Rheumatoid arthritis (RA) is a common autoimmune inflammatory disease of unknown cause, in which both genetic and environmental risk factors have been implicated 1. Since the genetic contribution to RA has been estimated to be between 50% and 60%, identification of genes contributing to disease is important for the understanding of underlying biological mechanisms 2.

In addition to human leucocyte antigen (HLA) 3 – the first identified genetic risk factor – and four other replicated regions – including the protein tyrosine phosphatase N22 gene (PTPN22) 4, the TNF receptor-associated factor 1 gene (TRAF1)/complement component factor 5 (C5) locus 567, the 6q23 locus near the TNFα-induced protein 3 gene (TNFAIP3) 89, and the signal transducer and activator of transcription 4 (STAT4) gene 1011 – a new genetic region associated with RA was described in the Dutch population 12. This region encompassing KIAA1109/Tenr/IL2/IL21 is contained in a large block (480 kb) of linkage disequilibrium located on chromosome 4q27 and includes the IL2 and IL21 genes, which are both plausible functional candidate loci for RA. Five SNPs (rs4505848, rs11732095, rs6822844, rs4492018 and rs1398553) of the block were investigated in a sample set consisting of 1,047 RA patients and 929 controls, showing a significant association of the rs6822844-T allele and the rs6822844-GT genotype (P = 0.0002, odds ratio (OR) = 0.72, 95% confidence interval (CI) = 0.61 to 0.86; and P = 0.0009, OR = 0.71, 95% CI = 0.58 to 0.87, respectively) and of a specific haplotype (AATGG) (P = 0.00025, OR = 0.70, 95% CI = 0.57 to 0.85) with RA 12.

Furthermore, in the North American Rheumatoid Arthritis Consortium and in the Epidemiological Investigation of Rheumatoid Arthritis populations, rs6822844 and rs1398553 SNPs were significantly associated with RA (P = 0.01, OR = 0.84, 95% CI = 0.74 to 0.96; and P = 0.003, OR = 1.17, 95% CI = 1.05 to 1.30, respectively) 6. In addition, the last meta-analysis performed with the North American Rheumatoid Arthritis Consortium, the Epidemiological Investigation of Rheumatoid Arthritis and the Wellcome Trust Case Control Consortium studies, with 3,393 RA patients and 12,462 controls, observed evidence of association of 4q27 with RA in an independent replication, suggesting that 4q27 is a true-positive association 13.

Given that association studies compare the frequencies in patients versus healthy individuals, unknown biases in control frequencies may lead to spurious associations. Family-based association studies therefore remain important to definitively establish association, especially when small effect sizes as well as variability in allele frequency in different populations are observed. With the aim to further substantiate the association at the KIAA1109/Tenr/IL2/IL21 gene region, we therefore took advantage of one of the largest reported European family resources dedicated to RA family-based studies.

A recent case–control study in the Dutch population has shown association of RA, celiac disease and type 1 diabetes with the 4q27 gene region. In that study the rs6822844-T allele was reported to be a perfect proxy for a haplotype that is highly associated with autoimmune diseases with frequencies of 0.13 in cases versus 0.19 in North European controls 12.

In addition, a study from the Wellcome Trust Case Control Consortium identified this 4q27 region in a search for risk factors for type 1 diabetes 22. In a follow-up study, some support for this association with type 1 diabetes was provided 23. The last meta-analysis of data from three genome-wide association studies of type 1 diabetes – testing 305,090 SNPs in 3,561 type 1 diabetes cases and 4,646 controls of European ancestry – obtained further support for 4q27 type 1 diabetes association (P = 1.9 × 10^-8), indicating that this is a true type 1 diabetes locus 24. The same region was studied in inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Four SNPs were genotyped in three cohorts concerning 4,782 cases (3,194 ulcerative colitis, 1,588 Crohn's disease) and 2,616 controls. All four SNPs were strongly associated with ulcerative colitis and moderately associated with Crohn's disease 25. The 4q27 locus was also reported to be associated with celiac disease 26. Recent evidence is also provided of a role for this region in psoriasis and psoriatic arthritis 27. Finally, a genetic association with systemic lupus erythematosus and two SNPs located within the IL-21 gene was found in a case–control study (1,318 cases and 1,318 controls) 28. All of these studies provide evidence that 4q27 seems to be a common locus for multiple forms of autoimmune diseases.

