Meta-analysis inmore than 17 , 900 cases of ischemic stroke reveals a novel association at 12 q 24 . 12

Objectives: To perform a genome-wide association study (GWAS) using the Immunochip array in 3,420 cases of ischemic stroke and 6,821 controls, followed by a meta-analysis with data from more than 14,000 additional ischemic stroke cases. Methods: Using the Immunochip, we genotyped 3,420 ischemic stroke cases and 6,821 controls. After imputation we meta-analyzed the results with imputed GWAS data from 3,548 cases and 5,972 controls recruited from the ischemic stroke WTCCC2 study, and with summary statistics from a further 8,480 cases and 56,032 controls in the METASTROKE consortium. A final in silico “look-up” of 2 single nucleotide polymorphisms in 2,522 cases and 1,899 controls was performed. Associations were also examined in 1,088 caseswith intracerebral hemorrhage and 1,102 controls. Results: In an overall analysis of 17,970 cases of ischemic stroke and 70,764 controls, we identified a novel association on chromosome 12q24 (rs10744777, odds ratio [OR] 1.10 [1.07–1.13], p5 7.123 10211) with ischemic stroke. The association was with all ischemic stroke rather than an individual stroke subtype, with similar effect sizes seen in different stroke subtypes. There was no association with intracerebral hemorrhage (OR 1.03 [0.90–1.17], p 5 0.695). Conclusion: Our results show, for the first time, a genetic risk locus associated with ischemic stroke as a whole, rather than in a subtype-specific manner. This finding was not associated with intracerebral hemorrhage. Neurology® 2014;83:678–685 GLOSSARY GWAS 5 genome-wide association study; ICH 5 intracerebral hemorrhage; LD 5 linkage disequilibrium; MAF 5 minor allele frequency; OR 5 odds ratio; QC 5 quality control; SNP 5 single nucleotide polymorphism; WTCCC2 5Wellcome Trust Case Control Consortium 2. Genetic variation is now thought to play an important role in many diseases, including stroke. Genome-wide association studies (GWAS) have been applied to ischemic stroke directly, with HDAC9 being identified as the first genetic risk factor specific to large artery ischemic stroke in the Wellcome Trust Case Control Consortium 2 (WTCCC2) study. A 6p21.1 locus has also been associated with large artery stroke in a GWAS from Australia. Subsequent replication of these associations in a large meta-analysis by the METASTROKE consortium confirmed that all were specific to individual ischemic stroke subtypes. GWAS arrays are designed to provide broad coverage of the entire human genome for non– hypothesis-driven studies. To bridge the gap between full GWAS arrays and targeted candidate gene studies, a smaller series of custom arrays has been developed. One such example is the Immunochip, which offers a targeted genome-wide array comprising;200,000 genetic variants spanning a range of immune-related genes. Development of the Immunochip also included approximately 3,000 single nucleotide polymorphisms (SNPs) associated with ischemic stroke from an early-stage analysis of WTCCC2 data. However, inflammatory processes have been implicated in the pathogenesis of Laura L. Kilarski, PhD Sefanja Achterberg, MD William J. Devan, BS Matthew Traylor, MSc Rainer Malik, PhD Arne Lindgren, MD Guillame Pare, MD Pankaj Sharma, FRCP Agniesczka Slowik, MD, PhD Vincent Thijs, MD, PhD Matthew Walters, MD Bradford B. Worrall, MD, MSc Michele M. Sale, PhD Ale Algra, MD, PhD L. Jaap Kappelle, MD Cisca Wijmenga, PhD Bo Norrving, MD Johanna K. Sandling, PhD Lars Rönnblom, MD An Goris, PhD Andre Franke, PhD Cathie Sudlow, FRCPE Peter M. Rothwell, FRCP Christopher Levi, MBBS Elizabeth G. Holliday, PhD Myriam Fornage, PhD Bruce Psaty, MD, PhD Solveig Gretarsdottir, PhD Unnar Thorsteinsdottir, PhD Sudha Seshadri, MD Braxton D. Mitchell, PhD Steven Kittner, MD Robert Clarke, MD Jemma C. Hopewell, PhD Joshua C. Bis, PhD Giorgio B. Boncoraglio, MD James Meschia, MD M. Arfan Ikram, MD, PhD Bjorn M. Hansen, MD Joan Montaner, MD Gudmar Thorleifsson, PhD Kari Stefanson, PhD, Drmed Jonathan Rosand, MD, MSc Paul I.W. de Bakker, PhD Martin Farrall, FRCPath Author list continued on next page *These authors jointly directed this work. Author affiliations are provided at the end of the article. Coinvestigators are listed on the Neurology® Web site at Neurology.org. