Metabolic syndrome is a prevalent disease characterized by concurrent manifestation of insulin resistance, obesity, dyslipidemia, hypertension and other hemodynamic and metabolic disorders. It has multifactorial type of inheritance and its resultant phenotype is determined by both environmental and genetic factors as well as their interactions. That is the main reason why comprehensive analysis of the genetic component of this syndrome is complicated in human population. Genetically designed experimental animal models are significant tools for analysis of genetic architecture of human complex conditions including the metabolic syndrome. The aim of this Thesis is utilization of functional and comparative genomic tools to uncover pathogenesis of metabolic syndrome aspects and their genetic determinants. We also studied pharmacogenetic interactions of these genetic determinants with drugs affecting particular components of the metabolic syndrome. Establishing and utilizing several genetically designed congenic rat strains, we undertook four different research projects focusing on pharmacogenetic interaction of all-trans retinoic acid and ondansetron with differential segment of rat chromosome 8, pharmacogenetic interaction of differential segment of rat chromosome 4 and dexamethasone, determining Plzf...

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.

Metabolic syndrome is a prevalent disease characterized by concurrent manifestation of insulin resistance, obesity, dyslipidemia, hypertension and other hemodynamic and metabolic disorders. It has multifactorial type of inheritance and its resultant phenotype is determined by both environmental and genetic factors as well as their interactions. That is the main reason why comprehensive analysis of the genetic component of this syndrome is complicated in human population. Genetically designed experimental animal models are significant tools for analysis of genetic architecture of human complex conditions including the metabolic syndrome. The aim of this Thesis is utilization of functional and comparative genomic tools to uncover pathogenesis of metabolic syndrome aspects and their genetic determinants. We also studied pharmacogenetic interactions of these genetic determinants with drugs affecting particular components of the metabolic syndrome. Establishing and utilizing several genetically designed congenic rat strains, we undertook four different research projects focusing on pharmacogenetic interaction of all-trans retinoic acid and ondansetron with differential segment of rat chromosome 8, pharmacogenetic interaction of differential segment of rat chromosome 4 and dexamethasone, determining Plzf...

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be quite separate temporally from time of intake) and the more temporally “relevant” effects (primarily olfactory and taste) that bridge the time from intake to the onset of the pharmacological effects. Dissociating these effects is essential to untangling the neurologic underpinnings of alcohol abuse and dependence. Intravenous self-administration of ethanol allows for controlled and precise dosing, bypasses first order absorption kinetics allowing for a faster onset of pharmacologic effects, and eliminates the confounding “non-pharmacological” effects associated with oral consumption. Intravenous self-administration of ethanol has been reliably demonstrated in both mouse and human experimental models; however, consistent intravenous self-administration of pharmacologically relevant levels of ethanol remains elusive in the rat. Previous work has demonstrated reliable elevated intravenous ethanol self administration using a compound reinforcer of oral sucrose and intravenous ethanol. The present study sought to elucidate the role of each component of this reinforcer complex using a multiple schedule study design. Male P rats had free access to both food and water during all intravenous self-administration sessions and all testing was performed in conjunction with the onset of the dark cycle. Once animals achieved stable operant responding on both levers for an orally delivered 1% sucrose solution (1S) on a FR4 schedule, surgery was conducted to implant an indwelling jugular catheter. Animals were habituated to the attachment of infusion apparatus and received twice daily sessions for four days to condition each lever to its associated schedule. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5 minute components. During one component only oral 1S was presented, while in the second component a compound reinforcer of oral 1S + IV 20% ethanol was presented (25 mg/kg/injection). Both levers were extended into the chamber during the session, with the active lever/schedule alternating as the session progressed across components. Average ethanol intake was 0.47 ± 0.04 g/kg. A significant increase in sucrose only reinforcers and sucrose lever error responding was found suggesting that sucrose not ethanol is responsible for driving overall responding. The current findings suggest that the existing intravenous ethanol self-administration methodology remains aversive in the rat.

