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G M Lenoir

Publications and source records attributed to G M Lenoir.

At least 19 recordsLinked to original sources
PubMed2000

Differential expression and subcellular localization of murine BRCA1 and BRCA1-delta 11 isoforms in murine and human cell lines.

BRCA1 mutations are involved in breast and ovarian cancer predisposition in humans. The biological functions of the murine BRCA1 gene have been extensively studied but little is known about murine BRCA1 proteins. To better characterize these proteins, we have cloned the full-length murine BRCA1 cDNA and a splice variant deleted of exon 11, BRCA1-delta 11, by RT-PCR method. Three polyclonal antibodies raised against various parts of murine BRCA1 were used in our study: D16, M20 and 5MO, which were generated in our laboratory. This allowed us to analyze the expression and subcellular localization of both isoforms in murine and human cell lines by immunoblotting, immunoprecipitation, cell fractionation and immunofluorescence. Endogenous BRCA1 was detected in murine cell lines but not splice variant BRCA1-delta 11, whereas both ectopically expressed murine isoforms were detected in transfected human Bosc 23 cells. Subcellular fractionation and immunofluorescence results showed that the BRCA1 protein was mainly located in the nucleus, whereas BRCA1-delta 11 was preferentially cytoplasmic. The conservation of exon 11 splicing and the differential subcellular localization of BRCA1 and BRCA1-delta 11 in human and mouse suggest that these proteins could play distinct roles and that they could differentially act in the pathological mechanisms leading to the development of breast and ovarian cancer. The characterization of the murine BRCA1 proteins and antibodies will be useful to further study BRCA1 functions in murine models.

3T3 Cells↗
PubMed2000

Screening for genomic rearrangements in families with breast and ovarian cancer identifies BRCA1 mutations previously missed by conformation-sensitive gel electrophoresis or sequencing.

The frequency of genomic rearrangements in BRCA1 was assessed in 42 American families with breast and ovarian cancer who were seeking genetic testing and who were subsequently found to be negative for BRCA1 and BRCA2 coding-region mutations. An affected individual from each family was tested by PCR for the exon 13 duplication (Puget et al. 1999a) and by Southern blot analysis for novel genomic rearrangements. The exon 13 duplication was detected in one family, and four families had other genomic rearrangements. A total of 5 (11. 9%) of the 42 families with breast/ovarian cancer who did not have BRCA1 and BRCA2 coding-region mutations had mutations in BRCA1 that were missed by conformation-sensitive gel electrophoresis or sequencing. Four of five families with BRCA1 genomic rearrangements included at least one individual with both breast and ovarian cancer; therefore, 4 (30.8%) of 13 families with a case of multiple primary breast and ovarian cancer had a genomic rearrangement in BRCA1. Families with genomic rearrangements had prior probabilities of having a BRCA1 mutation, ranging from 33% to 97% (mean 70%) (Couch et al. 1997). In contrast, in families without rearrangements, prior probabilities of having a BRCA1 mutation ranged from 7% to 92% (mean 37%). Thus, the prior probability of detecting a BRCA1 mutation may be a useful predictor when considering the use of Southern blot analysis for families with breast/ovarian cancer who do not have detectable coding-region mutations.

BRCA2 Protein↗
PubMed2000

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.

Animals↗
PubMed2000

An Alu-mediated 7.1 kb deletion of BRCA1 exons 8 and 9 in breast and ovarian cancer families that results in alternative splicing of exon 10.

Constitutive large deletions and duplications of BRCA1 resulting from Alu-mediated recombination account for a significant proportion of disease-causing mutations in breast and/or ovarian cancer families. Using Southern blot analysis and a protein truncation test (PTT), we have identified a 7.1 kb germline deletion in two families with breast and ovarian cancer. This deletion, which includes exons 8 and 9 and leads to a frameshift at the mRNA level, appears to result from homologous recombination between closely related Alu repeats, one in intron 7 and one in intron 9. In addition to the transcript without exons 8 and 9, analysis of RNA by protein truncation test from individuals with the deletion also identified the presence of alternative splicing of exon 10 from the mutant allele, which results in a transcript that lacks exons 8, 9, and 10. Of interest is that the two American families who carry this deletion are of northern European ancestry and share a common haplotype, suggesting that this deletion may represent a founder mutation. Genes Chromosomes Cancer 28:300-307, 2000.

