Exome Sequencing in Familial Cardiovascular Malformations

描述（由申请方提供）：关于大多数心血管畸形（CVM）的特定病因学，存在知识空白。 这个缺口代表了一个重要的问题，因为除非它被解决，否则潜在的分子机制将在很大程度上是不可穿透的。 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。 该R21申请的总体目标是发现导致一个亚组的CVM、左心室流出道（LVOT）畸形（包括主动脉瓣狭窄、主动脉缩窄和左心发育不良综合征）的遗传变异。 这些畸形有很大的遗传成分，但很少有易感基因已被确定。 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，使用表现出孟德尔遗传模式的LVOT畸形的家庭来鉴定。 拟议研究的基本原理是，确定LVOT畸形的遗传原因有可能为一组畸形提供更好的风险咨询，这些畸形导致大部分婴儿因出生缺陷而死亡。 在支持这一前提的最新文献和拥有独特的家族队列的指导下，将通过追求一个单一的特定目标来测试中心假设，即对所有已知人类基因的外显子使用基因组分区捕获技术，然后对捕获的靶标进行大规模平行测序。 这将适用于受影响的个人从四个家系表现出常染色体显性遗传模式。 不太可能致病的变异将根据现有的变异数据库和医学上可用的个人基因组序列数据被过滤掉。 将使用生物信息学（预测突变效应、基因表达数据等）来识别致病变异体 将通过桑格测序确认变体，并通过表达试验研究其功能效应。 然后将筛选更大的队列，以确定基因中的突变。 这种做法是创新的。 它将采用新技术快速识别多重家庭中的致病基因，绕过以前的全基因组连锁分析、精细定位和候选基因选择方法，在基因定位方法中创造一个范式转变。 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异。 这项研究意义重大，因为它有望通过确定LVOT畸形的第一个致病基因来垂直推进该领域。 这项工作的自然进展将导致R01应用程序，以研究所确定的变体的分子机制。 在当前的胎儿诊断和干预时代，以及许多CVM是遗传的理解，这些知识可能被用于提供更好的家庭风险咨询，新的预防措施，子宫内早期诊断和靶向治疗。 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重要原因。 这项计划中的研究有可能增加对疾病发病机制的基本了解，并及早识别出有风险的个体，以便进行可能的干预。 因此，拟议的研究与《国家HLBI战略计划》的目标1有关，即通过确定导致对特定疾病易感性的关键遗传变异，增加对健康和疾病的分子和生理基础的了解，也与国家儿童健康与发展研究所的科学目标有关，即通过对出生缺陷的遗传研究，了解疾病易感性的遗传学以及正常和异常发育。

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.