La pharmacogénomique (PGx) étudie le concept selon lequel les déterminants génétiques peuvent aider à prédire la réponse clinique d’un patient aux médicaments. Les concentrations plasmatiques de ces derniers sont essentielles pour déterminer l’exposition, les profils pharmacocinétiques (PK), les effets cliniques et éventuellement les doses des médicaments, dont la plupart sont métabolisés par des enzymes hépatiques, les cytochromes P450 (CYPs). Néanmoins, la plupart des découvertes en matière de PGx concernant la prédiction des profils de concentrations des médicaments ont généralement recours à des plans d’études PK traditionnels avec une approche fonctionnelle. Bien qu’utile, cette méthodologie comporte des limites pour les études PGx, notamment le nombre restreint de sujets inclus, qui réduit la puissance statistique des associations PGx et limite l’identification de nouveaux variants génétiques moins fréquents. À l’inverse, les grandes cohortes observationnelles sont largement utilisées pour identifier des marqueurs génétiques physiopathologiques. Cette thèse de doctorat visait donc à 1) synthétiser les données publiées concernant les effets cliniques des polymorphismes génétiques de l’enzyme CYP2D6 sur le traitement au métoprolol, un agent β-bloquant. Les concentrations plasmatiques de métoprolol ont montré à plusieurs reprises qu’elles étaient fortement influencées par la PGx du CYP2D6; 2) développer une nouvelle méthode bioanalytique capable de quantifier les concentrations chirales de métoprolol des patients dans un contexte clinique; 3) mener une étude clinique en utilisant une grande cohorte observationnelle, ou biobanque, comme preuve de concept pour recréer l’association précédemment établie entre les phénotypes inférés des génotypes du CYP2D6 et les concentrations plasmatiques de métoprolol. Ces projets sont présentés en tant que chapitres de thèse et sous forme de manuscrits publiés. Le premier projet consistait en une revue systématique qui a permis d’extraire toutes les études relatives à la PGx du métoprolol-CYP2D6. La synthèse qualitative a suggéré que les métaboliseurs lents du CYP2D6, dépourvus de capacité enzymatique, avaient des valeurs plus élevées concernant les réductions de la fréquence cardiaque et de tension artérielle, ainsi que la survenue d’épisodes bradycardiques relativement aux autres phénotypes. Une méta-analyse ultérieure a confirmé la significativité de ces associations. Le deuxième projet a combiné des techniques bioanalytiques telles que la dérivation, l’extraction en phase solide et la chromatographie liquide avec spectrométrie de masse en tandem. Une méthode permettant de surmonter les limites analytiques antérieures a été validée avec succès pour mesurer les concentrations plasmatiques de (S)-métoprolol, l’énantiomère pharmacologiquement actif, et de son métabolite spécifique au CYP2D6. L’applicabilité d’une telle méthode a ensuite été démontrée grâce aux échantillons d’un groupe de patients issus de la Cohorte Hospitalière de l’Institut de Cardiologie de Montréal (ICM). Puis, le troisième projet présente la réalisation de l’étude LEVEL-PGx (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing : A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). L’étude portait sur un échantillon de >1000 patients sélectionnés dans la cohorte hospitalière de l’ICM, incluant leur génotypage pour CYP2D6 et la quantification du métoprolol racémique et de son métabolite spécifique au CYP2D6 dans des échantillons provenant de la Biobanque de l’ICM. Un seul échantillon unique et aléatoire par patient a été utilisé. Le recours à des modèles multivariables a validé le concept selon lequel de grandes cohortes transversales recueillant des échantillons biologiques pouvaient être utilisées afin d’identifier des associations PGx de concentrations de médicaments et ce, à des valeurs satisfaisant les seuils de significativité d’essais pangénomiques. D’autres analyses de cette cohorte ont indiqué que cette méthodologie parvenait à identifier des associations PGx qui influençaient la fréquence cardiaque au repos et la posologie du métoprolol à-travers les phénotypes du CYP2D6 et pour les déterminants génétiques uniques, même en présence de co-médications. Cependant, ces associations PGx avec les paramètres cliniques n’ont pas atteint une significativité applicable aux seuils pangénomiques. En résumé, par la reproduction d’une association PGx préalablement démontrée, l’ensemble des travaux présentés dans cette thèse suggère que l’identification et la découverte de nouveaux