THE GENETIC BASIS OF CONGENITAL HEART DEFECTS (CHD)

先天性心脏缺陷 (CHD) 的遗传基础

• 批准号: 7381934
• 负责人: Aoy Tomita-Mitchell
• 金额: $ 6.02万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381934
• 关键词: GENETIC; BASIS; CONGENITAL; HEART; DEFECTS

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。先天性心脏病是最常见的出生缺陷之一。先天性心脏病婴儿通常需要多次医疗和/或手术干预，但即使没有死亡率，也会出现严重的发病率。尽管其患病率和临床意义，冠心病的病因仍然在很大程度上是未知的。普遍的观点是，这种疾病是异质性和复杂的，既有环境因素，也有遗传的遗传风险因素。一些心脏发育基因的突变，如NKX2.5、GATA 4和TBX5，被认为是遗传性风险因素。我们建议使用一种新的，高灵敏度的突变检测技术，称为恒定变性毛细管电泳（CDCE）结合高保真PCR（HiFi PCR），以确定每个候选基因中的突变是否在患者人群中富集相比，一个大的控制人口。这项新技术可以通过一次汇集和筛选96个个体的DNA，对大规模人群进行快速和具有成本效益的分析。大规模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应关系是必要的。一旦确定了富集突变，我们将检查每个突变的潜在生物学效应。这些突变可能会或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，有越来越多的证据表明，即使是沉默突变和多态性也可以通过改变前体mRNA剪接而产生表型影响。另一层复杂性是，已经提出了先天性心脏病的单独遗传机制。最近在超过95%的间隔缺损心脏中发现了NKX2.5和/或TBX5的体细胞突变。这些突变是在间隔缺损中发现的，在取自同一心脏的未受影响的区域中未发现，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学的。此外，在大多数先天性心脏病患者中，在同一患者中观察到多个突变以及多个单倍型。在一个基因中发生两个或两个以上的体细胞突变是极其罕见的事件，表明基因组极度不稳定，这让人想起在癌症中观察到的情况。在这个提议中，我们将研究NKX2.5，GATA 4和TBX5中遗传和体细胞突变对心脏畸形的遗传贡献。具体来说，我们将：具体目标1：检查NKX2.5，GATA 4和TBX5的分子缺陷对CHD病因的贡献。a.使用CDCE/HiFi PCR检验NKX2.5、GATA 4和TBX5突变增加CHD风险的假设。B。检验NKX2.5、GATA 4和TBX5的选择性剪接增加或降低CHD易感性的假设。C.确定CHD患者中NKX2.5、GATA 4和TBX5中观察到的沉默序列变异是否导致选择性剪接。D.继续确定具有CHD特定目标2的大型、特征良好的受试者队列：检查体细胞突变对CHD病因的贡献。a.验证心脏发育基因的体细胞突变是冠心病病因的假设。B。测定正常和畸形心脏心脏发生过程中心肌细胞的突变率。C.使用突变光谱法发现正常和畸形心脏的主要致突变途径。我们的目标是鉴定与CHD有因果关系的遗传突变，确定它们增加先天性心脏病风险的机制，并检验体细胞突变是先天性心脏病病因的假设。我们将评估在这些基因中发现的突变的生物学意义，目的是确定改进的疾病管理策略。