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Objective: To perform a genome wide association study (GWAS) of stroke recovery phenotypes as defined by the trajectories of patients’ NIH stroke scale scores over time. Background: Stroke recovery research entails tracking multiple domains of neurologic impairment over time that are measured on differing scales. This presents challenges for analytic approaches commonly used to study stroke genetics. Here, we present a novel approach using a network science-based method, Trajectory Profile Clustering (TPC), to define stroke recovery phenotypes for GWAS. Design/Methods: We analyzed data from 3,679 patients in the VISP dataset (Vitamin Intervention for Stroke Prevention) on 15 NIHSS impairment measures at 6 time points spanning 24 months post-stroke. The 4 identified TPC profiles (Figure, Panel A), were used as phenotypes for a subsequent GWAS on the 2,099 genotyped VISP patients. We used a multinomial generalized regression with TPC profiles as the response variable. The model adjusted for age, sex, treatment group, and the first ten principal components. Results: TOPMed imputation and strict quality control resulted in 6,392,745 SNPs. Although none of our loci reached genome wide significance, the GWAS identified several suggestive loci (p Conclusion: Phenotypes defined using TPC, even in this limited sample, identified multiple suggestive genetic associations with biological relevance for both stroke risk and stroke recovery.

Introduction: Social and environment factors are associated with increased stroke risk. It is unclear if favorable cardiovascular health may attenuate this risk. We evaluated the impact of neighborhood socioeconomic characteristics on stroke incidence, and if levels of cardiovascular health modify this relationship. Methods: We conducted a longitudinal cohort study of Black and White participants using data from the Reasons for Geographic and Racial Differences in Stroke. We included all stroke-free participants and geocoded their home addresses to the census-tract level. Our primary exposure was neighborhood socioeconomic condition index (nSES; in quartiles) constructed using data from the U.S. Census on income, education, and occupation of residents by census tract. We evaluated cardiovascular health based on the American Heart Association’s Life Simple 7 (ideal, adequate, inadequate). We used a Cox proportional hazards model to regress nSES and its interaction with Life Simple 7 on stroke incidence, adjusting for individual demographics, socioeconomic status, and clinical characteristics. Results: We included 26,941 adults with a mean follow-up of 11.6 years (SD ± 5.2). During the study, 1,596 (5.9%) participants had a first incidence of stroke. Participants within the lowest nSES quartile, compared to those in the highest, were more likely to be Black, have lower household income, and an inadequate Life Simple 7 score. In multivariable models, there was a non-significant increased incidence of stroke (HR-1.13; 95% CI: 0.95-1.35) between the lowest and highest nSES quartiles. The interaction between nSES and Life Simple 7 was also non-significant for stroke incidence with worse cardiovascular health across all quartiles. Discussion: Among a national cohort of Black and White adults, we found a non-significant difference in stroke incidence between adults who live in worse neighborhood socioeconomic conditions and have inadequate cardiovascular health. Characterizing the potential contribution of social, environmental, and health behaviors could inform clinical and public health strategies to mitigate stroke risk.

Introduction: Genetic reference panels and imputation approaches have improved greatly in the last 10 years. The development of the TOPMed reference plane has led to enhanced imputation quality and quantity of single nucleotide polymorphisms (SNPs) due to greater sample diversity among various population ancestries. We revisited our prior GWAS of recurrent stroke by utilizing the TOPMed imputation server. Methods: This GWAS used a Cox proportional hazards model of time to recurrent stroke with the Vitamin Intervention for Stroke Prevention clinical trial cohort. There were 2,100 genotyped patients (64% male) in total with an average age of 67.2 (±10.8) years and ancestry distribution of 1,725 (82%) European, 258 (12%) African, and 117 (6%) Other or Mixed ancestry. Genotyped samples underwent a strict quality control process. We utilized TOPMed for imputation which totaled 10,467,887 biallelic SNPs which was 14 times greater in number compared the original analysis. Results: Recurrent stroke was observed in 182 (8.7%) patients. We identified seven novel SNPS on chromosome 1 in addition to our previous finding, rs6664786. Interestingly all chromosome 1 SNPs were located within the LINCO1362 gene. This gene has an acute change in expression in the presence of smoking even after adjustment of relevant clinical factors. Two novel SNPs were found on chromosome 16 located in gene desert nearest the pseudo-gene RNU6-21P in an intergenic region downstream of the Cadherin-8 (CDH8) gene. Both SNPs and RNU6-21P have no previously reported clinical relevance, except for their relative position to CDH8. CDH8 is highly expressed in brain tissue. Conclusions: We identified several novel SNPs associated with recurrent stroke. Capitalizing on genetic imputation advancements allows potential new insights and discoveries with past trial cohorts. Understanding these insights may provide further mechanistic knowledge of recurrent stroke to develop potential therapeutic targets.

