Genomic and Functional Architecture of Congenital Heart Disease

 描述(申请人提供)：先天性心脏病(CHD)是导致新生儿非感染性死亡的最常见原因。尽管有证据支持遗传病因学，但已知的遗传突变和拷贝数变异只解释了一小部分疾病负担。然而，在临床上，严重的冠心病经常零星发生并损害生殖适合性，这表明从头突变在其病因中扮演着关键角色。通过使用下一代外显子组测序，我们最近报道了在发育中的小鼠心脏中高水平表达的基因的从头突变的高负担(Zaidi等人，《自然》，2013年；PMCID#3706629)。我们发现，在涉及H3K4或H3K27甲基化或H2BK120泛素化的9个染色质修饰基因中，这种突变明显过剩。由于心脏形态发生的遗传控制缺陷是大多数CHD的基础，一旦发现新的致病基因，我们就必须在动物模型中研究它们的影响。非洲爪哇心脏可以快速研究基因剂量对心脏形态发生的特定阶段的影响。这项建议的总体目标是更全面地描述严重冠心病的基因组和功能结构。为此，我们假设到目前为止尚未确定的从头开始和遗传突变将通过下一代测序来识别，并且新识别的基因将在形态发生的特定阶段作用于破坏正常的心脏发育。在特定的目标1中，使用我们自己编写的、经过验证的贝叶斯算法，我们将识别在先证者中存在但在父母中不存在的新的从头突变。我们已经从另外445例病例(NHLBI的儿科心脏基因组学联合会)和645名对照三人组(来自Simon Simplex孤独症集合的自闭症病例的未受影响的兄弟姐妹)中获得样本。这些新的三人组将用于扩大和独立验证我们最初的队列。根据我们的能力计算，另外445个三元组应该识别44个新的风险相关基因(95%可信区间，22-66)。在具体目标2中，我们将通过将我们的队列与CEU HapMap3(欧洲)个体进行聚类，并使用人群等位基因频率为&lt；0.1%和/或d1%的群体等位基因频率研究显性和隐性遗传稀有变异，并对突变类型或心脏表达进行分层，来调查罕见传播变异对CHD的贡献。在具体目标3中，我们将研究敲除9个组蛋白修饰基因中的3个，即WDR5(H3K4甲基化)、Kdm5b(H3K4去甲基化)和RNF20(H2BK120泛素化)对非洲爪哇心脏发育的影响。我们将检查心脏循环、左右模式、数量、结构和功能 纤毛、心脏形态和功能。HHMI遗传学系和我的导师Richard Lifton博士提供了一个影响深远的智力环境，包括浓缩活动以及生物统计、生物信息和技术支持，这些共同应该确保成功完成。我们预计，这些研究不仅应该提高我们对心脏发育的理解，而且还可能转化为改善患者护理。

 DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common cause of non-infectious death in newborns. Although there is evidence to support a genetic etiology, only a small fraction of the disease burden is explained by known inherited mutations and copy number variations. Clinically, however, severe CHD often occurs sporadically and impairs reproductive fitness, suggesting that de novo mutations play a crucial role in its etiology Using next generation exome sequencing, we recently reported a high burden of de novo mutations in severely affected CHD patients in genes expressed at high levels in the developing murine heart (Zaidi et al., Nature, 2013; PMCID# 3706629). We found a marked excess of such mutations in nine chromatin- modifying genes involved in H3K4 or H3K27 methylation or H2BK120 ubiquitination. As defects in the genetic control of cardiac morphogenesis underlie a majority of CHD, it is essential that once new causative genes are discovered we study their effect in an animal model. The Xenopus heart allows the rapid study of effects of gene dosage on specific stages of cardiac morphogenesis. The overall goal of this proposal is to more fully characterize the genomic and functional architecture of severe CHD. Towards this end, we hypothesize that hitherto yet uncharacterized de novo and inherited mutations will be identifiable by next generation sequencing, and that the newly identified genes will act at specific stages of morphogenesis to disrupt normal heart development. In Specific Aim 1, using our self-written, validated Bayesian algorithm, we will identify novel de novo mutations that are present in probands, but are absent in parents. We have obtained samples from a further 445 case (NHLBI's Pediatric Cardiac Genomics Consortium) and 645 control trios (unaffected siblings of autism cases from Simon Simplex Autism Collection). These new trios will be used to expand and independently validate our original cohort. Per our power calculations, an additional 445 trios should identify 44 new (95% CI, 22-66) risk-associated genes. In Specific Aim 2, we will investigate the contribution of rare transmitted variants to CHD by clustering our cohort with CEU HapMap3 (European) individuals, and studying both dominantly and recessively inherited rare variants using population allele frequencies of <0.1% and/or d1%, with or without stratifying for mutation type or heart expression. In Specific Aim 3, we will study the effect of knocking down three of the nine histone-modifying genes, namely Wdr5 (H3K4 methylation), Kdm5b (H3K4 demethylation), and Rnf20 (H2BK120 ubiquitination), on development of the Xenopus heart. We will examine for heart looping, left-right patterning, number, structure, and function of cilia, and heart morphology and function. The Department of Genetics, HHMI, and my mentor, Dr. Richard Lifton, have provided a far- reaching intellectual environment, including enrichment activities, and biostatistical, bioinformatic, and technical support, which together should ensure successful completion. We envisage that the studies should not only improve our understanding of heart development, but may also translate into improved patient care.