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Notre génome contient des centaines de milliers de variants génétiques, qui pour la plupart, n’ont aucun impact sur notre santé. Après séquençage, il faut les filtrer pour ne conserver que ceux qui sont potentiellement impliquées dans une maladie. On utilise des annotateurs qui prédisent l’impact des variants. Ces prédictions sont faites sans tenir compte des variants en cis dans le même gène. Pourtant, des variants neutres peuvent, lorsqu’ils sont réunis chez un individu, devenir délétères. J’ai donc développé l’outil bioinformatique GEMPROT qui permet de visualiser l’effet des variants génétiques sur la séquence protéique et de mettre en évidence les combinaisons de variants touchant un même domaine fonctionnel.J’ai ensuite étudié l’impact de deux variants associés à la p.Phe508del (508del) sur la protéine CFTR.Le variant p.Val470M est présent sur tous les haplotypes portant la délétion mais pas sur la séquence de référence, qui est généralement utilisée pour la construction de plasmides. Nous avons montré des différences de fonction de la protéine CFTR selon l’acide aminé en position 470. La fonction est augmentée avec une Valine et il convient donc de s’assurer, lors de la construction de plasmides, que le contexte haplotypique des variants étudiés est bien respecté. Le variant p.Ile1027Thr conduit à une dégradation de la fonction de la protéine 508del.Ce variant n’est présent que sur une partie des haplotypes 508del et pourrait donc avoir un effet modificateur de l’expression de la délétion. En conclusion, nous montrons l’importance de la prise en compte des contextes haplotypiques dans l’étude des maladies et proposons un outil bioinformatique pour le faire. / We all carry hundreds of thousands genetic variations in our genome that, for the most of them, have no impact on our health. After sequencing, they must be filtered to only retain those potentially involved in a disease. We use annotators that predict the impact of variants.These predictions are done for each variant taken independently without considering cis variants in the same gene. However, neutral variants can become deleterious when associated together. I have developed the bioinformatics tool GEMPROT, which makes it possible to visualize the effect of genetic variants on the protein sequence and to highlight combinations of variants affecting the same functional domain.I then studied the impact of two variants associated with p.Phe508del (508del) on CFTR protein function.The variant p.Val470M is present on all carrying deletion haplotypes but not on the reference sequence, which is generally used for the construction of plasmids. We have shown differences in the function of the mutated CFTR protein 508del according to the amino acid at position 470. The function is increased with a Valine and it is therefore necessary to ensure, when constructing plasmids, that the haplotype context of the studied variants is well respected.The variant p.Ile1027Thr leads to a degradation of the function of the 508del protein. This variant is present only on a portion of the 508del haplotypes and could therefore have a modifying effect on deletion expression. In conclusion, we show the importance of considering haplotype contexts in the diseases studies and propose a bioinformatics tool to do so.

Notre patrimoine génétique dévoile, de plus en plus, les passerelles démogénétiques d’une susceptibilité plus accrue de certains individus à des maladies infectieuses complexes. En vue d’une caractérisation de la variabilité génétique des populations ouest-africaines, nous avons analysé 659 chromosomes X au locus dys44 qui comprend, 35 SNPs et un microsatellite distribués sur 2853 pb en amont et 5034 pb en aval de l’exon 44 du gène de la dystrophine en Xp21.3. Les génotypes obtenus, par ASO dynamique et électrophorèse sur gel d’acrylamide, ont servi à la détermination des haplotypes. Des paramètres comme la diversité haplotypique (G) et l'indice de fixation (Fst) ont été calculés. Des analyses en composantes principales ainsi que multidimensionnelles ont été réalisées. Sur 68 haplotypes détectés, 26 sont nouveaux, et cette région, avec une diversité haplotypique moyenne (Gmoy) de 0,91 ± 0,03, se révèle beaucoup plus hétérogène que le reste du continent (Gmoy = 0,85 ± 0,04). Toutefois, malgré l’existence de disparités sous régionales dans la distribution des variants du marqueur dys44, l’AMOVA montre d’une manière générale, une faible érosion de l’éloignement génétique entre les populations subsahariennes (Fst = 1,5% ; p<10-5). Certains variants tel que l’haplotype eurasien B006 paraissent indiquer des flux transsahariens de gènes entre les populations nord-africaines et celles subsahariennes, comme l’exemplifie le pool génétique de l’une des populations ubiquitaires de la famille linguistique Nigéro-congolaise : Les Fulani. Nos résultats vont aussi dans le sens d’un héritage phylétique commun entre les Biaka, les Afro-américains et les populations de la sous-famille de langues Volta-Congo. / The unravelling of our genetic heritage has revealed a demogenetic segueway leading to an increased susceptibility of certain individuals to complex infectious diseases. In order to characterize