Abstract Body: Background: Genetic susceptibility to coronary artery disease (CAD) is associated with variants at more than 300 genomic loci, among which the 9p21.3 locus shows the strongest and most consistent association. Despite its prominence, the mechanisms by which this locus influences vascular cell behavior remain poorly understood. This ~60 kb gene desert contains ~80 single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium, defining two major haplotypes: risk and non-risk. We previously demonstrated that the 9p21.3 CAD risk haplotype promotes an osteochondrogenic phenotype in vascular smooth muscle cells (VSMCs). Notably, many genes dysregulated in risk VSMCs are involved in mRNA splicing. We therefore hypothesize that the 9p21.3 risk haplotype drives widespread alterations in RNA splicing, leading to aberrant VSMC phenotypic modulation.
Methods: Induced pluripotent stem cells (iPSCs) heterozygous at the 9p21.3 locus were genome edited to generate haplotype-biased hemizygous lines. iPSCs were differentiated into VSMCs and analyzed using long-read RNA sequencing to quantify transcript expression, isoform usage, and splicing patterns. CRISPR interference (CRISPRi) was used to functionally validate candidate splicing regulators in mature VSMCs.
Results: The 9p21.3 risk allele altered the expression of more than 800 transcripts, including genes critical for vascular homeostasis, remodeling, and CAD pathogenesis. Long-read RNA sequencing revealed distinct transcriptional programs driven by the two haplotypes. The risk haplotype drives extensive genome-wide reprogramming of mRNA splicing resulting in disrupted transcript isoform expression and usage, frequently independent of total gene expression changes, promoting aberrant VSMC phenotypic modulation. Among the affected genes, we prioritized and validated the RNA helicase DDX5 as a downstream effector of the 9p21.3 locus.
Conclusion: The 9p21.3 CAD risk haplotype exerts a previously unrecognized role in regulating genome-wide RNA splicing in VSMCs, affecting both gene expression and alternative isoform usage of multiple CAD-related genes. This study provides the first comprehensive isoform-level comparison of the two major 9p21.3 haplotypes and identifies allele-specific transcriptional programs that reshape VSMC splicing landscapes and phenotypic plasticity. We propose a 9p21.3–DDX5 axis as a key regulator of VSMC modulation and a potential therapeutic target for reducing cardiovascular risk.