In our family-based study, compared with the Dutch study, we have detected a similar undertransmission of the rs6822844-T allele, the rs6822844-GT genotype and the AATGG haplotype, but without significance. Furthermore, we observed evidence of association between RA and the rs4505848-AG genotype, but no significance was found after Bonferroni correction. These results exclude the 4q27 locus from being a major RA genetic region, but a minor association should be not excluded and needs to be tested in a larger RA trio sample. Furthermore the already cited last RA meta-analysis, suggesting a 4q27 true-positive association, was performed in a large number of individuals. For the rs4572894 SNP tested, there was only a 2% variation between RA and non-RA minor allele frequency (27% versus 29%, respectively) with a P value of 1.1% 13. The putative effect of the 4q27 locus in RA is therefore very small and difficult to replicate in our RA family sample. In addition, a two-stage genome-wide association study of RA in the Spanish population did not found 4q27 as a RA susceptibility gene region 29.

The long region of linkage disequilibrium at chromosome 4q27 contains several genes: testis nuclear RNA-binding protein, a gene encoding a protein of unknown function (KIAA1109), and genes encoding the IL-2 and IL-21 cytokines. Testis nuclear RNA-binding protein is expressed primarily in the testis and KIAA1109 transcripts are ubiquitous, hence their roles in autoimmunity are not particularly compelling. Both IL-2 and IL-21 belong to the type 1 cytokine family, share a large degree of homology, and possess pleotropic functions in immune cells 26. These two genes are both plausible functional candidates as genetic modifiers of autoimmunity, and thus have particular interest to RA.

IL-2 exerts its effects on many cell types, the most prominent of which is the T lymphocyte. Accordingly, a major function of IL-2 is to promote proliferation and expansion of both antigen-specific clones of CD4⁺ and CD8⁺ T cells as well as to induce production of other cytokines. In CD4⁺ T cells, IL-2 plays a nonredundant role in the development of CD4⁺ CD25⁺ regulatory T cells. Accumulating evidence supports CD4⁺ CD25^high regulatory T cells playing an essential role in controlling and preventing autoimmunity 30. In addition, the IL2 receptor (CD25) susceptibility locus has recently been reported to be associated with RA 22.

IL-21 is involved in both cell-mediated and humoral responses and has a pleiotropic effect on a variety of immune and nonimmune cells. In RA, the synovial fluid and tissue have enhanced inflammatory responses to IL-21 and elevated IL-21 receptor expression. In two animal models – collagen-induced arthritis and adjuvant-induced arthritis – treatment with an IL-21-blocking agent ameliorated disease and/or reversed established disease, and also lowered levels of IL-6 and IL-17 31.

In the present study, using a family-based approach, we have provided a trend for the association of the KIAA1109/Tenr/IL2/IL21 gene region with RA in European descent populations. Nevertheless, we failed to replicate a significant association of this region in our RA family sample. Further investigation of this region, including detection and testing of all variants, is required to confirm RA association.

CI: confidence interval; IL: interleukin; OR: odds ratio; RA: rheumatoid arthritis; SNP: single nucleotide polymorphism.

The authors declare that they have no competing interests.

VHT carried out the molecular genetic studies. VHT and EP-T carried out the design of the study and drafted the manuscript. VHT, EP-T, BP and CP performed analysis of the data. PM, AB, RW, PB, HA, CV, MF, DP-S, SB, JD, TRR, PVR, LvdP, AL-V, TB and FC contributed to the recruitment of families and to the acquisition of clinical data. All authors read and approved the final manuscript.

The European Consortium on Rheumatoid Arthritis Families initiated with funding from the European Commission (BIOMED2) is coordinated by FC.