This is a competitive supplemental request for grant # PO1 HD39942-02 """"""""Comparative Genomics to Correct Human Lung Hopoplasia"""""""" which expands at least four-fold the patient base for mutational analysis in project IV """"""""Gene Mutation and Rescue in Human Diaphragmatic Hernia"""""""". In this supplemental grant, we will be able to capture a large percentage of all the cases of congenital diaphragmatic hernia (CDH) born in the Boston and New England region. The reviewers of the grant anticipated that the number of cases accrued at the MGH would be too small for meaningful interpretation. However, initiating Projects I, II, III, and establishing a database for Project IV, has convinced us of the value of this Program Project and its likelihood of success. Completion of the database has demonstrated that the number of patients is even smaller then anticipated. Thus, we will add the impressive professional experience and patient numbers of the Children's Hospital Medical Center (CHMC) to the proposal, as suggested by the original reviewers of the Program Project and fortunately, agreed to by Dr. Jay Wilson and his team at the CHMC. The advantage of adding Dr. Wilson's program to that of the MGH is his unique multidisciplinary follow-up clinic where we can interact directly with a large number of CDH patients and families in one locale. A database will be established for all CDH patients and their families who come to the Boston CHMC for prospective care, as well as for all of those patients cared for by the CHMC Pediatric Surgical Service since 1990 (Aim I) and cared for by the multidisciplinary CDH clinic. These will be added to those generated at MGH. These patients and families giving consent will provide blood and tissue from which cell lines will be immortalized for chromosomal and genetic analysis. As with the original study, we will use the same previously collected and prospective CDH and normal fetal lungs to study the expression of genes known to be associated with human CDH, gene knockouts which have strong CDH phenotypes, and candidates that arise from Drosophila, chick, or rodent screens. Replaced genes which show complementation which corrects phenotypes of CDH in these models (Aim II) will satisfy the final and most stringent selection criteria necessary but not absolutely essential to be carried forward for extensive mutational analysis. We will clone or obtain human homologs and design PCR probes from the coding regions of the most promising candidates for mutational scanning of the anticipated much larger number of CDH patients and their families (Aim HI). Loss of homozygosity scanning will be done for 15q22-ter, 12q, and 8q regions (Aim IV) and candidate genes revealed by the scans will be tested for expression in the discarded fetal normal and CDH lungs and for complementation of CDH phenotype in the animal models. Genes which are abnormal in CDH lungs can be complemented in the various small animal models, and show abnormalities on mutational analyses of the patients, will be used to design treatment strategies for humans with CDH after appropriate testing in larger animal models. ? ?
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