PubMed Health⌕ Search

Biomedical subjects

D Vidaud

Publications and source records attributed to D Vidaud.

At least 37 records · Page 2Linked to original sources
PubMed1999

Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

Adult↗
PubMed1999

TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency.

BACKGROUND: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy. METHODS: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of approximately 1 in individuals without the deletion, whereas a ratio of approximately 0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals. RESULTS: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472-0.556 (SD, 0.013-0.078). In the five individuals without deletions, the ratios were 0.919-1.019 (SD, 0.006-0.075). CONCLUSIONS: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.

Carrier Proteins↗
PubMed1999

Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications.

BACKGROUND: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations. METHODS: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA. RESULTS: The dynamic range was >1000-fold. The relationship between C(t) and log starting concentration was linear (r(2) >/=0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification. CONCLUSIONS: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.

Adult↗
PubMed1998

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

Breast Neoplasms↗
PubMed1998

Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors.

Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.

Adult↗
PubMed1998

Novel recurrent nonsense mutation causing neurofibromatosis type 1 (NF1) in a family segregating both NF1 and Noonan syndrome.

Neurofibromatosis type 1 (NF1), a genetic disorder with neuroectodermal involvement, demonstrates phenotypic overlap in some patients with Noonan syndrome (NS), ultimately resulting in the so-called neurofibromatosis-Noonan syndrome (NF-NS). A strong association of the two phenotypic traits was recently illustrated by a four-generation family, although NF1 and NS were eventually demonstrated to segregate independently on the basis of polymorphic DNA markers [Bahuau et al., 1996: Am J Med Genet 66:347-355]. Identification of the causal NF1 mutation seemed a prerequisite to further dissecting this singular familial association. Using the protein truncation assay, a nonsense mutation (C2446T-->R816X) of the neurofibromin gene was evidenced. This mutation occurred on a CpG dinucleotide within exon 16 and 5' to the GAP domain-specifying region of the gene. R816X creates a recognition site for endonuclease HphI, absent in 2 individuals with NS only. Screening 184 unrelated NF1 patients, three novel occurrences of the mutation were found in individuals diagnosed with classical NF1. Based on the assumption of genotype-phenotype correlation in these individuals, clinical and molecular analyses of this four-generation family demonstrated that the NF-NS phenotype was additive, being the result of both classical NF1 and NS. This particular observation also suggests the presence of an NS locus on 17q, which might be of interest for further linkage studies.

DNA Mutational Analysis↗
PubMed1998

A novel missense mutation D513G in exon 10 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene identified in a French CBAVD patient. Mutations in brief no. 175. Online.

Congenital bilateal absence of the vas deferens (CBAVD) with obstructive azoospermia is a congenital reproductive disorder that affects one in 10000 male individuals. The observation that many men presenting with CBAVD have mutations in their CFTR genes had led to the proposal that CBAVD may be a primary genital form of cystic fibrosis. We report here one novel mutation located in exon 10 of the CFTR gene. This mutation, named D513G (A-->G at position 1670), has been found in one of 83 patients with CBAVD from France, the analysis of exon 10 using a chemical clamp DGGE assay allowed us to identify three CF mutations AEF508 (37/166; 22%), AE1507 (1/166; 0/6%) and D513G (1/166; 0.6%), and two variants M470V and E528E (1716 G>A). The novel D513G mutation has not been found in more than 200 non-CF chromosomes and in a sample of 300 CF chromosomes from French classical CF patients.

Animals↗
PubMed1997

Familial aggregation of malignant melanoma/dysplastic naevi and tumours of the nervous system: an original syndrome of tumour proneness.

A five-generation family is here reported in which several members developed malignant melanoma, dysplastic naevi, astrocytoma in all grades, benign or malignant schwannoma, neurofibroma, or meningioma in a single instance. Significant cosegregation of skin and nervous tumours, preclusion of allelism to type 1 neurofibromatosis and phenotypic departure from known syndromes of hereditary proneness to cancer make one suggest an original familial predisposition to both malignant melanoma and central/peripheral nervous tumours.

Adult↗
PubMed1996

Exclusion of allelism of Noonan syndrome and neurofibromatosis-type 1 in a large family with Noonan syndrome-neurofibromatosis association.

A large four-generation family with Noonan syndrome (NS) and neurofibromatosis-type 1 (NF1) was studied for clinical association between the two diseases and for linkage analysis with polymorphic DNA markers of the NF1 region in 17q11.2. Nonrandom segregation between NS and NF1 phenotypes was observed. Neurofibromatosis was tightly linked to NF1 markers, whereas Noonan syndrome was found not be allelic to NF1. These results suggest that two mutations at two independent but closely linked loci are the cause of neurofibromatosis-Noonan syndrome (NF-NS) association in this family.

Alleles↗
PubMed1996

A new series of mycobacterial expression vectors for the development of live recombinant vaccines.

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.

Antigens↗
PubMed1996

Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease.

