestive CAPS and SS patients, especially for those with negative genetic test, to (1) confirm the diagnosis and to (2) guide the therapeutic choice.

    Disclosure of Interest

    None Declared

    P2002 Vaccination safety and coverage in a single cohort of autoinflammatory syndromes in Italy

    Leonardo O. Mendonca, Enrica Toniolo, Stefano Volpi, Roberta Caorsi, Marta Bustaffa, Matteo D'Alessandro, Sara Signa, Marco Gattorno
    International Center for Autoinflammatory Diseases and Primary Immunodeficienceis, Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Leonardo O. Mendonca

    Introduction: Vaccine-preventable diseases are emerging after anti-vaccine movements appear. In AID, vaccine triggered-disease is a well known phenomena for Hyper-IgD/Mevalonate-Kinase Deficiency (MKD). However, recent publications stressed out doubts in physicians and families, regarding severe flares after pneumococcus vaccine in CAPS or that PFAPA patients does not achieve sufficient and protective levels of antibodies.

    Objectives: This work aims to evaluate the vaccination coverage of Italian Vaccination Schedule and the prevalence of adverse reactions or disease induced flare to vaccine in a single cohort of AID in Italy.

    Methods: An anamnestic questionnaire was applied to outpatients, followed at AID Unit, Istituto Giannina Gaslini from august 2017 to august 2018. Data acquired was revised for quality of information. Data of triggers in Autoinflammatory disease from EUROFEVER registry was obtained for statistical reference.

    Results: Triggers in AID Eurofever Registry: In august 2018 a total of 3783 patients were enrolled in EUROFEVER registry and 50,43%, 1908 were female. The average of age symptoms started was 7.04 years (standard deviation of 9,48 years, minimal of 0 and maximum of 75,92 years).The distribution among the periodic hereditary fevers were: 28,75% were FMF (n=1081); 17,66% were PFAPA (n=666); 7,85% (n=297) were CAPS; 7,16% were TRAPS (n=271) and 5,39% were MKD (n=204). Vaccine triggered disease was most common in MKD with 70%. PFAPA, TRAPS and UND presented with similar prevalence of reactions, around 20%. CAPS had 12,34% of vaccine reactions identified. FMF and inflammatory bone disorders carried 6% and 3%, respectively. Excluding other causes of reactions, and isolating just vaccine as cause, MKD still remains the top of the rank (7,14%). Just PFAPA and UND had more than 1% and CAPS, TRAPS, FMF and inflammatory bone disorders had all less than 1%. IGG cohort: 150 questionnaires were distributed with 70% rate of response. Quality of data was 100% for coverage and adverse reactions. Data of 105 patients could be obtained distributed as PFAPA (n=26); CAPS (n=5); TRAPS (n=6); FMF (n=14); MKD (n=8); Inflammatory Bone Disorders (CRMO and PAPA, n=4) and Undefined Inflammatory Diseases, UND, (n=41). Rate of converage was lower than 90% for Hib3 (83,11%) , all measles containing vaccine MMR/MMRV (88,9%) and for Rota C (1,85%). For DTP3, Hep3, PCV3 and IPV the rate of coverage was higher than 90% for all vaccines. A total of 13 severe/serious reactions was observed following: 5 after DTPA+IPV (1 PFPAPA; 2 TRAPS, 1 MKD and 1 UND); 2 after Hep B (TRAPS); 1 after Hib (PFAPA); 1 after P10/13 (PFAPA); 4 after MPR (1 PFAPA, 1 TRAPS, 1 MKD and 1 UND) and 1 afterHepatitis C (1 PFAPA). The general rate of severe reaction per shot was 7,7 for every 1000 shots and no severe infection, death, persistent or significant disability or life-threatening condition was observed. Just one patient, MKD, with severe/serious flare required hospitalization due to disease activity following pneumocoocal vaccine.

    Conclusion: Vaccination in AID is a distinct and a potential public health problem. Specific recommendations for vaccination in AID are extremely necessary as well as further investigations for immunologic protection.

    Disclosure of Interest

    None Declared

    P2003 Peripheral T regulatory lymphocytes and apoptosis expression in a large group of autoimmune, multifactorial and autoinflammatory diseases reveals specific clinical manifestations

    Leonardo O. Mendonca1, Marta Bustaffa1, Caterina Matucci-Cerinic1, Ignazia Prigione1, Stefano Volpi1, Roberta Caorsi1, Sabrina Chiesa1, Francesca Schena1, Alice Grossi2, Paola Terranova3, Isabella Ceccherini2, Maurizio Miano3, Marco Gattorno1
    1International Center for Autoinflammatory Diseases and Primary Immunodeficienceis; 2UOC Medical Genetics; 3Pediatric Hematology Unit, Istituto Giannina Gaslini, Genoa, Italy
    Correspondence: Leonardo O. Mendonca

    Introduction: The application of the semantical immunological division into innate and adaptive immunity to systemic inflammatory syndromes segregates autoinflammatory from autoimmune diseases. Nonetheless a large number of conditions falls down into undefined systemic inflammatory syndromes and some share clinical characteristics with immunedysregulation syndromes such as Autoimmune Lymphoproliferative Syndromes (ALPS) characterized by activation of apoptosis pathway.

    Objectives: Characterize clinically, immunological, molecular and therapeutically a group of patients with high expression of apoptosis pathway. Describe the main diferences expressions of regulatory T cells in a large group of autoinflammatory, autoimune and multifactorial diseases.

    Methods: Retrospective review of all patients submitted, from 2015 to 2018, to flow citometry, for specific ALPS markers and markers of lymphocyte apoptosis: CD3+TCRαβ+CD4-CD8-(αβ-DNTCs), CD19+CD27+(Memory B), TCR αβ+ B220+, CD3CD25+/CD3HLA-DR+ RATIO. Furthermore NK cells, NKT-like cells (CD3+CD16+CD56+), B cells TCRγδ+, Treg, Tnaive and Tmemory cells , sFAS ligand, interleukin 18, interleukin 10, FAS-apoptosis were also studied. A similar flow cytometry composed of healthy Italian controls was used as reference. All clinical and laboratory results were statistically analyzed.

    Results: A total 2089 “ALPS-flow” was realized between 2015-2018. Data that could not be accessed or repeated was ruled out. Finally, 562 flow cytometry was selected and revised. Results divided by age resulted in (number ;female ratio ; average age flow was performed): 1-12 months (9; 55,5%; 0,78); 1-5 years (174; 43,67%; 3,13); 5-10 years (150; 43,3%; 7,25) and > 10 (220 ; 62,33%; 19,5) and no difference between healthy controls were observed. High levels (>1,8) Double negative T cells (CD4-CD8-(αβ-DNTCs) were present in 38 patients carrying the diagnosis of ALPS/ALPS-like, 14 patients with PFAPA, 30 patients with undefined syndromes and in 9 patients with classical autoinflamamtory diseases. 13 patients fulfilled the four flow criteria for apoptosis (1 ALPS, 1 PFAPA, 1 AID, 6 UIS, 1 autoimmune and 1 SJIA). Clinical and immunological comparison was done between those who had complete response to sirolimus or micofenolate. 28 patients were genetically sequenced for large genetic pannels.

    Conclusion: The “alps flow” is an useful instrument to guide clinical evaluation in undefined inflammatory syndromes and can guide specific therapy. Further evaluations are necessary to understand the immunological observation of apoptosis activation in this group of patients.

    Disclosure of Interest

    None Declared

    P2004 Association of gene polymorphism with autoinflammatory syndromes in patients with palindromic rheumatism and intermittent hydrathrosis

    Takako Miyamae, Yumi Tani, Takayuki Kishi, Manabu Kawamoto, Yasushi Kawaguchi, Atsuo Taniguchi, Hisashi Yamanaka
    Institute of Rheumatology, Tokyo Women's Medical University, Tokyo, Japan
    Correspondence: Takako Miyamae

    Introduction: Palindromic rheumatism (PR) is defined as articular and para- articular inflammation that lasts from a few hours to several days, which resolves spontaneously and is associated with increase in inflammatory markers during active disease. It is characterized by recurrent joint manifestations without residual joint destruction. Intermittent hydrarthrosis (IH) is a chronic condition of unknown cause characterized by recurring, temporary episodes of fluid accumulation in the knee. The cause is unknown but allergic and auto-inflammatory mechanisms have been proposed. More recently, specific association with the Mediterranean fever gene, MEFV, has been proposed. So, with some individuals carrying gene mutations (MEFV and also TRAPS-related genes), the native immune system seems to plays a role in the development of IH.

