Genetic associations implicate aberrant activation and regulation of autoreactive T-cells as central to RA. In addition to the established human leukocyte antigen locus DRB1, other genes more recently confirmed (either through wide replication or combined analysis at a genome-wide level of significance, P ≤ 10^-8) as playing a role in the development of RA are the protein tyrosine phosphatase non-receptor 22 gene (PTPN22) 1 , cytotoxic T-lymphocyte associated 4 (CTLA4) 2 , an intergenic region on human chromosome 6 3 4 , signal transducer and activator of transcription 4 (STAT4) 5 6 , the TNF receptor-associated factor 1 region (TRAF/C5) 3 7 8 , CD40 9 10 , B-lymphocyte kinase (BLK) and the NF-kB family member c-Rel 11 . Aside from HLA-DRB1 and PTPN22, the effects are weak (odds ratio (OR) < 1.3). Most of these loci are also implicated as risk factors in other autoimmune phenotypes 12 .

The KIAA1109-TENR-IL2-IL21 region has been associated with a number of autoimmune phenotypes including type 1 diabetes (T1D) 13 , ulcerative colitis 14 , Crohn's disease 15 , celiac disease 16 , Graves' disease (GD) 13 , systemic lupus erythematosus (SLE) 17 , psoriatic arthritis 18 , and juvenile idiopathic arthritis 19 (Table 1). There have been several studies testing this region for association with RA in European Caucasian sample sets, with varying levels of supporting evidence (0.24 > P > 2.8 × 10^-4) 6 12 20 21 . There is extensive linkage disequilibrium across the region, hampering fine-mapping efforts 13 , however it is clear that there are two independent autoimmune associated regions within the KIAA1109-TENR-IL2-IL21 gene cluster. Here, we aimed to consolidate all available data on two SNPs independently associated with autoimmunity within the KIAA1109-TENR-IL2-IL21 gene cluster: rs6822844 (minor allele protective) and rs17388568 (minor allele susceptible), each into a single meta-analysis of association with RA that included previously published data, new genotype data from Australasia, and publicly-available data from the Wellcome Trust Case Control Consortium (WTCCC) 22 .

We examined rs6822844, rs17388568 and rs907715. The latter SNP was chosen because it had been associated with risk of the systemic autoimmunity SLE in a single study 17 (OR = 0.78, P = 0.002), was in weak LD with both rs6822844 and rs17388568 and we hypothesized that it could represent a possible third effect within the KIAA1109-TENR-IL2-IL21 cluster, perhaps specific to systemic autoimmunity. The imputed genotype data from the WTCCC RA case-control sample revealed no significant association between rs6822844 and RA (Table 2; OR = 0.91 (0.82 to 1.02); P = 0.10) (the WTCCC samples did not overlap with those analysed in Barton et al. 12 ). SNP rs6822844 had been genotyped in the NARAC sample set 3 , revealing nominal evidence for a protective effect of the minor allele (Table 2; OR = 0.84 (0.74 to 0.96), P = 0.011). We then genotyped rs6822844 across the Australasian case-control sample set, finding no evidence for association between rs6822844 and RA (Table 2; OR = 0.95 (0.80 to 1.12), P = 0.54) although, consistent with the other association studies, the OR was less than one. Meta-analysis of all available data was undertaken (Figure 1). Zhernakova et al. 21 and Coenen et al. 28 both reported association of the KIAA1109-TENR-IL2-IL21 region with RA in overlapping Dutch case-control cohorts. We used data from the former study, as it was the only one to type rs6822844. The meta-analysis provided very strong (genome-wide) support for rs6822844 playing a role in the development of RA (OR = 0.86 (0.82 to 0.91), P = 8.8 × 10^-8). The NARAC GWAS data (OR _rs6822844 = 0.84 (0.74-0.96), P = 0.011) 7 were combined with the meta-analysis result, yielding P = 2.1 × 10^-8.

For rs17388568, the genotype data from the WTCCC case-control sample revealed a weak association between rs17388568 and RA, with a susceptibility effect of the minor allele (Table 3; OR = 1.14 (1.04 to 1.25), P = 0.005). We genotyped rs17388568 across the Australasian case-control sample set, finding no evidence for association between rs17388568 and RA (Table 3; OR = 1.03 (0.90 to 1.19), P = 0.66), a result that was consistent with a study of a UK case-control sample set by Barton et al. 12 (Table 3; OR = 0.97 (0.91 to 1.05), P = 0.47). Meta-analysis of all the available data was done (Figure 2), with the combined analysis showing no significant role for rs17388568 in the development of RA (OR = 1.03, (0.98 to 1.09), P = 0.22). At rs907715, the imputed WTCCC genotype data also revealed a weak association (Table 3; OR = 0.92 (0.84 to 1.00), P = 0.055). Genotyping of the Australasian sample set and combined analysis with the WTCCC data (Table 3; Figure 3) slightly weakened the evidence for association of rs907715 with RA (OR = 0.93 (0.87 to 1.00), P = 0.07). Neither rs17388568 nor rs907715 nor any surrogate SNP was present in the NARAC data. Given the moderate LD between rs907715 and rs6822844 (Table 1), rs907715 was tested for association in the combined WTCCC and Australasian samples conditional on genotype at rs6822844. This revealed that the trend towards association seen for rs907715 was not independent of the association seen at rs6822844 (P = 0.38).

