Integrative analysis of liver-specific non-coding regulatory SNPs associated with the risk of coronary artery disease

Abstract

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.

Keywords: CRISPR; GWAS; SNP; STARR-seq; cholesterol; coronary artery disease; enhancer; functional genomics; hepatocyte; liver.