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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.

Thiopurine methyltransferase (TPMT) risk alleles (mainly *2,*3B, *3C and *3A) are the major determinants of interindividual differences in the severe toxicity or efficacy of 6-mercaptopurine (6MP) during the treatment of childhood acute lymphoblastic leukemia (ALL). The frequencies of these risk alleles, known to functionally impair TPMT activity, were investigated among 167children with ALL and 206 healthy adult controls in Turkish population by using allele specific PCR and PCRRFLP methods. TPMT*3A and TPMT*3C were the only deficiency alleles detected in Turkish population with an allele frequency of 0.5% for both. The total frequency of mutant TPMT alleles in Turkish population (1.0%) was found to be significantly lower than those of other Caucasian populations (5.3-7.0%), but it was found to be very similar to Kazak population (1.2%) which is also Caucasian in ethnic origin. v In the patient group, two individuals were found to be heterozygote for *3C and *3A allele. One individual was homozygous mutant (*3B/*3C). In this study, the clinical histories of the patients with TPMT defects were examined retrospectively from hospital records. The patients with heterozygous or homozygous mutant genotypes had systematically developed severe neutropenia, infection and some other specific conditions (like lesions around mouth, oral herpes and high fever) when they were administered with 6MP during the therapy. This study provides the first data on the frequency of common TPMT risk alleles in the Turkish population, based on analysis of pediatric patients with ALL. The results would contribute valuable information to the public health, as more clinicians and patients become aware of the importance of TPMT polymorphisms, less patients will suffer from 6MP related adverse effects. In addition, in this study two genes EPHX1-microsomal epoxide hydrolase (exon 3 and exon 4 polymorphisms) and SULT1A1*2 variant &ndash / sulfotransferase 1A1, either alone or in combination were investigated as risk modifiers in the development of childhood acute lymphoblastic leukemia due to their dual role (activation/detoxification) in the metabolism of various carcinogens. Also interactions of these polymorphisms with non-genetic risk factors (parental smoking exposure and parental age at conception) were investigated. The conclusion inferred from results was that only genetically reduced EPHX1 activity (homozygous mutant genotype for EPHX1 exon 3 polymorphism and some specific genotype combinations with exon 4 polymorphism) was found to be significantly associated with the risk of childhood ALL.

Les thiopurines sont des médicaments cytotoxiques et immunosuppresseurs largement prescrits, notamment dans les maladies inflammatoires chroniques de l’intestin (MICI). Ils représentent l’un des meilleurs exemples d’application clinique de la pharmacogénétique avec le dépistage du déficit en thiopurine S-méthyltransférase (TPMT), enzyme clé du métabolisme des thiopurines. La variabilité interindividuelle de la réponse à ces médicaments rend nécessaire leur optimisation thérapeutique. Ce travail de thèse a d’une part, analysé les relations entre activité TPMT et concentrations des métabolites thiopuriniques, et d’autre part, recherché des facteurs associés à la résistance aux thiopurines. A l’aide d’une base de données pharmacogénétiques hospitalière et d’une étude « PheWAS » à partir d’un entrepôt de données cliniques, nous avons analysé la distribution et la corrélation génotype-phénotype pour la TPMT, en lien avec les concentrations des métabolites thiopuriniques. Nous avons observé qu’une activité TPMT très élevée (phénotype « ultra-rapide ») était associée à des paramètres clinico-biologiques reflétant une maladie évolutive et un traitement inefficace dans les MICI. De plus, une étude clinique rétrospective dans les MICI pédiatriques a permis d’identifier des facteurs associés à la lymphopénie observée sous thiopurines. Enfin, à partir d’un modèle in vitro fondé sur des lignées cellulaires lymphoblastoïdes (LCL) sélectionnées, nous avons établi une signature transcriptomique, incluant 32 gènes, prédictive de la résistance aux thiopurines. Une analyse fonctionnelle bioinformatique a abouti à l’identification de voies métaboliques liées à la protéine p53 et au cycle cellulaire, ainsi que des mécanismes moléculaires associés à la résistance aux thiopurines. En conclusion, ce travail de thèse, qui a exploré la variabilité de réponse aux thiopurines et tout particulièrement la résistance à ces médicaments, propose des hypothèses pour l’individualisation et l’optimisation thérapeutique des thiopurines. / Thiopurines are cytotoxic and immunosuppressive drugs widely prescribed, mainly in inflammatory bowel disease (IBD). They constitute one of the best success story of pharmacogenetic implementation into clinical practice based on the screening of thiopurine S-methyltransferase (TPMT) deficiency, a key enzyme in thiopurine metabolism. Optimization of thiopurine response is challenging because of its large interindividual variability such as inefficacy and toxicities. This thesis has explored, on one hand, the relationships between TPMT activity and metabolite concentrations, and on the other hand, factors associated with thiopurine inefficacy. Using a primary care pharmacogenetic database, we first analyzed TPMT distribution and genotype-phenotype correlation, in relation with thiopurine metabolites in a large population. Using a PheWAS study based on a clinical data warehouse we then reported that a very high TPMT activity (“ultra-rapid” phenotype) was associated with parameters of active IBD and poor response to thiopurines. Furthermore, a retrospective study in pediatric IBD identified factors predicting the occurrence of lymphopenia during thiopurine therapy. Finally, using a lymphoblastoid cell line (LCL) in vitro model, we established a transcriptomic signature, including 32 genes predicting thiopurine cellular resistance. A bioinformatic functional analysis identified metabolic pathways in relation with p53 and cell cycle, as well as molecular mechanisms associated with thiopurine resistance. To conclude, this research work, focusing on the variability of thiopurine response and mainly therapeutic resistance, provides new hypotheses to individualize and optimize therapeutic response to thiopurines.

