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Orientadores: Iscia Teresinha Lopes-Cendes, Fernando Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T21:09:18Z (GMT). No. of bitstreams: 1 Silva_MarianaSaragiottoda_D.pdf: 6874265 bytes, checksum: 8205182499c4fdeb6cca8cbc2c6e6dea (MD5) Previous issue date: 2010 / Resumo: A Epilepsia do Lobo Temporal Mesial (ELTM) representa a mais freqüente síndrome de epilepsia focal, sendo comumente associada à chamada esclerose mesial temporal (EMT). A ELTM tem grande importância clínica devido à alta frequencia de pacientes refratários ao tratamento com drogas antiepilépticas (DAEs). Vários fatores genéticos e ambientais têm sido atribuídos à refratariedade medicamentosa nesses pacientes. Dentre as causas genéticas, acredita-se que variações alélicas, dadas por polimorfismos, em genes metabolizadores e transportadores de drogas podem ser responsáveis pela atuação e metabolização ineficientes das drogas, levando à refratariedade medicamentosa. Deste modo, o objetivo principal deste trabalho é avaliar se polimorfismos em alguns genes candidatos podem estar relacionados com a refratariedade medicamentosa na ELTM. Para tanto, foram selecionados polimorfismos de nucleotídeo único (do inglês single nucelotide polimorphisms - SNPs) em duas categorias funcionais de genes candidatos: i) genes transportadores de drogas: 43 SNPS selecionados em 3 genes, ABCB1, ABCC2, e RLIP76(RALBP1) e ii) genes metabolizadores de drogas pertencentes ao sistema do citocromo P450: 95 SNPs em nove genes, CYP1B1, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2E1, CYP1A1, CYP1A2, CYP2D6. Foram genotipados dois grupos de pacientes não relacionados, com ELTM, sendo 78 pacientes responsivos ao tratamento com DAEs (responsivos às DAEs) e 164 farmacoresistentes (resistentes às DAEs). Para assegurar que ambos os grupos poderiam ser comparáveis do ponto de vista de estrutura genética, todos os pacientes foram genotipados para 119 SNPs adicionais não relacionados aos genes candidatos de interesse. Os cálculos de AMOVA e Fst entre os grupos, mostram que não há estruturação populacional, portanto, ambos os grupos selecionados podem ser comparados entre si em estudos de associação. Após cálculo das frequências gênicas para os 141SNPs genotipados nos genes candidatos, 75 SNPs tiveram que ser descartados pois não se encontravam em equilíbrio de Hardy-Weinberg e apresentaram MAF (do inglês, minor allele frequency) menor que 5%. No grupo dos genes transportadores nossos resultados mostraram associação com um SNP sinônimo rs3740066 (Ile1324Ile), presente no exon 28 do gene ABCC2, onde o alelo C é mais freqüente no grupo de pacientes resistentes às DAE. Além disso, experimentos adicionais mostraram que a expressão do gene ABCC2 em hipocampo de pacientes resistentes às DAEs está aumentada em relação ao hipocampo de indivíduos sem ELTM, provenientes de autópsia. Para os genes do CYP450, foram encontradas associações em SNPs intrônicos presentes nos genes CYP1B1 e CYP1A2, para as variantes genotípicas rs2551188-TT e rs12904742GG, respectivamente, além de duas variantes associadas no gene CYP2C9: rs2153628AA e rs4086116CT, sendo que todas as variantes citadas acima são mais freqüentes nos pacientes resistentes às DAEs. Também foi encontrada associação para variantes alélicas nos genes CYP2C9: rs4086116T, CYP1A2: rs12904742G e CYP2C9: rs1934963C. Do mesmo modo, estes alelos são mais freqüentes em pacientes resistentes às DAEs. Há diferença estatisticamente significativa quanto à presença de atrofia hipocampal entre os grupos, sendo esta mais freqüente no grupo de farmacoresistentes (p=0,0000068), sendo que, através do cálculo de regressão passo a passo, a presença de atrofia hipocampal contribui com 9% de influência na refratariedade medicamentosa no grupo de pacientes analisados. Já os fatores genéticos associados à farmacoresistência analisados neste trabalho contribuem com 12% para o fenótipo da farmacoresistência nestes pacientes. Portanto, pode-se concluir que os 79% restantes de influência na refratariedade medicamentosa em pacientes com ELTM pode ser atribuído a outros fatores ainda não identificados. Finalmente, pode-se afirmar que nosso trabalho fornece fortes evidências indicando que a refratariedade medicamentosa em pacientes com ELTM é de causa poligênica e multifatorial, já que comprovamos a evidente influência genética e ambiental. / Abstract: Mesial temporal lobe epilepsy (MTLE) is the most frequent syndrome of focal epilepsy and it is often associated with mesial temporal sclerosis (MTS). MTLE has great clinical importance because it is associated with a significant proportion of patients who do not respond to treatment with antiepileptic drugs (AEDs). Several genetic and environmental factors have been attributed to failure in response to AEDs in these patients. Among the genetic causes, it is believed that allelic variations in drug-transporter and metabolizing genes may be responsible for inefficient performance and metabolism of drugs, leading to failure in drug response. Thus, the main objective of this study is to verify whether polymorphisms in candidate genes may be related to drug refractory MTLE. To accomplished this objective we selected single nucleotide polymorphisms (SNPs) in two functional categories of candidates genes: i) drug-transporter genes: 43 SNPS in three selected