It has previously been shown that, in heavy drinkers, serum apolipoprotein A-I (ApoA-I) levels are closely related to the degree of liver injury: they are at a maximum in patients with steatosis, begin to decrease in patients with fibrosis, and reach a minimum in patients with severe cirrhosis. In contrast with serum ApoA-I variations, serum apolipoprotein B (ApoB) levels are stable. The assessment of messenger RNA (mRNA) levels of ApoA-I and ApoB using a quantitative competitive polymerase chain reaction (PCR) method was performed in 18 alcoholic patients: 8 with normal livers or steatosis (group I), 6 with fibrosis or nonsevere cirrhosis (group II), and 4 with severe cirrhosis (group III). For ApoA-I, group I had higher serum levels (208.4 +/- 37.6 mg/dL mean +/- SE) and mRNA levels (0.51 +/- 0.12) than group II (serum 116 +/- 19 mg/dL, P < .01, mRNA 0.40 +/- 0.11, P < .09) or group III (serum 56.5 +/- 28.6 mg/dL, P < .01, mRNA 0.27 +/- .02, P = .008). For ApoB, the three groups had similar serum ApoB levels: 129.9 +/- 37.7, 121 +/- 51, 120.7 +/- 57.4 mg/dL. Group I presented higher levels of ApoB mRNA than those of group III (0.68 +/- 0.21 vs. 0.41 +/- 0.18, P < .06). There was a significant correlation between serum and mRNA levels of ApoA-I (r = .65, P = .003) but no correlation between serum and mRNA levels of ApoB (r = .19, NS). We suggest that (1) steatosis is associated with increased ApoA-I mRNA; (2) fibrosis is associated with decreased serum ApoA-I, probably caused by a posttranscriptional mechanism; (3) severe alcohol-induced cirrhosis is associated with a nonspecific decrease in ApoA-I and ApoB mRNA; and (4) in contrast to ApoA-I mRNA, the ApoB mRNA level makes a slight contribution to the ApoB serum concentration.

Adult↗
PubMed1996

Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a prevalence of around 1 in 3500, affecting all ethnic groups. The clinical manifestations of the disease are variable, even among members of the same family, and affect a variety of tissues and cell types, including skin, iris, central and peripheral nervous systems and skeletal system. It has been reported that the majority of sporadic mutations in NF1 arise in paternally inherited alleles. We present here a collaborative study of the parental origin and type of mutation in individuals with de novo NF1, who account for up to a half of all cases of clinically diagnosed NF1. We have studied intragenic and extragenic markers in 470 NF1 families. In 32 of these families it was possible to assess the parental origin of a de novo NF1 mutation either by linkage analysis (in families with three generations) or by the detection of an intragenic deletion in a sporadic NF1 case. Eleven of these 32 families have three generations (the second and third generation being affected), with the mutation (not a large deletion) being of paternal origin in 82% of them (P < 0.05). In the other 21 families an intragenic deletion was detected, in 76% being in the maternal chromosome and in 24% in the paternal one (P < 0.05). Our results suggest that in NF1 the majority of deletions occur in oogenesis, while other types of mutations should account for the paternally derived NF1 mutations.

Chromosome Mapping↗
PubMed1996

Role of liver extracellular matrix in transcriptional and post-transcriptional regulation of apolipoprotein A-I by hepatocytes.

Apolipoprotein A-I, a protein produced mainly by hepatocytes, is of major importance in prevention of atherosclerosis. Its serum level varies according to the degree of liver fibrosis and the mechanism of this regulation is unknown. The aim of this study was to investigate the role of extracellular matrix in the regulation of apolipoprotein A-I by the liver. Primary mouse hepatocytes were cultured on different extracellular matrix components. The apolipoprotein A-I mRNA level was quantified in these different culture conditions by a sensitive quantitative RT-PCR procedure and compared according to the extracellular matrix component used as substrate. A significant decrease in the apolipoprotein A-I mRNA level was observed when cells were plated on fibronectin by comparison with cells cultured on all other components. Potential binding of apolipoprotein A-I to the different matrix components was also studied in vitro. We demonstrated that apolipoprotein A-I significantly bound to fibronectin in a concentration-dependent, saturable and specific manner. Thus, fibronectin, a major liver extracellular matrix component, can interact with apolipoprotein A-I both by downregulating its mRNA level in liver cells and by binding this molecule after its secretion in the extracellular space. Since fibronectin is the first matrix component to be produced in excess and deposited in liver fibrosis, it could be involved in the decrease in serum apolipoprotein A-I in alcoholic patients with liver fibrosis and cirrhosis.

Animals↗
PubMed1995

[Exclusive nodular plexiform neurofibroma. An unusual case of neurofibromatosis type 1].

INTRODUCTION: Type 1 neurofibromatous tumours (NF) are benign skin tumours which include cutaneous, subcutaneous and plexiform neurofibromas. Plexiform neurofibromas are either diffuse or nodular, the latter form being much more frequent. CASE REPORT: We observed a particular form of neurofibroma in an 18-year-old patient who developed large deep subcutaneous which histology examination revealed to be exclusively nodular plexiform neurofibromas. The patient also had 6 café au lait spots leading to the diagnosis of sporadic NF 1. He did not have acoustic neuronoma, schwannoma or posterior cataract, eliminating NF 2. COMMENTS: In NF 1, subcutaneous neurofibromas develop in 5 p. 100 of the patients. These lesions are termed nodular plexiform neurofibromas when they form long formations along nerve branches. The exclusive nature of the nodular plexiform neurofibromas in our case was exceptional. It could be hypothesized that the particular phenotype in our patient might correspond to a particular anomaly of the NF 1 gene.

Adolescent↗