Frequency of occurrence of selected single nucleotide polymorphisms of CYP2C8 and MDR1 in the Czech population and their influence on the effect of amiodarone Introduction: Variability in drug response is sometimes conditioned by genetic differences in the metabolism and the transport of drugs. Interindividual differences are often caused by polymorphisms affecting biotransformation activity of enzymes and expression of transporters. In the thesis we paid attention to the cytochrome P450 CYP2C8 and MDR1. First, we described the frequency of occurrence of selected variant alleles CYP2C8 * 2, CYP2C8 * 3 (2 substitution in exon 3 and 8, CYP2C8 and CYP2C8 * 3G416A * 3A1196G), CYP2C8 * 4, CYP2C8 P404A in the healthy Czech population and MDR1 variant alleles in these exons: 26 C3435T, 21 G2677A/T, 12 C1236T a 17 T-76A. Subsequently, we studied the influence of these polymorphisms on effects of amiodarone in the selected group of patients. Methods: We determined genotypes MDR1 a CYP2C8 by PCR-RFLP by using restriction enzymes and specific primers. We determined the frequency of MDR1 genotypes in 189 healthy volunteers and CYP2C8 in 161 healthy subjects. Further we included into the study 63 patients treated with amiodarone for longer than two months. Their treatment was assessed from medical records and...

INTRODUÇÃO: evidências sugerem a participação de fatores genéticos na susceptibilidade para o desenvolvimento das complicações renais em pacientes portadores de diabetes mellitus tipo 1 (DM1). Vários genes relacionados às vias bioquímicas induzidas pela hiperglicemia têm sido investigados e o estresse oxidativo foi reconhecido como o principal mecanismo patogênico responsável pelo dano celular causado pela hiperglicemia no DM. Assim, genes que codificam enzimas que participam de vias antioxidantes endógenas são candidatos a conferirem susceptibilidade, ou proteção, contra as complicações renais. Os sistemas da glutationa, glutarredoxina, tiorredoxina e a enzima transcetolase são importantes mecanismos de defesa celular contra o estresse oxidativo. OBJETIVOS: avaliar a associação entre os seguintes polimorfismos de um único nucleotídeo (SNP) e a doença renal em pacientes diabéticos tipo 1: -2030 T/G (rs34071297) e +718C/T (rs713041) no gene que codifica a glutationa peroxidase 4 (GPX4); -3310 G/C (rs10427424) no gene que codifica a glutationa sintetase (GSS); -247 A/G (rs2978668) no gene que codifica a glutationa redutase (GSR); -2763 A/G (rs6556885) no gene que codifica a glutarredoxina (GLRX); -224 T/A (rs2301242) no gene que codifica a tiorredoxina (TXN); +402 T/C (rs7211) no gene que codifica a proteína de interação com a tiorredoxina (TXNIP); -192 G/A (rs3788319) no gene que codifica a tiorredoxina redutase 2 (TXNRD2) e -3787 T/G (rs7637934) e -1410 T/C (rs11130365) no gene que codifica a transcetolase (TKT). CASUÍSTICA E MÉTODOS: 443 pacientes (192 do sexo masculino e 251 do sexo feminino) com DM1 com mais de 10 anos de diagnóstico foram classificados conforme a presença ou ausência das seguintes complicações: (1) nefropatia diabética franca (ND), definida por macroalbuminúria a proteinúria persistente; (2) nefropatia diabética estabelecida (NDE), definida por macroalbuminúria a proteinúria persistente ou RFGe < 60 mL/min/1,73 m2 ou pacientes em terapia de substituição renal e (3) ritmo de filtração glomerular estimado (RFGe) ou < que 60 mL/min/1,73 m2. O teste de Pearson 2 foi usado para comparar as frequências dos genótipos e a magnitude de associação foi estimada pelo cálculo do odds ratios (OR). A OR ajustada foi estimada por regressão logística para possíveis fatores de confusão (sexo, idade ao diagnóstico, tempo de diabetes, HbA1c, concentrações plasmáticas de colesterol e triglicérides e presença de hipertensão arterial). Pacientes controle não diabéticos também foram incluídos para avaliar se os SNPs não confeririam susceptibilidade para o DM1. RESULTADOS: A presença de pelo menos um alelo T do polimorfismo +718C/T no gene GPX4 conferiu proteção para a presença de ND estabelecida (OR=0,41; IC 95% 0,19-0,83, p= 0,0146) e ND franca (OR=0,37; IC 95% 0,15- 0,85; p= 0,021) na população masculina mesmo após ajuste para os