genes, ABCB1, ABCC2, and RLIP76 (RALBP1) and ii) drug-metabolizing genes belonging to the cytochrome P450 system: 95 SNPs in nine genes, CYP1B1, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2E1, CYP1A1, CYP1A2, CYP2D6. Were genotyped two groups of unrelated patients with MTLE, 78 patients who responded to treatment with antiepileptic drugs (AED-responsive) and 164 pharmacoresistants patients (AED-pharmacoresistant), classified according to previously determined clinical criteria. Moreover, to ensure that both groups could be comparable in terms of genetic structure, all patients were genotyped for 119 additional SNPs not related to the candidate genes. Using the AMOVA and Fst calculation we showed that there is no population structure in both groups (AED-responsive and AED-farmacoresistentes); therefore, they can be compared in association studies. After calculation of gene frequencies of the 141 SNPs selected and genotyped in candidate genes, 75 SNPs were eliminated because there were not in Hardy-Weinberg equilibrium (p <0.05) and showed minor allele frequency (MAF) lower than 5%. In the group of transporter genes our results showed an association with a synonymous SNP rs3740066 (Ile1324Ile), present in exon 28 of ABCC2 gene, where the C allele is more frequent in the group of refractory patients. In addition, further experiments showed that expression of the ABCC2 gene in the hippocampus of farmacoresistentes patients is increased when compared to the hippocampus of individuals without MTLE, from autopsy. For drug metabolizing CYP450 genes, association was found with intronic SNPs present at genes CYP1A2 (rs2551188-TT) and CYP1B1 (rs12904742GG), as well as with 2 additional SNPs at gene CYP2C9 gene: rs2153628AA and rs4086116CT. All variants mentioned above are more frequent in pharmacoresistant patients. In addition, we found association for allelic variants at CYP2C9: rs4086116T, CYP1A2: rs12904742G and CYP2C9: rs1934963C, likewise, the alleles described are more frequent in pharmacoresistant patients. There is a statistically significant difference regarding the presence of hippocampal atrophy between the groups, being more frequent in group farmacoresistentes (p = 0.0000068). The stepwise regression analysis shows that the presence of hippocampal atrophy contributes with 9% of the total variability influencing pharmacoresistance in these MTLE patients. When analyzing the combined effect of the genetic factors which were found to be associated with pharmacoresistance in this study we conclude that their overall contribution to pharmacoresistance was 12%. Therefore, we can conclude that the remaining 79% of variability influencing pharmacoresistance in our group of patients with MTLE can be attributed to other factors not yet identified. Finally, our results show strong evidence indicating that pharmacoresistance in patients with MTLE is polygenic and multifactorial, since it shows clear genetic and environmental influences. / Doutorado / Neurociencias / Doutor em Fisiopatologia Medica

Indiana University-Purdue University Indianapolis (IUPUI) / Indoleamine 2,3-dioxygenase (IDO) is a rate limiting enzyme in tryptophan catabolism that has been implicated in the pathogenesis of a number of diseases. Large interindividual variability in IDO activity in the absence of stimuli and as the result of therapy induced changes has been reported. This variability has the potential to contribute to susceptibility to disease and to interindividual variability in therapeutic response. To identify genetic variations that might contribute to interindividual variability in IDO activity, we resequenced the exons, intron/exon borders and 1.3 kb of the 5’-flanking region of the INDO gene in 48 African-American (AA) and 48 Caucasian-American (CA) subjects from the Coriell DNA Repository. A total of 24 INDO variants were identified. Seventeen of these were in exons, introns, or exon/intron boundries, while seven were within 1.3 kb upstream of the translation start site. Seventeen are novel and 7 were previously identified. When transiently expressed in COS-7 or HEK293 cells the amino acid sequence change in Arg77His resulted in significant decrease in activity, and it reduced the Vmax of IDO. The Arg77His variant and the 9 bp deletion resulted in nearly complete loss of enzyme activity and a lack of detectable protein expression. The function of the Arg77His variant IDO was restored in a dose dependent manner by the heme analog hemin; but there was no associated increase in IDO protein. Cellular heme concentration was higher in cells transfected with the wild type and Ala4Thr variant constructs, but not in cells transfected with the Arg77His variant. The heme synthesis inhibitor, succinylacetone (SA), blocked IDO activity in cells transfected with Arg77His. We identified 22 putative transcription binding sites within the 1.3 kb upstream of the translation start site. Two of the SNPs were located in GATA3 and FOXC1 sites. A specific 3-SNP haplotype reduced promoter activity when transiently transfected into 2 different cell lines. We conclude that there are naturally occurring genetic variants in the INDO gene which affect both expression and activity. These results make clear that interindividual variability in IDO activity at baseline or in response to therapy may be in part due to inherited genetic variability.

