Gene Mutation and Rescue in Human Diaphragmatic Hernia

总体方案 摘要 我们对“人类膈疝的基因突变和拯救”计划项目的研究策略是 1）从基因组分析中识别先天性膈疝患者的基因变异，2）测试 用于体外和离体以及动物模型中验证和功能的高优先级候选物，3）确定 通过整合已知的和新的候选基因定义的分子途径，和4）选择分子途径， 从这些目标中构思先天性髋关节脱位的产后治疗策略。CDH是一种出生缺陷， 1/3000活产，死亡率和发病率高，幸存者需要高技术驱动 介入性围产期护理在三级或四级新生儿重症监护病房。40%的CDH患者 除了CDH之外，还有多种异常，但多达60%是孤立的，可能会增加 如果我们能改善导致其高发病率的相关肺发育不全，的社会和 CDH的个人负担有望通过减少由CDH引起的患者和家庭压力来减轻。 与这种出生缺陷同时发生的是相当高的发病率和死亡率以及护理费用。如果实质性 肺分化可以诱导出生后发生在肺中， 氧合（ECMO）和积极的呼吸管理可能会减少，从而保护脆弱的肺 继发性气压伤和支气管肺发育不良 为了实现有益的改变，我们计划继续采用基因组方法，如全外显子组 测序（WES）、阵列比较基因组杂交（aCGH）和全基因组测序 (WGS)，并将不同的发现与来自发育中的膈肌和肺的表达分析的数据相结合 小鼠模型中的组织，以及来自人类CDH患者的隔膜活检。WES、WGS和aCGH 数据将在临床特征良好的单例队列中持续确定和分析， 父母/孩子三人组，以及罕见的大家庭。本P01的目标是利用这一不断增长的队列和我们的 分析方法，以产生候选基因，将产生重要的线索，作为分子发病机制 CDH，特别是在将最高优先级的候选物整合到分子途径中之后。 项目I：基因组和基因表达分析，以发现人类CHD基因和途径。 项目II：小鼠模型将阐明CDH和相关肺缺陷的遗传学， 临床相关目标。 项目III：CDH患者基因变异体在体外和选定动物模型中的功能评价。

OVERALL PROGRAM ABSTRACT Our research strategy for the Program project “Gene Mutation and Rescue in Human Diaphragmatic Hernia” is to 1) identify gene variants in patients with Congenital Diaphragmatic Hernia from genomic analyses, 2) test high priority candidates for validation and function in vitro and ex vivo, and in animal models, 3) determine molecular pathways defined by integrating already known and new candidate genes, and 4) select molecular targets from which to conceive postnatal treatment strategies for CDH. CDH is a birth defect which occurs in 1/3000 live births, and has a high mortality and morbidity, with survivors requiring high technology-driven interventional perinatal care in tertiary or quarternary neonatal intensive care units. 40% of CDH patients have multiple anomalies in addition to CDH, but as many as 60% are isolated with the potential for increased survival if we can improve the associated lung hypoplasia responsible for its high morbidity. The societal and individual burden of CDH can hopefully be lessened by reducing the patient and family stresses caused by the considerable morbidity and mortality, as well as the cost of care, coincident with this birth defect. If substantial pulmonary differentiation can be induced to occur in the lung postnatally, the use of Extra Corporeal Membrane Oxygenation (ECMO) and aggressive ventilatory management may be reduced, thus protecting fragile lungs from secondary barotrauma and bronchopulmonary dysplasia. To affect a salutary change, we plan to continue to employ genomic methods such as Whole Exome Sequencing (WES), array Comparative Genomic Hybridization (aCGH), and Whole Genomic Sequencing (WGS), and to integrate variant findings with data from expression analyses of developing diaphragm and lung tissues in mouse models, and with diaphragm biopsies from human CDH patients. WES, WGS, and aCGH data will be continually ascertained and analyzed on a clinically well characterized cohort of singletons and parent/child trios, as well as rare extended families. It is the goal of this P01 to use this growing cohort and our analytic approach to generate candidate genes that will yield important clues as to the molecular pathogenesis of CDH, particularly after integrating the highest priority candidates into molecular pathways. PROJECT I: Genomic and gene expression analyses to discover human CHD genes and pathways. PROJECT II: Mouse models will elucidate genetics of CDH and associated pulmonary defects and identify clinically relevant targets. PROJECT III: Functional evaluation of CDH patient gene variants in vitro and in select animal models.