Functional noncoding SNPs in human endothelial cells fine-map vascular trait associations

Abstract

Functional consequences of genetic variation in the noncoding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional noncoding SNPs with effects in human endothelial cells (ECs), we utilized our previous molecular quantitative trait locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional noncoding SNPs. Together with information from genome-wide association studies (GWASs) for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5711 variants validated, whose enriched attributes included: (1) mutations to TF binding motifs for ETS or AP-1 that are regulators of the EC state; (2) location in accessible and H3K27ac-marked EC chromatin; and (3) molQTL associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of noncoding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlighted evidence suggesting mechanisms by which noncoding SNPs at two loci affect risk for pulse pressure/large artery stroke and abdominal aortic aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH. Together, we highlight the attributes and context dependence of functional noncoding SNPs and provide new mechanisms underlying vascular disease risk.

Single cell ‘omic profiles of human aortic endothelial cellsin vitroand human atherosclerotic lesionsex vivoreveals heterogeneity of endothelial subtype and response to activating perturbations

“…Finally, GWAS studies have established that hundreds of independent common genetic variants in human populations affect risk for CAD, yet discovering the causal mechanisms remains a major challenge given that most of the risk is in non-coding regions of the genome. One approach to prioritize causal variants in regulatory elements is through integration of open chromatin regions from the cell type and states of interest followed by expression quantitative trait loci (eQTL) or other linking evidence to target gene (75, 76). In the current study, we find significant enrichment for CAD-risk variants in open chromatin regions across the entire dataset (“panEC”) as well as specifically for EC2 and EC4 subpopulations (Figure 6B ; Table S15-17 in the Data Supplement ).…”

Coronary Artery Disease risk variant dampens the expression of CALCRL by reducing HSF binding to shear stress responsive enhancer in endothelial cells

“…TeloHAEC and HepG2 cell lines were used for the experiment. STARR-Seq library and experiment have been described previously in 22,24,25 . To assess transcriptional activity of haplotypes of the candidate regulatory regions, STARR-Seq RNA read data was UMI-deduplicated, depth-normalized to library size, and normalized for haplotype abundance in the plasmid DNA (input) library.…”

“…TeloHAEC were cultured using Vascular Cell cocktail by pipetting up and down several times and incubated on ice for 15 minutes. 25 μl detergent (10% NP40) was added and vortexed for 10 seconds at highest setting.…”

Using Genomics to Identify Novel Therapeutic Targets for Aortic Disease

“…This study identified 2 SNPs at a GWAS locus for AAA located at the 3ʹ end of the LDAH gene; targeted deletion of the enhancer region containing both SNPs resulted in lower LDAH expression, without affecting other genes in the locus. 47 To our knowledge, similar studies have not yet been published for thoracic aortic variants; however, these initial reports offer a roadmap for combining traditional fine-mapping with sophisticated epigenomic techniques as well as high-throughput reporter assays to inform causal variant discovery.…”