Adult↗
PubMed2000

Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG-->ATG) mutation.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. METHODS: We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. RESULTS: We identified a mutation in codon 804 in exon 14 (GTG-->ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. CONCLUSIONS: Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative.

Adult↗
PubMed2000

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.

Azacitidine↗
PubMed1999

Germline brca2 sequence variants in patients with ocular melanoma.

On the basis, chiefly, of anecdotal reports of cases of ocular melanoma (OM) occurring in families with inherited susceptibility to breast cancer due to brca2 germline mutations, we examined the frequency of brca2 alterations in a series of 62 ocular melanoma cases. These cases were preferentially selected on the basis of reported family history of breast or ovarian cancer, or OM, although the series also included a randomly selected set of cases without family history of cancer. A total of 7 germline alterations were found, of which 3 were likely to be associated with disease. While all 3 deleterious mutations were found in patients who also had a personal history of breast cancer, only 1 of the 3 families had a family history of breast/ovarian cancer or OM. Although germline brca2 mutations may account for a small proportion of all OM cases, there may be additional loci that contribute to familial aggregation of OM and to the familial association between OM and breast cancer.

Eye Neoplasms↗
PubMed1999

BRCA1 expression during prenatal development of the human mammary gland.

Germ-line alterations of BRCA1 are associated with elevated risk of breast cancer. Evidence for the involvement of Brca1 in cellular differentiation and morphogenesis has been obtained in mouse models during embryogenesis. Although the presence of well-conserved functional domains might suggest a similar function for both human and mouse genes, very few data on BRCA1 expression in human fetal tissues are available. We have, therefore, investigated the expression of BRCA1 in the mammary gland from human female fetuses aged between 15 and 33 weeks. Quantification of BRCA1 transcripts, using a competitive reverse transcriptase PCR method, indicates a progressive decrease in BRCA1 expression with increasing fetal age between the 15th and 30th week of gestation. Subsequently, the amount of BRCA1 transcripts becomes similar to that found in adult mammary gland. Analysis of BRCA1 protein revealed, in fetal samples, a 220 kDa band corresponding to the 220 kDa BRCA1 protein described in human cell lines. These later experiments confirm that the relative level of the 220 kDa BRCA1 protein is highest in the early stages of mammary gland development. The temporal patterns of BRCA1 expression in human fetuses suggest a role for BRCA1 in the morphogenesis and differentiation of the human mammary gland.

Adult↗
PubMed1999

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions.

Most previous BRCA1 mutation screening studies conducted on breast cancer families were aimed at identifying mutations in the coding sequence and splice sites. Mutations in the promoter and untranslated regions, and large rearrangements are missed by standard mutation detection strategies. To look specifically for such germ-line mutations in the BRCA1 gene, we have analyzed a series of 27 American and 51 French breast cancer families in which no BRCA1 mutation was identified by classical techniques. No mutations were detected in either the promoter or untranslated regions, and we did not find any deletion of the whole gene. Four families were found to carry distinct deletions. Two of them, probably generated by Alu-mediated homologous recombination, were internal deletions of 3 and 23.8 kb, encompassing exon 15 and exons 8-13, respectively. These alterations both lead to a frameshift in the mutant mRNA and to premature stop codon-mediated mRNA decay. The other two deletions encompass exons 1 and 2. On the basis of previous and present analyses, rearrangements represent 8% (3/37) of all mutations in our set of BRCA1 American families. Consequently, the search for rearrangements appears mandatory in BRCA1 mutation screening studies.

3' Untranslated Regions↗
PubMed1999

Co-segregation of MEN2 and Hirschsprung's disease: the same mutation of RET with both gain and loss-of-function?

