Open Access

TLR4 rs41426344 increases susceptibility of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) in a central south Chinese Han population

  • Yan Wang1, 2, 3,
  • Lianghui Chen1, 2, 3,
  • Fang Li1, 2,
  • Meihua Bao1, 2,
  • Jie Zeng1, 2,
  • Ju Xiang1, 2,
  • Huaiqing Luo1, 2, 3,
  • Jianming Li1, 4 and
  • Liang Tang1, 2Email author
Pediatric Rheumatology201715:12

DOI: 10.1186/s12969-017-0137-5

Received: 1 November 2016

Accepted: 19 January 2017

Published: 21 February 2017

Abstract

Background

The aim of the study was to determine whether polymorphisms in toll-like receptor 4 (TLR4) confer susceptibility to rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) in a central south Chinese Han population.

Methods

Genotyping for six well studied polymorphisms (rs4986790, rs4986791, rs10759932, rs41426344, rs11536889 and rs7873784) in TLR4 gene were conducted in 1074 unrelated patients with RA and 1692 healthy control subjects, as well as in 217 unrelated patients with JIA and 378 healthy control subjects using direct sequencing technique. Comparisons between cases and controls in alleles, genotypes and haplotypes were carried out using Fisher’s exact test.

Results

Significant genetic associations were detected between the 3’UTR rs41426344C and RA (p < 0.001, p adj < 0.001, OR = 2.24) and JIA (p < 0.001, p adj < 0.001, OR = 2.05). In addition, rs4986790G was found to be significantly associated with the susceptibility for RA (p = 0.005, p adj = 0.03, OR = 3.43), but not for JIA (p = 0.06, p adj = 0.36, OR = 2.65). Furthermore, significant increasing in the distributions of haplotypes H4 and H10 in RA (H4: p = 0.001, OR = 1.13; H10: p = 0.001, OR = 1.15) and JIA (H4: p = 0.04, OR = 2.06; H10: p = 0.02, OR = 2.47) were also found. Moreover, the frequency of rs41426344C significantly increased in RF-positive and anti-CCP positive subjects both in RA (RF+: p <0.0001, OR = 2.33; anti-CCP+: p =0.008, OR = 2.79) and JIA (RF+: p =0.02, OR = 2.91; anti-CCP+: p = 0.02, OR = 2.78).

Conclusions

Our study suggested that rs41426344 and rs4986790 of TLR4 might contribute to RA, and rs41426344 might contribute to JIA pathogenesis in central south Chinese Han population.

Keywords

Genetic association Toll like receptors 4 (TLR4) Rheumatoid arthritis (RA) Juvenile idiopathic arthritis (JIA) Chinese Han population

Background

Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive particular damage caused by inflammatory cells and synoviocytes and was thought to be caused by complex interaction of multiple susceptibility genes and environmental factors [1]. It affects approximately 0.32% Chinese Han population and 1% Caucasian respectively. Juvenile idiopathic arthritis (JIA) refers to a group of chronic childhood arthropathies of unknown aetiology [2]. Chronic arthritis is a common feature of RA and JIA. Familiar and twins studies have provided robust evidence for the role of genetic factors in these diseases [3, 4].

Toll-like receptors (TLRs) play important roles in the recognition of inflammatory diseases caused by invading microorganisms. They have been also increasingly suggested to have important roles in RA and JIA [5, 6]. There are 13 structurally unique members identified in TLRs family. Toll-like receptor 4 (TLR4), one of the important member of TLRs, plays a key role in the process of the innate immune response, and activates the nuclear factor-κB (NF-κB) signaling pathway by binding to lipopolysaccharide (LPS), which was identified to be an important mechanism in the development of rheumatic diseases [711].

The TLR4 gene consisting of three exons is located on chromosome 9q32–33 [12]. Previous studies have reported that some polymorphisms in the TLR4 coding/non-coding region, in particular Asp299Gly polymorphism, are associated with a blunted receptor activity and a subsequently diminished inflammatory response in humans [1316]. Furthermore, variants in the TLR4 were also reported to be associated with lymphoid tissue lymphoma [17], Hodgkin lymphoma [17], cancer [18] and ischemic cerebrovascular disease [19]. Surprisingly, relatively few genetic studies reported significant associations of polymorphisms in TLR4 with RA and JIA susceptibility. Most studies have focused on the correlation between two well known TLR4 polymorphisms (Asp299Gly and Thr399Ile) and RA and JIA, while inconclusive or contradictory results were observed [20, 21]. To our knowledge, only three studies with relatively small sample size have investigated the association between variants in the TLR4 and RA in Chinese Han population [2224], and negative result was also reported [23, 24]. In addition, no research conducted on the association between TLR4 polymorphisms and JIA in central Chinese Han population was found. Thus, the role of TLR4 in RA and JIA in central Chinese Han population remains unclear.

In present study, we aimed to examine the possible associations of TLR4 polymorphisms with auto-antibody levels in RA and JIA susceptibility in a central south Chinese Han population.

