Background Modern neonatal care has improved the survival rate of extremely preterm infants. These infants are at high risk of malnutrition and growth failure during 3-4 months of hospital care. The objectives of this study was to investigate nutritional intakes during hospitalization and explore associations between nutritional intakes, postnatal growth and retinopathy of prematurity (ROP). Perioperative nutrition in infants undergoing surgery for patent ductus arteriosus (PDA) was also investigated. Methods This is a population-based study of Swedish extremely preterm infants (<27 weeks) born during 2004-2007 (n=602). Detailed data on nutritional supply and anthropometric measurements during hospitalization were retrospectively retrieved from hospital records. Comprehensive data on cohort characteristics, neonatal morbidity and infant mortality were obtained from the Extremely Preterm Infants in Sweden Study (EXPRESS). Results During the first 70 days of life, intakes of energy, protein and several micronutrients, with the exception of iron and some vitamins, were less than estimated requirements, and infants showed severe postnatal growth failure. Energy and protein intake predicted growth in all anthropometric outcomes even when adjusting for severity of illness, and fat intake was positively associated with head growth. Low folate intake was positively correlated with poor weight and length gain while high iron intake, mainly explained by blood transfusions, was negatively associated with poor length gain. Furthermore, a low energy intake was associated with severe ROP (stage 3-5). An increased energy intake of 10 kcal/kg/d was associated with 24% decrease in severe ROP (p=0.01). During the first month, 99% of the infants were exclusively fed human milk. Infants who underwent surgery for PDA (n=140) were malnourished, with energy and macronutrient intakes below minimum estimated requirements before, during and after surgery. Conclusions The severe postnatal growth failure observed in Swedish extremely preterm infants may be prevented by improved intakes of energy, protein, fat and folate and a reduction of the number of blood transfusions. Human milk is the main enteral food source and analyses of human milk macronutrient contents facilitates individualized fortification. Provision of adequate energy intakes during the first four weeks of life may be an effective way to reduce the risk of severe ROP. Perioperative nutrition in infants undergoing PDA surgery needs to be improved. The study results have important implications for nutritional regimens, postnatal growth and health outcome in this new generation of survivors.

The mitochondrial folate transport protein (MFT) functions to transport folates into the mitochondrial matrix. The MFT is a member of a mitochondrial carrier family (MCF) of proteins that have a high degree of sequence and structural similarities, yet they transport vastly different substrates at high specificities. In this dissertation research, the folate-specific transport mechanism of the MFT was explored using experimental and computational techniques. MFT residues that differed from MCF consensus residues in conserved PxD/ExxK/R motifs and at a predicted substrate-binding site common to all MCF proteins were investigated. Site-directed mutagenesis of these anomalous residues in the MFT revealed that these residues were adapted for optimal folate transport, and that the MCF consensus residues at these positions were incompatible with folate transport. The structure of the MFT was predicted by homology modeling using the solved crystallographic structure of the ADP/ATP carrier as a template and this model was subjected to ~75 ns of molecular dynamics simulations. These simulations predicted a stepwise descent for the folate substrate into the MFT transport cavity and implicated several aromatic and basic residues in folate recognition and orientation. A predicted set of interactions at the base of the transport cavity between the MCF PxD/ExxK/R conserved motif residues did not appear static as previously hypothesized; these interactions appeared to be induced in the presence of the folate substrate. Therefore, we believe it is unlikely that these interactions form a barrier at the base of the transport cavity. We also investigated the role of the MFT in the compartmentalization of folate metabolism. Cell lines were created that could be induced with doxycycline to express either the cytosolic or mitochondrial isoform of the enzyme folylpoly-γ-glutamate synthetase (FPGS). The constructed cell lines were used to study the flux of folylpolyglutamates across the mitochondrial membrane. It appeared that cellular folylpolyglutamates are not transported across the mitochondrial membrane in either direction. We also demonstrated that many antifolates, including methotrexate and pemetrexed, impaired mitochondrial folate uptake. We believe that these folate analogs competitively inhibit the MFT and have purified the MFT protein for future analysis in reconstituted transport systems.

