Aims While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation. Methods and results We carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1β (vs. vehicle), and associated with smooth muscle cell migration in vitro. Conclusions A large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies (GWAS) revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs Methods and Results: We quantified twelve atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multi-ethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed GWAS for twelve atherosclerosis-relevant phenotypes and identified four genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD GWAS loci with our dataset and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk SNP rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 in VSMCs resulted in lower proliferation, consistent with human genetics findings. Further, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Further, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Rationale: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cis-regulatory elements (CREs) but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation. Objective: Single nucleus chromatin accessibility profiling of the human atherosclerotic lesions was used to investigate cell type-specific patterns of CREs, to understand transcription factors establishing cell identity and to interpret CAD-relevant, non-coding genetic variation. Methods and Results: We used single nucleus ATAC-seq to generate DNA accessibility maps in > 7,000 cells derived from human atherosclerotic lesions. We identified five major lesional cell types including endothelial cells, smooth muscle cells, monocyte/macrophages, NK/T-cells and B-cells and further investigated subtype characteristics of macrophages and smooth muscle cells transitioning into fibromyocytes. We demonstrated that CAD associated genetic variants are particularly enriched in endothelial and smooth muscle cell-specific open chromatin. Using single cell co-accessibility and cis-eQTL information, we prioritized putative target genes and candidate regulatory elements for ~30% of all known CAD loci. Finally, we performed genome-wide experimental fine-mapping of the CAD GWAS variants using epigenetic QTL analysis in primary human aortic endothelial cells and STARR-Seq massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal SNP(s) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci. Conclusions: These findings highlight the potential of applying snATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by non-coding GWAS variants.

Background A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1-subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. Methods Gene and protein expression were analyzed using quantitative polymerase chain reaction (qPCR) and immunoblotting, respectively. Putative allele-specific transcription factors were identified using in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed using reporter gene assays. To assess the effect of ZEB1, siRNA-mediated knockdown as well as overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes were analyzed using vascular smooth muscle cell (VSMC) migration assays and platelet aggregation analyses. Results Whole blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the non-risk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of non-risk allele promoter activity, as well as a significant reduction of endogenous GUCY1A3 expression. Ex vivo studied platelets from homozygous non-risk allele carriers displayed enhanced inhibition of adenosine diphosphate-induced platelet aggregation by the NO donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil as compared to homozygous risk allele carriers. Moreover, pharmacologic stimulation of sGC led to reduced migration only in VSMC homozygous for the non-risk allele. In the Hybrid Mouse Diversity Panel higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. Conclusions Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the non-risk allele leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.