Abstract Body: Cardiovascular disease (CVD) is the leading cause of death worldwide. Despite current lipid-lowering therapies, residual CVD risk in patients maximally treated with lipid-lowering agents remains a significant unmet clinical need. Large-scale genetic studies have identified >300 coronary artery disease (CAD) associated loci, yet the majority of causal genes that drive atherosclerosis remain unknown. Vascular smooth muscle cells (SMC) play an essential role in atherosclerosis, but even less is known about CAD-associated loci that control SMC function and identity during atherosclerosis progression. To identify novel SMC causal CAD genes, we developed a gene discovery pipeline that integrates large-scale human WGS/WES studies, rare variant analysis of human “knockouts” (homozygous for predicted loss-of-function variants pLoFs) from the Pakistani Genomics Resource (PGR), and previous SMC single cell profiling previously published by our lab. Starting at non-lipid CAD loci from a large multi-ethnic meta-analysis (1.1 million participants), we prioritized genes with high expression in SMC scRNA-seq, bulk RNAseq, and eQTL SNP datasets during atherosclerosis. Through gene burden testing of rare pLoFs and damaging variants in the largest CAD WES/WGS meta-analyses, including PGR (114,596 CAD cases and 481,375 controls), we identified multiple SMC causal candidate genes, including HHIPL1, RAB23, and SERPINH1. Initial replication analyses across independent cohorts validated the association between rare HHIPL1 pLoF variants and CAD. Moreover, analysis of FinnGen, a geographically and genetically unique cohort, independently validated the causal association of HHIPL1 pLoFs and CAD, supporting allelic and ancestral generalizability. Phenome-wide association studies (PheWAS) showed a significant association between HHIPL1 and atherosclerosis phenotypes, reinforcing disease specificity. Leveraging high consanguinity in PGR, we identified and recruited multiple families harboring rare HHIPL1 pLoF variants for recall and mechanistic studies. In silico structural modeling of PGR HHIPL1 pLoF protein variants revealed disruption of a highly conserved domain predicted to bind SHH, thus providing insights into the possible mechanism underlying HHIPL1-CAD function. In conclusion, we have developed a novel discovery pipeline for SMC-specific genes causative for CAD that has identified multiple candidates, including HHIPL1 and RAB23, both of which modulate vascular SHH signaling.