Extended report Power calculations were performed using the Quanto V.0.5 software (Department of Preventive Medicine University of Southern California, California, USA), considering minor allele frequencies and ORs similar to those found in previous studies 4 5 11 12 at the 5% signiﬁcance level. RESULTS We selected four polymorphisms previously reported as RA sus- ceptibility markers for testing in the current study: (1) rs4810485, located in the second intron of the CD40 gene; (2) rs2812378, mapping to the 5’ untranslated region of CCL21; (3) rs42041, positioned in a CDK6 intron11; (4) rs6682654, located in CD244, which is an almost perfect proxy of the functional RA marker for the Japanese popu- lation within this gene, rs3766379 (D′=1, r2=0.97).12 Genotype frequencies conformed to Hardy–Weinberg expec- tations in patients and controls for all the polymorphisms under study. The estimated power of the study was 86% for rs481048, 77% for rs2812378, 60% for rs42041 and 99% for rs6682654. Table 1 shows the results of this validation study. Allele fre- quencies were similar to those previously described in a US pop- ulation for the CD40, CCL21 and CDK6 markers.11 We found that the minor T allele of CD40 rs4810485 was signiﬁcantly increased in healthy controls compared to patients with RA (p=2×10−4, OR 0.86, 95% CI 0.79 to 0.93). This is in accordance with the previous study by Raychaudhuri et al. When we analysed CCL21 rs2812378, we observed a higher frequency of the minor C allele in patients with RA as previ- ously described,11 although this skewing seemed to have a more modest effect in our population (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). With regard to CDK6 rs42041, no evidence of associa- tion with RA was found in our UK population (table 1). Additionally, we examined the functional CD244 vari- ant rs6682654, which was previously shown to be associated with RA in a Japanese population.12 Linkage disequilibrium (LD) between this SNP and the other marker within CD244, rs3766379, also reported to be associated with RA in the Japanese study, is higher in the Caucasian population (r2=0.97, D′=1) than in the Japanese population (r2=0.87, D′=1).15 When we compared the distribution of allele and genotype frequen- cies of rs6682654 between patients and controls, we did not ﬁnd statistically signiﬁcant differences (table 1). Allele frequencies of the reference G allele were lower in the UK population than in the previously described Japanese population (0.43 and 0.44 in patients and controls, respectively, for the UK population vs 0.66 and 0.59 in patients and controls, respectively, for the Japanese population), which is in accordance with the data deposited in HapMap (http://www.hapmap.org). Stratiﬁcation analysis of the four polymorphisms under study showed that the rs4810485 CD40 SNP was strongly associated with the presence of RF and anti-CCP antibodies (p=8.10×10−6, OR 0.82, 95% CI 0.75 to 0.89 and p=9.52×10−5, OR 0.81, 95% CI 0.73 to 0.90, respectively). CCL21 rs2812378 also showed this trend, but p values for the association with seropositive dis- ease were modest (p=0.02, OR 1.10, 95% CI 1.01 to 1.18 and p=0.03, OR 1.10, 95% CI 1.01 to 1.21, for RF and anti-CCP RA, respectively) (supplementary table 2). Meta-analysis of these markers was undertaken, pooling data from the previously published RA GWASs,4 5 11 together with data from a validation cohort from the US11 and our validation study in a UK cohort. p Values for the Breslow–Day test were 0.31 for rs4810485, 0.81 for rs2812378 and 0.02 for rs42041 (table 2). The meta-analysis revealed strong evidence of associa- tion with the CD40 variant and a more modest, but suggestive association with the CCL21 variant. The evidence for associa- tion with CDK6 was relatively weak. The rs6682654 SNP mapping to CD244 and showing evi- dence for association with RA in a Japanese population12 was not tested in the meta-analysis by Raychaudhri et al.11 In order to investigate whether a weak effect on susceptibility could be detected in a UK population, we performed a meta-analysis of the variant, combining imputed data from the WTCCC dataset with that derived from direct genotyping in the current valida- tion study. Pooled analysis revealed no association with RA in the UK population (p=0.87, OR 1.004, 95% CI 0.95 to 1.06). Joint analysis of the Japanese and UK cohorts was not feasible, due to the high degree of genetic heterogeneity between the populations (pBreslow–Day=1.8×10−6). DISCUSSION In this large UK cohort, we have found strong association of the rs4810485 CD40 variant with susceptibility to RA, and weaker evidence for association with RA in the CCL21 locus. We found no evidence for association with either the CDK6 locus or the Table 1 Validation study of novel rheumatoid arthritis (RA) susceptibility markers in a UK population Locus SNP CD40 rs4810485 T/G CCL21 rs2812378 G/A CDK6 rs42041 G/C CD244 rs6682654 G/A mMainjoorr/ MAF MAF RA allele RA controls 11 12 Genotype frequency, no (%) Controls 22 11 12 22 p For allele p For OR (95% CI) for p For 12+22 allele genotypes vs 11 0.23 0.37 0.25 0.43 0.25 0.35 0.25 0.44 165 (4.5) 1334 (36.2) 2184 (59.3) 173 (5.7) 1189 (39.1) 1681 (55.2) 2×10−4 0.86 (0.79 to 0.93) 0.001 478 (12.9) 1752 (47.2) 1478 (39.9) 377 (12.5) 1339 (44.5) 1294 (43) 0.04 1.08 (1.01 to 1.16) 0.03 228 (6.1) 1402 (37.8) 2079 (56.1) 182 (6.1) 1125 (38) 1655 (55.9) 0.91 0.99 (0.92 to 1.08) 0.99 638 (18.1) 1756 (49.8) 1132 (32.1) 571 (19.9) 1394 (48.5) 909 (31.6) 0.20 0.96 (0.89 to 1.02) 0.19 0.02 0.65 0.99 0.07 MAF, minor allele frequency; SNP, single nucleotide polymorphism. Meta-analysis of newly identiﬁed rheumatoid arthritis (RA) markers in populations of European ancestry GWAS meta-analysis11 US validation11 UK validation Meta-analysis SNP rs4810485 rs2812378 rs42041 p Value 2.4×10−7 6.9×10−5 5.5×10−5 OR 0.83 1.13 1.15 p Value 0.003 9.7×10−4 0.01 OR 0.91 1.10 1.08 p Value 2×10−4 0.04 0.91 OR (95% CI) 0.86 (0.79 to 0.93) 1.08 (1.01 to 1.16) 0.99 (0.92 to 1.08) p Value 7.8×10−8 1.2×10−4 0.002 OR (95% CI) 0.87 (0.83 to 0.92) 1.09 (1.04 to 1.14) 1.08 (1.03 to 1.14) Breslow–Day p value 0.31 0.81 0.02 GWAS, genome-wide association studies; SNP, single nucleotide polymorphism. Table 2 Locus CD40 CCL21 CDK6 814 Ann Rheum Dis 2010;69:813–816. doi:10.1136/ard.2009.109579