Molecular mechanisms of diaphragm development: implications for congenital diaphragmatic hernia

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Congenital abnormalities of the diaphragm, specifically congenital diaphragmatic hernia (CDH), affect 1 in 3,000 live births and are associated with substantial morbidity and mortality. Evidence in humans and animal models support genetic causation. The etiology of most cases, however, remains unknown, though is likely polygenic in the majority. Another impediment to uncovering CDH etiology is that collection of human specimens at the onset of diaphragm development (4 weeks gestation) is not feasible. Identification of key transcriptional programs involved in normal diaphragm development is needed to gain a more comprehensive understanding of the genetic etiology of CDH. As an initial step, unique transcriptome-wide data sets are generated from laser-captured mouse embryonic diaphragms at three key time-points of diaphragm development. These data sets provide the first unbiased perspective on genes and signaling pathways active during early and late diaphragm morphogenesis. Moreover, using a curated list of known CDR-associated genes as "baits", the normal expression data sets are filtered for identification of biologically relevant CDH candidate genes. By this approach, 27 novel CDH candidate genes are described, among which, pre-B cell leukemia transcription factor 1 (Pbxl) is selected to validate the prioritization approach. Diaphragm defects resembling the human CDH phenotype, never previously appreciated, are observed among Pbxl mutant mice at E15.5. Secondly, a human family containing several affected individuals with CDH is used for gene discovery. A multiplatform analysis approach, coupled with external filtering with the normal diaphragm expression data, provide a mechanism for identification of candidate modifier loci. This integrative strategy, which identifies a FOG2 deletion and several potential modifiers of the CDH phenotype, is described. The design of a customized diagnostic/discovery capture array for next-generation sequencing of a large CDH patient cohort uses the set of CDR-candidate genes identified herein.2031-01-0