The applicant's laboratory has discovered several genetic alterations on 11p15 that suggest a crucial role for this region in Wilms tumor (WT) and other embryonal cancers. These include loss of heterozygosity (LOH) of 11p15; genetic linkage to 11p15 of Beckwith-Wiedemann syndrome (BWS), which causes fetal overgrowth and predisposition to a wide variety of embryonal tumors, including WT and rhabdomyosarcoma; and loss of imprinting (LOI), a novel class of genetic alteration causing biallelic expression of genes normally expressed from a single parental allele. In order to localize a BWS gene, the applicant has molecularly cloned 7 germline balanced chromosomal arrangement breakpoints from BWS patients. Surprisingly, these breakpoints occur in two separate regions of 11p15 separated by 4 Mb. The applicant has also developed a novel general genetic complementation strategy to localize and isolate tumor suppressor genes, using subchromosomal transferable fragments (STFs) that can be introduced into any mammalian cell. The applicant has applied this strategy to isolate a region that causes in vitro growth arrest of rhabdomyosarcoma cells. This region overlaps the more telomeric BWS breakpoint cluster. These results suggest that multiple genes on 11p15 are involved in the pathogenesis of embryonal tumors. The applicant is now identifying genes within the more telomeric BWS breakpoint cluster. One of these is p57KIP2, a recently identified cyclin-dependent kinase inhibitor, and a second is a novel gene likely involved in nucleosome assembly. The applicant will continue to isolate genes within the two BWS germline chromosomal rearrangement breakpoint clusters that they have cloned. He will use their novel genetic complementation approach of STFs to determine whether there is a second embryonal tumor suppressor gene on 11p15 corresponding to the more centromeric group of BWS breakpoints. Yeast artificial chromosomes will be transferred directly into tumor cells, in order to further delimit the regions harboring these genes. Alterations in candidate genes in BWS and embryonal tumors will be also identified. Finally, the applicant will determine the normal function of these genes, including their tissue and cellular localization, their developmental pattern of expression, the effect of their expression on normal and tumor cells, and the consequences of gene alteration both in vitro and in vivo. These studies should provide novel insights into how multiple genetic alterations in a single large chromosomal domain give rise to embryonal tumors.
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