In vivo Characterization of Regulatory Variant Pathogenicity in Congenital Heart Disease

项目总结 先天性心脏病(CHD)是一组严重的出生缺陷，共同代表着主要的原因 与出生缺陷相关的疾病和死亡。尽管临床基因检测和整个外显子组的广泛使用 测序(WES)，目前只有不到三分之一的CHD病例可以由蛋白质编码的突变来解释 基因。许多剩余的、目前尚未解释的情况被认为是由于非编码序列造成的 改变心脏发育必需基因表达的变异体。中发现非编码变体 冠心病患者，国家心肺血液研究所的B2B和TopMed计划 正在对大量的CHD患者队列使用全基因组测序(WGS)，主要是针对先证者 WES未能发现一个可能的致病编码变体。来自B2B队列的1,831个患者-父母三人组的WGS 目前可以使用，目前正在对数百个额外的三重奏进行测序。~750例的初步分析 先驱们已经在预测的胎儿心脏增强剂中发现了2000多个新变种，以及 在统计上显著过剩的遗传基因座(27个基因比预期的3.7个基因，p=1x10-5) 邻近的人类胎儿心脏增强剂在病例中显示出多种从头开始的变异。这表明CHD 风险是通过这些增强子各自的靶基因调控失调而产生的。然而，因果关系 这些变异在冠心病中的作用以及它们潜在致病性的分子基础仍有待研究。 演示了。基于我们之前在绘制和表征心脏增强剂方面的广泛工作，请访问 规模，我们建议对来自CHD的从头序列变体进行系统的体内功能验证 居住在预测的心脏增强剂中的患者揭示了导致心脏病病因的增强子突变 先心病。我们将使用预测的人类心脏增强剂、基因和 表观基因组分析工具，以及诱导分化的心肌细胞的大量平行报告分析 多能干细胞(IPSC-CMS)用于鉴定和优先考虑携带新基因突变体的心脏增强剂 CHD患者，2)使用我们世界级的小鼠转基因管道与新型单细胞相结合 测试200个优先增强子的参考等位基因和变异等位基因的特征方法(400个等位基因在 在心脏发育的适当阶段，评估风险等位基因如何改变体内的增强子功能 在细胞分辨率上，3)使用CRISPR/Cas9基因组工程为 改变增强子活性的人类CHD变异等位基因和匹配的人类参考等位基因 单细胞转录组分和心脏移植联合应用对心脏结构和功能的影响 表型鉴定。拟议研究的成功完成将提供对以下方面作用的基本见解 在最常见的严重人类出生缺陷中，识别非编码调控序列的具体例子 人类增强子变体最终与疾病有关，并提供了对其机制的初步见解 各自的行动模式，为探索未来的治疗提供新的途径。

PROJECT SUMMARY Congenital heart disease (CHD) is a group of severe birth defects that collectively represent the leading cause of birth defect-associated illness and death. Despite the extensive use of clinical genetic testing and whole exome sequencing (WES), less than a third of CHD cases can currently be accounted for by mutations in protein-coding genes. Many of the remaining, currently unexplained cases are assumed to be due to non-coding sequence variants that alter the expression of genes essential for cardiac development. To uncover non-coding variants in CHD patients, the National Heart, Lung, and Blood Institute's Bench to Bassinet (B2B) and TopMed programs are using whole genome sequencing (WGS) on large CHD patient cohorts, principally for probands whose prior WES failed to uncover a likely causative coding variant. WGS of 1,831 patient-parent trios from the B2B cohort is currently available, with several hundred additional trios currently being sequenced. Initial analyses of ~750 probands have already identified over 2,000 de novo variants in predicted fetal human heart enhancers, along with a statistically significant excess of genetic loci (27 genes versus 3.7 expected, p=1x10-5) at which the neighboring human fetal heart enhancers showed multiple de novo variants in cases. This suggests that CHD risk is conferred through dysregulation of the respective target genes of these enhancers. However, the causality of these variants in CHD, as well as the molecular underpinnings of their potential pathogenicity, remain to be demonstrated. Building on our extensive previous work in mapping and characterizing cardiac enhancers at scale, we propose to perform systematic in vivo functional validation of de novo sequence variants from CHD patients that reside in predicted heart enhancers to reveal enhancer mutations that contribute to the etiology of CHD. We will 1) use a combination of comprehensive maps of predicted human heart enhancers, genetic and epigenomic analysis tools, and massively parallel reporter assays in cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) to identify and prioritize cardiac enhancers harboring de novo variants from CHD patients, 2) use our world-class mouse transgenesis pipeline in combination with novel single-cell characterization methods to test the reference and variant alleles of 200 prioritized enhancers (400 alleles in total) at appropriate stages of cardiac development to assess how the risk alleles alter enhancer function in vivo at cellular resolution, 3) use CRISPR/Cas9 genome engineering to generate 20 knock-in mouse models for human CHD variant alleles that alter enhancer activity and matched human reference alleles to assess their impact on the structure and function of the heart using a combination of single-cell transcriptomics and cardiac phenotyping. Successful completion of the proposed studies will provide foundational insights into the role of non-coding regulatory sequences in the most common severe human birth defect, identify specific examples of human enhancer variants conclusively implicated in disease, and provide initial mechanistic insights into their respective mode of action to provide new avenues for exploring future therapeutics.