Tuberous sclerosis complex (TSC) is an autosomal dominant human disease characterized by abnormal growths in a variety of tissues. Genetic linkage analysis has implicated the distal portion of the long arm of chromosome 9 in the etiology of TSC1. This proposal is aimed at a detailed characterization of this region of chromosome 9q34, refinement of the critical region, identification and characterization of the affected gene. Initial studies will involve construction of a high resolution map of the region represented in cosmids. The resulting overlapping cosmid clones, which are more amenable to manipulation in molecular biological studies, will be used for polymorphic marker isolation and gene identification in the candidate region. The polymorphic markers generated will potentially serve to narrow the region by analyzing meiotic recombinations that define the critical region. Genes or gene fragments isolated from this region will be used to search for alterations in patient lymphoblast DNA and RNA samples; this will initially entail Southern hybridization, and single-stranded conformation polymorphism (SSCP) detection and direct sequencing of RNA/PCR products. Subsequent to identification of the TSC1 gene, its genomic structure will be fully characterized, making possible complete and efficient mutation screening for epidemiological studies and affected tissue analysis. Analysis of TSC1 expression pattern in developing and adult tissues, as well as in affected tissues, is likely to be a critical first step in understanding of its role in normal and disease states. To this end, we will determine TSC1 expression patterns at the mRNA and protein levels. For more comprehensive developmental studies of TSC1 mRNA expression, the murine homologue will be isolated and used in in situ hybridization experiments using sections of staged mouse embryos. For protein expression studies, a panel of polyclonal and monoclonal antibodies will be raised for the TSC1 protein , or specific domains of it, to determine in more detail its expression pattern in both normal and affected tissues. Identification of the gene mutated at the TSC1 locus will lead to a better understanding of the mechanisms by which TSC arises, as well as significant improved molecular diagnostics for the disease. In addition, the information and resources generated in these studies will be invaluable in understanding the biochemical role of the gene in normal and abnormal cellular and developmental processes, and may lead to effective treatments for the disease.
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