In vivo Characterization of Regulatory Variant Pathogenicity in Congenital Heart Disease

项目摘要 先天性心脏病（CHD）是一组严重的出生缺陷，共同代表的主要原因 与出生缺陷相关的疾病和死亡。尽管临床基因检测和全外显子组的广泛使用 根据基因测序（WES），目前只有不到三分之一的CHD病例可以归因于蛋白质编码突变。 基因.许多剩余的，目前无法解释的情况下，被认为是由于非编码序列 改变心脏发育所必需的基因表达的变异。为了发现非编码变异， CHD患者，国家心脏、肺和血液研究所的B2B和TopMed项目 正在对大型CHD患者队列使用全基因组测序（WGS），主要针对先前 WES未能发现可能的致病编码变体。来自B2B队列的1，831例患者-父母三人组的WGS 目前有数百个其他的trios正在测序中。初步分析~750 先证者已经在预测的人类胎儿心脏增强子中鉴定出超过2,000种从头变异，沿着 具有统计学上显著过量的遗传位点（27个基因与预期的3.7个基因，p= 1x 10 -5）， 邻近的人胎儿心脏增强子在一些情况下显示出多种从头变异。这表明CHD 通过这些增强子的各自靶基因的失调而产生风险。然而，因果关系 CHD中这些变异的分子基础，以及它们潜在致病性的分子基础，仍有待进一步研究。 演示。基于我们之前在绘制和表征心脏增强剂方面的广泛工作， 规模，我们建议进行系统的体内功能验证的从头序列变异，从冠心病 存在于预测的心脏增强子中的患者，以揭示有助于心脏病病因学的增强子突变。 冠心病我们将1）使用预测的人类心脏增强剂的综合图谱，遗传和 表观基因组分析工具和大规模平行的报告基因测定， 多能干细胞（iPSC-CM），以识别和优先考虑心脏增强子， CHD患者，2）使用我们世界一流的小鼠转基因管道与新型单细胞 测试200个优先化增强子的参考和变体等位基因的表征方法（400个等位基因， 总数）在心脏发育的适当阶段评估风险等位基因如何改变体内增强子功能 3）使用CRISPR/Cas9基因组工程产生20个基因敲入小鼠模型， 改变增强子活性的人CHD变体等位基因和匹配的人参考等位基因，以评估其 使用单细胞转录组学和心脏的组合对心脏结构和功能的影响 表型分析成功完成拟议的研究将提供对以下作用的基本见解： 非编码调控序列在最常见的严重的人类出生缺陷，确定具体的例子， 人类增强子变体最终与疾病有关，并为其提供了初步的机制见解。 各自的作用模式，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.