Gene Mutation and Rescue in Human Diaphragmatic Hernia

先天性膈疝（CDH）是一种常见的，往往是致命的隔膜缺陷与肺发育不全引起的各种因素，我们假设主要是遗传，但异质性。项目IV将使用临床，分子，发育，基因组和细胞遗传学策略的组合来识别导致CDH的突变，希望阐明受干扰的生化途径，然后可以作为药物干预或治疗的功能靶标。 目标一：我们将从两个主要的临床地点，MGH和波士顿儿童医院招募一组经过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身上建立细胞系，并进入患者的死亡人群，多个受影响成员的家庭，近亲家庭和单卵双胞胎。将从各种来源提取DNA，用于候选基因的突变分析、基于阵列的比较基因组杂交（aCGH）、杂合性缺失研究（洛）和亚端粒FISH的中期制备。 目标二：高分辨率的细胞遗传学工具，如1 Mb阵列CDH，亚端粒FISH和多重配体定向探针扩增（MLPA）将用于识别微缺失，微重复，平衡和不平衡易位，然后通过断点分析，以确定这些区域中的基因突变引起CDH。 目的III：将确定的非同义SNP作为可能的致病突变进行研究。全基因组SNP分析将用于揭示近亲Donnai巴罗家系和不协调单卵双胞胎中的洛缺失区域。 目标四：将研究来自动物模型或来自人CDH洛区域的候选基因在人CDH肺或横膈膜中的异常表达，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靶点，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.