Regulatory T cells (Tregs) are crucial in the maintenance of the immune tolerance and seem to have an important role in systemic sclerosis (SSc). The interleukin 2 receptor α (IL2RA) is an important Treg marker, and polymorphisms of IL2RA gene are associated with a number of autoimmune diseases. Therefore, we aimed to investigate for the first time the association of the IL2RA locus in SSc. For this purpose, a total of 3023 SSc patients and 2735 matched healthy controls, from six European Caucasian cohorts, were genotyped for the IL2RA gene variants rs11594656, rs2104286 and rs12722495 using the TaqMan allelic discrimination technology. The overall meta-analysis reached statistical significance when the three polymorphisms were tested for association with SSc, the limited subtype (lcSSc) and anti-centromere auto-antibodies (ACAs). However, no significant P-values were obtained when the ACA-positive patients were removed from the SSc and lcSSc groups, suggesting that these associations rely on ACA positivity. The strongest association signal with ACA production was detected for rs2104286 (PFDR=2.07 × 10−4, odds ratio=1.30 (1.14–1.47)). The associations of rs11594656 and rs12722495 were lost after conditioning to rs2104286, and allelic combination tests did not evidence a combined effect, indicating that rs2104286 best described the association between IL2RA and ACA presence in SSc.
The interleukin 2 (IL2) signalling is involved in T-cell proliferation, survival and differentiation of effector cells, being essential for the maintenance of the immune tolerance.1 A key component of this pathway is the high-affinity heterotrimeric IL2 receptor (IL2R), which is composed of the interleukin 2 receptor α (IL2RA), β (IL2RB) and γ (IL2RG) subunits. IL2RA (also known as CD25) is an important marker of CD4+CD25+ regulatory T cells (Tregs), which have a central role in protecting an individual from autoimmunity.2, 3 Several polymorphisms surrounding the IL2RA locus at 10p15.1 have been reported to be associated with different autoimmune diseases, including type I diabetes (T1D), rheumatoid arthritis, multiple sclerosis, Graves’ disease and systemic lupus erythematosus (SLE).4, 5, 6, 7
Autoimmune diseases are complex polygenic disorders in which environmental factors, and common and rare genetic variants contribute to their susceptibility, each with generally modest effects independently. There are increasing evidences pointing to a shared underlying immunological mechanism in immune-related pathologies.8 The association of IL2RA with multiple autoimmune disorders clearly supports this idea. In addition, it is known that different autoimmune diseases may sometimes present similar genetic backgrounds. For example, most of the genetic associations of systemic sclerosis (SSc) are shared with SLE.9, 10 SSc, also called scleroderma, is a chronic multisystem disease of unknown aetiology, characterised by fibrotic events, vasculopathy and production of auto-antibodies against nuclear self-antigens, including anti-centromere auto-antibodies (ACAs) and anti-topoisomerase antibodies (ATA).11 Two main clinical subsets of the disease have been described based on the extent of skin involvement: limited cutaneous scleroderma (lcSSc) and diffuse cutaneous scleroderma (dcSSc).12 Interestingly, elevated serum levels of soluble IL2RA have been associated with the progression of the disease in SSc patients.13, 14
Taking into account the above, we aimed to investigate whether the IL2RA locus is involved in the genetic predisposition and clinical manifestations of SSc. For this purpose, we genotyped in a large cohort of European Caucasian origin three independent single-nucleotide polymorphisms (SNPs) of the IL2RA gene, rs11594656, rs2104286 and rs12722495, that have been previously reported to be involved in autoimmunity.4, 5, 6, 15, 16, 17
The IL2RA genetic variants rs11594656, rs2104286 and rs12722495 were in Hardy–Weinberg equilibrium in all analysed control cohorts (all P-values <0.05 were considered significant).
Independent allele tests
In a first step, we investigated the possible implication of the IL2RA SNPs in SSc susceptibility and clinical manifestations in a large Spanish cohort of Caucasian origin (Supplementary Tables 1–3, upper rows). Although no significant differences between the IL2RA rs11594656 allele frequencies of SSc cases and controls were detected (Supplementary Table 1), the phenotype analysis evidenced a weak but significant association of this SNP with the ACA presence (PFDR=0.018, odds ratio (OR)=0.77 (0.64–0.93)), as well as a suggestive OR in the lcSSc analysis (PFDR=0.126, OR=0.88 (0.74–1.04)). On the other hand, the analysis of rs2104286 (Supplementary Table 2) showed statistically significant P-values when the control population was compared with the SSc cohort (PFDR=0.017, OR=1.25 (1.07–1.46)), with the limited subtype subgroup (PFDR=0.025, OR=1.28 (1.07–1.53)), and with the ACA-positive subgroup (PFDR=0.018, OR=1.29 (1.06–1.58)). Finally, no evidence of association was observed for the IL2RA variant rs12722495 either with the global disease or a specific phenotype (Supplementary Table 3).
