Biomedical subjects

M Vidaud

Publications and source records attributed to M Vidaud.

At least 73 records · Page 4Linked to original sources

PubMed1999

Expression of growth hormone and its receptor in the placental and feto-maternal environment during early pregnancy in sheep.

In a previous study we showed the existence of GH in the ovine placenta. We now supplement the information available on placental GH and describe the presence and distribution of GH receptor (GH-R) messenger RNA (mRNA) in uterine, fetal, and placental tissues during early pregnancy. GH mRNA was not detected in the placenta before day 27 (d27). Its expression peaked between d40 and d45 and fell after d55. GH mRNA was localized in the trophectoderm and syncytium. During the d35-d50 period, concentrations of GH in the maternal circulation were not increased. In umbilical blood, however, GH was detected from d35 and was presumed to be of placental origin, because GH mRNA was not detected in the fetal pituitary gland on d40. We report on GH-R mRNA expression in the placenta between d20-d120. The relative abundance of GH-R transcripts increased significantly between d25-d43. In the endometrium, GH-R mRNA was detected from d8-d120 of pregnancy and from d4-d16 of the cycle. GH-R mRNA was localized in the trophectoderm, fetal mesoderm, and maternal uterine stroma. In the fetal liver, GH-R mRNA was first detectable on d35. The results of this study indicate that between d35-d50 of pregnancy, the endometrium, placenta, and fetus are all potential targets for the placental GH.

Allantois↗

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

Insulin-like 4 (INSL4) gene expression in human embryonic and trophoblastic tissues.

Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues.

Gene Expression Regulation, Developmental↗

PubMed1998

Slow progression rate of fibrosis in hepatitis C virus patients with persistently normal alanine transaminase activity.

In hepatitis C virus (HCV) patients with persistently normal alanine transaminase (ALT), the progression rate of fibrosis is unknown. The aims of this study were: 1) to compare HCV patients with normal ALT (group I) with HCV patients with elevated ALT (group II) matched on independent factors associated with fibrosis; and 2) to assess the progression rate of fibrosis. One hundred two HCV patients were included in each group. Histological lesions were staged using the METAVIR score. We defined fibrosis progression per year as the ratio of the fibrosis stage in METAVIR units to the duration of infection. In group I, ALT values were normal, and lower than in group II (25 vs. 127 IU/L; P < .0001). HCV RNA was present less frequently in group I (66% vs. 97%; P < .0001). There were no significant differences for viremia and genotypes. Histological activities were lower in group I (0.6 vs. 1.38; P < .0001). The stage of fibrosis was lower in group I (0.95 vs. 1.8; P < .001). The median progression rate of fibrosis was lower in group I (0.05 vs. 0.13; P < .001). In group I, after exclusion of negative HCV-RNA patients, the median progression rate of positives remained lower (0.05 vs. 0.13; P < .001). In group I, all cirrhotic patients (n = 3) were heavy drinkers. HCV patients with normal ALT showed weaker histological activity and lower fibrosis scores, and the progression rate of fibrosis was twice as slow as in HCV patients with elevated ALT. In these patients, severe fibrosis was associated with high alcohol consumption.

Adolescent↗

PubMed1998

Clonal analysis of macronodules in cirrhosis.

Several arguments suggest that most hepatocellular carcinomas (HCCs) occurring in human cirrhotic livers arise from large hepatocellular nodules or macronodules. Except for nodules with obvious features of HCC, there exist no consistent criteria enabling the differentiation between benign regenerative and neoplastic, potentially malignant macronodules. Surrogate markers able to accurately discriminate those lesions that will evolve toward a HCC are required. In this study, we investigated the clonality of 26 macronodules isolated from eight cases of explanted cirrhotic livers in women by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each macronodule, a large set of pathological features was evaluated and used to classify the macronodules into four groups: entirely benign-looking nodule (type 1), low-grade dysplastic nodule (type 2), high-grade dysplastic nodule (type 3), and HCC (type 4). Clonal analysis showed that 14 macronodules (54%) were monoclonal and 12 (46%) were polyclonal. Monoclonality was detected in 5 of 11 (45%) nodules from groups of entirely benign-looking and low-grade dysplastic nodules (types 1 and 2) and in 9 of 15 (60%) nodules from the group of high-grade dysplastic nodule and HCC (types 3 and 4). Neither the etiology of cirrhosis nor the size or histological classification of macronodules was correlated with the clonal status. In conclusion, clonal analysis of macronodules enables the differentiation between mono- and polyclonal macronodules in cirrhosis. Because monoclonal macronodules are prone to evolve to HCC, the determination of the clonal status of a macronodule could provide additional information for evaluating the prognosis of these lesions.

