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Biomedical subjects

C Férec

Publications and source records attributed to C Férec.

At least 55 records · Page 3Linked to original sources
PubMed1999

Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay.

The risk of contracting hepatitis from blood transfusions is estimated to be 1 in 63000 in the case of hepatitis B virus (HBV) and 1 in 103000 in the case of hepatitis C virus (HCV). In some countries (Germany, USA and England, for example), molecular protocols are evaluated to detect viral genomes in blood donations in order to reduce the seroconversion period. However, no such method is available currently to screen large series samples for HBV and HCV. While strategies involving the pooling of plasma samples have been proposed and tested in Germany, there is the question of sensitivity. We developed a novel approach to screen for HBV and HCV based on the TaqMan technology that allows for the quantification of an amplified fragment during PCR analysis (Lee et al., 1993). This approach is more sensitive than other quantification methods. As a first step primers and probes were designed to detect the different sub-types of HBV and HCV genomes. We then optimized the reaction conditions in order to screen for the two viruses at the same time. The observed sensitivity is less than 50 molecules per ml for HBV and less than 50 molecules per ml for HCV. This assay is, to our knowledge, the first that allows the simultaneous detection of DNA and RNA viral genomes. In conclusion, this TaqMan approach could be used as a single test to screen for HBV and HCV genomes in a series of 96 samples in less than 5 h. Such an approach is a first step for development of automation allowing a systematic screening of blood donations.

Blood Donors↗
PubMed1999

Screening for HBV, HCV and HIV genomes in blood donations: shortcomings of pooling revealed by a multicentre study simulating real-time testing.

This study was undertaken in order to determine whether screening of viremic blood donations by testing of pooled donor samples could constitute a technically feasible transfusional safety measure. A pilot study of real-time simulation, on a day-to-day basis, of screening of three viral genomes (hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) was conducted by five French Blood Centers on plasma samples collected from blood donors and studied within undiluted samples and within sample pools of various sizes. This study was carried out within time conditions compatible with the release of platelets. For the detection of HCV and HIV genomes, the five laboratories achieved a sensitivity that decreased with the size of the sample pool. Four were successful in detecting all undiluted samples. In the 1/10 diluted samples, four failed to detect one HIV or HCV sample. In the 1/100 diluted samples, all laboratories failed to detect one or more HIV or HCV samples. For HBV genome, no participating laboratories detected all of the samples of the panel, even undiluted samples, and the sample pooling considerably affected sensitivity. The improvement and standardization of assays needs to be attained, and training of laboratories appears to be a step crucial for routine screening of viral genomes in blood donations.

Blood↗
PubMed1999

Blood immunoreactive trypsinogen concentrations are genetically determined in healthy and cystic fibrosis newborns.

Newborns with cystic fibrosis (CF) have increased blood immunoreactive trypsinogen concentrations. When screening for CF in the newborn by immunoreactive trypsinogen measurement, an abnormally high proportion of healthy deltaF508 carriers is found among false-positive neonates, suggesting that a relationship could exist between immunoreactive trypsinogen concentration at birth and the genetic status. Therefore, this study analysed the possible relationships between neonatal blood immunoreactive trypsinogen concentrations and genotype in 1842 healthy newborns and 111 CF patients detected by a neonatal screening programme. A close correlation was found between immunoreactive trypsinogen and deltaF508: the probability of a healthy newborn being a carrier of this mutation increased regularly with the neonatal immunoreactive trypsinogen concentration. In CF patients, there was a significant difference between deltaF508 homozygotes and deltaF508/X (X = other mutation) compound heterozygotes with respect to the mean neonatal blood immunoreactive trypsinogen concentration. CF neonates with two mutations affecting the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator protein had significantly higher mean immunoreactive trypsinogen concentrations than patients with one mutation affecting a membrane-spanning domain. The data strongly suggest that the neonatal immunoreactive trypsinogen concentration is, in part, genetically determined, with a wide range of variations, similar to the features which have been shown for the relations between the genotype and clinical phenotypes of CF patients.

Case-Control Studies↗
PubMed1999

Blood concentrations of pancreatitis associated protein in neonates: relevance to neonatal screening for cystic fibrosis.