genetic variability among the West African populations, we analyzed 659 X chromosomes at the dys44 locus which comprises 35 SNPs and a microsatellite spanning a region 2853 bp upstream and 5034 bp downstream of exon 44 of the dystrophine gene in Xp21.3. The resulting genotypes, obtained by dynamic allele specific oligonucleotide hybridization and acrylamide gel electrophoresis, were used for haplotype construction. Gene diversity parameters such as the haplotypic diversity (G) and fixation indexes (Fst) were estimated. Multidimensional analysis of the data, including principal component analysis was also performed. Of the 68 distinct haplotypes detected in our data set, 26 were novel. The mean haplotypic diversity (Gmoy) was 0.91 ± 0.03 for this West African region which was shown to be more heterogeneous than the rest of the continent (Gmoy = 0.85 ± 0.04). However, despite certain sub-regional differences in the distribution of dys44 variants, the analysis of molecular variance showed an overall decline in the genetic distance between Sub-Saharan populations (Fst = 1.5% ; p<10-5). Certain variants, such as the Eurasian-specific haplotype B006, appear to suggest a Trans-Saharan gene flux between North African and Sub-Saharan populations as exemplified by the observed genetic pool of one of the ubiquitous populations of the Nigerian-Congolese linguistic family: The Fulani. Our results are also in agreement with a phyletic heritage between the Biaka, the Afro-Americans and the populations of the Volta-Congo language subfamilies.

Advances in high-throughput genomic technologies have facilitated the collection of DNA information for thousands of individuals, providing unprecedented opportunities to explore the genetic architecture of complex disease. One important finding has been that the majority of variants in the human genome are low in frequency or rare. It has been hypothesised that recent explosive growth of the human population afforded unexpectedly large amounts of rare variants with potentially deleterious effects, suggesting that rare variants may play a role in disease predisposition. But, importantly, rare variants embody a source of information through which we may learn more about our recent evolutionary history. In this thesis, I developed several statistical and computational methods to address problems associated with the analysis of rare variants and, foremost, to leverage the genealogical information they encode. First, one constraint in genome-wide association studies is that lower-frequency variants are not well captured by genotyping methods, but instead are predicted through imputation from a reference dataset. I developed the meta-imputation method to improve imputation accuracy by integrating genotype data from multiple, independent reference panels, which outperformed imputations from separate references in almost all comparisons (mean correlation with masked genotypes r²&GT;0.9). I further demonstrated in simulated case-control studies that meta-imputation increased the statistical power to identify low-frequency variants of intermediate or high penetrance by 2.2-3.6%. Second, rare variants are likely to have originated recently through mutation and thereby sit on relatively long haplotype regions identical by descent (IBD). I developed a method that exploits rare variants as identifiers for shared haplotype segments around which the breakpoints of recombination are detected using non-probabilistic approaches. In coalescent simulations, I show that such breakpoints can be inferred with high accuracy (r²&GT;0.99) around rare variants at frequencies &LT;0.05%, using either haplotype or genotype data. Third, I show that technical error poses a major problem for the analysis of whole-genome sequencing or genotyping data, particularly for alleles below 0.05% frequency (false positive rate, FPR=0.1). I therefore propose a novel approach to infer IBD segments using a Hidden Markov Model (HMM) which operates on genotype data alone. I incorporated an empirical error model constructed from error rates I estimated in publicly available sequencing and genotyping datasets. The HMM was robust in presence of error in simulated data (r²&GT;0.98) while nonprobabilistic methods failed (r²&LT;0.02). Lastly, the age of an allele (the time since its creation through mutation) may provide clues about demographic processes that resulted in its observed frequency. I present a novel method to estimate (rare) allele age based on the inferred shared haplotype structure of the sample. The method operates in a Bayesian framework to infer pairwise coalescent times from which the age is estimated using a composite posterior approach. I show in simulated data that coalescent time can be inferred with high accuracy (rank correlation &GT;0.91) which resulted in a likewise high accuracy for estimated age (&GT;0.94). When applied to data from the 1000 Genomes Project, I show that estimated age distributions were overall conform with frequency-dependent expectations under neutrality, but where patterns of low frequency and old age may hint at signatures of selection at certain sites. Thus, this method may prove useful in the analysis of large cohorts when linked to biomedical phenotype data.

