SummaryBackgroundVarious genome-wide association studies (GWAS) have been done in ischaemic stroke, identifying a few loci associated with the disease, but sample sizes have been 3500 cases or less. We established the METASTROKE collaboration with the aim of validating associations from previous GWAS and identifying novel genetic associations through meta-analysis of GWAS datasets for ischaemic stroke and its subtypes.MethodsWe meta-analysed data from 15 ischaemic stroke cohorts with a total of 12 389 individuals with ischaemic stroke and 62 004 controls, all of European ancestry. For the associations reaching genome-wide significance in METASTROKE, we did a further analysis, conditioning on the lead single nucleotide polymorphism in every associated region. Replication of novel suggestive signals was done in 13 347 cases and 29 083 controls.FindingsWe verified previous associations for cardioembolic stroke near PITX2 (p=2·8×10−16) and ZFHX3 (p=2·28×10−8), and for large-vessel stroke at a 9p21 locus (p=3·32×10−5) and HDAC9 (p=2·03×10−12). Additionally, we verified that all associations were subtype specific. Conditional analysis in the three regions for which the associations reached genome-wide significance (PITX2, ZFHX3, and HDAC9) indicated that all the signal in each region could be attributed to one risk haplotype. We also identified 12 potentially novel loci at p<5×10−6. However, we were unable to replicate any of these novel associations in the replication cohort.InterpretationOur results show that, although genetic variants can be detected in patients with ischaemic stroke when compared with controls, all associations we were able to confirm are specific to a stroke subtype. This finding has two implications. First, to maximise success of genetic studies in ischaemic stroke, detailed stroke subtyping is required. Second, different genetic pathophysiological mechanisms seem to be associated with different stroke subtypes.FundingWellcome Trust, UK Medical Research Council (MRC), Australian National and Medical Health Research Council, National Institutes of Health (NIH) including National Heart, Lung and Blood Institute (NHLBI), the National Institute on Aging (NIA), the National Human Genome Research Institute (NHGRI), and the National Institute of Neurological Disorders and Stroke (NINDS).

Genetic factors have been implicated in stroke risk but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) in ischemic stroke and its subtypes in 3,548 cases and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 cases and 6,281 controls. We replicated reported associations between variants close to PITX2 and ZFHX3 with cardioembolic stroke, and a 9p21 locus with large vessel stroke. We identified a novel association for a SNP within the histone deacetylase 9 (HDAC9) gene on chromosome 7p21.1 which was associated with large vessel stroke including additional replication in a further 735 cases and 28583 controls (rs11984041, combined P = 1.87×10−11, OR=1.42 (95% CI) 1.28-1.57). All four loci exhibit evidence for heterogeneity of effect across the stroke subtypes, with some, and possibly all, affecting risk for only one subtype. This suggests differing genetic architectures for different stroke subtypes.

Summary Introduction The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading approach to the identification of novel biological pathways for human disease. To date, GWAS have had been limited by relatively small sample sizes and yielded relatively few loci associated with ischemic stroke The National Institute of Neurological Disorders Stroke Genetics Network (NINDS-SiGN) is an international consortium that has taken a systematic approach to phenotyping and produced the largest ischemic stroke GWAS to date. Methods In order to identify genetic loci associated with ischemic stroke, we performed a two-stage genome-wide association study. The first stage consisted of 16,851 cases with state-of-the-art phenotyping and 32,473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtyped by centrally trained and certified investigators using the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identify samples genotyped on (nearly) identical arrays and of similar genetic ancestral background. Data was cleaned and imputed using dense imputation reference panels generated from whole-genome sequence data. Genome-wide testing was performed within each stratum for each available phenotype, and summary level results were combined using inverse variance-weighted fixed effects meta-analysis. The second stage consisted of in silico look-ups of 1,372 SNPs in 20,941 cases and 364,736 stroke-free controls, with cases previously subtyped using the TOAST classification system according to local standards. The two stages were then jointly analyzed in a final meta-analysis. Findings We identified a novel locus at 1p13.2 near TSPAN2 associated with large artery atherosclerosis (LAA)-related stroke (stage I OR for the G allele at rs12122341 = 1·21, p = 4.50 × 10−8; stage II OR = 1·19, p = 1·30 × 10−9). We also confirmed four loci robustly associated with ischemic stroke and reported in prior studies, including PITX2 and ZFHX3 for cardioembolic stroke, and HDAC9 for LAA stroke. The 12q24 locus near ALDH2, originally associated with all ischemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke. Other loci, including NINJ2, were not confirmed. Interpretation Our results identify a novel LAA-stroke susceptibility gene and now indicate that all loci implicated by GWAS to date are subtype specific. Follow-up studies will be necessary to determine whether the locus near TSPAN2 yields a novel therapeutic approach to stroke prevention. Given the subtype-specificity of these associations, the rich phenotyping available in SiGN is likely to prove vital for further genetic discovery in ischemic stroke. Funding National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH).

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries.