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Cloning and sequencing of 5.5kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines has revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5kb sequence in 494 normal lymphocyte samples showed heterozygous deletion in 28.3% of African- American ancestry samples but only in 4.8% of Caucasian samples (p

A transcription map of a 1200-kb region encompassing the MEN1 locus was constructed by direct cDNA selection and mapping ESTs. A total of 29 genes were mapped. Ten transcripts were identified by cDNA selection of a focused 300-kb genomic region telomeric to the MEN1 consensus region. Since many of the sequences cloned by cDNA selection also identified ESTs from the region, 19 additional RH-mapped ESTs were mapped to the entire contig region by PCR amplification of genomic clones. Nine known genes, 2 putative human homologues to mouse genes, and 18 novel transcripts map to the region. Transcripts that map to the MEN1 interval PYGM –D11S449 include SGC35223, IB1256, AA147620, ZFM1, FAU, and CAPN1. The latter 3 known genes have already been excluded as candidate MEN1 genes. The 2 putative human homologues of mouse genes Ltbp2 and Spa-1 may be candidate tumor suppressor genes, but they map telomeric to D11S449. Although both of these genes map outside the MEN1 consensus region they may play a role in sporadic endocrine tumors independent of the MEN1 gene or in other tumors, such as breast cancer, that have loss of heterozygosity within this region.

Linkage analysis and loss of heterozygosity studies have shown that the gene responsible for the multiple endocrine neoplasia type-1 (MEN1) syndrome localizes to a small interval between D11S427 and D11S460 on chromosome 11q13. As an initial step to clone this tumor suppressor gene, our group is the first to map the MEN1 region physically using yeast artificial chromosome, bacterial artificial chromosome (BAC), and cosmid contigs. The 1.5-Mb high-resolution, contiguous map extends from PYGM to 300 kb telomeric of D11S460. Of this, the 1.2-Mb interval between PYGM and D11S460 is isolated in cosmids and BACs and will be useful for the development of genomic sequences and transcription maps of this important region. Nine new sequence-tagged sites (STS) are also characterized from this region. The physical map and the STSs will be valuable tools for the cloning of the MEN1 gene.

Nonrandom deletion of chromosome 11q13 sequences is a significant event in a number of human tumors. We have recently identified a 300-kb minimal area of deletion in primary cervical tumors that overlaps with deletions observed in endocrine and nasopharyngeal tumors. We have also observed a 5.7-kb homozygous deletion within this interval in HeLa cells (a cervical cancer cell line), HeLa cell-derived tumorigenic hybrids, and a primary cervical tumor, suggesting the presence of a tumor suppressor gene in this region. In the present investigation, we have constructed a 700-kb contig map encompassing the 300-kb deletion using the human genome sequence database and confirmed the map using various STS markers from the region. Our map also shows the overlap of a previously published rare, heritable fragile site, FRA11A, with the cervical cancer deletion locus. The mapped region contains highly repetitive GC-poor sequences. We have identified and characterized eight different polymorphic microsatellite markers from the sequences within and surrounding the deletion. Further, expression studies performed with 18 different ESTs localized adjacent to the homozygous deletion showed the presence of a transcript for only one of the ESTs, AA282789. This EST mapping within the homozygous deletion is also expressed in HeLa cells, thereby excluding the EST as the putative tumor suppressor gene. Additionally, analysis of four candidate genes (SF3B2, BRMS1, RIN1, and RAB1B) from the region showed expression of the expected size message in both the nontumorigenic and the tumorigenic HeLa cell hybrids, thereby excluding them as the putative tumor suppressor gene(s). However, Northern blot analysis with a fifth candidate gene, PACS1 (phosphofurin acidic cluster sorting protein), mapped to the deletion/FRA11A overlap region showed the expression of an 8-kb transcript in HeLa and five other tumor cell lines in addition to the expected 4.5-kb transcript. Since the gene shows abundant expression in normal tissues and an altered transcript is observed in tumor cell lines, we hypothesize that this gene could represent sequences of the putative tumor suppressor gene. Finally, we have observed a perfect 48-bp CAG/CCG repeat 99 kb proximal to D11S913, the marker linked to the neurodegenerative disorder spinocerebellar ataxia 5. The physical and transcription maps and the microsatellite markers of the 700-kb region of chromosome 11q13 should be helpful in the cloning of the cervical cancer tumor suppressor gene.