Lack of association of TLR4 polymorphisms with susceptibility to rheumatoid arthritis and ankylosing spondylitis: A meta-analysis

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Lack of association of TLR4 polymorphisms with susceptibility to rheumatoid arthritis and ankylosing spondylitis: A meta-analysis Wang-Dong Xu 1 , Shan-Shan Liu 1 , Hai-Feng Pan , Dong-Qing Ye ∗ Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, 81, Meishan Road, Hefei, 230032 Anhui, PR China

a r t i c l e

i n f o

Article history: Accepted 10 February 2012 Available online xxx Keywords: Rheumatoid arthritis Ankylosing spondylitis Toll-like receptor 4 Polymorphism Meta-analysis

a b s t r a c t Objective: The aim of the study was to determine whether polymorphisms of toll-like receptor 4 (TLR4) confer susceptibility to rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Methods: A meta-analysis was conducted on the association between the D299G/T399I polymorphisms and RA/AS (when available) using (1) the allelic contrast, (2) the recessive, (3) the dominant, and (4) the additive models. Results: A total of eleven relevant studies met the inclusion criteria were identified, including RA: D299G (six studies), T399I (three studies); AS: D299G (five studies), T399I (four studies). Meta-analysis was performed with fixed/random effect models. Overall, no significant RA/AS risk was found in all genetic models when all studies were pooled into the meta-analysis (P > 0.05). Conclusions: The present study might suggest that TLR4 D299G/T399I polymorphisms are not associated with RA/AS susceptibility. © 2012 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.

1 Introduction Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation in synovial joints. It ranges in severity from mild arthritis to severe systemic disease affecting internal organs [1]. Human leukocyte antigen (HLA), the major genetic susceptibility locus, has been demonstrated with susceptibility to RA, but HLA is considered to contribute less than one-third of the total genetic susceptibility component [2–4], and other non-HLA genes might contribute to RA susceptibility. Ankylosing spondylitis (AS) is a chronic autoimmune disease, characterized by pain and stiffness in the spine and sacroiliac joints [5,6]. Although the etiologies of RA and AS have not been fully unraveled, epidemiological evidence suggests that both of them are likely due to a genetic component. Toll-like receptors (TLRs) are a phylogenetically conserved receptors family, mainly expressed on dendritic cells and macrophages that recognize pathogen-associated molecular patterns and play an important role in both innate and adaptive immune responses [7,8]. TLR4, the first human TLR among the now expanding members of the TLR family, has been recognized as the receptor for Gram-negative lipopolysaccharide (LPS) as well as some endogenous ligands, such as fibronectin [9], fragments of hyaluronic acid [10] and heatshock proteins (HSPs) [11–13].

∗ Corresponding author. Tel.: +86 551 5167726; fax: +86 551 5161171. E-mail address: [email protected] (D.-Q. Ye). 1 Wang-Dong and Shan-Shan Liu contributed equally to this work and should be considered co-first authors.

There are two functional variants present in a putative coreceptorbinding region of TLR4 in linkage disequilibrium, an adenine for guanine substitution at 896 nucleotides from the transcription start site of TLR4 cDNA (+896) causes an amino acid (aa) change at aa 299 of aspartic acid to glycine P (D299G: rs4986790). This mis-sense mutation within the fourth exon alters the extracellular domain of the TLR4 receptor interrupting TLR4 mediated signaling leading to endotoxin hyporesponsiveness. Another mis-sense mutation replacing threonine with isoleucine at aa 399 (T399I: rs4986791) in the TLR4 gene has been described that reduces the response of this receptor to stimulation with LPS and probably endogenous ligands [14,15] (Table 1). Evidence has suggest that TLR4 may play a role in the pathogenesis of autoimmune diseases such as RA, in which TLR4 was highly expressed in the invading pannus of RA [16] and stimulation of synovial fibroblasts from patients with RA with TLR4 ligands could enhance the synthesis of the proinflammatory cytokines interleukin (IL)-15, IL-17 [17]. Similarly, the expression of TLR4 by peripheral blood cells is upregulated in patients with AS and associates with the levels of several inflammatory markers [18]. Therefore, aberrant functions of TLR4 involved in host defense may induce the loss of self-tolerance and triggering of autoimmune diseases. Recently, a number of case-control studies have been conducted to investigate the association of these two polymorphisms with RA/AS. However, studies have shown inconclusive or contradictory results. This inconsistency may attribute to studies with small sample size, inadequate statistical power, racial and ethnic differences, or uncorrected multiple hypothesis testing. Meta-analysis is a means of augmenting the effective sample size through the pooling of data from individual association

