The overall goals of the project are (1) to understand the biology, genetics, and pathogenesis of diseases associated with deficiency of human chromosome 15q11-q13 and the homologous region of mouse chromosome 7, both of which contain numerous imprinted genes, and (2) to study selected basic aspects of imprinted gene expression. Paternal deficiency for human 15q11-q13 causes Prader-Willi syndrome (PWS) while maternal deficiency causes Angelman syndrome (AS). The first specific aim is to extend mutational analysis in AS patients in whom no molecular defect is identified to date with particularly emphasis on identifying additional missense mutations in UBE3A, the locus that is mutated in AS and encodes E6-AP ubiquitin protein ligase, and on identifying mutations in cis regulatory elements essential for expression of UBE3A. The second specific aim is to perform detailed phenotypic analysis of Ube3a knockout mice comparing maternal deficiency (AS) model, paternal deficiency, and homozygous deficiency with wild type mice. Emphasis will be on behavioral and learning studies, electrophysiological analysis of long-term potentiation, and immunohistochemical studies. The third specific aim is to perform an extensive genetic analysis of the PWS/AS region in the mouse by preparing a series of null mutations for Snrpn, Ipw, Gabrb3, Ndn, and newly identified genes. In addition, deletions will be prepared across intervals in the region with a focus on determining which loci account for the perinatal lethal phenotype associated with paternal deficiency for the region (potential equivalent to PWS) and on identifying cis regulatory elements upstream or downstream of Ube3A. The fourth specific aim is to define the putative imprinting center (IC) by constructing deletions upstream of Snrpn in the mouse to obtain mutations that prevent switching between paternal and maternal epigenotypes. The fifth specific aim is to develop a novel and broadly applicable method for visualizing regulation of gene expression through coat color variation and screen for cis- and trans- acting mutations that affect imprinted gene expression. Imprinted expression of the agouti cDNA under the control of the Snrpn promoter has been obtained, and ENU mutagenesis will be utilized. This project is directly relevant to the mental retardation found in PWS and AS, and the Ube3a mutant mouse has the potential to increase our understanding of learning and memory. The use of a coat color marker as a reporter for analysis of gene regulation in vivo in combination with mutagenesis has general applicability beyond imprinted gene expression.
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