To confirm that IL2RA represents a susceptibility locus in SSc, we decided to test for associations in five independent Caucasian cohorts from five different European countries. Despite their different geographical origin, the combinability test according to Breslow–Day method showed no significant heterogeneity in the ORs of the different sets (PBD for rs11594656=0.14, PBD for rs2104286=0.93 and PBD for rs12722495=0.17). Therefore, we conducted a pooled OR analysis using the Mantel–Haenszel test under fixed effects. Interestingly, the meta-analysis of the replication sets (Supplementary Tables 1–3, lower rows) showed a significant association of rs11594656 with the lcSSc subtype (PFDR=0.016, OR=0.84 (0.74–0.95)) and a trend with the global disease (PFDR=0.064, OR=0.89 (0.80–0.99)), and a considerable P-value for rs2104286 in the ACA+/control comparison (PFDR=5.98 × 10−3, OR=1.30 (1.10–1.53)). In addition, the meta-analysis of rs12722495 in the replication cohort also reached statistical significance when the limited subtype and the ACA positivity were tested (PFDR=0.041, OR=1.23 (1.02–1.49), and PFDR=0.014, OR=1.35 (1.07–1.70), respectively).
As no significant heterogeneity was neither observed when including the Spanish cohort to the pooled European set (Breslow–Day P=0.21, 0.69 and 0.27 for rs11594656, rs2104286 and rs12722495, respectively), we performed an overall meta-analysis including all six independent cohorts to increase the statistical power and, hence, to obtain more accurate estimates of susceptibility (Table 1). This analysis showed consistent associations of the three IL2RA SNPs with lcSSc (rs11594656: PFDR=5.11 × 10−3, OR=0.85 (0.77–0.94); rs2104286: PFDR=9.83 × 10−3, OR=1.15 (1.03–1.28); rs12722495: PFDR=8.53 × 10−3, OR=1.24 (1.06–1.44)) and ACA positivity (rs11594656: PFDR=9.48 × 10−3, OR=0.86 (0.76–0.96); rs2104286: PFDR=2.07 × 10−4, OR=1.30 (1.14–1.47); rs12722495: PFDR=9.48 × 10−3, OR=1.27 (1.06–1.52)). Although moderate, the combined analysis of the global SSc also evidenced statistically significant differences in the allele frequencies of cases and controls (rs11594656: PFDR=0.017, OR=0.90 (0.83–0.98); rs2104286: PFDR=0.017, OR=1.14 (1.04–1.25); rs12722495: PFDR=0.017, OR=1.19 (1.04–1.36)).
Considering that the strongest association signal was detected in the ACA analysis (rs2104286: PFDR=2.07 × 10−4), we decided to further investigate whether the associations observed with the global disease and the limited subtype were a consequence of the presence of ACA-positive patients in those groups. With this purpose, we excluded the ACA-positive patients from the SSc and lcSSc groups, and tested for associations with the control cohort (Supplementary Table 4). No statistical significance was detected between the allele frequencies of the control population and the new groups composed of the ACA-negative SSc patients (rs11594656: PFDR=0.113, OR=0.90 (0.81–0.99); rs2104286: PFDR=0.851, OR=1.01 (0.90–1.13); rs12722495: PFDR=0.251, OR=1.12 (0.95–1.32)), and the ACA-negative lcSSc patients (rs11594656: PFDR=0.092, OR=0.86 (0.76–0.99); rs2104286: PFDR=0.714, OR=0.97 (0.84–1.13); rs12722495: PFDR=0.103, OR=1.21 (0.98–1.48)). Hence, the associations observed with SSc and lcSSc are likely to rely on the ACA positivity. To confirm this assumption, we compared the allele frequencies of lcSSc patients with those of dcSSc patients (Supplementary Table 5, upper rows) and found no statistically significant differences between them (rs11594656: PFDR=0.324, OR=0.94 (0.82–1.07); rs2104286: PFDR=0.324, OR=1.08 (0.93–1.25); rs12722495: PFDR=0.324, OR=1.16 (0.94–1.44)).