AIM: To determine whether pancreatitis associated protein (PAP) is a marker for cystic fibrosis which could be used in neonatal screening for the disease. METHODS: PAP was assayed on screening cards from 202,807 neonates. Babies with PAP > or = 15 ng/ml, or > or = 11.5 ng/ml and immunoreactive trypsinogen (IRT) > or = 700 ng/ml were recalled for clinical examination, sweat testing, and cystic fibrosis transmembrane regulator (CFTR) gene analysis. RESULTS: Median PAP value was 2.8 ng/ml. Forty four cases of cystic fibrosis were recorded. Recalled neonates (n = 398) included only 11 carriers. A receiver operating characteristic curve analysis showed that PAP above 8.0 ng/ml would select 0.76% of babies, including all those with cystic fibrosis, except for one with meconium ileus and two with mild CFTR mutations. Screening 27,146 babies with both PAP and IRT showed that only 0.12% had PAP > 8.0 ng/ml and IRT > 700 ng/ml, including all cases of cystic fibrosis. CONCLUSION: PAP is increased in most neonates with cystic fibrosis and could be used for CF screening. Its combination with IRT looks promising.

Acute-Phase Proteins↗
PubMed1999

Prevalence of GBV C/HGV RNA and GBV C/HGV antibodies in French volunteer blood donors: results of a collaborative study.

BACKGROUND AND OBJECTIVES: Posttransfusion hepatitis still occurs at an incidence of about 1 in 118,000 for HBV and 1 in 220,000 for HCV. This collaborative study aimed to determine the prevalence of a novel flavivirus, GBV-C/HGV, even though its role in transfusion-associated hepatitis is uncertain. MATERIALS AND METHODS: GBV-C/HGV RNA was detected by PCR using either the Boehringer detection kit or by primers previously described. HGV antibodies were detected by a serological assay from Boehringer. RESULTS: The observed GBV-C/HGV RNA frequency was 3.4%. HGV antibodies occurred in 9.5% of donors. CONCLUSION: In our study, 12. 9% of the donors had been in contact with the GBV-C/HGV virus.

Adolescent↗
PubMed1999

Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis.

Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion -28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.

Cations↗
PubMed1998

Cationic phosphonolipids as nonviral gene transfer agents in the lungs of mice.

With the aim of developing new gene transfer tools for treating CF with gene therapy, we have synthesized a novel family of molecules named cationic phosphonolipids. The most efficient among them were selected by in vitro screening to compare their activities in vivo in mouse lungs. We used a reporter gene whose activity was measured cytofluorimetrically (FACS-Gal assay) and by means of a chemiluminescence technique. These tests allowed us to identify the percentage of transfected cells and to quantify total beta-galactosidase in the lungs. This enabled us to identify two molecules, significantly efficient in comparison with DNA alone: GLB73 (p = 0.0015) and GLB253 (p = 0.007). Their use resulted in a time lag between transfection and maximum efficiency: maximum efficiency was observed 4 days after transfection with GLB73, whereas it was noticeable only on day 7 with GLB253. Moreover, from toxicity studies carried out in vivo, GLB73 seems to be nontoxic. In vivo results were correlated with in vitro results obtained with CF epithelial cell lines. Consequently, GLB73 is a potential candidate for phase I clinical trials in humans.

Animals↗
PubMed1998

Complete identification of cystic fibrosis transmembrane conductance regulator mutations in the CF population of Saguenay Lac-Saint-Jean (Quebec, Canada).

Over the past few years, we have conducted a systematic study of 230 cystic fibrosis (CF) chromosomes in the Saguenay Lac-Saint-Jean (SLSJ) population which has a high CF incidence (1/936 live births). We identified 11 mutations accounting for 100% of the CF chromosomes found in patients born in SLSJ. Our results indicate that denaturing gradient gel electrophoresis (DGGE) is a powerful method of identifying CF mutations. They have also considerable implications for genetic counselling and molecular characterization of doubtful patients. They make carrier screening technically feasible in this population.

Cystic Fibrosis↗
PubMed1998

Screening blood donations for viral genomes: multicenter study of real-time simulation using pooled samples on the model of hepatitis C virus RNA detection.