déterminants génétiques prédictifs des concentrations et des doses des médicaments pourrait s’effectuer par le biais de grandes cohortes observationnelles à l’échelle du génome. Ces approches permettraient de développer des modèles prédictifs plus précis de l’exposition et de la réponse aux médicaments, ce qui pourrait favoriser les découvertes PGx et, dans certains cas, éventuellement développer le potentiel translationnel d’une approche thérapeutique personnalisée selon le profil génétique des patients. / Pharmacogenomics (PGx) studies the concept that genetic determinants can help predict a patient’s clinical response to therapies. Drug concentrations are an essential component to determining the exposure, pharmacokinetic (PK) profiles, clinical effects, and potentially drug doses, most of which are metabolized through the cytochrome P450 (CYPs) liver enzymes. Nevertheless, most PGx discoveries regarding the prediction of drug concentration profiles have generally resorted to traditional PK study designs with a functional approach. Though useful, this methodology contains limitations for gene-drug interaction studies, most notably the restricted number of subjects included, which reduces the statistical power for PGx associations and limits the identification of new, less frequent genetic variants. On the opposite, large observational cohorts have long been utilized for identifying genetic markers of disease. This doctoral thesis therefore aimed to 1) synthesize published data regarding the clinical effects of CYP2D6 genetic polymorphism on metoprolol therapy. A β-blocker, metoprolol plasma concentrations have shown repeatedly to be heavily influenced by the PGx of the CYP2D6 enzyme; 2) develop a new bioanalytical method able to quantify patients’ chiral concentrations of metoprolol in a clinical setting; 3) conduct a clinical study using a large observational cohort, or biobank, as a proof of concept to recreate the previously established association between CYP2D6 genotype-inferred phenotypes and metoprolol plasma concentrations. Those projects are presented as thesis chapters in the form of published manuscripts. The first project was a systematic review that allowed us to find all studies pertaining to the PGx of metoprolol. The qualitative synthesis suggested that CYP2D6 poor metabolizers (PMs), without enzymatic capacity, had greater values regarding reductions in heart rate, blood pressures, and occurrences in bradycardia relative to non-PMs. A subsequent meta-analysis confirmed the significance of those associations. The second project combined bioanalytical techniques such as derivatization, solid phase extraction, and liquid chromatography-tandem mass spectrometry. A method overcoming previous analytical shortcomings was successfully validated to measure (S)-metoprolol plasma concentrations and its CYP2D6-specific metabolite. Its application was later demonstrated in a group of patients from the Montreal Heart Institute (MHI) Hospital Cohort. Then, the third project presents the conduct of the LEVEL-PGx study (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing: A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). The study implicated a sample of >1000 selected patients selected from the MHI Hospital Cohort, along with the genotyping of CYP2D6, and the quantification of racemic metoprolol and its CYP2D6-specific metabolite in samples from the MHI Biobank. A single, random sample per patient was used. Multivariable modeling validated the concept that large observational cohorts collecting biospecimens could be utilized to identify PGx associations of drug concentrations with genome-wide significance. Further analyses in our cohort indicated that the tested PGx associations influenced resting heart rate and metoprolol daily drug dosage across CYP2D6 phenotypes and for single genetic determinants, regardless of interfering comedications. However, such PGx associations with clinical parameters could not achieve genome-wide significance. In summary, the body of work presented in this thesis suggested that, using a previously validated PGx association, the identification of novel genetic determinants predictive of drug concentrations and dosage could be discovered and identified at the genome-wide level with large observational cohorts. These approaches would help develop more accurate predictive models of drug exposure and response, which could favor PGx discoveries and the translational potential of a personalized approach to treatments according to a patient’s genetic profile.