Methods

Sample collection

The study was approved by the Ethical Committee at Changsha Medical University (EC/14/013, 06/11/2014). Written, informed consents for genetic analysis were obtained from all subjects or their guardians. A total of 1074 unrelated patients (Female/Male: 842/232; age: 41.7 ± 11.6 years) who met the American College of Rheumatology (The American Rheumatism Association) 1987 revised criteria for RA [25] and 217 unrelated patients (boy/girl: 178/39; age: 6.3 ± 3.1 years) who fulfilled the EULAR JIA criteria were recruited from the first affiliated hospital, Changsha Medical University. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) status were determined for all the patients. The erythrocyte sedimentation rate (ESR) was tested by Westergren method. The auto-antibody levels were detected by Enzyme-linked immunosorbent assay (ELISA). In addition, 1692 unrelated control subjects without the history of RA and 378 unrelated control subjects without the history of JIA (matched for ethnicity, gender and age) for this study were also enrolled. The control subjects were healthy individuals who took the health examination in the first affiliated hospital, Changsha Medical University. All participants were Chinese Han population in origin.

Genotyping

A combination of 6 well-studied informative TLR4 SNPs (Two functional variants [rs4986790 (Asp299Gly) and rs4986791 (Thr399Ile) in exon 3, one variant (rs10759932) in 5’UTR and three variants (rs41426344, rs11536889 and rs7873784) in 3’UTR were genotyped in RA, JIA and healthy controls. Genomic DNA was extracted from peripheral leukocytes using the standard phenol–chloroform method [26]. The multiplex PCR was carried out on the ABI Veriti Thermal Cycler (Applied Biosystems, Foster City, CA). Genotyping was conducted using direct sequencing by the ABI 3730XL DNA Sequencer (Applied Biosystems, Foster City, CA). The PCR primers and sequencing probes were shown in Additional file 1: Table S1.

Statistical analysis

Hardy-Weinberg equilibrium (HWE) was tested in the cases and controls using a classic chi-square test with 1° of freedom. The statistical analysis was performed using SHESIS (http://analysis.bio-x.cn/SHEsisMain.htm). Individual analyses of associations between TLR4 polymorphisms and RA and JIA, as well as clinical features were performed by comparing alleles and genotypes in cases and controls using Fisher’s exact test. The corresponding ORs and 95% confidence intervals (CI) were assessed using a standard logistic regression analysis. Bonferroni correction was applied to adjust the p value (P adj) in multiple comparisons. Analysis of haplotype diversity was performed using the expectation-maximization algorithm (EM). Specific P values and ORs and 95% confidence intervals (CI) were obtained by comparing each haplotype with the more common haplotype in the population using Fisher’s exact test. Statistical significance was set at p < 0.05.

Results

Clinical features such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), IgA, IgG, IgM were shown in Table 1. For JIA, the patients can be classified into five subtypes (systemic JIA, polyarticular (RF+ and RF) JIA, pauciarticular JIA, psoriatic JIA and other JIA). There were 21 (9.6%), 52 (23.7%) (RF+: 15 (6.9%); RF: 37 (16.8%)), 111 (51.2%), 28 (12.9%) and 5 (2.3%) separately for each subtype.
Table 1

Clinical characteristics of RA and JIA patients and heathy controls

Clinical characteristics

RA (Mean ± SD)

Control (Mean ± SD)

p

JIA (Mean ± SD)

Control (Mean ± SD)

p

Sex ratio (Female/Male)

3.63 (842/232)

3.47 (1314/378)

0.17

4.56 (178/39)

4.72 (312/66)

0.89

Age (years)

41.7 ± 11.6

39.6 ± 13.2

0.75

6.3 ± 3.1

6.7 ± 2.5

0.75

Onset age, years

49.5 ± 7.9

7.5 ± 4.9

Bone erosions n (%)

421 (39.1%)

46 (21.3%)

Shared epitope

467 (43.5%)

79 (36.4%)

DAS28

4.7 ± 1.3

3.4 ± 1.1

ESR (mm/h) (0–10 mm/h)

33.2 ± 12.5

4.5 ± 2.2

<0.001

34.5 ± 19.3

4.3 ± 2.4

<0.001

CRP (mg/l) (0.8–8 mg/l)

25.8 ± 12.2

4.9 ± 1.3

<0.001

19.3 ± 37.5

3.77 ± 1.4

<0.001

IgA mg/mL (0.71–3.35 mg/mL)

12.2 ± 2.7

2.88 ± 1.4

<0.001

10.2 ± 2.4

2.59 ± 1.7

<0.001

IgG mg/mL (7.6–16.6 mg/mL)

45.7 ± 5.4

10.3 ± 4.1

<0.001

29.6 ± 5.1

9.7 ± 1.3

<0.001

IgM mg/mL (0.48–2.12 mg/mL)

6.6 ± 1.3

1.78 ± 2.1

<0.001

5.4 ± 1.7

1.88 ± 1.02

<0.001

RF+, %

826 (76.9%)

0 (0%)

 

37 (16.9%)

0 (0%)

 

CCP+, %

766 (71.3%)

0 (0%)

 

33 (15.4%)

0 (0%)

 

Abbreviation: SD Standard Deviation, ESR erythrocyte sedimentation rate, CRP C-reactive protein, RF rheumatoid factor, JIA juvenile idiopathic arthritis, RA Rheumatoid arthritis

Disease activity score 28(DAS28): a score for evaluation of RA activity by assessing the state of 28 joints; anti-CCP: anti-cyclic citrullinated peptide