Η μεθυλοτρανσφεράση της θειοπουρίνης (TPMT) είναι ένα ένζυμο του κυτταροπλάσματος, που καταλύει τη μεθυλίωση των θειοπουρινικών φαρμάκων, όπως η 6-μερκαπτοπουρίνη (6-MP), η οποία έχει καθιερωθεί στη θεραπεία συντήρησης παιδιών με ΟΛΛ. Ένα πολυμορφικό στοιχείο (VNTR) στον υποκινητή του γονιδίου ΤΡΜΤ, φαίνεται να επηρεάζει τα επίπεδα δραστικότητας του ενζύμου που παράγεται. Πρόκειται για διαδοχικές επαναλήψεις τριών διαφορετικών αλληλουχιών (A, B και C), ο αριθμός των οποίων κυμαίνεται από τρεις έως εννέα. Η περιοχή VNTR είναι πλούσια σε επαναλήψεις των βάσεων GC, οι οποίες αποτελούν πιθανές θέσεις πρόσδεσης μεταγραφικών παραγόντων. Μερικοί ασθενείς, οι οποίοι ακολουθούν θεραπεία με 6-ΜΡ, δεν είναι ανθεκτικοί στην καθιερωμένη δόση του φαρμάκου και παρουσιάζουν μυελοτοξικότητα ως αποτέλεσμα της ανεπάρκειας του ενζύμου TPMT. Στην παρούσα εργασία πραγματοποιήθηκε μία εκτεταμένη μελέτη της περιοχής VNTR, με απώτερο σκοπό την αξιολόγησή της ως πιθανό φαρμακογονιδιωματικό δείκτη, που να σχετίζεται με την πρόβλεψη ανεπιθύμητων παρενεργειών σε ασθενείς με ΟΛΛ, οι οποίοι λαμβάνουν 6-ΜΡ. Αρχικά, προσδιορίστηκε η συχνότητα των διαφορετικών VNTR αλληλομόρφων στον ελληνικό πληθυσμό, σε ένα σύνολο υγιών ατόμων, με σκοπό να αντιπαραβάλλουμε τις συχνότητες αυτές με δεδομένα από άλλες πληθυσμιακές ομάδες. Τα αποτελέσματά που προέκυψαν συμφωνούν με τα αντίστοιχα στοιχεία που υπάρχουν για άλλους πληθυσμούς. Οι μέθοδοι γονοτύπησης που εφαρμόσθηκαν περιλαμβάνουν την αντίδραση της PCR και τον προσδιορισμό αλληλουχίας κατά Sanger. Στη συνέχεια μελετήθηκε η επίδραση συγκεκριμένων μοτίβων VNTR στον υποκινητή του γονιδίου υγιών ατόμων, στη μεταγραφή του γονιδίου, καθώς και στην έκφραση του ενζύμου ΤΡΜΤ. Η ικανότητα μεταγραφής εκτιμήθηκε με τη μέθοδο της ποσοτικής Real Time PCR, ενώ τα επίπεδα έκφρασης του ενζύμου προσδιορίστηκαν μέσω της αντίδρασης χημειοφωταύγειας, με χρήση κατάλληλων αντισωμάτων. Ο μικρός αριθμός δειγμάτων δεν επέτρεψε την εξαγωγή ασφαλών συμπερασμάτων για το ρόλο της VNTR περιοχής, στην μεταγραφή του γονιδίου ΤΡΜΤ. Σύμφωνα με τα αποτελέσματά μας, διακρίνεται μία τάση συσχέτισης, η οποία όμως απαιτεί περαιτέρω διερεύνηση. / Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of thiopurine drugs, such as 6-mercaptopurine (6-MP), which is typically used as maintenance therapy in pediatric acute lymphoblastic leukemia (ALL). A variable number of tandem repeat (VNTR) within the TPMT promoter has been reported to “modulate” levels of this enzyme activity. The VNTR region architecture is defined by three types of repeats (A, B and C) rang¬ing from three to nine (VNTR*3 to VNTR*9). These repeats are GC rich, and are putative binding sites of various transcription factors. Some patients do not tolerate standard doses of 6-MP and develop myelotoxicity as a consequence of inherited TPMT deficiency. In this study, we performed an extensive analysis of VNTR region, in order to evaluate it as a putative pharmacogenomic marker to predict 6-MP toxicity, in ALL patients. The frequency of TPMT alleles bearing different VNTR architectures was investigated in the Hellenic population. The results of this study were consistent with data, already existed, from other populations. The number and type of tandem repeats were determined by PCR amplification followed by direct re-sequencing. Moreover, we explored the influence of specific VNTR motifs, within the TPMT promoter of healthy individuals, on transcription and expression of TPMT gene. Quantitative Real-Time PCR was performed in order to investigate the influence of VNTR architecture on TPMT gene transcription. The expression levels of TPMT enzyme were estimated by chemiluminescence reaction, using appropriate antibodies. The role of VNTR region on TPMT gene transcription was not clarified through this study, owing to small cohort of samples included. A statistical trend of association has been observed, requiring, however, further investigation.