Multiple endocrine neoplasia type 2 (MEN2) and Hirschsprung's disease (HSCR) are two dominantly inherited neurocristopathies ascribed to mutations in the RET gene [Chakravarti, 1996; Pasini et al., 1996; Eng and Mulligan, 1997]. MEN2 is a cancer syndrome comprising three related clinical subtypes: (1) MEN type 2A (MEN2A; MIM# 171400) characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo), and hyperparathyroidism; (2) MEN type 2B (MEN2B; MIM# 162300), which includes MTC, Pheo, mucosal neuromas, ganglioneuromatosis of the digestive tract, and skeletal abnormalities; and (3) familial MTC (FMTC; MIM# 155240), defined by the sole occurrence of MTC. HSCR (MIM# 142623) is a congenital malformation caused by the absence of enteric plexuses in the hindgut, leading to bowel obstruction in neonates. The RET gene (MIM# 164761) codes for a transmembrane tyrosine kinase, a component of a multimeric complex that also comprises one of four members of a novel family of glycosylphosphatidylinositol (GPI)-anchored receptor, GFRalpha((1-4) (e.g., GFRA1, MIM# 601496; references are detailed in Baloh et al. [1998]. Four structurally related soluble factors-glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin-are the ligands of these multimolecular receptors in which the nature of the GFRalpha determines the ligand specificity of the complex [see Baloh et al., 1998, for references]. It is well documented that RET/GFRalpha-1/GDNF delivers a signal critical for the survival of the early neural crest-derived precursors that colonize the intestine below the rostral foregut and give rise to the enteric nervous plexuses [Gershon, 1997; Cacalano et al., 1998; Enomoto et al., 1998].

Drosophila Proteins↗
PubMed1999

Psychosocial impact of genetic testing in familial medullary-thyroid carcinoma: a multicentric pilot-evaluation.

BACKGROUND: Many crucial problems are associated with the diagnosis of inherited cancer susceptibility. One of the most important is related to the psychosocial consequences of the knowledge by the patients and their relatives of their own genetical status. Little data are available in the literature, mainly from studies including small numbers of selected and motivated patients. PATIENTS AND METHODS: From January till December 1997, we studied the psychometric and quality of life parameters of 77 subjects followed in two French specialized centers. These subjects had been treated for either sporadic or familial or were at risk for medullary thyroid carcinoma. All patients had previously attended genetic counselling with detection of germline Ret-mutations, were informed on their own genetic risk, had good short-term prognosis and performance status and did not receive recent cancer treatment. Each patient was invited to answer two questionnaires, the hospital anxiety and depression scale (HADS) and the subjective quality of life profile (SQLP). RESULTS: We report herein the descriptive results of this study (HADS and SQLP scores and distributions) and describe the individual clinical covariates that might explain the observed differences between subgroups of individuals. Although psychometric scores appeared similar in these subgroups, quality of life scores were lower in Ret-mutation carriers. Genetically-predisposed patients were less satisfied and expressed more expectations for favourable change in their quality of life. CONCLUSION: This finding suggests a high level of frustration and latent unsatisfaction related either to the management of the genetic information given by the clinicians and its psychosocial consequences or simply to the knowledge of the genetic risk of cancer. Further studies on the individual consequences of genetic testing, information delivery and when necessary psychotherapeutic interventions, are needed to insure the quality of presymptomatic genetic testing in this field of oncology.

Adult↗
PubMed1999

Two distinct mutations of the RET receptor causing Hirschsprung's disease impair the binding of signalling effectors to a multifunctional docking site.

The RET gene codes for a transmembrane tyrosine kinase which is a subunit of a multimeric complex that acts as a receptor for four structurally related molecules: the glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephin. Germline mutations of RET cause a dominantly inherited dysgenesis of the enteric nervous system known as Hirschsprung's disease (HSCR; aganglionosis megacolon). The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function. Two novel distinct mutations of RET that led either to the deletion of codon 1059 (denoted Delta1059) or to the substitution of a Pro for Leu1061 have been identified in five HSCR families. In one large pedigree, two children born from asymptomatic consanguineous parents presented a severe form of HSCR and were found to carry the mutation at codon 1061 in the homozygous state. A tyrosine residue at position 1062 is an intracytoplasmic docking site that enables RET to recruit several signalling molecules, including the Shc adaptor protein. We now report that both HSCR mutations impair the fixation of Shc to RET and consequently prevent its phosphorylation. In addition, quantitative analysis in PC12 cells reveals that mutation Delta1059 inactivates the ability of RET to transduce a downstream signal whereas mutation L1061P only partially inhibits the signalling of RET. Finally, we provide evidence that these effects are partly mediated via the disruption of the RET/Shc interaction. Collectively, these results demonstrate that HSCR can be ascribed to mutations of RET which interfere with the binding of transduction effectors, such as Shc, and further provide a biochemical explanation for the phenotype of patients carrying a homozygous mutation at codon 1061. Finally, these data indicate that Y1062 is a multifunctional docking site that confers to RET the capacity to engage downstream signalling pathways which exert a crucial role during enteric neurogenesis.

