Systemic inflammation and chronic kidney injury in a patient due to RNASEH2B defect


 Background

Aicardi-Goutières (AGS) is a rare immune dysregulated disease due to mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1 or IFIH1. Severe systemic inflammation is not typically persistent in AGS. Chronic kidney injury (CKI) has been never reported in this syndrome. Herein, we report a patient presenting with systemic inflammation and CKI to broaden the clinical phenotype spectrum of RNASEH2B defect.
Methods

Clinical data extracted included medical history, clinical manifestations, laboratory results, radiological findings, management, and prognosis. Whole exome sequencing was performed on whole peripheral blood cells. After exposure to cGAMP in vitro for 24 hours, mRNA expression of twelve IFN-stimulated cytokine genes in PBMCs was assessed. Serum cytokine levels were detected by Milliplex.
Results

A 11-year-old female patient presented with recurrent aseptic fever, arthritis, chilblains, failure to thrive, mild hearing loss, and neurological manifestations. Laboratory and immunologic findings revealed lymphopenia, low complement levels, positive autoantibodies, elevated levels of acute phase reactants and inflammatory cytokines. Renal biopsy showed glomerular sclerosis in three of fourteen glomeruli, infiltration of lymphocytes and other mononuclear cells. Whole exome sequencing (WES) revealed a homozygous and heterozygous mutations in RNASEH2B.Over-expression of IFN-stimulated cytokine genes was observed in the patient, including IFI44, IFI27, IFIT1, IFIT2, IFIT3, ISG15, OAS1, and SIGLEC1.
Conclusions

Systemic autoinflammation and chronic renal injury may expand the clinical phenotype spectrum of RNASEH2B defect.
Trial registration:
 Not applicable; this was a retrospective study.


Subjects
This study was approved by the Ethics Committee of Shenzhen Children's hospital. All human subjects (or their guardians) provided written informed consent. Clinical data of a patient with both homozygous and heterozygous variants in RNASEH2B was collected. Fifteen healthy volunteers were included as healthy controls. Venous blood (3 mL) was collected from each study subject.

Whole Exome Sequencing (WES)
Genomic DNA was extracted from peripheral blood cells isolated from the patient and her parent. The exonic regions and anking splicing or intronic junctions of the whole genome were captured and sequenced using an Illumina HiSeq 2000 sequencer conducted by MyGenostics (Beijing, China). The FASTQ les were mapped to the human reference genome (hg19). The functional effects of variants were predicted using three algorithms (PolyPhen-2, SIFT, and MutationTaster), and amino acid conservation among species was analyzed. Sanger sequencing was used to con rm pathogenic variants. The primers used to target human RNASEH2B included (forward: CAGGGATTTGAAGCTCTTTGG) and (reverse: TAGTGCTCTGTCCTGCACTGG).

Real-time PCR
After exposure to cGAMP in vitro for 24 hours, mRNA expression of twelve IFN-stimulated cytokine genes in PBMCs was assessed. Total RNA was extracted from PBMCs isolated from the patient and ve HCs by RNA isolation kit(DP424, TIANGEN). cDNA was derived following the GoScript Reverse Transcription System kit(A5001, Promega). Quantitative reverse transcription PCR analysis was performed with the GoTaq qPCR Master Mix (A6002, Promega). Primers for PCR included were described in the supplementary material.

Quanti cation Of Cytokine Levels
Plasma samples were isolated from the patient and fteen HCs. Cerebrospinal uid (CSF) sample was collected from the patient. Blood samples were collected in vacutainers containing sodium heparin. Plasma cytokine analyses were determined on a bead-based immunoassay (Milliplex, HCYTOMAG-60K, Millipore, USA) according to the manufacturer's protocol.

Statistical analysis
Data were analyzed using an unpaired two-tailed Student t-test. All statistical analyses were conducted in GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA).

Clinical manifestations
The patient presented with recurrent fever, arthritis, movement limitation, and growth retardation at the age of 11 years. At the age of 2 years, she began to suffer from recurrent aseptic fever with an intermittent resolution by traditional Chinese medicine. At the age of 5 years, she began to present with arthritis accompanied by mild hearing loss. She was born to a non-consanguineous healthy parent. At birth, her weight was 3 Kg, crown to heel length was 49 cm, and head circumference was 34 cm. She had standard motor and language development. Manifestations of failure to thrive had been signi cant since she was three years old. Physical examination revealed short stature with 106 cm top(<-3SD) (Fig. 1B3), macrocephaly with 54 cm head width, chilblains on elbows and lower limbs (Fig. 1B2), swelling and deformation of inter-phalangeal and knee joints (Fig. 1B1). Her Intelligence Quotient (IQ) test value was 108. Her EPQJ, CBCL, Conners, and HAMA scale tests did not demonstrate any social and psychological problems. Knee magnetic resonance imaging (MRI) revealed a thickness of the synovial capsule without invasive bone destruction (Fig. 1B4). Cerebral MRI showed cerebral atrophy and white matter abnormalities (Fig. 1B6). Intracranial calci cation was further identi ed at the basal ganglia and cerebellum by CT scanning (Fig. 1B5 and Fig. 1B7). Laboratory ndings revealed hyper-in ammation and chronic kidney insu ciency ( Fig. 2B and Fig. 2F ). Screening tests for fungal, bacteria and Mycobacterium tuberculosis infection were all negative. Pathology of the renal biopsy showed glomerular sclerosis in three of fourteen glomeruli, a mild proliferation of mesangial cells without deposits of any amyloid, immunoglobulin or immune complex, expansion of the tubular lumen, partial tubular atrophy, mild tubular brosis, in ltration of lymphocytes and other mononuclear cells (Fig. 1C). Granule degeneration and calcium deposition were visible in renal tubules.