Single-locus association

All variants in cases and controls were in Hardy-Weinberg equilibrium (HWE) (p > 0.05). Genotype data for the 6 TLR4 SNPs successfully typed in the central south Chinese Han population cases and controls were examined by single-marker analysis (Tables 2 and 3). Genotype analysis showed that the distribution of rs41426344 CC was significantly higher in RA and JIA patients compared with controls, even after the Bonferroni’s correction (RA: p < 0.001, p adj < 0.001, OR [CI95%]: 3.75 [2.51–5.6]; JIA: p = 0.0002, p adj = 0.0006, OR [CI95%]: 4.79 [1.97–11.67]). The frequencies of rs41426344C in RA and JIA were 0.21 and 0.25 separately. Significant associations between rs41426344C and RA and JIA were observed in further allelic analysis (RA: p < 0.001, p adj < 0.001, OR [CI95%]: 2.24 [1.76–2.85]; JIA: p < 0.001, p adj < 0.001, OR [CI95%]: 2.05 [1.52–2.77]).
Table 2

Allele distributions of TLR4 gene polymorphisms in RA, JIA and healthy controls

   

RA

   

JIA

   

SNPs (MAF)

Region

Position

Case (freq.)

Control (freq.)

P

P a adj

OR [95%CI]b

Case (freq.)

Control (freq.)

P

P a adj

OR [95%CI]b

rs10759932(C)

5’UTR

27786349

0.32

0.29

0.37

1.10 [0.89–1.34]

0.26

0.26

0.86

1.03 [0.78–1.34]

rs4986790 (G)

Exon 3

27796507

0.02

0.006

0.005

0.03

3.43 [1.39–8.45]

0.02

0.008

0.06

2.65 [0.93–7.49]

rs4986791 (T)

Exon 3

27796807

0.05

0.04

0.03

0.18

1.75 [1.09–2.82]

0.07

0.05

0.12

1.46 [0.90–2.37]

rs41426344 (C)

3’UTR

27799138

0.21

0.13

<0.001

<0.001

2.24 [1.76–2.85]

0.25

0.14

<0.001

<0.001

2.05 [1.52–2.77]

rs11536889 (C)

3’UTR

27799336

0.22

0.19

0.07

1.24 [0.98–1.56]

0.23

0.19

0.19

1.21 [0.91–1.61]

rs7873784 (C)

3’UTR

27800141

0.15

0.12

0.03

0.18

1.35 [1.03–1.77]

0.13

0.14

0.68

0.93 [0.66–1.31]

Abbreviation: SNP, single nucleotide polymorphism, MAF minor allele frequency, OR odds ratio, 95% CI 95% confidence intervals, not calculated, RA Rheumatoid arthritis, JIA juvenile idiopathic arthritis, Freq frequency; padj, padjusted

aThe Bonferroni’s correction was carried out to adjust the p value

bOR and 95% CI are calculated for the minor allele of each SNP

Table 3

Distribution of the genotypes of TLR4 gene polymorphisms in RA and JIA cases and controls

SNPs

Genotype

Control no.

RA no.

OR [95%CI]

p a

p b adj

Control no.

JIA no.

OR [95%CI]

p a

p b adj

rs10759932 (C)

TT

844

498

0.87 [0.74–1.01]

0.07

204

125

1.16 [0.83–1.62]

0.39

 

CT

696

466

1.09 [0.94–1.28]

0.24

151

73

0.76 [0.54–1.08]

0.13

 

CC

143

108

1.21 [0.93–1.57]

0.15

23

19

1.48 [0.79–2.79]

0.22

rs4986790 (G)

AA

1671

1029

0.28 [0.17–0.49]

<0.001

<0.001

372

208

0.37 [0.13–1.06]

0.06

 

GA

21

45

3.47 [2.06–5.87]

<0.001

<0.001

6

9

2.68 [0.94–7.64]

0.06

 

GG

0

0

0

0

rs4986791 (T)

CC

1590

986

0.72 [0.53–0.96]

0. 03

0.09

399

186

0.58 [0.35–0.97]

0.04

0.12

 

CT

100

85

1.37 [1.01–1.84]

0.04

0.12

39

30

1.65 [0.99–2.73]

0.05

 

TT

2

3

0

1

rs41426344 (C)

GG

1287

615

0.42 [0.36–0.49]

<0.001

<0.001

280

127

0.49 [0.35–0.70]

<0.001

<0.001

 

GC

369

378

1.94 [1.64–2.31]

<0.001

<0.001

91

72

1.57 [1.08–2.26]

0.02

0.06

 

CC

36

81

3.75 [2.51–5.6]

<0.001

<0.001

7

18

4.79 [1.97–11.67]

0.0002

0.0006

rs11536889 (C)

GG

1113

647

0.79 [0.67–0.92]

0.003

0.009

242

133

0.89 [0.63–1.26]

0.51

 

GC

518

372

1.20 [1.02–1.41]

0.03

0.09

125

70

0.96 [0.67–1.37]

0.84

 

CC

60

54

1.44 [0.99–2.09]

0.06

11

14

2.3 [1.02–5.16]

0.04

0.12

rs7873784 (C)

GG

1308

790

0.81 [0.68–0.97]

