Association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) Polymorphisms and Graves' Disease Risk: A Meta-Analysis of 11 Case-Control Studies

Background Data on the association between the interleukin-1 (IL-1) gene polymorphisms and Graves' disease (GD) risk were conflicting. A meta-analysis was undertaken to assess this association. Methods We searched for case-control studies investigating the association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk. We extracted data using standardized forms and calculated odds ratios (OR) with 95% confidence intervals (CI). Results A total of 11 case-control studies were included in this meta-analysis. Available data indicated that the IL1B (-511) polymorphism was associated with GD risk in the overall populations (Caucasians and Asians) in homozygote model (TT vs. CC, OR = 0.86, 95% CI: 0.76–0.97, Pz = 0.015), but not in dominant and recessive models (TT+TC vs. CC: OR = 0.95, 95% CI: 0.81–1.12, Pz = 0.553 and TT vs. TC+CC: OR = 0.82, 95% CI: 0.60–1.12, Pz = 0.205, respectively). No association between the IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk was found in the overall populations in any of the genetic models. In subgroup analyses according to ethnicity, the IL1B (-511) polymorphism was associated with GD risk in Asians in recessive and homozygote models (TT vs. TC+CC: OR = 0.68, 95% CI: 0.55–0.84, Pz<0.001 and TT vs. CC: OR = 0.81, 95% CI: 0.70–0.93, Pz = 0.003, respectively), but not in dominant model (TT+TC vs. CC: OR = 0.92, 95% CI: 0.77–1.11, Pz = 0.389). No association between the IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk was indicated in Asians, and we found no association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk in Caucasians in any of the genetic models. Conclusion The IL1B (-511) polymorphism, but not the IL1B (+3954) and IL1RN (VNTR) polymorphisms was associated with GD risk in Asians. There was no association between these polymorphisms and GD risk in Caucasians.


Introduction
Graves' disease (GD) is an autoimmune thyroid disorder characterized by hyperthyroidism. It is commonly seen in young and middle-aged people, affecting 0.5-1.0% of the general population [1]. In GD, circulating autoantibodies are produced, which target well-defined thyroidal antigens, such as thyroid peroxidase, thyroglobulin, and the thyroid-stimulating hormone receptor (TSHR) [2]. Stimulatory autoantibodies binding to TSHR on thyroid follicular cells induce over-synthesis of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). The systematic effects of elevated T3/T4 manifests as a sped up metabolism and its correlated symptoms. In addition to hyperthyroidism, some patients with GD have clinical involvement of the eyes resulting in Graves' Ophthalmopathy (GO) and the skin resulting in localized myxoedema [3,4].
The etiology of GD remains unknown; however, it is considered to be caused by an interplay between genetic and environmental factors [5,6]. In common with other autoimmune diseases, GD has a complex genetic basis, with numerous different genes each contributing in various degrees to the inherited susceptibility. Using both the candidate gene approach and whole genome linkage studies, some major susceptibility genes to GD have been identified, including human leukocyte antigen (HLA) on chromosome 6p21, Cytotoxic T-Lymphocyte Antigen (CTLA) -4 on chromosome 2q33 and lymphoid tyrosine phosphatase (LYP) on chromosome 1p13 [7][8][9][10]. Besides these, it is believed that additional genes contribute to the genetic susceptibility to GD, as well as to its different phenotypes. Much interest has currently centered around the exploration of additional susceptibility genes to GD, which may help elucidate signaling pathways involved in the pathogenesis of GD, and help develop novel therapeutic strategies for the disease.
The interleukin-1 (IL-1) family is a group of related cytokines including two agonist proteins (IL-1A and IL-1B) and an antagonist protein (IL-1RA). The genes encoding for these molecules are located close to one another on chromosome 2q13-14, which are candidate genes for GD. There are several common polymorphisms that have been most frequently investigated. The IL1B gene has two single nucleotide polymorphisms (SNP) at position -511 in the promoter region (rs16944) and at position +3954 in the fifth exon (rs1143634), respectively [11,12]. In the IL1RN gene, the second intron contains a variable number of tandem repeats (VNTR) of 86 base pairs (rs2234663) [13]. Many genetic association studies have intensively investigated the association of these SNPs with GD risk; however, results from individual studies are not consistent [14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Here we conducted a meta-analysis with pooled data from 11 genetic association studies to determine risk of GD associated with these three polymorphisms in the IL-1 gene.

Search strategy and selection of studies
We conducted a literature search for studies reporting on the association between IL-1 gene polymorphisms and GD risk using the electronic database PubMed, Medline and Web of Science from January 1990 up to the end of July 2013. The search results were limited to publications in English. The search strategy used the key words ''IL-1, Graves' disease, thyroid disease, genetic, association, polymorphism, susceptibility''. We also screened references of retrieved publications. Inclusion criteria were as follows: (a) case-control design; (b) sufficient data provided to reconstruct two by two tables or determine odds ratio and confidence intervals. Studies were excluded if one of the following existed: (a) no control; (b) no usable data reported.