fatores de confusão e a presença de dois alelos polimórficos A no polimorfismo -224 T/A no gene TXN conferiu risco para a presença de ND franca na população feminina após ajuste para os fatores de confusão (OR= 4,06; IC 95% 1,59-10,6, p= 0,0035). O genótipo TT para o SNP +402 T/C do gene da TXNIP foi mais frequente nos pacientes portadores de DM1 em relação aos controles não diabéticos. O genótipo CC do SNP TXNIP +402 T/C conferiu proteção para a presença de ND estabelecida em homens mesmo após ajuste para os fatores de confusão (OR=0,45; IC 95% 0,22-0,91; p= 0,02). CONCLUSÕES: Os SNPs +718C/T (rs713041) no gene GPX4, -224 T/A (rs2301242) no gene TXN e +402 T/C (rs7211) no gene TXNIP, modulam o risco para o comprometimento renal na população de portadores de DM1 estudada / INTRODUCTION: there is evidence suggesting that genetic factors are involved in the susceptibility to the development of renal complications in patients with type 1 diabetes mellitus (DM1). Several genes related to the mechanisms of hyperglycemia-induced cell damage have been investigated. Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. Thus, genes that encode enzymes involved in endogenous antioxidant pathways may be candidates for conferring risk or protection against renal complications. The glutathione, glutaredoxin, and thioredoxin systems and transketolase enzyme are important mechanisms of cellular defense against oxidative stress. OBJECTIVES: to evaluate the association between the following single nucleotide polymorphisms (SNPs) and renal disease in type 1 diabetic patients: -2030 T/G (rs34071297) and +718C/T (rs713041) in the gene encoding glutathione peroxidase 4 (GPX4); -3310 G/C (rs10427424) in the gene encoding glutathione synthetase (GSS); -247 A/G (rs2978668) in the gene encoding glutathione reductase (GSR); -2763 A/G (rs6556885) in the gene encoding glutaredoxin (GLRX); -224 T/A (rs2301242) in the gene encoding thioredoxin (TXN); +402 T/C (rs7211) in gene encoding thioredoxin interacting protein (TXNIP); -192 G/A (rs3788319) in the gene encoding thioredoxin reductase 2 (TXNRD2); - 3787 T/G (rs7637934) and -1410 T/C (rs11130365) in the gene encoding transketolase (TKT). MATERIALS AND METHODS: 443 patients (192 males and 251 females) with type 1 diabetes duration > 10 years were grouped according to presence or absence of the following complications: (1) overt diabetic nephropathy (DN) defined by persistent macroalbuminuria to proteinuria; (2) established diabetic nephropathy (EDN), defined by persistent macroalbuminuria or proteinuria or estimated glomerular filtration rate (RFGe) < 60 mL/min/1.73 m2 or patients under renal replacement therapy and (3) RFGe or < 60 mL/min/1.73 m2. Pearsons 2 test was performed to compare the genotype frequencies and magnitude of association was estimated using odds ratios (OR). Adjusted OR was estimated by logistic regression for possible confounders (sex, age at diagnosis, diabetes duration, HbA1C, cholesterol and triglyceride concentrations and the presence of hypertension). Nondiabetic subjects were also included. RESULTS: The presence of at least one T polymorphic allele of the SNP GPX4 +718 C/T was protective against EDN (OR = 0.41, CI 95% 0.19- 0.83, p= 0.0146) and against overt DN (OR=0.37; IC 95% 0.15-0.85; p= 0.021) in the male population even after adjustment for possible confounders. The presence of two polymorphic alleles of the SNP TXN -224 T/A conferred independent risk for the presence of overt DN in the female population after adjustment for possible confounders (OR = 4.06, CI 95% 1.59- 10.6, p= 0.0035). The TT genotype for the SNP TXNIP +402 T/C was more frequent in patients with type 1 diabetes compared to nondiabetic controls. The genotype CC of the SNP TXNIP +402 T/C was protective against EDN in male population even after adjustment for possible confounders (OR=0.45; IC 95% 0.22-0.91; p= 0.02) CONCLUSIONS: The SNPs GPX4 +718C/T (rs713041), TXN -224 T/A (rs2301242) and TXNIP +402T/C (rs7211) modulate the risk for renal disease in the studied population of type 1 diabetes patients