Supplemental Digital Content is available in the text., Background and Purpose: Metabolic traits affect ischemic stroke (IS) risk, but the degree to which this varies across different ethnic ancestries is not known. Our aim was to apply Mendelian randomization to investigate the causal effects of type 2 diabetes (T2D) liability and lipid traits on IS risk in African ancestry individuals, and to compare them to estimates obtained in European ancestry individuals. Methods: For African ancestry individuals, genetic proxies for T2D liability and circulating lipids were obtained from a meta-analysis of the African Partnership for Chronic Disease Research study, the UK Biobank, and the Million Veteran Program (total N=77 061). Genetic association estimates for IS risk were obtained from the Consortium of Minority Population Genome-Wide Association Studies of Stroke (3734 cases and 18 317 controls). For European ancestry individuals, genetic proxies for the same metabolic traits were obtained from Million Veteran Program (lipids N=297 626, T2D N=148 726 cases, and 965 732 controls), and genetic association estimates for IS risk were obtained from the MEGASTROKE study (34 217 cases and 406 111 controls). Random-effects inverse-variance weighted Mendelian randomization was used as the main method, complemented with sensitivity analyses more robust to pleiotropy. Results: Higher genetically proxied T2D liability, LDL-C (low-density lipoprotein cholesterol), total cholesterol and lower genetically proxied HDL-C (high-density lipoprotein cholesterol) were associated with increased risk of IS in African ancestry individuals (odds ratio per doubling the odds of T2D liability [95% CI], 1.09 [1.07–1.11]; per standard-deviation increase in LDL-C, 1.12 [1.04–1.21]; total cholesterol: 1.23 [1.06–1.43]; HDL-C, 0.93 [0.89–0.99]). There was no evidence for differences in these estimates when performing analyses in European ancestry individuals. Conclusions: Our analyses support a causal effect of T2D liability and lipid traits on IS risk in African ancestry individuals, with Mendelian randomization estimates similar to those obtained in European ancestry individuals.

Background and Purpose— von Willebrand factor (vWF) plays an important role in thrombus formation during cerebrovascular damage. We sought to investigate the potential role of circulating vWF in recurrent cerebrovascular events and identify genetic contributors to variation in vWF level in an ischemic stroke population. Methods— We analyzed the effect of circulating vWF on risk of recurrent stroke using survival models in the VISP trial (Vitamin Intervention for Stroke Prevention) and the use of vWF in reclassification over traditional factors. We conducted a genome-wide association study) with imputation, based on 1000 Genomes Project data, for circulating vWF levels and then interrogated loci previously associated with vWF levels. We performed expression quantitative trait locus analysis for vWF across different tissues. Results— Elevated vWF levels were associated with increased risk for recurrent stroke in VISP. Adding vWF to traditional clinical parameters also improved recurrent stroke risk prediction. We identified single-nucleotide polymorphisms significantly associated with circulating vWF at the ABO locus ( P −8 ) and replicated findings from previous genetic associations of vWF levels in humans. Expression quantitative trait locus analyses demonstrate that most associated ABO single-nucleotide polymorphisms were also associated with vWF gene expression. Conclusions— Elevated vWF levels are associated with recurrent stroke in VISP. In the VISP population, genetic determinants of vWF levels that impact vWF gene expression were identified. These data add to our knowledge of the pathophysiologic and genetic basis for recurrent stroke risk and may have implications for clinical care decision making.

