Two independent alleles at 6q23 associated with risk of rheumatoid arthritis

Abstract

To identify susceptibility alleles associated with rheumatoid arthritis, we genotyped 397 individuals with rheumatoid arthritis for 116,204 SNPs and carried out an association analysis in comparison to publicly available genotype data for 1,211 related individuals from the Framingham Heart Study. After evaluating and adjusting for technical and population biases, we identified a SNP at 6q23 (rs10499194, approximately 150 kb from TNFAIP3 and OLIG3) that was reproducibly associated with rheumatoid arthritis both in the genome-wide association (GWA) scan and in 5,541 additional case-control samples (P = 10(-3), GWA scan; P < 10(-6), replication; P = 10(-9), combined). In a concurrent study, the Wellcome Trust Case Control Consortium (WTCCC) has reported strong association of rheumatoid arthritis susceptibility to a different SNP located 3.8 kb from rs10499194 (rs6920220; P = 5 x 10(-6) in WTCCC). We show that these two SNP associations are statistically independent, are each reproducible in the comparison of our data and WTCCC data, and define risk and protective haplotypes for rheumatoid arthritis at 6q23.

Figure 1

Q-Q plots of GWA analyses in unrelated individuals: influence of missing genotype data and population stratification. We conducted GWA analysis of BRASS rheumatoid arthritis cases compared to unrelated FHS controls. Light blue diamonds indicate SNPs within the extended MHC region (defined as chromosome 6, 25–35 Mb), pink diamonds indicate non-MHC SNPs and red diamonds indicate non-MHC SNPs following correction by dynamic genomic control (corr). (a,b) 88,000 (88K) SNP panel (a; >90% call rate) and 41K SNP panel (b; >99% call rate) with no attempt to correct for population stratification. P values were generated by 2 × 2 contingency tables of allele frequency (χ² test). The 88K SNP panel captures ∼30% and the 41K panel ∼18% of common HapMap variants at an r² > 0.80. (c) 41K SNP panel (>99% call rate), with correction for population stratification with PLINK CMH. Few non-MHC SNPs are observed in the tail of the statistical distribution, and λ_GC = 1.04, indicating adequate control of bias. (d,e) 80K SNP panel (>95% call rate) in unrelated FHS controls (d) and related FHS controls (e), obtained by applying a linear model fit for missing data and minor allele frequency interaction (dynamic genomic control). MHC SNPs have been excluded, and correction for population stratification has been applied with PLINK CMH. After applying dynamic genomic control (red diamonds), few non-MHC SNPs are observed in the tail of the statistical distribution, and λ_GC = 1.08. A similar pattern is observed in analysis of related individuals (and after correction for inflation due to relatedness among controls). Many (5 of 8) of the non-MHC SNPs with P < 10⁻⁵ were rare alleles (MAF < 0.05). In contrast, when call rate is uncorrected by the linear model, deviation from the null is observed at P < 0.01. The 80K SNP panel captures ∼29% of common HapMap variants.

Figure 2

Case-control association results and linkage disequilibrium (LD) structure at 6q23. Results for SNPs genotyped across 1 Mb as part of the original GWA scan in 397 CCP⁺ rheumatoid arthritis cases and 1,211 related controls (gray diamonds), as well 17 SNPs genotyped in additional replication samples (2,283 unrelated CCP⁺ rheumatoid arthritis cases and 3,258 unrelated controls). In the replication samples, the color of each diamond is based on r² (CEU HapMap) with the most significant SNP in our study (rs10499194). The blue diamond indicates the P value for all samples in our study (original GWA scan plus replication samples), as determined by Fisher's method of combining P values (EIGENSTRAT in both original GWA scan and replication samples). The recombination rate based on CEU HapMap is shown in light blue along the x axis (scale on the right); the red line indicates a 63-kb region of strong LD used to construct haplotypes. The green arrows indicate gene location; the associated SNP is ∼185 kb from either TNFAIP3 or OLIG3.

Figure 3

Haplotype analysis in our replication samples and in the WTCCC study of ∼2,000 individuals with rheumatoid arthritis and ∼3,000 controls. Haplotype analysis with 17 genotyped SNPs and 3 imputed SNPs across a 63-kb region of strong LD in our replication samples (2,283 unrelated CCP⁺ rheumatoid arthritis cases and 3,258 unrelated controls) yielded six haplotypes with population frequency >5% (constituting 96% of all observed haplotypes). When expressed relative to the minor allele, two haplotypes tagged by rs10499194 are ‘protective’ (haplotypes E and F) and a single haplotype tagged by rs6920220 provides ‘risk’ (haplotype B). (a) The haplotype group, risk category and frequency of all samples are shown. The P value (P) and odds ratio (OR) for each haplotype were calculated by comparing each haplotype to all others, using the statistical program WHAP. The highlighted SNPs (in order: rs1878658, rs675520, rs9376293, rs10499194, rs6920220 (imputed)) define the six common haplotypes. The 11 SNPs within the box were used to define haplotype phylogeny in b. (b) Five SNPs served to uniquely identify the phylogeny of the six common haplotypes. Haplotype frequencies (cases and controls) and P values from single-marker analysis in our replication samples or from the WTCCC study (where rs13207033 is the WTCCC SNP) are shown.