Abstract Body: Introduction: Genome-wide association studies (GWAS) have identified 300+ loci associated with coronary artery disease (CAD) risk, with variable effects across human populations. The 9p21.3 locus was the first discovered and remains the strongest genetic risk factor for CAD. This region lacks protein-coding genes and only partially overlaps with the long non-coding RNA ANRIL. Approximately 80 single-nucleotide polymorphisms (SNPs) in strong linkage disequilibrium (LD) define two major haplotypes at 9p21.3: risk (R) and non-risk (N). Notably, individuals of African genetic ancestry exhibit low LD at this locus and no association with CAD. We hypothesize that the 9p21.3 locus contains smaller functional regions that differentially regulate vascular smooth muscle cell (VSMC) phenotypes, contributing to population-specific disease susceptibility.
Methods: We used a diverse panel of induced pluripotent stem cells (iPSCs) derived from individuals of European descent (high LD) and African descent (low LD). iPSCs were differentiated into VSMCs and assayed with gene expression profile and functional assays. CRISPR-mediated genome editing was employed to target distinct regions of the 66 kb 9p21.3 locus to evaluate their functional contributions.
Results: Low LD at 9p21.3 observed in individuals of African descent was recapitulated in our iPSC cohort. iPSC-derived VSMCs with low LD did not express previously described CAD risk–associated transcriptional signatures, including short ANRIL isoforms, and instead resembled non-risk VSMCs. Expression of long ANRIL isoforms was inversely correlated with the number of risk SNPs, suggesting a protective role against CAD-associated cellular phenotypes. Moreover, VSMC migration exhibited an additive relationship with the number of risk SNPs. Based on these findings, we employed six CRISPR-based genome editing strategies, generating the first collection of human iPSC lines designed to dissect functional subregions of the 9p21.3 locus and assess the cumulative effects of multiple variants.
Conclusions: Leveraging iPSCs to explore the genetic diversity at the 9p21.3 CAD risk locus, we demonstrate how ancestry-specific variation influences VSMC gene expression and function. Our CRISPR-engineered iPSC collection enables systematic dissection of functional domains within this locus and provides insight into mechanisms underlying population-specific CAD risk, with potential implications for future gene-based therapeutic strategies.