Data extraction
Information on general study characteristics including first author, year of publication, country, ethnicity, sample size, and genotype distribution in cases and controls was collected from the published papers.

Discussion
Since 1995, many genetic association studies have assessed the relation of the IL1B (-511) and (+3954), and IL1RN (VNTR) polymorphisms with GD risk; however, the findings remain inconclusive. This may partly be owing to a small sample size of individual studies and distinct genetic background. Meta-analysis is a quantitative statistical analysis that combines the results of previous separate but related studies in order to test the pooled data for statistical significance. It has been increasingly utilized in assessing associations between genetic variants and autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes, and GD [30]. In the present study, we undertook a meta-analysis of 11 published case-control studies to investigate the association between the three main IL-1 gene polymorphisms and GD risk. The main findings were as follows: (1) the IL1B (-511) polymorphism was associated with GD risk in homozygote model in the overall populations; (2) the IL1B (+3954) and IL1RN (VNTR) polymorphisms were not associated with GD risk in any of the genetic models in the overall populations; (3) in Asians, the IL1B (-511) polymorphism was associated with GD risk in recessive and homozygote models, while the IL1B (+3954) and IL1RN (VNTR) polymorphisms were not associated with GD risk; (4) no association between the IL1B (-511), IL1B (+3954), and IL1RN Figure 3. Meta-analysis with a random effects model for the association between the IL1B (-511) polymorphism and GD risk in recessive model. Each study is shown by the point estimate of the odds ratio, and a horizontal line denotes the 95% confidence interval. The pooled odds ratio is represented by a diamond. The area of the grey squares reflects the weight of the study in the meta-analysis. doi:10.1371/journal.pone.0086077.g003 Table 3. Meta-analysis of polymorphisms in the IL-1 gene and GD risk. (VNTR) polymorphisms and GD risk was observed in any of the genetic models in Caucasians. IL-1B is a pluripotential proinflammatory cytokine that primarily produced by B lymphocytes, monocytes and fibroblasts. It is an important mediator for the inflammatory responses and play key roles in the triggering of immune functions, effecting nearly every cell type. IL-1B influenced thyroid cells via a number of underlying mechanisms, including down-regulation of thyroid peroxidase gene expression [31], induction of dissociation of the junctional complex [32], and inhibition of cyclic adenosine monophosphate (cAMP) and thyroglobulin production [33]. In the development of GD, infiltration of the thyroid by activated immune cells results in local release of IL-1B. It has been observed that IL-1B induces the production of IL-6, IL-8, intercellular adhesion molecule-1 (ICAM-1), and other inflammatory mediators [34][35][36]. IL-1B also enhances T cell-dependent antibody production by augmenting CD40 ligand and OX40 expression on T cells [37]. IL-1B was shown to promote differentiation of T-helper 17 (Th17) cell, the proportion of which was reported to be higher in intractable GD than that of GD in remission [38]. Therefore, IL1B may play a role in the pathogenesis of thyroid autoimmunity. In this meta-analysis, we observed that the IL1B (-511) TT genotypes were protective against GD in Asians. This SNP may affect protein expression and function [27,39], resulting in down-regulation of inflammatory responses and resistance to develop GD. Several reasons may account for the discrepancy of results between Asians and Caucasians. First, distinct genetic background may play a role. Second, GD is a complex disease which is also related to environmental factors. Genetic susceptibility to GD may be modified by a variety of environmental exposures, leading to differences in genetic associations. Between-study heterogeneity was identified in several pooled analyses. However, when we undertook subgroup analyses according to ethnicity, heterogeneity was greatly reduced, suggesting that ethnicity was the main source of heterogeneity. Other potential factors, such as study design, sample size and genotyping methods, might also contribute to heterogeneity.
Some limitations should be acknowledged when interpreting this meta-analysis. First, our study only included publications written in English. It is possible that some relevant papers written in other languages were missed. Second, all included case-control studies were conducted in Caucasians and Asians. Therefore, the results of this meta-analysis were only applicable to these two ethnic groups. For providing additional evidence of the association between the IL-1 gene polymorphisms and GD risk, future studies should be performed in other ethnic groups, for example in Latin Americans. Third, although we combined data from all included published case-controls assessing the IL1B (-511), IL1B (+3954) and IL1RN (VNTR) polymorphisms, the sample size of this metaanalysis was still relatively small, which limited the statistical power to achieve a definitive conclusion. Fourth, haplotype analysis using haplotypes constructed by these SNPs could not be conducted because relevant data provided by included studies were limited. Future association studies are needed to evaluate haplotype associations with GD risk, which will provided more insights for the effects of gene-gene interaction among these SNPs on GD risk.
In summary, our meta-analysis suggested that the IL1B (-511) polymorphism, but not the IL1B (+3954) and IL1RN (VNTR) polymorphisms was associated with GD risk in Asians, and there was no association between these polymorphisms and GD risk in Caucasians. Future studies should be performed using large sample numbers to establish a more definitive conclusion

Supporting Information
Checklist S1 PRISMA Checklist.