Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene (ChIPseq). Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene (ChIPseq)

Genome-wide association studies (GWAS) have identified 100s of loci associated with coronary artery disease (CAD) and blood pressure (BP)/hypertension. Many of these loci are not associated with traditional risk factors, nor include obvious candidate genes, complicating their functional characterization. We hypothesized that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis (e.g. selective barrier, inflammation, hemostasis, vascular tone) and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. We generated an integrated map of gene expression (RNA-sequencing), open chromatin regions (ATAC-sequencing), and 3D interactions (Hi-C) in resting and TNFα-treated human endothelial cells. We showed that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We used physical loops identified by Hi-C to link open chromatin peaks that include CAD or BP SNPs with the promoter of genes expressed in endothelial cells. This analysis highlighted 4,548 combinations of regulatory elements-promoters, including 108 pairs that involve a differentially open chromatin site and a differentially expressed gene following TNFα treatment. At a CAD locus, we validated one of these pairs by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measuring an effect on the expression of the novel CAD candidate gene AIDA. Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD or hypertension Overall design: H3K27ac ChIP-seq of TNF-alpha treated endothelial cells (teloHAEC) for 0, 4h or 24h

全基因组关联研究（Genome-wide association studies, GWAS）已识别出数百个与冠状动脉疾病（coronary artery disease, CAD）及血压（blood pressure, BP）/高血压相关的基因座。此类基因座中，多数与传统风险因子无关联，亦未包含明确的候选基因，这为其功能表征工作带来了极大复杂性。我们提出假说：诸多与血管疾病相关的GWAS基因座可调控内皮细胞功能。内皮细胞在维持血管稳态（如选择性屏障、炎症反应、止血功能、血管张力）中发挥关键作用，而内皮功能障碍是动脉粥样硬化与高血压的标志性病理特征。我们对静息状态及肿瘤坏死因子α（TNFα）处理后的人内皮细胞，构建了涵盖基因表达（RNA-sequencing）、开放染色质区域（ATAC-sequencing）以及三维相互作用（Hi-C）的整合图谱。我们证实，与CAD及BP相关的遗传变异在内皮细胞的开放染色质区域中显著富集。我们借助Hi-C识别的物理环结构，将包含CAD或BP相关单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）的开放染色质峰与内皮细胞中表达基因的启动子进行关联。该分析共筛选出4548个调控元件-启动子组合，其中108对涉及TNFα处理后出现开放染色质位点差异变化与基因表达差异的配对。在一个CAD基因座中，我们通过成簇规律间隔短回文重复序列相关蛋白9（CRISPR/Cas9）靶向敲除TNFα敏感型调控元件，并检测其对新型CAD候选基因AIDA表达的影响，以此验证了其中一对关联。本研究数据证实，血管内皮细胞中的遗传变异可通过调控相关通路改变个体间CAD或高血压的患病风险，该机制发挥着重要作用。整体实验设计：对经TNF-α处理0、4或24小时的内皮细胞（teloHAEC）进行H3K27ac染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）