Finally, we carried out another meta-analysis comparing the ACA-positive SSc patients versus those with the disease but without this type of auto-antibodies (Supplementary Table 5, lower rows). Interestingly, the meta-analysis of the ACA+/ACA− comparison only showed statistically significant differences for rs2104286 (PFDR=1.22 × 10−3, OR=1.29 (1.12–1.48)), which definitively confirms that this specific IL2RA genetic variant is consistently associated with the ACA presence.
To analyse the possible interaction between rs11594656, rs2104286 and rs12722495, an allelic combination test was performed on the pooled data from SSc, lcSSc and ACA-positive patients (Table 2). Although some combinations were significantly associated with SSc, lcSSc and ACA presence, particularly that containing the three protective alleles TTT (SSc: PFDR=2.60 × 10−3, OR=0.86 (0.79–0.95), lcSSc: PFDR=5.49 × 10−4, OR=0.82 (0.74–0.92), ACA: PFDR=1.74 × 10−3, OR=0.81 (0.71–0.92)), the strength of these associations did not seem to differ substantially from those observed in the independent analyses (Table 1).
Conditional logistic regression
We used the pair-wise conditioning method on ACA data (which showed the strongest signal) to check whether one of the three analysed IL2RA genetic variants tags the other two (Table 3). The association of rs2104286 with ACA presence remained significant when conditioned to rs11594656 (P=1.06 × 10−3) and rs12722495 (P=2.43 × 10−3), whereas the corresponding associations of rs11594656 and rs12722495 were lost after conditioning to rs2104286 (P=0.208 and P=0.541, respectively), thus suggesting that rs2104286, or any other SNP within its haplotypic block, is the causal variant of the association of IL2RA with ACA production.
The recent identification of CD4+CD25+ Tregs as important regulators of the self-tolerance maintenance has led to new perspectives in the study of autoimmunity.3 Functional studies have demonstrated that the IL2/IL2R pathway is directly involved in the differentiation and homoeostasis of Tregs.2, 3 Mice lacking IL2RA and IL2RB chains develop autoimmune-like pathologies with age due to Treg misregulation.18, 19
Although the aetiology of SSc is not yet understood, there are evidences strongly suggesting that misregulation of Tregs may underlie the immune dysfunction in the progression of this disease.20, 21 Interestingly, it has been proposed that skin-specific Treg anomalies could be crucial in the SSc pathogenesis.21 As IL2RA has an important role in Treg regulation,2 genetic alteration of its encoding gene could have serious consequences in the function of this cell type by altering the IL2/IL2R signalling.
Recent genome-wide association and candidate gene studies on different autoimmune disorders clearly suggest a shared genetic architecture.8 The IL2RA gene is an example of common susceptibility locus in autoimmunity, as it has been associated with a number of autoimmune diseases.4, 5, 6, 7
Studies on T1D suggested that the IL2RA association with this disease could be narrowed down to three independent haplotypic blocks spanning a genomic region around the 5′ end of gene.4, 15, 17, 22 In this regard, the IL2RA genetic variants that we have analysed (rs11594656, rs12722495 and rs2104286) represent the highest association signals for T1D susceptibility that best define the common disease-associated IL2RA haplotypes, and it has been hypothesised that the causative variants of these associations may alter the protein levels.22 However, heterogeneity in the genetic associations has been observed between T1D and multiple sclerosis at the IL2RA region, and there are evidences pointing to a correlation between multiple IL2RA variants and sIL-2RA levels independently.15
The present study reports for the first time that the Treg marker IL2RA is also involved in the genetic susceptibility to SSc, likely by having a role in ACA production, as the three SNPs analysed showed consistent association signals. Interestingly, one of these SNPs, rs11594656, has been previously associated with the SSc immune-related disease SLE.6 The fact that the IL2RA rs11594656 minor allele shows a protective effect in both SLE6 and SSc (the current study) should also be noted, because these diseases have a very similar genetic component and most of their shared associations are in the same direction.9, 10 Moreover, low circulating levels of soluble IL2RA have been found to be associated with the IL2RA rs11594656 major allele in T1D patients,4 consistent with the protective effect of the rs11594656 minor allele that we have observed in SSc.
However, our data suggest that, despite being in different haplotypic blocks, the SNP rs2104286 could tag both rs11594656 and rs12722495 Il2RA variants and, hence, the associations observed for the latter SNPs may be a consequence of the LD structure in the studied population. Another possible explanation could be that rs11594656 and rs12722495 have a hypothetical independent role on a specific group of SSc patients with a clinical phenotype not analysed in this study that overlap with the ACA subgroup. In any case, the association reported here between rs2104286 and SSc could be considered controversial in principle, because this IL2RA variant was not found to be associated with SLE.6 Nevertheless, this association relies on ACA production, which is an SSc-specific auto-antibody. Consequently, it would be interesting to perform an analysis on SLE patients stratified accordingly with the auto-antibody status, to investigate whether rs2104286 has a role in the production of auto-antibodies in SLE.
