Variation of iron loading expression in C282Y homozygous haemochromatosis probands and sib pairs.
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Adult↗
Biomedical subjects
C Férec
Publications and source records attributed to C Férec.
Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC.
BACKGROUND: Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutations, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and further defined whether HC probands with an incomplete HFE genotype carry uncommon mutations. METHODS: Analytical conditions for each coding exon were determined by a combination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and optimize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA samples harboring the C282Y, H63D, or S65C mutations and 17 artificially created positive controls that carried either 1 of the 14 other known HFE mutations or 3 selected polymorphisms. RESULTS: Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more complex melting profile and required two analysis temperatures. DHPLC detected all known HFE mutations as well as the three selected polymorphisms. CONCLUSIONS: DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation.
Chromatography, High Pressure Liquid↗
Cation substitution in cationic phosphonolipids: a new concept to improve transfection activity and decrease cellular toxicity.
Cationic lipids have been shown to be an interesting alternative to viral vector-mediated gene delivery into in vitro and in vivo model applications. Prior studies have demonstrated that even minor structural modifications of the lipid hydrophobic domain or of the lipid polar domain result in significant changes in gene delivery efficiency. Previously, we developed a novel class of cationic lipids called cationic phosphonolipids and described the ability of these vectors to transfer DNA into different cell lines and in vivo. Up until now, in all new cationic lipids, nitrogen atoms have always carried the cationic or polycationic charge. Recently we have developed a new series of cationic phosphonolipids characterized by a cationic charge carried by a phosphorus or arsenic atom. In a second step, we have also examined the effects of the linker length between the cation and the hydrophobic domain as regards transfection activity. Transfection activities of this library of new cationic phosphonolipids were studied in vitro in different cell lines (HeLa, CFT1, K562) and in vivo using a luciferase reporter gene. A luminescent assay was carried out to assess luciferase expression. We demonstrated that cation substitution on the polar domain of cationic phosphonolipids (N --> P or As) results in significant increase in transfection activity for both in vitro and in vivo assays and decrease of cellular toxicity.
Alanine Transaminase↗
Neonatal screening for cystic fibrosis in Brittany, France: assessment of 10 years' experience and impact on prenatal diagnosis.
BACKGROUND: Neonatal screening for cystic fibrosis has been a subject of debate over the past few years. This study assesses 10 years of neonatal screening in Brittany, France, and examines its impact on prenatal screening of subsequent pregnancies in couples with an affected child. METHODS: The study included all the neonates screened for cystic fibrosis in Brittany from Jan 1, 1989, to Dec 31, 1998. The screening consisted of an immunoreactive trypsinogen assay from dried blood spots, plus, from 1993, mutation analysis. Data were collected on incidence of cystic fibrosis, and genotypic and biochemical characteristics. The use of prenatal screening of subsequent pregnancies in affected families was also investigated. FINDINGS: Of the 343,756 neonates screened, 118 children with cystic fibrosis were identified, giving an incidence of one in 2913. All mutated alleles were characterised: 34 different mutations resulting in 36 genotypes were detected. The introduction of DNA analysis into the protocol greatly reduced the recall rate and increased the sensitivity of the test. The mean cost of the screening programme was US$2.32 per screened child. 39 (34%) of the families identified by neonatal screening opted for subsequent prenatal diagnosis at least once. 12 couples would have benefited from this procedure while their first child was still symptom-free. 42 healthy children were born, and 18 pregnancies were terminated (therapeutic abortion rate of 100%). INTERPRETATION: We have shown the feasibility of neonatal screening for cystic fibrosis in Brittany. Through the detection of a large range of mutations, neonatal screening provides the opportunity for more reliable prenatal diagnosis and cascade screening. The neonatal screening programme described here could provide a good model for other countries intending to initiate such a scheme.
Cystic Fibrosis↗
Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency.
Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.
Animals↗
Cationic phosphonolipids as nonviral vectors: in vitro and in vivo applications.
Since the development of the concept of gene therapy using cationic lipids as nonviral vectors by Felgner's group in 1987, numerous molecules have been synthesized. Such vectors were first proposed to avoid viral vector-induced drawbacks. But, it quickly became clear that a thorough knowledge of their physical and chemical characteristics was fundamental to use them under optima conditions. Over the last years our laboratory has developed a family of cationic lipids called phosphonolipids whose structure is based on that of natural phosphonolipids; compared with other vectors, these compounds had to be well-tolerated by biologic membranes. Some of our synthesized molecules exhibited an interesting potential for gene transfer, both in vitro and in vivo. Structural changes in the different parts (hydrophobic, hydrophilic, and intermediary domains) of these vectors were evaluated in vitro on different cell-lines; these studies led us to select some of these molecules to carry out in vivo tests. So, the plasmid/phosphonolipid complexes were first administered to mice by intratracheal and aerosol routes with a beta-galactosidase plasmid as reporter gene. In a second set of experiments, we explored the possibilities offered by intravenous injection; in these studies, we used a luciferase plasmid as reporter gene because of its high sensibility. These experiments revealed a transgene expression essentially localized in the lungs. In a further study, we compared systemic administration with local ones; we, then, observed that the optimum formulation of a given molecule depended on its route of administration.
