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Biomedical subjects

T Bienvenu

Publications and source records attributed to T Bienvenu.

At least 91 records · Page 5Linked to original sources
PubMed1992

Rapid and direct detection of the most frequent Mediterranean beta-thalassemic mutations by multiplex allele-specific enzymatic amplification.

A rapid nonradioactive method for the diagnosis of the most frequent Mediterranean beta-thalassemic mutations is described based on a multiplex allele-specific polymerase chain reaction (PCR). This method allows direct detection of normal or mutated alleles on genomic DNA. We have used this approach to detect the most frequent Mediterranean mutations: IVS-1 nt 110 (G----A) and 39 nonsense (C----T). For each mutation three allele-specific oligonucleotides were used: one common upstream primer and two downstream primers differing in their terminal 3' nucleotide (one specific for the normal allele and one for the mutant allele). For each sample two PCR reactions were performed in parallel using in one case IVS-1 nt 110 and codon 39 normal primers and in the second case using the corresponding mutated primers. In both cases the different PCR fragments were visualized. After optimization these primers directed only amplification of their complementary allele. A single blind study was performed on the DNA of 18 individuals who were homozygous or heterozygous for these mutations. In comparison with a parallel investigation, using oligonucleotide probes, all the results were unambiguous. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows the screening of a population group, including heterozygotes, which is required from an epidemiological and anthropological point of view. It could be extended to the large series screening of haplotypes before targeted diagnosis of various genetic diseases.

Algeria↗
PubMed1991

[Plasma homocysteine assay in the exploration of thrombosis in young subjects].

Homocystinuria is a rare inherited metabolic disease transmitted as an autosomal recessive trait. Arterial and venous thromboembolic events are frequent and life-threatening complications in homocystinuric patients. It has been suggested that mild homocysteinemia could be a risk factor for vascular disease. We measured total plasma L-homocysteine concentrations by radioisotopic assay in 32 subjects with arterial (n = 15) or venous (n = 17) thrombosis. Twelve subjects exceeded the upper normal limit (2.70 SD above the mean), i.e. 14.1 mumol/l. Seven had arterial thrombotic disease and five had deep vein thrombosis. In 18 subjects thrombosis developed in the absence of any of the hitherto recognized risk factors; 6 of these subjects had mild homocysteinemia. Thus, homocysteine metabolism must be investigated in patients with thromboembolism. An increase of homocysteinemia could be a risk factor for thromboembolic events, and the possible benefit of vitamin therapy should be discussed.

Adolescent↗
PubMed1991

Antithrombin III activity is not related to plasma homocysteine concentrations.

Arterial and venous thromboembolic events represent frequent and life-threatening complications in homocystinuric patients and are responsible for their early deaths. Reduced levels of antithrombin III activity in homocystinuric patients have recently been reported. So, high plasma L-homocysteine concentration could play a role in the low antithrombin III activity level. In the present study, we have studied the relationship between total plasma homocysteine and inhibitors of blood coagulation levels in 16 patients with malignancies who received bone marrow grafts. There were no correlations between homocysteine values and inhibitors of blood coagulation levels. So, while the defect in amino acid transsulfuration that is responsible for homocystinuria can directly affect the synthesis or activity of some clotting factors, homocysteine concentration is not responsible for this effect.

Adult↗
PubMed1991

A simple non-invasive procedure for the investigation of cytochrome P-450 IIIA dependent enzymes in humans.

