▶ Additional data (supplementary information and supplementary tables) are published online only at http:// ard.bmj.com/content/vol69/ issue5 1arc Epidemiology Unit, The University of Manchester, Manchester, UK 2School of Medicine and Biomedical Sciences, The University of Shefﬁeld, Shefﬁeld, UK 3NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK 4Clinical and Academic Rheumatology, Kings College Hospital NHS Foundation Trust, Denmark Hill, London, UK 5Bone Research Group, Department of Medicine and Therapeutics, University of Aberdeen, UK 6University of Oxford Institute of Musculoskeletal Sciences, Botnar Research Centre, Oxford, UK Correspondence to Dr Gisela Orozco, arc Epidemiology Unit, Faculty of Medical and Human Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK; gisela.orozco@manchester. ac.uk Accepted 3 May 2009 Published Online First 11 May 2009 Extended report Association of CD40 with rheumatoid arthritis conﬁrmed in a large UK case-control study Gisela Orozco,1 Steve Eyre,1 Anne Hinks,1 Xiayi Ke,1 Wellcome Trust Case Control Consortium, YEAR consortium, Anthony G Wilson,2 Deborah E Bax,2 Ann W Morgan,3 Paul Emery,3 Sophia Steer,4 Lynne Hocking,5 David M Reid,5 Paul Wordsworth,6 Pille Harrison,6 Wendy Thomson,1 Anne Barton,1 Jane Worthington1 ABSTRACT Objective A recent meta-analysis of published genome- wide association studies (GWAS) in populations of European descent reported novel associations of markers mapping to the CD40, CCL21 and CDK6 genes with rheumatoid arthritis (RA) susceptibility while a large- scale, case-control association study in a Japanese population identiﬁed association with multiple single nucleotide polymorphisms (SNPs) in the CD244 gene. The aim of the current study was to validate these potential RA susceptibility markers in a UK population. Methods A total of 4 SNPs (rs4810485 in CD40, rs2812378 in CCL21, rs42041 in CDK6 and rs6682654 in CD244) were genotyped in a UK cohort comprising 3962 UK patients with RA and 3531 healthy controls using the Sequenom iPlex platform. Genotype counts in patients and controls were analysed with the χ2 test using Stata. Results Association to the CD40 gene was robustly replicated (p=2×10−4, OR 0.86, 95% CI 0.79 to 0.93) and modest evidence was found for association with the CCL21 locus (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). However, there was no evidence for association of rs42041 (CDK6) and rs6682654 (CD244) with RA susceptibility in this UK population. Following a meta- analysis including the original data, association to CD40 was conﬁrmed (p=7.8×10−8, OR 0.87 (95% CI 0.83 to 0.92). Conclusion In this large UK cohort, strong association of the CD40 gene with susceptibility to RA was found, and weaker evidence for association with RA in the CCL21 locus. INTRODUCTION Genome-wide association studies (GWAS) have led to a breakthrough in the search for complex dis- ease susceptibility markers.1 Speciﬁcally for rheu- matoid arthritis (RA), these studies have detected a number of new susceptibility loci, including the 6q23,2–4 TRAF1/C5,5–9 PRKCQ, KIF5A and IL2RB genetic regions.10 Recently, a meta-analysis of the Wellcome Trust Case Control Consortium (WTCCC) GWAS and a US GWAS4 5 conﬁrmed association to PRKCQ, KIF5A and MMEL and iden- tiﬁed additional RA-associated markers mapping to the CD40, CCL21 and CDK6 loci,11 that have yet to be conﬁrmed in an independent UK population. In addition, a recent case-control association study in a Japanese population identiﬁed two functional vari- ants mapping to the CD244 gene, rs3766379 and rs6682654, that were associated with RA, again yet to be tested for association in a UK population.12 These studies therefore add to the recent explo- sion in the number of loci reported to be associated with RA susceptibility. In order for these loci to be conﬁrmed as risk factors for RA, well powered val- idation studies are required to support these initial ﬁndings. The aims of this study were, therefore, ﬁrst to validate the additional RA markers derived from the recent meta-analysis (CD40, CCL21 and CDK6) in an independent UK population and, second, to investigate a functional CD244 variant reported to be associated with RA in the Japanese study.11 12 METHODS Patients with RA (n=3962) were recruited from six centres (Manchester, Shefﬁeld, Leeds, Aberdeen, Oxford and London) across the UK. All patients were Caucasian of North European ancestry and all fulﬁlled the 1987 American College of Rheumatology classiﬁcation criteria modiﬁed for genetic studies.13 14 Clinical and demographic characteristics of the cohort are detailed in supple- mentary table 1. Brieﬂy, 71.8% participants were women, 72.1% were rheumatoid factor positive and 67.9% carried anti-citrullinated protein anti- bodies (ACPA) as recognised by the anti-cyclic cit- rullinated protein (CCP) antibody test. This cohort is therefore representative of a hospital-based series of patients with RA. Autoantibody status data was available for 3723 and 2281 patients for rheumatoid factor (RF) and anti-CCP, respectively. Healthy con- trols (n=3531) were recruited from ﬁve of the six centres (patients only recruited from London). All participants were recruited after providing informed consent and the study was approved by the North West Research Ethics Committee (MREC 99/8/84). Genotyping was performed using the Sequenom iPlex platform (http://www.sequenom.com) accord- ing to the manufacturer’s instructions. All genotyp- ing was performed at the arc Epidemiology Unit, Manchester. Only samples and single nucleotide polymorphisms (SNPs) exceeding a 90% success rate were used in the analysis. Genotype counts in patients and controls were analysed using Stata software (Stata, College Station, Texas, USA) with the χ2 test. For the meta-analysis, the Breslow–Day test for homoge- neity and the Cochran Mantel–Haenszel test were performed using Stata. For all the tests performed, p values <0.0125 were considered statistically sig- niﬁcant, after applying Bonferroni correction for four comparisons. Ann Rheum Dis 2010;69:813–816. doi:10.1136/ard.2009.109579 813