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Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression [lentiMPRA]

ABSTRACT: Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279300 | GEO | 2024/10/14

REPOSITORIES: GEO

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Project description:Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

Project description:Coronary artery disease (CAD) is the leading cause of death worldwide. A recent meta-analysis of genome-wide association studies (GWAS) identified 175 loci associated with CAD. The majority of these loci are in non-coding regions and are predicted to regulate gene expression. We hypothesized that a subset of the CAD GWAS loci was associated with the regulation of transcription in vascular smooth muscle cells (SMCs), which play critical roles in the development of CAD. We measured gene expression in SMCs isolated from the ascending aortas of 151 ethnically diverse heart transplant donors in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single nucleotide polymorphism (SNP) markers across the genome. We identified 4,910 expression and 4,412 splice quantitative trait loci (sQTL) that represent regions of the genome associated with transcript abundance and splicing. 3,660 of the expression quantitative trait loci (eQTL) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 of the eQTLs were SMC- and sex-specific, respectively. To identify the effector transcripts regulated by the CAD GWAS loci, we used four distinct colocalization approaches and identified 84 eQTL and 164 sQTLs that colocalized with CAD loci, suggesting a more significant role for the genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. Two of the CAD loci colocolized with SMC eQTL showed sex-specific (AL160313.1 and TERF2IP) and one of the loci colocalized with SMC-specific eQTLs (ALKBH8). 27% and 37% of the sQTLs were unique to quiescent or proliferative SMCs, respectively. The most significantly associated CAD locus, 9p21, was an sQTL for the long non-coding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, these results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in vascular smooth muscle cells.'

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Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression [lentiMPRA]

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