Urinary 6 beta-hydroxycortisol (6 beta OHF) is a specific marker of the induction of the cytochrome P-450 IIIA. Often, the production of 6 beta OHF is expressed from 24-h urine collection as the ratio 6 beta OHF/17-hydroxycorticosteroids or even better 6 beta OHF/free cortisol in order to adjust for minor day to day variation in adrenal cortisol production. A 24-h urine collection is a non-invasive method applicable to clinical studies in adult man, but more difficult to realize in newborn and infants. Therefore, we have tested the validity of a single urine sampling for the determination of the 6 beta OHF/free cortisol ratio as a clinical test to estimate cytochrome P-450 IIIA enzyme activity. Urinary 6 beta OHF and free cortisol have been measured in 19 healthy volunteers by high performance liquid chromatography methods using the same extraction procedure. A good correlation was found between 6 beta OHF/free cortisol ratio in morning urine samples and in 24h-collections (r = 0.899). Thus, a morning spot urine sampling is a simple procedure that may be helpful for investigations on cytochrome P-450 IIIA enzyme activity in humans, particularly in newborn and in children.

Adult↗
PubMed1990

Effect of hypophysectomy on caffeine elimination in rats.

Two groups of 8-week-old Sprague-Dawley male rats were used: 8 hypophysectomized (H[-]), operated on day 0, treated by daily sc tetracosactid (ACTHs: 10 micrograms), and thyroxine (T4:5 micrograms/100 g); 7 sham-operated, treated by sc saline solution. ACTHs, T4, saline solution were administered on days 7-16. The animals received po caffeine (CAF) 4 mg/kg as citrate salt on day 15. Ten blood samples were drawn from the tail. Plasma CAF concentrations were determined by HPLC. CAF apparent clearance and apparent volume of distribution were lower in H(-) rats than in controls: 0.281 +/- 0.072 vs 0.455 +/- 0.165 l/kg/h (-38%; P less than 0.05) and 0.520 +/- 0.239 vs 1.28 +/- 0.266 l/kg (-59%; P less than 0.01) respectively. CAF half-life was lower in H(-) rats than in controls: 1.33 +/- 0.621 vs 2.12 +/- 0.676 h (-37%; P less than 0.01). CAF is a drug with a low hepatic extraction ratio and low plasma protein binding. CAF clearance is therefore primarily dependent on intrinsic clearance, which depends on the activity of the enzymes involved in CAF metabolism. These data suggest that hepatic CAF metabolism is reduced in H(-) rats treated by SC ACTHs and T4. The decrease in CAF apparent volume of distribution is probably related to dehydration, as suggested by increase in urine flow and hematocrit. The CAF half-life was probably low because the volume of distribution was proportionally more decreased than the clearance. Our results suggest that the pituitary gland plays a role in the regulation of hepatic CAF metabolizing enzymes.

Adrenocorticotropic Hormone↗
PubMed1990

[Radioisotopic assay of total L-homocysteine in plasma and urine: application to serial determinations].

The authors report here a simple radioenzymatic determination of total L-homocysteine in plasma and urine. L-homocysteine-containing disulfides were reduced with dithioerythritol. L-homocysteine was then condensed by S-adenosyl L-homocysteine hydrolase to 14C-adenosine to form S-14C adenosyl L-homocysteine that was separated from 14C-adenosine by paper descendant chromatography. The concentration of the total plasma L-homocysteine of 45 normal subjects was 8.04 +/- 0.26 (mean +/- SEM) mumol/l and total L-homocysteine concentration in urine was 0.59 +/- 0.06 mumol/mmol of creatinine (25 subjects). This method is as sensitive than the other methods described in the literature but more rapid and less expensive.

Adult↗
PubMedDate not supplied

Effect of pregnandiol on caffeine metabolism in female rats.

Three groups of six 5-week-old Sprague Dawley female rats received i.p. injections of pregnandiol, 1.25, 2.50 or 5 mg/kg, respectively, in triolein daily for 7 days. Caffeine metabolism was studied in liver slices on day 8 by HPLC. Only primary metabolites were formed. N-1 demethylation was the most important pathway (theobromine represented 51% of total dimethylxanthines). Unlike in human in vitro or in vivo, 1,3,7-DAU (6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil) was an important metabolite (9.7% of total caffeine metabolites). Pregnandiol inhibited N-1, N-3 and N-7 demethylation in vitro (-33%, -33% and -28%, respectively, at 5 mg/kg/day), but it had no effect on N-1 demethylation at 1.25 or 2.50 mg/kg/day. Pregnandiol at all doses had no effect on 1,3,7-trimethyluric acid and 1,3,7-DAU formation. These results are consistent with the hypothesis that C-8 hydroxylation and demethylation of caffeine are mediated by different isoenzymes. They indicate that pregnandiol is a potent inhibitor of microsomal drug metabolism, specifically of cytochrome P450 IA, which could explain the immaturity of some metabolic pathways of caffeine in neonates.