Submitted by Camila Urbano Braz null (camila_urbano@yahoo.com.br) on 2016-08-29T20:11:34Z No. of bitstreams: 1 Tese_Camila_Urbano_Braz.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-08-30T18:25:24Z (GMT) No. of bitstreams: 1 braz_cu_dr_jabo.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) / Made available in DSpace on 2016-08-30T18:25:24Z (GMT). No. of bitstreams: 1 braz_cu_dr_jabo.pdf: 2227912 bytes, checksum: 63cf8f89ad9895f8c74ed5d890e0b2f9 (MD5) Previous issue date: 2016-07-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O mercado consumidor tem sido cada vez mais exigente quanto à qualidade da carne e, portanto, a pecuária precisa melhorar sua eficiência e fornecer produtos diferenciados, padronizados e com qualidade. Entre as características de qualidade de carne, a maciez é a que mais influencia na satisfação dos consumidores. Considerando a importância dada à maciez da carne, pesquisas têm sido desenvolvidas para melhor compreender os mecanismos relacionados à expressão fenotípica desta característica. Os estudos de genes candidatos têm possibilitado a identificação de polimorfismos que modificam as estruturas das proteínas ou ainda, que estejam em desequilíbrio de ligação com alterações funcionais no DNA. Com a automatização dos polimorfismos de nucleotídeo único (SNPs) muitas regiões ao longo do genoma foram identificadas como responsáveis pela variação fenotípica da maciez da carne. No entanto, os marcadores SNPs podem ter baixa capacidade de identificar locos que atuam nas características quantitativas (QTL) por serem, na grande maioria, bi-alélicos e, mesmo que aconteçam mutações, as mudanças nas frequências alélicas dos SNPs podem permanecer quase inalteradas. Por outro lado, com a utilização de haplótipos, mutações tendem a causar mudanças nas frequências dos haplótipos, aumentando as chances de identificação dos QTL. Sendo assim, este estudo teve como objetivos detectar QTL e mutações causais em genes candidatos e identificar QTL por meio de uma análise de associação genômica ampla, para a característica maciez da carne em bovinos da raça Nelore, utilizando haplótipos como unidades fundamentais dos testes de associação. Foram utilizadas informações fenotípicas, genotípicas e de pedigree de animais provenientes de fazendas que integram os programas de melhoramento genético DeltaGen e PAINT. Cinquenta e dois genes candidatos foram escolhidos para serem analisados utilizando haplótipos construídos com base no desequilíbrio de ligação, utilizando 1.616 animais. Do total de haplótipos, dois foram significativos, sendo que os éxons próximos e dentro destes foram sequenciados visando buscar a mutação causal. O sequenciamento foi realizado com 298 animais e os SNPs identificados foram imputados para os 1.318 animais remanescentes. Foram realizadas análises de associação utilizando haplótipos construídos com base na metodologia de janelas sobrepostas, sendo que seus efeitos foram estimados pelo método Genomic Best Linear Unbiased Predictor (GBLUP). Os valores genéticos dos animais foram estimados para cada haplótipo e SNP e, após, as variâncias genéticas aditivas foram calculadas. Utilizando haplótipos construídos com base em janelas sobrepostas, verificou-se que o aumento do número de SNPs no haplótipo permitiu capturar maior proporção da variância genética aditiva da característica maciez da carne. Seis possíveis QTL foram identificados explicando as maiores proporções de variância genética aditiva para maciez da carne, dos quais um está no gene CAPN1 e cinco no gene ASAP1. Não houve evidências de que a mutação causal para a maciez da carne tenha sido identificada nos dois genes. Uma análise de associação genômica ampla foi realizada utilizando haplótipos construídos com base na metodologia de janelas sobrepostas de tamanhos variados. Foram utilizados nesta análise 1.405 animais genotipados com o painel Illumina Bovine HD e 1.756 animais genotipados com painel de menor densidade (70 K Neogen) e, posteriormente, imputados para o painel HD, em um total de 3.161 animais analisados. Os efeitos dos haplótipos e SNPs foram estimados pelo método GBLUP e as variâncias genética aditivas de cada haplótipo e SNP foram calculadas. Os genes NOS1AP, SUCLG1, PHLDB2 e LOC107132946 foram associados com a característica maciez da carne em bovinos da raça Nelore, por meio de análises de associação genômica ampla utilizando