A azatioprina e a 6 mercaptopurina (6-MCP) são drogas muito utilizada no tratamento das doenças inflamatórias intestinais (DII), porém estão associadas a vários efeitos colaterais. A determinação prévia do genótipo da tiopurina metiltransferase (TPMT) pode identificar pacientes de maior risco de toxicidade a droga. Os objetivos deste estudo foram avaliar a prevalência dos polimorfismos do gene da TPMT em pacientes com DII acompanhados no Hospital Universitário Pedro Ernesto (HUPE) da UERJ, comparando com a prevalência em outras populações e correlacionar a presença desses polimorfismos com a toxicidade às drogas. Foram avaliados 146 pacientes com doença de Crohn (DC) e 73 com retocolite ulcerativa idiopática (RCUI). A pesquisa dos principais genótipos da TPMT (*2, *3, *3C) foi realizada por técnicas de PCR (alelo específico e RFLP). Os achados clínicos foram correlacionados com a genotipagem e avaliados por análises multivariadas. Dentre os pacientes que estavam em uso de azatioprina, 14 apresentaram pancreatite ou elevação de enzimas pancreáticas, 6 apresentaram hepatoxicidade e 2 evoluíram com neutropenia. Os polimorfismos do gene da TPMT foram observados em 37 dos 219 pacientes (8 foram heterozigotos para o genótipo *2, 11 heterozigotos para *3A e 18 foram heterozigotos para o polimorfismo *3C). Não foi observado nenhum homozigoto polimórfico. Uma correlação positiva foi observada entre a elevação de enzimas pancreáticas e os genótipos *2 e *3C. A prevalência dos polimorfismos neste estudo (16,89%) foi maior que a descrita para população caucasiana e em outros estudos brasileiros. Apesar do predomínio do genótipo *3C, não houve ocorrência exclusiva de um polimorfismo, conforme observado em outras populações. A população brasileira devido à sua miscigenação têm características genotípicas próprias diferentes do outros países do mundo. Dois polimorfismos da TPMT (*2 e *3C) estiveram associados à toxicidade ao uso da azatioprina em pacientes com DII no sudeste do Brasil. O teste genético pode auxiliar na escolha da melhor droga e na dose ideal para os pacientes portadores de DII antes do início do tratamento. / Although azathioprine is commonly used for treating patients with inflammatory bowel disease (IBD), there is still concern about potential severe side effects. Determining the Thiopurine-Methyl-Transferase (TPMT) genotype is expected to identify patients predisposition in respect of toxicity prior to azathioprine administration. The aim of this study was to analyze the prevalence of TPMT genotypes and association with drug toxicity in IBD patients from south-eastern Brazil, constituted by a heterogeneous population. We analyzed 146 Crohns disease (CD) and 73 ulcerative colitis (UC) patients, for the presence of the major TPMT genetic variants (TPMP*2, *3A, *3C) by means of specific allele and RFLP PCR. Clinical data were systematically recorded and correlated with the genotype results, and analyzed in multivariate models. Among the side effects recorded from patients taking azathioprine, 14 presented pancreatitis and/or elevation of pancreatic enzymes, while 6 had liver toxicity, and 2 had neutropenia. TPMT polymorphisms were detected in 37/219 patients (8 heterozygous for *2, 11 heterozygous for *3A and 18 heterozygous for *3C). No homozygotic polymorphisms were found. A positive correlation was found between the elevation of pancreatic enzymes and either TPMT*2 or TPMT*3C. The prevalence of polymorphisms in this study (16,89%) was greater than described with caucasian population or other Brazilian studies. Despite the prevalence of genotype *3C, it was not the only polymorphism shown as observed in other populations. Two genetic variants of TPMT gene (*2 and *3C) are associated with azathioprine toxicity in IBD patients from a south-eastern Brazilian population. Genetic testing may help in the selection of the most appropriate medication prior to treatment initiation.