    Objectives: We evaluated polymorphisms of the genes responsible for AID and clinical characteristics in patients diagnosed as PR with IH.

    Methods: Among patients who visited Institute of Rheumatology, Tokyo Women’s Medical University, those who fulfills thediagnostic criteria of PR by Gonzal-Lopez L, et al. and manifested IH were enrolled in this study. Polymorphisms of AID genes, clinical manifestations, findings of blood examinations and joint images, treatment responses, and outcome were evaluated retrospectively. Genomic DNA was isolated from the patients’ peripheral blood and a polymerase chain reaction (PCR) was used to amplify the indicated exons of 12 genes: MEFV (exons 1-10), TNFRSF1A (exons 2-4), MVK (exons 9-11), NLRP3 (exon 3-6), NOD2 (exon 4), LI1RN (exons 2-4), IL36RN (exons 2-5), PSMB8 (exons 2, 3, and 5), NALP12 (exons 3 and 9), PSTPIP1 (exons 10 and 11), TNFAIP3, and NLRC4. After cleaning the PCR products, cycle sequencing was carried out using the Big Dye® Terminator v3.1 kit and analyzed with an ABI 3130xl Prism Genetic Analyzer. Genetic polymorphisms within the above-mentioned genes were examined.

    Results: Out of six patients (2 males and 4 females, median age: 47.1 years, median age at disease onset: 20 years, median disease duration:12 years), all manifested intermittent knee hydrathrosis lasting for several days with 1-4 weeks of attack intervals. Rheumatoid factor and anti-cyclic citrullinated peptides antibody were not detected in anyone. Knee joint X-ray showed no abnormality in all cases whereas increased synovial fluid and synovium tissue proliferation with positive vascularity by ultrasonography. Polymorphisms of AID genes were identified in all 7 cases as following; MEFV (n=4, all non-exon10), CIAS1(n=1) and TNFRSFIA (n=1). Regarding efficacy of medical treatments, although csDMARDs were not effective in all cases, colchicine applied to 4 with MEFV polymorphism was effective in all the four cases. TNF inhibitors was effective in one of two. During observation, no patients developed rheumatoid arthritis and one patient was complicated with Bechet’s disease.

    Conclusion: Association of polymorphisms of the genes responsible for auto-inflammatory diseases should be considered in cases manifesting PR, especially IH, though systemic manifestations were not noted. Colchicine is promising medication in these conditions.

    Disclosure of Interest

    None Declared

    P2005 Report of 65 patients with SAID in Argentina

    Ileana Moreira, Analía G. Seminario, Agostina Llarens, Lina R. Riaño Cardozo, Lorena Regairaz, Liliana Bezrodnik
    Immunología, Centro de Inmunología Clínica Dra. Bezrodnik, Buenos Aires, Argentina
    Correspondence: Ileana Moreira

    Introduction: Systemic auto-inflammatory diseases (SAID) can be defined as a group of immunodeficiencies marks by excessive inflammation that is predominantly mediated by increase response to known or unknown triggers by cells and molecules of the innate immune system. Most patients have recurrent systemic inflammation episodes with fever, acute phase reactants elevated and several clinical manifestations, with healthy intervals. SAIDs have, in most cases, a genetic background, with highly penetrant mutations of single genes, but some cases are polygenic with strong environmental influence that can modulate the phenotype.

    Objectives: Report 65 Argentinian patients with different SAID.

    Methods: Retrospective analysis of medical records of 65 patients with SAID.

    Results: The patients have: Family mediterranean fever (FMF):22 patients (p), Hiper IgD Syndorme (HIDS): 1p. Familial Cold Autoinflammatory Syndrome (CAPS): 1p. Chronic atypical neutrophilic dermatitis with lipodystrophy (CANDLE) like: 1p. Syndrome of periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA syndrome): 30p and 10p are unclassified.

    Most of them are female and only 5/63 has a relative affected.

    The median age of the first symptom was 2 yo (0-16 yo) and the median age at first immunology consult was 10 yo (0.1-48y).

    The most frequent clinical manifestation was periodic fever and skin compromise. Other symptoms include: abdominal pain, arthralgia, aphthas, adenomegalies, pharyngitis, headache and chronic diarrhea.

    During the fever episodes most of them had neutrophilia and elevated CRP and ESR.

    Four patients have humoral compromise (2 with FMF and the ones with CAPS and Candle like).

    Among the FMF, five patients have homozygous pathogenic variants and three have heterozygous compound variants. There are 14 patients with heterozygous variants, but all of them have periodic fever with several associated symptoms and also all of them present good response to oral colchicines treatment.

    The CAPS patient has a heterozygous variant in NLRP12 and he is doing well with IVIG and oral budesonide. The patient with Candle-like syndrome has a poor response to steroids, anti-TNF and anti IL-1β. She has a homozygous variant in SAMD9L. After diagnosis, she received a full matched non related donor HSCT with full engraftment.

    Among the patients with PFAPA syndrome, most of them have good response to oral steroids during crisis and half of them required also daily colchicine. All of them improve around 6 yo without new episodes.

    None of patients have currently amyloidosis.

    Conclusion: The symptoms between the different diseases can be similar, with predominant skin involvement, in addition to fever. All patients with heterozygous variants in MEFV gen were symptomatic and improved with the treatment. Only in 1 patient (SAMDL9) needed biological treatment, all the others had have good responses to colchicine or steroid treatment. There is a significant difference between the age of onset of the symptoms and the age of the first visit to the immunologist. We believe that the diagnosis is important because of potential implications for therapy, monitoring for the development of secondary amyloidosis, and the need for genetic counseling. Diffusion of SAID between pediatricians and clinicians would improve the age of diagnosis.

    Disclosure of Interest

    None Declared

    P2006 Multisystemic inflammatory disease due to DNASE2 mutations: from physiopathology to new therapeutic approaches

    Valentina Moressa1, Alessandra Tesser2, Andrea Trombetta1, Sergio Ghirardo1, Marco Bobbo1, Elisa Piscianz1, Flavio Faletra2, Stefano Volpi3, Alberto Tommasini2, Serena Pastore2, Andrea Taddio2
    1University of Trieste, Italy; 2IRCCS Burlo Garofolo, Trieste, Italy, Trieste; 3IRCCS Istituto G. Gaslini, Genova, Italy
    Correspondence: Valentina Moressa

    Introduction: We recently described the case of a boy with a novel interferonopathy due to DNase2 deficiency. He presented at birth a TORCH-like picture with hepatitis and cytopenias, self-healing in the following months. In his early years, he presented unexplained fever episodes, growth retardation, and poly-articular arthritis. In the subsequent years he developed lipodystrophy, skin rash and lupus pernio. The disease was refractory to several antirheumatic drugs and steroids. At the age of 15 years, DNase2 deficiency was diagnosed by exome analysis (1). Functional data showed that the disease is due to cGAS-dependent interferon inflammation, paving the way for improved treatments based on the inhibition of the hyperactive pathway.

    Objectives: To describe how the diagnosis impacted on medical choices and overall clinical history of a patient.

    Methods: Review of clinical sheets and analysis of interferon signature over the 4 years after genetic diagnosis.

    Results: At the time of the genetic diagnosis, the patient complained of failure to thrive, severe poly-articular arthritis with impairment in walking ability, almost requiring a wheelchair assistance, and severe headache (NRS 9-10) requiring daily administration of major analgesics like tramadol. Treatment included: prednisone, Ramipril, lansoprazole, calcium, celecoxib, tramadole and abatacept.