3T3 Cells↗
PubMed1998

Down-regulation of BRCA1 in human sporadic breast cancer; analysis of DNA methylation patterns of the putative promoter region.

Germ-line alterations of BRCA1 are responsible for about 50% of familial breast cancers. Although its biological function(s) has not yet been fully determined, it has been suggested that it may act as a tumor suppressor gene in breast and ovarian cancers. In sporadic breast cancers alterations of BRCA1 have not been detected and in vitro experiments have indicated that BRCA1 negatively regulates cellular proliferation. The present study was designed to identify and quantify, the BRCA1 mRNA levels, in normal and neoplasic human breast tissue. BRCA1 mRNA molecules were quantified using competitive reverse transcriptase PCR assays. DNA methylation patterns of this gene have been analysed by Southern blot experiments using methylation sensitive restriction enzymes. We found that BRCA1 mRNA levels were significantly lower in sporadic breast cancers (37 cases analysed, 24 cases of invasive ductal carcinomas not otherwise specified (NOS), two lobular carcinomas in situ two medullary carcinomas, four invasive lobular carcinomas, two invasive mucinous carcinomas and three invasive ductal carcinomas with predominantly in situ component) compared with normal breast tissues (P=0.0003). This down-regulation of BRCA1 is observed in all histologic types analysed. In invasive ductal carcinomas NOS, this down-regulation does not correlate with any of the prognostic factors studied (tumor size, node status, histologic grade, hormone receptor status). In the samples analysed, alterations of DNA methylation patterns were not dectected in the vicinity of the major transcription start site. These data suggest the involvement of BRCA1 in the carcinogenesis of these histologic types.

BRCA1 Protein↗
PubMed1998

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines.

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.

3T3 Cells↗
PubMed1998

Effect of smoking on breast cancer in carriers of mutant BRCA1 or BRCA2 genes.

BACKGROUND: Smoking has carcinogenic effects, and possibly antiestrogenic effects as well, but it has not been found to be a risk factor for breast cancer in women in the general population. However, hereditary breast cancer is primarily a disease of premenopausal women, and interactions between genes and hormonal and environmental risk factors may be particularly important in this subgroup. METHODS: We conducted a matched case-control study of breast cancer among women who have been identified to be carriers of a deleterious mutation in either the BRCA1 or the BRCA2 gene. These women were assessed for genetic risk at one of several genetic counseling programs for cancer in North America. Information about lifetime smoking history was derived from a questionnaire routinely administered to women who were found to carry a mutation in either gene. Smoking histories of case subjects with breast cancer and age-matched healthy control subjects were compared. Odds ratios for developing breast cancer were determined for smokers versus nonsmokers by use of conditional logistic regression for matched sets after adjustment for other known risk factors. RESULTS: Subjects with BRCA1 or BRCA2 gene mutations and breast cancer were significantly more likely to have been nonsmokers than were subjects with mutations and without breast cancer (two-sided P = .007). In a multivariate analysis, subjects with BRCA1 or BRCA2 mutations who had smoked cigarettes for more than 4 pack-years (i.e., number of packs per day multiplied by the number of years of smoking) were found to have a lower breast cancer risk (odds ratio = 0.46, 95% confidence interval = 0.27-0.80; two-sided P = .006) than subjects with mutations who never smoked. CONCLUSIONS: This study raises the possibility that smoking reduces the risk of breast cancer in carriers of BRCA1 or BRCA2 gene mutations.

Breast Neoplasms↗