L’insuffisance rénale chronique (IRC) est un problème majeur fréquemment rencontré chez les greffés cardiaques. Les inhibiteurs de la calcineurine, pierre angulaire de l’immunosuppression en transplantation d’organes solides, sont considérés comme une des principales causes de dysfonction rénale postgreffe. Plusieurs autres éléments tels que les caractéristiques démographiques, cliniques et génétiques du receveur contribuent également au phénomène, mais il demeure plutôt difficile de déterminer quels sont les patients les plus à risque de développer une IRC après la transplantation. Ainsi, la découverte de nouveaux marqueurs génétiques de dysfonction rénale pourrait un jour mener à l’individualisation de la thérapie immunosuppressive selon le profil génétique de chaque patient. Or, on ne connaît pas les opinions des greffés à l’égard des tests pharmacogénomiques et l’on ne sait pas si celles-ci diffèrent des opinions exprimées par les individus en bonne santé. Cette thèse de doctorat a donc pour objectifs : 1- De décrire l’évolution de la fonction rénale à très long terme après la transplantation et d’identifier les marqueurs démographiques et phénotypiques associés à l’IRC postgreffe cardiaque; 2- D’identifier les marqueurs génétiques associés à la néphrotoxicité induite par les inhibiteurs de la calcineurine; 3- D’évaluer et de comparer les attitudes des patients et des individus en bonne santé par rapport à l’intégration clinique potentielle des marqueurs pharmacogénomiques. Trois projets ont été réalisés pour répondre à ces questions. Le premier repose sur une analyse rétrospective de l’évolution de la fonction rénale chez les patients greffés au sein de notre établissement entre 1983 et 2008. Nous y avons découvert que le déclin de la fonction rénale se poursuit jusqu’à 20 ans après la transplantation cardiaque et que les facteurs de risque d’IRC incluent entre autres l’âge avancé, le sexe féminin, la dysfonction rénale prégreffe, l’hypertension, l’hyperglycémie et l’utilisation de la prednisone. Le deuxième projet est une étude pharmacogénomique s’intéressant aux déterminants génétiques de la néphrotoxicité induite par les inhibiteurs de la calcineurine. Elle nous a permis d’illustrer pour la première fois qu’un polymorphisme génétique lié à PRKCB (gène codant pour la protéine kinase C-β) est associé avec la fonction rénale des patients greffés cardiaques, alors que cela n’est probablement pas le cas pour les polymorphismes de TGFB1 (gène codant pour le transforming growth factor-β1). La troisième section de cette thèse rapporte les résultats d’un questionnaire dont le but était de comparer les attitudes envers les tests pharmacogénomiques parmi un groupe de personnes en bonne santé, de patients greffés cardiaques et de patients souffrant d’insuffisance cardiaque. Cette étude a démontré que, bien que l’enthousiasme pour la pharmacogénomique soit partagé par tous ces individus, les craintes liées à la confidentialité et aux répercussions potentielles sur l’emploi et les assurances sont plus prononcées chez les personnes en bonne santé. En résumé, les travaux issus de cette thèse ont révélé que l’identification précoce des patients greffés cardiaques les plus susceptibles de présenter une détérioration de la fonction rénale ainsi que l’adoption d’une approche thérapeutique individualisée reposant notamment sur les applications cliniques de la pharmacogénomique pourraient éventuellement permettre de freiner cette complication postgreffe. / Chronic kidney disease (CKD) is a major problem frequently observed in cardiac transplant recipients. Calcineurin inhibitors, which have become the cornerstone of immunosuppressive treatments in solid organ transplantation, are considered a major cause of post-transplant renal dysfunction. Several other factors such as recipients’ demographic, clinical and genetic characteristics also contribute to this phenomenon, but it remains rather difficult to determine which patients present the highest risk of CKD after transplantation. Discovery of new genetic markers of renal dysfunction could one day lead to individualization of immunosuppressive therapy according to each patient’s genetic profile. However, transplant patients’ opinions towards pharmacogenomic testing remain unknown, and it is unclear whether these differ from healthy individuals’ opinions. This doctoral thesis thus aims: 1- To describe the very long-term evolution of renal function after transplantation and to identify demographic and phenotypic markers associated with postheart transplant CKD; 2- To identify genetic markers associated with calcineurin inhibitor-induced nephrotoxicity; 3- To assess and to compare the attitudes of patients and healthy individuals concerning the potential integration of pharmacogenomic markers in clinical practice. Three projects have been conducted to answer these questions. The first one relies on a retrospective analysis of the evolution of renal function in patients who received a heart transplantation at our institution between 1983 and 2008. We discovered that deterioration of renal function continues up to 20 years after transplant and that risk factors of CKD include, among others, advanced age, female gender, pretransplant renal dysfunction, hypertension, hyperglycemia and use of prednisone. The second project is a pharmacogenomic study looking at genetic determinants of calcineurin inhibitor-induced nephrotoxicity. We were able to illustrate for the first time that a genetic polymorphism related to PRKCB (gene encoding protein kinase C-β) is associated with renal function in heart transplant patients, whereas it is probably not the case for polymorphisms in TGFB1 (gene encoding transforming growth factor-β1). The third section of this thesis reports the results of a questionnaire whose purpose was to compare the attitudes towards pharmacogenomic testing in a group of healthy volunteers, cardiac transplant recipients and heart failure patients. This study demonstrated that, although enthusiasm regarding pharmacogenomics is shared equally among these individuals, preoccupations related to confidentiality and potential impacts on employment and insurance are more important in healthy volunteers. In summary, the work presented in this thesis showed that early identification of heart transplant patients who are most likely to develop renal dysfunction as well as adoption of an individualized therapeutic approach involving clinical applications of pharmacogenomics could potentially help to prevent this post-transplant complication.