Aerosols↗
Phosphonocationic lipids in protein delivery to mice lungs.
Cationic liposomes constitute one of the main approaches currently investigated to introduce a gene with therapeutic properties into a cell. Another alternative consists in directly introducing the normal protein of concern to, for example, restore the deleted function. We report here on in vitro and in vivo results obtained with GLB73, one of the phosphonolipids investigated as gene transfer agents. In previous studies this cationic lipid had shown its DNA-transfer efficacy in vitro and in vivo. We also confirmed the feasibility of protein/cationic lipid delivery in epithelial cells of mice lungs after intratracheal administration by use of a reporter gene (beta-galactosidase). Two quantitative tests (i.e., a chemiluminescent assay and a flow cytometry assay) were used to determine the amount of beta-galactosidase found in the lungs and the percentage of transfected cells. They showed that 50% of the cells of mice lungs were still positive at day 4 after protein/GLB73 delivery. Immunohistochemistry and electron microscopy studies allowed us to determine the spatial distribution and visualize the penetration of our complex into the lungs.
Animals↗
Clinical and molecular aspects of juvenile hemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, canada).
We report the clinical, biochemical, and genetic characteristics of 13 hemochromatosis patients from Saguenay-Lac-Saint-Jean in whom the first symptoms appeared before age 30. Although the mean age at onset of the first symptoms was 21. 5 years, their mean age at diagnosis was 23.8 years; the diagnosis was particularly delayed among women. Seventy-seven percent of the patients had hypogonadotrophic hypogonadism and 69% heart failure and/or cardiac arrhythmias. Genetic analysis of the HFE gene revealed heterozygosity for the C282Y mutation in 2 patients and for the S65C mutation in 2 others and homozygosity for the H63D mutation in 1 patient. The remaining 8 patients had no identified mutation in the HFE gene, although sequencing of all seven codons and intron-exon junctions was performed (5 patients). All 13 patients fulfill the clinical criteria of juvenile hemochromatosis and represent the largest cluster thus far reported.
Adolescent↗
Nramp2 analysis in hemochromatosis probands.
The mechanism that leads to iron overload in hereditary hemochromatosis is not yet fully understood and genes other than HFE may be involved. Nramp2 is an intestinal iron transporter, upregulated by dietary iron deficiency, which also colocalizes with transferrin in recycling endosomes. The purpose of the present study was to analyze the coding region of the Nramp2 gene in 14 hemochromatosis probands which did not carry any HFE mutations on both chromosomes. We confirmed the existence of a polymorphism (1254 T --> C), which presumably is not associated with hereditary hemochromatosis, but we did not find any mutation. On the other hand, we identified 17 splice variants of the Nramp2 mRNA. Eight corresponded to activation of cryptic splicing sequences between exons 3 and 4. They were observed in a majority of hemochromatosis probands and control subjects. This indicates the existence of an important splicing instability in this region. At this stage, the biological significance of these variants is unclear. Our study did not find evidence for the involvement of the Nramp2 gene in hereditary hemochromatosis. The remaining question is whether hemochromatosis probands in our study have iron overload because of environmental factors or due to mutation in gene(s) other than HFE and Nramp2.
Alternative Splicing↗
Relation between HFE mutations and mild iron-overload expression.
The identification of the HFE gene involved in hemochromatosis allows genetic tests based on mutation analysis to be performed. However, discrepancies in the correlation between HFE genotypes and iron-loading status have arisen. We investigated 708 patients with various signs or symptoms suggesting a putative iron overload that, nevertheless, did not reach the current criteria for hemochromatosis diagnosis. Most of the patients (91.4%) included in our study displayed one of three classical iron marker values above the threshold defined for iron overloading. HFE mutation analysis allowed us to identify 45.7% of carrier chromosomes in the studied group of patients that showed higher frequencies of HFE mutations compared with controls. In addition, the frequencies of compound C282Y/H63D heterozygous, H63D/H63D homozygous, and C282Y heterozygous genotypes were higher than those in HH probands and controls; they accounted for 16, 5.6, and 22.5% of the patients, respectively. All genotypic groups had a significantly higher value of serum ferritin concentration compared to the normal value; only the C282Y homozygotes and compound heterozygotes with H63D had a transferrin saturation significantly higher than the normal value. On the whole the H63D homozygous and compound heterozygous patients constitute an intermediate phenotypic group between HH and controls. Some of them may reach the critical overloading defined for HH diagnosis along with a potential risk of developing complications, whereas others only show a partial phenotypic expression.
Adult↗
Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay.
The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.
Flaviviridae↗
Mutation analysis in the HFE gene in patients with hereditary haemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, Canada).
