The monoamine oxidases, MAO-A and MAO-B, are the primary enzymes involved in degradation of biogenic amines in mammals. They control levels of amine neurotransmitters, such as dopamine, norepinephrine and serotonin, in the nervous system and metabolize amines that can act as false transmitters or neurotoxins. Humans inherit wide variations in levels of these enzymes. Normal variations are thought to influence the susceptibility of individuals to pathogenic processes involving amines, such as Parkinson disease (PD). Loss of activity is hypothesized to affect the development of the nervous system and possibly to be responsible for a type of X-linked mental retardation (XLMR). The proposed studies are designed to elucidate structural differences in alleles for the MAOA gene that control levels of enzyme activity. Sequence variations in the MAOA gene will be assessed in genomic DNA from male, cultured skin fibroblast lines which vary over 100- fold in activity. This will be carried out by PCR amplification of exons and 5' flanking sequence, followed by analysis of single strand conformational polymorphisms (SSCPs) and direct sequencing of variant fragments. Correlations will be made between specific sequence differences and levels of MAO-A activity and mRNA; the latter will be determined by northern and slot blot analysis, as well as evaluation of mRNA turnover by 4-thiouridine incorporation and affinity chromatography. Critical polymorphisms in the MAOA gene that control levels of activity will be assessed in control and PD males, where differences in allele frequencies have been noted in preliminary studies. To screen for an MAO deficiency state, genomic DNA from several hundred undiagnosed XLMR patients will be assessed for structural integrity of the MAO genes by multiplex PCR analysis of all exons, and SSCP analysis of conserved exons. In order to understand the effects of MAO deficiency on neuronal development, MAOA and MAOB genes will be disrupted in mice by homologous recombination in embryonic stem cells, formation of chimeric embryos and subsequent evaluation and breeding of mice. These studies will elucidate means by which the MAOA gene itself controls levels of activity, the extent of genetic variation in the MAO genes in the human population, and the effects of this variation on neuronal development and susceptibility to neuronal degeneration.
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