The Australasian sample set was further examined by stratifying rs6822844 according to gender, RF, CCP and SE status (Table 4). This revealed no specific association to any particular sub-phenotypes analysed (P > 0.05). It should be noted that the power to detect association with sub-phenotype within the Australasian sample set was limited; for example, in the analysis with the largest amount of data available (RF) there was adequate power (> 70%) to detect an allele frequency difference between RF positive and negative cases only when the difference was equivalent to an OR > 1.5.

Here we have combined published studies testing association of two SNPs, rs6822844 and rs17388568, located within the KIAA1109-TENR-IL2-IL21 region with RA: the former with two Dutch 6 21 , one UK 12 , and one Western Europe sample set 20 ; and the latter with one UK sample set 12 - along with new Australasian data, and data from the WTCCC 22 . Meta-analysis (Figure 1) showed a consistent protective effect for the minor allele of rs6822844 with RA (OR = 0.86, P = 8.8 × 10^-8). A similar protective effect was also observed in the NARAC GWAS for rs6822844 (OR = 0.84, P = 0.011), with combined evidence for association with RA of P = 2.1 × 10^-8 when combined with our meta-analysis P- value. Our analysis used the same European Caucasian RA sample sets recently meta-analysed by Maita et al. 29 , with the addition of the WTCCC, Barton et al. 12 , Australasian and NARAC 3 data. Meta-analysis of the T1D-associated SNP rs17385868 (Figure 2) on the other hand, did not reveal any significant association between the minor allele of rs17388568 and RA (OR = 1.03, P = 0.22). Note that SNP rs4505848, in strong LD with rs17385868, is more strongly associated with T1D (Table 1). Whilst there was weak evidence for association at rs907715 (Table 3), in a direction consistent with that previously observed in SLE 17 , this effect appears to be dependent on rs6822844. When this paper was under review a genome-wide association scan meta-analysis in RA was published, in 5,539 autoantibody positive cases and 20,169 controls of European descent 30 , with P = 7 × 10^-4 at rs6822844. We meta-analysed these data with data presented in Table 2 (removing the overlapping WTCCC samples), yielding OR = 0.86 (0.82 to 0.89), P < 1 × 10^-10. The rs6822844 data provide compelling evidence supporting a role for the KIAA1109-TENR-IL2-IL21 locus in etiology of RA in Caucasian populations. Including the HLA region and the 10 loci confirmed by Stahl et al. 30 (note that Stahl et al. did not confirm KIAA1109-TENR-IL2-IL21 at a genome-wide level of significance), KIAA1109-TENR-IL2-IL21 is the 20^th locus associated with RA at a genome-wide level of significance (P ≤ 5 × 10^-8). The association at rs6822844 dominates at this locus, with no evidence for an independent effect at rs17385868, as is seen in T1D (Table 1) 13 .

The KIAA1109-TENR-IL2-IL21 region was first implicated in autoimmunity after a GWAS in T1D 13 22 and has since been associated, also with a genome-wide level of support, with celiac disease 16 and ulcerative colitis 14 and, with lower supporting evidence, with SLE, psoriatic arthritis, Graves' disease and juvenile idiopathic arthritis 13 17 18 19 (Table 1). The region is characterized by a high degree of linkage disequilibrium 13 , meaning that the underlying disease-causing variant(s) and gene(s) have not yet been determined. Collectively, these data point to at least two independent associations within the KIAA1109-TENR-IL2-IL21 region, with the pattern of association differing between autoimmune phenotypes (Table 1); one marked by rs17388568 (in the TENR gene) and the other by rs6822844 (which maps between IL-2 and IL-21). There is no appreciable linkage disequilibrium between rs17388568 and rs6822844 (r² = 0.07 in HapMap CEU samples). Different patterns of association are evident in the different autoimmune phenotypes. For example (referring to the risk conferred by the minor allele), susceptibility at rs17388568 and protection at rs6822844 is observed in T1D 13 21 , some evidence for protection is seen at rs17388568 in Graves' disease 13 , there is no evidence for association of rs17388568 (or markers in high LD) with RA (Figure 2) whereas rs6822844 confers protection (Table 1). The studies in Crohn's disease, ulcerative colitis, celiac disease, psoriatic arthritis, and JIA are consistent in reporting the rs6822844-mediated minor allele protective effect 14 15 16 18 19 (Table 1), with little data available on rs17388568 in comparison to rs6822844. The single study in SLE 17 did not include SNPs in LD with rs6822844, however there was evidence for a susceptibility effect at rs17388568 (using rs2221903 which is in strong LD with rs17388568, Table 1). Collectively, these studies point to heterogeneity at rs17388568 between RA and other autoimmune phenotypes (T1D, GD, SLE) with which RA shares other genes and clinical features.