SNPs individuais e haplótipos. A análise utilizando SNPs identificou QTL diferentes das análises com haplótipos e, em alguns casos, SNPs apresentaram variâncias genéticas aditivas maiores do que as apresentadas pelos haplótipos. A análise que utilizou haplótipos construídos com cinco SNPs identificou mais QTL do que as análises de haplótipos construídos com sete e nove SNPs. Sugere-se que análises utilizando haplótipos, baseados em janelas sobrepostas, sejam realizadas para complementar análises de SNPs individuais em estudos de associação genômica ampla. / The consumer market has been increasingly demanding about the meat quality and therefore livestock needs to improve its efficiency and provide differentiated products, standardized and with quality. Considering the importance given to the meat tenderness, research has been undertaken to better understand the mechanisms related to the phenotypic expression of this trait. The candidate gene studies have allowed the identification of polymorphisms that change the structures of the proteins or that are in linkage disequilibrium with functional alterations in the DNA. With the advent of single nucleotide polymorphisms (SNPs) throughout many regions of the genome have been identified as responsible for phenotypic variation in meat tenderness. However, SNPs markers may have low ability to identify mutations, because SNPs are commonly bi-allelic and even when mutations have occurred, allelic frequencies can remain unaltered. On the other hand, using haplotype, mutations tend to cause major changes in haplotype frequencies, increasing the chances of identification of QTL. Thus, this study aimed to detect QTL and causal mutations by the approach of candidate genes and identify possible QTL through a genome-wide association analysis, for the trait meat tenderness in Nelore cattle using haplotypes as fundamental units of association tests. Information of the phenotypic, genotypic and pedigree were used from farms that belong to the breeding programs DeltaGen and PAINT. Fifty-two candidate genes were chosen for analysis using haplotypes constructed based on linkage disequilibrium using 1,616 animals. Two haplotypes were significant, and the exons near and within these haplotypes were sequenced to search for the causal mutation. The sequencing was performed using 298 animals and the identified SNPs were imputed for 1,318 remaining animals. Association analysis using haplotypes constructed based on the method of overlapping sliding windows were carried out and the SNPs and haplotypes effects were estimated using Genomic Best Linear Unbiased Predictor (GBLUP) method. The breeding values were estimated for each haplotype and SNPs and the additive genetic variances were calculated. Using haplotypes constructed based on overlapping sliding windows, we found that increasing the number of SNPs in the haplotype allowed to capture a greater proportion of additive genetic variance of meat tenderness. Six putative QTL were identified with the greatest additive genetic variances for meat tenderness, which one was in CAPN1 gene and five in ASAP1 gene. There was no evidence that the causal mutation for meat tenderness trait has been identified in these genes. A genome-wide association analysis was performed using haplotypes constructed based on the methodology of overlapping sliding windows of varying sizes. In this analysis, 1,405 animals genotyped with the Illumina Bovine HD panel and 1,756 genotyped animals with lower density panel (Neogen 70 K) were used and then, the genotypes of the 1,756 were imputed to the HD panel, in a total of 3,161 animals analyzed. The haplotypes and SNPs effects were estimated by the method GBLUP and the additive genetic variances were calculated for each haplotype and SNP. The NOS1AP, SUCLG1, PHLDB2 and LOC107132946 genes were associated with the meat tenderness trait in Nelore cattle through genome-wide association analysis using individual SNPs and haplotypes. The analysis using SNPs identified different QTL of the haplotype analyzes, and in some cases, the SNPs showed additive genetic variance greater than those presented by the haplotypes. The analysis used haplotypes constructed with five SNPs identified more QTL than analysis of haplotypes constructed with seven and nine SNPs. Analyzes using haplotypes based on overlapping sliding windows, should be conducted as additional analyzes for individual SNPs in genome-wide association studies. / FAPESP: 2013/00035-9