En dépit des progrès réalisés ces dernières décennies, le cancer demeure un problème majeur de santé publique. La plupart des chimiothérapies conventionnelles exploitent les propriétés cytotoxiques d'agents non sélectifs qui affectent les cellules à division rapide, qu'elles soient cancéreuses ou non, ce qui peut aboutir à une toxicité importante et de sévères effets secondaires. Pour pallier à ce problème, l'utilisation de ligands de ciblage pour délivrer spécifiquement l'agent cytotoxique aux cellules malignes tout en épargnant les tissus sains est une approche prometteuse. Parmi les différents ligands exploités, l'acide folique fait l'objet d'une attention particulière due à la surexpression préférentielle de son récepteur par de nombreux types de cellules cancéreuses. Cependant, les essais cliniques menés sur des molécules reposant sur cette stratégie indiquent que leur efficacité dépend surtout du niveau d'expression du récepteur ciblé. Dans ce contexte, j'ai développé deux approches permettant d'augmenter l'efficacité du ciblage des récepteurs à l'acide folique. D'une part, j'ai réalisé la validation biologique de nouveaux conjugués conçus pour délivrer conjointement deux agents anticancéreux. D'autre part, j'ai mis au point un traitement capable d'augmenter, in vitro et in vivo, l'expression des récepteurs à l'acide folique à la surface des cellules tumorales. Cela aboutit à l'internalisation d'une plus grande quantité de vecteur, qui se traduit in vivo, par une inhibition plus importante de la croissance tumorale, sans toxicité sur les cellules saines. / Despite significant advances obtained during the last decades, cancer is still lack of effective treatments. Indeed, most conventional chemotherapies relies on non-selective agents that kill indifferently healthy and tumor cells with high rate of division. This can lead to elevated toxicity and severe side effects. In this context, therapeutic approaches that exploit targeting ligands to selectively deliver cytotoxic drugs to malignant cells are currently promising. Among these different ligands, folate conjugates are subject to special attention due to the preferential overexpression of the folate receptor on several human cancer cell types that can mediate specific attachment and internalization of folate-derived imaging and therapeutic agents. However, clinical trials carried on such molecules revealed that their efficiency is mainly governed by folate receptor expression level on tumor cells. In this respect, I developed two approaches to increase efficacy of folate receptor targeting. On one hand, I realized biological validation of new targeting devices designed for the simultaneous delivery of two therapeutic agents. On the other hand, I developed a treatment to enhance in vitro and in vivo folate receptor expression at the surface of malignant cells. This treatment allows the internalization of a larger amount of a folic acid conjugate, previously synthesized and validated in our laboratory. That results in a drastically improvement of the in vivo tumor growth inhibition, without toxicity toward healthy cells.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFNγ), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFNγ, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFNγ do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFNγ. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFNγ e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFNγ), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFNγ, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFNγ gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFNγ gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFNγ and Stratifin genes.