O Transtorno Bipolar (TB) tipo I é uma doença caracterizada por episódios de mania e depressão recorrentes com importante prejuízo do funcionamento global e comprometimento das funções cognitivas. Além disso, sabe-se que o número de episódios de humor patológico ao longo da vida pode também influenciar o funcionamento cognitivo destes sujeitos. Neste cenário, ocorreu a necessidade de se investigar marcadores genéticos para disfunção cognitiva no TB com o objetivo de estudar este fenômeno. Dentre os potenciais genes responsáveis por influenciar a cognição destacam-se os polimorfismos funcionais do fator neurotrófico derivado do cérebro (BDNF), da catecol-O-metiltransferase (COMT), da apolipoproteína-E (APOE) e do canal de cálcio de baixa voltagem subunidade 1-C (CACNA1C). Sabe-se, também, que no TB os marcadores de estresse oxidativo estão aumentados durante todas as fases da doença, entretanto, não é claro qual impacto destes na disfunção cognitiva de indivíduos com TB. O objetivo dessa tese foi avaliar o desempenho cognitivo de pacientes jovens com bipolaridade tipo I e sua associação com o genótipo de BDNF, COMT, APOE e CACNA1C e também com os níveis plasmáticos de oxidação da guanosina (8-OHdG) e citosina (5-Mec) durante os episódios de humor, eutimia e em controles. Para investigar essa associação foram incluídos 116 pacientes (79 em episódio de humor patológico e 37 eutímicos) com diagnóstico de TB tipo I (DSMIV-TR); 97 controles saudáveis foram submetidos à avaliação neuropsicológica e coleta de sangue para extração de DNA visando genotipagem para BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 e rs7412), CACNA1C (rs1006737), 8-OhdG e 5-Mec. A análise dos dados obtidos revelou que pacientes portadores do genótipo Met/Met rs4680/rs165599 do COMT apresentam comprometimento cognitivo mais grave (função executiva, fluência verbal, memória e inteligência) comparado ao genótipo Val/Met ou Val/Val durante episódios maníacos ou mistos. Na mesma direção destes resultados, verificou-se que pacientes portadores do alelo Met rs4680 do COMT apresentam comprometimento do reconhecimento de emoções faciais em episódios de mania e depressão. Nenhum efeito do COMT foi observado em controles. O alelo de risco Met do CACNA1C se associou a um pior comprometimento executivo independente dos sintomas maníacos ou depressivos no TB, porém nenhum efeito se observou nos controles. O alelo Met do BDNF rs6265 ou a presença do alelo 4 da APOE não representa um fator que identifique um grupo com desempenho cognitivo diferenciado durante as fases do TB ou em controles. Sujeitos com TB apresentaram níveis mais elevados de 8-OHdG e tais níveis eram diretamente proporcionais ao número de episódios maníacos ao longo da vida, sugerindo um papel dos episódios hiperdopaminérgicos na oxidação das bases de DNA. Concluiu-se que a genotipagem para COMT e CACNA1C em pacientes com TB pode identificar um grupo de pacientes associados a pior disfunção cognitiva durante as fases maníacas e mistas do TB. Tal dado pode ser um indicador do envolvimento do sistema dopaminérgico e dos canais de cálcio de baixa voltagem na fisiopatologia da disfunção cognitiva no TB e deve ser explorado em outros estudos / Bipolar I disorder (BD) is a disease whose main features include severe mood swings that cause severe impairment in global functioning and cognitive domains. Moreover, the number of mood episodes throughout patients life is also associated with deterioration in cognitive functions. In this context, it is important to study genetic markers for the cognitive dysfunction observed in BD to elucidate the physiopathology of this phenomenon. The main candidates for genetic modulation of cognition are the genes brain derived neurotrophic factor (BDNF), catechol-o-methyltransferase (COMT), apolipoprotein E (APOE) and 1-C subunit of the L-type voltage-gated calcium channel (CACNA1C). Furthermore, elevated levels of oxidative stress have been reported in BD for all types of mood episodes but no data is available on their impact on cognitive functioning of BD patients. The aim of this thesis was to investigate whether cognitive functioning of BD patients is influenced by BDNF, COMT, APOE, CACNA1C genotypes or by levels of oxidative damage to the DNA base guanosine (8-OHdG) and