Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.

Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.

Coronary artery disease (CAD) is the most prevalent disorder and the leading cause of death worldwide. There are a number of CAD medications, which are effective and safe in most patients, but have been associated with adverse reactions such as angioedema induced by angiotensin I-converting enzyme inhibitors (AE-ACEi). In this study, we identified aminopeptidase P (APP) activity as an endophenotype for AE-ACEi, which is a heritable quantitative trait (heritability =0.336 +/- 0.251 SD) and is significantly reduced in a majority of our cases. Although initial mutation screening did not reveal any coding variants in XPNPEP2, which encodes membrane-bound APP, subsequent linkage analysis of APP activity in eight families provided a maximum LOD score (3.75) for this locus. Sequencing of additional cases identified a splice variant (314_431del) and a non-coding polymorphism (rs3788853) in this locus, which cosegregate with low plasma APP activity. The latter accounts for the linkage signal and is associated with AE-ACEi (P = 0.036). In addition, we identified other potential loci for APP activity and demonstrated that certain ACEi (Captopril and Enalapril) non-specifically inhibit APP activity. Furthermore, we detected polymorphisms associated with reduced APP and ACE activities among females with estrogen-dependent inherited angioedema. / We also conducted a genetic investigation of depression among CAD patients to identify common susceptibility loci which might explain the correlation between these diseases. Our candidate gene association study identified a polymorphism (rs216873) in the von Willebrand factor gene that was significantly associated (P = 7.4 x 10-5) with elevated depressive symptoms in our CAD cohort. These results suggest that risk factors for atherosclerosis also underlie susceptibility to depression among CAD patients. / This dissertation contributes to the field of genetics and pharmacogenomics of CAD. A better understanding of the toxic effects of CAD drugs will assist in the development of safer and more effective treatments. In addition, our results may facilitate clinical assays to identify individuals who are susceptible to angioedema prior to ACEi or estrogen therapy. Finally, our genetic investigation of depression in CAD patients reveals a novel drug target (VWF) for treatment of depression in cardiac cases.

Kiekvieno vaisto sukeltas tiek farmakologinis (veiksmingumo), tiek toksikologinis poveikis kiekvienam pacientui yra skirtingas, todėl gana dažnai vaistų skyrimas ir vartojimas tampa labai komplikuotas. Prancūzijoje 3.2% hospitalizacijos atvejų yra sąlygoti vaistų. Tai sudaro 320 milijonų eurų sumą per metus. Genetiniai faktoriai, sąlygojantys vaistų farmakokinetiką ir farmakodinamiką, dalinai paaiškina skirtingą vaistų poveikį žmogui. Tyrimo tikslas: 1. Patikrinti galimybę gausinti serume ir plazmoje esančią DNR nauju būdu ir atlikti genetinius tyrimus. 2. Patvirtinti realaus laiko polimerazės grandininės reakcijos (PGR) metodiką aromatazės genui, panaudojus ląsteles, kurių šio geno raiška yra pakankama. Rezultatai: Patikrinta galimybė gausinti serume ir plazmoje esančią DNR, sekvenavus gautą DNR ir sekas palyginus su esančiomis duomenų bazėje : rezultatai teigiami. Nustatyta, kad, ekstrahuojant druskiniu metodu, gaunama daugiau DNR, bet ji blogesnės kokybės, nei naudojant QIAGEN kolonėles. Patvirtinta realaus laiko PGR metodika aromatazės genui, naudojant krūties vėžio ląsteles. / There is much variability in the manner individuals respond to drugs, such that the management of some drugs is problematic. In France, the incidence of hospital admissions related to adverse drug reactions is estimated to be 3.2 %, at an annual cost of over 300 millions euros. Genetic factors affecting the pharmacokinetics and pharmacodynamics of drugs partly explain interindividual variability in drug response. Aim of experiment: 1. verify, if it is possible to amplify serum and plasma DNA using new method, and make a genetic research. 2. verify real time polymerase chain reaction to aromatase gene. Find cell line, whish have a sufficient expression of this gene. Results : We verified the possibility to amplify serum and plasma DNA using new method. We made the sequencing of DNA extracts and we compared results in data base : results are positives. It`s important, that QIAGEN extracts are cleanner than salt extracts, but there are few of DNA. We confirmed real time PCR method to aromatase gene, using breast cancer cells.

