""""""""Comparative Genomes to Correct Human Lung Hypoplasia"""""""" takes advantage of the conservation across species of molecular pathways responsible for normal lung development. We plan to use Drosophila, avian, rodent, and humans to discover aberrant molecular pathways which contribute to the lung hypoplasia responsible for the high mortality of congenital diaphragmatic hernia (CDH). Candidate genes uncovered by the screens, as well as those identified at chromosomal breakpoints in rare cases of human CDH, will be tested for abnormal expression in human CDH fetal lungs. Those that complement the lung hypoplasia phenotype in Drosophila, chick, or rodent assays will be considered candidate genes for mutational scanning and mapping in CDH patients and families. In Project I, Dr. Roberts performed an extensive chick lung bud screens for genes expressed in early lung development, and has four candidates. She will test these lung with factors known to cause lung hypoplasia when mutated in mice, and those obtained from the Drosophila screen in Project II, by using retroviral expression in the developing avian lung in ovo. If mutated forms cause lung hypoplasia phenotypes in the chick these will be tested in rodents and Drosophila complementation assays and for aberrant expression in her tissue bank of normal human and CDH lungs. In Project II, Dr. Perkins has already screened 60% of the Drosophila genome for intervals, which contain enhancers and suppressors of a hypomorphic corkscrew mutant which alter tracheal development. She will identify and characterize the tracheal modifiers, and determine if their homologues affect or avian or mouse lung development or are expressed in human fetal lung. She will then use the Drosophila model to screen for potential lung maturing therapeutics. In Project III, since we have noted a salutary effect of anti-oxidant on fetal lung development and on phosphorylation on ERK1 and 2 in the MAP kinase pathway. Drs. Schintzer and Kinane propose to use the rodent organ culture assay to define the molecular mechanism responsible for thee events and to and to elucidate other key genes or pathways responsible for lung hypoplasia. Candidate genes that correct lung hypoplasia will be studied for aberrant expression in human CDH fetal lungs. In Project IV, Drs. Holmes and Donahoe will create a database, perform karyotyping, and establish and immortalize cell lines on all CDH patients and their families since 1970. Candidate genes that cause lung hypoplasia in mouse knockouts or are associated with human CDH and are found by Drs. Donahoe and Roberts to be aberrantly expressed in human CDH lungs will be studied in mutational scans with Dr. MacDonald who will also study loss of heterozygosity in regions of CDH associated chromosomal aberrations. These interactive projects depend on each other for reagents and assays and will share valuable tissue bank material as well as phenotype ans candidate gene databases. The unique environment of the MGH, which integrates clinical, genetic, and developmental expertise, may, using results from these projects, produce novel pharmacotherapeutic treatments in utero to improve the outcome of CDH and it's attendant lung hypoplasia.
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