    After the diagnosis, a treatment with hydroxychloroquine and mepacrine was started, based on proofs of an inhibitory action of these drugs on cGAS (2). With such therapy, he was able to reduce the dosage of steroids and to improve motor capacity. To obtain a more complete control of the disease, ruxolitinib (JAK1/2 inhibitor) was added at the dosage of 10 mg, resulting in a rapid improvement of the headache and of the articular range of motion. In the subsequent six month, the lipodystrophy also showed some improvement, allowing further steroids tapering. However, he developed fatigue, tachycardia and peripheral cyanosis. Cardiologic assessment detected pulmonary arterial hypertension (PAH) and thus the boy started treatment with sildenafil, iloprost, and ambisentan.

    Considering a possible adverse effect of ruxolitinib, as supported by a single case report (3), we interrupted all immunosuppressive therapies except for steroid boluses, but without any improvement. Moreover, he developed progressive cytopenia and increased interferon signature. Therefore he started again ruxolitinib at an higher dose, together with antimalarials, obtaining a marked drop of pulmonary pressure and an improvement in interferon signature, as described in published cases ofPAH (4). In few months he got well again, being able to taper steroids. Recently, ruxolitinib was switched to baricitinib, without any evident change in clinical efficacy.

    Conclusion: The overall clinical improvement and the possibility of reducing the corticosteroid dosage suggest that treatment of our patient can be considered a precision, tailored therapy.

    Nevertheless, due to the novelty and the rarity of such cases, hard is to disentangle between manifestations of the disease and the effects of treatments that are tailored to each patient without a consistent previous experience, as it is evident for orphan disease.

    1. Rodero MP, et al. Nat Commun. 2017 Dec 19;8(1):2176.

    2. An J, et al. The Journal of Immunology, 194(9), 4089–4093.

    3. Low AT, et al. Haematologica, 100(6), e244–e245.

    4. Sanchez GAM, et al. J Clin Invest. 2018 Jul 2;128(7):3041-3052.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P2007 Pregnancy and other gynecological aspects in mevalonate kinase deficiency

    Catharina Mulders-Manders, Anna Simon
    Radboud University Medical Center, Nijmegen, Netherlands
    Correspondence: Catharina Mulders-Manders

    Introduction: Mevalonate kinase deficiency (MKD) is one of the classic monogenic autoinflammatory diseases. Only two pregnancies in MKD patients, have been reported, both with favorable outcome. Little is known on the influence of MKD on pregnancy or other gynecological aspects.

    Objectives: To gain further insight in pregnancy, lactation and gynaecological aspect in MKD.

    Methods: All adult female MKD patients from the Radboud university medical center, a Dutch expertise center on MKD, were asked to fill out an online questionnaire regarding pregnancy, lactation, menses and anticonceptive use, and their influence on frequency, duration and symptoms of MKD attacks.

    Results: Pregnancy and lactation

    Of twenty adult female MKD patients, eleven responded to the questionnaire (55.0%). Median age was 28 years (range 21-55 years). Two patients were treated with anakinra (18.2%) and six with canakinumab (54.5%).

    Four participants indicated that they had ever wanted to become pregnant, and all of them conceived spontaneously. One patient reported one pregnancy, and the other three all reported two pregnancies. Median age at first pregnancy was 27 years (range 24-30 years). During all pregnancies, patients remained attack-free without treatment. One of the four patients reported an MKD attack following delivery, and more frequent attacks in the first three months thereafter.

    During pregnancy, one patient was treated because of pre-eclampsia during the first, and hypertension during her second pregnancy. Five other pregnancies were uncomplicated. All pregnancies resulted in birth of a healthy living child without congenital abnormalities.

    One patient reported two periods of lactation in two children: one week in the first and 23 weeks in her second child. During lactation attack frequency was unchanged, but attacks were shorter.

    Other gynaecological aspects

    Four of the eleven patients reported more frequent MKD attacks after menarche. Two reported increased attack severity. In four patients, attacks were triggered by menstruation and during menstruation, two patients experienced longer attack duration. During the use of hormonal anticonceptive drugs, one out of eight patients experienced more frequent attacks, while four reported a decrease in frequency. Two patients reported shorter attack duration, while the other six did not notice any change in duration.

    Two patients were in menopause, one of whom experienced more frequent attacks after menopause. One patient reported a shorter attack duration, while the duration increased in the other patient.

    Five patients had ever experienced vaginal or vulvar ulcers.

    Conclusion: During seven pregnancies in female MKD patients, no MKD attacks occured. In one pregnancy delivery triggered an MKD attack, and during the only episode of lactation, MKD attacks were shorter than usual. All seven children were born healthy. As our cohort size was small, it would be interesting to confirm these positive findings in a larger number of patients.

    Disclosure of Interest

    None Declared

    P2008 Early onset sarcoidosis associated with NOD2 mutation: familial cases report

    Roberta Naddei1, Francesca Orlando1,2, Carolina Porfito1, Teresa Lastella1, Marinabiondina Amico1, Maria Alessio1
    1Pediatric Rheumatology Unit, Mother and Child Department, University of Naples Federico II; 2Department of Pediatrics, Santobono-Pausilipon Children’s Hospital, Naples, Italy
    Correspondence: Roberta Naddei

    Introduction: Early onset sarcoidosis is characterized by a triad of polyarthritis, uveitis and rash. The familial cases, manifesting the classic clinical triad and an autosomal transmission pattern, have been termed Blau syndrome (BS). The mutation involved the NOD2 gene.

    Objectives: To describe clinical features of a family with a history of uveitis, early onset arthritis and skin rash.

    Methods: Case report.

    Results: The propositus (patient 1) is a 7-year-old female referred to our Unit at the age of 2 years. The family history revealed a mother with rheumatoid arthritis and visual loss due to panuveitis. The clinical history disclosed that at the age of 12 and 15 months the patient had two episodes of diffused rash spontaneously resolved in two weeks. From the age of 18 months recurrent hip pain, defined as transient synovitis of the hip, was reported. It was treated successfully with non-steroideal antinflammatory drugs (NSAID) but pain would reappear once stopped the treatment. Clinical evaluation showed polyarticular symmetrical arthritis of wrists, elbows, knees, proximal interphalangeal joints. Polyarticular Juvenile Idiopathic Arthritis (JIA) diagnosis was made and treatment with NSAID and Methotrexate was started without significant improvement; therefore, Etanercept was added and NSAID stopped. Screening of uveitis was performed every six months. Persisting arthritis of right wrist after 7 months of therapy, ultrasound was performed showing tenosynovitis. Therefore, she underwent intra-articular injection of long-acting glucocorticoids with partial response. At the age of 4 years, she presented papuloerythematous rash, mainly on the trunk, infiltrated and confluent in plaques; skin biopsy was performed resulting consistent with granulomatous disease sarcoid-like. Suspecting BS, molecular analysis was done. At the age of 5 years, she presented scotoma and visual loss; the ocular assessment found bilateral anterior uveitis and posterior bilateral synechiae. She started systemic glucocorticoid therapy and shifted from Etanercept to Adalimumab with improvement of both ocular and articular disease. The molecular analysis of NOD2 showed missense mutation p.R334W (c.1000C<T) consistent with BS. Currently, the articular and cutaneous manifestations are controlled by the combined therapy with Adalimumab and Methotrexate; the ocular disease evolved into irido-lenticular synechiae, some mutton-fat keratic precipitates, band keropathy. She started topical treatment with improvement.

    Her brother (patient 2, age 3 years) at the age of 34 months, developed boggy tenosynovitis and face papuloerythematous rash; inflammatory cellular deposits at anterior segment were disclosed at ophthalmic evaluation. Therefore, treatment with Adalimumab and Methotrexate was started.

    The molecular analysis of NOD2 gene in patient 2, in a youngest asymptomatic brother and in the mother is ongoing.