Introduction: Several related neurovascular phenotypes, including cervical artery dissection, fibromuscular dysplasia, large artery stroke, hypertension, and migraine headache are associated with a single nucleotide polymorphism (rs9349378) in the phosphatase and actin regulator-1 ( PHACTR1 ) gene recently identified as a distal regulator of the endothelin-1 ( EDN1 ) gene. Vascular endothelial cells release EDN1, a potent vasoconstrictor. We hypothesize that atheromatous and normal carotid tissue will differentially express PHACTR1 and EDN1 . Methods: We obtained gene expression data from the publically available Gene Expression Omnibus Carotid Atheroma Dataset (GSE43292). This resource contains 32 paired samples of carotid atheroma and distant macroscopically intact carotid tissue from the same individual and gene expression data from the Affymetrix Human Gene 1.0 ST Array. We compared log transformed count of signal intensity of PHACTR1 and EDN1 expression in the 32 paired samples using a two-sample t-test assuming equal variance with SPSS (IBM, Armonk, NY). Results: PHACTR1 expression was lower in atheromatous compared to normal carotid tissue (7.152 vs. 7.399, delta 0.248, 95%CI 0.180 - 0.315, p=1.9 E -8 ). EDN1 expression did not differ (6.514 and 6.339, delta 0.176, 95%CI -0.004 - 0.356, p = 0.055). with genetic data associating the rs9349378[G] allele with lower levels of expression and atherosclerotic vascular diseases. The lack of differential EDN1 expression may reflect the small sample size or may challenge the assertion that EDNI is the mediator of the PHACTR1 association. Our data support the hypothesis that this pathway may play an important and potentially variable role in neurovascular phenotypes including large artery atherosclerotic stroke and non-atherosclerotic arteriopathies.

Introduction: Cervical artery dissection (CeAD) of the carotid and vertebral arteries is a prevalent cause of stroke in young adults. While the pathogenesis of incident CeAD remains poorly understood, clinical and genetic associations suggest a systemic arteriopathy at time of event. Hypothesis: Individuals suffering CeAD express a distinct gene expression profile at time of event compared to (1) age and sex-matched controls and (2) convalescent profiles within the same individual at a later time point. Methods: We enrolled 37 consecutive patients with carotid or vertebral artery dissection (with and without ischemic stroke) from 2013-2016, excluding concurrence of major trauma. Cases were age and sex matched to non-CeAD ischemic stroke controls (n=16) and healthy controls (n=11). Whole blood samples were collected within four weeks from time of event, and again at 3 and 6-month follow-up. We used IlluminaHT-12 microarrays to assess differential gene expression at time of CeAD relative to controls, and relative to gene expression at >3 month follow-up within CeAD cases. Mixed effects regression models included relevant covariates of age, sex, race/ethnicity, time of enrollment, and occurrence of stroke. We used a False Discovery Rate (FDR) cutoff of 5% to account for multiple testing as well as a 1.5 fold change cutoff to identify robust, differential gene expression. Results: We identified 538 differentially expressed genes between CeAD patients and healthy controls with 30 of these genes reaching our predetermined 1.5x fold change limit. Within CeAD cases, 1,238 genes showed differential expression at a 5% FDR compared to time points at 3-6 month follow-up, with 31 genes showing at least a 1.5 fold change. Compared to controls and convalescent profiles, 12 genes were significant using the 5% FDR and 1.5 fold change cutoffs. Preliminary gene-ontology analysis indicate the largest differential expression for genes associated with oxygen and gas transport. Conclusion: In this consecutive cohort of individuals with CeAD, we identified a distinct gene expression profile associated with incident dissection. These data are limited by small sample size and results are hypothesis generating. Replication in larger, independent cohorts is warranted.

Introduction: Recent success identifying genetic variation in ischemic stroke (IS) has yet to translate into actionable targets or pathways. Weighted Gene Co-Expression Network Analysis (WGCNA) uses an unsupervised approach to cluster genes into modules based on co-expression profiles. Modules likely contain biologically related genes. The correlation between a gene’s expression and the module’s overall expression pattern (eigengene) defines connectivity. Genes with high connectivity may inform and supplement genome-wide association studies (GWAS). Methods: Gene Expression in Carotid Artery GSE43292 (GEO) resource contains publically available Affymetrix Human GeneChip Gene 1.0 ST data from 34 carotid atherosclerotic plaques and paired normal carotid tissue from the same individual. WGCNA resulted in 16 co-expression modules tested against the atherosclerosis phenotype. Significant modules had strong correlation between module eigengene and atherosclerosis. We took the top genes from significantly associated modules and looked them up in two existing IS genetic datasets: Stroke Genetic Network (SiGN) (17,000 IS cases & 32,000 controls) and METASTROKE (10,307 cases & 19,326 controls). We conducted a secondary GWAS informed by WGCNA for only known functional variants in METASTROKE with replication in SiGN. Results: Five of 16 modules showed evidence of association with two standouts (“Turquoise” and “Brown”) both enriched for genes associated with vascular disease related traits (carotid IMT, coronary artery disease, etc.) and contain genes associated with IS, especially large artery atherosclerotic stroke (including HDAC9). We took the top 20 genes from these modules, not currently known to associate with IS, and looking them up in SiGN and METASTROKE, we found association with variants in three genomic regions (SLC7A7, ATXN2/SH2B3, ALDH2). The informed GWAS approach identified ADAMTSL3 as a potential novel translational target. Conclusion: Leveraging expression data in a convergent genomics approach holds promise to supplement on-going GWAS and accelerate identification of potential translational targets. Gene ontology analyses of genes from the top modules are underway.

