Regulatory variants of blood pressure genes (Omni-C)

Genome-wide association studies (GWAS) have identified blood pressure-related loci, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4608 genetic variants in linkage with blood pressure loci in vascular smooth muscle cells (VSMCs) and cardiomyocytes (CMs) by massively parallel reporter assays (MPRAs). Regulatory variants are in non-conserved loci, enriched in repeats, and alter trait-relevant transcription factor binding sites. Higher-order genome organization indicates that loci harboring regulatory variants converge in spatial hubs to control specific signaling pathways required for proper cardiovascular function. Modelling different variant allele frequencies by CRISPR prime editing led to expression changes of KCNK9, SFXN2, and PCGF6. We provide mechanistic insights into how regulatory variants converge their effects on blood pressure genes (i.e. ULK4, MAP4, CFDP1, PDE5A, 10q24.32), and cardiovascular pathways. Our findings support advances in molecular precision medicine to define functionally relevant variants and the genetic architecture of blood pressure genes. MPRA pool of 232,975 oligonucleotides, 1 sample for input library and then 5 replicates for CMs and 4 for VSMCs. The plasmid pool as DNA input and each TagSeq replicate (CMs = 5 replicates, VSMCs = 4 replicates) was sequenced on the HiSeq 2500 platform (Illumina), single end with 50 bp. Prime Editing (CRISPR/Cas9) using HEK293 with 3 replicates per allelic frequency of each SNP.

全基因组关联分析（Genome-wide association studies, GWAS）虽已识别出与血压相关的基因座，但针对其因果关系及相关分子机制的功能认知仍相对滞后。本研究通过大规模平行报告基因检测（massively parallel reporter assays, MPRAs），对血管平滑肌细胞（vascular smooth muscle cells, VSMCs）和心肌细胞（cardiomyocytes, CMs）中与血压相关基因座连锁的4608个遗传变异进行了功能表征。调控变异位于非保守基因座中，且富集于重复序列区域，同时会改变与性状相关的转录因子结合位点。高阶基因组结构分析显示，携带调控变异的基因座会聚集在空间枢纽区域，以此调控维持正常心血管功能所需的特定信号通路。通过CRISPR引物编辑（CRISPR prime editing）模拟不同变异等位基因频率的实验，导致KCNK9、SFXN2以及PCGF6的表达发生改变。本研究为调控变异如何协同作用于血压相关基因（即ULK4、MAP4、CFDP1、PDE5A、10q24.32）及心血管通路提供了机制层面的认知。本研究结果可为分子精准医学的发展提供支撑，助力鉴定具有功能相关性的变异以及血压相关基因的遗传结构。本研究构建了包含232975条寡核苷酸的MPRA文库池，其中1份样本作为输入文库，心肌细胞（CMs）设置5次生物学重复，血管平滑肌细胞（VSMCs）设置4次生物学重复。以质粒池作为DNA输入样本，所有TagSeq重复样本（CMs=5次重复、VSMCs=4次重复）均在Illumina公司的HiSeq 2500测序平台上进行单端50bp测序。针对每个单核苷酸多态性（single nucleotide polymorphism, SNP）的不同等位基因频率，本研究使用HEK293细胞开展CRISPR引物编辑实验，每组设置3次生物学重复。