Contexte: Bien que plusieurs algorithmes pharmacogénétiques de prédiction de doses de warfarine aient été publiés, peu d’études ont comparé la validité de ces algorithmes en pratique clinique réelle. Objectif: Évaluer trois algorithmes pharmacogénomiques dans une population de patients qui initient un traitement à la warfarine et qui souffrent de fibrillation auriculaire ou de problèmes de valves cardiaques. Analyser la performance des algorithmes de Gage et al., de Michaud et al. ainsi que de l’IWPC quant à la prédiction de la dose de warfarine permettant d’atteindre l’INR thérapeutique. Méthodes: Un devis de cohorte rétrospectif fut utilisé afin d’évaluer la validité des algorithmes chez 605 patients ayant débuté une thérapie de warfarine à l’Institut de Cardiologie de Montréal. Le coefficient de corrélation de Pearson ainsi que l’erreur absolue moyenne ont été utilisés pour évaluer la précision des algorithmes. L’exactitude clinique des prédictions de doses fut évaluée en calculant le nombre de patients pour qui la dose prédite était sous-estimée, idéalement estimée ou surestimée. Enfin, la régression linéaire multiple a été utilisée pour évaluer la validité d’un modèle de prédiction de doses de warfarine obtenu en ajoutant de nouvelles covariables. Résultats : L’algorithme de Gage a obtenu la proportion de variation expliquée la plus élevée (R2 ajusté = 44 %) ainsi que la plus faible erreur absolue moyenne (MAE = 1.41 ± 0.06). De plus, la comparaison des proportions de patients ayant une dose prédite à moins de 20 % de la dose observée a confirmé que l’algorithme de Gage était également le plus performant. Conclusion : Le modèle publié par Gage en 2008 est l’algorithme pharmacogénétique le plus exact dans notre population pour prédire des doses thérapeutiques de warfarine. / Background: Although numerous genotype-based warfarin dosing algorithms have been published, there is little data comparing the predictive ability of these algorithms in real clinical practice. Objectives: Our goal was to evaluate the performance of pharmacogenetic algorithms in an unselected patient population initiating warfarin treatment for atrial fibrillation or valve disease in a real-world clinical setting. The principal objective of the analysis was to determine if Gage’s, Michaud’s, and IWPC algorithms could predict the dose achieving the therapeutic International normalized ratio (INR). Methods: Data from a retrospective cohort study of 605 patients initiating warfarin therapy at the Montreal Heart Institute was used. We compared the dose predicted by the algorithms to the dose achieving the therapeutic INR. Pearson’s correlation coefficient and mean absolute error (MAE) were used to evaluate the predictive accuracy of the algorithms. Clinical accuracy of the predictions was assessed by computing the proportion of patients in which the predicted dose was under-estimated, ideally estimated, or overestimated. Finally, we used multiple linear regression analysis to evaluate the accuracy of a predictive model obtained by adding additional covariables in predicting therapeutic warfarin doses. Results: The proportion of variation explained (adjusted R2) was the highest for Gage’s algorithm (R2 = 44 %) and the mean absolute error was the smallest for the predictions made by Gage’s algorithm (MAE = 1.41 ± 0.06). Moreover, when we compared the proportion of patients whose predicted doses are within ± 20 % of the observed stable dose, Gage’s algorithm also performed the best overall. Conclusion: The algorithm published by Gage et al. in 2008 is the most accurate pharmacogenetically based equation in predicting therapeutic warfarin dose in our study population.