Within the KIAA1109-TENR-IL2-IL21 gene cluster, IL-21 is of particular interest in the context of RA, and the Th1/Th17 axis in which IL-23R is involved. IL-21 is required for differentiation of naïve human CD4⁺ T cells into Th17 cells 31 , whereas IL-23 is critical in the expansion and maintenance of Th17 cells 32 33 . It is important to note that Th17 cells produce a variety of cytokines including IL-17A, IL-17F, IL-21 and IL-22. Human studies have demonstrated that IL-21 receptor (IL-21R)-positive cells are significantly increased in inflamed synovial tissues of RA patients compared to controls and that IL-21 enhances local T-cell activation, proliferation and proinflammatory cytokine secretion 34 35 . Alongside these findings, animal studies demonstrate that IL-21R deficient mice have normal T-cell and NK cell development but fail to develop spontaneous autoimmune disease suggesting that IL-21 plays a vital role in the development of autoimmune disease in rodents. Studies using arthritic mice and rats also demonstrate that inhibition of IL-21 expression correlates with modulation of serum IL-6 levels and improvements in disease severity 36 . However, it remains to be determined whether inhibition of IL-21 in humans with RA will have a similar beneficial effect given the significant differences between Th17 cell biology in mice and men.

Given the importance of Th17 cells in autoimmunity 37 , the differential genetic effects observed in various autoimmune phenotypes mediated by the IL23R and KIAA1109-TENR-IL2-IL21 regions, evidence for genetic interaction between the KIAA1109-TENR-IL2-IL21 region and IL23R (in ulcerative colitis at least) 38 , there are reasonable grounds for considering the hypothesis that genetic control of the Th1/Th17 axis is centered on cytokines (and their receptors) important in Th17 biology. It is important to note that not all RA patients have evidence of IL-17A within synovial tissue 39 and the role of IL-17A appears to be as an amplifier of inflammation rather than an absolute requirement for inflammation in RA. One possible explanation is genetic variation in the KIAA1109-TENR-IL2-IL21 locus that results in non-functional IL-21 and hence lack of IL-17A or vice versa. Regulation of this axis may be an important factor in determining the risk to particular autoimmune phenotypes, which may have implications for selection of targeted biological therapies within an individual. What will be important in understanding molecular control of autoimmunity will be association studies in large sample sets from different autoimmune phenotypes that comprehensively capture common variation in the IL23R and KIAA1109-TENR-IL2-IL21 loci, fine-mapping of the genetic effects and analysis of interaction between the disease-associated variants, both within and between loci.

Genotyping of an Australasian RA case-control sample set, and meta-analysis with published and publicly-available data confirm at a genome-wide level of significance the rs6822844 SNP within the KIAA1109-TENR-IL2-IL21 locus to be a risk factor in RA (P = 2.1 × 10^-8; OR = 0.86). There was no evidence for an independent effect on RA mediated by other variants within the KIAA1109-TENR-IL2-IL21 locus, as is seen in type 1diabetes.

BLK: B-lymphocyte kinase; CCP: cyclic citrullinated peptide; GD: Graves' disease; GWAS: genome-wide association scan; IL: interleukin; LD: linkage disequilibrium; NZ: New Zealand; OR: odds ratio; NARAC: North America Rheumatoid Arthritis Consortium; RA: rheumatoid arthritis; RF: rheumatoid factor; SE: shared epitope; SLE: systemic lupus erythematosus; SNP: single nucleotide polymorphism; STAT4: signal transducer and activator of transcription 4; T1D: type 1 diabetes; WTCCC: Wellcome Trust Case Control Consortium;

The authors declare that they have no competing interests.

JEH-M, MC-X and TRM planned the study design and oversaw its execution. RT provided technical and analytical support. JEH-M, MC-X, ND, PJG, AAH, JH, PBBJ, MN, MDS, AR, GJ and LKS took part in clinical recruitment and data acquisition. JEH-M, MC-X, RT, ND, PJG, AAH, JH, PBBJ, MN, MDS, AR, GJ, LKS and TRM prepared the manuscript.