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. The Article Processing Charge was paid by the Wellcome Trust as funders of this study. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 678 © 2014 American Academy of Neurology cardiovascular disease and stroke, suggesting the nonstroke content of the Immunochip may provide additional information when considering the stroke phenotype. We report here the use of the Immunochip as the initial phase of a targeted GWAS, followed by metaanalysis with full GWAS data fromWTCCC2 and an international collaboration of ischemic stroke GWAS data (METASTROKE). This is followed by in silico replication (i.e., ascertainment from previous data without the need for de novo genotyping) with data from the INTERSTROKE and VISP studies. METHODS Study design and participating studies. The discovery sample consisted of 6 cohorts of patients of European ancestry with ischemic stroke. Participating centers were based in Belgium, Germany, the Netherlands (the PROMISe Study), Poland, Sweden, and the UK (2 cohorts, one from London [Imperial College; the BRAINS study] and one from Glasgow). All cohorts provided geographically and ancestry-matched controls. For the purposes of meta-analysis, the UK cohorts were treated as a single center in line with previous analyses undertaken in WTCCC2. Analysis plan. The analysis plan for this study was to perform a single meta-analysis of available data as follows: (1) association analysis of imputed Immunochip data; (2) meta-analysis with HAPMAP2imputed WTCCC2 data and METASTROKE consortium data for which summary statistics were available; and (3): in silico look-up of significant SNPs from meta-analysis in the INTERSTROKE cohort and the VISP cohort. The populations used in both WTCCC2 and METASTROKE have been previously reported. The WTCCC2 data have also been contributed to METASTROKE. Therefore, for this analysis the WTCCC2 data were removed from METASTROKE to prevent duplication of individuals, as was the BRAINS dataset, which overlapped with BRAINS cases contributing to the Immunochip discovery cohort. Table 1 includes full details of the discovery cohorts and outlines details of the WTCCC2, METASTROKE, INTERSTROKE, VISP, and intracerebral hemorrhage (ICH) cohorts. A GWAS standard a priori significance threshold of 5 3 10 was considered as a significant finding prior to analysis. We additionally determined whether genome-wide associated SNPs from this analysis were also associated with primary ICH by in silico replication in GWAS data from a meta-analysis of 1,088 ICH cases and 1,102 controls (Genetics of Cerebral Hemorrhage with Anticoagulation [GOCHA] study). Full population details and demographics of all consortia are available in their original publications. Full details of the Immunochip cohorts and the analysis methodology are available in the online supplementary material on the Neurology® Web site at Neurology.org. Data genotyping, imputation, and statistical analysis. The Immunochip consortium developed as an immune-related targeted GWAS array comprising ;200,000 SNPs. As part of Martin Dichgans, MD* Hugh S. Markus, MD, FRCP* Steve Bevan, PhD* On behalf of the GARNET Collaborative Research Group, Wellcome Trust Case Control Consortium 2, Australian Stroke Genetic Collaborative, the METASTROKE Consortium, and the International Stroke Genetics Consortium Correspondence to Dr. Bevan: snb31@medschl.cam.ac.uk