0.02

0.06

278

165

1.14 [0.78–1.68]

0.50

 

GC

358

268

1.24 [1.03–1.48]

0.02

0.06

96

48

0.83 [0.56–1.23]

0.37

 

CC

25

16

1.01 [0.54–1.89]

1.00

4

4

1.76 [0.43–7.09]

Abbreviation: SNP single nucleotide polymorphism, OR odds ratio, 95% CI 95% confidence intervals; −, not calculated, RA Rheumatoid arthritis, JIA juvenile idiopathic arthritis

a P value were calculated using Fisher’s exact test

bThe Bonferroni’s correction was carried out to adjust the P value

The distribution of the rs4986790GA in RA cases was significantly higher than that in controls (p < 0.001, p adj < 0.001, OR [CI95%]: 3.47 [2.06–5.87]). And allelic analysis of the RA cohort revealed that the frequency of the rs4986790G was significantly higher in patients (2%) compared with controls (0.06%) with an OR equal to 3.43 (p = 0.005, p adj = 0.03, OR [CI95%]: 3.43 [1.39–8.45]), which indicated that G allele in rs4986790 might reveal a strong risk factor for RA in central south Chinese Han population.

No association was detected between other SNPs in the 3’UTR (rs11536889 and rs7873784) and 5’UTR (rs10759932) of the TLR4 gene and RA and JIA (p > 0.05). And no notable association was detected between both genotypes and alleles in rs4986790 and JIA (p > 0.05).

Haplotype analysis

Haplotypes were predicted for 6 SNPs using PLINK 1.09 (http://pngu.mgh.harvard.edu/~purcell/plink/). Ten haplotypes in RA and JIA separately with a frequency > 1% were predicted in both cases and controls accounting for > 90% of all the haplotypes. The haplotype 1(H1) (TACGGG) containing rs10759932T, rs4986790A, rs4986791C, rs41426344G, rs11536889G, rs7873784G was the most common haplotype with a frequency of approximately 43% in RA and 42% in JIA. However, no association was found between H1 and RA and JIA (p > 0.05). Additionally, we observed a marginally significant increase in the distribution of H4 (TGTCCG) and H10 (CGTCCG) in RA compared with that in the controls (H4: p = 0.001, OR [95%CI] = 1.13 [0.77–1.26]; H10: p = 0.001, OR [95%CI] = 1.15 [1.02–1.56]) (Table 4). Similar results were found in H4 and H10 in JIA and controls (TGTCCG: p = 0.04, OR [95%CI] = 2.06[1.01–4.21]; H10: p = 0.02, OR [95%CI] = 2.47[1.11–5.49]) (Table 4).
Table 4

Haplotype analysis of RA and JIA cases and the healthy controls in the TLR4 genes

NO.

haplotypea

RA

OR [95CI%], P b

JIA

OR [95CI%], P b

Control (freq.)

Case (freq.)

Control (freq.)

Case (freq.)

H1

T A C G G G

0.47

0.43

1.03 [0.99–1.17],0.29

0.45

0.42

0.72 [0.52–1.01],0.06

H2

T A C G G C

0.06

0.12

1.07 [0.83–1.33],0.07

0.06

0.10

1.74 [0.95–3.21],0.07

H3

T A C G C G

0.11

0.09

0.54 [0.22–1.11],0.13

0.10

0.07

0.66 [0.36–1.23],0.19

H4

T G T C C G

0.02

0.08

1.13 [0.77–1.26],0.001

0.04

0.08

2.06 [1.01–4.21] 0.04

H5

T A C C C C

0.003

0.01

1.09 [0.76–1.45],0.06

0.003

0.006

1.75 [0.11–2.86],0.39

H6

C A C G G G

0.06

0.06

1.01 [0.99–1.03],0.89

0.04

0.06

1.54 [0.72–3.31],0.26

H7

C A C G C G

0.01

0.02

1.10 [0.98–1.24],0.57

0.02

0.02

0.87 [0.26–2.93],0.78

H8

C A C G C C

0.003

0.001

0.86 [0.63–1.05],0.69

0.002

0.003

1.74 [0.11–2.04],0.54

H9

C A C C G G

0.14

0.11

0.98 [0.97–0.99],0.07

0.15

0.11

0.70 [0.42–1.16],0.17

H10

C G T C C G

0.03

0.13

1.15 [1.02–1.56],0.001

0.03

0.07

2.47 [1.11–5.49],0.02

Abbreviation: SNP single nucleotide polymorphism, OR odds ratio, 95% CI 95% confidence intervals; −, not calculated, RA Rheumatoid arthritis, JIA juvenile idiopathic arthritis, Freq frequency

aHaplotype structure of TLR4 for RA and JIA were rs4986790, rs4986791, rs10759932,rs41426344, rs11536889, rs7873784

b P value were calculated using Fisher’s exact test

Allelic/Genotypic distribution of RF and anti-CCP in RA and JIA

Data were available on autoantibody levels including information on circulating RF and anti-CCP. Carriage of rs41426344C significantly increased in RF-positive (RF+ vs. RF : 0.17 vs. 0.08) and anti-CCP positive (anti-CCP+ vs. anti-CCP : 0.15 vs. 0.06) subjects in RA (RF+: p <0.0001, OR [95%CI] = 2.33 [1.65–3.01]; anti-CCP+: p =0.008, OR [95%CI] = 2.79[1.28–6.11]) and JIA (RF+ vs. RF : 0.19 vs. 0.08; anti-CCP+ vs. anti-CCP : 0.16 vs. 0.05) (RF+: p =0.02, OR [95%CI] = 2.91 [1.11–7.56]; anti-CCP+: p =0.02, OR [95%CI] = 2.78 [1.21–6.74]) (Table 5). Allele and genotype frequencies were not different after stratification by anti-CCP status for rs4986790 that was shown to be associated with RA and JIA in our study (Table 5).
Table 5