Abnormality In Clinical Immunologic Phenotype
Analysis of peripheral blood leukocyte revealed persistent lymphopenia with normal subset ratios ( Fig. 2A). Except for rheumatoid factor (RF) and anti-cyclic peptide containing citrulline (anti-CCP), other auto-antibodies including anti-nuclear antibody (ANA) and anti-neutrophil cytoplasmic antibody (ANCA) were all negative. Other abnormal clinical immunologic phenotypes included intermediate elevation of IgM and IgA levels (Fig. 2C)and reduction of C3 and C4 levels (Fig. 2D).

Over-expression Of IFN-stimulated Cytokine Genes
After exposure to cGAMP in vitro for 24 hours, mRNA expression of IFN-stimulated cytokine genes in PBMCs was detected by real-time PCR. In contrast to ve healthy controls, over-expression of IFNstimulated cytokine genes was observed in the patient, including IFI44, IFI27, IFIT1, IFIT2, IFIT3, ISG15, OAS1, and SIGLEC1. Normal mRNA expressions were found in IFNβ1 and IRF9 (Fig. 3B).

Treatment And Outcome
A one-year course of growth hormone showed no response to improve her short stature. She had received long-term treatment of ibuprofen, methotrexate, folic acid, and prednisone for more than ve years.
Aseptic fever relapsed intermittently. Tocilizumab was started for the high dose dependence of glucocorticoids and elevated pro-in ammatory cytokine levels, including IL-6. Following a 48-week course of tocilizumab, the prednisone dose was gradually reduced to 0.2 0.3 mg/Kg.d, and some abnormal laboratory ndings had been improved( Fig. 2B and Fig. 2F). While urine β2 microglobulin level sustained elevated signi cantly (Fig. 2E). Tocilizumab was discontinued. She began to receive tofacitinib for the over-expression of IFN-stimulated cytokine genes. The 12-week course of tofacitinib led to partial improvement of lymphocytes, C3 and C4 levels ( Fig. 2A and Fig. 2D), failing to improve chronic renal injury. This patient has demonstrated a later onset of AGS with average intelligence, presenting with chilblains, cerebral atrophy, white matter abnormalities, intracranial calci cation, and over-expression of Interferonstimulated genes. Besides, this patient has exhibited persistent systemic in ammation and chronic renal dysfunction, which are uncommon in AGS (Table 2). Systemic juvenile idiopathic arthritis (SoJIA), and later onset chronic infantile neurologic, cutaneous, and arthritis (CINCA) syndrome were once suspected. Different from the clinical manifestations in this patient, chilblains and intracranial calci cation are not present in SoJIA or CINCA; leukocytosis, destructive arthritis, or macrophage activation syndrome (MAS) are noted in SoJIA [3][4][5]; visual impairment, sensor neural deafness or progressive chronic meningitis have been commonly reported in CINCA [4]. Chronic renal dysfunction due to amyloidosis has been rarely reported in SoJIA, which is common in CINCA (Table 2) [7]. Renal biopsy in this patient revealed glomerular sclerosis and tubular injury without amyloidosis. Human IFN-alpha is ltrated by the kidney, primarily reabsorbed, most probably catabolized within the tubular epithelium, and excreted in negligible amounts with the urine [8]. Hence, a fairly high IFN-a level within the tubular epithelium due to a persistently elevated IFN-a level in plasma may amplify the activation of the interferon pathway, leading to the in ltration of lymphocytes and mononuclear cells, and local chronic in ammation. Further investigations will help to explore the distinct pathogenesis underlying chronic renal dysfunction in RNASEH2B defect. IL-6 is one of the downstream effector cytokines in the IFN signaling pathway. IL-6 blockade has good e cacy in a patient with a cerebral vasculopathy due to a homozygous SAMHD1 mutation. His cerebral vasculopathy was mostly reversed after tocilizumab treatment [9]. Tocilizumab has partial e cacy in this patient, leading to a reduction of acute-phase reactants. While it has failed to improve chronic renal tubular injury. Further structural clinical trials are required to clarify the e cacy of tocilizumab in AGS.
On the other hand, ruxolitinib failed to prevent the onset of clinical signs in a patient with RNASEH2B mutation [18]. Tofacitinib only demonstrated a partial response in this patient, failing to reduce autoin ammation and ameliorate chronic renal injury. Hence, based on limited case reports, the e cacy of JAK inhibitors in AGS remains uncertain. The currently ongoing trial conducted at the Children's Hospital of Philadelphia (ClinicalTrials.gov number, NCT03921554) will help to explore the e cacy and safety of baricitinib in AGS and AGS-related interferonopathies.

Conclusions
We have described a patient with both homozygous and heterozygous variants in RNASEH2B, revealing a synergistic pathogenic effect among variants in the same gene. Her systemic autoin ammation and chronic renal injury will expand the clinical phenotype spectrum of this syndrome. The pathogenesis underlying chronic renal dysfunction in this patient remains poorly understood. The e cacy of tocilizumab and JAK inhibitors in AGS remains uncertain, and further clinical researches are needed.

Availability of data and materials
Clinical datasets were collected from medical records of the participated patient in Shenzhen Children's hospital.
Ethics approval and consent to participate All participated family members were enrolled upon approval of the ethics committee of Shenzhen Children's hospital and written consent of all the families.

Consent for publication
Written consent for publication of this anonymous information was obtained from the patient's parents.

Con ict of Interests
All authors declare no con ict of interest.