Research is proposed to identify, clone and characterize potential tumor or growth suppressor genes that, through inactivation, contribute to the development of a large variety of cancers, including those exhibiting increased prevalence in Beckwith- Wiedemann Syndrome (BWS). The chromosome 11p15.5 interval between the anonymous DNA marker D11S12 and the insulin-like growth factor 2 (IGF2) gene is implicated in tumor suppression and BWS. We have mapped into this region two chromosome rearrangements (one from a rhabdoid tumor and the other associated with BWS) that may disrupt and therefore pinpoint a growth or tumor suppressor gene(s). Long- range restriction mapping indicates that there is little more than 2200 kb separating D11S12 and IGF2, a genomic length easily amenable to cloning in overlapping YACs. Using a battery of 37 probes (one probe per 60 kb) cognate YACs will be isolated from a 4-fold chromosome 11 YAC library and contigs assembled by probe content analysis and walking using YAC-derived Alu-PCR products. A significant advantage of using this YAC library for a walking approach is the rare incidence of human-human chimeric clones (<1%). YAC clones found to map in the chromosome rearrangement breakpoint region will be used to analyze metaphase chromosomes containing the rearrangements by fluorescence in situ hybridization (FISH). Those YACs found to """"""""cross"""""""" the breakpoints will be used to isolate corresponding cosmid clones which will then be screened by exon amplification for the presence of gene coding sequences. These exons will then be used to isolate cDNAs. Alternatively, YACs that span breakpoints or corresponding cosmids will be used to identify encoded genes by a cDNA selection technique. Mutation assays for candidate genes will be used to analyze tumors and additional BWS patients. The cloning and characterization of putative growth or tumor suppressor genes in this chromosome region will help address questions pertaining to the number and type of genes involved in the wide spectrum of tumors showing loss of heterozygosity (LOH) in this genomic segment. Furthermore, molecular assays of both diagnostic and prognostic value may be developed once genes contributing to tumorigenesis are characterized.
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