Rs4986790 and rs41426344 allele/genotype frequencies and autoantibody levels in patients with RA and JIA

SNPs

Allele/Genotypes

RA

P a ,OR [95%CI]

JIA

P a ,OR [95%CI]

RA

P a ,OR [95%CI]

JIA

P a ,OR [95%CI]

RF+ (freq.)

RF (freq.)

RF+(freq.)

RF (freq.)

Anti-CCP+(freq.)

Anti-CCP(freq.)

Anti-CCP+(freq.)

Anti-CCP(freq.)

rs4986790

G

0.11

0.13

 

0.15

0.14

 

0.25

0.21

 

0.13

0.11

 
 

A

0.89

0.87

0.74,0.94 [0.65–1.36]

0.85

0.86

0.82,1.07 [0.59–1.97]

0.75

0.79

0.55,1.19 [0.66–2.15]

0.87

0.89

0.84,1.26 [0.97–1.93]

 

GG

0.0

0.0

0.0

0.0

0.0

0.0

 

GA

0.33

0.29

0.51,1.17 [0.73–1.86]

0.31

0.30

0.84,1.06 [0.57–1.99]

0.34

0.32

0.75,0.88 [0.42–1.86]

0.29

0.27

0.64,1.22 [0.79–1.88]

 

AA

0.67

0.71

0.92,0.98 [0.63–1.52]

0.69

0.70

0.84,0.94 [0.50–1.76]

0.66

0.68

0.84,0.93 [0.45–1.90]

0.71

0.73

0.79,0.46 [0.22–0.97]

rs41426344

C

0.17

0.08

 

0.19

0.08

 

0.15

0.06

 

0.16

0.05

 
 

G

0.83

0.92

<.0001,2.33 [1.65–3..01]

0.82

0.92

0.02,2.91 [1.11–7.56]

0.85

0.94

0.008,2.79 [1.28–6.11]

0.84

0.95

0.02,2.78 [1.21–6.74]

 

CC

0.04

0.01

0.01,1.45 [0.48–4.26]

0.04

0.015

0.001,3.23 [0.39–26.79]

0.03

0.01

–,2.34 [0.27–20.45]

0.02

0.01

–,2.45 [0.36–18.75]

 

CG

0.30

0.13

<.0001,2.82 [1.90–4.19]

0.30

0.13

0.009,2.76 [1.26–6.05]

0.23

0.09

0.003,3.37 [1.48–9.43]

0.22

0.10

0.002,2.94 [1.39–8.25]

 

GG

0.66

0.86

<.0001,0.37 [0.25–0.53]

0.65

0.85

0.002,0.32 [0.15–0.69]

0.74

0.90

<.0001,0.21 [0.09–0.48]

0.76

0.89

<.0001,0.34 [0.11–0.85]

Abbreviation: SNP single nucleotide polymorphism, OR odds ratio, 95% CI 95% confidence intervals; −, not calculated, RF rheumatoid factor, anti-CCP anti-cyclic citrullinated peptide

a P value were calculated using Fisher’s exact test

Discussion

In the current study, 1074 RA, 217 JIA and 2070 healthy controls were genotyped for six polymorphisms in the TLR4 gene that was previously reported to be associated with autoimmune diseases. The data showed that the frequencies of TLR4 rs4986790G in RA cases, as well as rs41426344C in JIA cases significantly increased than that in the controls, which was, to our knowledge, the first study to demonstrate associations between the two common polymorphisms and RA and JIA in central Chinese Han population using case-control design.

TLRs play important roles in both innate and adaptive immune responses that invading by microorganisms [27]. The chronic inflammation and the well-recognized interactions of TLRs with numerous endogenous ligands have implicated this pathway in a number of disease states including RA and JIA [27, 28]. As a member of TLRs, TLR4 has been considered to recognize not only the LPS component of gram-negative bacteria but also the mouse mammary tumor virus [29, 30]. In particular, TLR4 has been identified as an important part of investigation in understanding arthritides pathogenesis. It has been also demonstrated that TLR4 is over-expressed in RA synovium [31]. Investigations using animal models of inflammatory arthritis also implicate TLR4 in RA. Mice with non-functional TLR4 or mice deficient of MyD88 are protected from inflammatory arthritis [32]. As for JIA, Donn R et al. indicated that the macrophage migration inhibitory factor (MIF) have been reported to be associated with JIA [33]. And a relationship between MIF and TLR4 was found in a study of MIF-deficient mice [34], which supported the hypothesis that TLR4 is a risk factor for investigation in JIA.

