Chen, Wei‐Min

Introduction: Estrogens have been associated with risk of stroke in both men and women. Estradiol and estrone are synthesized from testosterone via the aromatase enzyme in both men and postmenopausal women. Genetic variation affecting function of the aromatase gene (CYP19A1) has been linked to elevated estradiol levels. We tested the hypothesis that these variants, as well as a modifying gene, are associated with ischemic stroke in three cohorts: the Vitamin Intervention for Stroke Prevention trial (VISP), Women’s Health Initiative (WHI), and Genetic Susceptibility to Early-Onset Stroke study (GEOS). Methods: Genomic data for individuals of European descent with ischemic stroke was compared against an equal number of controls: VISP (1723 cases - 65% men), WHI (436 cases - all postmenopausal women), GEOS (557 cases - all men age <55). Genotyping was funded through the Genomics and Randomized Trials Network (GARNET) and Gene Environment Association Studies (GENEVA) consortia. All cohorts used the Illumina Omni1-Quad chip for genotyping with imputation as needed. Eight candidate single nucleotide polymorphisms (SNPs) in CYP19A1 on chromosome 15 and an additional regulatory SNP in a modifier locus on chromosome 8 were chosen for analysis due to coding status or previous association with estradiol levels. Analyses were adjusted for age, hormone therapy use (women), and the top three principal components. Association results were determined as odds ratios for individual SNPs (effect allele = minor), with significance level of p<0.05. Results: GWAS results are tabulated. There were no significant differences in allele frequency for four SNPs in CYP19A1 among stroke patients and controls. The additional four SNPs were monomorphic in all three populations. The modifier gene SNP (rs1864729) was less common among young men with prior stroke in GEOS, but not men in VISP or in women. Conclusions: Aromatase genetic variation was not associated with risk of stroke in these three cohorts. One SNP that modifies aromatase function was less common among young, but not older men with stroke. Replication in other studies is necessary.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified a number of novel SNPs associated with blood pressure and hypertension-related traits although studies were very large (approaching 80K participants) and effect sizes were modest with per allele effect sizes as small variants as a single millimeter of mercury difference in blood pressure. However, because hypertension is the most potent modifiable risk factor for ischemic stroke, lowering blood pressure by a few mm Hg can translate into measurable decrease in stroke risk. METHODS: We investigated 22 hypertension (HTN) associated SNPs in a case-control analysis of ischemic stroke in multiple stroke studies including the Siblings With Ischemic Stroke Study (SWISS), the Ischemic Stroke Genetics Study (ISGS), the Bio-Repository of DNA in Stroke (BRAINS), the Vitamin Intervention for Stroke Prevention (VISP) trial, and the Women's Health Initiative (WHI). SWISS/ISGS/BRAINS were analyzed together and included subtype information. Genotyping was funded through the individual studies, the Genomics and Randomized Trials Network (GARNET) consortium, and the SHARe (SNP Health Association Resource). All studies had both raw and imputed GWAS data. Analyses included the following phenotypes: all ischemic stroke, stroke subtypes, and age-at-stroke-onset and were stratified by race. Analyses were minimally adjusted for age, sex and principal components 1 & 2. Alpha was set at 0.05. RESULTS: Eleven of the SNPs from the HTN GWAS were associated with ischemic stroke in European ancestry from one or more of the study populations. Two SNPs on chromosome 12 and one on chromosome 21 were associated in multiple European ancestry cohorts. No SNP was associated in all studies. A SNP previously associated with HTN in African ancestry populations was associated with stroke in African-Americans. Analysis of subtypes found associations with small vessel disease and cardioembolic stroke but not large artery disease. The case-only age-at-stroke-onset analysis also found associations for the two SNPs on chromosome 12. CONCLUSION: These data suggest that candidate SNPs from a HTN GWAS may be associated with ischemic stroke and warrant further investigation. Racial differences were observed. Replication in populations from the other SiGN (Stroke Genetics Network), GENEVA (Gene Environment Association Studies) consortium, and the ISGC (International Stroke Genetics Consortium) studies are underway.

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 B6 and B12 reduced recurrent cerebral infarction. Baseline post-methionine load homocysteine levels were predictive of recurrent stroke risk in VISP. We have conducted a genome-wide association study (GWAS), investigating 2100 VISP stroke cases required to have homocysteine levels greater than the 25th percentile at enrolment. Using imputation with 1000 genomes and principal component analysis, we were able to detect an association of p≤6.70x10-17 for SNPs within, or flanking, the glycine N-methyltransferase (GNMT) gene and post-methionine load test homocysteine level. Change in homocysteine levels pre and post-methionine load test also resulted in significant GWAS scores in the chromosome 6 region (p = 2.54x10-23, rs7760250). GNMT catalyzes the conversion of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine and sarcosine via the folate one-carbon metabolism pathway. In addition to being an essential component in the conversion of methionine to homocysteine, GNMT plays a significant role in the global epigenetic state as homocysteine is an important methyl donor. This region displays significant linkage disequilibrium, warranting close analysis of the entire GNMT locus in search of both rare and common variants. Cases who carry the minor allele haplotype at associated loci had a higher level of post-methionine load homocysteine (37.69µM/L) than those who carry the major allele haplotype (27.33µM/L), inferring functional differences due to GNMT variants. Taken together these data suggest a clear and important role for GNMT in post-methionine load test homocysteine level, epigenetic state and stroke risk.

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.

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 <10 −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 <10 −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 <0.05) with stroke in COMPASS. 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.