Gene Mutation and Rescue in Human Diaphragmatic Hernia

先天性膈疝 (CDH) 是一种常见且往往致命的膈肌发育缺陷，与肺发育不全有关，由多种因素引起，我们假设这些因素主要是遗传性的，但具有异质性。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，希望阐明受到干扰的生化途径，然后将其作为药物干预或治疗的功能靶标。 目标 I：我们将从麻省总医院 (MGH) 和波士顿儿童医院这两个主要临床中心招募一组经过仔细表型分析的孤立且复杂的 CDH 患者，在每位患者及其父母和兄弟姐妹上建立细胞系，并输入已故患者群体、有多位受影响成员的家庭、近亲家庭和同卵双胞胎。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)、杂合性丢失研究 (LOH) 以及亚端粒 FISH 的中期制备。 目标 II：高分辨率细胞遗传学工具，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、微重复以及平衡和不平衡易位，然后进行断点分析，以识别这些区域中存在导致 CDH 的突变的基因。 目标 III：将鉴定出的非同义 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 Donnai Barrow 近亲亲属和不一致的同卵双胞胎中的 LOH 区域。 目标 IV：将研究来自动物模型或人类 CDH LOH 区域的候选基因在人 CDH 肺或隔膜中的异常表达，并在果蝇或小鼠细胞或器官培养测定中进行测试，以确定功能意义。这些研究发现的 CDH 基因缺陷将作为药理学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.