Contexte. L’insuffisance cardiaque (IC) touche 1 à 2% de la population et sa prévalence menace d’augmenter dans la population vieillissante. L’IC et ses nombreux facteurs de risque ont une forte composante héréditaire. Il existe plusieurs phénotypes d’IC qui se démarquent par leurs mécanismes physiopathologiques et leur pronostic. Objectif. Ce mémoire vise dans un premier temps à faire état des connaissances actuelles sur la pharmacogénomique de l’IC et, dans un deuxième temps, à décrire une cohorte de patients atteints d’IC montée dans la cadre d’une étude cas-contrôle. Les caractéristiques descriptives de différents sous-groupes de patients sont présentées et comparées avec les données épidémiologiques actuelles. Résultats. 829 patients atteints d’IC qui ont participé au projet de la Biobanque de l’Institut de cardiologie de Montréal étaient éligibles à notre étude. L’âge moyen de ces cas est de 66,1 ± 10,2 ans et 76,6% sont des hommes. La fraction d’éjection du ventricule gauche (FEVG) médiane est 38% (intervalle interquartile : 28–52%) et 55,0% des cas sont d’étiologie ischémique. L’IC à FEVG réduite représente 55,4% des sujets; les phénotypes d’IC à FEVG intermédiaire et préservée représentent 11,2% et 33,4%, respectivement. Conclusion. Le phénotype d’IC à FEVG préservée est sous-représenté dans notre cohorte par rapport à ce qui est décrit dans la littérature scientifique, avec un nombre plus élevé d’hommes et l’étiologie ischémique majoritaire. Ceci s’explique par le fait que l’Institut de cardiologie est un centre référent de greffe. Toutefois, les caractéristiques des principaux phénotypes d’IC concordent avec la littérature scientifique, ce qui suggère que les phénotypes d’IC dans notre cohorte sont représentatifs de ces sous-populations. / Context. Heart failure (HF) affects 1 to 2% of the population and its prevalence is expected to increase in the aging population. Recent evidence suggests that HF and associated risk factors are heritable. HF has multiple phenotypes each associated with different risk factors, pathophysiology and prognosis. Objective. This project presents the current knowledge on HF pharmacogenomics found in the scientific literature and describes a cohort of HF patients who participated in a case-control study. The characteristics of different subgroups are presented and compared to current epidemiologic data. Results. We recruited 829 HF patients who participated in the Montreal Heart Institute biobank project. The mean age is 66,1 ± 10,2 years and 76,6% are male. The median left ventricular ejection fraction (LVEF) is 38% (interquartile range: 28–52%) and ischemic etiology accounts for 55,0% of HF cases. The HF with a reduced LVEF phenotype represents 55,4% of participants while 11,2% and 33,4% of cases have HF with a mid-range LVEF and HF with a preserved LVEF, respectively. Conclusion. The HF with a preserved LVEF phenotype was underrepresented in our cohort compared to what is expected in the scientific literature. We report more men and ischemic etiology, which could be explained by the fact that the Montreal Heart Institute is a reference center for transplantation. Characteristics of the major HF phenotypes are consistent with those reported in the scientific literature, thus suggesting that our cohort is representative of these sub-groups.

