During the first five years of this grant we have defined the first polymorphic enzyme marker system for a solid human tumor. The esterase D (EsD) locus is closely linked to the locus for both the deletion and the dominantly inherited forms of retinoblastoma (Rb) allowing, in informative families, either prenatal or postnatal screening for the presence or absence of the retinoblastoma predisposing genotype. Our biochemical and cytogenetic investigation of Rb tumors has revealed that locus of chromosome 13 or deletion of region 13q14 is a non-random cytogenetic event in this tumor. This observation suggests that the Rb gene is recessive at the cellular level and that loss or inactivation of both wild type alleles at the Rb locus is associated with tumorigenesis. Using restriction fragment length polymorphisms (RFLPs), collaborators demonstrated that gross chromosomal changes leading to homozygosity or hemizygosity at 13q14 is, in fact, a common event in Rb tumors. Primary objectives of the renewal grant are: 1) to prove that the 13 chromosome carrying the wild type allele (Rb+) at the Rb locus is lost or deleted in the tumor as predicted to result in the homozygosity observed at this locus; 2) to determine if similar chromosomal mechanisms involving 13q14 are found in second primary tumors, the cause of death of more than 80% of the survivors of hereditary Rb; 3) to determine if tumor chromosomal karyotyping is useful, as preliminary data suggest, in identifying multifocal (i.e. hereditary) tumors among patients with unilateral disease, and finally 4) to determine the role of other non-random chromosomal changes found in Rb, namely iso 6 p and double minutes (evidence of gene amplification). We plan to meet our objectives by obtaining fresh tumor specimens from preselected patients in whom the 13 chromosome containing the Rb susceptibility allele can be identified. These tumors, obtained through a nationwide network of referring pediatric ophthalmologists, along with second primary tumors will be examined for loss of the wild type allele either cytogenetically or through the use of EsD or DNA RFLPs. The objectives of this renewal grant are directed toward continued improvement of genetic counseling for families with retinoblastoma, understanding the function of the Rb gene, and the ultimate cloning of this human cancer gene.
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