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-03-16T15:28:47Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) RogerioSantosRosa_Tese.pdf: 1740026 bytes, checksum: aa346f64c34419c4b83269ccb99ade6a (MD5) / Made available in DSpace on 2016-03-16T15:28:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) RogerioSantosRosa_Tese.pdf: 1740026 bytes, checksum: aa346f64c34419c4b83269ccb99ade6a (MD5) Previous issue date: 2015-03-12 / Haplotype information has a central role in the understanding and diagnosis of certain illnesses, and also for evolution studies. Since that type of information is hard to obtain directly, computational methods to infer haplotype from genotype data have received great attention from the computational biology community. Unfortunately, haplotype inference is a very hard computational biology problem and the existing methods can only partially identify correct solutions. I present neural network models that use different properties of the data to predict when a method is more prone to make errors. I construct models for three different Haplotype Inference approaches and I show that our models are accurate and statistically relevant. The results of our experiments offer valuable insights on the performance of those methods, opening opportunity for a combination of strategies or improvement of individual approaches. I formally demonstrate that Linkage Disequilibrium (LD) and heterozygosity are very strong indicators of Switch Error tendency for four methods studied, and I delineate scenarios based on LD measures, that reveal a higher or smaller propension of the HI methods to present inference errors, so the correlation between LD and the occurrence of errors varies among regions along the genotypes. I present evidence that considering windows of length 10, immediately to the left of a SNP (upstream region), and eliminating the non-informative SNPs through Fisher’s Test leads to a more suitable correlation between LD and Inference Errors. I apply Multiple Linear Regression to explore the relevance of several biologically meaningful properties of the genotype sequences for the accuracy of the haplotype inference results, developing models for two databases (considering only Humans) and using two error metrics. The accuracy of our results and the stability of our proposed models are supported by statistical evidence. / Haplótipos têm um papel central na compreensão e diagnóstico de determinadas doenças e também para estudos de evolução. Este tipo de informação é difícil de obter diretamente, diante disto, métodos computacionais para inferir haplótipos a partir de dados genotípicos têm recebido grande atenção da comunidade de biologia computacional. Infelizmente, a Inferência de Halótipos é um problema difícil e os métodos existentes só podem predizer parcialmente soluções corretas. Foram desenvolvidos modelos de redes neurais que utilizam diferentes propriedades dos dados para prever quando um método é mais propenso a cometer erros. Foram calibrados modelos para três abordagens de Inferência de Haplótipos diferentes e os resultados validados estatisticamente. Os resultados dos experimentos oferecem informações valiosas sobre o desempenho e comportamento desses métodos, gerando condições para o desenvolvimento de estratégias de combinação de diferentes soluções ou melhoria das abordagens individuais. Foi demonstrado que Desequilíbrio de Ligação (LD) e heterozigosidade são fortes indicadores de tendência de erro, desta forma foram delineados cenários com base em medidas de LD, que revelam quando um método tem maior ou menor propensão de cometer erros. Foi identificado que utilizando janelas de 10 SNPs (polimorfismo de um único nucleotídeo), imediatamente a montante, e eliminando os SNPs não informativos pelo Teste de Fisher leva-se a uma correlação mais adequada entre LD e a ocorrência de erros. Por fim, foi aplicada análise de Regressão Linear para explorar a relevância de várias propriedades biologicamente significativas das sequências de genótipos para a precisão dos resultados de Inferência de Haplótipos, estimou-se modelos para duas bases de dados (considerando apenas humanos) utilizando duas métricas de erro. A precisão dos resultados e a estabilidade dos modelos propostos foram validadas por testes estatísticos.

