Multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci.

Genome-wide association studies (GWASs) have identified hundreds of risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWASs, and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotypes to identify quantitative trait loci for expression (eQTLs) and splicing (sQTLs) in coronary arteries from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary artery; 19% exhibited cell-type-specific expression. Colocalization revealed subgroups of eGenes unique to CAD and blood pressure GWAS. Fine-mapping highlighted additional eGenes, including TBX20 and IL5. We also identified sQTLs for 1,690 genes, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing to accurately identify disease-relevant isoform expression. Our work provides a patient-derived coronary artery eQTL resource and exemplifies the need for diverse study populations and multifaceted approaches to characterize gene regulation in disease processes.

Integrative multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci.

Genome-wide association studies (GWAS) have identified hundreds of genetic risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWAS and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotype information to identify quantitative trait loci (QTL) for gene expression and splicing in coronary arteries obtained from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary arteries and 19% exhibited cell-type-specific expression. Colocalization analysis with GWAS identified subgroups of eGenes unique to CAD and blood pressure. Fine-mapping highlighted additional eGenes of interest, including TBX20 and IL5 . Splicing (s)QTLs for 1,690 genes were also identified, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing events to accurately identify disease-relevant gene expression. Our work provides the first human coronary artery eQTL resource from a patient sample and exemplifies the necessity of diverse study populations and multi-omic approaches to characterize gene regulation in critical disease processes.

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

Exploring the causal inference of shear stress associated DNA methylation in carotid plaque on cardiovascular risk.

BACKGROUND AND AIMS: Atherosclerosis is a lipid-driven inflammatory disease presumably initiated by endothelial activation. Low vascular shear stress is known for its ability to activate endothelial cells. Differential DNA methylation (DNAm) is a relatively unexplored player in atherosclerotic disease development and endothelial dysfunction. Previous studies showed that the expression of 11 genes was associated with differential DNAm due to low shear stress in murine endothelial cells. We hypothesized a causal relationship between DNAm of shear stress associated genes in human carotid plaque and increased risk of cardiovascular disease. METHODS: Using Mendelian randomisation (MR) analysis, we explored the potential causal role of DNAm of shear stress associated genes on cardiovascular disease risk. We used data from the Athero-Expression Biobank Study for the discovery of methylation quantitative trait loci (mQTLs) in 442 advanced carotid plaques. Next, we performed MR analysis using these mQTLs and publicly available GWAS summary statistics of coronary artery disease (CAD) and ischemic stroke (IS). RESULTS: We discovered 9 mQTLs in plaque in the promoters of shear stress associated genes. We found no significant effect of shear stress gene promoter methylation and increased risk of CAD and IS. CONCLUSIONS: Differential methylation of shear stress associated genes in advanced atherosclerotic plaques in unlikely to increase cardiovascular risk in human.