Therefore, it seems that the association between IL2RA and autoimmune disease is relatively complex, and it is possible that a combination of shared and disease-specific alleles may contribute to different autoimmune phenotypes.
To summarise, we have identified IL2RA as a novel susceptibility locus for SSc, and have determined that the rs2104286 genetic variant is specifically associated with the presence of ACAs. Taking into account the key role of IL2RA in the regulatory function of Tregs, which in turn seems to be tightly linked with the progression of SSc, this association may shed light into the complex genetic network underlying SSc aetiopathogenesis.
Patients and methods
An initial discovery cohort of 1081 SSc patients and 882 healthy controls from Spanish Caucasian origin, and a replication set composed of five independent European Caucasian populations (comprising a total of 1942 SSc patients and 1853 healthy controls from Germany, The Netherlands, Belgium, Norway and UK) were included in the study. All the control and case cohorts were recruited in the same geographical regions and matched by age, sex and ethnicity. Patients fulfilled either the American College of Rheumatology SSc classification criteria for this disease23 or the criteria proposed for early SSc.24 Case sets were further subdivided based on skin involvement and autoantibody status into lcSSc, dcSSc, ACA+ and ATA+ subgroups.12 Measurement of autoantibodies was performed using standard procedures. Clinical characteristics of the patients have been described before.25 Approval from the local ethical committees and written informed consent from all participants were obtained.
Genomic DNA was obtained from peripheral blood cells using standard methods. Samples were genotyped for the IL2RA variants, rs11594656, rs2104286 and rs12722495, using a TaqMan 5′ allele discrimination assay (IDs: C__11646696_10, C__16095542_10 and custom AHHSQ33, respectively) in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
Power Calculator for Genetic Studies 2006 software26 was used to calculate the overall statistical power of the study, which was <98% to detect associations with OR=1.3 at 5% significant level for all SNPs. All statistical analyses of allele frequencies were performed using the Linux software PLINK v1.07.27 To test for associations, P-values were obtained by performing 2 × 2 contingency tables and χ2 test and/or Fisher's exact test, when appropriate. OR and 95% confidence intervals were calculated according to Woolf's method. In addition, combined P-values and ORs were calculated by Mantel–Haenszel test under fixed effects, and Breslow–Day and Cochran's Q test methods were used to estimate the homogeneity among the different cohorts. Additionally, the Benjamini and Hochberg28 step-up false discovery rate (FDR) control correction for multiple testing was applied to the P-values.
Dependency of association between each SNP and every studied genetic variant was determined by conditional logistic regression analysis as implemented in PLINK. Allelic combination tests were carried out using PLINK and Haploview (V.4.2),29 and the meta-analyses of the different allelic combinations were performed using PLINK and StatsDirect (V.2.6.6; StatsDirect, Altrincham, UK). A P-value <0.05 was considered significant.
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We thank Sofía Vargas, Sonia García and Gema Robledo for their excellent technical assistance, and all the patients and healthy controls for kindly accepting their essential collaboration. Banco Nacional de ADN (University of Salamanca, Spain) and The Norwegian Bone Marrow Donor Registry are thanked for supplying part of the control material. We are also thankful to EUSTAR and the German Network of Systemic Sclerosis for the facilitation of this project. This work was supported by the following grants: JM was funded by GEN-FER from the Spanish Society of Rheumatology, SAF2009-11110 from the Spanish Ministry of Science, CTS-4977 and CTS-180 from Junta de Andalucía, and in part by RETICS Program, RD08/0075 (RIER) from Instituto de Salud Carlos III (ISCIII), Spain. FDC was supported by Consejo Superior de Investigaciones Científicas (CSIC) through the program JAE-DOC. TRDJR was funded by the VIDI laureate from the Dutch Association of Research (NWO) and Dutch Arthritis Foundation (National Reumafonds). CF is supported by ‘The Raynaud's and Scleroderma Association’ and ‘The Scleroderma Society’. JM and TRDJR were sponsored by the Orphan Disease Program grant from EULAR. TW was granted by DFG WI 1031/6.1.
The authors declare no conflict of interest.
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Martin, J., Carmona, F., Broen, J. et al. The autoimmune disease-associated IL2RA locus is involved in the clinical manifestations of systemic sclerosis. Genes Immun 13, 191–196 (2012). https://doi.org/10.1038/gene.2011.72
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