BACKGROUND: The systematic screening for several blood-borne viral genomes in blood donations is a complementary safety measure planned or discussed by the national authorities of several countries. STUDY DESIGN AND METHODS: To appreciate the feasibility of such screening using pooled samples, a multicenter study of real-time simulation has been performed on the model of hepatitis C virus (HCV) infection. Four blood transfusion center laboratories and two research and diagnosis laboratories simultaneously screened, by several HCV RNA polymerase chain reaction assays, a panel of plasma sample pools of different sizes (500, 100, and 10 samples), collected from HCV-infected or HCV-uninfected blood donors. One viremic plasma was introduced in each pool. HCV RNA was detected by in-house polymerase chain reaction procedures or by standardized manual or semi-automated polymerase chain reaction assays. RESULTS: The results indicate the feasibility of sample pooling, which renders the screening for viral genomes by molecular biology techniques applicable in the short term and the importance of the experience of laboratory personnel in the use of molecular biology tools. CONCLUSION: The improvement of standardized assays needs to be continued, and training of laboratory staff members appears to be a crucial step before systematic screening of blood donations for viral genomes by molecular biology techniques can occur.

Blood Donors↗
PubMed1998

Absence of mutations in the interspecies conserved regions of the CFTR promoter region in cystic fibrosis (CF) and CF related patients.

This study was aimed at testing if a 5.2 kb untranslated region on both sides of the first CFTR exon, shown to contain regulatory elements, could carry mutations responsible for cystic fibrosis (CF) or CF related phenotypes. Selection of the DNA segments studied within this region was based upon the identification of conserved sequences throughout evolution (phylogenetic footprints, PFs). Comparison of the CFTR sequences in eight species representing four orders of mammals (man, gibbon, rhesus monkey, squirrel, monkey, rabbit, cow, rat, and mouse) identified four clusters of PFs within the 3.9 kb of DNA sequence upstream from the initiation codon, as well as two nearby PFs at +1 kb within intron 1. Six DNA segments containing PFs were scanned for mutations by denaturing gradient gel electrophoresis (DGGE) in patients with CF (n = 29), congenital bilateral absence of the vas deferens (n = 143), or disseminated bronchiectasis (n = 33), for whom only one or no mutations had been identified despite extensive DGGE analysis of the 27 CFTR exons and exon/intron boundaries. Only one polymorphism (-966 T-->G) was identified with a frequency of 2.2% and no other sequence variations were found. This study reinforces the idea that the promoter region in the CFTR is not frequently mutated.

Animals↗
PubMed1998

Prevalence of 20210 A allele of the prothrombin gene in venous thromboembolism patients.

BACKGROUND: The 20210 A allele variation in the 3' -untranslated region of the prothrombin gene was recently identified as a risk factor as regards deep venous thrombosis. AIM: To assess the frequency of the variation in unselected patients with a proven venous thromboembolism (VTE). METHODS: The presence of the prothrombin variation was determined in all consecutive patients referred from July 1994 to August 1997 for a clinical suspicion of VTE, and in whom the diagnosis was confirmed. A control group consisted of bone marrow volunteer donors. RESULTS: Of the 366 patients included, 17 (4.6%) were carriers of the 20210 A allele (95% CI, 2.4% to 6.7%). The mutation was present in 1.0% of the 400 controls. Odds ratio for having VTE in the presence of the 20210 A allele was 4.8 (95% CI, 1.5 to 19.8). Forty-six (12.5%) patients had the mutation of the factor V gene and five (1.4%) patients shared both mutations. After excluding the carriers of the factor V mutation, odds ratio for having VTE in the presence of the 20210 A allele was 3.7 (95% CI, 1.1 to 13.6). Mean age at admission as well as mean age of the first VTE episode were both significantly higher in patients free from the two mutations studied, as compared to carriers of the 20210 A allele (p = 0.04 and 0.01, respectively). CONCLUSION: Our findings in a large series of patients (1) confirm the 20210 A allele prothrombin gene as a risk factor for VTE. (2) suggest that its association with the factor V Leiden is not uncommon.

Adult↗
PubMed1997

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene.

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.

Alleles↗
PubMed1997

Cationic phosphonolipids as non viral vectors for DNA transfection in hematopoietic cell lines and CD34+ cells.

