Development of the human brain during early embryogenesis has been studied almost exclusively on a descriptive level. Analysis of genes and gene products necessary for the morphogenesis of the central nervous system will contribute to our understanding not only of normal brain development, but also of the etiology of abnormal formation as seen in congenital brain anomalies. Isolation of genes involved in early embryonic brain formation can be accomplished through molecular studies of cells from individuals with abnormal brain development. The holoprosencephaly (HP) sequence is such a structural anomaly characterized by abnormal midline development of brain and face. The clinical spectrum is well described varying from severe forms with a single brain ventricle and cyclopia, which are incompatible with postnatal life, to milder craniofacial abnormalities such as cleft lip and palate. The genetic basis of holoprosencephaly is heterogeneous with both familial occurrence and sporadic cases due to specific chromosome anomalies. A set of related hypotheses are proposed: First, genes necessary for normal brain development are located in chromosomal regions non-randomly associated with holoprosencephaly. Second, gene rearrangements, e.g. deletions, alter gene expression, leading to the clinical features of the holoprosencephaly sequence. Third, expression of these genes is assumed to occur during the gastrulation stage, which involves the conversion of the two-layered into a three-layered embryo. To address these hypotheses the proposed research will concentrate on a form of holoprosencephaly associated with a minute chromosomal deletion in 2p2l. The following specific experiments will be completed: 1. Cell lines from patients with holoprosencephaly will be established and studied for microdeletions and other rearrangements in 2p2l by cytogenetic and molecular genetic analysis and flow cytometry; 2. Human cell lines with 2p2l anomalies will be fused to hamster cells to separate normal and abnormal copies of chromosome 2 into different somatic cell hybrid clones; 3. DNA probes will be isolated and mapped in 2p2l, and used to identify large fragments of human DNA cloned into yeast artificial chromosomes (YACs); 4. cDNA libraries from early mouse embryos will be screened with 2p2l specific YACs that contain evolutionarily conserved DNA sequences. cDNA clones derived from gastrulation stage libraries that map to chromosomal region 2p2l are candidates for genes necessary for normal brain development. Analysis of expression and function of these genes will elucidate the basic DNA defect which programs abnormal craniofacial development as seen in holoprosencephaly.
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