Rare, Low-Frequency, and Common Variants in the Protein-Coding Sequence of Biological Candidate Genes from GWASs Contribute to Risk of Rheumatoid Arthritis  Dorothée Diogo, Fina Kurreeman, Eli A. Stahl, Katherine P. Liao, Namrata Gupta, Jeffrey D. Greenberg, Manuel A. Rivas, Brendan Hickey, Jason Flannick, Brian Thomson, Candace Guiducci, Stephan Ripke, Ivan Adzhubey, Anne Barton, Joel M. Kremer, Lars Alfredsson, Shamil Sunyaev, Javier Martin, Alexandra Zhernakova, John Bowes, Steve Eyre, Katherine A. Siminovitch, Peter K. Gregersen, Jane Worthington, Lars Klareskog, Leonid Padyukov, Soumya Raychaudhuri, Robert M. Plenge  The American Journal of Human Genetics  Volume 92, Issue 1, Pages 15-27 (January 2013) DOI: 10.1016/j.ajhg.2012.11.012 Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 1 Description of the Study Design Our study used two sources of data: (1) we sequenced the coding exons of 25 genes located within RA risk loci identified by GWASs, leading to the identification of 281 protein-coding variants (top panel: distribution shown for MAF in controls); and (2) we used integrated Immunochip and GWAS data for 10,609 seropositive RA cases and 35,605 controls and focused only on the protein-coding variants from these same 25 genes. We performed three types of analyses: (1) To test for association, we investigated burden association signals driven by an accumulation of rare variants (frequency < 0.5%). (2) We assessed the role of low-frequency (0.5%–5%) and common (frequency > 5%) variants with weak effect in RA by adjusting for the common SNP identified by GWASs. (3) For detailed analysis, we selected the CD2 locus, which showed suggestive evidence of an independent signal of association in the conditional analysis. The American Journal of Human Genetics 2013 92, 15-27DOI: (10.1016/j.ajhg.2012.11.012) Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 2 Accumulation of Coding Rare Variants in IL2RA and IL2RB (A) Burden association signal driven by nonsynonymous variants. Two types of tests were performed: unweighted (UW) tests and tests weighted (W) with PolyPhen scores. For these two genes, we did not obtain any result by using the C-alpha method because it did not include singletons. (B) Accumulation of rare variants exclusive to RA cases in IL2RA and IL2RB. (C and D) Distribution of variants across IL2RA and IL2RB. Missense, nonsense, and synonymous variants are shown in red, brown, and green, respectively. For the missense variants, PolyPhen prediction is indicated (B, benign; d, potentially damaging; and D, probably damaging). Variants included in the collapsing tests and burden tests (i.e., variants not described in the 1000 Genomes Project or in dbSNP) are highlighted with a star. The American Journal of Human Genetics 2013 92, 15-27DOI: (10.1016/j.ajhg.2012.11.012) Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 3 Enrichment of Nominal Association Signal Driven by Nonsynonymous and Synonymous Variants in the Conditional Analysis The numbers of nonsynonymous variants (A) and synonymous variants (B) reaching the p < p threshold in our conditional analysis or after 1,000 permutations of the phenotypes are shown. Significant enrichment of SNPs with the p < p threshold in our conditional analysis was assessed by a Fisher’s exact test (∗p < 0.05 and ∗∗p < 0.01). The American Journal of Human Genetics 2013 92, 15-27DOI: (10.1016/j.ajhg.2012.11.012) Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 4 Evidence of an Independent Signal of Association at the CD2 Locus (A and B) Association results from the meta-analysis (A) and the conditional analysis (B). In these analyses, missense SNP rs699738 is represented by a group of SNPs, including rs798036 (highlighted in blue), in perfect LD (r2 = 1). In each analysis, the best signal of association is indicated by a diamond. Only SNPs present in more than five collections are shown. (C) ORs and 95% confidence interval in the independent cohorts and the meta-analysis. (D) Results from the haplotype analysis using the best signal in the meta-analysis (rs624988) and rs798036. In this analysis, only genotype data were used (7,222 RA cases and 15,870 controls). With this subset of samples, rs624988 and rs798036 reached p = 1.3 × 10−5 and p = 2 × 10−3 in the meta-analysis, respectively. The overall CD2 variation due to rs624988 and rs798036 contributed to RA with p = 4 × 10−6. The RA risk allele is highlighted in red. The American Journal of Human Genetics 2013 92, 15-27DOI: (10.1016/j.ajhg.2012.11.012) Copyright © 2013 The American Society of Human Genetics Terms and Conditions