    Conclusion: Patient 1 presented typical clinical manifestations of pediatric sarcoidosis, confirmed by skin biopsy. R334W is the most common mutation reported in the international Blau registry. The presence of typical clinical manifestations, NOD2 mutation and family history led to the diagnosis of Blau Syndrome. Clinical surveillance and molecular analysis is essential in first-degree relatives of patients with Blau Syndrome in order to recognize clinical manifestations precociously and start appropriate therapy.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P2009 Extending the clinical spectrum of Blau syndrome, a case report with liver cirrhosis

    Mohammed Nashawi1, Heiko Brennenstuhl1, Barbara Bangol2, Thomas Lutz1
    1Center for Pediatrics and Adolescent Medicine, University Hospital Heidelberg, Heidelberg; 2Genetic Department, Center for Human Genetics and Laboratory Diagnostics, Martinsried, Germany
    Correspondence: Mohammed Nashawi

    Introduction: Blau Syndrome is a rare auto-inflammatory disease with variants in NOD2 and an important differential diagnosis for periodic fever syndrome. Patients typically present in the first year of life with fever and a triad (skin manifestation, arthritis and uveitis). The patients could develop pan-uveitis, cardiac manifestation or even renal diseases.

    Objectives: Our objective is to expand the differential diagnosis of periodic fever syndrome by characterizing a patient with atypical clinical features of Blau Syndrome harboring the most common known genetic variant.

    Methods: We used standard laboratory assessment of inflammatory markers, MRI and liver biopsy. To confirm the diagnosis we used next generation sequencing to identify NOD2 variants.

    Results: A 2-year-old male child presented to the hospital with recurrent fevers and decompensating heart failure due to arterial hypertension. He could not walk because of swelling in both ankles and additionally had swelling of the wrists. The past and the family history were unremarkable. On clinical examination, his weight and length were below the 3rd percentile. Skin examination showed erythematous exanthema on the trunk. There were also hepatosplenomegaly and a cardiac murmur present. At that time, he had been treated for heart failure and hypertension. After his medical condition was stable, further work-up had been done to look for the cause of his symptoms. CRP was massively increased. Infections and malignancy were excluded. There were no signs of immunodeficiency and no signs of uveitis. With the presented symptoms and signs the child was finally diagnosed with systemic juvenile idiopathic arthritis. Initially, he was started on Prednisolone 2 mg/kg/d. He started to get better. Methotrexate (MTX) had been also introduced and titrated until 15 mg s.c. once weekly. His symptoms improved under therapy, but he was still not in remission, with recurrent fevers, arthritis and high inflammatory markers. In this condition he was transferred to our center. Here, we started the treatment according to the following table:

    However, there was no full remission, so whole body MRI was performed, which showed signs of liver cirrhosis, which was not explained by the disease and medications. Liver biopsy showed liver fibrosis with signs of inflammation and granulation formation. We did a molecular genetic analysis (fever panel) which showed a heterozygous mutation in NOD2 gene (c.1001G>A; p.Arg334Gln, Exon 4), which causes Blau Syndrome. Currently, he is responding well to the therapy with Adalimumab.

    Conclusion: Here, we report a case of Blau Syndrome with liver cirrhosis extending the phenotypic spectrum of this rare disease. In our case the patient presented with heart failure, hypertension, arthritis, skin rash and also hepatomegaly. Episodic fever was noted. Our patient did not develop uveitis, under the treatment with MTX and the other medications which are all effective against uveitis. The recommended therapy for Blau Syndrome is to use steroid daily in addition to immunosuppressants. Different types of immunosuppressants have been used with a controversial effect, but TNF-alpha inhibitors have been shown to be most effective in treating patients with or without uveitis. Our patient showed partial response under the therapy with MTX, IL-1β antagonist and IL-6 antagonist. For the first time, under treatment with TNF-alpha inhibitor (specifically Adalimumab), he is in full clinical and laboratory remission.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    Table 1 (abstract P2009). See text for description

    P2010 Diagnosis and management of hereditary recurrent fevers: 20-year experience at a single Italian referral centre

    Laura Obici1, Grazia Bossi2, Roberto Caporali3, Roberta Mussinelli1, Simona Casarini1, Sara Monti3, Isabella Ceccherini4, Francesco Caroli4, Antonio Vergori2, Eleonora Di Buduo1, Claudia Sforzini1, Marco Gattorno5, Giampaolo Merlini1
    1Amyloidosis Research and Treatment Centre; 2Pediatrics Unit; 3Rheumatology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia; 4Medical Genetics Unit; 5Pediatric Rheumatology Unit, IRCCS Giannina Gaslini Institute, Genova, Italy
    Correspondence: Laura Obici

    Introduction: Disease awareness, diagnostic tools and therapeutic options for hereditary periodic fevers (HRF) have significantly improved in recent years but whether patients’ diagnostic pathway and outcome have consistently ameliorated remains to be elucidated.

    Objectives: To investigate presentation, genotypes, clinical characteristics, natural history and response to treatment in patients with HRF referred to our centre between 1998 and 2018.

    Methods: We conducted a retrospective study of all patients diagnosed with HRF according to genetics and/or clinical Eurofever classification criteria. All patients underwent genetic analysis and full clinical evaluation including investigation for possible amyloid disease.

    Results: 213 patients (FMF 166, TRAPS 33, CAPS 5, MKD 9) were included, 10% of them are of non-Caucasian ancestry and 41% had a positive family history. Overall 61 genotypes were identified, with 3 novel gene variants, not reported to date. 87% of FMF patients had ³ 1 clearly pathogenic variant, the most common being p.Met694Val. 79% of TRAPS patients had a clearly pathogenic mutation, affecting a cysteine residue in 15 cases. Median age at disease onset was 12 years (range 0.5-51). Median age at diagnosis was 34 years (2-78). 79 % of patients presented in adult age, of which 35 (31 FMF and 4 TRAPS) reported symptom onset after the age of 20 (median 30, range 21-51). Median time from disease onset to diagnosis in patients referred in adult age was 25 years (0.5-61). Fever was the most common reported symptoms (in 96% of patients), followed by abdominal flares. Median number of inflammatory attacks was 8 in FMF (1-24). A chronic disease course was seen in 50% of TRAPS patients. AA amyloidosis was diagnosed in 22 patients (10.3%). Median age at AA onset was 40 years (range 12-77) with a median time from HRF onset to appearance of renal amyloidosis of 33.5 (8-56). In 19 cases (86%) the diagnosis of HRF was established after AA amyloidosis presentation. Regarding diagnostic delay, presenting features and rate of AA amyloidosis, no significant differences was observed throughout different time periods (1998-2004; 2005-2011; 2012-2018). In patients with FMF, complete response to colchicine is observed in 88%, with a median dose of 1 mg/day (range 0.5-2); partial response and/or limited tolerability was reported in 29 patients (22.7%). 7 pts were shifted to anti-IL1 agents, including two kidney transplanted patients due to AA amyloidosis, with complete response and good safety profile

    Conclusion: Diagnostic delay hasn't significantly reduced over time in spite of increasing awareness and optimized diagnostic tools. A substantial proportion of patients is still recognized in adult age and therefore remain at high risk ofdeveloping long-term renal complications.

    Disclosure of Interest

    None Declared

    P2011 RNF213 gene variants in patients with Takayasu arteritis or vasculopathy

    Ebun Omoyinmi1, Annette Keylock1, Dara McCreary1, Sarka Fingerhutova2, Pavla Dolezalova2, Seza Ozen3, Sylvia Kamphuis4, Cheryl Hemingway5, Despina Eleftheriou1, Paul Brogan1
    1Infection, Inflammation and Rheumatology Section, UCL Great Ormond Street Institute of Child Health, London, United Kingdom; 2Paediatric Rheumatology and Autoinflammatory Diseases Unit, General University Hospital, praha, Czech Republic; 3Department of Paediatric Rheumatology, Hacettepe University, Ankara, Turkey; 4Department of Paediatric Rheumatology, Sophia Children's Hospital, Erasmus University Medical Centre, Rotterdam, Netherlands; 5Paediatric neuroradiology department, Great Ormond Street Hospital for Children NHS foundation Trust, London, United Kingdom
    Correspondence: Ebun Omoyinmi

    Introduction: RNF213 (Ring Finger Protein 213) was first identified as a major susceptibility gene for moyamoya arteriopathy, which is a progressive steno-occlusive disease of the cerebral arteries and the development of abnormal basal collateral vessels. Several other studies have since reported an association of RNF213 variants with various vasculopathies, pulmonary hypertension, coronary artery disease and stroke. In vivo experiments demonstrated abnormal development of brain vessels in rnf213 knockdown zebrafish, while suppression of both arteriogenesis and angiogenesis were observed in knockout mice.Consequently, RNF213 may not be just a susceptibility gene for moyamoya, but could also play a role in systemic vasculopathy, and be an important mimic of Takayasu arteritis (TA) of the young.

