POSITIONAL CLONING OF MEN1 GENE

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The purpose of this project is to identify the gene and the mutations responsible for the predisposition to MEN1, an autosomal dominant disease linked to markers at 11q13. Our efforts using interphase-FISH, somatic and radiation-reduced cell hybrids as well as construction of genomic clone contigs have allowed us to generate a physical map for the 2.5 Mb MEN1 interval defined by the markers, D11S480 and D11S913. We have constructed a YAC contig, a bacterial clone (PAC/BAC/P1) contig and a cosmid contig for this genomic region. Nearly 100 STSs developed primarily from the end sequences of these clones have allowed us to generate a high-density STS-content map. This contig provides templates for sequencing, transcript identification, and transcript fine mapping. Polymorphic markers isolated from these clones have been helpful in our continued efforts to narrow the interval by LOH (loss of heterozygo-sity) studies in MEN1 tumors and linkage disequilibrium studies in MEN1 pedigree. Our current MEN1 interval is 1.3 Mb (PYGM-D11S913). A transcript map (currently nearing 20 transcripts including 11 known genes) is under construction for the 1.3MB MEN1 region. Efforts are being made to obtain full length cDNA sequence of each transcript and to analyze their expression and exon-intron organization. So far, 6 of the 20 transcripts have been analyzed for mutations in MEN1 patients by Southern blot, Northern blot and by SSCP/DDF methods, and eliminated as candidate genes responsible for MEN1.