In this study, we found significant evidence of association of a polymorphism within the IL2RA/CD25
gene with JIA in 2 independent cohorts. SNPs within the IL2RA/CD25
gene have previously been associated with a number of other autoimmune diseases, including type 1 DM (10
), Graves' disease (20
), and MS (9
). In addition, a rare mutation of the IL2RA/CD25
gene can lead to the development of a severe autoimmune disease characterized by decreased numbers of peripheral T cells displaying abnormal proliferation, with extensive lymphocytic infiltration of tissues accompanied by tissue atrophy and inflammation, but normal B cell development (21
). The finding of an association with JIA further extends the evidence that variation across this gene predisposes to autoimmunity in general.
The concept of “autoimmune genes” is not novel. Previous analysis of linkage studies in a number of autoimmune diseases showed evidence of overlap between regions of linkage (22
). Furthermore, there is precedent for this because the PTPN22
gene has been associated with a variety of autoimmune diseases, although, interestingly and unlike the IL2RA/CD25
gene, not with MS.
Three SNPs were selected for genotyping based on their association with other autoimmune diseases, and 2 of these SNPs were associated with JIA in the current study. The strongest effect was seen with rs2104286 (OR for minor allele 0.76 [95% CI 0.66–0.88]), which is comparable to the ORs calculated for MS (OR 0.81 [95% CI 0.74–0.89]) and RA (OR 0.81 [95% CI 0.73–0.89]). This SNP maps to intron 1 of the IL2RA/CD25
gene, and was selected for genotyping because recent genome-wide association studies of both RA and MS found an association with the same variant (rs2104286). In addition, the MS study also found an association with a SNP in strong linkage disequilibrium with it, rs12722489 (9
The other 2 SNPs genotyped in this study of JIA were selected based on recent studies of the IL2RA/CD25
region in type 1 DM (10
). Fine-mapping studies detected an association with 2 independent groups of SNPs spanning overlapping regions of 14 kb and 40 kb, encompassing IL2RA/CD25
intron 1 and the 5′-intergenic region between IL2RA/CD25
. The first region contains a group of 8 genetically indistinguishable SNPs, the most strongly associated being rs41295061, while the second group comprised 3 indistinguishable SNPs, the most strongly associated being rs11594656. Both of these SNPs are potentially functional, given that genotypes at each have been correlated with lower concentrations of soluble IL-2Rα/CD25. This suggests that a reduction in immune activation may predispose to type 1 DM (10
) and, indeed, other autoimmune diseases.
Interestingly, in the present study, only 1 of the 2 SNPs associated with type 1 DM (rs41295061) showed a weak association with JIA. The evidence of association with this SNP was stronger when genotype frequencies from the JIA cases were compared with those in the large type 1 DM control cohort (n = 6,855) (OR for the allele 0.69 [95% CI 0.55–0.85], P for the genotype = 0.002), while that of the second SNP (rs11594656) remained nonsignificant (OR for the allele 0.94 [95% CI 0.82–1.08], P for the genotype = 0.4). Association of the type 1 DM intron 1 SNP (rs41295061) with JIA was not as strong as with SNP rs2104286, and this association requires validation in an additional cohort. Hence, it will be vital to determine whether the genotype at the rs2104286 locus also correlates with soluble IL-2Rα/CD25 levels or any other functional effect.
Examination of linkage disequilibrium between the SNPs tested in this study suggested that there is low linkage disequilibrium between rs2104286 and the 2 SNPs (rs41295061 and rs11594656) identified in the type 1 DM fine-mapping study (r2 = 0.27 and r2 = 0.03, respectively). However, logistic regression analysis of the 3 SNPs conditioning on the most-associated SNP, rs2104286, suggests that this is the SNP that drives the association with JIA. The association with rs41295061 is due to linkage disequilibrium with rs2104286. Further investigation of SNPs across the IL2RA/CD25 gene will now be required to identify the causal variant.
The association with rs2104286 was confirmed in an independent cohort of North American JIA cases and controls (OR 0.84 [95% CI 0.65–0.99], P for trend = 0.05). Meta-analysis of the 2 data sets strengthened the effect (OR 0.76 [95% CI 0.62–0.88], P = 4.9 × 10−5), thereby providing further support for the role of IL2RA/CD25 in susceptibility to JIA.