Orientador: Jose Eduardo Tanus dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T02:13:15Z (GMT). No. of bitstreams: 1 Syllos_RogerWilliamCruzde_M.pdf: 523113 bytes, checksum: 7a17db80334615a2d5ead89d954e6305 (MD5) Previous issue date: 2006 / Resumo: Polimorfismos no gene da sintase endotelial do óxido nítrico (eNOS) têm sido associados com desenvolvimento ou severidade da retinopatia diabética em pacientes com diabetes mellitus tipo 1(DM1). Entretanto, existem poucos estudos investigando esta associação em portadores de diabetes mellitus tipo 2 (DM2). Além disso, não há estudo prévio que tenha analisado a combinação de marcadores genéticos (haplótipos) e aparecimento e/ou gravidade das complicações vasculares do DM2 . Foram estudados três polimorfismos genéticos: Polimorfismo de base única (SNP) na região promotora (T 786 C), no exon 7 (Glu298Asp) e um número variável de repetições sequenciais (VNTR) no intron 4 (b/a), em 103 voluntários saudáveis e 170 pacientes portadores de diabetes mellitus tipo 2, classificados em diabéticos sem retinopatia (114) e com retinopatia (56). Outrossim, examinamos a associação dos haplótipos genéticos da eNOS com diabetes mellitus tipo 2 e retinopatia diabética. Não foram encontradas diferenças na freqüência dos genótipos e dos alelos dos três polimorfismos entre os três grupos testados. Entretanto, um determinado haplótipo "C - Glu - b" foi mais comum no grupo de indivíduos saudáveis (24%) do que nos pacientes diabéticos (7%) (P=0.0001). Quando comparamos diabéticos com retinopatia diabética com aqueles sem retinopatia, não foi identificada diferença significativa na distribuição da freqüência dos haplótipos da eNOS (P=0,7372). Estes achados sugerem que não há associação entre retinopatia diabética e haplótipos individuais da eNOS em pacientes portadores de diabetes mellitus tipo 2. O haplótipo "C - Glu - b" , no entanto, pode possuir algum efeito protetor contra o desenvolvimento de diabetes mellitus tipo 2. Novos estudos devem se conduzidos para tentar identificar uma base molecular para tal efeito / Abstract: Background: Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been associated with the development of diabetic retinopathy (DR) in patients with type 1 diabetes mellitus (T1DM), but not with T2DM. However, no previous study has analyzed combinations of genetic markers (haplotypes), which can be more informative. Methods: We studied three eNOS genetic polymorphisms: a single nucleotide polymorphism in the promoter region (T-786C), in exon 7 (Glu298Asp), and a variable number of tandem repeats in intron 4 (b/a) in 103 healthy controls, and in 170 patients with T2DM (without DR, N=114; with DR, N=56). We also examined the association of eNOS gene haplotypes with T2DM and with DR. Results: No differences were found in the frequencies of genotypes and alleles of the three polymorphisms among the three groups of subjects. Conversely, the "C-Glu-b" haplotype was more common in healthy controls (24%) than in T2DM patients (7%) (P=0.0001). Finally, no significant difference in the distribution of eNOS haplotypes frequencies was found when T2DM patients with or without DR were compared (P=0.7372). Conclusions: These findings suggest no association between DR and individual eNOS haplotypes in T2DM patients. The "C-Glu-b" haplotype, however, may have a protective effect against T2DM. Further studies should be conducted to address the molecular basis for such an effect / Mestrado / Mestre em Farmacologia

A associação não aleatória entre alelos de diferentes lócus caracteriza o que é chamado de desequilíbrio de ligação (DL) entre eles. A extensão do DL nas populações humanas pode ser influenciada por muitos fatores, tais como taxa de recombinação, características demográficas (idade, tamanho e taxa de crescimento) e fatores evolutivos (deriva genética, efeito fundador, gargalos populacionais, mutação, seleção e fluxo gênico). Portanto, o conhecimento dos padrões do DL fornecem dados que auxiliam na descrição dos eventos demográficos e evolutivos sofridos pelas populações. O objetivo deste estudo foi descrever os padrões de DL de quatro populações brasileiras e correlacioná-los com suas respectivas histórias demográficas, uma vez que estas populações experimentaram alguns dos eventos evolutivos que geram ou retardam o decréscimo do DL, como fundação por poucos indivíduos, miscigenação no momento da fundação e posterior isolamento. Foram analisadas amostras de três populações remanescentes de quilombos do Estado do Piauí, Gaucinha (GAU, n = 14), Mimbó (MIB, n = 15) e Sítio Velho (STV, n = 15) e da população urbana de Teresina, Piauí (TES, n = 15), além de sete amostras populacionais do projeto HapMap (CEU, CHB, JPT, ASW, LWK, MKK, YRI, todas com n = 15). Foram genotipados mais de 250 mil SNPs (Single Nucleotide Polymorphisms) utilizando-se o GeneChip® Human Mapping 250K Nsp I Array - Affymetrix® nas amostras das quatro populações brasileiras. Os dados brutos das populações do HapMap para este array foram obtidos na página do projeto. Os genótipos para todas as amostras foram determinados pelo algoritmo CRLMM após comparação com o algoritmo BRLMM, e as análises de DL e determinação dos blocos de haplótipos foram realizadas com o uso do programa Haploview. Considerando-se o número de blocos de haplótipos detectados em cada população estudada, padrão semelhante foi observado em todos os autossomos. Em geral, a população europeia (CEU) e as duas populações asiáticas (CHB e JPT) do HapMap apresentaram os maiores números de blocos, enquanto que os menores números foram observados nos quilombos GAU e MIB e na população TES. As populações africanas LWK, MKK e YRI e a população afro-americana ASW apresentaram os valores intermediários e a população afro-brasileira STV, apresentou um número de blocos apenas inferior a CEU, CHB e JPT. A grande contribuição africana nos quilombos GAU e MIB pode explicar o menor DL observado nestas comunidades. Por outro lado, o menor DL em TES se deve, provavelmente, à sua fundação, que envolveu um maior número de indivíduos e foi seguida por um rápido crescimento. A possível explicação para o maior DL observado em STV, em relação aos demais quilombos, consiste em sua peculiar história demográfica: esta comunidade experimentou uma miscigenação no momento de sua fundação, que foi seguida por um crescimento lento e pouca diferenciação. Assim, foi demonstrado como os eventos demográficos de cada população influenciam seus respectivos padrões de DL. / The non-random association between alleles of different loci characterizes what is called linkage disequilibrium (LD) between them. The LD extent in human populations can be influenced by many factors, such as recombination rate, demographic features (age, size and growth rate) and evolutionary events (genetic drift, founder effects, population bottlenecks, mutation, selection and gene flow). Therefore, knowledge of the LD patterns provides data that assists in describing the evolutionary and demographic events experienced by populations. The aim of this study was to describe the LD patterns of four Brazilian populations and correlate these patterns with their respective demographic histories, since these populations have experienced some of the evolutionary events that produce or retard the LD decrease, such as foundation by few individuals, admixture at the founding moment and subsequent isolation. Samples from three quilombo remnants populations of the Piauí State, Gaucinha (GAU, n = 14), Mimbó (MIB, n = 15) and Sítio Velho (STV, n = 15) and the urban population of Teresina, Piauí (TES, n = 15), and seven population samples from the HapMap Project (CEU, CHB, JPT, ASW, LWK, MKK, YRI, all with n = 15) were analyzed. More than 250 thousand SNPs (Single Nucleotide Polymorphisms) were genotyped using the GeneChip ® Human Mapping 250K Nsp Array I - Affymetrix ® in the samples of the four Brazilian populations. Raw data of the HapMap population samples for this array were obtained from the HapMap homepage. Genotypes for all samples were determined by CRLMM algorithm after comparison with the BRLMM algorithm. LD analyzes and determination of haplotype blocks were performed using the Haploview software. Considering the number of haplotype blocks detected in each population, a consistent pattern was observed for all autosomes. The European population (CEU) and the two Asian populations (CHB and JPT) of the HapMap showed the highest numbers of blocks, while the lowest numbers were observed in the GAU and MIB quilombos and in the TES population. The African populations, LWK, MKK and YRI, and the African-American ASW exhibited intermediate values and the African-Brazilian population STV, presented a number of blocks smaller than that observed for CEU, CHB and JPT. The great African contribution in the GAU and MIB quilombos may explain the lower LD observed in these communities. On the other hand, the lower LD in TES is probably due to its foundation that involved a larger number of individuals and was followed by a fast growth. A possible explanation for the higher LD observed in STV, compared to other quilombos, consists in its particular demographic history: this community experienced admixture at the time of its foundation, which was followed by slow growth and low differentiation. Thus, it was shown how the demographic events of each population influence their respective LD patterns.

