THE GENETIC BASIS OF CONGENITAL HEART DEFECTS (CHD)

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 先天性心脏病(CHD)是最常见的出生缺陷之一。婴儿出生时患有 CHD通常需要多种内科和/或外科干预，但即使不是死亡率，也会有很高的发病率。尽管它们的患病率和临床意义，冠心病的病因仍然很大程度上是未知的。流行的观点认为，这种疾病是异质性和复杂性的，既有环境因素，也有遗传风险因素。一些心脏发育基因的突变，如NKX2.5、GATA4和TBX5，已被认为是遗传风险因素。我们建议使用一种新的、高灵敏的突变检测技术，称为恒变性毛细管电泳法(CDCE)和高保真聚合酶链式反应(HiFi PCR)相结合，以确定与大量对照人群相比，每个候选基因的突变是否在患者群体中丰富。这项新技术通过一次汇集和筛选96个人的DNA，使快速和具有成本效益的大群体分析成为可能。对大量人群的分析对于发现低频率的因果突变和量化突变与疾病之间的低效关系是必要的。一旦鉴定出丰富的突变，我们将检查每个突变的潜在生物学效应。这些突变可能会也可能不会通过无义或错义突变导致编码蛋白质的明显变化。事实上，越来越多的证据表明，即使是翻译沉默的突变和多态也可以通过改变前mRNA剪接而产生表型影响。另一层复杂性是，已经提出了一种单独的先天性心脏病的遗传机制。最近在NKX2.5和/或TBX5中发现了体细胞突变 超过95%的心脏有间隔缺陷。这些突变是在间隔缺陷内发现的，在取自同一心脏的未受影响的区域中没有发现，这表明这些突变具有体细胞和嵌合体的性质，并表明这些突变是病因。此外，在大多数先天性心脏病患者中，观察到同一患者体内存在多种突变和多种单倍型。一个基因中出现两个或两个以上的体细胞突变是一种极其罕见的事件，这表明了一种极端的基因组不稳定状态，这让人想起在癌症中观察到的情况。 在这项建议中，我们将研究遗传和体细胞的遗传贡献 NKX2.5、GATA4和TBX5基因突变与心脏畸形的关系具体来说，我们会： 具体目标1：研究NKX2.5、GATA4和TBX5的分子缺陷对 冠心病的病因学。 A.验证NKX2.5、GATA4和TBX5基因突变会增加冠心病风险的假设 采用CDCE/HiFi聚合酶链式反应。 B.测试NKX2.5、GATA4和TBX5的选择性剪接可增加或降低冠心病易感性的假设。 C.确定在CHD中是否观察到NKX2.5、GATA4和TBX5中的沉默序列变异 患者会产生选择性剪接。 D.继续确定一大批特征良好的冠心病受试者 具体目的2：研究体细胞突变在冠心病病因学中的作用。 A.检验心脏发育基因的体细胞突变是心脏病的病因的假设 先心病。 B.测定正常和畸形心脏在心脏发生过程中的心肌细胞突变率。 使用突变光谱分析在正常和畸形心脏中发现主要的诱变途径。 我们的目标是识别与冠心病有因果关系的遗传突变，确定 他们可以通过哪些机制增加先天性心脏病的风险，并检验体细胞突变是先天性心脏病的病因的假设。我们将评估在这些基因中发现的突变的生物学意义，以确定改进的疾病管理策略。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Congenital heart disease (CHD) is one of the most common birth defects. Infants born with CHD often require multiple medical and/or surgical interventions and will nonetheless experience significant morbidity if not mortality. Despite their prevalence and clinical significance, the etiology of CHD remains largely unknown. The pervading view is that the disease is heterogeneous and complex, with bothenvironmental and inherited genetic risk factors. Mutations in a few cardiac development genes, such as NKX2.5, GATA4, and TBX5 have been implicated as inherited risk factors. We propose using a novel, highly sensitive mutation detection technology called Constant Denaturant Capillary Electrophoresis (CDCE) combined with High-Fidelity PCR (HiFi PCR) to determine whether mutations in each candidate gene are enriched in the patient population as compared to a large control population. This new technology enables the rapid and cost-effective analysis of large populations by pooling and screening DNA from 96 individuals at a time. The analyses of large populations are necessary to discover low frequency causal mutations and to quantitate low effect relationships between mutations and disease. Once enriched mutations are identified, we will examine the potential biological effect of each mutation. These mutations may or may not lead to obvious changes in the encoded proteins via nonsense or missense mutations. Indeed, there is increasing evidence that even translationally silent mutations and polymorphisms can have a phenotypic affect by altering pre-mRNA splicing. An additional layer of complexity is that a separate genetic mechanism for congenital heart disease has been proposed. Somatic mutations were recently identified in NKX2.5 and/or TBX5 in greater than 95% of hearts with septal defects. These mutations were identified within the septal defects and were not found in unaffected regions taken from the same heart, indicating a somatic and mosaic nature for these mutations and suggest that these mutations are etiologic. Moreover, multiple mutations as well as multiple haplotypes were observed within the same patient in a majority of these congenital heart disease patients. The occurrence of two or more somatic mutations in a gene is an exceedingly rare event and suggests a condition of extreme genomic instability, reminiscent of that observed in cancer. In this proposal, we will examine the genetic contribution of both inherited and somatic mutations in NKX2.5, GATA4, and TBX5 to cardiac malformations. Specifically, we will: Specific Aim 1: Examine the contribution of molecular deficits in NKX2.5, GATA4, and TBX5 to the etiology of CHD. A. Test the hypothesis that mutations in NKX2.5, GATA4, and TBX5 increase risk for CHD using CDCE/HiFi PCR. B. Test the hypothesis that alternative splicing of NKX2.5, GATA4, and TBX5 confer an increased or decreased susceptibility to CHD. C. Determine if silent sequence variants observed in NKX2.5, GATA4, and TBX5 in CHD patients result in alternative splicing. D. Continue the ascertainment of a large, well-characterized cohort of subjects with CHD Specific Aim 2: Examine the contribution of somatic mutations to the etiology of CHD. A. Test the hypothesis that somatic mutations in cardiac developmental genes are etiologic in CHD. B. Determine the mutation rate of cardiomyocytes during cardiogenesis in normal and malformed hearts. C. Use mutational spectrometry to discover primary mutagenic pathways in normal and malformed hearts. Our goal is to identify inherited mutations that are causally related to CHD, determine the mechanisms by which they can increase risk of congenital heart disease, and test the hypothesis that somatic mutations are etiologic in congenital heart disease. We will assess the biological significance of mutations found in these genes with the goal of identifying improved management strategies for the disease.