Animals↗
PubMedDate not supplied

Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. Recent studies have identified large gene rearrangements that escape the common PCR-based mutation screening strategy and mutations in a novel MeCP2 isoform (named MECP2B). We have collected the results of MECP2 mutational analysis concerning 424 RTT patients conducted in eight laboratories in France. In total, 121 different MECP2 mutations were identified. R168X (11.5%) is the most common of MECP2 mutations, followed by R270X (9%), R255X (8.7%), T158 M (8.3%) and R306C (6.8%). Only eight mutations had relative frequency>3%. Large and complex rearrangements not previously detected using only a PCR-based strategy represent 5.8% of MECP2 mutations. On the contrary, mutation in exon 1 appears to be rare (less than 0.5%). These data demonstrate the high allelic heterogeneity of RTT in France and suggest that routine mutation screening in MECP2 should include quantitative analysis of the MECP2 gene. This study represents an important instrument for molecular diagnosis strategy and genetic counseling in RTT families.

Cohort Studies↗
PubMedDate not supplied

Determination of the gene structure of human oligophrenin-1 and identification of three novel polymorphisms by screening of DNA from 164 patients with non-specific X-linked mental retardation.

We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.

Amino Acid Substitution↗
PubMedDate not supplied

Reduction in the DAZ gene copy number in two infertile men with impaired spermatogenesis.

Microdeletions of Yq are associated with azoospermia and severe oligozoospermia. Recently, we described a new molecular genetic strategy based on the denaturing gradient gel electrophoresis (DGGE) to rapidly identify deletions of the Y chromosome that include the DAZ locus. Using this approach, we have shown not a complete deletion but only a reduction in the number of copies of the DAZ gene by PCR-DGGE in two oligozoospermic patients. These results have been confirmed by Southern blot analysis. This finding suggests that partial deletion of the DAZ cluster could be cause of impaired spermatogenesis.

Adult↗
PubMedDate not supplied

[Molecular detection of Y chromosome microdeletions: a new approach based on the denaturing gradient gel electrophoresis].

Ten percent of couples trying for a child fail to conceive. In approximately 50% of cases, infertility is due to the inability of the male partner to produce spermatozoa in sufficient numbers to effect conception. Over the past 5 years, molecular studies have suggested that interstitial microdeletions in Yq11 represent an etiological factor for male infertility. Y-microdeletions have been detected in 12% of non-obstructive azoospermia and 6% of severe oligozoospermia. In general, microdeletions were detected by separate multiplex-polymerase chain reaction (PCR) reactions using primer pairs for single tagged sites (STSs) of all three azoospermia factor (AZF) regions (AZFa, AZFb and AZFc). This review describes the molecular methods and laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. The diagnostics of Y-chromosomal microdeletions should be performed in two steps: in any case, the routine diagnostic should include six STS loci, two STS loci localised in each AZF region, and once a deletion is detected, the analysis can be extended to STS loci known to cross the proximal and the distal borderlines of each AZF region. Other molecular techniques such as DGGE, Southern blot should be performed to detect partial deletions of gene copies or mosaicism. These different molecular approaches should allow explaining 10% of male infertility, to evaluate the risk to pass the defect onto their male offspring (by intracytoplasmic sperm injection) and improve the genetic counselling of couples undergoing micromanipulative assisted reproduction.

Blotting, Southern↗