Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci

Rationale: Genome-wide association studies have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cisregulatory elements 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 cisregulatory elements, to understand transcription factors establishing cell identity, and to interpret CAD-relevant, noncoding genetic variation. Methods and Results: We used single-nucleus ATAC-seq (assay for transposase-accessible chromatin with sequencing) to generate DNA accessibility maps in >7000 cells derived from human atherosclerotic lesions. We identified 5 major lesional cell types including endothelial cells, smooth muscle cells, monocyte/macrophages, natural killer/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 coaccessibility and cis-expression quantitative trait loci information, we prioritized putative target genes and candidate regulatory elements for approximate to 30% of all known CAD loci. Finally, we performed genome-wide experimental fine-mapping of the CAD variants identified in genome-wide association studies using epigenetic quantitative trait loci analysis in primary human aortic endothelial cells and self-transcribing active regulatory region sequencing (STARR-Seq) massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal single-nucleotide polymorphisms (SNPs) 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 single-nucleus ATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by noncoding genome-wide association study variants.</p

Genetic regulation of circular RNA expression in human aortic smooth muscle cells and vascular traits

Summary: Circular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type-specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease. In this study, we employed a systems genetics approach to identify SMC circRNA transcripts and their relevance in cardiovascular traits across the genome. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from donors of various genetic ancestries. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 differentially expressed circRNAs. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs that were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not quiescent SMCs. We identified six SNPs that had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease-related genome-wide association studies loci. We identified 14 blood pressure, one myocardial infarction, and three coronary artery disease loci, which were associated with a circRNA transcript but not an mRNA transcript. Overall, our results provide insight into the genetic basis of vascular disease traits mediated by circRNA expression

Effect of Differences in the Microbiome of Cyp17a1-Deficient Mice on Atherosclerotic Background

CYP17A1 is a cytochrome P450 enzyme that has 17-alpha-hydroxylase and C17,20-lyase activities. Cyp17a11 deficiency is associated with high body mass and visceral fat deposition in atherosclerotic female ApoE knockout (KO, d/d or −/−) mice. In the present study, we aimed to investigate the effects of diet and Cyp17a1 genotype on the gut microbiome. Female Cyp17a1 (d/d) × ApoE (d/d) (DKO) and ApoE (d/d) (controls) were fed either standard chow or a Western-type diet (WTD), and we demonstrated the effects of genetics and diet on the body mass of the mice and composition of their gut microbiome. We found a significantly lower alpha diversity after accounting for the ecological network structure in DKO mice and WTD-fed mice compared with chow-fed ApoE(d/d). Furthermore, we found a strong significant positive association of the Firmicutes vs. Bacteroidota ratio with body mass and the circulating total cholesterol and triglyceride concentrations of the mice when feeding the WTD, independent of the Cyp17a1 genotype. Further pathway enrichment and network analyses revealed a substantial effect of Cyp17a1 genotype on associated cardiovascular and obesity-related pathways involving aspartate and L-arginine. Future studies are required to validate these findings and further investigate the role of aspartate/L-arginine pathways in the obesity and body fat distribution in our mouse model

Media 1: Ultrahigh-resolution, high-speed spectral domain optical coherence phase microscopy

Originally published in Optics Letters on 01 January 2014 (ol-39-1-45