Chang X. Zhang

Germ-Line Mutation Analysis in Patients with Multiple Endocrine Neoplasia Type 1 and Related Disorders

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.

Expression analysis of endogenous menin, the product of the multiple endocrine neoplasia type 1 gene, in cell lines and human tissues

We have investigated the endogenous expression of menin, a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1). Western blot analysis showed strong expression of menin as a 68 kDa protein in all of 7 human and primate cell lines tested. In a panel of 12 fetal human tissue extracts, 68 kDa menin was readily detected in brain cortex, kidney, pituitary, testis and thymus and weakly detected in thyroid. Reproducible bands other than 68 kDa were observed in adrenal and heart, whereas menin was undetectable in liver, lung, pancreas and skin. Analysis of synchronized HeLa cells revealed no variation in the amount or size of menin throughout the cell cycle. Protein expression was compared between lymphoblastoid cell lines from healthy controls and MEN1 patients carrying nonsense mutations on 1 allele. No truncated protein was detected in either cytoplasmic or nuclear fractions in mutation‐carrying cells. The expression level and cellular location of full‐length menin did not differ between cell lines derived from MEN1 patients and healthy donors. This suggests that the wild‐type allele has been up‐regulated in mutation‐carrying cells to compensate for the loss of 1 functional allele. Int. J. Cancer 85:877–881, 2000.

Expression and chromosomal localization of the Requiem gene

Abstract. Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.

The European Consortium on MEN1 - Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Abstract Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1.

Deregulation of anti-Mullerian hormone/BMP and transforming growth factor-β pathways in Leydig cell lesions developed in male heterozygous multiple endocrine neoplasia type 1 mutant mice

Multiple endocrine neoplasia type 1 (MEN1) results from the mutation of the predisposing gene, MEN1. Heterozygous Men1 mutant mice previously generated by several laboratories, including ours, mimic largely MEN1 pathology. Interestingly, our heterozygous Men1 mutant mice exhibit not only the endocrine tumours commonly seen in MEN1 patients, but also Leydig cell tumours (LCT) with high frequency, accompanied systematically by loss of the wild-type Men1 allele. As there exists a similarity of tumour phenotype between these mice and those mutated for the components of anti-Mullerian hormone (AMH)/bone morphogenic protein (BMP) pathway belonging to transforming growth factor-beta (TGF-beta) family, we investigated the expression and the activity of this pathway, known to have an important biological role in Leydig cells. Here, we report that the expression of AMH receptor type 2 is reduced in Men1 LCTs. Both immunostaining and western blot analyses also demonstrate a markedly decreased nuclear expression of Smad1, 3, 4 and 5 in the tumours. More interestingly, we show that the reconstituted menin expression in Men1-deficient Leydig cells derived from LCTs can significantly increase the transcriptional activity of a BMP pathway target promoter, XVent2. Furthermore, we found that the expression of p18, p27 and cyclin dependant kinase 4 (Cdk4), targets of TGF-beta pathways, is altered in the Leydig cell lesions. Our data provide the evidence of the deregulation of AMH/BMP and TGF-beta pathways in mouse Men1 LCTs, highlighting their involvement in tumorigenesis of Leydig cells due to Men1 inactivation.

Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1).

Abstract The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing a contig, we have identified the location of the gene encoding the B56β subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations in the PP2A-B56β coding region, together with the 5′ and 3′ untranslated regions in six MEN1 patients. DNA sequence abnormalities were not identified and thus the PP2A-B56β gene is excluded as the candidate gene for MEN1. However, our precise localisation of PP2A-B56β to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this gene-rich region.