The purpose of this project is to determine the role of oncogenes in the pathogenesis of malignancies occurring in neurofibromatosis (NF). Dominantly inherited syndromes predisposing to neoplasia constitute an important resource for study because individuals at high risk for malignancies of specific types are identifiable years in advance. The sequence of mutations leading to malignancies may be identifiable by comparison of malignant tissue, unaffected tissue from the same individual, and tissue from unaffected individuals. Of inherited syndromes predisposing to neoplasia, neurofibromatosis is the most suitable for analysis because it is dominant, common, and involves multiple tumors of multiple types at accessible sites. When NF patients have malignancies resected, malignant and nontumor tissue will be obtained and DNA and RNA purified. In order to determine which oncogenes are hyperexpressed in neurofibromatosis, poly A+ RNA will be purified from tumors and control tissue and specific oncogene transcripts will be quantitated by RNA slot blotting. For any specific oncogene which is hyperexpressed in the tumor compared to control tissue, size will be determined by Northern blotting. For any specific oncogene which is hyperexpressed, gene number will be determined by DNA slot blotting in order to determine whether the gene is amplified. To look for structural rearrangements, including large deletions, insertions, or translocations, restriction mapping will be performed. If an abnormality in the restriction map is found, sequencing will be performed. To pursue our preliminary finding that sis and N-ras may be hyperexpressed in NF neurofibrosarcomas, gene products will be assayed by their activity (PDGF) or immunoreactivity (ras-like), restriction fragment patterns of NF tumor tissue, NF non-tumor tissue, and unaffected relatives will be compared to investigate the site of germline and somatic mutations, and mutations in homologous genes will be investigated by hybridization under conditions of reduced stringency. Transforming activity of DNA from NF malignancies will be assayed in mouse 3T3 fibroblasts, human NF fibroblasts, and human non-NF fibroblasts. Identification of oncogene abnormalities in neurofibromatosis may elucidate the nature and sequence of mutational events, permit correlation of clinical and cellular phenotypes with molecular genotypes, permit early diagnosis in suspected cases, lead to prenatal diagnosis, and validate a strategy applicable to other phakomatoses and dominantly inherited predispositions to cancer.
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