Gene Mutation and Rescue in Human Diaphragmatic Hernia

先天性膈疝（CDH）是一种常见且经常致命的膈缺损伴肺发育不全的发育状况，由多种因素引起，我们假设这些因素主要是遗传的，但异质性的。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，希望阐明受干扰的生化途径，然后作为药物干预或治疗的功能靶点。目的1：我们将从MGH和波士顿儿童医院两个主要临床站点招募一组经过仔细表型分析的分离和复杂CDH患者，在每位患者、父母和兄弟姐妹身上建立细胞系，并进入已故患者人群、有多名患病成员的家庭、近亲家庭和同卵双胞胎。DNA将从各种来源提取，用于候选基因的突变分析、基于比较基因组杂交（aCGH）的排列、杂合性缺失研究（LOH）和亚端粒FISH的中期准备。目标二：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）将用于鉴定微缺失、微重复、平衡和不平衡易位，随后进行断点分析，以鉴定这些区域中具有导致CDH突变的基因。目的三：非同义snp鉴定将研究作为似是而非的致病突变。全基因组SNP分析将用于揭示近亲Donnai Barrow家族和不一致的同卵双胞胎的LOH区域。目的四：将研究来自动物模型或来自人类CDH的LOH区域的候选基因在人类CDH肺或膈中的异常表达，并在果蝇或小鼠细胞或器官培养试验中进行测试，以确定功能意义。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，以改善或预防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.