Mechanisms of Abnormal Diaphragm and Cardiac Development

项目摘要 先天性横隔膜疝气(CDH)是一种危及生命的出生缺陷，占所有主要 先天畸形。在CDH与心血管畸形(CVM)并存的情况下，死亡率 发病率从30%上升到60%，长期发病率很常见。我们的目标是找出导致 CDH和CDH/CVM，并发现它们控制横隔膜和心脏发育的机制。 从大的候选基因列表中正确识别与特定表型有关的基因 研究和临床研究中产生的数据是这一领域和其他研究领域取得进展的主要障碍。 在特定目标#1中，我们将通过生成排名的CDH和CDH/CVM致病力来应对这一挑战 使用机器学习算法对所有RefSeq基因进行评分，该算法集成了大规模基因组数据 知识来源。我们将利用这些分数从细胞遗传学中鉴定新的CDH和CDH/CVM基因 定义关键区域并形成大型下一代测序数据库，作为我们多方面工作的一部分 新基因发现的途径。我们还将加快人类疾病基因研究的步伐 通过免费提供这些分数和我们的机器学习算法而发现的。 8p23.1包含GATA4和SOX7的微缺失是最常见的原因之一 专用宿主机/云服务器。在rna-seq研究中，我们发现了几个与cdh相关的基因在 E15.5Gata4-Flox/Flox的横隔膜；PrX1-Cre胚胎。这些胚胎是理想的囊状疝气模型 占人类CDH病例的20%。在具体目标#2中，我们将结合来自RNA-SEQ和生物芯片-SEQ的数据 利用AIM#1中生成的优先级分数进行分析，以识别其主要的GATA4靶基因 调节失调参与了SAC CDH的发生发展。然后我们将确定表达式中的更改是否 这些靶基因可以引起CDH，或者可以改变GATA4缺陷小鼠的CDH表型。 我们已经证明SOX7缺乏通过减少内皮细胞到间充质细胞而导致间隔缺陷 发育中心脏的过渡(EMT)。在RNA-seq和原位杂交研究中，我们已经表明 在E9.5 Sox7/-心脏中，心内膜EMT的关键调节因子Wnt4的表达严重减少。 在具体目标#3中，我们将确定WNT4是否是SOX7的主要目标，以及WNT4或 其下游效应器可以挽救SOX7相关的心脏表型。 多条证据表明，Wnt4是人类新发现的CDH/CVM基因。来确认这一点 并了解更多关于WNT4在横隔膜和心脏发育中的作用，我们将确定 Wnt4缺乏导致小鼠CDH/CVM，如果Sox7和Wnt4在发育过程中存在遗传交互作用 CDH/CVM和IF SOX7调节Wnt4在发育中的横隔膜中的转录。 通过这些研究，我们将发现新的CDH和CDH/CVM基因和途径。生物信息学 我们在这笔赠款中开发的工具将加强多个研究领域的基因发现努力。

Project Summary Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that accounts for 8% of all major congenital anomalies. In cases where CDH co-occurs with cardiovascular malformations (CVMs), mortality rates increase from 30 to 60%, and long-term morbidity is common. Our goal is to identify genes that cause CDH and CDH/CVM, and to discover the mechanism by which they control diaphragm and heart development. Correctly identifying genes that contribute to specific phenotypes from the large list of candidate genes data generated in research and clinical studies is a major obstacle to progress in this and other research fields. In Specific Aim #1, we will address this challenge by generating ranked CDH and CDH/CVM pathogenicity scores for all RefSeq genes using a machine-learning algorithm that integrates data from large-scale genomic knowledge sources. We will use these scores to identify novel CDH and CDH/CVM genes from cytogenetically defined critical regions and form large next-generation sequencing databases as part of our multifaceted approach to novel gene discovery. We will also accelerate the pace at which human disease genes are discovered by making these scores and our machine-learning algorithm freely available. 8p23.1 microdeletions that encompass GATA4 and SOX7 are among the most frequently identified causes of CDH/CVM. In RNA-seq studies, we identified several CDH-associated genes that are dysregulated in the E15.5 diaphragms of Gata4flox/flox;Prx1-Cre embryos. These embryos are an ideal model of the sac hernias that comprise 20% of human CDH cases. In Specific Aim #2, we will combine data from RNA-seq and BioChIP-seq analyses with the priority scores generated in Aim #1 to identify primary GATA4 target genes whose dysregulation contribute to the development of sac CDH. We will then determine if alterations in the expression of these target genes can cause CDH, or can modify the CDH phenotypes of GATA4-deficient mice. We have shown that SOX7 deficiency causes septal defects by decreasing endothelial-to-mesenchymal transition (EMT) in the developing heart. In RNA-seq and in situ hybridization studies we have shown that the expression of Wnt4—a key regulator of EMT in the endocardium—is severely decreased in E9.5 Sox7-/- hearts. In Specific Aim #3 we will determine if Wnt4 is a primary target of SOX7, and whether modulation of WNT4 or its downstream effectors can rescue SOX7-related cardiac phenotypes. Several lines of evidence suggset that WNT4 is a novel CDH/CVM gene in humans. To confirm this association, and learn more about the role of WNT4 in diaphragm and heart development, we will determine if WNT4 deficiency causes CDH/CVM in mice, if Sox7 and Wnt4 interact genetically in the development of CDH/CVM and if SOX7 regulates Wnt4 transcripition in the developing diaphragm. Through these studies we will identify novel CDH and CDH/CVM genes and pathways. The bioinformatic tools we develop in this grant will enhance gene discovery efforts across multiple research fields.