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Please cite this article in press as: Xu W-D, et al. Lack of association of TLR4 polymorphisms with susceptibility to rheumatoid arthritis and ankylosing spondylitis: A meta-analysis. Joint Bone Spine (2012), doi:10.1016/j.jbspin.2012.02.012

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Table 1 Brief information of the polymorphisms of TLR4 included in the analysis. mRNA D299G

Asp299Gly: rs4986790

T399I

Thr399Ile: rs4986791

Accession NM 138554.3 mRNA Accession NM 138554.3

Position 1187

Allele change GAT→GGT

Position 1487

Allele change ACC→ATC

studies and enhancing the statistical power of the analysis for the estimation of genetic effects [19]. In this study, a comprehensive meta-analysis was performed to assess the importance of TLR4 D299G/T399I polymorphisms for RA/AS susceptibility. 2 Methods 2.1 Identification of eligible studies and data extraction All of the studies discussing the relationship between TLR4 D299G/T399I and RA/AS were fully considered and carefully selected in November 2011. A search of the literature was made using MedLine and PubMed to identify available articles, references in the studies were reviewed to find additional studies not indexed by MedLine or PubMed, with the text words “toll-like receptor 4”, “TLR4”, “rheumatoid arthritis”, “RA”, “ankylosing spondylitis”, “AS” and “polymorphism”. We only recruited data from the full-published paper, not any conference abstract or meeting. In addition, a study was a case-control study and published up to November 25, 2011. We excluded the followings: • studies that contained overlapping data; • studies in which family member had been studied because of the analysis based on linkage considerations; • genotype distribution of the control population is not in HardyWeinberg equilibrium (HWE). Each study had been extracted as followings: author, year of publication, ethnicity of the study population, numbers of cases and controls, and genotype and allele frequency information for TLR4 polymorphisms. 2.2. Meta-analysis methods This meta-analysis was performed with fixed/random effect models on: (1) allelic contrast, on (2) recessive, (3) dominant, and

(4) additive models. Point estimates of risk, odds ratio (OR), and 95% confidence interval (95% CI) were estimated for each study. The between-study heterogeneity was evaluated through the Chi-square test-based Q-statistic [20]. If a significant Q-statistic (P <0.10) indicated heterogeneity across studies, the random effect model was selected. In addition, another measure, I2 = 100% × (Qdf)/Q was used to quantify the effect of heterogeneity [21]. The I2 -statistic measures the degree of inconsistency in the studies by computing what percentage of the total variation across studies was due to heterogeneity rather than by chance. The overall or pooled estimate of risk (OR) was obtained by a fixed effect model (Mantel-Haenszel) or a random effect model (DerSimonian and Laird) [22,23]. Pooled OR in the metaanalysis was performed weighting individual OR by the inverse of their variance. The significance of the pooled OR was determined by the Z-test. Allele frequency and genotype frequency at the TLR4 D299G/T399I polymorphisms from the respective study were determined by the allele counting method. 2.3. Evaluation of publication bias An estimate of potential publication bias was investigated with the funnel plot, where the standard error of logOR of each study was plotted against its OR and funnel plot asymmetry was further assessed by Egger’s test. Analyses were performed via the software Review Manager 5.0 (Cochrane Collaboration, http:www.cc-ims.net/RevMan/relnotes.htm) and Stata version 11 (Stata Corp LP, College Station, TX, USA). All the P values were two sided, a P value < 0.05 was considered statistically significant. 3. Results 3.1. Studies included in the meta-analysis Characteristics of studies investigating the association of TLR4 D299G/T399I polymorphisms with RA/AS were present in Table 2. A total of eleven relevant studies met the inclusion criteria were identified, including RA: D299G (six studies), T399I (three studies); AS: D299G (five studies), T399I (four studies) [5,24–33]. Because of not enough samples to ethnicity-specific, stratified analysis based on ethnicity was not done for allele and genotype frequency calculating. The distribution of the genotype in the control

