A genome-wide association study of rheumatoid arthritis in 2418 cases and 4504 controls from North America identified an association at the REL locus, encoding c-Rel, on chromosome 2p13 (rs13031237, p=6.01 × 10-10). Replication in independent case-control datasets comprising 2604 cases and 2882 controls confirmed this association, yielding an allelic OR=1.25 (95%CI 1.177-1.318, p = 3.08 × 10-14) for marker rs13031237 and an allelic OR= 1.21 (95%CI 1.150-1.282, p = 2.60 × 10-11) for marker rs13017599 in the combined dataset. The combined dataset also provides definitive support for associations at both CTLA4 (rs231735; OR=0.85, 95% CI 0.81-0.90; p= 6.25 × 10-9) and BLK (rs2736340; OR= 1.19, 95% CI 1.125-1.268; p=5.69 × 10-9 ). C-Rel is an NF-kappaB family member with distinct functional properties in hematopoietic cells, and its association with RA suggests disease pathways that involve other recently identified RA susceptibility genes including CD40, TRAF1, TNFAIP3, and PRKCQ1,2.
Rheumatoid arthritis [RA (MIM 180300)] is a common autoimmune disorder affecting approximately 1% of populations of European origin and whose predominant manifestation is inflammation with bone and cartilage destruction in diarthrodial joints. The genetic basis for RA is complex, with at least six genes generally accepted as associated with disease in populations of European origin, including HLA-DRB1, PTPN22, STAT4, TRAF1, and TNFAIP31. A number of additional loci have recently been reported as a result of expanded genome-wide association studies3 and metanalyses2. Many of these are likely to reflect true associations, although a convincing demonstration often requires very large samples sizes, given that many of the associations at these loci are quite modest. In most cases the causative allele(s) have not been identified, and therefore the actual contribution to disease risk at these loci is unknown.
The diagnosis of rheumatoid arthritis is based on clinical criteria established over two decades ago4. However, these criteria do not yet include antibody reactivity to cyclic citrullinated peptides (CCP), the presence of which is a highly sensitive and specific marker for the diagnosis of RA; between 50 and 80% of patients meeting standard criteria for RA exhibit anti-CCP antibodies5. Remarkably, the classical HLA-DRB1 associations with RA are entirely restricted to this phenotypic subgroup6, as are many of the other reported genetic associations. In addition to this phenotypic heterogeneity, there is evidence for genetic heterogeneity in risk for RA among different racial groups7, and this has complicated efforts at replication. Given these considerations, it is apparent that additional risk genes for RA remain to be discovered. For these reasons we undertook an expansion of our previous genome-wide association study of rheumatoid arthritis8, restricted to North American cases of European origin that were overwhelmingly (~95%) anti-CCP antibody positive. We also assembled a large case-control population for replication studies.
All new genotyping of case samples for this study was performed on Illumina HapMap370 BeadArray typing platforms, and after quality control filtering (see Supplementary Methods), a combined dataset of 2418 RA cases and 4504 controls was available for WGA analysis that had been genotyped on 278502 SNPs that passed all quality control filters applied to each set of data. The cases are derived in part from affected sibling pair families of the North American Rheumatoid Arthritis Consortium (NARAC) previously reported8 (one case per family), as well as new collections from both the U.S. and Canada (see Supplementary Table 1A). The genome-wide lambda value inflation of chi-square values was calculated to be 1.21, allowing for the large sample size. Structured association was therefore applied to correct for population stratification by matching homogeneous clusters of cases and controls, as implemented in PLINK. The genome-wide lambda value after adjustment was calculated to be 1.06.
Figure 1 displays a graphical summary of the results of the genome-wide analysis as implemented in PLINK, after conditioning on clusters. As noted in previous studies, the largest association signal is in an extended region within the MHC achieving a maximal level significance of p=9.50 × 10-104 in this study. (The y axis is truncated at - log p=27 in Figure 1). In addition, the previously reported associations8 are confirmed at PTPN22 (rs2476601, p= 1.62 × 10-21) and the TRAF1/C5 regions (maximal association at rs881375, p= 4.09×10-8). However, after PTPN22 the second most significant association is observed on chromosome 2 in the region of REL, where two markers provide highly significant evidence of association (rs13031237, allele specific p=6.01 × 10-10 and rs13017599, allele specific p=9.05 × 10-9), as shown in Table 1. This is a novel finding, since REL has not been brought forward as a candidate region for RA risk by any of the previous association studies in RA, although a recent publication has reported an association with Coeliac disease9. At lower levels of significance, we also noted evidence of association (Table 1) with CTLA4 (rs 6748358, p= 8.24 × 10-5) and a SNP marker in the region of the BLK locus (rs2736340, p= 6.06 × 10-7). A complete list of results at significance level p<0.01 for the entire dataset is provided (Supplementary Table 2).