    Objectives: To review the phenotype of vasculopathy/vasculitis in a cohort of patients referred for genetic testing where rare or novel variants in RNF213 were identified.

    Methods: RNF213 variants were screened as part of a next-generation sequencing screening strategy for patients with suspected vasculitis or autoinflammation. Variants that were either novel or less than 1% in public genetic databases were considered as candidate for the purposes of this study.

    Results: We identified 54/272 patients with at least one novel or rare RNF213 variant. Fifteen/54 had vasculopathy or suspected vasculitis without clearly defined aetiology (Table). Amongst these cases, there were two children with TA, and a third case with familial moyamoya arteriopathy with 3 affected family members. The first case was a Turkish girl, with a novel heterozygous RNF213 mutation p.L5003V; predicted to be damaging, who presented with hypertension at the age of 4 years, with vascular phenotype suggestive of TA, without cerebral involvement. The second TA case was a Dutch girl presenting with systemic inflammation and occlusive vasculopathy of multiple arterial beds, including renal and brain. She has 2 heterozygous variants in RNF213: p.P1721L and p.K4732E. The p.P1721L variant has previously been reported in cohort of patients with moyamoya arteriopathy, and is predicted to be damaging. The third case was a child from a family investigated for suspected familial moyamoya arteriopathy affecting 3 family members. The proband had a heterozygousp.D4013N RNF213 missense mutation, previously reported in association with moyamoya. He had typical occlusive cerebral arteriopathy and collateral vessel formation. This mutation segregated with the same phenotype in the affected father and sister. Vasculopathies observed in the other cases are summarised in the Table.

    Conclusion: These observations could support a role for mutations in RNF213 as the cause of a monogenic but heterogeneous vasculopathy mimicking vasculitis such as TA, or stenotic vasculopathy associated with moyamoya arteriopathy.

    Disclosure of Interest

    None Declared

    Table 1 (abstract P2011). RNF213 variants in patients with Takayasu arteritis or vasculopathy

    P2012 Clinical and molecular features of 3 children with neonatal onset multisystem inflammatory disease: an experience from a setting where there is lack of access to anti-IL1 agents

    Vignesh Pandiarajan1, Deepti Suri2, Sagar Bhattad1, Anju Gupta1, Amit Rawat1, Raphaela Goldbach-Mansky3, Marco Gattorno4, Surjit Singh1
    1Allergy Immunology Unit, Pediatrics; 2Postgraduate Institute of Medical Education and Research, Chandigarh, India; 3Translational Autoinflammatory Diseases Section, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, United States; 4UO Pediatria II, Istituto G. Gaslini, Genoa, Italy
    Correspondence: Vignesh Pandiarajan

    Introduction: Neonatal-onset multisystem inflammatory disease (NOMID) is characterized by sterile inflammation of multiple organs due to molecular defect in NLRP3. Clinical manifestations usually start from early infancy and include fever, urticaria-like rash, arthropathy, and aseptic meningitis. Development of targeted therapies against interleukin-1 (IL1) has revolutionized the management of this clinical condition.

    Objectives: To describe 3 cases of NOMID from a tertiary care centre in North India.

    Methods: Case records of the patients followed up in our Pediatric Rheumatology Clinic were reviewed. Children with clinical features suggestive of of NOMID and proven molecular defect in NLRP3 were included after informed consent from the parents.

    The first child (P1) was symptomatic since early infancy in form of recurrent episodes of fever and urticaria-like rash. She developed frontal bossing and progressive arthropathy of knees over years, and the diagnosis was established at age of 13 years. Second child (P2) had swelling of knees and recurrent urticaria-like rash over trunk since early infancy. He developed sensorineural hearing loss, progressive enlargement of knees and contractures of both hips and knees. He developed renal amyloidosis and obstructive hydrocephalus at the age of 11 years. Radiograph of knees showed metaphyseal cupping, epiphyseal enlargement with sclerosis and prominent patella. Third child (P3) developed nephrotic syndrome at the age of 6.5 years. She received multiple immunosuppressive medications for her treatment- cyclophosphamide, tacrolimus, and mycophenolate mofetil. Renal biopsy done at the age of 10 years revealed AA amyloidosis. She had occasional urticarial rash in early infancy which went unnoticed. All three children had persistent neutrophilic leukocytosis, thrombocytosis, elevation in erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP).

    Results: Thalidomide (3 mg/kg) produced significant reduction in inflammatory burden of the first child (P1). She is now 16 years old and doing well. The second child (P2) was started on thalidomide since 4 years of age, however, no response was noted. Second (P2) and third child (P3) succumbed to renal failure secondary to amyloidosis, at the age of 13 and 11 years, respectively. Molecular defects identified in NLRP3 in children P1, P2, and P3 were p.Asp305His, p.Thr349Ile, and p.Ala352Val, respectively.

    Conclusion: Delay in diagnosis, lack of awareness, and lack of access to anti-IL1 agents contributed to significant morbidity and mortality in our patients. Thalidomide can be tried to reduce the inflammatory burden in patients with NOMID in settings where there is no access to anti-IL1 agents

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P2013 Long-term outcomes and treatment efficacy in patients with TNF receptor-associated autoinflammatory syndrome (TRAPS): a series of 290 cases from the Eurofever/EUROTRAPS international registry

    Riccardo Papa1, Thirusha Lane2, Taryn Youngstein2, Tamer Rezk2, Charalampia Papadopoulou3, Nicolino Ruperto1, Paul A. Brogan3, Philip N. Hawkins2, Patricia Woo3, Marco Gattorno1, Helen J. Lachmann2
    1Paediatric Rheumatology Clinic, IRCCS Giannina Gaslini Institute, GENOVA, Italy; 2National Amyloidosis Centre, Division of Medicine, Royal Free Campus, University College London; 3Department of Infection, Inflammation and Rheumatology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
    Correspondence: Riccardo Papa

    Introduction: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is one of the best known monogenic auto-inflammatory disorder resulting from an autosomal dominant variation in the TNF super family receptor 1A (TNFRSF1A) gene.

    Objectives: To define best treatment approach in patients with TRAPS and effect on long-term outcomes.

    Methods: We reviewed all data on patients with TNFRSF1A variants enrolled in the Eurofever/EUROTRAPS international registry.

    Results: Data on 290 patients were available. Patients with R92Q, P46L or intronic variants (49%) displayed milder disease than 147 patients with mutations affecting other coding regions, with less frequent abdominal pain and skin rashes (P<0.01), higher efficacy rate of colchicine as maintenance treatment, and none developed AA amyloidosis. Almost 90% of patients with exon mutations required maintenance therapy. Anti-interleukin (IL) 1β drugs were the most frequently used (47 patients), with the highest efficacy rate (>90% complete response), while Etanercept was less effectively used and discontinued in 72% of patients. No patients on anti-IL1β treatment developed amyloidosis and 10 patients with amyloidosis have been successfully treated with anti IL-1 agents with preservation of native renal function in 7 and excellent long-term transplant function in 2. Nine women had a history of failure to conceive and seven had successful pregnancies without fertility treatment following complete disease control with anti-IL1β drugs. Long term safety profiles for anti IL-1 agents were excellent even in the presence of comorbidity.

    Conclusion: Anti-IL1β drugs are the best maintenance treatment in TRAPS with potential to reverse the most serious disease complications of AA amyloidosis and infertility. The diagnosis of TRAPS should be considered very carefully in patients carrying R92Q, P46L or intronic TNFRSF1A variants.

    Disclosure of Interest

    None Declared

    P2014 A child with Majeed syndrome from India

    Pallavi Pimpale Chavan, Raju P. Khubchandani
    Pediatric Rheumatology Clinic,Department of Pediatrics, Jaslok Hospital and Research Centre, Mumbai, India
    Correspondence: Pallavi Pimpale Chavan

    Introduction: Majeed syndrome is a very rare autosomal recessive, autoinflammatory disorder characterized by a triad of chronic recurrent multifocal osteomyelitis, congenital dyserythropoietic anemia and neutrophilic dermatosis caused by LPIN2 gene mutation. 14 mutation positive cases identified till 2017 have been reported from the Middle East.

