Beckwith-Wiedemann Syndrome (BWS) causes prenatal overgrowth, midline birth defects, and a wide variety of embryonal tumors. Our laboratory previously mapped BWS to llpl5 by genetic linkage analysis and also demonstrated frequent loss of heterozygosity (LOH) of the same region in embryonal tumors. In the past grant period, in order to identify the genes involved in BWS and LOH, we molecularly cloned genes within and surrounding a cluster of balanced germline chromosomal rearrangement breakpoints from BWS patients termed BWSCRJ. Surprisingly, within this region we identified at least 8 genes which are imprinted, i.e., show preferential expression of a specific parental allele. Several of these genes, which span 1 Mb of lip 15, show genetic or epigenetic alterations in BWS patients. These include p57/KIP2, KvLQT1, H19, IGF2, and LITI, a novel antisense orientation untranslated RNA that we found is within, and imprinted and transcribed oppositely to KvLQT1. This multigene domain was itself divided into two separate imprinted subdomains, with nonimprinted genes between them. Genetic complementation experiments mapped an embryonal tumor suppressor gene to this nonimprinted interval, although rare mutations were also found as well in a novel imprinted gene, TSSC5. Based on our identification of specific genetic alterations that cause BWS, we will now determine the relationship between genotype and phenotype in BWS, and the genetics of transmission of BWS in families. With the assistance of the MIT/Whitehead Genome Center, we will obtain sequence of the entire 1.2 Mb homologous region in mouse, and identify the conserved genes, CpG islands, and other potential intergenic regulatory elements within it. We will determine the functional role of these sequences in normal cells, as well as alterations in BWS patients, including those who appear to show altered imprinting affecting the entire imprinted gene domain. We will identify the gene(s) that suppress the growth of embryonal tumors and explore the mechanism of their alteration, including the possibility that aberrant imprinting leads to inactivation of one copy. Finally, we will determine the normal function of these genes and regulatory sequences using transgenic mice. These studies should continue to provide novel insights into the role of these genes in birth defects and cancer, as well as an exciting species comparative approach to understanding the regulation of multiple genes within a large imprinted domain.
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