The ability to transfer genes into a hematopoietic stem cell and to achieve regulation of their expression in lymphoid or myeloid lineages should open many new therapeutic opportunities. Besides gene transfer mediated by virus vectors like retrovirus or adenovirus, non viral systems have the theoretical advantage of being safe and easy to manage. We developed a new family of cationic lipids called phosphonolipids, synthesized 24 new molecules, and then in a first step we tested their potential to transfer genes in human hematopoietic cell lines (K562 and TF1). A LacZ plasmid under the control of a strong viral promoter was used as a reporter gene and a FACS-Gal assay and a quantitative test CPRG assay evaluated the beta gal expression. The targeted cells were analyzed 48 hours after transfection. The present work shows that seven novel molecules display a high transfer efficiency. One of them is nine-fold more efficient than the commercially available cationic lipids. The results obtained ex vivo on CD34 cells with the FACS-Gal assay show that at day 10 after transfection, 45 percent of cells are expressing gal.

Antigens, CD34↗
PubMed1997

Factor V Leiden prevalence in venous thromboembolism patients.

BACKGROUND: Recent findings have demonstrated a high frequency of activated protein C resistance in patients suffering from deep venous thrombosis (DVT). This abnormality has been related to a mutation in the factor V gene (at nucleotide position 1,691, guanine to adenine [G-->A] substitution). AIM: To assess the frequency of the mutation in unselected inpatients with a proved DVT. To study the clinical characteristics of such patients. METHODS: All consecutive patients admitted to the hospital because of a clinical suspicion of DVT were eligible. Diagnosis of DVT with the help of venous ultrasound imaging or venography. Ventilation and perfusion lung scan was performed in all patients, and interpreted according to the Prospective Investigation of Pulmonary Embolism Diagnosis criteria; in patients with a low- or intermediate-probability lung scan, pulmonary angiography was requested. Polymerase chain reaction amplification was performed in patients with a proved DVT. A control group consisted of bone marrow volunteer donors. RESULTS: From July 1994 to November 1995, 165 patients were included. Thrombosis was considered as distal in 77 and proximal in 88; an associated pulmonary embolism (PE) was found in 75 patients. Of 165 patients, 24 (14.5%) showed the factor V gene mutation (95% confidence interval, 9.4 to 19.8); the mutation was present in 3.5% of 200 bone marrow volunteer donors; odds ratio for having DVT in the presence of the mutation was 4.1. No difference in the level of DVT, or the presence of an associated PE was observed according to the presence of the mutation. Patients with the mutation have a significantly more frequent history of DVT (p=0.04) and more previous reported episodes (1.1 vs 0.6; p=0.04). CONCLUSION: The factor V mutation is frequent in unselected DVT patients. No difference in the severity of the thrombosis episode was observed in these patients.

Adult↗
PubMed1997

Clinical features of cystic fibrosis patients with rare genotypes in Saguenay Lac-Saint-Jean (Quebec, Canada).

We describe the clinical features of six cystic fibrosis (CF) patients from Saguenay Lac-Saint-Jean who bear rare genotypes. Two patients with a delta F508/I148T genotype had pancreatic insufficiency, as did two patients compound heterozygous for the 621 + 1G-->T mutation who also had a major growth retardation. One CF adult who carried a delta F508/Q890X genotype had meconium ileus and bronchiectasis. The sixth patient (A455E/R117C) had borderline sweat chloride concentrations; the diagnosis of cystic fibrosis had remained doubtful until the molecular analysis showed the presence of two CF mutations. The seventh patient with a delta F508/R1158X genotype experienced several complications and is now 43 years old.

Adult↗
PubMed1996

Molecular analysis of the TP53 gene in Barrett's adenocarcinoma.

The TP53 gene is the most frequently mutated gene in human cancers. Barrett's esophagus provides an excellent model by which to understand the genetic events that lead from dysplasia to cancer. We screened for mutations in the TP53 gene by a combination of denaturing gradient gel electrophoresis and DNA sequencing in ten cases of adenocarcinoma arising in Barrett's mucosa. We have identified missense mutations in five of the ten samples, three transitions (R282W, G245S, R248W) and two transversions (E286Q and C176F). In one case we have analyzed biopsy specimens taken from the same site, one year before the patient developed an intra mucosal carcinoma. The mutation that was identified in this high grade dysplastic area was identical to that detected in the cancer. This would suggest TP53 mutations occur as an early genetic event in the development of Barrett's adenocarcinoma.

Adenocarcinoma↗
PubMed1996

Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers.

We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, delta F508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A-->G, R1162X, and 3849 + 10kbC-->T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

Cystic Fibrosis↗