Volume 3 Supplement 7
A search for non-chromosome 6 susceptibility loci contributing to rheumatoid arthritis
© Suarez et al; licensee BioMed Central Ltd. 2009
Published: 15 December 2009
We conducted a search for non-chromosome 6 genes that may increase risk for rheumatoid arthritis (RA). Our approach was to retrospectively ascertain three "extreme" subsamples from the North American Rheumatoid Arthritis Consortium. The three subsamples are: 1) RA cases who have two low-risk HLA-DRB1 alleles (N = 18), 2) RA cases who have two high-risk HLA-DRB1 alleles (N = 163), and 3) controls who have two low-risk HLA-DRB1 alleles (N = 652). We hypothesized that since Group 1's RA was likely due to non-HLA related risk factors, and because Group 3, by definition, is unaffected, comparing Group 1 with Group 2 and Group 1 with Group 3 would result in the identification of candidate susceptibility loci located outside of the MHC region. Accordingly, we restricted our search to the 21 non-chromosome 6 autosomes. The case-case comparison of Groups 1 and 2 resulted in the identification of 17 SNPs with allele frequencies that differed at p < 0.0001. The case-control comparison of Groups 1 and 3 identified 23 SNPs that differed in allele frequency at p < 0.0001. Eight of these SNPs (rs10498105, rs2398966, rs7664880, rs7447161, rs2793471, rs2611279, rs7967594, and rs742605) were common to both lists.
Rheumatoid arthritis (RA) is a chronic inflammatory disorder in which the articular joints are gradually destroyed. Occasionally there is systemic involvement, which can include pulmonary fibrosis and vasculitis in various organs. The etiology of RA is complex, with significant genetic and environmental components.
Among the genetic components, genes in the MHC region on chromosome 6p21.3 are widely acknowledged to be the major player, with the HLA-DRB1 locus as the leading suspect [1, 2]. Five alleles dramatically increase risk (DRB1*0401, *0404, *0405, *0408, and *0409), while other alleles appear to confer a moderate increase in risk (DRB1*0101, *0102, *0104, *0105, *1001, *1402, and *1406). In what follows we denote the high-risk alleles as H, the moderate risk alleles as M and the low-risk alleles as L. Most of the high-risk alleles possess a shared epitope (SE) of five amino acids at positions 70-74 in the third hypervariable region of the DRβ1chain . Genotypes consisting of alleles *0401/DRX (where DRX denotes a non-SE allele) and *0404/DRX are estimated to increase the relative risk of RA by 4.7-fold and 5.0-fold, respectively, while the *0401/*0401 genotype carries a relative risk of 18.8 and the compound heterozygote *0401/*0404 has a relative risk of 31.0 [4, 5]. We included in the high-risk group individuals whose genotype was reported as 4/4 or 4/*0401.
Not all persons with RA possess one or two high-risk SE alleles, however. It is estimated that in persons of European ancestry, approximately 30% do not carry an SE-encoding allele . In the data made available to the Genetic Analysis Workshop 16 participants, the percentage of cases carrying two low risk DRB1 alleles is much smaller than 30%. We hypothesize that RA cases with two non-SE encoding DRB1 alleles constitute a subgroup of patients that are enriched for other susceptibility alleles located elsewhere in the genome. Accordingly, we undertook a genome-wide association (GWA) analysis that compares single-nucleotide polymorphism (SNP) allele frequencies in two groups of cases-those whose DRB1 genotype contains two high-risk alleles (HH) and those whose DRB1 genotype contains two low-risk alleles (LL)-and one group of controls whose DRB1 genotype also consists of two LL alleles.
Sample size for various subdivisions of the NARAC data
There is some evidence for population substructure in this sample-perhaps occasioned by the fact that patients were drawn from rheumatology clinics across North America, while all of the controls were selected from participants who were part of the New York Cancer Project . We searched for systematic differences in the clinical variables that might distinguish the two case subsamples we compared in this study. We also undertook an analysis of substructure for the two case samples based on SNPs. We selected every 50th SNP from all of the autosomes, except chromosome 6, for a sample of 9,920 SNPs, and performed an EIGENSTRAT analysis .
Distribution of three clinical variables in high- and low-risk cases
Mean (± SD)
Sex ratio (Male:female)
Results of the GWA analyses for SNPs that differed between the comparison groups at p < 0.0001: minor allele frequency in LL cases
Physically closes gene
LL cases vs. HH cases
LL cases vs. LL cases
We are, of course, aware that a sample of N = 18 cases is extremely small by today's standards. Nonetheless, we are mindful that even small samples can be useful if the genetic effects are large enough. For instance, Cudworth and Woodrow  were able to confirm the involvement of the MHC in type I diabetes using a sample of only 17 affected sib-pair families. Moreover, many of the early gene expression studies employed very small sample sizes, yet provided useful insights into various expression patterns.
Although it is customary to report SNP allele frequencies in terms of the MAF (as we did for the LL cases in Table 3), our hypothesis predicts higher allele frequencies for non-chromosome 6 susceptibility genes in RA subjects who have LL genotypes at the HLA-DRB1 locus. For all of the SNPs listed in Table 3, the MAF in the LL cases are less than the comparison group. Accordingly, we would predict that the opposite SNP allele in the LL cases is probably in linkage disequilibrium with alleles at a functional gene.
Some of the SNPs listed in Table 3 are either in the same gene (e.g., TRIO, where all of the listed SNPs are intronic) or are close to the same gene (e.g., SLC6A15 and FBXL17). We carried out an analysis of linkage disequilibrium and estimated the r2 values between these adjacent SNPs from the NARAC control panel and obtained estimates of r2 = 1.0 for the two SNPs near FBXL17, r2 = 0.384, 0.949, 0.821, 0.801, and 0.734 for the five pairs of adjacent SNPs from TRIO (from rs173948 to rs27114), and r2 = 0.975, 0.837, 0.997, and 0.960 for the four pairs of adjacent SNPs near SLC6A15.
Previously identified non-chromosome 6 loci or regions that contribute to the risk of RA
Type of variation
Tokuhiro et al. 
Tokuhiro et al. 
Production of RA-specific autoantibodies
Suzuki et al. 
Non-synonymous coding SNP
Begovich et al. 
SNP in the type III of the MHC class II transactivator
Swanberg et al. 
Remmers et al. 
A 100-kb region
Plenge et al. 
We carried out a GWA study using an extreme sampling design on three subsets of the NARAC data. We identified 17 SNPs and 23 SNPs that distinguished the LL cases from the HH cases, and the LL cases from the LL controls at p < 0.0001, respectively. Eight SNPs are common to both lists, although the presence of significant linkage disequilibrium suggests that the actual overlap would be less. We used a variety of sources to identify the nearest gene, or predicted gene [15–19]. For some of these SNPs, however, there are multiple genes in their vicinity.
List of abbreviations used
genome wide association
Minor allele frequency
North American Rheumatoid Arthritis Consortium
The Genetic Analysis Workshops are supported by NIH grant R01 GM031575 from the National Institute of General Medical Sciences. Additional support was obtained from the Urological Research Foundation and from NIH grants K01 AA015572, K25 GM069590, R03 DA023166, and IRG-58-010-50 from the American Cancer Society.
This article has been published as part of BMC Proceedings Volume 3 Supplement 7, 2009: Genetic Analysis Workshop 16. The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/3?issue=S7.
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