A azatioprina e a 6 mercaptopurina (6-MCP) são drogas muito utilizada no tratamento das doenças inflamatórias intestinais (DII), porém estão associadas a vários efeitos colaterais. A determinação prévia do genótipo da tiopurina metiltransferase (TPMT) pode identificar pacientes de maior risco de toxicidade a droga. Os objetivos deste estudo foram avaliar a prevalência dos polimorfismos do gene da TPMT em pacientes com DII acompanhados no Hospital Universitário Pedro Ernesto (HUPE) da UERJ, comparando com a prevalência em outras populações e correlacionar a presença desses polimorfismos com a toxicidade às drogas. Foram avaliados 146 pacientes com doença de Crohn (DC) e 73 com retocolite ulcerativa idiopática (RCUI). A pesquisa dos principais genótipos da TPMT (*2, *3, *3C) foi realizada por técnicas de PCR (alelo específico e RFLP). Os achados clínicos foram correlacionados com a genotipagem e avaliados por análises multivariadas. Dentre os pacientes que estavam em uso de azatioprina, 14 apresentaram pancreatite ou elevação de enzimas pancreáticas, 6 apresentaram hepatoxicidade e 2 evoluíram com neutropenia. Os polimorfismos do gene da TPMT foram observados em 37 dos 219 pacientes (8 foram heterozigotos para o genótipo *2, 11 heterozigotos para *3A e 18 foram heterozigotos para o polimorfismo *3C). Não foi observado nenhum homozigoto polimórfico. Uma correlação positiva foi observada entre a elevação de enzimas pancreáticas e os genótipos *2 e *3C. A prevalência dos polimorfismos neste estudo (16,89%) foi maior que a descrita para população caucasiana e em outros estudos brasileiros. Apesar do predomínio do genótipo *3C, não houve ocorrência exclusiva de um polimorfismo, conforme observado em outras populações. A população brasileira devido à sua miscigenação têm características genotípicas próprias diferentes do outros países do mundo. Dois polimorfismos da TPMT (*2 e *3C) estiveram associados à toxicidade ao uso da azatioprina em pacientes com DII no sudeste do Brasil. O teste genético pode auxiliar na escolha da melhor droga e na dose ideal para os pacientes portadores de DII antes do início do tratamento. / Although azathioprine is commonly used for treating patients with inflammatory bowel disease (IBD), there is still concern about potential severe side effects. Determining the Thiopurine-Methyl-Transferase (TPMT) genotype is expected to identify patients predisposition in respect of toxicity prior to azathioprine administration. The aim of this study was to analyze the prevalence of TPMT genotypes and association with drug toxicity in IBD patients from south-eastern Brazil, constituted by a heterogeneous population. We analyzed 146 Crohns disease (CD) and 73 ulcerative colitis (UC) patients, for the presence of the major TPMT genetic variants (TPMP*2, *3A, *3C) by means of specific allele and RFLP PCR. Clinical data were systematically recorded and correlated with the genotype results, and analyzed in multivariate models. Among the side effects recorded from patients taking azathioprine, 14 presented pancreatitis and/or elevation of pancreatic enzymes, while 6 had liver toxicity, and 2 had neutropenia. TPMT polymorphisms were detected in 37/219 patients (8 heterozygous for *2, 11 heterozygous for *3A and 18 heterozygous for *3C). No homozygotic polymorphisms were found. A positive correlation was found between the elevation of pancreatic enzymes and either TPMT*2 or TPMT*3C. The prevalence of polymorphisms in this study (16,89%) was greater than described with caucasian population or other Brazilian studies. Despite the prevalence of genotype *3C, it was not the only polymorphism shown as observed in other populations. Two genetic variants of TPMT gene (*2 and *3C) are associated with azathioprine toxicity in IBD patients from a south-eastern Brazilian population. Genetic testing may help in the selection of the most appropriate medication prior to treatment initiation.

Cette étude a pour objectifs 1) d'évaluer l'activité érythrocytaire de l'inosine triphosphate pyrophosphatase (ITPA) et de l'inosine monophosphate deshydrogénase (IMPDH) en lien avec le suivi des métabolites thiopuriques et le phénotypage de la thiopurine S-méthyltransférase (TPMT), 2) d'étudier le lien entre l'activité de l'ITPA et la survenue d'effets indésirables observés sous azathioprine (AZA).L'étude rétrospective réalisée sur une large population de sujets adultes et enfants, sains et atteints de maladies auto-immunes, a permis d'identifier une distribution quadrimodale de l'activité de l'ITPA à l'aide d'un modèle de mélange gaussien ainsi qu'une faible variabilité intra-individuelle de cette activité. Dans la population pédiatrique, une activité de l'ITPA basse est corrélée à une augmentation des dérivés méthylés suggérant un risque d'hépatotoxicité. Le lien observé entre l'activité ITPasique, le volume globulaire moyen et les gammaglobulines chez les enfants atteints de maladie inflammatoire chronique de l'intestin sous AZA suggère que la mesure de l'activité de l'ITPA permettrait d'anticiper la persistance d'un syndrome inflammatoire chez les sujets à activité élevée.L'étude phénotypique de l'IMPDH montre une importante variabilité inter-individuelle de l'activité de cette enzyme et l'absence d'influence de l'âge, du sexe et du traitement par AZA sur cette activité. Une distribution bimodale de l'activité de l'IMPDH érythrocytaire a été observée ainsi qu'un lien entre cette activité et les dérivés méthylés.En perspective, l'étude combinée des activités de l'ITPA, l'IMPDH et de la TPMT sur la variabilité de la réponse aux traitements thiopuriques sera envisagée / The aims of this study are 1) to evaluate inosine triphosphate pyrophosphatase (ITPA) and inosine monophoshate dehydrogenase (IMPDH) activities in red blood cells (RBCs) in relation to the monitoring of thiopurine metabolites and the phenotyping of thiopurine S-methyltransferase (TPMT), 2) to investigate a possible link between ITPA activity and the occurence of adverse effects observed under azathioprine (AZA) treatment.The retrospective study was carried out on a large population of healthy adults and children as well as those suffering from immunological diseases. A quadrimodal distribution of ITPA was identified among the population using a gaussian mixture model. A weak intraindividual variability of ITPA activity was found. In the paediatric population, a low ITPA activity is correlated with increased levels of methyl nucleotides, suggesting a risk of hepatotoxicity. The relationship observed between ITPA activity and both mean corpuscular volume and gammaglobulin levels in IBD children on AZA therapy suggests that the determination of ITPA activity may allow the prediction of a persistent inflammatory process in subjects with elevated ITPA activity.The phenotypic study of IMPDH shows no influence of age, sex and AZA therapy on the activity of IMPDH. Moreover a large interindividual variability in the activity of IMPDH was found. A bimodal distribution of IMPDH activity in RBCs was observed as well as a relation between this activity and the methyl nucleotides.Further study on the combined effect of the three enzymes ITPA, IMPDH and TPMT on the variability of response to thiopurine therapy will be considered

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.