PROJECT SUMMARY/ABSTRACT Genetic predisposition is a significant risk factor for coronary artery disease (CAD), the leading cause of mortality. Most CAD risk variants identified in genome-wide association studies (GWAS) are in noncoding DNA, which poses a major challenge in identifying the target genes in the disease-critical cell types. Using the Activity-by-Contact Model to predict cell type-specific gene-enhancer pairs, we identified cis-regulatory elements in the chromosome 10q23 locus harboring multiple CAD-risk variants. Our preliminary studies show that these enhancers in the 10q23 locus regulate tetraspanin 14 (TSPAN14) gene expression, specifically in vascular endothelial cells (ECs) and monocytes. We have established strong population genetics evidence that higher TSPAN14 expression reduces CAD risk and there is a consistent protective effect for the minor alleles at the lead variants. TSPAN14 is an adaptor protein that aids in trafficking proteins like ADAM10, a Notch receptor activator, to the plasma membrane. The role of Notch pathway activation imparted by this TSPAN14- ADAM10 interaction in CAD pathogenesis has not been explored. In addition, we performed RNA-seq and identified differentially expressed genes in TSPAN14-deficient cells that will help discover Notch-independent TSPAN14 functions. These findings provide a premise for the central hypothesis that the genetic regulation of TSPAN14 by variations in the 10q23 enhancer sequences affects CAD pathogenesis through Notch- dependent and -independent mechanisms in vascular ECs and monocytes. In Aim 1, Dr. Lee-Kim will determine the cell type-specific effect of 10q23 enhancer sequence variations on TSPAN14 expression regulation. In Aim 2, she will determine the Notch-dependent effects of TSPAN14 expression in vascular and immune cells. In Aim 3, she will characterize the Notch-independent TSPAN14 functions in vascular and immune cells. The results from these studies will functionally validate the gene target for noncoding variants associated with CAD-risk in the disease-relevant cell types and elucidate how TSPAN14 functions in CAD pathogenesis. These studies will be conducted under the supervision of mentors, Dr. Rajat Gupta and Dr. Stephen Blacklow, and an advisory committee dedicated to Dr. Lee-Kim’s success. With additional support from the MOSAIC UE5 awardee sponsored professional development opportunities, continued training in the K99 phase will prepare Dr. Lee-Kim for successful transition to independence.

PROJECT NARRATIVE Cardiovascular disease continues to be a leading cause of mortality, despite effective therapies available for known risk factors such as high cholesterol and high blood pressure. This proposal uses a genetic approach to understand the mechanism by which the gene TSPAN14 is involved in coronary disease, and thus may reveal new therapeutic targets. We will use cell and animal models to understand how genetic variation between humans control the function of this unstudied gene in healthy and diseased blood vessels.