cytosine (5-Mec). One hundred sixteen patients (79 during mood episode and 37 euthymic) with BD type I (mania, depression or euthymia) and 97 healthy controls were submitted to neuropsychological evaluation and blood collection for DNA analysis. All subjects were genotyped for BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 and rs7412), CACNA1C (rs1006737), DNA levels of 8-OHdG and 5-Mec were also measured. Our results revealed that BD subjects that carried the rs4680/rs165599 Met/Met genotype had more severe cognitive dysfunction (executive function, verbal fluency, memory and intelligence) than carriers of other genotypes during manic or mixed episodes. Moreover, patients carrying the COMT rs4680 Met allele had worse performance on facial emotion recognition tests during manic and depressive episodes. BD carriers of the Met allele of CACNA1C had more severe executive dysfunction than non-carriers, regardless of manic or depressive symptoms. No effect of CACNA1C or COMT genotypes was observed in controls. The genotypes of BDNF or APOE were not associated with cognitive dysfunction in BD patients or controls. The BD group exhibited higher levels of 8-OHdG than the control group and these levels were influenced by the lifetime number of manic episodes, suggesting that hyperdopaminergic episodes may influence the oxidation of DNA bases. In summary, the genotype of COMT and CACNA1C may represent a useful tool for identifying BD subjects at risk of developing more severe cognitive dysfunction in all mood states of the disease. This evidence associating dopamine catabolism and calcium channels to degree of cognitive dysfunction in BD should be further explored by future research

O Líquen Plano Oral (LPO) é uma doença inflamatória crônica que pode apresentar quadros sintomáticos por longos períodos, afetando a qualidade de vida dos portadores desta doença. Sua etiologia ainda é desconhecida, desse modo os tratamentos disponíveis atualmente se restringem ao controle dos períodos sintomáticos. Acredita-se que a patogênese do LPO possa estar relacionada com alterações imunológicas e genéticas que levam a uma aberrante ativação das vias de sinalização dos receptores Toll-like (TLR). O objetivo deste estudo foi verificar a expressão do TLR2 e duas variantes genéticas deste receptor (rs1898830 e rs4696480) em estudo caso-controle envolvendo 91 amostras de pacientes com LPO e 83 amostras de indivíduos controles, pareados por sexo e idade. Não foram observadas diferenças na expressão do TLR2 entre grupo caso e controle, e nenhuma das variantes avaliadas foi relacionada com relação de risco para o desenvolvimento de LPO. Poucos estudos avaliaram os TLR no LPO, e os dados da literatura sugerem que a cronicidade da lesão e alterações na tolerância oral poderiam estar associadas a resposta imunológica via TLR2, que apresenta sua expressão inalterada no LPO, de forma que estudos mais aprofundados são necessários para se confirmar a real participação deste grupo de receptores no LPO. / Oral lichen planus (OLP) is a chronic inflammatory disease, which can be symptomatic for long periods, affecting the patient quality of life. LPO etiology still unknown, thus the treatments currently available are limited to symptomatic period. Probably the pathogenesis of OLP is related to immunological and genetic changes that provide aberrant Toll-like receptors (TLR) signaling. The aim of this study was to investigate the expression of TLR2 and two genetic variants of this receptor (rs1898830 and rs4696480) in a case-control study involving 91 samples from OLP patients and 83 samples from control subjects, matched for age and sex. No differenc es were observed in TLR2 expression between case and control groups, and neither of the variants was associated with an increased risk ratio for LPO development. Few studies have been conducted on TLR and OLP, and the literature suggests that the chronicity of the lesion in addition to the changes in oral tolerance may be associated with immune response through TLR2 stimulation, which shows unchanged expression in LPO. Further studies are required to confirm the actual participation of this group of receptors in OLP.

