In vivo Characterization of Regulatory Variant Pathogenicity in Congenital Heart Disease

先天性心脏病调节变异致病性的体内表征

基本信息

批准号：
10390962
负责人：
Len Alexander Pennacchio
金额：
$ 76.59万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10390962
关键词：
Affect Alleles Biological Assay CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiac development Cells Cessation of life Code Collection Congenital Abnormality DNA Data Diagnosis Disease Distant Embryo Embryonic Development Engineering Enhancers Etiology Fetal Heart Foundations Future Gene Expression Gene Transfer Techniques Genes Genetic Genetic Enhancer Element Genome Genome engineering Genomics Heart Heart Abnormalities Heart Diseases Histologic Human Human Genetics Knock-in Mouse Letters Life Link Maps Medical Genetics Methods Molecular Mus Mutation National Heart, Lung, and Blood Institute Parents Pathogenicity Patient Care Patients Phenotype Physiological Population Proteins Regulator Genes Reporter Research Resolution Resources Risk Role Site Structure Techniques Testing Therapeutic Tissues Trans-Omics for Precision Medicine Transgenic Mice Untranslated RNA Validation Variant Work cardiogenesis cell type cohort congenital heart disorder de novo mutation epigenomics exome sequencing falls fetal follow-up functional genomics gene function genetic testing genetic variant genome sequencing genomic locus heart disease risk heart function human model in vivo induced pluripotent stem cell insight mouse model next generation novel proband programs risk variant single-cell RNA sequencing tool transcriptomics whole genome

项目摘要

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.

项目摘要先天性心脏病（CHD）是一组严重的先天缺陷，共同代表了主要原因与先天缺陷相关的疾病和死亡尽管广泛使用了临床基因检测和整个外显子组测序（WES），目前不到三分之一的冠心病病例可以通过蛋白质编码的突变来解释基因。剩下的许多目前无法解释的情况被认为是由于非编码序列造成的改变基因表达对心脏发育必不可少的变体。发现非编码变体冠心病患者，国家心脏，肺和血液研究所的卧床板凳（B2B）和额外的程序在大型冠心病患者同类中使用整个基因组测序（WGS），主要用于先验 Wes未能发现可能的因果编码变体。来自B2B队列的1,831名患者父母三重奏的WGS 目前可用，目前正在测序几百个三重奏。〜750的初始分析概率已经确定了预测的胎儿人心脏增强剂中的2,000多个从头变体具有统计学意义的遗传基因座（27个基因与3.7的预期，p = 1x10-5），其中邻近的人类胎儿心脏增强剂在病例中显示出多种从头变体。这表明CHD 风险通过这些增强子的各个靶基因失调而赋予风险。但是，因果关系这些冠心病中的这些变体以及其潜在致病性的分子基础，仍然是证明。在我们以前的广泛映射和表征心脏增强剂的绘制基础上建立比例尺，我们建议从CHD进行从头序列变体进行系统的体内功能验证驻留在预测心脏增强剂中以揭示增强子突变的患者冠心。我们将1）结合预测的人心脏增强子，遗传和表观基因组分析工具和与诱导的心肌细胞中的大规模平行记者测定法多能干细胞（IPSC-CMS）识别和优先级的心脏增强剂，这些心脏增强剂具有从头开始的变体冠心病患者，2）使用我们的世界一流小鼠转基因管道与新型单细胞结合测试200个优先增强剂的参考和变体等位基因的表征方法（400位等位基因总计）在心脏发展的适当阶段，以评估风险等位基因如何改变体内增强子功能在蜂窝分辨率下，3）使用CRISPR/CAS9基因组工程生成20个敲门鼠标模型改变增强子活性并匹配的人类参考等位基因的人类冠心病变异等位基因评估其评估使用单细胞转录组和心脏的组合对心脏的结构和功能的影响表型。成功完成拟议的研究将提供有关其作用的基本见解在最常见的严重人类出生缺陷中的非编码调节序列，确定人类增强子变体最终与疾病有关，并为其提供初始的机械见解各自的行动方式为探索未来治疗学提供新的途径。