O receptor scavenger classe B tipo I (SR-BI) media a captação seletiva do colesterol da lipoproteina de alta densidade (HDL) e participa no effluxo do colesterol livre para aceptores lipoprotéicos. A HDL tem um importante rol aterogênico associado com sua participação no transporte reverso do colesterol. Polimorfismos no gene que codifica para o SR-BI (SCARB1) foram relacionados com alterações do perfil lipídico sérico e outros fatores de risco associados com doença cardiovascular. As estatinas são inibidores da síntese do colesterol utilizados no tratamento da dislipidemia. Vários polimorfismos em genes envolvidos no metabolismo intermediario de lipideos foram relacionados com diferenças na resposta a hipolipemiantes. Com a finalidade de avaliar o efeito de polimorfismos do SCARB1 sobre o perfil lipídico sérico, expressão gênica e a resposta a estatinas, foram selecionados 185 indivíduos normolipidêmicos (NL) e 147 pacientes hipercolesterolêmicos (HC). Os pacientes HC foram tratados com atorvastatina (10 mg/dia/4 semanas). DNA e RNA foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G4A, In5C>T e Ex8C>T foram detectados por PCR-RFLP. A expressão de RNAm do SCARB1 em células mononucleares de sangue periférico (CMSP) foi analisada por PCR em tempo real usando o gene da Ubiquitina c (UBC) como referência endógena. Nos indivíduos HC, as freqüências dos alelos raros G4A (12%), In5C>T (7%) e Ex8C>T (40%), no grupo HC, foram similares às encontradas no grupo NL (4A: 15%, In5T: 7%, e Ex8T: 35%, p>0,05). O alelo SCARB1 4A (genótipos GA + AA) foi associado com valores diminuídos de apoAI no grupo NL. O alelo In5T foi associado com maior concentração LDL-C sérico (p=0,029), em NL, e com apoB e razão apoB/apoAI elevadas (p>0,05) no grupo HC. O SNP SCARB1 Ex8C>T não foi relacionado com o perfil lipídico sérico basal, embora os portadores do genótipo Ex8CC foram associados com resposta reduzida ao tratamento com atorvastatina mostrando menor variação de colesterol total, LDL-C, apoB e razão apoB/apoAI. O SNP Ex8C>T foi associado com maior probabilidade (OR=3,1; 95% IC: 1,00-9,5; p=0,044) de ter uma resposta à atorvastatina diminuída. Os SNPs SCARB1 In5C>T e Ex8C>T estão em desequilíbrio de ligação. O haplótipo G1C5C8/G1T5C8 foi associado com concentrações basais elevadas de triglicérides e VLDL-C em NL e diminuídas de HDL-C e apoAI em HC. Os haplótipos G1C5C8/A1C5C8 e C5C8/C5C8 tiveram variação diminuída da apoB quando comparados com os outros haplótipos, G1C5C8/A1C5C8 e o diplótipo C5C8/C5C8 também apresentou uma variação reduzida da razão apoB/apoAI. Os SNPs G4A e In5C>T estão associados com diminuição da expressão gênica do SCARB1 em NL. O tratamento com atorvastatina não modifica a expressão de RNAm do SCARB1 em CMSP nos HC. Esses resultados são sugestivos de que os polimorfismos no SCARB1 estão associados com valores basais do perfil lipídico sérico e de expressão de RNAm do SCARB1, assim como de resposta à atorvastatina. / The scavenger receptor class B type I (SR-BI) mediates the selective uptake of the high density lipoprotein (HDL) cholesterol and it participates in the free cholesterol efflux to lipoprotein acceptors. HDL has an important antiatherogenic role associated with important activity in the cholesterol reverse transport. Polymorphisms in the SR-BI gene (SCARB1) have been related to variations on plasma lipoprotein profile and other risk factors for cardiovascular disease. Statins are potent inhibitors of cholesterol synthesis prescribed for treatment of the dislipidemia. Several polymorphisms in genes involved in intermediary metabolism of lipids have been related to differences in response to lowering-cholesterol drugs. In order to evaluate the effect of SCARB1 polymorphisms on serum lipids, gene expression and lipid-lowering response to atorvastatin, 185 normolipidemic (NL) and 147 hypercholesterolemic (HC) individuals were selected. HC individuals were treated with atorvastatin (10 mg/day/4 weeks). DNA and RNA were extracted from peripheric blood mononuclear cells (PBMC). SCARB1 mRNA expression was analyzed by real time PCR using ubiquitin c gene (UBC) as endogenous reference. The frequencies of the rare alleles in HC group (G4A: 12%; In5C>T: 7%, and ExC>T: 39%) were similar to those found in NL individuals (4A: 15%, In5T: 7%, and Ex8T: 35%, p>0.05). The SCARB1 4A allele (GA+AA genotypes) was associated with lower apoAI concentration in NL. The In5T allele was associated with higher serum LDL-C (p=0,029) in NL individuals, and with higher apoB and apoB/apoAI ratio (p>0,05) in HC group. SCARB1 Ex8C>T SNP was not related to serum lipids profile, however Ex8CC genotype carriers had lower variation of total cholesterol, LDL-C, apoB and apoB/apoAI ratio in response to atorvastatin. SCARB1 Ex8C>T was associated with higher chance to have a lower atorvastatin response (OR=3.1, 95% CI: 1.00-9.5; p=0.044). SCARB1 In5C>T and ExC>T were in linkage disequilibrium. G1C5C8/G1T5C8 SCARB1 haplotype was associated with higher level of triglycerides and VLDL-C in NL and lower HDL-C and apoAI levels in HC individuals. G1C5C8/A1C5C8 haplotype and C5C8/C5C8 diplotype had lower variations on apoB than the other haplotypes, and G1C5C8/A1C5C8 had also lower variation on apoB/apoAI ratio. G4A and In5C>T SNPs are associated with lower SCARB1 mRNA expression in PBMC of NL individuals. Atorvastatin therapy did not modify the expression level of the SCARB1 transcript in HC. Our results suggest that SCARB1 polymorphisms are associated with basal serum lipids profile, mRNA SCARB1 expression and atorvastatin response.

