Super-Enhancer-Associated Aortic Valve Stenosis Risk Locus 1p21.2 Alters NFATC2 Binding Site and Promotes Fibrogenesis [ATAC-Seq]. Super-Enhancer-Associated Aortic Valve Stenosis Risk Locus 1p21.2 Alters NFATC2 Binding Site and Promotes Fibrogenesis [ATAC-Seq]

Genome-wide association studies identified a strong signal for non coding variants at the 1p21.2 locus associated with calcific aortic valve stenosis (CAVS). Regulation at the locus and impact on the biology of the aortic valve is presently unknown. Integrative mapping of genetic association data was performed. The locus was evaluated by 3D genome mapping, analysis of atomic resolution data, DNA-binding assay and CRISPR activation (CRISPRa). Weighted gene co-expression network analysis (WGCNA) was performed to determine regulatory network in CAVS. The functional impact of the locus was assessed in isolated VICs. Fine-grained mapping at 1p21.2 risk locus identified rs6702619 as being located in open chromatin and a distant-acting super-enhancer including chromatin interaction with the promoter of PALMD in VICs. Atomic-level data showed that the risk variant modified base readout and DNA shape, which prevented the recruitment of NFATC2, a transcription factor involved in heart valve morphogenesis, and lowered the expression of PALMD. CRISPRa confirmed that rs6702619 exerts a control on the expression of PALMD. WGCNA performed in 233 calcified aortic valves identified a co-expression network encompassing PALMD, which was enriched in actin-based process. In LC-MS/MS and co-immunoprecipitation assay, actin was identified as a protein interacting with PALMD. Lower expression of PALMD in VICs promoted the polymerization of actin, the activation of myocardin-related transcription factor and fibrogenesis. Risk allele at rs6702619 disrupts a NFATC2 binding site and decreases enhancer-mediated expression of PALMD, which results in actin polymerization, a myofibroblast-like phenotype in VICs and fibrogenesis, a key underpinning process in the pathogenesis of CAVS. Overall design: ATAC-seq in human aortic valve interstitial cells.

全基因组关联研究（Genome-wide association studies）已在1p21.2位点发现与钙化性主动脉瓣狭窄（calcific aortic valve stenosis, CAVS）相关的非编码变异强信号。目前该位点的调控机制及其对主动脉瓣膜生物学的影响尚不明确。本研究对遗传关联数据开展了整合定位分析，通过三维基因组定位、原子分辨率数据分析、DNA结合实验及CRISPR激活（CRISPRa）技术对该位点进行评估。采用加权基因共表达网络分析（Weighted gene co-expression network analysis, WGCNA）解析钙化性主动脉瓣狭窄中的调控网络，并在分离得到的主动脉瓣间质细胞（valve interstitial cells, VICs）中评估该位点的功能影响。

对1p21.2风险位点的精细定位分析显示，rs6702619位于开放染色质区域及远距离作用的超级增强子内，该增强子可与主动脉瓣间质细胞中PALMD基因的启动子发生染色质相互作用。原子级数据分析表明，该风险变异可改变碱基读出信号与DNA构象，进而阻碍参与心脏瓣膜形态发生的转录因子NFATC2的募集，并下调PALMD基因的表达水平。CRISPRa实验证实，rs6702619可调控PALMD基因的表达。

对233份钙化性主动脉瓣膜样本开展WGCNA分析，发现包含PALMD的共表达网络显著富集于肌动蛋白相关生物学过程。在液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）及免疫共沉淀实验中，肌动蛋白被鉴定为与PALMD相互作用的蛋白质。主动脉瓣间质细胞中PALMD表达下调可促进肌动蛋白聚合、心肌素相关转录因子活化及纤维化进程。

rs6702619的风险等位基因可破坏NFATC2结合位点，降低增强子介导的PALMD表达，进而引发肌动蛋白聚合、主动脉瓣间质细胞出现肌成纤维细胞样表型及纤维化进程——这正是钙化性主动脉瓣狭窄发病机制中的核心支撑过程。

整体实验设计：对人主动脉瓣间质细胞进行转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）。