Pharmacogenetics (PGx) and personalised medicine are new disciplines that, gathering the existing knowledge about the genetic and phenotypic factors that underpin drug response, aim to deliver more targeted therapies that avoid the existing problems of adverse drug reactions or lack of drug efficacy. PGx and personalised medicine imply a shift in the way drugs are prescribed, as they require introducing diagnostic tools and implementing pre-screening mechanisms that assess patients' susceptibility to new or existing drugs. The direct benefit is an improvement in drug safety and/or efficacy. However, neither pharmacogenetics nor personalised medicine, are widely used in clinical practice. Both technologies face a number of controversies that hamper their widespread use in clinical practice. This thesis investigates the scientific; technological; social; economic; regulatory and ethical implications of PGx and personalised medicine, to understand the enablers and barriers that drive the process of technology diffusion in three conditions: autoimmune diseases, acute lymphoblastic leukaemia and non-small cell lung cancer.The thesis uses concepts of the sociology of science and a qualitative approach, to explore the arguments for and against the use of the technology by different actors (pharmaceutical and biotechnology companies, researchers, clinicians, regulators and patient organisations). The core of this analysis lies in the understanding of how, diagnostic testing (TPMT testing in the case of autoimmune diseases, acute lymphoblastic leukaemia, and EGFR testing in the case of non-small-cell lung cancer) may affect the existing drug development and service delivery mechanisms, with a particular focus on the user-producer interactions and feedback mechanisms that underpin diffusion of medical innovations and technological change in medicine.The thesis concludes by identifying gaps in knowledge and common issues among TPMT and EGFR testing, which might be used, in the future, to inform policy on how to improve PGx service delivery through a public Health System such as the NHS.

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.