Les donneurs de méthyles (B12 et folates) régulent le cycle des monocarbones qui joue un rôle primordial dans les régulations épigénétiques/épigénomiques par méthylation. Une carence en donneurs de méthyles produit un retard de croissance intra-utérine et favorise les anomalies du développement, principalement du système nerveux central. De plus, des taux élevés d'homocystéine associés à une telle carence constituent un facteur de risque pour diverses pathologies neurodégénératives. Nous avons étudié les conséquences d'une carence péri-conceptionnelle et gestationnelle sur le développement cérébral embryonnaire de rats Wistar. L'étude morphométrique a montré un retard de croissance des embryons carencés qui affectait également le cerveau, avec une atrophie de structures telles que l'hippocampe, le cortex et la zone subventriculaire. En raison de la forte sensibilité de l'hippocampe, les effets de la carence ont par ailleurs été étudiés sur un modèle cellulaire de progéniteurs neuronaux hippocampiques. L'utilisation de ces deux modèles a permis de montrer in vivo et in vitro la régulation négative par la carence de la voie Stat3, qui influence prolifération et survie, via une régulation épigénomique post-transcriptionnelle impliquant miR-124. La dérégulation du programme neurogénique impliquant les histones désacétylases affecte la différenciation cellulaire. Par ailleurs, nous avons démontré que la carence en donneurs de méthyles était associée à une modification post-traductionnelle correspondant à une N-homocystéinylation irreversible de protéines neuronales, en particulier associées au cytosquelette. Cette modification induit l'agrégation des protéines, phénomène impliqué dans de nombreuses maladies neurodégénératives. La combinaison de ces différents mécanismes apporte un éclairage nouveau sur les défauts de développement et les troubles cognitifs associés à une carence précoce en donneurs de méthyles, soulignant l'importance de la « programmation foetale » dans la survenue de certaines pathologies neurologiques / Methyl donors (B12 and folate) regulate the one-carbon cycle that plays an important role in the epigenetic/epigenomic regulations by methylation. Methyl donor deficiency (MDD) leads to intrauterine growth retardation and promotes neurodevelopmental abnormalities. Also, high levels of homocysteine associated with such a deficiency are a risk factor for various neurodegenerative diseases. We have studied the consequences of a periconceptional and gestational deficiency on the development of the embryonic brain of Wistar rats. Morphometric studies showed retardation in the development of deficient embryos which also affected the brain, with an atrophy of some structures including hippocampus, cortex and subventricular zone. Given the high sensitivity of the hippocampus, the effects of MDD have been additionally studied in a cellular model of hippocampal neuronal progenitors. Using these two models, we showed both in vivo and in vitro the downregulation of Stat3 pathway regulating cell proliferation and survival, through an epigenomic post-transcriptional process involving miR-124. Disruption of the neurogenic program implying histone deacetylases was shown to alter cell differentiation. Furthermore, we showed that methyl donor deficiency was associated with a post-translational modification corresponding to an irreversible N- homocysteinylation of neuronal proteins, especially those associated with the cytoskeleton. Such a process leads to protein aggregation, a phenomenon involved in many neurodegenerative diseases. The combination of these different mechanisms provides new insights into developmental defects and cognitive impairment associated with an early MDD, highlighting the importance of "fetal programming" in the occurrence of some neurological diseases

Made available in DSpace on 2016-01-26T12:51:31Z (GMT). No. of bitstreams: 1 gustavohenriquemarucci_dissert.pdf: 2122770 bytes, checksum: 2efb8990fe737368548a02b38fdef073 (MD5) Previous issue date: 2011-12-12 / Down syndrome (DS) is a complex genetic disease resulting mainly from the presence of three copies of chromosome 21. It is the most frequent human chromosomal abnormality and, in most cases (about 95%) results from maternal chromosome nondisjunction, which occurs during meiosis I. Recent studies suggest that the etiology of maternal risk for DS in young mothers is associated with polymorphisms in genes of folate/homocysteine metabolism. The proper function of folate metabolism is essential for the synthesis of methyl groups necessary for DNA methylation. The deficiency of this metabolite has resulted in DNA hypomethylation, chromosomal breakage and aneuploidies. Objectives: Evaluate the influence of the C1420T polymorphism in serine hidroximetiltransferase (SHMT) gene