Determination of Cytochrome P450 2D6 (CYP2D6) Gene Copy Number by Real-Time Quantitative PCR

Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number). Using the 2(−ΔΔCt) calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies), and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication

Collection of Human Genomic DNA From Buccal Cells for Genetics Studies: Comparison Between Cytobrush, Mouthwash, and Treated Card

Alternative sources such as buccal cells have already been tested for genetic studies and epidemiological investigations. Thirty-seven volunteers participated in this study to compare cytology brushes, mouthwash, and treated cards for DNA collection. Quantity and quality of DNA and cost and feasibility were assessed. The mean DNA yield at 260 nm was found to be 3.5, 4, and 2.6 μg for cytobrushes, mouthwashes, and treated cards, respectively. A second quantification technique by fluorescence showed differences in the DNA yield with 1.1 and 5.2 μg for cytobrushes and mouthwash, respectively. All buccal samples allowed isolation of DNA suitable for polymerase chain reaction. According to the procedure of sample collection, the yield and purity of collected DNA, and storage conditions, the use of cytobrush appears to be the more appropriate method for DNA collection. This protocol has been validated and is currently applied in three large-scale multicentric studies including adults or children

Les biais cognitifs : comment les interpréter pour mieux les comprendre

Disponible en français dans EDUQ.info sous le titre "Les biais cognitifs : comment les interpréter pour mieux les comprendre

Impact of aspirin and clopidogrel interruption on platelet function in patients undergoing major vascular surgery.

AIMS: To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption. METHODS/RESULTS: We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; p = 0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; p = 0.02), compared to baseline. CONCLUSIONS: Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management

Pharmacogenetic assessment of toxicity and outcome in patients with metastatic colorectal cancer treated with LV5FU2, FOLFOX, and FOLFIRI: FFCD 2000-05

L’objectif de ce travail était la recherche de biomarqueurs moléculaires prédictifs de la tolérance et de l’efficacité des chimio– thérapies utilisées dans le colorectal (CCR) métastatique. Nous avons effectué le génotypage de 20 polymorphismes présents au sein de 9 gènes connus ou suspectés d’être impliqués dans la voie du 5FU, de l’oxaliplatine, ou de l’irinotécan, à partir de l’ADN extrait du sang de 346 patients traités dans le cadre d’un essai de phase III. Cet essai comparait une chimiothérapie séquentielle par 5FU (schéma LV5FU2) suivie d’une association 5FU plus oxali– platine (schéma FOLFOX) à une chimiothérapie combinée de type FOLFOX d’emblée en première ligne de traitement. Nous avons trouvé un risque de toxicité hématologique sévère sous FOLFOX significativement augmenté chez les patients porteurs de l’allèle ERCC2-K751QC. La présence de l’allèle TS-5’UTR3RG du gène de la thymidylate synthase était associée à un taux de réponse significativement plus élevé sous LV5FU2. Le taux de réponse au FOLFOX en 2e ligne était significativement supérieur chez les patients porteurs de l’allèle ERCC1-IVS3+74G, et chez ceux ayant au moins un allèle de GSTT1 présent. L’analyse prédictive a montré un effet dépendant du traitement de certains polymorphismes. En effet, une survie sans progression significativement allongée par l’ajout de l’oxaliplatine en 1re ligne a été observée uniquement chez les patients ayant un génotype TS-5’UTR2R/2R ou 2R/3R, suggérant l’absence de bénéfice d’une bithérapie par FOLFOX d’emblée en première ligne chez les patients TS-5’UTR3R/3R. Ces résultats montrent que l’étude des polymorphismes constitutionnels permettent de prédire non seulement la toxicité mais aussi l’efficacité des chimiothérapies antitumorales du cancer colorectal, et ainsi (sous réserve d’une validation sur une population indépendante) d’orienter la stratégie thérapeutique à l’échelle de l’individu

Pooled analysis of iron-related genes in Parkinson's disease: Association with transferrin