The TLR4 Asp299Gly (rs4986790) is a functional allele located in the exon 3 region of TLR4 gene and was known to cause an aspartic acid to glycine replacement, which alter its extracellular domain and potentially modify its binding affinity. The strong association between TLR4 Asp299Gly polymorphism and RA disease susceptibility has been reported in a Dutch cohort [35], but not in Irish, British and Spanish populations [3537]. And no positive results was found between TLR4 Asp299Gly and JIA in UK Caucasian and Indian [5, 38]. In our study, the frequency of Asp299Gly polymorphism in central south Chinese Han population was higher than that in other Chinese Han population populations [23, 39, 40], but was similar with that in Caucasian populations [4144]. And a significant association was detected between TLR4 Asp299Gly and RA in central south Chinese Han population compared to healthy controls. To our knowledge, this is the first study that a significant association between TLR4 Asp299Gly and RA in Chinese Han population was reported. Interestingly, negative result was shown by Zheng [23] and Yuan [24]. The complex genetic ethnic specificity in Chinese Han populations might contribute to the difference.

Notable, the rs41426344 appeared to be significantly associated with both RA and JIA in central south Chinese Han population. Both rs41426344C allele and CC genotype are increased in RA cases, which was similar with the result reported by Zheng [22]. There were already evidences suggesting that the rs41426344 may act as susceptibility loci with diseases [44]. Cheng et al. suggested that rs41426344 may be a functional site, which could attenuate the LPS-induced transmembrane signaling through the alteration of post-transcriptional regulation of 3’UTR and target gene expression [41]. In addition, no significant association was found between other two 3’UTR SNPs (rs11536889 and rs7873784) and RA and JIA. Though the absence of association in these two loci was detected in our present study, we cannot exclude the possible effect of these two SNPs on RA and JIA development in other populations for genetic polymorphisms often vary between ethnic groups. Thus, replication in other populations is needed before these results can be generalized.

Conclusions

We observed significant associations between RA and JIA disease susceptibility and a TLR4 variant (rs41426344) in a set of RA and JIA patients, as well as rs4986790 in RA patients and healthy individuals in central south Chinese Han population. Our finding needs to be confirmed in other larger numbers of Chinese Han population cohorts. And to identify the potential mechanisms by which variant in rs41426344 and rs4986790 affects TLR4 and RA and JIA is necessary.

Declarations

Acknowledgements

We are grateful to the patients and control individuals for participating in this study. In addition, we should thanks Dr. Bifeng Chen from Chinese University of Hongkong and Peter R. Patrylo, Ph. D from Departments of Physiology and Anatomy Southern Illinois University School of Medicine for Language polishing.

Funding

This study was supported in part by the Foundation of the Education Department of Hunan (11C0141, 15C0513, 16C0162), the key Foundation of the Education Department of Hunan (15A023,16A027), National Science Foundation of Hunan province (2015JJ6010), Foundation of the health department of Hunan (B2016096) and the construct program of the key discipline in Hunan province.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Authors’ contributions

LT and JML designed the experiments and drafted the manuscript. YW, LHC, FL and JZ collected the samples and carried out the genotyping. MHB and JX contributed to the statistical analysis. JML and HQ L are project leader and planned the study. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Consents to publish have obtained from all subjects or their guardians.

Ethics approval and consent to participate

The study was approved by the Ethical Committee at the Changsha Medical University (EC/14/013, 06/11/2014). Written informed consents for genetic analysis were obtained from all subjects or their guardians.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Human Anatomy, Histology and Embryology, Institute of Neuroscience, Changsha Medical University
(2)
School of Basic Medical Science, Changsha Medical University
(3)
Experiment center for Function, Changsha Medical University
(4)
Department of Neurology, Xiang-ya Hospital, Central South University