Adult↗

PubMed1998

Clonal analysis of renal sporadic angiomyolipomas.

Renal angiomyolipomas are benign tumors composed of variable amounts of mature fat, smooth muscle, and thick-walled blood vessels. They occur either sporadically or in association with tuberous sclerosis. Such tumors are considered as hamartomas, but few data are available concerning their pathogenesis. Indeed, it is not known whether angiomyolipoma is a congenital malformation or a neoplastic process. To answer this question, we assessed the clonality of sporadic angiomyolipomas using molecular analysis. Seven women (mean age, 59 years) with renal angiomyolipomas were included. DNA of the tumor and the normal adjacent kidney was extracted from archival paraffin-embedded tissue. DNA methylation pattern at a polymorphic site on the HUMARA gene was studied by polymerase chain reaction (PCR) amplification after methylation-sensitive enzyme digestion. This procedure enables the differentiation between polyclonal and monoclonal lesions according to their X-chromosome inactivation pattern. Five of the seven women included were informative for the HUMARA gene. The mean size of the angiomyolipomas was 53 mm (range, 18 to 110). In one case, a tumor thrombus was observed in the inferior vena cava. Clonal analysis showed that all of the angiomyolipomas and the tumor thrombus studied were monoclonal. This study shows that sporadic angiomyolipomas are monoclonal lesions consistent with neoplastic disorders. This result strongly supports the hypothesis that angiomyolipomas arise from the clonal proliferation of an uncommited cell, which will further evolve toward different cell types.

Adult↗

PubMed1998

The INSL4 gene maps close to WI-5527 at 9p24.1-->p23.3 clustered with two relaxin genes and outside the critical region for the monosomy 9p syndrome.

The insulin like growth factor 4 (INSL4) gene belongs to the insulin gene superfamily and has been mapped by fluorescent in situ hybridization to 9p24. Expression of INSL4, of unknown function, has been recently detected in the perichondrium and ligaments of the human embryo. Here we have mapped INSL4 within the framework of a partial YAC contig covering the distal part of 9p to find out whether this gene lies within the critical region defined for the monosomy 9p syndrome. The gene was also located using a human-rodent radiation hybrid panel. INSL4 was found to be distal to the del(9p) critical region and excluded as a candidate for the syndrome. In addition, the positions of two relaxin genes (RLN1, RLN2) that belong to the same superfamily as INSL4 and which have also been mapped to chromosome 9, were refined. We have shown that the three genes are clustered in the same region.

Animals↗

PubMed1998

Prognostic value of chorionic gonadotropin beta gene transcripts in human breast carcinoma.

The beta subunit of human chorionic gonadotropin is potentially encoded by six genes, which can be categorized into two types based on a sequence change at codon 117: GCC for the type I and GAC for the type II genes. We previously showed that, whereas type I genes were exclusively expressed in normal breast tissues, expression of type II genes was associated with malignant transformation (Bellet, D., et al. Cancer Res., 57: 516-523, 1997). We designed a simple and robust test (the CG117 assay) that measures the percentage of type II over both types of chorionic gonadotropin beta mRNAs. Normal breast tissues consistently had a negative CG117 index, whereas cancer breast tissues showed indexes ranging from 0 to 100%. The prognostic significance of the CG117 index was investigated in a series of 99 unilateral invasive primary breast cancer patients with known long-term outcome (median follow-up, 9 years). The CG117 index was positive in 48 (48.5%) of the 99 tumor mRNA samples. The index was not significantly associated with standard prognostic parameters, including clinical and macroscopic tumor size, histopathological grade, and lymph node status or steroid receptor status. Patients with a positive CG117 index in primary tumor mRNA had significantly shorter metastasis-free survival (P = 0.014) and overall survival (P = 0.038) after surgery, compared to patients with a negative index. The prognostic significance of the CG117 index persisted in Cox multivariate regression analysis, both for metastasis-free survival (P = 0.008) and overall survival (P = 0.016), together with lymph node status (P = 0.027 and P = 0.009, respectively). These findings indicate that the CG117 index may contribute to the identification of high-risk breast cancer patients.

Adult↗

PubMed1997

Malignant transformation of nontrophoblastic cells is associated with the expression of chorionic gonadotropin beta genes normally transcribed in trophoblastic cells.

