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 (PLAK) gene. The human homologue of mouse FLJ22041 gene is internally located on 122F4 and in the opposite transcriptional orientation. This gene contains 10 exons that span 12kb. The gene is transcribed as 2.6 kb RNA species that is expressed in the normal mammary gland. It encodes an immunphilin that is an intracellular receptor for rapamycin and FK506 and has peptidyl-proline cis-trans isomerase activity (PPPIase). No mutations were found in PLAK or FLJ22041 in tumor DNAs having LOH at 17q21 by single strand conformation polymorphism (SSCP) or nucleotide sequence analysis. On 122F4 the nucleotide sequences corresponding to the R61279 human EST were identified. It is expressed in the normal mammary gland. However, only the 3' end of R61279 was present in 122F4. After screening a P1 library using the 5' portion of R61279 as a probe, another P1 phage clone (23877) containing the complete gene was identified. Currently, breast tumor DNAs are being analyzed for mutations in the R61279 gene. Two other genes, SC65 and FLJ10572, were identified by nucleotide sequence analysis of the 23877 P1 phage clone. SC65 is composed of seven exons and is transcribed as a 2.3 kb RNA species in several tissues including the breast. It encodes a protein that is localized uniformly throughout the granular component of the nucleolus and on the surface of chromosomes during mitosis. Interestingly the R61279 and FLJ22041 genes are located in the fifth intron of SC65. The FLJ10572 gene is composed of 3 exons that are transcribed as a 2.3kb RNA species. It encodes a protein that is weakly similar to Ring Canal Protein. FLJ10572 is expressed in several tissues, but not in the breast. No overlap between 50H1 and 122F4 or 23877 P1 phage clones has been found. 50H1 and 122F4 were thought to be overlapping since they were both reported to be positive for the 122F4 3' STS marker (100). This result has not been confirmed in my laboratory. Based on my earlier work on the breast tumor DNAs it seemed likely that the target gene for LOH is located between D17S746 and D17S846. D17S746 has been found on 122 P1 phage in the region in which PLAK is located. D17S846 is on 50H1 but it has not been localized yet since the nucleotide sequence of this P1-phage clone has not been completed. Nucleotide sequence analysis of 50HI has found the Gastrin (GAS) and NEUROAN1 genes. 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.