False-positive associations can arise as a result of population stratification. The WTCCC study (8
) previously established that there was very little evidence of this across the UK. We did not test the North American data for population stratification. However, there was no evidence of heterogeneity between the 2 populations as assessed by the Breslow-Day test. Furthermore, the fact that the genotype/allele frequencies for the associated markers were very similar in the UK and the North American case and control populations suggests that the detected association is genuine.
JIA is a phenotypically heterogeneous disease. Thus, although it may be argued that stratification analysis may result in inflated Type I error rates because of multiple testing, the creation of more homogenous subgroups may increase the power to detect an association. In the current study, evidence of an association with rs2104286 was strongest in the oligoarthritis subtypes, female cases, and JIA cases with ANAs. The association with all these subgroups is not surprising, since the phenotypes are correlated. The same subgroup effect does appear to be true for rs41295061. However, it should be noted that the small sample sizes in some of the ILAR subtypes may have limited the ability to draw robust conclusions about association of the IL2RA/CD25 SNPs with those phenotypes. Comparison of rs2104286 SNP allele frequencies between the 5 subtypes suggests that they are not statistically different (P = 0.28). Therefore, larger sample sizes will be required to be able to clarify association across the individual subtypes.
The IL2RA/CD25 gene was a strong candidate JIA susceptibility gene not only because of the previous evidence of association with a variety of other autoimmune diseases, but also because of its role in the T cell signaling pathway, which is thought to play a key role in mediating the inflammatory autoimmune response in JIA. IL-2, the T cell growth factor, exerts its effect on T cells through binding with the IL-2R. The IL-2 receptor is composed of 3 chains, α, β, and γ, the α-chain being encoded by the IL2RA/CD25 gene. The α and β chains are involved in the binding of IL-2, and the β and γ chains are involved in signal transduction once IL-2 has bound. IL-2Rα/CD25 is central to immune regulation via its role in the development and function of regulatory T cells. These cells are increasingly seen as playing a central role in self tolerance. CD4+CD25+ Treg cells suppress activated T cells, and this immune suppression is vital in preventing autoimmune diseases. Hence, variation in the gene encoding IL-2Rα/CD25 could have profound effects in the predisposition to autoimmune disease.
A recent study investigated CD4+CD25+ Treg cells in children with persistent oligoarthritis (1–4 joints affected at diagnosis and throughout the disease course) and extended oligoarthritis (a more destructive disease course in which the number of affected joints increases beyond 4 within 6 months of disease onset), hypothesizing that Treg cells may play a role in the reversal of the autoimmune disease process in patients with persistent oligoarticular JIA (23
). It was shown that the number of CD4+
Treg cells at the site of inflammation (i.e., the joints) was enriched compared with the number in the peripheral blood. In addition, there was a greater frequency of CD4+
Treg cells in patients with persistent oligoarthritis as compared with those with extended oligoarthritis. CD4+
Treg cells in the synovial fluid (SF) showed higher expression of many activation markers, such as FoxP3, CTLA-4, and HLA–DR, suggesting that they are more functionally mature than those in the peripheral blood. These cells also showed an increased suppressive capacity in vitro compared with cells from the peripheral blood (23
). A more recent study also found that the ratio of Treg cells to activated T cells was higher in patients with oligoarthritis than in patients with polyarthritis (24
). Similar findings in RA have been reported, with higher numbers of Treg cells and concomitantly increased suppressive activity found in SF compared with peripheral blood cells (25
). Thus, there appear to be functional differences in Treg cells across JIA subtypes.
Interestingly, when we analyzed the IL2RA/CD25 SNPs by JIA subtype, the strongest association was in both oligoarthritis subtypes, although the limited sample sizes of some of the other subtypes make conclusions about subtype-specific associations difficult. In future studies, we will examine correlations between Treg cell function and IL2RA/CD25 genotype.
The IL-2 receptor has already been a potential target of immunologic therapy in autoimmune diseases. For example, IL-2–directed immunotherapies have been successful in ameliorating disease in experimental autoimmune encephalomyelitis, an animal model of MS (26
). In human MS, a monoclonal antibody that binds to the α-chain of IL-2R has been shown to have some therapeutic benefits, although its mechanism of action is unknown (27
In summary, we identified a putative novel JIA susceptibility gene, IL2RA/CD25. The association of IL2RA/CD25 with a fifth autoimmune disease suggests that this gene, along with the PTPN22 gene, appears to play a vital role in immune regulation and function as well as predisposition to autoimmunity in general. Further investigation of the gene using both genetic and functional approaches is now required.