References

  1. Davidson A, Diamond B. Autoimmune diseases. N Engl J Med. 2001;345:340–50.View ArticlePubMedGoogle Scholar
  2. Goulielmos GN, Chiaroni-Clarke RC, Dimopoulou DG, Zervou MI, Trachana M, Pratsidou-Gertsi P, Garyfallos A, Ellis JA. Association of juvenile idiopathic arthritis with PTPN22 rs2476601 is specific to females in a Greek population. Pediatr Rheumatol. 2016;14:25.View ArticleGoogle Scholar
  3. Kirchner M, Sonnenschein A, Schoofs S, Schmidtke P, Umlauf VN, Mannhardt-Laakmann W. Surface expression and genotypes of Toll-like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus. Pediatr Rheumatol. 2013;11:9.View ArticleGoogle Scholar
  4. Malievsky VA, Viktorova TV, Danilko KV, Nazarova LS. PReS-FINAL-2113: The association of the NFKB1 gene polymorphism with systemic onset juvenile idiopathic arthritis in Russia. Pediatr Rheumatol. 2013;11 Suppl 2:125.View ArticleGoogle Scholar
  5. Lamb R, Zeggini E, Thomson W, Donn R. Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis. Ann Rheum Dis. 2005;64:767–9.View ArticlePubMedGoogle Scholar
  6. Kuuliala K, Orpana A, Leirisalo-Repo M, Kautiainen H, Hurme M, Hannonen P, Korpela M, Mo¨tto¨nen T, Paimela L, Puolakka K, Karjalainen A, Repo H. Polymorphism at position +896 of the toll-like receptor 4 gene interferes with rapid response to treatment in rheumatoid arthritis. Ann Rheum Dis. 2006;65:1241–3.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Ospelt C, Brentano F, Rengel Y, Stanczyk J, Kolling C, Tak PP, Gay RE, Gay S, Kyburz D. Overexpression of Toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis. Toll-like receptor expression in early and longstanding arthritis. Arthritis Rheum. 2008;58:3684–92.View ArticlePubMedGoogle Scholar
  8. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001;2:675–80.View ArticlePubMedGoogle Scholar
  9. Schröder NW, Schumann RR. Single nucleotide polymorphisms of toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis. 2005;5:156–64.View ArticlePubMedGoogle Scholar
  10. Candore G, Colonna-Romano G, Balistreri CR, Di CD, Grimaldi MP. Biology of longevity: role of the innate immune system. Rejuvenation Res. 2006;9:143–8.View ArticlePubMedGoogle Scholar
  11. Turner MW, Hamvas RM. Mannose-binding lectin: structure, function, genetics and disease associations. Rev Immunogenet. 2000;2:305–22.PubMedGoogle Scholar
  12. Buraczynska M, Baranowicz-Gaszczyk I, Ksiazek A. Toll-like receptor 4 gene polymorphism and early onset of diabetic retinopathy in patients with type 2 diabetes. Hum Immunol. 2009;70:121–4.View ArticlePubMedGoogle Scholar
  13. Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet. 2000;25:187–91.View ArticlePubMedGoogle Scholar
  14. Hemming NV, Blanco JCG, Segal DM, Medvedev SA, Lorenz E, Arditi M, Rallabhandi VGP, Bell J, Boukhvalova MS. Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. J Immunol. 2006;177:322–32.View ArticlePubMedGoogle Scholar
  15. Kinane DF, Shiba H, Stathopoulou PG, Zhao H, Lappin DF, Singh A, Eskan MA, Beckers S, Waigel S, Alpert B, Knudsen TB. Gingival epithelial cells heterozygous for toll-like receptor 4 polymorphisms Asp299Gly and Thr399ile are hypo-responsive to Porphyromonas gingivalis. Genes Immun. 2006;7:190–200.View ArticlePubMedGoogle Scholar
  16. Montes AH, Asensi V, Alvarez V, Valle E, Ocaña MG, Meana A, Carton JA, Paz J, Fierer J, Celada A. The toll-like receptor (Asp299Gly) polymorphism is a risk factor for gram-negative and haematogenous osteomyelitis. Clin Exp Immunol. 2006;143:404–13.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Nuolivirta K, Hurme M, Halkosalo A, Koponen P, Korpp M. Gene polymorphism of IFNG+ 874 T/A and TLR4 + 896A/G and recurrent infections and wheezing in toddlers with history of bronchiolitis. Pediatr Infect Dis J. 2009;28:1121–3.View ArticlePubMedGoogle Scholar
  18. Zhang K, Zhou B, Wang Y, Rao L, Zhang L. The TLR4 gene polymorphisms and susceptibility to cancer: a systematic review and meta-analysis. Eur J Cancer. 2013;49:946–54.View ArticlePubMedGoogle Scholar
  19. Yin YW, Li JC, Li BH. Toll-like receptor 4 gene Asp299Gly polymorphism in ischemic cerebrovascular disease: a meta-analysis. Int J Neurosci. 2014;124:252–60.View ArticlePubMedGoogle Scholar
  20. Xu WD, Liu SS, Pan HF, Ye DQ. Lack of association of TLR4 polymorphisms with susceptibility to rheumatoid arthritis and ankylosing spondylitis: A meta-analysis. Joint Bone Spine. 2012;79:566–9.View ArticlePubMedGoogle Scholar
  21. Tizaoui K, Naouali A, Kaabachi W, Hamzaoui A, Hamzaoui K. Association of Toll like receptor Asp299Gly with rheumatoid arthritis risk: A systematic review of case–control studies and meta-analysis. Pathol Res Pract. 2015;211:219–25.View ArticlePubMedGoogle Scholar
  22. Yang HJ, Wei CY, Li Q, Shou T, Yang Y, Xiao CJ, Yu M, Li M, Yang ZL, Zhang JY, Zheng BR. Association of TLR4 gene non-missense single nucleotide polymorphisms with rheumatoid arthritis in Chinese Han population. Rheumatol Int. 2013;33:1283–8.View ArticlePubMedGoogle Scholar