Populations of Black-African descent have slower rates of nicotine and cotinine metabolism, smoke fewer cigarettes (~10 cigarettes/day), and have higher incidences of tobacco-related illnesses compared to Caucasians. Cytochrome P450 2A6 (CYP2A6) is the main enzyme involved in the metabolism of nicotine and its proximal metabolite cotinine, as well as tobacco-specific nitrosamines. Genetic polymorphisms in CYP2A6 contribute to the large variability observed in rates of nicotine metabolism. Reduced CYP2A6 activity has been associated with fewer cigarettes smoked, higher quit rates, and lower lung cancer risk in predominantly moderate to heavy-smoking (~20–30 cigarettes/day) Caucasians. CYP2A6 genetic variants and their impact on smoking behaviours have not been well studied among individuals of Black-African descent. The main objectives herein were to identify and characterize new CYP2A6 variants that may explain the slower rates of metabolism, and determine whether CYP2A6 variation is a predictor of smoking phenotypes in this population. Furthermore, we examined whether previously validated biomarkers of tobacco exposure have limitations among individuals of Black-African descent given their low and sporadic smoking patterns. A new CYP2A6 variant (CYP2A6*23) was found in individuals of Black-African descent recruited for a nicotine pharmacogenetic-pharmacokinetic study. CYP2A6*23 reduced activity towards nicotine and coumarin in vitro and was associated with slower rates of CYP2A6 kinetics in vivo. In a clinical trial of African-American light smokers, CYP2A6 slow metabolizers were more successful at smoking cessation compared to normal metabolizers, although no differences in cigarette consumption were found. Two common biochemical markers of tobacco smoke exposure, cotinine and exhaled carbon monoxide, were weakly correlated with self-reported cigarette consumption. These biomarkers were not substantially affected by variables previously shown to alter amount smoked and/or rates of cotinine metabolism such as gender, age, body mass index or smoking menthol cigarettes. However, CYP2A6 slow metabolizers had significantly higher cotinine without smoking more cigarettes. Identification and characterization of novel variants adds to our understanding of nicotine pharmacokinetic differences between racial/ethnic minority groups and improves accuracy of CYP2A6 genotype groupings for genetic association studies. Furthermore, better insight into the biological factors associated with smoking behaviours will aid in the development of more efficacious targeted treatments for this understudied population.

Populations of Black-African descent have slower rates of nicotine and cotinine metabolism, smoke fewer cigarettes (~10 cigarettes/day), and have higher incidences of tobacco-related illnesses compared to Caucasians. Cytochrome P450 2A6 (CYP2A6) is the main enzyme involved in the metabolism of nicotine and its proximal metabolite cotinine, as well as tobacco-specific nitrosamines. Genetic polymorphisms in CYP2A6 contribute to the large variability observed in rates of nicotine metabolism. Reduced CYP2A6 activity has been associated with fewer cigarettes smoked, higher quit rates, and lower lung cancer risk in predominantly moderate to heavy-smoking (~20–30 cigarettes/day) Caucasians. CYP2A6 genetic variants and their impact on smoking behaviours have not been well studied among individuals of Black-African descent. The main objectives herein were to identify and characterize new CYP2A6 variants that may explain the slower rates of metabolism, and determine whether CYP2A6 variation is a predictor of smoking phenotypes in this population. Furthermore, we examined whether previously validated biomarkers of tobacco exposure have limitations among individuals of Black-African descent given their low and sporadic smoking patterns. A new CYP2A6 variant (CYP2A6*23) was found in individuals of Black-African descent recruited for a nicotine pharmacogenetic-pharmacokinetic study. CYP2A6*23 reduced activity towards nicotine and coumarin in vitro and was associated with slower rates of CYP2A6 kinetics in vivo. In a clinical trial of African-American light smokers, CYP2A6 slow metabolizers were more successful at smoking cessation compared to normal metabolizers, although no differences in cigarette consumption were found. Two common biochemical markers of tobacco smoke exposure, cotinine and exhaled carbon monoxide, were weakly correlated with self-reported cigarette consumption. These biomarkers were not substantially affected by variables previously shown to alter amount smoked and/or rates of cotinine metabolism such as gender, age, body mass index or smoking menthol cigarettes. However, CYP2A6 slow metabolizers had significantly higher cotinine without smoking more cigarettes. Identification and characterization of novel variants adds to our understanding of nicotine pharmacokinetic differences between racial/ethnic minority groups and improves accuracy of CYP2A6 genotype groupings for genetic association studies. Furthermore, better insight into the biological factors associated with smoking behaviours will aid in the development of more efficacious targeted treatments for this understudied population.