on the maternal risk for DS, and investigate the association between this polymorphism and variation in the concentration of serum folate, plasma Hcy and methylmalonic acid (MMA); investigate the impact of 19 base pairs (pb) deletion polymorphism of the dihydrofolate reductase (DHFR) gene and the C1420T polymorphism of the SHMT gene on serum folate concentrations, plasma Hcy and MMA in individuals with DS. Material and methods: 105 mothers of individuals with free trisomy of chromosome 21 and 185 mothers of individuals without the syndrome (no history of miscarriage), and 85 individuals with free trisomy of 21 chromosome were included in this study. The polymorphism of DHFR gene was evaluated by Polymerase Chain Reaction (PCR) by fragment size difference, and the polymorphism of SHMT gene was analyzed by real-time PCR allelic discrimination. Results: The SHMT CC and CT genotypes were associated with decreased maternal risk for DS (CC: P= 0.0002; 95% CI: 0.20 0.60; OR: 0.35. CT: P < 0.0001; 95% IC: 0.11 - 0.39; OR: 0.21). The different genotypes did not influence the concentrations of metabolites studied. In individuals with DS, the combined genotypes DHFR II/ SHMT TT and DHFR DD/ SHMT TT were associated, respectively, with increased concentrations of Hcy (P<0.001) and folate (P<0.001). Moreover, individuals with DHFR II/ SHMT CT genotypes presented a reduction of folate concentration (P= 0.01). Conclusion: The CC and CT genotypes of SHMT C1020T polymorphism has a protector effect for maternal risk for DS. This polymorphism does not seem to influence the folate, Hcy and MMA concentrations. The del 19pb DHFR and SHMT C1420T polymorphisms present a synergic effect in modulating folate and Hcy concentrations in individuals with DS. / A síndrome de Down (SD) é uma doença genética complexa resultante, principalmente, da presença de três cópias do cromossomo 21. É a cromossomopatia humana mais frequente e, na maioria dos casos (cerca de 95%), decorrente da não disjunção cromossômica materna, ocorridas durante a meiose I. Recentes estudos sugerem que a etiologia do risco materno para a SD em mães jovens está relacionada com polimorfismos em genes do metabolismo do folato/homocisteína (Hcy). O funcionamento adequado do metabolismo do folato é essencial para a síntese de grupos metil necessários para a metilação do DNA. A deficiência deste metabólito tem como resultado, a hipometilação do DNA, quebras cromossômicas e aneuploidias. Objetivos: Avaliar a influência do polimorfismo C1420T do gene serina hidroximetiltransferase (SHMT) no risco materno para a SD e nas concentrações de folato sérico, Hcy e ácido metilmalônico (MMA) plasmáticos ; investigar o impacto dos polimorfismos de deleção de 19 pares de base (pb) do gene dihidrofolato redutase (DHFR) e de transição C1420T do gene SHMT nas concentrações de folato sérico, Hcy e MMA plasmáticos em indivíduos com SD. Casuística e métodos: Foram incluídas no estudo 105 mães de indivíduos com trissomia livre do cromossomo 21 e 185 mães de indivíduos sem a síndrome (sem história prévia de aborto espontâneo), e 85 indivíduos com trissomia livre do cromossomo 21. O polimorfismo do gene DHFR foi avaliado por meio da Reação em Cadeia da Polimerase (PCR) por diferença de tamanho de fragmentos, e o polimorfismo SHMT C1420T foi analisado por PCR em tempo real. Resultados: Os genótipos CC e CT do polimorfismo SHMT C1420T foram associados à redução do risco materno para SD (CC: P = 0,0002; 95% IC: 0,20 0,60; OR: 0,35. CT: P < 0,0001; 95% IC: 0,11 0,39; OR: 0,21). Os diferentes genótipos não influenciaram as concentrações dos metabólitos estudados. No grupo de indivíduos com SD, os genótipos combinados DHFR II/SHMT TT e DHFR DD/SHMT TT foram associados, respectivamente, com concentrações aumentadas de Hcy (P < 0,001) e de folato (P < 0,001). Além disso, indivíduos com os genótipos DHFR II/SHMT CT apresentaram concentração reduzida de folato (P = 0,01). Conclusão: Os genótipos CC e CT do polimorfismo SHMT C1420T conferem um efeito materno protetor para SD. Este polimorfismo parece não influenciar as concentrações de folato, Hcy e MMA. Os polimorfismos del 19pb DHFR e C1420T SHMT apresentam um efeito sinérgico na modulação das concentrações de folato e Hcy de indivíduos com SD.

