Exome Sequencing in Familial Cardiovascular Malformations

描述(由申请人提供)：关于大多数心血管畸形(CVM)的具体病因，知识存在差距。这一差距代表着一个重大问题，因为在它被解决之前，潜在的分子机制将在很大程度上是不可渗透的。我们的长期目标是更好地了解导致CVM的潜在遗传原因和分子机制。R21应用的总体目标是发现导致CVM亚群的基因变异，即包括主动脉瓣狭窄、主动脉缩窄和左心发育不良综合征在内的左室流出道(LVOT)畸形。这些畸形有很大的遗传成分，但几乎没有发现易感基因。中心假设是，致病基因可以通过高度选择性的全基因组方法识别，使用表现出左心室流出道畸形孟德尔遗传模式的家系。这项拟议研究的基本原理是，确定左心室流出道畸形的遗传原因有可能为一组畸形提供更好的风险咨询，这些畸形导致很大比例的婴儿因出生缺陷而死亡。在最近支持这一前提的文献的指导下，并拥有一个独特的家族队列，中心假设将通过追求一个单一的特定目标来检验，即使用基因组分割捕获技术来获取所有已知人类基因的外显子，然后对捕获的目标进行大规模并行测序。这将适用于来自四个表现常染色体显性遗传模式的家系的受影响个体。根据现有的变异数据库和公开可用的个人基因组序列数据，不太可能导致疾病的变异将被过滤掉。将使用生物信息学(预测的突变效应、基因表达数据等)。以确定因果变量。变异体将通过Sanger测序得到确认，其功能效应将通过表达分析进行研究。然后，将对更大的队列进行筛查，以确定已识别基因的突变。这种方法是创新的。它将使用新技术来快速识别多基因家族中的致病基因，绕过以前的全基因组连锁分析、精细定位和候选基因选择的方法，创造基因定位方法的范式转变。此外，还开发了独特的方法来过滤大量的预期数据并确定可能的因果变量。这项拟议的研究意义重大，因为它有望通过识别第一个左心室流出道畸形的原因基因来垂直推进这一领域。这项工作的自然进展将导致R01应用于研究所识别的变体的分子机制。在当前的胎儿诊断和干预时代，以及对许多CVM是遗传的理解，这些知识可能被用来提供更好的家庭风险咨询、新的预防措施、宫内早期诊断和有针对性的治疗。 公共卫生相关性：拟议的研究与公共卫生相关，因为它解决了婴儿期发病率和死亡率的一个重要原因。这项有计划的研究有可能增加对疾病发病机制的基本了解，并及早识别高危个体以进行可能的干预。因此，拟议的研究与NHLBI战略计划的目标1有关，目的是通过确定导致特定疾病易感性的关键基因变异，增加对健康和疾病的分子和生理基础的了解，并与NICHD通过出生缺陷的基因研究了解疾病易感性的遗传学和正常和异常发育的科学目标相关。

DESCRIPTION (provided by applicant): There is a gap in knowledge regarding the specific etiologies for most cardiovascular malformations (CVMs). This gap represents a significant problem, because until it is solved, the underlying molecular mechanisms will be largely impenetrable. Our long term goal is to better understand the underlying genetic causes and molecular mechanisms leading to CVMs. The overall objective of this R21 application is to discover the genetic variation leading to a subgroup of CVMs, the left ventricular outflow tract (LVOT) malformations that includes aortic valve stenosis, coarctation of the aorta, and hypoplastic left heart syndrome. There is a large genetic component to these malformations, but few susceptibility genes have been identified. The central hypothesis is disease causing genes can be identified by highly selective genome-wide approaches, using families exhibiting Mendelian inheritance patterns for LVOT malformations. The rationale for the proposed research is that identifying the genetic causes of LVOT malformations has the potential to provide better risk counseling for a group of malformations that contribute to a large proportion of infant mortality due to birth defects. Guided by recent literature supporting this premise and possession of a unique cohort of families, the central hypothesis will be tested by pursuing a single specific aim to use genome partitioning capture technology for the exons of all known human genes followed by massively parallel sequencing of the captured targets. This will be applied to affected individuals from four pedigrees exhibiting an autosomal dominant inheritance pattern. Unlikely disease causing variants will be filtered out, based on existing variant databases and publically available personal genome sequence data. Bioinformatics will be used (predicted mutation effect, gene expression data, etc.) to identify the causal variant. Variants will be confirmed by Sanger sequencing and their functional effects investigated by expression assays. A larger cohort will then be screened for mutations in the identified genes. This approach is innovative. It will employ new technology to rapidly identify the causative gene in multiplex families, by-passing previous methods of genome wide linkage analysis, fine mapping, and candidate gene selection, creating a paradigm shift in the approach to gene mapping. In addition, unique methods have been developed to filter the large amount of expected data and identify the likely causal variant. The proposed research is significant because it is expected to vertically advance the field by identifying the first causal genes for LVOT malformations. The natural progression of this work will lead to an R01 application to investigate the molecular mechanisms of the variants identified. In the current era of fetal diagnosis and intervention, as well as the understanding that many CVMs are inherited, this knowledge can potentially be used to provide better family risk counseling, novel preventive measures, early in utero diagnosis, and targeted therapies. PUBLIC HEALTH RELEVANCE: The proposed study is relevant to public health as it address a significant cause of morbidity and mortality in infancy. This planned research has the potential to increase the fundamental understanding of disease pathogenesis, and identify at risk individuals early for possible interventions. Thus, the proposed research is relevant to Goal 1 of the NHLBI Strategic Plan, to increase understanding of the molecular and physiological basis of health and disease, by determining key genetic variants that account for susceptibility to specific disease, and to the NICHD's scientific goals of understanding genetics of disease susceptibility and normal and abnormal development by genetic studies of birth defects.