| This dissertation describes the characterization of a holoprosencephaly locus on human chromosome 14q, designated HPE8, with the ultimate objective to identify and characterize one or more candidate genes for this disease. Holoprosencephaly is the most common congenital defect in the development of the human forebrain and associated facial structures. A spectrum of mild to severe forebrain and facial anomalies occur in this disorder. A panel of patients, who are carriers of proximal chromosome 14q rearrangements, was collected through worldwide collaborations. These were used for mapping to define an approximately 2 Mb minimal critical region for holoprosencephaly on chromosome 14q13. Both YAC and BAC contigs were built by standard methods, including chromosome walking. With the availability of human draft genomic sequence from the Human Genome Project and Celera Genomics, the sequence was annotated for 34 putative genes using bioinformatic approaches. Three genes (C14ORF11, NPAS3, SNX6), expressed in human fetal brain, were selected for characterization. These three candidate genes were characterized in humans in order to obtain insight into the biological function. These included the isolation of the human cDNA, putative properties using extensive bioinformatic analyses, and determination of the genomic structure and expression profile and cellular location of the protein. The murine orthologues of these three candidate genes were also studied for the temporal and spatial expression profile in developing mouse embryos. C14ORF11, SNX6 and NPAS3 are suggested to be excellent candidates for holoprosencephaly based on expression in the developing human fetal brain, expression during the correct spatio-temporal periods of neurulation in mice embryogenesis, and/or role in developmental pathways implicated in the holoprosencephaly phenotype. In order to further identify phenotypes associated with specific genes on chromosome 14, analysis of the translocation chromosome breakpoint junctions is carried out on three patients who demonstrated phenotypes partly similar to the deletion cases analyzed, or had a breakpoint junction in the HPE8 minimal critical region. This research contributes to understanding the molecular pathogenesis of holoprosencephaly that may prove useful in the diagnosis of those affected. |