Les études d'association sur un génome complet (GWAS) sont conçues pour découvrir les combinaisons de points de polymorphisme (SNP) associées à des maladies. La découverte de ces associations permet d'élaborer de meilleures stratégies pour détecter, traiter ou prévenir les maladies. Récemment, l'utilisation de techniques d'extraction de patterns discriminatif a été investiguée dans le cadre de problématiques GWAS. Toutefois, la découverte de combinaisons de SNP dans de grands jeux de données GWAS est encore difficile à cause de la complexité des algorithmes utilisés. La thèse se propose donc d'améliorer l'état de l'art des approches d'extraction de motifs discriminants, dans le cadre d'extraction de combinaisons de SNP corrélées à un phénotype d'intérêt. Plusieurs solutions ont été proposées, s'attaquant aux problèmes majeurs en GWAS : évaluation de la force d'association, découverte efficace de combinaisons de SNP et visualisation de ces combinaisons. Les approches proposées sont également prometteuses pour d'autres tâches de bioinformatique comme la découverte d'expressions génique, la détection de motifs de phosphorylation et la détection de motifs de régulation. / Discovering high-order SNP combinations associated with diseases is an important task of bioinformatics. Once new genetic associations are identified, they can be used to develop better trategies to detect, treat and prevent the diseases. Recently, this issue has been effectively tackled with discriminative pattern mining algorithms. However, the number of SNPs is often very large, discovering of SNP combinations remains many challenges. To address these challenges this thesis has been advanced the state-of-the-art discriminative pattern mining techniques to discover SNP combinations associated with interesting phenotype. Different solutions have been proposed in this thesis to tackle GWAS analysis. These solutions focus on efficient association strength evaluation, statistically significant discriminative SNP combinations discovery and interesting SNP combinations visualization. The solutions proposed in this thesis are also promising for other tasks of bioinformatics such as differential gene expression discovery, phosphorylation motifs detection and regulatory motif combination mining.

4 Abstract The prevalence of obesity and its related cardiometabolic complications in children remains high across the world. Obesity is a multifactorial disease caused by interaction between genes and environmental factors. Genome-wide association studies have discovered several single nucleotide polymorphisms associated with obesity. A causal role of infection in the pathogenesis of obesity has also been considered, particularly the role of adenovirus 36 (Adv36). The aim of the Ph.D. thesis was to investigate the associations of obesity susceptibility loci (TMEM18, SH2B1, KCTD15, PCSK1, BDNF, SEC16B, MC4R, FTO) and Adv36 infection with obesity-related characteristics and complications in the Czech adolescent population. The results are described in eight publications, of which six are original papers and two are reviews. Studies were performed on a cohort of Czech adolescents recruited either from the general population (1,533 individuals from the epidemiological study) and from in-patient or outpatient weight management clinics (562 overweight/obese individuals underwent an intervention). The results demonstrated an association of TMEM18, SEC16B and FTO gene variants with obesity. Some variants of the genes involved in hypothalamic regulation of energy homeostasis − MC4R, BDNF, PCSK1 − were related to...

Magister Scientiae - MSc / The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South Africa; a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot® and Multiplex AS-PCR genotyping assays, and also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the Cape Metropolitan area. Two SNaPshot® Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific – PCR (MAS-PCR) genotyping system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan large numbers of samples for novel genetic variations. / South Africa

The tilapias are a group of African and Middle Eastern cichlid fish that are widely cultured in developed and developing countries. With many different species and sub-species, and extensive use of interspecies hybrids, identification of tilapia species is of importance in aquaculture and in wild populations where introductions occur. This research set out to distinguish between tilapia species and sub-species by retrieving species-specific nuclear DNA markers (SNPs) using two approaches: (i) sequencing of the coding regions of the ADA gene; and (ii) next-generation sequencing, both standard RADseq and double-digest RADseq (ddRADseq). The mitochondrial DNA (mtDNA) marker cytochrome c oxidase subunit I (COI) was used to verify tilapia species status. ADA gene sequence analysis was partially successful, generating SNP markers that distinguished some species pairs. Most species could also be discriminated using the COI sequence. Reference based analysis (RBA: using only markers found in the O. niloticus genome sequence) of standard RADseq data identified 1,613 SNPs in 1,002 shared RAD loci among seven species. De novo based analysis (DBA: based on the entire data set) identified 1,358 SNPs in 825 loci and RBA detected 938 SNPs in 571 shared RAD loci from ddRADseq among 10 species. Phylogenetic trees based on shared SNP markers indicated similar patterns to most prior phylogenies based on other characteristics. The standard RADseq detected 677 species-specific SNP markers from the entire data set (seven species), while the ddRADseq retrieved 38 (among ten species). Furthermore, 37 such SNP markers were identified from ddRADseq data from a subset of four economically important species which are often involved in hybridization in aquaculture, and larger numbers of SNP markers distinguished between species pairs in this group. In summary, these SNPs are a valuable resource in further investigating hybridization and introgression in a range of captive and wild stocks of tilapias.