Les folates sont des vitamines indispensables à tous les âges, particulièrement pendant la grossesse et l’enfance, étant donné leur fonction dans la division cellulaire. Le régime alimentaire en Afrique est essentiellement basé sur les céréales, consommées toujours après transformation. La fermentation est l’un des moyens de transformation des céréales pouvant augmenter les folates dans les aliments. L’objectif de ce travail était d’utiliser la fermentation pour augmenter les ingérés en folates des populations africaines via la consommation d’aliments céréaliers fermentés. Sept aliments céréaliers fermentés couramment consommés en Afrique de l’Ouest ont été investigués. La plupart des aliments avaient des teneurs en folates (1,8–31,3 µg/100g matière fraîche) inférieures à celles des céréales de départ (13,8-73,4 µg/100g matière fraîche). Des pertes en folates ont lieu au cours de certaines étapes de procédés dont le décorticage, le séchage, le trempage, le broyage et la filtration. Toutefois, la fermentation a permis une augmentation de la teneur en folates dans certains aliments. La bioaccessibilité des folates, évaluée à l'aide d'un modèle de digestion statique in vitro, variait de 23% à 81%. Elle était influencée par la matrice alimentaire et la stabilité des folates au cours de la digestion. Il a été calculé qu’au maximum 8% des besoins journaliers en folates des jeunes enfants consommant l’un des aliments étudiés pourraient être couverts. Des essais d’inoculation de bouillies fermentés à base de mil avec des souches de bactéries lactiques sélectionnées pour leurs propriétés nutritionnelles (synthèse de folates, hydrolyse de l’amidon) permettaient d’augmenter significativement les teneurs en folates (jusqu’à 8,7 µg/100 g matière fraîche) par rapport à leur équivalent préparées de manière traditionnelle (2,5-5,4 µg/100 g matière fraîche). L’inoculation par un pied-de-cuve provenant d’une fermentation spontanée permettait aussi une augmentation significative des teneurs folates (jusqu’à 7,4 µg/100 g matière fraîche). La caractérisation de la diversité bactérienne de 7 aliments céréaliers fermentés du Burkina Faso, d’Ethiopie et de la Finlande montrait que les bactéries lactiques du genre Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus et Streptococcus étaient les principaux acteurs de la fermentation de ces aliments. Toutefois, une présence non négligeable d’autres microorganismes potentiellement pathogènes des genres Bacillus, Pseudomonas, Staphylococcus, Erwinia, Klebsiella, Escherichia et Acinetobacter a été identifiée. Cette contamination était liée à certaines étapes du procédé de transformation des céréales dont le stockage et broyage dans les moulins publics. Ces microorganismes pathogènes étaient réduits par fermentation et finalement éliminés après l'étape de cuisson. / Folates represent an essential vitamin in the human diet at all ages, particularly during pregnancy and infancy, as it is required for the production of new cells. In many African countries, the main staple foods are based on cereals, which are always consumed after processing. Fermentation is one of the processing, which could increase folate contents in foods. The objective of this work was to increase folates intake of African people through the consumption of cereal-based fermented foods using fermentation. Seven types of cereal-based fermented foods (CBFF), commonly consumed in West Africa, were investigated in this study. Total folate content of cereal-based fermented ranged between 1.8 and 31.3 µg/100g fresh weight, and was almost always lower than in the raw material (13.8-73.4 µg/100g fresh weight). Folate losses occurred during some processing steps like debranning, soaking and drying steps. However, fermentation was able to increase the folate content in some CBFF. Folate bioaccessibility was assessed using a static in vitro digestion model, and ranged from 23% to 81%. The bioaccessible folate content was influenced by total folate content, the structure of food matrices that modulated folate release, and folate stability during digestion process. Calculations of the contributions of CBFF to the reference nutrient intake for folate showed that folate intakes from these foods would cover a maximum of 8% of the folate requirements for young children. Porridges prepared with starter cultures of lactic acid bacteria selected for the nutritional properties (folate synthesis, starch hydrolysis) had significantly higher folate contents (up to 8.7 µg/100 g fresh matter) than the porridge prepared using the traditional process (2.5-5.4 µg/100 g fresh matter). Back slopping using an inoculum from a spontaneous fermentation also enabled an interesting increase in folate contents (up to 7.4 µg/100 g fresh matter). The bacterial diversity of seven cereal-based fermented foods from Burkina Faso, Ethiopia and Finland were assessed using 16S rRNA amplicon sequencing. Lactic acid bacteria genus, including Lactobacillus, Enterococcus, Weissella, Pediococcus, Lactococcus and Streptococcus were the main bacteria present in cereal-based fermented foods. Several potentially pathogenic bacteria, namely, Bacillus, Pseudomonas, Staphylococcus, Erwinia, Escherichia, Klebsiella and Acinetobacter were also found in some intermediary products resulting from storage and wet milling. These microorganisms were reduced by fermentation and finally removed after the cooking step.