We have undertaken a program that is aimed at determining, on a molecular level, those genetic alterations in primary breast tumor DNA that have a statistically significant association with the patients history, characteristics of the tumor, and the patients prognosis. The most frequent type of mutation is loss of heterozygosity (LOH) at specific regions of the cellular genome in tumor DNA. Approximately 20 such regions have been identified in breast tumor DNAs. Several studies indicate that there are at least three regions on the long arm of chromosome 17 that are affected by LOH. Previous studies of primary human breast tumors showed that chromosome 17q21 was affected by loss of heterozygosity (LOH) in 30% of the tumors. In addition, deletions of this region have a significant correlation with the clinical parameters of aggressive breast cancer. The hereditary breast cancer gene BRCA1 has previously been localized to chromosome 17q21. This gene, however, is not the target for LOH sporadic breast cancer. Seventeen polymorphic sequences tagged site markers were examined in 130 sporadic tumor samples between the D17S250 and D17S579 loci to screen for deletion as measured by loss of heterozygosity (LOH). The smallest common region deleted occurred in the approximately 120-kilobases interval between the 17S846 and 17S746 loci. These loci are located on two overlapping recombinant P1 bacteriophage clones: 122F4 and 50H1. To identify the target gene for LOH, we are determining the nucleotide sequence of the two P1 phage clones. The 5 end of 122F4 contains the Plakoglobin gene. The human homologue of mouse FK506BP gene is located further down 122F4 and in the opposite transcriptional orientation. This gene, contains 11 exons spanning 12kb which encodes a 2.2 kb RNA species that is expressed in the normal mammary gland. On 122F4 we also found nucleotide sequence corresponding to the R61279 human EST which is expressed in the normal mammary gland. However, only the 3 end of this gene was present in 122F4, suggesting that this P1 phage clone contains an internal deletion. After screening a P1 library using the 5 portion of the gene as a probe, we found another P1 phage clone containing the complete gene. Nucleotide sequence analysis of 50HI showed that its 5 end contains exons 2 ?10 of the FK506BP gene. We also found the Gastrin (GAS) and neuroan1genes. The latter gene encodes a protein that is related to the Hap1 protein (Huntington protein). Neither of these genes is expressed in normal mammary tissue. Using nucleotide sequence analysis we have not detected any mutations in the hPlakoglobin and hFK506BP genes in tumors having LOH at 17q21. Currently we are analyzing breast tumor DNAs for mutations in the R61279 gene and continuing our nucleotide sequence analysis of the P1 phage clones to detect additional known or new gene.
