Male germ cell tumors (GCTs) exhibit 12q deletions in more that 30 percent of cases and high frequency of loss of heterozygosity (LOH) at 12q13 and 12q22 indicating sites of putative tumor suppressor genes (TSGs). Homozygous deletions were also found in one tumor with probes D12S7 and MGF mapped to the 12q22. We further characterized the 12q22 deleted region by analysis of LOH utilizing 16 microsatellite markers which identified a minimally deleted region suggesting the site of TSG. We generated a physical map across the region by constructing a YAC contig and a radiation hybrid maps, which estimated the size of the deletion to about 2 Mb. We propose here to identify the TSG at 12q22 with the following specific aims: 1: Narrowing the common region of deletion at 12q22 to less than 1 Mb: A small insert genomic contig in the deleted region will be generated by isolating cosmids/PACs that will provide reagents to generate new polymorphic markers. The newly generated polymorphic markers will be integrated into the contig map and analyzed for LOH on a panel of normal-tumor DNAs to further narrow the boundaries of the common region of deletion to less than 1 Mb. 2. Isolation of transcribed sequences in the commonly deleted region and identification of the putative TSG: Transcribed sequences will be isolated from the cosmids and PACs generated in aim 1 by employing a combination of methods such as exon trapping, cDNA selection and screening for CpG-rich islands. The derived sequences will be utilized to isolate full-length cDNA clones. 3. Characterization of the candidate TSG of interest and verification of its role: The candidate cDNA sequences identified from aim 2 will be tested for gene expression, and mutations. Loss of expression, present of mutations, and correction of phenotype by transfection of cDNA in expression vectors corroborate that the identified gene is a TSG. Subsequently, the TSG will be characterized as to its evolutionalry conservation, genomic organization, normal function, and specific role in tumorigenesis.
