THE GENETIC BASIS OF CONGENITAL HEART DEFECTS (CHD)

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。先天性心脏病（CHD）是最常见的出生缺陷之一。出生时患有冠心病的婴儿通常需要多种医疗和/或手术干预，尽管如此，即使没有死亡率，也会出现显著的发病率。尽管其患病率和临床意义，冠心病的病因在很大程度上仍然未知。普遍的观点是，这种疾病是异质和复杂的，既有环境风险因素，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被认为是遗传危险因素。我们建议使用一种新的，高度敏感的突变检测技术，称为恒定变性毛细管电泳（CDCE）结合高保真PCR （HiFi PCR）来确定每个候选基因的突变是否在患者群体中比在大型对照人群中丰富。这项新技术通过一次收集和筛选96个个体的DNA，使大规模人群的快速和经济有效的分析成为可能。为了发现低频率的因果突变和量化突变与疾病之间的低影响关系，有必要对大量人群进行分析。一旦富集突变被确定，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性也可以通过改变mrna前剪接而产生表型影响。另一个复杂的层面是，已经提出了先天性心脏病的单独遗传机制。最近在95%以上的室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陷中发现的，而在取自同一心脏的未受影响的区域未发现，表明这些突变具有体细胞和马赛克性质，并表明这些突变是病因性的。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和多个单倍型。在一个基因中出现两个或两个以上的体细胞突变是极其罕见的事件，这表明一种极端的基因组不稳定状态，让人想起在癌症中观察到的情况。在本研究中，我们将研究NKX2.5、GATA4和TBX5基因的遗传和体细胞突变对心脏畸形的影响。具体而言，我们将：具体目标1：检查NKX2.5， GATA4和TBX5分子缺陷对冠心病病因的贡献。A.利用CDCE/HiFi PCR验证NKX2.5、GATA4和TBX5基因突变增加冠心病风险的假设。B.验证NKX2.5、GATA4和TBX5的选择性剪接是否会增加或减少冠心病的易感性。C.确定在冠心病患者的NKX2.5、GATA4和TBX5中观察到的沉默序列变异是否导致选择性剪接。D.继续确定一个大的、特征明确的冠心病受试者队列。具体目标2：检查体细胞突变对冠心病病因的贡献。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突变途径。我们的目标是确定与冠心病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，并验证体细胞突变是先天性心脏病病因学的假设。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.