Table 2 Characteristics of studies investigating the association of TLR4 polymorphisms with RA/AS. ID

Study

Year

Ethnic group

Disease

1

Alvarez-Rodriguez

2011

Spain

RA

2

Emonts

2011

Dutch

RA

3 4 5

Jaen Radstake Sanchez

2009 2004 2003

France Dutch Spain

RA RA RA

6 7

Sheedy Adam

2008 2006

UK UK

RA AS

8

Jr

2006

Hungary

AS

9

Na

2008

Korea

AS

10 11

Paardt Snelgrove

2005 2007

Dutch Canada

AS AS

Polymorphism

rs4986790 rs4986791 rs4986790 rs4986791 rs4986790 rs4986790 rs4986790 rs4986791 rs4986790 rs4986790 rs4986791 rs4986790 rs4986791 rs4986790 rs4986791 rs4986790 rs4986790 rs4986791

Sample size Case

Control

93 93 370 370 100 282 224 224 937 193 184 138 138 200 200 113 100 101

126 126 432 434 100 314 199 199 965 125 113 140 140 197 197 170 98 100

Frequencies of alleles

Frequencies of genotypes

HWE(P)

Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available

Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available Available

1.000 0.594 0.831 1.000 0.827 0.325 0.815 0.812 0.998 0.984 1.000 0.594 0.555 NA NA 0.597 0.756 1.000

RA: rheumatoid arthritis; AS: ankylosing spondylitis; HWE: Hardy–Weinberg equilibrium of genotypes of controls; NA: none available.

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Table 3 Meta-analysis of TLR4 polymorphisms in RA/AS. Diseases

RA (D299G)

(T399I)

AS (D299G)

(T399I)

Comparison

A AA AA AA + AG C CC CC CC + CT A AA AA AA + AG C CC CC CC + CT

No of studies

Sample size

vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.

G GG AG + GG GG T TT CT + TT TT G GG AG + GG GG T TT CT + TT TT

Case

control

1877 1758 1758 1999 643 601 601 686 711 684 684 741 595 571 571 621

1983 1838 1838 2128 709 664 664 754 701 677 677 727 530 513 513 548

6 6 6 6 3 3 3 3 5 5 5 5 4 4 4 4

Test of association

Test of heterogeneity

Model

OR(95%CI)

Z

P

␹2

P

I2

F F F F F F F F F F F F F F F F

1.11(0.87–1.42) 1.08(0.43–2.72) 1.14(0.95–1.36) 1.07(0.42–2.70) 1.03(0.68–1.56) 2.73(0.55–13.41) 1.00(0.73–1.36) 2.73(0.55–13.43) 0.96(0.57–1.63) 1.05(0.27–4.00) 0.96(0.65–1.44) 1.06(0.28–4.06) 0.91(0.50–1.65) 1.15(0.23–5.71) 0.91(0.58–1.43) 1.17(0.24–5.85)

