Patrick Gaudray

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.

Detailed map of a region commonly amplified at 11q13→q14 in human breast carcinoma

Amplification of loci present on band q13 of human chromosome 11 is a feature of a subset of estrogen receptor positive breast carcinomas prone to metastasis. As many as five distinct amplification units have been described on 11q13. They include particularly a genomic area encompassing the GARP gene at 11q13.5-->q14.1. We have reassessed our current knowledge of this region, located telomeric to CCND1 and EMS1, which is amplified in 7-10% of mammary tumors. The loose definition of the driving forces of these amplification events led us to map accurately the boundaries of the amplifiable region, and thus to contribute a physical and transcriptional map of a 3-Mb region of chromosome 11. Four new genes were placed on the regional map, namely CBP2, CLNS1A, UVRAG, and PAK1. We have narrowed the core of the 11q13-->q14 amplicon to a 350-kb area encompassing D11S533, mostly on its telomeric side. The map reported here represents an indispensable step toward sequencing the entire region, and thus toward uncovering gene(s) which play(s) a critical role in breast cancer progression.

Characterization of the mouse Men1 gene and its expression during development

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized mostly by tumors of the parathyroids, pancreas and anterior pituitary. The gene responsible, MEN1, encodes Menin, a 610 aminoacid nuclear protein with no sequence homology to other proteins. Although a mouse knock-out model is available, the function of Menin is still elusive. Proteins of known function are shown to interact with Menin: JunD, nuclear factor-KappaB, Smad3, Pem, Nm23H1, glial fibrillary acidic protein, Vimentin, and probably P53. Their partnership with Menin may correspond to a regulation of their activity, but their relevance to the various traits of MEN1 pathogenicity is not established. This raises fundamental issues on the regulation pathways implicated in this complex endocrine disease.

Experimental assessment of the detection limit of genomic amplification by comparative genomic hybridization CGH

Artificial amplicons of known size, constructed by use of YACs featuring human 8p12 and 12q13, were analyzed by comparative genomic hybridization (CGH). A minimum of 15 Mb of overrepresented DNA sequences could be detected. The sensitivity is (1) not dependent on the chromosome site and (2) related to the size of the amplicon, decreasing with decreasing size.

New gene in the homologous human 11q13–q14 and mouse 7F chromosomal regions

Alterations in the chromosomal region 11q13–11q14 are involved in several pathologies in which most of the key genes remain to be identified. In an effort to isolate as many candidates as possible, we are cloning genes from this region. We report here the mapping for a new sequence from 11q13.5–11q14. This sequence, designated D11S833E, putatively encodes a new gene, provisionally named GARP. We cloned its homologous sequence in the mouse and located it on Chromosome (Chr) 7, region F. The human and mouse genes belong to a conserved group of synteny. This, together with the similar conservation of the FGF and TYR genes, indicates that the human 11q13–q14 and mouse 7E-7F regions share homology.

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.

Two polyoma virus gene functions involved in the expression of the transformed phenotype in FR 3T3 rat cells I. Localization of a transformation maintenance function in the proximal half of the large T coding region

Abstract The hypothesis is presented that the expression of a fully transformed phenotype in FR 3T3 rat fibroblast cells transformed with polyoma virus requires two viral gene products. One is the membrane-associated middle T protein (as previously recognized in a number of laboratories). A second necessary function is dependent on an N-terminal region of the large T protein. It can be exerted either by the full-size large T protein (105K), or by truncated forms deleted in the C terminal region. This hypothesis is supported by the results of a complementation analysis using the tsa mutant, which affects only the complete large T protein, and a cloned segment of the viral DNA that codes only for the N-terminal part of large T (BamHI-EcoRI fragment of an hrt (NG 18) deletion). Results show that this subgenomic fragment is able to complement the tsa defect for the maintenance of the transformed state, but not for its establishment.

Cytogenetics of malignant gliomas: I. The autosomes with reference to rearrangements

Autosomal chromosome abnormalities are far from always detectable and, when detected, far from fully consistent in malignant gliomas. In 15 of 41 malignant gliomas, we found autosomal chromosome aberrations ranging from solitary trisomy to a wildly abnormal polyploid complement. The sequence of chromosome events appears to proceed from the normal to the near-diploid state (via structural and numerical changes) to near-tetraploidy (via polyploidization), and finally toward near-triploidy (via chromosome loss and additional rearrangements). Characteristic chromosome changes of trisomy 7 and monosomy 10 were repeatedly found, usually together in the same cell clones. In only one case was trisomy 7 an isolated change. We observed structural rearrangements of chromosomes 7 and 10 which may be of some use in mapping specific genes duplicated or deleted by the whole-chromosome changes of chromosomes 7 and 10. Nonrandom structural changes of other autosomes, including chromosomes 1, 5, and 11, fit with the model of malignant glioma as a process involving multiple genes. An unusual concentration of breakpoints in 12q13, juxtaposing it to at least five other regions, reflects the presence of genetic information in 12q13 important to the development of malignant gliomas.

D11S146 and BCL1 are physically linked but can be discriminated by their amplification status in human breast cancer

Band q13 of chromosome 11 is frequently altered in a number of human cancers. We have undertaken physical mapping in this region, starting with D11S146, an anonymous 11q13 DNA fragment. This probe has been used by others as a landmark to locate MEN1, a locus of predisposition to multiple endocrine neoplasia. Long-range restriction mapping locates D11S146 within approximately 400 kb of the BCL1 translocation breakpoint involved in certain B-cell malignancies. BCL1 and two proto-oncogenes, INT2 and HST, were previously found to be coamplified in approximately 1/5 breast carcinomas. Although close to BCL1, D11S146 is present in less than 3/4 of these amplification units and delimits their centromeric boundary. Therefore, we propose that D11S146 defines two genetic regions. The centromeric region--PYGM/D11S146--contains MEN1. The telomeric one includes the D11S146/BCL1/INT2/HST area and is relevant to DNA amplification in carcinomas and to B-cell translocations.