Gene Mutation and Rescue in Human Diaphragmatic Hernia

先天性横隔膜疝气(CDH)是一种常见的、通常是致命的发育状况，与肺发育不全相关的横隔膜缺陷是由多种因素引起的，我们假设这些因素主要是遗传的，但不同的。项目IV将结合临床、分子、发育、基因组和细胞遗传学策略来识别导致CDH的突变，希望阐明扰乱的生化途径，然后将其作为药物干预或治疗的功能靶点。目的I：我们将从MGH和波士顿儿童医院两个主要临床地点招募一组仔细分型的孤立和复杂的CDH患者，在每个患者及其父母和兄弟姐妹上建立细胞系，并进入患者死亡人群、多个患病成员的家庭、血缘关系家庭和同卵双胞胎。DNA将从各种来源提取，用于候选基因的突变分析、基于阵列的比较基因组杂交(ACGH)、杂合性丢失研究(LOH)以及亚端粒FISH的中期准备。目的II：高分辨率细胞遗传学工具，如1Mb阵列CDH、亚端粒FISH和多重配体定向探针扩增(MLPA)将被用来识别微缺失、微复制、平衡和不平衡易位，然后进行断点分析，以确定这些区域中具有导致CDH的突变的基因。目的III：已发现的非同源SNPs将作为可能的致病突变进行研究。全基因组SNP分析将被用来揭示血缘关系Donnai Barrow亲属和不协调单卵双胞胎的LOH区域。目的：研究来自动物模型或人类CDH缺失区域的候选基因在人类CDH肺或横隔膜中的异常表达，并在果蝇、小鼠细胞或器官培养实验中进行测试，以确定其功能意义。这些研究发现的CDH基因缺陷将作为药物或其他治疗干预的目标，以改善或防止这种严重的出生畸形。

Congenital Diaphragmatic Hernia (CDH) is a frequent and often fatal developmental condition of diaphragm defects associated with lung hypoplasia caused by diverse factors that we hypothesize are predominantly genetic, but heterogeneous. Project IV will use a combination of clinical, molecular, developmental, genomic and cytogenetic strategies to identify mutations causing CDH, with the hope of elucidating perturbed biochemical pathways that can then serve as functional targets for pharmacological intervention or treatment. Aim I: We will recruit a cohort of carefully phenotyped isolated and complex CDH patients from two major clinical sites, MGH and Children's Hospital Boston, establish cell lines on each patient, and parents,and siblings, and enter deceased populations of patients, families with multiple affected members, consanguineous families, and monozygotic twins. DNA will be extracted from various sources for mutational analysis of candidate genes, arrayed based Comparative Genomic Hybridization (aCGH), loss of heterozygosity studies (LOH), and metaphase preparations for subtelomeric FISH. Aim II: High resolution cytogenetic tools such as 1 Mb array CDH, subtelomeric FISH, and multiplex ligand-directed probe amplification (MLPA) will be used to identify microdeletions, microduplications, and balanced and unbalanced translocations followed by breakpoint analyses to identify genes in these regions that have mutations causative for CDH. Aim III: nonsynonomous SNPs identified will be studied as plausible causative mutations. Genome wide SNP analysis will be used to reveal regions of LOH in consanguineous Donnai Barrow kindreds and in discordant monozygotic twins. Aim IV: Candidate genes from animal models or from LOH regions with CDH in human will be studied for abnormal expression in human CDH lungs or diaphragms and tested in Drosophila or mouse cell-based or organ culture assays to determine functional significance. CDH gene defects uncovered by these studies will serve as targets for pharmacological or other therapeutic interventions to ameliorate or prevent this severe birth malformation.