O receptor scavenger classe B tipo I (SR-BI) media a captação seletiva do colesterol da lipoproteina de alta densidade (HDL) e participa no effluxo do colesterol livre para aceptores lipoprotéicos. A HDL tem um importante rol aterogênico associado com sua participação no transporte reverso do colesterol. Polimorfismos no gene que codifica para o SR-BI (SCARB1) foram relacionados com alterações do perfil lipídico sérico e outros fatores de risco associados com doença cardiovascular. As estatinas são inibidores da síntese do colesterol utilizados no tratamento da dislipidemia. Vários polimorfismos em genes envolvidos no metabolismo intermediario de lipideos foram relacionados com diferenças na resposta a hipolipemiantes. Com a finalidade de avaliar o efeito de polimorfismos do SCARB1 sobre o perfil lipídico sérico, expressão gênica e a resposta a estatinas, foram selecionados 185 indivíduos normolipidêmicos (NL) e 147 pacientes hipercolesterolêmicos (HC). Os pacientes HC foram tratados com atorvastatina (10 mg/dia/4 semanas). DNA e RNA foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G4A, In5C>T e Ex8C>T foram detectados por PCR-RFLP. A expressão de RNAm do SCARB1 em células mononucleares de sangue periférico (CMSP) foi analisada por PCR em tempo real usando o gene da Ubiquitina c (UBC) como referência endógena. Nos indivíduos HC, as freqüências dos alelos raros G4A (12%), In5C>T (7%) e Ex8C>T (40%), no grupo HC, foram similares às encontradas no grupo NL (4A: 15%, In5T: 7%, e Ex8T: 35%, p>0,05). O alelo SCARB1 4A (genótipos GA + AA) foi associado com valores diminuídos de apoAI no grupo NL. O alelo In5T foi associado com maior concentração LDL-C sérico (p=0,029), em NL, e com apoB e razão apoB/apoAI elevadas (p>0,05) no grupo HC. O SNP SCARB1 Ex8C>T não foi relacionado com o perfil lipídico sérico basal, embora os portadores do genótipo Ex8CC foram associados com resposta reduzida ao tratamento com atorvastatina mostrando menor variação de colesterol total, LDL-C, apoB e razão apoB/apoAI. O SNP Ex8C>T foi associado com maior probabilidade (OR=3,1; 95% IC: 1,00-9,5; p=0,044) de ter uma resposta à atorvastatina diminuída. Os SNPs SCARB1 In5C>T e Ex8C>T estão em desequilíbrio de ligação. O haplótipo G1C5C8/G1T5C8 foi associado com concentrações basais elevadas de triglicérides e VLDL-C em NL e diminuídas de HDL-C e apoAI em HC. Os haplótipos G1C5C8/A1C5C8 e C5C8/C5C8 tiveram variação diminuída da apoB quando comparados com os outros haplótipos, G1C5C8/A1C5C8 e o diplótipo C5C8/C5C8 também apresentou uma variação reduzida da razão apoB/apoAI. Os SNPs G4A e In5C>T estão associados com diminuição da expressão gênica do SCARB1 em NL. O tratamento com atorvastatina não modifica a expressão de RNAm do SCARB1 em CMSP nos HC. Esses resultados são sugestivos de que os polimorfismos no SCARB1 estão associados com valores basais do perfil lipídico sérico e de expressão de RNAm do SCARB1, assim como de resposta à atorvastatina. / The scavenger receptor class B type I (SR-BI) mediates the selective uptake of the high density lipoprotein (HDL) cholesterol and it participates in the free cholesterol efflux to lipoprotein acceptors. HDL has an important antiatherogenic role associated with important activity in the cholesterol reverse transport. Polymorphisms in the SR-BI gene (SCARB1) have been related to variations on plasma lipoprotein profile and other risk factors for cardiovascular disease. Statins are potent inhibitors of cholesterol synthesis prescribed for treatment of the dislipidemia. Several polymorphisms in genes involved in intermediary metabolism of lipids have been related to differences in response to lowering-cholesterol drugs. In order to evaluate the effect of SCARB1 polymorphisms on serum lipids, gene expression and lipid-lowering response to atorvastatin, 185 normolipidemic (NL) and 147 hypercholesterolemic (HC) individuals were selected. HC individuals were treated with atorvastatin (10 mg/day/4 weeks). DNA and RNA were extracted from peripheric