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.
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