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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.

SUBMITTER: Selvarajan I

PROVIDER: S-EPMC8008493 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Coronary artery disease (CAD) is the leading cause of mortality and morbidity driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with CAD and myocardial infarction (MI) susceptibility in multi-ethnic populations. The majority of these variants reside in non-coding regulatory regions and are co-inherited with hundreds of candidate regulatory SNPs. Herein, we use integrative genomic, epigenomic, and transcriptomic fine-mapping in human coronary artery smooth muscle cells (HCASMC) and tissues to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps we prioritize 65 candidate variants and perform allele-specific binding and expression analyses on 7 top candidates. We validate our findings in two independent cohorts of diseased human arterial expression quantitative trait loci (eQTL), which together demonstrate fundamental links between CAD associations and regulatory function in the appropriate disease context. We performed ATAC-seq, ChIP-seq, and RNA-seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182). For ATAC-seq and RNA-seq we performed stimulations with growth factors (TGF-B1, PDGF-BB, PDGF-DD) versus serum-free control. We conducted two biological replicates for each condition using independent donors. For ATAC-seq experiments, sequencing was completed on an Illumina Hiseq 2500, paired-end 50bp reads. For ChIP-seq we performed immunoprecipitations using H3K27ac (Abcam ab4729). We conducted two biological replicates using HCASMC from independent donors, and also did an IgG control for these studies. For RNA-seq we also conducted two replicates using HCASMC from independent donors. For both ChIP-seq and RNA-seq experiments, sequencing was completed on an Illumina HiSeq 2500, paired-end 100bp reads. We also performed ex-vivo ATAC-seq on frozen tissues (isolated media) from normal and atherosclerotic human coronary arteries, using three independent donors for each. Sequencing was also completed on an Illumina HiSeq 2500, paired end 50bp reads.

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

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