The beta subunit of human chorionic gonadotropin (hCGbeta) is encoded by four nonallelic CGbeta genes. An assay was developed for distinguishing type I CGbeta allelic genes beta7 and beta6, which possess a GCC codon corresponding to an alanine at position 117 of hCGbeta, from type II CGbeta genes beta8, beta5, and beta3 and its allele beta9, which possess a GAC codon corresponding to an aspartic acid at the same position. In normal trophoblast, hCGbeta is encoded by type II CGbeta genes, whereas normal nontrophoblastic tissues of differing histological origin (breast, prostate, skeletal muscle, bladder, adrenal glands, thyroid, colon, and uterus) express only type I CGbeta genes. We studied the expression of CGbeta genes in 86 tumor specimens collected from patients with breast, bladder, prostate, and thyroid cancer and found that up to 61% of these nontrophoblastic tumors expressed type II CGbeta genes. Experiments performed on tumor tissues and their normal counterparts confirmed that the malignant transformation of nontrophoblastic cells is associated with the expression of type II CGbeta genes. These findings provide the basis for a simple test (the CG117 assay) that may be useful for the diagnosis of the most frequent malignancies.

Cell Line↗

PubMed1997

[Mechanisms and consequences of genetic mutations].

Identification of mutations responsible for human monogenic hereditary diseases has lately undergone rapid development. It is now possible to establish the mutation spectrum for a given genetic disease and international databases are now available on the Internet. New mutational mechanisms have been identified:transposition, inversion, trinucleotide repeat expansion. At the disease level, knowledge of molecular defects has allowed to better delimit mechanisms of gain or loss of function, to understand dominant or recessive mode of inheritance, to explain anticipation phenomena, to identify founder effect amid a selected population, to establish, in some privileged cases, phenotype-genotype correlations and above all to elicit allelic and genetic heterogeneity hitherto unsuspected. Identification of deleterious mutations is also the basis of diagnosis at the gene level which today has a growing impact on genetic counselling and prenatal diagnosis.

Alleles↗

PubMed1997

Evidence for the polyclonal nature of focal nodular hyperplasia of the liver by the study of X-chromosome inactivation.

Focal nodular hyperplasia (FNH) of the liver is a benign tumor commonly considered as a reactive disorder related to a pre-existing vascular malformation. However, the pathogenesis of this lesion has been recently discussed. To determine whether FNH is a polyclonal or a clonal lesion, we investigated the inactivation pattern of the X chromosome, using molecular genetic analysis of the DNA methylation pattern at a polymorphic site on the human androgen receptor gene (HUMARA). Fifteen FNH were studied, and results were compared with those obtained from 7 hepatic adenomas (HA) and 2 hepatocellular carcinomas (HCC). DNA was extracted from both lesional and nonlesional livers, fixed, and paraffin-embedded. To assess the methylation pattern, we used a quantitative fluorescent polymerase chain reaction (PCR) procedure that allows for the accurate measurement of the peak intensities of each allele. Three patients were noninformative because they were homozygous at the HUMARA locus. According to the threshold for monoclonality established by a titration curve, all FNH showed a random pattern of X-chromosome inactivation consistent with a polyclonal lesion. In contrast, all but 1 hepatic adenoma and all hepatocellular carcinomas were clonal, as shown by the nonrandom pattern of X-chromosome inactivation observed in these cases. In conclusion, these results suggest that FNH should be considered as a reactive disorder rather than as a tumoral proliferation. Discordant results recently observed in the literature could be at least in part explained by methodological differences in the PCR procedure.

Adenoma↗

PubMed1997

Microsatellite in the beta-tubulin gene of Toxoplasma gondii as a new genetic marker for use in direct screening of amniotic fluids.

To examine the correlation between Toxoplasma gondii genotype and congenital human toxoplasmosis, the polymorphism of the microsatellite consisting of a dinucleotide (TG) repeat in the intron of the beta-tubulin gene was investigated by PCR. Thirty-four reference strains were studied, including 7 strains virulent in mice and 27 strains avirulent in mice. The seven virulent strains had a (TG)8 microsatellite, and the avirulent strains had a (TG)7 microsatellite. This confirms the dichotomy already observed for virulent and avirulent strains. Additionally, 37 samples of amniotic fluid from infected fetuses were tested. All of them had the (TG)7 microsatellite marker. This result confirms that most of the human cases of congenital toxoplasmosis are due to strains avirulent in mice. Nevertheless, their virulence in human fetuses was obvious, as numerous abnormalities were observed on ultrasonic examination. The new genetic marker is the first one directly used for typing T. gondii isolates without any bias due to cultivation of the parasite. This microsatellite marker is not sufficient to type the strains which are avirulent in mice; however, seeking more polymorphic microsatellites should be worthwhile to obtain new genetic markers for direct screening of biological samples.

Amniotic Fluid↗

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↗

PubMed1997

Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.

Animals↗