    Objectives: We describe our brief experience with the second family with a child afflicted with Majeed syndrome from India.

    Methods: A 4 year old male child born of a consanguineous marriage, presented with recurrent irregular episodes of fever with irritability and failure to thrive since 5 months of age. At 9 months of age parents noticed, small skin ‘boils’ over the trunk and limbs, which recurred every 15-20 days along with the fever and stayed for 4-5 days. At 18 months, parents noticed that he refused to bear weight and walk and would cry on handling.

    Gradually the episodes of painful small skin boils, limb pains which the parents could not localize and fever increased in frequency and after multiple pediatric, orthopaedic consults he was referred to us by a pediatric neurologist who had been referred the case to rule out Fabry disease. The pediatric neurologist had asked for whole exome sequencing and referred the case to us for clinical evaluation sensing a musculoskeletal rather than neurological disease.

    Results: On examination the weight (10 kg) and height (83 cm) were below 3rd centile. He had pallor, painful swelling over the left tibia, motor delay with hamstring tightness and mild bilateral knee contractures. Language and social milestones were normal. There were no skin lesions at the time of evaluation and systemic examination was normal.

    Review of his past investigations revealed a persistent, microcytic hypochromic anemia (Hemoglobin 8.3-10.6 gm/dl) with raised erythrocyte sedimentation rate (54-105 mm/hr), C-reactive protein (37-98 mg/l) and platelets (4.3-8.9 x103 cu mm).

    His genetic mutation for LPIN 2 returned positive with homozygous missense variation in exon 17 of the LPIN2 gene resulting in amino acid substitution of cysteine to arginine at codon 736 (p.Arg736Cys;ENST00000261596). Meanwhile a bone scintigraphy done, showed increased vascularity and osseous activity in proximal shaft of left tibia with mild osseous activity at D9 and D10 vertebrae. A bone marrow examination was not performed as a conclusive mutation diagnosis was obtained.

    He showed a dramatic response to Ibuprofen with abatement of fever, no skin boils and improvement of general mood and playfulness. With a view to attempt to omit NSAIDs, intravenous Pamidronate was commenced. He has received four doses of Pamidronate (cumulative dose 12 mg/kg) over one year and has gained weight (11.5kg) and height (96cm), remained afebrile with full physical activity and no new skin lesions.

    Conclusion: Paucity of awareness amongst the medical community about these rare illnesses are responsible for diagnostic delays and complex referral pathways. With the high rates of consanguinity in several parts of our country we believe that we are seeing just the tip of the iceberg of monogenic autoinflammatory diseases. Due to non availability of Interleukin-1 inhibitors in India, which are highly effective at controlling osseous and systemic inflammation in Majeed syndrome , we have limited treatment options to offer to our patient. We have offered educational resources and genetic counselling to the family.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P2015 Spondyloenchondrodysplasia – a case from India

    Pallavi Pimpale Chavan1, M Moreews2, Shashi Ranade3, A Belot2, Raju P. Khubchandani1
    1Section of Pediatric Rheumatology, Jaslok Hospital and Research Centre, Mumbai, India; 2Pediatric Rheumatology Clinic, National Referee centre RAISE (Rhumatism and AutoImmunity in children) & CIRI, INSERM U1111, Lyon, France, 3Consultant Paediatrician, Ranade Hospital, Karad, India
    Correspondence: Pallavi Pimpale Chavan

    Introduction: Spondyloenchondrodysplasia (SPENCD) is a very rare genetic immuno-osseous dysplasia characterized by skeletal anomalies (short stature, platyspondyly) and enchondromas in the long bones or pelvis. SPENCD may have a heterogeneous clinical spectrum with neurological involvement or autoimmune manifestations, such as systemic lupus erythematosus (SLE).

    Objectives: We describe a child from India with SPENCD, who presented with features of SLE , skeletal dysplasia and enchondromas

    Methods: A 9 year old male born of second degree consanguineous marriage presented with fever , oral ulcers, leg pain with morning stiffness since 1 month. He was worked up by his primary paediatrician for pyrexia of unknown origin and a summary of laboratory tests performed included, hemogram with hemoglobin 8.7 gm/dl, white cell count 5900 /cu mm platelet 1.85 x 103 cu mm, erythrocyte sedimentation rate 100 mm/hr, antinuclear antibody 4+.Suspecting connective tissue disease child was referred to us for further evaluation.

    Results: On further enquiry, there was past history of multiple medical visits for poor growth since infancy and a bony swelling of the right forearm leading to incomplete supination for which he was operated at age 5 years. No details were available. His milestones were appropriate for age with normal scholastic performance.His weight (16.9 kg) and height (113cm) were below the 3rd centile. On examination, he had pallor, facial rash, mucositis of the palate, generalised lymphadenopathy, triangular facies, prominent anteverted ears, prominent conjunctival blood vessels, supernumerary teeth, right elbow swelling, paucity of body hair On systemic examination abdomen was soft with hepatosplenomegaly and other systems were normal.

    On further investigation his dsDNA was 4+ , Direct Coombs test was positive, C3 - 19.1 mg/dl C4 - 3.36 mg/dl, urinalysis was normal, Anti Ro and lupus anticoagulant were negative and APLA IgG was positive and APLA IgM negative. The diagnosis of SLE was confirmed.

    Considering, consanguineous marriage, short stature and right elbow swelling with other subtle dysmorphisms described above, a syndromic form of lupus was suspected and a skeletal Xray analysis showed extensive areas of metaphyseal abnormalities with epiphyseal remodelling changes and platyspondyly. Hence a clinical diagnosis of Spondyloenchrondrodysplasia (SPENCD) was considered. Sanger sequencing of ACP5 encoding the protein TRAP revealed homozygous non sense mutation (c.849T>A, p.(Tyr283stop) confirming the diagnosis of SPENCD.He was further investigated for diseases associated with SPENCD, which revealed normal thyroid function, IGF – 1, immunoglobulin levels and 2DECHO.

    He was started on hydroxychloroquine (5mg/kg/day), oral prednisolone (0.6mg/kg/day), low dose aspirin, supplements and was advised sun protection. On follow up after a year, he is continued on hydroxychloroquine (5mg/kg/day), oral prednisolone (0.25mg/kg/day), low dose aspirin and supplements. He has gained weight (20.6 kg), height (114 cm), is afebrile with no arthritis, mucositis or rash.

    Conclusion: SPENCD is an immunosseous dysplasia and a very rare cause of monogenic lupus. This, is the second case of SPENCD reported from India. The entity should be suspected in children with SLE with a consanguineous background who have short stature and bony abnormalities. We expand the phenotype here with additional dysmorphic features comprising supernumerary teeth, triangular facies, prominent anteverted ears and prominent conjunctival blood vessels not described before to the best of our knowledge.

    Consent for publication has been obtained from patient

    Yes

    Disclosure of Interest

    None Declared

    P2016 Clinical profile of seven children with hereditary complement deficiency from a center in Mumbai, India

    Prajakta Ranade1, Y.J. Crow2, Pallavi Pimpale Chavan3, L Seabra4, G Rice5, Raju P. Khubchandani3
    1Paediatrics, St John's Medical College Hospital, Mumbai, India; 2Centre for Genomic And Experimental Medicine, Institute of Genetics and Molecular Medicine, Edinburg, United Kingdom; 3Section of Pediatric Rheumatology, Jaslok Hospital and Research Centre, Mumbai, India; 4Neurogenetics and Neuroinflammation, Laboratory of Neurogenetics and Neuroinflammation, Paris, France; 5Division of Evolution and Genomic Science, Manchester Academic Health Science Centre, Manchester, United Kingdom
    Correspondence: Pallavi Pimpale Chavan

    Introduction: While systemic lupus erythematosus (SLE) is classically considered as a multifactorial polygenic disease, hereditary monogenic forms are well recognized. Deficiencies in the early components of the classical pathway strongly predispose to the development of SLE, of which C1q deficiency is the commonest. Children with C1q deficiency are estimated to develop SLE in 93% of cases. We report our experience with this entity.