A asma brônquica é doença inflamatória crônica complexa das vias aéreas provocada pela interação de fatores genéticos e ambientais. O gene HLA-G (Antígeno Leucocitário Humano G) foi identificado como gene de susceptibilidade à asma, codificando uma molécula não clássica do complexo principal de histocompatibilidade (MHC, do inglês Major Histocompatibility Complex) de classe I com função moduladora das células do sistema imunológico. Nesse contexto, avaliamos o papel do HLA-G na asma afim de identificar genótipos, alelos e haplótipos associados com proteção ou susceptibilidade nas diferentes formas de apresentação da doença. Investigamos os sítios polimórficos da região 3\' não traduzida (3\'UTR-untranslated region) do HLA-G (14 pb Ins/Del, + 3001 C/T, +3003 C/T, +3010 C/G, +3027 A/C, +3035 C/T, +3142 C/G, +3187 A/G e +3196 C/G) em 118 pacientes asmáticos estratificados em asma leve ou moderada e grave e 183 indivíduos brasileiros saudáveis. Testes de associação foram realizados para avaliar as frequências dos genótipos, alelos e haplótipos da 3\'UTR do HLA-G na asma brônquica, considerada como grupo total ou estratificada de acordo com a gravidade da doença. Nossos resultados demonstraram que as frequências dos alelos +3001 C, +3003 C, +3035 C e +3196 C e do genótipo 14 bp DI estavam aumentadas no grupo total e nas diversas formas de apresentação da doença. Os alelos +3010 C e +3142 G e o genótipo +3010 CC estavam mais representados em pacientes com asma leve ou moderada. Por outro lado, os genótipos +3010 GG, +3142 CG e +3187 AG e o alelo +3010 G apresentaram maior frequência nos asmáticos graves, estando fortemente associados com o desenvolvimento da forma grave da asma. Além disso, os genótipos 14 pb II, +3010 CC e +3142 GG e o alelo +3010 C conferiram proteção à asma grave. Além disso, identificamos um haplótipo da 3\'UTR do HLA-G associado ao desenvolvimento de asma brônquica, a UTR-8, e um haplótipo que conferiu proteção contra a mesma, a UTR-7. Concluindo, neste estudo, observamos frequências diferenciais de sítios polimórficos do segmento 3\'UTR do HLA-G associados com predisposição à asma brônquica e, também, com a gravidade da doença / Bronchial asthma is a complex chronic inflammatory disease of the airways caused by the interaction of genetic susceptibility and environmental factors. The HLA-G (Human Leucocyte Antigen G) gene was identified as a susceptible marker for bronchial asthma, encoding a nonclassical Major Histocompatibility Complex (MHC) class I molecule, considered to be an important immune check point modulator. In the present study, we evaluated the role of HLA-G in bronchial asthma susceptibility and disease severity, evaluating HLA-G genotypes, alleles or haplotypes. We investigated the HLA-G 3\'Untraslated region (3\'UTR) polymorphic sites (14-bp INS/DEL, +3001, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G, and +3196C/G) in 118 asthmatic Brazilian patients, stratified according to disease severity into mild/moderate and severe asthma, and in 183 healthy individuals. HLA-G 3\'UTR variation sites were individually analyzed or lumped together as haplotypes. Our results showed that frequencies of +3001 C, +3003 C, +3035 C e +3196 C alleles and 14 pb ID genotype were increased in asthma group considered as a whole and in patients stratified according to disease severity. The +3010 C and .3142 G alleles and the +3010 CC genotype were overrepresented in patients with mild and moderate forms. Similarly, the +3010 GG, +3142 CG, +3187 AG genotypes and +3010 G allele presented increased frequency in severe asthmatic patients. In contrast, the 14 pb II, +3010 CC and +3142 GG genotypes and +3010 C allele conferred protection against severe asthma. In addition, we identified a 3\'UTR HLA-G haplotype that was associated with bronchial asthma development (UTR-8) and one haplotype that conferred protection against asthma (UTR-7). In conclusion, in this study, we observed differential frequencies at HLA-G 3\'UTR polymorphic sites that are associated with bronchial asthma predisposition and, also, with disease severity

Background: Type of 1 diabetes (T1D) is an autoimmune disease in which the cells of immune system attack the β-cells of pancreas. Consequently, destroyed β-cells do not produce insulin to reduce blood sugar levels. This disease is very complex, the pathogenesis is contributed by both genetic factors and environmental factors. In recent years, the number of individuals with T1D is increasing worldwide. Aims: The aim of this thesis was to investigate whether it is possible to predict T1D based on the expression profile of BACH2, CDC20, IGLL3P, EIF3A and TXNDC5 genes , which are involved in the development of immune system cells and insulin production. Another aim was to compare the expression of selected genes in children, in which the first detection of the disease may be done, and adults who suffer from prolonged T1D. The final goal was to compare the expression of individual selected genes in the HLA risk alleles DR04, DR03, DQA*05:01 a DQB*03:02. Methods: The DNA and RNA of patients with T1D and healthy individuals was isolated from blood. DNA was used to HLA genotyped. Isolated RNA was reverse transcribed into cDNA and then used in real-time PCR to determine the relative levels of gene expression. Conclusion: Significant results were obtained when the expression of BACH2, CDC20 and TXNDC5 genes...