CORE--PATIENT MUTATION ANALYSIS

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The Patient Mutation Analysis Core, is a blood processing, tissue culture and mutation analysis core. In this facility, we collect and process peripheral blood of WAS and XLT patients including quantifying and phenotyping blood cell subpopulations, isolation of mononuclear cells and lymphocytes, cryopreservation, and generation, maintenance and characterization of EBV-transformed B-cell lines that will be used in investigations in Projects 1 and 2. The Core contributes to diagnosis of WAS in newly referred patients by performing platelet sizing and, where family history is lacking, heterozygosity analysis of female relatives. The Core identifies the WAS gene mutation of diagnosed patients by isolating DNA and amplifying exon regions, analyzing amplified regions by single chain conformation polymorphism analysis (SCCP) followed be sequencing of the abnormal region. The Core characterizes and quantifies the mutated WASP protein expressed in patient cells by a combination of Western blotting and Phosphor-Imaging. The Core will determine whether addition of protease inhibitors, a potential therapeutic treatment, will increase the level of WASP protein in cultured cells of some patients. Finally the Core maintains a database of patient blood samples and cell lines as well as clinical and molecular information on participating patients and a separate database of published WAS defects.