A New Strategy to Identify the Genetic Causes of Isolated CDH

 描述（由申请人提供）：先天性膈疝（CDH）是一种毁灭性的出生缺陷，其特征是隔膜形成不完全，导致高发病率和死亡率。确定导致孤立性CDH的遗传因素对于改善结果和降低医疗费用至关重要。我们发表的数据显示，16个大家庭具有统计学显著的高突变率孤立CDH，其中4个大家庭的7名患者有5个共享染色体区域（SCR）。五个SCR中的两个含有GATA 4和NR 2F 2，这些基因先前在小鼠模型中与CDH相关。CDH易感性变异可能存在于非编码区，因为我们没有发现这些基因中的编码变异。SCRs定位与全基因组测序（WGS）相结合将为鉴定导致孤立性CDH的基因变异提供一种新的工具。在委员会认证的医学遗传学家的监督下，我们从>70名患有孤立性CDH的患者和>100名未受影响的家庭成员中收集了血液DNA样品。在目标1的准备中，我们从5个大家庭中选择了12名患者，并对其中7人进行了WGS。对于目标2，我们从一个三代高患病率的家庭中选择了五个人：一个未受影响的母亲和四个受影响的人（两个兄弟姐妹，一个父亲和一个祖父），并对所有人进行了全检查测序（WES）。我们还收集了大量的423例散发病例（57例来自犹他州，90个家庭来自贝勒医学院，276例来自哈佛大学），我们将在两个目标中作为重复队列进行筛选。我们研究的最终目标是确定孤立性CDH的易感性变异。该提案的中心假设是，我们使用基于家族的遗传方法的独特方法将识别孤立性CDH的分子原因。目的1：检验来自5个大家族的12例患者的孤立CDH易感性变异存在于SCR内的假设。在SCR分析和WGS之后，我们将进行WGS分析，重点是SCR中的编码和非编码变体。我们将通过使用变异注释、分析和搜索工具（VAAST）、pedigree-VAAST和Phevor分析我们的WGS数据来生成预测致病候选者的短名单;这三种新的从头开始疾病基因发现者由犹他州大学的Mark Yandell博士开发。候选人将进行分子验证，然后在这些家庭中进行分离测试。一旦候选基因被确认，我们将在我们的大型复制队列中筛选它们。目的2：在一个三代高致病率家族中导致孤立性CDH的图谱变异。WES数据的分析与目标1相似，但重点关注编码变体。这项研究计划将通过揭示孤立的CDH中涉及的新DNA变体来推动CDH领域的发展。在未来，我们的新型基因工程工具旨在加速小鼠中人类CDH变体的建模。

 DESCRIPTION (provided by applicant): Congenital diaphragmatic hernia (CDH) is a devastating birth defect characterized by incomplete formation of the diaphragm that results in high morbidity and mortality. Identifying the genetic factors leading to isolated CDH is essential for improving outcome and decreasing healthcare costs. Our published data revealed 16 extended families with statistically significant high-penetrance isolated CDH, and five shared chromosomal regions (SCRs) in seven patients from four of these extended families. Two of the five SCRs contain GATA4 and NR2F2, genes previously linked to CDH in mouse models. CDH susceptibility variants could reside in noncoding regions because we did not identify coding variants in these genes. Mapping SCRs in combination with whole-genome sequencing (WGS) will provide a new tool to identify gene variants causing isolated CDH Under the. oversight of board certified medical geneticists, we collected blood DNA samples from >70 patients with isolated CDH and >100 unaffected family members. In preparation for Aim 1, we selected 12 patients from five extended families, and performed WGS on seven of them. For Aim 2, we selected five individuals from a three-generation high-penetrance family: an unaffected mother and four affected (two siblings, a father and a grandfather), and performed whole-exam sequencing (WES) on all of them. We also have a large collection of 423 sporadic cases (57 trios from Utah, 90 families from Baylor College of Medicine, and 276 trios from Harvard University) which we will screen as a replication cohort in both Aims. The ultimate goal of our research is to identify susceptibility variants for isolated CDH. The central hypothesis of this proposal is that our unique approach, using family-based genetic methods, will identify the molecular causes of isolated CDH. Aim 1: Test the hypothesis that isolated CDH susceptibility variants reside within SCRs in 12 patients from five extended families. After SCR analysis and WGS, we will perform WGS analysis, focusing on coding and non-coding variants within SCRs. We will generate a short-list of predicted disease-causing candidates by analyzing our WGS data with the variant annotation, analysis, and search tool (VAAST), pedigree-VAAST and Phevor; three new ab initio disease-gene finders developed by Dr. Mark Yandell at the University of Utah. Candidates will undergo molecular validation, followed by testing for segregation in these families. Once candidate genes are confirmed, we will screen them in our large replication cohort. Aim 2: Map variants causing isolated CDH in a three-generation, high-penetrance family. WES data will be analyzed similarly to Aim 1 except for a focus on coding variants. This research program will move the CDH field forward by revealing new DNA variants involved in isolated CDH. In the future, our novel genetic engineering tools aim to accelerate modeling human CDH variants in mice.