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Testes de paternidade geralmente são feitos analisando amostras de DNA do suposto pai, mãe e criança. Para realizar esse exame antes de a criança nascer era preciso recorrer à métodos invasivos, tais como amniocentese e biópsia de vilo corial. Com a descoberta de DNA fetal livre (fcfDNA) no soro e plasma materno, hoje é possível utilizar técnicas que usem esse fcfDNA diminuindo assim os riscos à saúde do feto e da mãe. Testes de pa- ternidade que analisam Short Tandem Repeats (STRs) do fcfDNA, embora possíveis, não são confiáveis, pois muitas vezes há degradação do DNA. Por sua vez, Single Nucleotide Polymorphisms (SNPs) têm sido demonstrados como bons candidatos para identificação humana e podem ser obtidos de fragmentos pequenos de DNA (ou seja, mesmo com o DNA degradado). No entanto, SNPs possuem um número limitado de alelos diferentes (entre dois e quatro). Micro-haplótipos são segmentos cromossomais menores do que 200 pb (pares de bases), contendo dois ou mais SNPs que formam pelo menos três haplótipos distintos. Ao utilizá-los como marcadores genéticos, aumentamos o número de possíveis alelos formados a partir dos SNPs. Como o fcfDNA possui um tamanho de aproximada- mente 145 pb, isso é suficiente para conter micro-haplótipos que podem ser sequenciados usando tecnologia de Sequenciamento de Nova Geração (NGS). O objetivo desse projeto é determinar a probabilidade de paternidade usando SNPs dentro de micro-haplótipos. Os micro-haplótipos foram escolhidos com base em literatura prévia e as frequências relativas destes foram calculadas com base nos grupos étnicos dos dados do 1000 Genomes. Dados brutos de sequenciamento de três amostras de DNA são analisados: o suposto pai, a mãe e o plasma materno (mistura de DNA livre da mãe e do feto). Em seguida, desenvolvemos scripts para obter e analisar os genótipos do suposto pai e da mãe, para cada um dos micro-haplótipos escolhidos. Combinando informação genotípica, frequências populacio- nais e frações fetais (plasma), desenvolvemos um método para calcular a probabilidade de paternidade em casos de não exclusão da mesma. / Paternity tests are usually done by analyzing DNA samples from the alleged father, the mother, and the child. To perform this exam before the birth, invasive methods such as am- niocentesis and chorionic villus sampling are usually necessary. Fortunately, the discovery of fetal cell-free DNA (fcfDNA) in maternal plasma and serum, and the development of te- chniques to analyze this fcfDNA have allowed researchers to reduce the health risk for both fetus and mother. Although paternity tests that analyze Short Tandem Repeats (STRs) from fcfDNA are possible, they are not reliable because DNA degradation often occurs. Single Nucleotide Polymorphisms (SNPs) have been demonstrated as good candidates for human identification and they can be obtained from small DNA fragments (even from de- graded DNA). However, SNPs have a limited number of different alleles (between two and four). Microhaplotypes are chromosomal segments smaller than 200 bp (base pairs) con- taining two or more SNPs that form at least three distinct haplotypes. By using them as genetic markers, we increased the number of possible alleles formed from the SNPs. Since fcfDNA has approximately 145 bp, this is sufficient to contain microhaplotypes that can be sequenced using Next Generation Sequencing (NGS) technology. The aim of this project is to determine the probability of paternity using SNPs within microhaplotypes. Microha- plotypes were chosen based on previous literature review. The haplotype frequencies were calculated based on the ethnic groups from 1000 Genomes database. Raw DNA sequence data from three DNA samples were analyzed: the alleged father, the mother, and the maternal plasma (mixture of mother and fcfDNA). Then, we developed scripts to analyse and obtain the genotypes of the alleged father and mother, for each microhaplotype. By combining genotypic information, population frequencies, and fetal fractions (plasma), we developed a method to calculate the probability of paternity in cases of non-exclusion.