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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.

项目概要先天性心脏病（CHD）是一组严重的出生缺陷，共同代表了主要原因与出生缺陷相关的疾病和死亡。尽管广泛使用临床基因检测和全外显子组测序（WES），目前不到三分之一的冠心病病例是由蛋白质编码突变引起的基因。许多剩余的、当前无法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。发现非编码变体 CHD 患者、国家心肺血液研究所的 Bench to Bassinet (B2B) 和 TopMed 计划正在对大型 CHD 患者队列使用全基因组测序 (WGS)，主要针对先证者 WES 未能发现可能的致病编码变异。来自 B2B 队列的 1,831 名患者-家长三人组的全基因组测序目前可用，另外数百个三重奏目前正在测序。初步分析~750 先证者已经在预测的胎儿人类心脏增强剂中鉴定出超过 2,000 个从头变异，具有统计学上显着过量的遗传位点（27 个基因与预期的 3.7 个基因相比，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异。这表明冠心病风险是通过这些增强子各自的靶基因的失调而产生的。然而，因果关系 CHD 中的这些变异及其潜在致病性的分子基础仍有待进一步研究。证明了。以我们之前在绘制和表征心脏增强剂方面所做的大量工作为基础规模，我们建议对 CHD 的从头序列变异进行系统的体内功能验证存在于预测的心脏增强子中的患者揭示了导致病因学的增强子突变冠心病。我们将 1）结合使用预测的人类心脏增强剂、遗传和表观基因组分析工具，以及从诱导分化的心肌细胞中进行大规模平行报告分析多能干细胞（iPSC-CM）可识别并优先考虑含有从头变异的心脏增强剂 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相结合用于测试 200 个优先增强子（400 个等位基因）的参考和变体等位基因的表征方法总计）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在细胞分辨率下，3) 使用 CRISPR/Cas9 基因组工程生成 20 个敲入小鼠模型人类 CHD 变异等位基因改变增强子活性并匹配人类参考等位基因以评估其结合单细胞转录组学和心脏功能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为了解其作用提供基础见解最常见的严重人类出生缺陷中的非编码调控序列，确定以下具体例子人类增强子变异最终与疾病有关，并为其提供初步的机制见解各自的作用模式为探索未来治疗提供新途径。