Project Summary Stroke is the leading cause of death and disability in the US and disproportionally affects minority populations. Hispanics have a particularly high risk for stroke; this increased risk of stroke may be explained by specific genetic and non-genetic factors. Dominicans are the fastest growing Caribbean Hispanics in the US, and yet little is known about their genetic and non-genetic determinants of stroke and cardiovascular disease (CVD). For the past 15 years, our team has been investigating stroke genetic risk factors in Dominicans. We have assembled a cohort of Dominican families at high risk of stroke and focused on stroke precursor phenotypes (SPPs) to reduce phenotypic heterogeneity and complexity of stroke etiology. We investigated well-recognized SPPs including carotid intima-media thickness, carotid plaque, left ventricular mass, and left atrial diameter. We have successfully identified quantitative trait loci and DNA sequence variants associated with SPPs using high throughput genotyping and next generation sequencing. These findings account for a small portion of the inter- individual variation in SPPs. In this application, we propose to expand our investigations to conduct a methylome- wide-association-study (MWAS) to identify differential DNA methylation regions (DMRs) associated with SPPs, stroke and CVD. Methylation is an epigenetic process that regulates gene expression without changing DNA sequence. DNA methylation has been shown as a key process contributing to the development of vascular disease. The proposed investigations would provide new insights relevant to the development of novel clinical strategies for prevention and treatment of stroke and CVD, as our ultimate goal is to reduce stroke risk and race- ethnic disparities in stroke and CVD. We plan 4 aims: Aim 1 to identify DMRs associated with stroke SPPs; Aim 2 to assess the relative contribution of genetic and non-genetic factors to SPP-associated DMRs; Aim3 to evaluate the functional impact of DNA sequence variation and DMRs using CRISPR-Cas9 technology; and Aim 4 to examine the predictive effect of SPP-associated DMRs on vascular events. To achieve these aims we will leverage the rich data already collected in the Family Study and add new data collection to detect vascular events (stroke, myocardial infarction, vascular death). We will validate the findings in an independent sample from the ongoing longitudinal Northern Manhattan Study, from which the Family Study originated. Both studies have used the same assessment tools and collection instruments for obtaining SPPs, lifestyle risk factors and genome-wide SNP data. The innovative aspects of our proposal include novel discoveries of modifiable epigenetic sites for stroke and CVD, CRISPR-Cas9 technology to model specific genome-editing, a unique population of Dominicans and a family study design, and an available independent population of Dominicans for validation studies. Findings from our study may lead to the most promising molecular strategies for risk stratification, prevention and treatment of stroke.