In order to establish that these results are robust to various analytic approaches, we also performed analysis using Eigenstrat. Before performing the PCA, we removed markers in the region of chromosome 8p that shows inversions in Northern Europeans (8.135-11.936 Mb) and markers in the centromeric region of chromosome 17q21.31 (40-43 Mb) that is polymorphic in European origin populations10. We also removed markers around the HLA region that are related to European ancestry and also rheumatoid arthritis risk (from 24-36 Mb). These HLA-related markers were also removed for analyses that estimate inflation of the genome-wide inflation of test statistics that can arise from differences in population ancestry, since the HLA region includes many hundreds of markers that are associated with RA risk6. The association tests with all markers (including those on chromosomes 6p, 8p, and 17p) were performed adjusting for the eigenvectors derived using Eigenstrat to remove population admixture effects. The lambda value was 1.20 prior to PCA correction and this value reduced to a lambda value of 1.06 after PCA correction. Results from Eigenstrat analysis along with trend tests from PLINK are presented in supplementary table 3. Despite some genome-wide excess in the expected number of positive results from tests for association, the specific findings for REL did not appear to be influenced substantially by population structure. As shown in supplementary table 3, we found that without and with adjustment for population structure the p-values and odds ratios associating the SNPs we queried were quite similar. For example, for rs13031237, the odds ratio and p-value without adjustment for population structure in the combined genome-wide association analysis was 1.278 (p=5.2 × 10-11) versus an odds ratio of 1.268 with adjustment (p=6.0 × 10-10).
In order to confirm the associations with REL, we carried out a replication study on independent sets of 2604 cases and 2882 controls from the U.S. and Canada (Supplementary Table 1B). While complete serologic data were not available for all subjects, the majority of cases were seropositive (either rheumatoid factor or CCP+) in the replication datasets. In addition to selected SNPs from the REL locus, we also included candidate SNPs from the CTLA4 and BLK regions. For technical reasons, slightly different SNP panels were utilized for the replication studies in the Canadian and U.S. samples, based on tagging LD provided in the HapMap, and the results from the separate datasets are provided in Supplementary Table 2. The combined results with common SNPs markers across all the datasets are shown in Table 1 for REL, CTLA4 and BLK. Two highly associated SNPs at the REL locus are observed in the combined data using a Cochrane-Mantel-Haenszel analysis to allow for stratification among populations (rs13031237, OR=1.24, p = 3.08 × 10-14 and rs13017599, OR=1.21, p= 2.06 × 10-12). A graphical representation of the association results across the REL locus is shown in Figure 2. In addition, the data in Table 1 (and supplemental Table 2) provide definitive evidence for the previously suggested association with CTLA4 (rs 231735, OR=0.86, p=6.25 × 10-9). The data also support BLK as a new RA risk locus (rs2736340, OR 1.19, p=5.69 × 10-9), a finding of some interest given the recent association of this locus with systemic lupus11,12. Supplementary table 4 presents genotype-specific results, which show codominance for all the loci except CTLA4, which is nearly dominant.
The nuclear factor-κB (NF-κB)/REL family of transcription factors contain five members including c-Rel, p65/Rel-A, Rel-B, p50/NFkB-1 and p52/NFkB-2. These factors have a central role in coordinating the expression of a wide variety of genes that control immune responses and autoimmunity13. Therefore, the identification of REL, encoding c-Rel, as a new risk locus for RA has provoked us to consider how this observation may fit in with pathways suggested by the complex emerging landscape of genetic susceptibility for RA. While the various NF-κB subunits have complex overlapping functions, current data suggest some distinct roles for c-Rel. The production of IL12 and IL23 subunits by macrophages and dendritic cells are critically dependent on c-Rel14,15. Thus, c-Rel knockout animals exhibit deficiencies in Th1-type immune responses, although intrinsic T cell defects may also contribute to this phenotype16. Intriguingly, both c-Rel and another recently identified RA risk gene, PRKCQ, are specifically involved in the survival of activated CD8 cells, at least in part through the regulation of IL2 production by these cells. In addition, a variety of genes in T cells are regulated by c-Rel, including CD40 and TNFAIP3, both of which are now accepted RA susceptibility loci17.
In addition to effects on T cell and antigen presenting cell function, c-Rel has been shown to have a role in B cell proliferation and survival that particularly involves CD40 signaling pathways. Specifically, c-rel deficient B cells are susceptible to BCR induced apoptosis that cannot be prevented by activation through CD4018. This is due to reduced expression of the anti-apoptotic protein Bcl-XL, a gene that is known to be regulated by c-Rel. Interestingly, rescue from Fas induced apoptosis is normal in these cells, demonstrating the existence of distinct CD40 signaling pathways that are at least in part distinguished by the involvement of c-rel. A similar c-rel associated difference in CD40 signaling has been seen in patients with ectodermal dysplasia and hyperIgM syndrome due to mutations in the NF-κB essential modulator, NEMO, where c-Rel dependent IL4 responses are also impaired19. C-Rel is the only NF-kappaB family member with oncogenic activity and the gene is amplified in some B cell lymphomas. Intriguingly, in both tumors and normal B cells it has recently been reported that the CD40 and c-Rel proteins can physically interact and form a heterodimer that is translocated to the nucleus20 and has transcriptional regulatory activity for known c-rel target genes, including CD154, BLyS/BAFF, and Bfl-1/A1.
The associations of CD402, REL, TRAF18 and TNFAIP3 21are consistent with an important role for CD40 signaling pathways in RA pathogenesis. Indeed, CD40-CD40 ligand interactions have previously been identified as a potential target for therapy in autoimmune disease22, and clinical trials in lupus have shown promise23. Unfortunately, the clinical development of monoclonal antibody inhibitors of CD40L was cut short by adverse effects on the platelet function associated with the development of thromboembolic complications. Nevertheless, this pathway remains viable as a therapeutic target, and the current results mandate a thorough analysis of all the genes in this pathway to search for additional susceptibility alleles