Molecular studies of multiple endocrine neoplasia type 1 (MEN1)

Sammanfattning: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by tumors of the parathyroids, the endocrine pancreas and anterior pituitary. The MEN1 locus has been previously localized to chromosome 11q13 and subsequently a MEN1 minimum region was defined by a combination of linkage and tumor deletion studies. We performed cDNA selection on a bovine parathyroid library using a BAC containing PYGM from this region as a target, and isolated two novel genes termed SCG1 and SCG2. We demonstrated that SCG2 was ubiquitously expressed as a 2.9 kb transcript. Mutation analysis of this gene using SSCP on MEN1 individuals demonstrated the presence of inactivating frameshift, missense or nonsense mutations, which segregated with the disease. Accordingly, SCG2, subsequently named MEN1, was confirmed to be the gene responsible for MEN1, MEN1 consists of 10 exons and encodes a 610 amino acid (aa) protein termed menin. Initial analysis of the predicted amino acid sequence revealed no homologies to any proteins or sequence motifs. We then isolated and characterized the Men1 gene in mouse. Murine Men1 is smaller than its human counterpart, with a genomic size of 6.7 kb as compared to 9 kb. It contains 10 exons and is expressed co-dominantly as two transcripts, 2.8 and 3.2 kb. In situ hybridization analysis demonstrated that Men1 was strongly expressed at gestational day 14, at a later gestational age strong expression becomes more restricted. In mouse, menin is a 611 aa protein, which shows 97% identity to human menin. Using a polyclonal antibody directed against human menin, we studied the expression of menin in both rat and mouse tissues in Western blot. A single band was seen in all tissue tested. Cell specific expression was seen, with menin localizing to the nuclei of nerve cells in the brain, while in testis the staining was perinuclear, using immunohistochemistry. Using tBLASTX analysis, we identified and sequenced a zebrafish EST clone containing the MEN1 gene, which when translated displays a predicted protein of 617 aa. When aligned with human and rodent menin, the zebrafish protein showed high conservation. It has 66.3% identity and 75.2% similarity to human menin. When we compared zebrafish menin to those amino acids which were previously shown to be affected by MEN1-associated missense mutations, we found 90% conservation (compared with 100% in rodent). Menin expression was studied in human and primate cell lines, including lines derived from normal and MEN1-affected individuals. Menin was detected in nucleus and also in cytoplasm, albeit at lower levels. We found that the level of menin remained constant throughout the cell cycle, and displayed no size alteration. A strong signal at 68 kDa was observed in all lymphoblastoid cell lines, i.e. both normal and those carrying germline nonsense mutations. We did not detect a truncated protein nor was any obvious alteration in cellular localization or expression levels seen. Finally, in an attempt to identify the 5'-end of the MEN1 gene, we demonstrated strong heterogeneity of its transcripts, indicating that its expression is more complex than proviously believed. Altogether six transcripts with alternative first exons were identified, three of which derived from the published intron 1, from a region which shows significant homology between human and mouse. They strongly overlap with the position of exon I in mouse Men1 and may represent major MEN1 transcripts in human.