Alveolar rhabdomyosarcoma is an aggressive soft tissue tumor with striated muscle differentiation which usually occurs in the pediatric population. Diagnosis is often complicated by the paucity of features of striated muscle differentiation and its similarity to a large group of pediatric soft tissue tumors, which includes the embryonal subtype of rhabdomyosarcoma, Ewing's sarcoma, neuroblastoma, and lymphoma. Cytogenetic studies have identified a characteristic translocation of chromosomes 2 and 13, t(2;13)(q35;q14), in the majority of alveolar rhabdomyosarcomas. The consistency and specificity of this translocation indicate that this genetic alteration represents an important step in the pathogenesis of alveolar rhabdomyosarcoma and provides a specific molecular marker for clinical management We developed a physical mapping strategy to localize the t(2;13) translocation breakpoints on genetic maps of human chromosomes 2 and 13. Using this strategy, we have identified and cloned the genes involved in the translocation. Our findings indicate that the coding regions of the PAX3 gene on chromosome 2 and the FKHR gene on chromosome 13 are juxtaposed to produce a chimeric transcript and protein product consisting of the N-terminal PAX3 and C- terminal FKHR regions. Both genes encode members of transcription factor families, and the fusion product is hypothesized to be a chimeric transcription factor. In the proposed project, we will investigate the clinical applications of translocation detection, the functional properties of the fusion product, and the phenotypic consequences of the translocation event. The cloned regions and sequence data from our studies of PAX3 and FKHR will be used to design Southern blot, PCR, and in situ hybridization assays for detection of the t(2;13) translocation in clinical material. These sequence-based assays will be applied to a variety of types of pathologic specimens to establish their utility for differential diagnosis, prognosis, and minimal disease detection. The functional properties of the fusion protein product will be analyzed by DNA binding and transcriptional regulatory assays in conjunction with in vitro mutagenesis experiments to delineate the specific functional domains. Finally, cell culture assays for transformation and animal assays for tumorigenicity will be employed to examine the role of the translocation in neoplastic development and progression.
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