A mutation analysis of the HFE gene followed, when applicable, by sequencing was performed on 47 patients with hereditary haemochromatosis (HH) living in Saguenay-Lac-Saint-Jean. The C282Y and H63D mutations were present on 50% and 20.3% of the HH chromosomes respectively. These frequencies were very different from those found in other populations and could be, at least partially, the result of a founder effect. No new mutation was identified among the remaining 28.1% of the HH chromosomes. Five of the eight probands with no mutation in the HFE gene had a severe and early onset suggestive of juvenile haemochromatosis.
Alleles↗
High rate of GB virus type C/HGV transmission from mother to infant: possible implications for the prevalence of infection in blood donors.
BACKGROUND: Because GB virus type C(GBV-C)/HGV (GBV-C/HGV) is blood-borne and sexually transmitted, persons at risk of infection with such viruses have a high prevalence of GBV-C/HGV markers. However, adults with no apparent risk factors, such as blood donors, frequently are positive for GBV-C/HGV markers. Mother-to-infant transmission could explain this high prevalence, but it has been studied only through small series of GBV-C/HGV-infected mothers co-infected with HCV or HIV. STUDY DESIGN AND METHODS: To determine the rate of mother-to-infant transmission of GBV-C/HGV RNA in women who are HCV- or HIV-negative, a prospective study was performed in a cohort of 288 mothers screened for viral RNA and in the infants born to GBV-C/HGV-infected mothers. RESULTS: Thirteen mothers (4.5%) were found positive for GBV-C/HGV RNA. Of the infants in whom at least one blood sample was collected between the third and the ninth months of life, 89 percent were positive for viral RNA. The majority of these newborns were negative for GBV-C/HGV RNA at birth and positive after the third month. The viral RNA titers of infants born to GBV-C/HGV-infected mothers appeared as elevated as those of their mothers. All the GBV-C/HGV-infected infants remained positive for viral RNA during the entire study period. No clinical events possibly linked to a primary GBV-C/HGV infection were reported in infants. Serum ALT level and blood count remained within normal values throughout the follow-up of all GBV-C/HGV-infected infants. CONCLUSION: The frequency of mother-to-infant GBV-C/HGV transmission is elevated and could explain the high prevalence of GBV-C/HGV markers (viral RNA and E2 antibody) in adults at low risk for blood-borne or sexually transmitted viruses, such as blood donors.
Adult↗
Correlation between mutations and age in cystic fibrosis in a French Canadian population.
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Age Factors↗
Cationic Phosphonolipids Containing Quaternary Phosphonium and Arsonium Groups for DNA Transfection with Good Efficiency and Low Cellular Toxicity**
Replacing the ammonium polar head in cationic lipids 1 (A=N) by a phosphonium or an arsonium group (A=P, As) improves their properties as synthetic vectors for DNA transfection. The increased volume of the cationic head is supposed to modify the interactions of the vector with the solvent and DNA.
Journal Article↗
Myocardial infarction: absence of association with VNTR polymorphism of GP Ibalpha.
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Humans↗
HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.
Hereditary hemochromatosis (HH) is a common autosomal recessive genetic disorder of iron metabolism. The HFE candidate gene encoding an HLA class I-like protein involved in HH was identified in 1996. Two missense mutations have been described: C282Y, accounting for 80% to 90% of HH chromosomes, and H63D, which is associated with a milder form of the disease representing 40% to 70% of non-C282Y HH chromosomes. We report here on the analysis of C282Y, H63D, and the 193A-->T substitution leading to the S65C missense substitution in a large series of probands and controls. The results confirm that the C282Y substitution was the main mutation involved in hemochromatosis, accounting for 85% of carrier chromosomes, whereas the H63D substitution represented 39% of the HH chromosomes that did not carry the C282Y mutation. In addition, our screening showed that the S65C substitution was significantly enriched in probands with at least one chromosome without an assigned mutation. This substitution accounted for 7.8% of HH chromosomes that were neither C282Y nor H63D. This enrichment of S65C among HH chromosomes suggests that the S65C substitution is associated with the mild form of hemochromatosis.
Amino Acid Substitution↗
GBV-C/hepatitis G virus (HGV) RNA load in immunodeficient individuals and in immunocompetent individuals.
The aim of this study was to establish the mean plasma GBV-C/hepatitis G virus (HGV) RNA load in groups of GBV-C/HGV-infected individuals with varied immune status and to determine the most frequent patterns of evolution of the plasma GBV-C/HGV RNA load over time during the natural history of infection. The mean plasma GBV-C/HGV RNA load observed was, from the lowest to the highest: 5.21 log in immunodepressed multiply-transfused patients, 6.45 log in HIV-positive individuals, 6.66 log in immunocompetent multiply-transfused patients, and 6.71 log in blood donors. The difference was significant between the four groups (P < 0.0001). The most frequent pattern of evolution of the plasma GBV-C/HGV RNA load was as follows: after the primary GBV-C/HGV infection, the viral load was elevated from the onset; then, a high, persistent and relatively steady viral RNA level was the rule; and when it occurred, the loss of viremia was not preceded by a decrease before recovery from GBV-C/HGV infection.
Adolescent↗