0.85 0.16 1.38 0.14 0.13 1.23 0.02 1.23 0.14 0.07 0.18 0.08 0.31 0.17 0.41 0.19

0.39 0.87 0.17 0.89 0.90 0.22 0.98 0.22 0.89 0.94 0.86 0.93 0.76 0.86 0.68 0.85

3.21 2.18 6.11 2.13 1.41 0.42 2.10 0.38 2.93 1.22 5.82 1.11 2.50 1.06 3.66 0.97

0.67 0.70 0.30 0.71 0.50 0.81 0.35 0.83 0.40 0.75 0.12 0.78 0.29 0.59 0.16 0.62

0 0 18 0 0 0 5 0 0 0 48 0 20 0 45 0

RA: rheumatoid arthritis; AS: ankylosing spondylitis; OR: odds ratio; CI: confidence interval; F: fixed-effect mode.

group of each study in this meta-analysis was consistent with HWE. 3.2. Evaluation of D299G/T399I polymorphisms and association with RA/AS We did not find a significant association between TLR4 D299G/T399I polymorphisms with RA/AS between allele and genotype frequency comparisons (Table 3). There was no Q-test of heterogeneity between all comparisons, therefore, fixed effect models were conducted. 3.3. Evaluation of publication bias Funnel plot asymmetry was assessed through the method of Egger’s test. If there was asymmetry, the index to determine Egger’s test will show P < 0.05. It was shown that there was no publication bias between all of the comparisons (Table 4). 4. Discussion As is known, there is individual susceptibility to autoimmune diseases even with the same environmental exposure. Host factors, including gene polymorphisms involved in autoimmune diseases, might have interpreted this difference partly. Table 4 Tests for publication bias (Egger’s test) in population. Disease

Comparison

RA

(D299G)

(T399I)

AS

(D299G)

(T399I)

Egger’s test (P) A vs. G AA vs. GG AA vs. AG + GG AA + AG vs. GG C vs. T CC vs. TT CC vs. CT + TT CC + CT vs. TT A vs. G AA vs. GG AA vs. AG + GG AA + AG vs. GG C vs. T CC vs. TT CC vs. CT + TT CC + CT vs. TT

RA: rheumatoid arthritis; AS: ankylosing spondylitis.

0.727 0.533 0.710 0.525 0.436 0.565 0.451 0.578 0.763 0.718 0.696 0.700 0.592 0.813 0.622 0.819

Therefore, genetic susceptibility to autoimmune diseases has been a research focus in the scientific community. Recently, genetic variants of the TLR4 gene in the pathogenesis of some autoimmune diseases have fascinated much attention. Studies have suggested that the D299G/T399I polymorphisms in the TLR4 gene are emerging as susceptible alleles for autoimmune diseases, such as RA, AS [5,28,29,34]. However, the results were inconsistent. Radstake et al. [28] found TLR4 D299G functional variant was associated with RA disease susceptibility in Caucasians, the difference in genotype frequency between patients and controls was statistically significant (P = 0.025) and translated into an OR of 1.7 for the A/A genotype (95% CI: 1.1-2.8). On the contrary, Kang et al. [35] found none of the individuals in patient and control groups had D299G and T399I polymorphisms in a Korean population. Similarly, a study on the Finns with AS revealed over-transmission of the D299G/T399I TLR4 haplotype (P = 0.03) and showed an association with AS [34]. Furthermore, another case-control study in the Newfoundland population indicated that the D299G variant G allele frequency was significantly higher in AS cases compared to controls (7.5% vs 2.6%, respectively; OR = 3.10, P = 0.037) [30]. However, Jr et al. [32] discovered no significant differences in allele or genotype frequency of these two polymorphisms between AS patients and controls in a Hungarian population. Therefore, to better comprehend the relationship between these two polymorphisms and RA/AS, a pooled analysis with a large sample size, and heterogeneity explored is needed. Overall, the results of this meta-analysis suggest TLR4 D299G/T399I polymorphisms are not associated with RA/AS risk when all studies are pooled together. In deed, the population studies have indicated a specific geographical (ethnic) distribution of these two variants haplotype, wherein the African have the highest frequency of these two polymorphisms about 16% [36], Caucasian was 4–10% [2,37] and no presence of them was in Asian populations. It is possible that this pattern of TLR4 haplotype results from different selective pressures such as distinct pathogens and infectious diseases on the human population during the migration into Eurasia, Europe, Asia. Still, most of the negative case-control studies from Caucasian and Asian RA/AS patients have relatively low sample size and low observed powered values, it is reasonable that these polymorphisms can not be conclusively excluded as statistically associated alleles [26,32]. Moreover, most of the autoimmune diseases such as RA/AS are complicated multi-genetic diseases, distinct genetic backgrounds might contribute to this discrepancy. Our results also should be interpreted with caution owing to the limited number of studies