  23. Zheng BR, Li Q, Wei CY, Qin J, Shou T, Zhou R, Shao J, Yang Y, Xiao C. Lack of association of TLR4 gene Asp299Gly and Thr399Ile polymorphisms with rheumatoid arthritis in Chinese Han population of Yunnan Province. Rheumatol Int. 2010;30:1249–52.View ArticlePubMedGoogle Scholar
  24. Yuan M, Xia J, Ma L, Xiao B, Yang Q. Lack of the toll-like receptor 4 gene polymorphisms Asp299Gly and Thr399ile in a Chinese population. Int J Neurosci. 2010;120(6):415–20.View ArticlePubMedGoogle Scholar
  25. Arnett FC: The American Rheumatism Association. revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1987;1988(31):315–24.Google Scholar
  26. Di PF, Ortenzi F, Tilio M, Concetti F, Napolioni V. Genomic dna extraction from whole blood stored from 15- to 30-years at −20 °c by rapid phenol-chloroform protocol: a useful tool for genetic epidemiology studies. Mol Cell Probes. 2010;25:44–8.Google Scholar
  27. Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature. 2004;430:257–63.View ArticlePubMedGoogle Scholar
  28. Foell D, Wittkowski H, Roth J. Mechanisms of disease: a “DAMP” view of inflammatory arthritis. Nat Clin Pract Rheumatol. 2007;3:382–90.View ArticlePubMedGoogle Scholar
  29. Erridge C. Endogenous ligands of TLR2 and TLR4: agonists or assistants? J Leukoc Biol. 2010;87:989–99.View ArticlePubMedGoogle Scholar
  30. Dhaouadi T, Sfar I, Haouami Y, Abdelmoula L, Turki S, Hassine LB. Polymorphisms of toll-like receptor-4 and cd14 in systemic lupus erythematosus and rheumatoid arthritis. Biomarker Res. 2013;1:20.View ArticleGoogle Scholar
  31. Sanchez-Pernaute O, Filkova M, Gabucio A. Citrullination enhances the pro-inflammatory response to fibrin in rheumatoid arthritis synovial fibroblasts. Ann Rheum Dis. 2013;72:1400–6.View ArticlePubMedGoogle Scholar
  32. van den Berg WB, van Lent PL, Joosten LA, Abdollahi-Roodsaz S, Koenders MI. Amplifying elements of arthritis and joint destruction. Ann Rheum Dis. 2007;66 Suppl 3:iii45–8.PubMedPubMed CentralGoogle Scholar
  33. Donn R, Alourfi Z, Zeggini E, Lamb R, Jury F, Lunt M, the British Paediatric Rheumatology Study Group, Meazza C, De Benedetti F, Thomson W, Ray D. A functional promoter haplotype of macrophage migration inhibitory factor (MIF) is linked and associated with juvenile idiopathic arthritis. Arthritis Rheum. 2004;50:1604–10.View ArticlePubMedGoogle Scholar
  34. Roger T, David J, Glauser MP, Calandra T. MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature. 2001;414:920–4.View ArticlePubMedGoogle Scholar
  35. Kilding R, Akil M, Till S, Amos R, Winfield J, Iles MM, Wilson AG. A biologically important single nucleotide polymorphism within the toll-like receptor-4 gene is not associated with rheumatoid arthritis. Clin Exp Rheumatol. 2003;21:340–2.PubMedGoogle Scholar
  36. Sánchez E, Orozco G, López-Nevot MÁ, Jiménez-Alonso J, Martín J. Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus. Tissue Antigens. 2004;63:54–7.View ArticlePubMedGoogle Scholar
  37. Sheedy F, Marinou IL, Wilson A. The Mal/TIRAP S180L and TLR4 G299D polymorphisms are not associated with susceptibility to or severity of rheumatoid arthritis. Ann Rheum Dis. 2008;67:1328–31.View ArticlePubMedGoogle Scholar
  38. Myles A, Aggarwal A. Lack of association of single nucleotide polymorphisms in toll-like receptors 2 and 4 with enthesitis-related arthritis category of juvenile idiopathic arthritis in Indian population. Rheumatol Int. 2013;33:417–21.View ArticlePubMedGoogle Scholar
  39. Guo QS, Xia B, Jiang Y, Morre SA, Cheng L, Li J, Crusius JBA, Pen˜a AS. Polymorphisms of CD14 gene and TLR4 gene are not associated with ulcerative colitis in Chinese patients. Postgrad Med J. 2005;81:526–9.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Cheng L, Eng HL, Chou MH, You, Lin TM. Genetic polymorphisms of viral infection associated Toll-like receptors in Chinese population. Transl Res. 2007;150:311–8.View ArticlePubMedGoogle Scholar
  41. Radstake TRDJ, Franke B, Hanssen S, Netea MG, Welsing P, Barrera P, Joosten LAB, van Riel PLCM, van den Berg WB. The Toll-like receptor 4 Asp299Gly functional variant is associated with decreased rheumatoid arthritis disease susceptibility but does not influence disease severity and/or outcome. Arthritis Rheum. 2004;50:999–1001.View ArticlePubMedGoogle Scholar
  42. Adam R, Sturrock RD, Gracie JA. TLR4 mutations (Asp299Gly and Thr399Ile) are not associated with ankylosing spondylitis. Ann Rheum Dis. 2006;65:1099–101.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Paulus SC, Hirschfeld AF, Victor RE, Brunstein J, Thomas E, Turvey SE. Common human toll-like receptor 4 polymorphisms-role in susceptibility to respiratory syncytial virus infection and functional immunological relevance. Clin Immunol. 2007;123:252–7.View ArticlePubMedGoogle Scholar
  44. Hishida A, Matsuo K, Goto Y, Mitsuda Y, Hiraki A. Toll-like receptor 4 + 3725 G/C polymorphism, Helicobacter pylori seropositivity, and the risk of gastric atrophy and gastric cancer in Japanese. Helicobacter. 2009;14:47–53.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2017

Advertisement