Background: Gastric cancer is often diagnosed in the metastatic stage, and only 10% of patients survive for as long as 2 years. Current chemotherapy regimens show significant treatment-related toxicities. It is crucial to identify the patients that will benefit most from certain chemotherapy regimens in order to avoid unnecessary side effects. Patients and Methods: 2 patients with advanced gastric cancer repeatedly received 5-FU/cisplatin combination chemotherapy. Genomic DNA was extracted from tumor tissue and mononuclear blood cells. Genotype analysis of genes of metabolizing and DNA repair enzymes was carried out using a PCR-RFLP technique. Direct sequencing was used to identify mutations of the gene dihydropyrimidine dehydrogenase (DPD). Results: Prolonged survival of 51 and 29 months, respectively were observed in our 2 patients. Both patients were positive for genotypes of thymidylate synthase - the target enzyme of 5-FU - that are associated with improved drug response. DPD variants connected with increased toxicity were not observed. However, both patients also showed genotypes in cisplatin metabolizing enzymes which enhance the effect of the drug. Conclusion: Genotype analysis in drug metabolizing enzymes of 5-FU and cisplatin provide a possible explanation for extraordinary therapy effects observed in 2 patients with advanced gastric cancer. / Hintergrund: Das Magenkarzinom wird häufig im fortgeschrittenen Stadium diagnostiziert, und nur etwa 10% der Patienten überleben 2 Jahre. Aktuelle Chemotherapien zeigen eine hohe therapiebedingte Toxizität. Es ist daher von großer Bedeutung, diejenigen Patienten zu identifizieren, die von einer bestimmten Therapie profitieren, um anderen Patienten die Nebenwirkungen einer solchen Therapie zu ersparen. Patienten und Methoden: 2 Patienten mit fortgeschrittenem Magenkarzinom erhielten wiederholt eine Kombinationschemotherapie aus 5-FU/Cisplatin. Genomische DNS wurde aus Tumorgewebe und Leukozyten isoliert. Genotypanalysen von Genen, die am Metabolismus der Substanzen und am DNS-Reparaturprozess beteiligt sind, wurden mithilfe einer PCRRFLP-Methode durchgeführt. Das Gen der Dihydropyrimidindehydrogenase (DPD) wurde direkt sequenziert. Ergebnisse: Beide Patienten zeigten ein deutlich verlängertes Überleben von 51 bzw. 29 Monaten. Genotypen des 5-FU-Zielenzyms Thymidylatsynthase, die mit einem verbesserten Ansprechen assoziiert sind, konnten in beiden Patienten nachgewiesen werden. DPD-Varianten, die mit einer erhöhten Toxizität verbunden sind, wurden nicht beobachtet. Zusätzlich konnten bei beiden Patienten Genotypen in Cisplatin metabolisierenden Genen gefunden werden, die eine prolongierte Wirkung der Substanz bedingen. Schlussfolgerungen: Durch Genotypanalysen in Genen des 5-FU- und Cisplatin-Metabolismus konnte ein spezifisches pharmakogenetisches Profil identifiziert werden, das möglicherweise die Ursache eines außergewöhnlich guten Therapieeffektes in 2 Patienten mit fortgeschrittenem Magenkarzinom ist. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Pharmacogenetics is the study of variability in drug response attributed to genetic variation. Olanzapine (OLA) is a widely used antipsychotic drug for schizophrenia treatment. The pharmacokinetics of OLA display large inter-individual variation leading to multiple-fold differences in drug exposure between patients at a given dose. This variation in turn gives rise to the need of individualized dosing in order to avoid concentration-dependent adverse effects and therapeutic failure. The observed variability has been partially explained by environmental and physiological factors. Genetically determined differences in drug metabolism represent a less studied source of variability. Precluded contribution by cytochrome P450 (CYP) 2D6 calls for evaluation of the other major OLA metabolizing enzymes. The objective of this thesis was to study pharmacogenetic influence of flavin-containing monooxygenase (FMO) 1 and 3, CYP1A2 and uridine diphosphate-glucuronosyltransferase (UGT) 1A4 on therapeutic OLA exposure. We conducted genetic association studies applying gene re-sequencing and genotyping of candidate and tagging SNPs. Patients carrying the FMO1*6 allele displayed increased dose-adjusted concentrations (C/Ds) of OLA, in serum as well as cerebrospinal fluid. Patients who were homozygous for the FMO3 K158-G308 compound variant showed reduced C/Ds of OLA N-oxide metabolite, but no alteration in OLA exposure. This compound variant is expected to have clinical relevance primarily for non-African populations, since low frequencies were detected among native Africans. Deviation in OLA exposure was observed in carrier of a rare FMO3 mutation, predicted in silico to affect gene splicing. Reduced OLA exposure was observed in UGT1A4*3 carriers. The CYP1A2 -163(A) (CYP1A2*1F) variant was not associated with increase in CYP1A2-catalyzed OLA metabolism or reduction in OLA exposure. Correlations were detected for two cis-acting variants within the inter-genetic region of the CYP1A cluster and a trans-acting variant located upstream the locus encoding aryl hydrocarbon receptor. The inconsistent data reported for CYP1A2*1F could be explained by presence of ethnic specific haplotype structures incorporating the -163(A) variant. A continuously improved understanding of the wide range of factors that can influence pharmacokinetics and pharmacodynamics will increase the likelihood of achieving optimal treatment response for individual patients.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.