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included in this meta-analysis, which might also restrict further subgroup analyses. Further, as a matter of fact, it may be common that epidemiologic results are not in coincidence with the results of functional studies. With the presence of some other as-yet unidentified causal genes, which are involved in the development of autoimmune diseases, might defilade the influence of the alleles as well. The present study has some limitations should be considered. Firstly, publication bias may affect the analysis, because negative results might not have been published. Even though Egger’s test was performed, the possibility of bias could not be eliminated. Secondly, as a complex disease, RA/AS disease phenotype and disease susceptibility factors may exist heterogeneity [38]. Thirdly, meta-analysis remains a retrospective research, which is subject to the methodological deficiencies of the included studies [39]. In conclusion, the present study might not demonstrate that TLR4 D299G/T399I polymorphisms are associated with RA/AS because of not enough evidence. However, to reach a definitive conclusion, further gene-gene and gene-environment interactions studies based on larger sample size, case-control design and stratified by ethnicity are still needed. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement This work was partly supported by grants from the key program of National Natural Science Foundation of China (30830089) and the Anhui Provincial Natural Science Foundation (11040606M183). References [1] Karouzakis E, Neidhart M, Gay RE, et al. Molecular and cellular basis of rheumatoid joint destruction. Immunol Lett 2006;106:8–13. [2] Zheng B, Li Q, Wei C, et al. 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. [3] Wordsworth BP, Lanchbury JS, Sakkas LI, et al. HLA-DR4 subtype frequencies in rheumatoid arthritis indicate that DRB1 is the major susceptibility locus within the HLA classII region. Proc Natl Acad Sci 1989;86:10049–53. [4] Deighton CM, Walker DJ, Griffiths ID, et al. The contribution of HLA to rheumatoid arthritis. Clin Genet 1989;36:178–82. [5] Na KS, Kim TH, Rahman P, et al. Analysis of single nucleotide polymorphisms in Toll-like receptor 4 shows no association with ankylosing spondylitis in a Korean population. Rheumatol Int 2008;28:627–30. [6] Lea W.I, Lee F Y.H. The associations between interleukin-1 polymorphisms and susceptibility to ankylosing spondylitis: a meta-analysis. Joint Bone Spine, DOI:10.1016/j.jbspin.2011.06.010. [7] Richez C, Blanco P, Rifkin I, et al. Role for toll-like receptors in autoimmune disease: the example of systemic lupus erythematosus. Joint Bone Spine 2011;78:124–30. [8] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune response. Nat Immunol 2004;10:987–95. [9] Okamura Y, Watari M, Jerud ES, et al. The extra domain A of Wbronectin activates toll-like receptor 4. J Biol Chem 2001;276:10229–33. [10] Termeer C, Benedix F, Sleeman J, et al. Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 2002;195:99–111. [11] Poltorak A, He X, Smirnova I, et al. signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282:2085–8. [12] Hoshino K, Takeuchi O, Kawai T, et al. Toll-like receptor 4 (TLR4)–deWcient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52. [13] Ohashi K, Burkart V, Flohe S, et al. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 2000;164:558–61.

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Please cite this article in press as: Xu W-D, et al. Lack of association of TLR4 polymorphisms with susceptibility to rheumatoid arthritis and ankylosing spondylitis: A meta-analysis. Joint Bone Spine (2012), doi:10.1016/j.jbspin.2012.02.012