    Objectives: To describe the clinical and laboratory profile and follow up of a cohort of seven children with hereditary complement deficiency.

    Methods: We reviewed our data on seven children with hereditary complement deficiency and obtained a genetic diagnosis in 5 of these 7 patients using next-generation sequencing. Informed consent was taken from the parents and the data was tabulated in the study proforma.

    Serial no/Sex CSM Onset (m)/time to diagnosis (m) Clinical Positive antibody profile C3/C4/CH50 Genetics Treatment Follow up (m)/Outcome
    1/F N 10/7 Rash, mucositis, neuroregression ANA Ds DNA Anti Ro 175/23/42.1 Homozygous for a c.606delA / p.Gly204Alafs*78 variant in C1QA HCQ Sx AZT 28/Improved
    2/F Y 24/24 Rash, mucositis, arthritis, renal disease, herpes zoster ANA Anti Ro 203/73/54 Homozygous for a c.622C>T / p.Q208* STOP variant in C1QA HCQ,Sx,AZT,MMF Death (at 12 years of age)
    3/M Y 21/12 Rash, mucositis, delayed development, hyperreflexia ANA Ds DNA Anti Ro ACLA IgG 158/47.7/43 Homozygous for a c.622C>T / p.Q208* STOP variant in C1QA HCQ,Sx,AZT 16/Improved
    4/M Y 12/24 Rash, mucositis, hyperreflexia, seizures ANA Anti Ro 117/39/18.6 Homozygous for a c.171delT / p.Gly58Alafs*224 variant in C1QA HCQ,Sx,AZT,MMF 41/Improved
    5/F Y 27/4 Rash, mucositis, neuroregression, hemiparesis, cerebral atrophy, hyperreflexia, hypothyroidism ANA Anti Ro ACLA IgM Anti TPO 179/42.8/NA Homozygous for a c.79C>T / p.Arg27* variant in C1QA HCQ,Sx,Cyc,AZT 31/Improved
    6/M Y 40/0.5 Rash’ mucositis ANA ACLA IgM 114/NA/NA Afflicted sibling of Sr no 2 Not tested HCQ,Sx 22/Improved
    7/F Y 48/36 Rash, mucositis, arthritis, proteinuria, infections (varicella, scabies, impetigo), hypothyroidism ANA 30/NA/35.7 Demise before testing HCQ, Sx, Cyc MMF Death (at 12 years of age)
    1. Abbreviations: F: Female, M: Male, CSM: Consanguinity, Y: Yes, N: No, m: months, Sx: Steroids, AZT: Azathioprine, Cyc:Cyclophosphamide, MMF: Mycophenolate

    Results:

    Conclusion: Based on our study and others reported, hereditary complement deficiency (notably C1Q deficiency) is the commonest form of monogenic lupus in our part of the world. Afflicted children present most commonly with rash and mucositis below 5 years of age and there is a significant delay in diagnosis due to lack of physician awareness. Neurologic features are frequently seen while renal manifestations are not. Neuroregression responds to immunosuppressive therapy with children improving on their mental, motor and speech milestones. Except in one patient major life-threatening infections have not been a challenge despite high infection rates in the community coupled with immunosuppression.

    Disclosure of Interest

    None Declared

    P2017 Preliminary results of the Latin-American cohort of auto-inflammatory syndromes

    Daniela G. P. Piotto1, Clovis A. Silva2, Katia Kozu2, Ana Luiza Cunha3, Flavia Patricia Santos4, Ana Laura Tolin5, Sheila K. Oliveira6, Simone Appenzeller7, Claudia S. Magalhães8, Marcia Bandeira9, Flavio Sztajnbok10, Carlos NobreRabelo Jr11, Carmen Laurade Cunto12, Cristina De Battagliotti13, Blanca Bica14, P Munittis15, Liliana Bezrodnik16, Marta F. Rodrigues17, Tereza C. Robazzi18, Erica N. Matos19, Ricardo Russo20, Maria Teresa Terreri1
    1Division of Rheumatology, Department of Pediatrics, Universidade Federal de Sao Paulo; 2Division of Rheumatology, Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo; 3Division of Rheumatology, Department of Pediatrics, Hospital Infantil João Paulo II, Belo Horizonte; 4Division of Rheumatology, Department of Pediatrics, Universidade Federal de Minas Gerais, São Paulo, Brazil; 5Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics, Hospital Notti, Mendoza, Argentina; 6Division of Rheumatology, Department of Pediatrics, Federal University of Rio de Janeiro, Rio de Janeiro; 7Rheumatology Department, Faculty of Medical Sciences, UNICAMP, Campinas; 8Division of Rheumatology, Department of Pediatrics, Universidade Estadual de São Paulo - Faculdade de Medicina de Botucatu, Botucatu; 9Division of Rheumatology, Department of Pediatrics, Hospital Pequeno Principe, Curitiba; 10Division of Rheumatology, Department of Pediatrics, Hospital Universitário Pedro Ernesto, Rio de Janeiro; 11Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics, Universidade Federal de Fortaleza, Fortaleza, Brazil; 12Division of Rheumatology, Department of Pediatrics, Hospital Italiano, Buenos Aires; 13Division of Rheumatology, Department of Pediatrics, Hospital de niños Dr Orlando Alassia, Santa Fe, Argentina; 14Division of Rheumatology, Department of Pediatrics, Hospital Universitário Clementino Fraga FilhoUniversidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 15Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics, Hospital El Cruce, F. Varela; 16Servicio Inmunología HNRG., Hospital de Niños, Buenos Aires, Argentina; 17Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 18Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics,Universidade Federal da Bahia, Bahia; 19Division of Rheumatology, Department of Pediatrics,Hospital Universitário da Faculdade de Medicina da Universidade Federal de Mato Grosso do Sul, Mato Grosso do Sul, Brazil; 20Division of Rheumatology, Department of Pediatrics, Hospital Garrahan, Buenos Aires, Argentina
    Correspondence: Daniela G. P. Piotto

    Introduction: Auto-inflammatory syndromes (AIS) are rare and with large spectrum of clinical, genetical and laboratorial findings, which vary according to ethnic and geographical factors.

    Objectives: There is no report evaluating AIS solely in Latin–American (LA) cohorts. Therefore, the objective of this study is to characterize AIS in a LA population.

    Methods: Fourteen countries from all over Latin America were invited to participate in this observational and descriptive study collecting data on clinical, laboratory, genetic characteristics and treatment response of polygenic and monogenic AIS. Inclusion criteria were patients with clinical and/or genetic diagnosis of AIS. Data were prospectively collected in a web-based data bank.

    Results: At the time of this report, 162 patients from Brazil (78.8%) and Argentina (21.2%) had been included: 52% males, 75% Caucasians; median age at symptoms onset was 2.1 years, median age at diagnosis of 6.9 years, and median disease duration was 8.5 years. The main AIS diagnosis were: PFAPA (28%), CNO (18%), FMF (16%), PGA (10%), CAPS (9%), HIDS (7%), TRAPS (5%) and PAPA (3%).The diagnosis was made clinically in 62% of patients (either they were PFAPA or CNO patients or presented a negative genetic test); 33% of patients were diagnosed by both clinical features and genetic tests and 5% of patients had no clinical but only a positive genetic test that was performed due to family history. Clinical features were consistent with the genetic abnormalities found. Genetic testing had been done in different laboratories using either Sanger sequencing and/or NGS. One of both acute phase reactants (either ESR or CRP) elevation occurred in 91% patients. The most frequently used medication were corticosteroids (66%, partial response in more than a half of patients) and colchicine (29.6%, total or partial response in half of patients). Regarding biological agents, etanercept was used in 11.1% with a partial response in most patients. IL1 blockers were prescribed in 11.1% but with a total response in the majority who used them. Of note, 51% of patients achieved remission, but 46% were still active at the time of evaluation, 2% had deceased, MAS was observed in 1% and none of them had amyloidosis.

