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.

Agreement between TOAST and CCS diagnoses was moderate. Caution is warranted when comparing or combining results based on the 2 systems. Replication of study results, for example, genome-wide association studies, should utilize phenotypes determined by the same classification system, ideally applied in the same manner.

Background and Purpose NINDS Stroke Genetics Network (SiGN) is an international consortium of ischemic stroke studies that aims to generate high quality phenotype data to identify the genetic basis of etiologic stroke subtypes. This analysis characterizes the etiopathogenetic basis of ischemic stroke and reliability of stroke classification in the consortium. Methods Fifty-two trained and certified adjudicators determined both phenotypic (abnormal test findings categorized in major etiologic groups without weighting towards the most likely cause) and causative ischemic stroke subtypes in 16,954 subjects with imaging-confirmed ischemic stroke from 12 US studies and 11 studies from 8 European countries using the web-based Causative Classification of Stroke System. Classification reliability was assessed with blinded re-adjudication of 1509 randomly selected cases. Results The distribution of etiologic categories varied by study, age, sex, and race (p<0.001 for each). Overall, only 40% to 54% of cases with a given major ischemic stroke etiology (phenotypic subtype) were classified into the same final causative category with high confidence. There was good agreement for both causative (kappa 0.72, 95%CI:0.69-0.75) and phenotypic classifications (kappa 0.73, 95%CI:0.70-0.75). Conclusions This study demonstrates that etiologic subtypes can be determined with good reliability in studies that include investigators with different expertise and background, institutions with different stroke evaluation protocols and geographic location, and patient populations with different epidemiological characteristics. The discordance between phenotypic and causative stroke subtypes highlights the fact that the presence of an abnormality in a stroke patient does not necessarily mean that it is the cause of stroke.

Background and Purpose: Stroke is a complex disease with multiple genetic and environmental risk factors. Blacks endure a nearly 2-fold greater risk of stroke and are 2× to 3× more likely to die from stroke than European Americans. Methods: The COMPASS (Consortium of Minority Population Genome-Wide Association Studies of Stroke) has conducted a genome-wide association meta-analysis of stroke in >22 000 individuals of African ancestry (3734 cases, 18 317 controls) from 13 cohorts. Results: In meta-analyses, we identified one single nucleotide polymorphism (rs55931441) near the HNF1A gene that reached genome-wide significance ( P =4.62×10 −8 ) and an additional 29 variants with suggestive evidence of association ( P <1×10 −6 ), representing 24 unique loci. For validation, a look-up analysis for a 100 kb region flanking the COMPASS single nucleotide polymorphism was performed in SiGN (Stroke Genetics Network) Europeans, SiGN Hispanics, and METASTROKE (Europeans). Using a stringent Bonferroni correction P value of 2.08×10 −3 (0.05/24 unique loci), we were able to validate associations at the HNF1A locus in both SiGN ( P =8.18×10 −4 ) and METASTROKE ( P =1.72×10 −3 ) European populations. Overall, 16 of 24 loci showed evidence for validation across multiple populations. Previous studies have reported associations between variants in the HNF1A gene and lipids, C-reactive protein, and risk of coronary artery disease and stroke. Suggestive associations with variants in the SFXN4 and TMEM108 genes represent potential novel ischemic stroke loci. Conclusions: These findings represent the most thorough investigation of genetic determinants of stroke in individuals of African descent, to date.