We are conducting a positional cloning project to identify a putative tumor suppressor gene(s) on chromosome 3p12.2 or 3q25.3, which are regions previously identified by allelic deletion and cytogenetic analysis, to harbor candidate tumor suppressor genes. Koji Sasajima, a former LHC fellow, has identified a colorectal cancer patient with diffuse digestive tract polyposis and germ line abnormality in chromosome 3; inv(3)(p12.2q25.3). Therefore, we proposed the hypothesis that the gene(s) affected by the chromosome inversion predisposed the patient to diffuse polyposis and malignancy. The chromosomal 3 breakpoints of this patient have been isolated by positional cloning strategy. We and our collaborator microscopically dissected the chromosome fragments around the breakpoints. Using a DNA probe isolated from the dissected fragments, we made YAC contigs and YAC clones containing the breakpoint in 3p which were identified by FISH (fluorescent in situ hybridization). The cosmid contigs were made from these YACs, cosmid clones spanning the breakpoint were identified by FISH, and the nucleotide sequence at and around the breakpoint in 3p12.2 was determined. The breakpoint at 3p12.2 is located in the region having homology with L1 repetitive sequence. Chromosome inversion caused 2 bp deletion. A search for the gene disrupted by the inversion is in progress using an exon-trapping method. We have obtained a candidate sequence having no homology with any genes presently listed in the genomic databank. The breakpoint in 3q25.3 is being identified from the cosmid clones containing 3p12-3q25 sequence isolated from the genomic library made from the patient's DNA. The breakpoint at 3q25.3 contains a short deletion of 100-200 kb. At one edge of the deletion, tandem repeats composed of more than 10 copies of 100 bp-long sequence distantly related to alu repeats were found. We have constructed cosmid and P1 contigs spanning the deletion. Selected cosmids are being subjected to exon trapping to identify the putative tumor suppressor gene(s) involved.
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