Background and Purpose— The majority of genome-wide association studies (GWAS) of stroke have focused on European-ancestry populations; however, none has been conducted in African Americans, despite the disproportionately high burden of stroke in this population. The Consortium of Minority Population Genome-Wide Association Studies of Stroke (COMPASS) was established to identify stroke susceptibility loci in minority populations. Methods— Using METAL, we conducted meta-analyses of GWAS in 14 746 African Americans (1365 ischemic and 1592 total stroke cases) from COMPASS, and tested genetic variants with P −6 for validation in METASTROKE, a consortium of ischemic stroke genetic studies in European-ancestry populations. We also evaluated stroke loci previously identified in European-ancestry populations. Results— The 15q21.3 locus linked with lipid levels and hypertension was associated with total stroke (rs4471613; P =3.9×10 −8 ) in African Americans. Nominal associations ( P −6 ) for total or ischemic stroke were observed for 18 variants in or near genes implicated in cell cycle/mRNA presplicing ( PTPRG , CDC5L ), platelet function ( HPS4 ), blood–brain barrier permeability ( CLDN17 ), immune response ( ELTD1, WDFY4 , and IL1F10-IL1RN ), and histone modification ( HDAC9 ). Two of these loci achieved nominal significance in METASTROKE: 5q35.2 ( P =0.03), and 1p31.1 ( P =0.018). Four of 7 previously reported ischemic stroke loci ( PITX2, HDAC9, CDKN2A/CDKN2B , and ZFHX3 ) were nominally associated ( P Conclusions— We identified a novel genetic variant associated with total stroke in African Americans and found that ischemic stroke loci identified in European-ancestry populations may also be relevant for African Americans. Our findings support investigation of diverse populations to identify and characterize genetic risk factors, and the importance of shared genetic risk across populations.

The Vitamin Intervention for Stroke Prevention (VISP) trial was a multi-center, controlled, double blinded clinical trial, designed to determine whether the daily intake of high dose folic acid, vitamins B 6 and B 12 reduced recurrent cerebral infarction. As part of GARNET (the Genomics and Randomized Trials Network), we have performed a Genome-Wide Association Study (GWAS), using the Illumina Omni 1M SNP platform in 2,100 ischemic stroke patients from VISP. Extensive quality control (QC) measures were performed, resulting in a total of 737,081 SNPs for analysis. We have performed GWA analyses for baseline quantitative measures of creatinine, total cholesterol (TC), high density lipoprotein (HDL), triglycerides (TG), C-reactive protein (CRP), Factor 1+2 thrombin-antithrombin (TAT), tissue plasminogen activator (tPA), thrombomodulin (TM) and von Willebrand factor (vWF), using linear regression approaches implemented in PLINK. Principal component analysis (PCA), implemented in KING, was utilized to address confounders due to population substructure. Inverse normal transformation was performed for each of the quantitative traits, prior to analysis. Genome wide association analyses were performed using age, sex and the top 10 principal components as covariates. There is no inflation in our GWAS scan results (GC lambda ≤ 1.012 in all scans). We observed three associated loci that exceed genome wide significance (≤6.8×10 -8 ): in or near the CETP gene for HDL (6.5×10 -10 ), the CRP gene for CRP (7.4×10 -10 ), and at the ABO locus for vWF (8.7×10 -31 ). The most strongly associated SNPs for other traits were closest to the following genes: GABRA3 for creatinine ( rs994424 , 5.7×10 -6 ), SPATA13 for TC (rs9553189, 3.2×10 -7 ), between ARMCX2 and NXF5 genes on the X chromosome for TG (rs2213369, 1.7×10 -6 ); the ZIC4 gene for TAT (rs17565826, 4.1×10-6); LOC283089 for tPA (rs3011337, 3.6×10 -6 ); and JAZF1 for TM (rs10258132, 1.1×10 -6 ). Putative novel associations with these traits will require confirmation in larger independent samples. Our GWAS scan has successfully replicated associations for SNPs in the CETP, CRP and ABO genes for HDL, CRP and vWF respectively in an ischemic stroke population.