blood mononuclear cells (PBMC). SCARB1 mRNA expression was analyzed by real time PCR using ubiquitin c gene (UBC) as endogenous reference. The frequencies of the rare alleles in HC group (G4A: 12%; In5C>T: 7%, and ExC>T: 39%) were similar to those found in NL individuals (4A: 15%, In5T: 7%, and Ex8T: 35%, p>0.05). The SCARB1 4A allele (GA+AA genotypes) was associated with lower apoAI concentration in NL. The In5T allele was associated with higher serum LDL-C (p=0,029) in NL individuals, and with higher apoB and apoB/apoAI ratio (p>0,05) in HC group. SCARB1 Ex8C>T SNP was not related to serum lipids profile, however Ex8CC genotype carriers had lower variation of total cholesterol, LDL-C, apoB and apoB/apoAI ratio in response to atorvastatin. SCARB1 Ex8C>T was associated with higher chance to have a lower atorvastatin response (OR=3.1, 95% CI: 1.00-9.5; p=0.044). SCARB1 In5C>T and ExC>T were in linkage disequilibrium. G1C5C8/G1T5C8 SCARB1 haplotype was associated with higher level of triglycerides and VLDL-C in NL and lower HDL-C and apoAI levels in HC individuals. G1C5C8/A1C5C8 haplotype and C5C8/C5C8 diplotype had lower variations on apoB than the other haplotypes, and G1C5C8/A1C5C8 had also lower variation on apoB/apoAI ratio. G4A and In5C>T SNPs are associated with lower SCARB1 mRNA expression in PBMC of NL individuals. Atorvastatin therapy did not modify the expression level of the SCARB1 transcript in HC. Our results suggest that SCARB1 polymorphisms are associated with basal serum lipids profile, mRNA SCARB1 expression and atorvastatin response.

Indiana University-Purdue University Indianapolis (IUPUI) / Traditional treatment strategy development for diseases involves the identification of target proteins related to disease states, and the interference of these proteins with drug molecules. Computational drug discovery and virtual screening from thousands of chemical compounds have accelerated this process. The thesis presents a comprehensive framework of computational drug discovery using system biology approaches. The thesis mainly consists of two parts: disease biomarker identification and disease treatment discoveries. The first part of the thesis focuses on the research in biomarker identification for human diseases in the post-genomic era with an emphasis in system biology approaches such as using the protein interaction networks. There are two major types of biomarkers: Diagnostic Biomarker is expected to detect a given type of disease in an individual with both high sensitivity and specificity; Predictive Biomarker serves to predict drug response before treatment is started. Both are essential before we even start seeking any treatment for the patients. In this part, we first studied how the coverage of the disease genes, the protein interaction quality, and gene ranking strategies can affect the identification of disease genes. Second, we addressed the challenge of constructing a central database to collect the system level data such as protein interaction, pathway, etc. Finally, we built case studies for biomarker identification for using dabetes as a case study. The second part of the thesis mainly addresses how to find treatments after disease identification. It specifically focuses on computational drug repositioning due to its low lost, few translational issues and other benefits. First, we described how to implement literature mining approaches to build the disease-protein-drug connectivity map and demonstrated its superior performances compared to other existing applications. Second, we presented a valuable drug-protein directionality database which filled the research gap of lacking alternatives for the experimental CMAP in computational drug discovery field. We also extended the correlation based ranking algorithms by including the underlying topology among proteins. Finally, we demonstrated how to study drug repositioning beyond genomic level and from one dimension to two dimensions with clinical side effect as prediction features.