Pathologic features of Parkinson's disease (PD) include death of dopaminergic neurons in the substantia nigra, presence of α-synuclein containing Lewy bodies, and iron accumulation in PD-related brain regions. The observed iron accumulation may be contributing to PD etiology but it also may be a byproduct of cell death or cellular dysfunction. To elucidate the possible role of iron accumulation in PD, we investigated genetic variation in 16 genes related to iron homeostasis in three case-control studies from the United States, Australia, and France. After screening 90 haplotype tagging single nucleotide polymorphisms (SNPs) within the genes of interest in the US study population, we investigated the five most promising gene regions in two additional independent case-control studies. For the pooled data set (1289 cases, 1391 controls) we observed a protective association (OR. = 0.83, 95% CI: 0.71-0.96) between PD and a haplotype composed of the A allele at rs1880669 and the T allele at rs1049296 in transferrin (TF; GeneID: 7018). Additionally, we observed a suggestive protective association (OR. = 0.87, 95% CI: 0.74-1.02) between PD and a haplotype composed of the G allele at rs10247962 and the A allele at rs4434553 in transferrin receptor 2 (TFR2; GeneID: 7036). We observed no associations in our pooled sample for haplotypes in SLC40A1, CYB561, or HFE. Taken together with previous findings in model systems, our results suggest that TF or a TF- TFR2 complex may have a role in the etiology of PD, possibly through iron misregulation or mitochondrial dysfunction within dopaminergic neurons

Pharmacogénétique des anticoagulants oraux (variations génétiques du métabolisme et de la cible pharmacologique)

PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Pharmacogénétique des antagonistes de la vitamine K dans des populations particulières (l'exemple des patients âgés, des enfants et des patients résistants)

Les antivitamines K (AVK) sont difficiles à manier du fait d une marge thérapeutique étroite et d une importante variabilité interindividuelle de la réponse. Quasiment aucune donnée n était disponible sur la part des facteurs pharmacogénétiques et non génétiques dans la variabilité de la réponse aux AVK dans des populations particulières (patients âgés, enfants, patients résistants). Dans 2 cohortes de patients gériatriques traités par warfarine (n=300) ou fluindione (n=156), les facteurs génétiques sont les déterminants majeurs de la dose à l équilibre ( 20% de variabilité expliquée contre 10% pour les non génétiques). Les variables influençant significativement la dose à l équilibre sont VKORC1/CYP2C9/CYP4F2/EPHX1 et l âge pour la warfarine, VKORC1/ABCB1/CYP4F2, le poids et la prise d amiodarone pour la fluindione. VKORC1 est un marqueur d hypersensibilité à la warfarine en début de traitement. Après modélisation, VKORC1/CYP2C9 ont la meilleure valeur prédictive de la dose de warfarine à l équilibre avant traitement, tandis que leur apport est négligeable par rapport à l INR quand un schéma posologique standardisé est utilisé pour l initiation. Dans une cohorte d enfants (n=120), 70% de variabilité de la dose de warfarine à l équilibre sont expliqués, avec la taille (48%) et VKORC1/CYP2C9 (20%) comme principaux déterminants. Parmi les 100 cas de résistance analysés, 30 patients sont porteurs de mutations VKORC1 caractérisées sur le plan fonctionnel in vitro, suggérant l implication d autres causes génétiques de résistance. La pharmacogénétique apparaît comme une aide à la prescription chez des patients sélectionnés mais le génotypage systématique a-t-il sa place ?The use of vitamin K antagonists (VKA) is challenging because of their narrow therapeutic index and a large inter-individual variability. Pauci data were available regarding the relative contribution of pharmacogenetic and non-genetic factors to VKA response in special populations (elderly, children, resistant patients). In two cohorts of elderly patients receiving warfarin (n=300) or fluindione (n=156), genetic factors were the main determinants of the maintenance dose explaining 20% of the variability versus 10% for non-genetic factors. The variables significantly associated with the maintenance dose were VKORC1/CYP2C9/CYP4F2/EPHX1 and age for warfarin, and VKORC1/ABCB1/CYP4F2, weight and amiodarone intake for fluindione (multivariate analysis). During warfarin initiation, VKORC1 genotype had a strong predictive value for warfarin sensitivity. When building prediction models of the warfarin dose, VKORC1/CYP2C9 were the best predictors before initiation whereas their contribution was negligible once INR value was available after starting warfarin using a standardized regimen. In a children cohort (n=120), height and VKORC1/CYP2C9 were the main determinants of warfarin dose requirement explaining 70% of the variability, accounting for 48% and 20%, respectively. Among the 100 resistant patients referred to us for analysis, only 30 patients were carriers of VKORC1 mutations for which in vitro functional characterization was performed. Our results suggest the involvement of other genetic factors in VKA resistance. Pharmacogenetics will help for the development of personalized medicine, but what is the clinical usefulness of systematic genotyping in VKA management?PARIS-BIUP (751062107) / SudocSudocFranceF