Dissecting Genetic Mechanisms of Hypertrophic Cardiomyopathy by ENU Mutagenesis

描述（由申请人提供）：先前的人类遗传学研究已经确定了许多遗传性心血管疾病的病因。肥厚型心肌病（HCM）是最常见的遗传性心血管疾病（患病率1：500），至少有19个基因发生突变，其中大部分编码肌节蛋白，沿着少数编码Z盘和钙处理蛋白。然而，只有约50%的患者在先前鉴定的基因中检测到突变。因此，我们假设，在其他迄今未发现的基因，调节肌节，Z盘，或细胞钙稳态，包括代谢基因的变异，可以直接导致疾病。小鼠大规模正向遗传学是鉴定这些未发现基因的新方法。NHLBI资助了一项小鼠乙基亚硝基脲（ENU）诱变计划，该计划正在确定导致先天性心脏病的遗传途径。初步研究表明，6%的胎儿纯合子为一个给定的突变有孤立的心脏肥大。因此，我们的总体目标是通过ENU诱变程序利用我们可用的独特资源来恢复与HCM相关的新基因。我们的具体目标是：1.用HCM恢复ENU诱变的小鼠家系。将询问在胎儿中表现出纯合状态的HCM的家系是否存在成人中杂合状态的疾病表型，特别是超声心动图和HCM组织病理学特征的异常。在杂合状态下明显的表型更可能具有临床相关性。2.鉴定导致HCM的新基因突变。将使用序列捕获阵列对异常杂合子成人进行基因分型，以排除先前与HCM相关的基因和功能相关基因的突变。我们将选择~6个具有潜在新基因突变的家系（包括我们已经鉴定的HCM家系）。从其中2个家系中，我们将使用大规模并行全基因组测序来鉴定新基因中的突变。 这项探索性的提议旨在为未来的多项研究奠定基础：建立感兴趣的谱系库，供更广泛的科学界进一步分析;一旦证明，将这种策略扩展到更大的小鼠队列;进一步研究小鼠和其他模型系统中的新突变;确定新的遗传变异在我们的大型预先存在的HCM人类受试者队列中的作用。我们认为，我们必须继续进行拟议的项目，这样我们和其他研究人员就有机会研究由ENU诱变程序产生的HCM小鼠谱系。 公共卫生相关性：导致心肌异常增厚的遗传基因突变是猝死的最常见原因之一，特别是在年轻人中。虽然在过去的20年里，在确定这种疾病的遗传原因方面取得了巨大的进展，但大约一半的患者没有确定的原因。通过研究小鼠，该项目将确定遗传性心脏病的一些未知原因。这些发现将使医生能够确定谁是猝死的高风险，并及早干预，以防止死亡。从长远来看，这些发现将导致改进治疗的发展。

DESCRIPTION (provided by applicant): Previous human genetic studies have identified the etiology of many heritable cardiovascular disorders. Hypertrophic cardiomyopathy (HCM), the most common heritable cardiovascular disorder (prevalence 1:500), has been associated with mutations in at least 19 genes, most of which encode sarcomere proteins, along with a few encoding Z-disc and calcium handling proteins. However, mutations are detected in previously identified genes in only ~50% of patients. Therefore, we hypothesize that variants in other heretofore undiscovered genes that regulate the sarcomere, the Z-disc, or cellular calcium homeostasis, including metabolic genes, can directly cause disease. Large scale forward genetics in mice is a novel approach to identifying these undiscovered genes. The NHLBI has funded a mouse ethylnitrosourea (ENU) mutagenesis program that is identifying genetic pathways contributing to congenital heart disease. Preliminary studies show that 6% of fetuses homozygous for a given mutation have isolated cardiac hypertrophy. Therefore, our overall objective is to leverage the unique resource available to us through this ENU mutagenesis program to recover novel genes associated with HCM. Our specific aims are: 1. To recover ENU mutagenized mouse pedigrees with HCM. Pedigrees exhibiting HCM in the homozygous state in fetuses will be interrogated for the presence of the disease phenotype in the heterozygous state in adults, specifically, abnormalities on echocardiograms and histopathology characteristic of HCM. Phenotypes evident in the heterozygous state are more likely to be clinically relevant. 2. To identify novel genes with mutations leading to HCM. Abnormal heterozygous adults will be genotyped to exclude mutations in genes previously associated with HCM and functionally related genes, using sequence capture arrays. We will select ~6 pedigrees with potentially novel genetic mutations (including an HCM pedigree that we have already identified). From 2 of these pedigrees, we will identify mutations in novel genes using massively parallel whole genome sequencing. This exploratory proposal is meant to establish a foundation for multiple future studies: banking of pedigrees of interest for further analysis by the wider scientific community; expansion of this strategy, once proven, to larger cohorts of mice; further mechanistic studies of novel mutations in mice and other model systems; determination of the role of novel genetic variants in our large pre-existing cohorts of human subjects with HCM. We feel that it is imperative for us to pursue the proposed project, so that the opportunity for both us and other investigators to study mouse pedigrees with HCM that are generated by this ENU mutagenesis program is not wasted. PUBLIC HEALTH RELEVANCE: Inherited genetic mutations leading to abnormal thickening of the muscle of the heart are among the most common causes of sudden death, particularly in the young. Although enormous progress has been made in the past 20 years to identify the genetic causes of this disorder, approximately half of all patients do not have an identified cause. Studying mice, this project will identify some of these unknown causes of inherited heart disease. These findings will allow physicians to determine who is at high risk of sudden death and to intervene early to prevent death. In the long term, these findings will lead to the development of improved treatments.