published_or_final_version / Pathology / Master / Master of Medical Sciences

Interpatient variability in clinical response to antipsychotics (AP) is observed in the treatment of schizophrenia (SCZ) with evidence to support a genetic basis for this phenomenon. Dysfunction in the dopaminergic (DA) system as well as the glutamatergic (GLU) system have both been implicated in the formation of SCZ symptoms. Therefore, we explored the role of DA and GLU receptor variation on clinical response to CLZ, an AP of last resort. We focused on DA receptor genes DRD3, DRD4, and DRD5 having already previously published on DRD1 and DRD2 in this sample. N-methyl-D-aspartate receptor (NMDAR) subunit gene variants and epistatic effects between and among these variants and DA receptor gene variants were also studied. For DRD3, we extended a statistically significant meta-analysis on single nucleotide polymorphism (SNP) rs6280 (Ser9Gly) previously performed by Jonsson et al. (2003). We observed a consistent trend for the serine allele and poor CLZ response (six of seven studies with the same direction of effect). Eight other tagged DRD3 SNPs were also tested with two identified for future replication. For DRD4, associations were observed between the 4-repeat allele of the exon III variable number tandem repeat (VNTR) polymorphism and better CLZ response in Caucasians with a non-significant but same direction of effect in African-Americans; the 142/140-base pair (bp) genotype of the intron 1 guanine mononucleotide repeat ((G)n) polymorphism with poor CLZ response in the whole sample; and the 1-copy allele of the 5’-flanking region 120-bp tandem repeat polymorphism in African-Americans. Three other tagged SNPs across DRD4 and five across DRD5 were negative. Our investigation of GLU receptor gene variants focused on NMDAR subunit genes GRIN1, GRIN2A, and GRIN2B with negative findings. Examining gene-gene epistatic effects among and between NMDAR subunit and DA receptor gene polymorphisms identified several interactions with the strongest result being between the DRD1 rs686 G carrier/DRD3 Ser9Gly G non-carrier group with good response. Overall, our results suggest possible minor roles for DA receptor gene variants on CLZ response. Integrating this data with exciting new advances in technologies and bioinformatics will surely bring us closer to personalized medicine in psychiatry.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

Interpatient variability in clinical response to antipsychotics (AP) is observed in the treatment of schizophrenia (SCZ) with evidence to support a genetic basis for this phenomenon. Dysfunction in the dopaminergic (DA) system as well as the glutamatergic (GLU) system have both been implicated in the formation of SCZ symptoms. Therefore, we explored the role of DA and GLU receptor variation on clinical response to CLZ, an AP of last resort. We focused on DA receptor genes DRD3, DRD4, and DRD5 having already previously published on DRD1 and DRD2 in this sample. N-methyl-D-aspartate receptor (NMDAR) subunit gene variants and epistatic effects between and among these variants and DA receptor gene variants were also studied. For DRD3, we extended a statistically significant meta-analysis on single nucleotide polymorphism (SNP) rs6280 (Ser9Gly) previously performed by Jonsson et al. (2003). We observed a consistent trend for the serine allele and poor CLZ response (six of seven studies with the same direction of effect). Eight other tagged DRD3 SNPs were also tested with two identified for future replication. For DRD4, associations were observed between the 4-repeat allele of the exon III variable number tandem repeat (VNTR) polymorphism and better CLZ response in Caucasians with a non-significant but same direction of effect in African-Americans; the 142/140-base pair (bp) genotype of the intron 1 guanine mononucleotide repeat ((G)n) polymorphism with poor CLZ response in the whole sample; and the 1-copy allele of the 5’-flanking region 120-bp tandem repeat polymorphism in African-Americans. Three other tagged SNPs across DRD4 and five across DRD5 were negative. Our investigation of GLU receptor gene variants focused on NMDAR subunit genes GRIN1, GRIN2A, and GRIN2B with negative findings. Examining gene-gene epistatic effects among and between NMDAR subunit and DA receptor gene polymorphisms identified several interactions with the strongest result being between the DRD1 rs686 G carrier/DRD3 Ser9Gly G non-carrier group with good response. Overall, our results suggest possible minor roles for DA receptor gene variants on CLZ response. Integrating this data with exciting new advances in technologies and bioinformatics will surely bring us closer to personalized medicine in psychiatry.