    Conclusion: A Latin-American registry of AIS included patients especially from South America. A clear limitation in access to genetic diagnosis was noted. Recognizing the barriers to the awareness about expansion of this group is a priority to better understand these complex diseases, especially in Central America. Some features like absence of amyloidosis and medications used in treatment differentiate our cohort from American and European ones.

    Disclosure of Interest

    None Declared

    P2018 Chronic nonbacterial osteomyelitis: report of six cases

    Daniela G. P. Piotto1, André Aihara2, Artur Fernandes2, Gabriela Balbi1, Claudio Len1, Maria Teresa Terreri1
    1Division of Rheumatology and Unit of Pediatric Rheumatology - Department of Pediatrics; 2Department of Imaging Diagnosis, Universidade Federal de Sao Paulo, São Paulo, Brazil
    Correspondence: Daniela G. P. Piotto

    Introduction: Chronic nonbacterial osteomyelitis (CNO), also known as chronic recurrent multifocal osteomyelitis (CRMO), is a rare autoinflammatory bone disorder that causes multifocal aseptic lytic lesions in bone biopsy and is characterized by periodic exacerbations and remissions, mostly affecting children and adolescents.

    Objectives: To report and describe clinical features, laboratory and treatment of six cases of CNO.

    Methods: Retrospective descriptive review of six children and adolescents with CNO treated at a specialized center, between 2010 and 2018.

    Results: Four (66%) out of 6 patients were girls with mean age of 15.5 years (range 11-20.4) and mean follow-up time of 5.1 years (range 1.5-10.2). Mean age of first symptoms was 10.3 years (range 0.7–15.3) and mean age at diagnosis was 12.8 years (range 9.5-16). The most affected sites were metaphysis and diaphysis of long bones. Median number of initial bony lesions was 2.8 (range 1-7) at onset and 3.5 (range 1-8) during the follow up. Two (33.3%) patients had recurrence in clavicle, one (16.6%) in temporomandibular joint and two in mandible. One (16.6%) patient presented with fever and dyserythropoietic anemia, receiving diagnosis of Majeed syndrome.Five (83.3%) patients presented with arthralgia and bone pain, and two (33.3%) of them had acute migratory polyarthritis. All patients had increased acute phase reactants and radiographic and magnetic resonance imaging alterations.All patients received NSAIDs therapy (indomethacin) with clinical remission in half of the cases.Three (50%) patients received bisphosphonates (alendronate), and one took methotrexate associated with alendronate. All patients had good clinical response and two achieved clinical remission.

    Conclusion: The awareness of characteristic features of CNO is important for an early diagnosis and can help avoiding unnecessary diagnostic procedures and prolonged antibiotic therapy.

    Disclosure of Interest

    None Declared

    P2019 Familial Mediterranean fever: strong evidence for a functional effect of E148Q when combined with M694V

    Elon Pras1, Ori Eyal1, Yael Shinar2, Mordechai Pras3
    1Institute of Human Genetics; 2FMF Clinic; 3Heller Institute of Medical Science, Sheba Medical Center, Ramat Gan, Israel
    Correspondence: Elon Pras

    Introduction: The role of E148Q in the pathogenesis of familial Mediterranean fever (FMF) is controversial. The penetrance of the M69V/M694V genotype in adult FMF patients is close to 100%. Disease penetrance in patients with the M694V/E148Q and M694V/null (single M694V mutation) genotypes is unknown. A difference in the penetrance of the latter two may indicate functionality for E148Q.

    Objectives: To assess the penetrance of the M694V/E148Q and M694V/null genotypes in FMF

    Methods: The study was performed on North African Jewish (NAJ) population in which FMF is highly prevalent and mutation frequencies are well known. Combined carrier rates for M694V and E148Q were calculated from three previous studies. The expected frequencies of the M69V/M694V, M694V/E148Q and M694V/null genotypes were calculated. We constructed a cohort of 107 consecutive patients with definite FMF, all of NAJ decent who came for genetic analysis. The ratio between the calculated frequencies of the 3 genotypes and the actual frequency obtained from the patient cohort was used to determine the penetrance of M694V/E148Q and M694V/null.

    Results: In the patient cohort we found 75 patients with the M694V/M694V genotype, 15 with the M694V/E148Q an 16 with M694V/null. Pooled allele frequencies from 443 NAJ controls were 0.066 and 0.047 for M694V and E148Q, respectively.Considering 100% penetrance for adult FMF patients homozygous for M694V, calculations show a penetrance of 0.1405 and 0.00756 for M694V/E148Q and M694V/null, respectively.

    Conclusion: The penetrance of M694V/E148Q is more than 18 times higher than M694V/null indicating an active role for E148Q when combined with M694V.

    Disclosure of Interest

    None Declared

    P2020 Treatment of type 1 interferonopathy with Ciclosporin A and baricitinib in a 5 year old boy with heterozygous PSMB-8 mutation

    Christoph Rietschel1, Eduardo Salamano1, Min Ae Lee-Kirsch2, Kay Latta3
    1Pediatric Rheumatology, Clementine Kinderhospital, Frankfurt/Main; 2Molecular Pediatrics, Klinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, Dresden; 3General Pediatrics, Clementine Kinderhospital, Frankfurt/Main, Germany
    Correspondence: Christoph Rietschel

    Introduction: Type 1 Interferonopathy is a challenging autoinflammatory disease that can mimic different systemic diseases such as systemic Juvenile Idiopathic Arthritis. It shows a considerable clinical heterogeneity with neurological, cutaneous, arthritic and other symptoms and is commonly transmitted in an autosomal recessive manner. Different mutations have been discovered in recent years. Exceptionally, only one mutation can be found. The activation pattern of genes involved in the interferon pathway, the interferon signature, is the diagnostic hallmarkof the disease and provides an important tool for monitoring disease activity.

    Objectives: We present this interesting case to show the clinical spectrum, diagnostic workup, possible complications and the chosen treatment modalities in this particular disease.

    Methods: Our patient, born 03/2013, with an uneventful previous history, developed first symptoms in 09/2016. He showed persistent fever and a transient rash. From the beginning he suffered from strong, unexplained abdominal pain. His CRP values and leukocytes were only moderately elevated. He was first treated in another hospital for infection, but did not respond to broad spectrum antibiotics. He was transferred for further rheumtologic evaluation. Initial blood testing yielded only slightly elevated CrP levels but high ferritine >2000ug/l. Bone marrow aspiration showed strong granulopoiesis, no malignant infiltration or MAS. Ultrasound revealed hepatosplenomegaly, moderate serositis and cervical lymphadenopathy. Due to ongoing doubt on the diagnosis of SJIA a second bone marrow aspirate, lymph node and skin biopsy were performed, which were nonspecific. In the course of the disease the general condition deteriorated,CrP levels rose to >200mg/l and ferritine to >12.000ug/l. Liver enzymes were elevated, no cytopenia was seen. We assumed that our patient was at constant risk of full-blown MAS.

    Results: Treatment with Anakinra was started at 2mg/kg/d and raised to 4mg/kg/d but was not effective. High dose methylprednisolonetherapy showed good clinical and laboratory response. On tapering of corticosteroids (CS) however, a rapid relapse resulted and the patient was put on tocilizumab 12mg/kg/2w and ultimately canakinumab 4mg/kg/4w, both of which were not effective. Ciclosporin A (CSA) 5mg/kg/d was added 01/17 due to incomplete control of inflammation and persistent risk of MAS. Further laboratory evaluation was obtained. IL-18 levels were above the detectable level of 20.000pg/ml. Panel diagnostic for autoinflammatory diseases showed a heterozygous mutation of unknown relevance in the PSMB-8 gene, a gene hosting several known mutations responsible for interferonopathies. Interferon signature in our patient showed a very high score, ultimately leading to the hypothesis of type 1 interfonopathy. We started a treatment with baricitinib (BAR) 05/17 and continued CSA and CS. The patient showed a rapid response, BAR was continuously raised to a dose of 4x2mg/day with complete remission. CS were tapered carefully and stopped 08/17. Ultimately CSA was tapered and stopped 09/17. Six weeks after discontinuation of CSA while on monotherapy with BAR 4x2mg/kg, a moderate clinical relapse occured, the interferon signature showing an extremely high score. Thus CSA was restarted and a course of CS was