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-10-03T18:49:30Z No. of bitstreams: 1 daniellabalduinovictorino_dissert.pdf: 2037812 bytes, checksum: ec4892555795968e4470a593baca8ebc (MD5) / Made available in DSpace on 2016-10-03T18:49:30Z (GMT). No. of bitstreams: 1 daniellabalduinovictorino_dissert.pdf: 2037812 bytes, checksum: ec4892555795968e4470a593baca8ebc (MD5) Previous issue date: 2014-10-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Introduction Down syndrome (DS) is caused by the presence of three copies of chromosome 21 in consequence to chromosome nondisjunction in maternal meiosis observed in about 95% of cases. Genetic polymorphisms involved in folate metabolism were associated with the maternal risk for DS. However, the results are contradictories. Objectives To perform a systematic review and meta-analysis in order to evaluate the association between Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, Methionine synthase reductase (MTRR) A66G, Methionine synthase (MTR) A2756G, Reduced folate carrier 1 (RFC1) A80G, Cystathionine β-synthase (CβS) 844ins68, Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) G1958A and Transcobalamin 2 (TC2) C776G genetic polymorphisms and the maternal risk for DS. Methods Studies were searched up to May 2014 on MEDLINE, EMBASE, LILACS, hand searched reference lists of published articles and conference meetings and personal communication. Case-control studies that evaluated the association between genetic polymorphisms in case mothers (DSM) and controls mothers (CM) were included. DSM are considered mothers that have gave birth to children with free trisomy of 21 chromosome and CM are considered mothers that have gave birth to children without chromosomal abnormality, syndrome or malformation. Studies with mothers of DS individuals with translocation or mosaicism, case reports, editorials and review articles were excluded. Data extraction and quality assessment were performed independently by two investigators. Meta-analysis assesses the associations between each genetic polymorphism and maternal risk for DS by dominant, recessive, codominant and allelic genetic models. Dichotomous outcome measures were pooled using fixed and random effects models and the results were expressed by odds ratio (OR) with 95% confidence intervals (95% CI). Heterogeneity between studies was evaluated using Q test and the I2 and subgroup and sensitivity analyses were performed in order to investigate the potential sources of heterogeneity. Publication bias was estimated using funnel plot and linear regression test. Results Collectively, 30 case-control studies including 3,101 DSM and 3,967 CM were included. Significant association between MTHFR C677T and MTRR A66G polymorphisms and maternal risk for DS was found when all population is considered. Subgroup and sensitivity analyses according ethnicity showed significant associations for the MTHFR C677T polymorphism in Caucasians, Brazilians and Asians and for the MTRR A66G polymorphism in Caucasians. Additionally, the results of the RFC1 A80G polymorphism demonstrated significant association, it was also found in Asians and maternal age less than 35 years at conception subgroups analyses. Finally, MTHFD1 1958GA genotype was revealed as maternal risk factor for DS when only studies with control group in Hardy-Weinberg equilibrium were considered. No association among MTHFR A1298C, MTR A2756G, CβS 844ins68 and TC2 C776G polymorphisms and maternal risk for DS was found. Conclusions MTHFR C677T, MTRR A66G, RFC1 A80G and MTHFD1 1958GA polymorphisms are associated with maternal risk for DS. / Introdução Síndrome de Down (SD) é atribuída à presença de três cópias do cromossomo 21, decorrente da não-disjunção cromossômica meiótica materna em 95% dos casos. Polimorfismos genéticos maternos envolvidos no metabolismo do folato foram associados ao nascimento de indivíduos com a SD, porém os resultados dos estudos são contraditórios. Objetivos Avaliar, por meio de revisão sistemática e metanálise, a associação entre os polimorfismos genéticos maternos Metilenotetrahidrofolato redutase (MTHFR) C677T e A1298C, Metionina sintase redutase (MTRR) A66G, Metionina sintase (MTR) A2756G, Carreador de folato reduzido 1 (RFC1) A80G, Cistationina β-sintase (CβS) 844ins68, Metilenotetrahidrofolato desidrogenase 1 (MTHFD1) G1958A e Transcobalamina 2 (TC2) C776G e o nascimento de indivíduos com a SD. Métodos As buscas bibliográficas foram realizadas anteriormente a maio de 2014 e os bancos de dados utilizados foram: PUBMED, EMBASE, LILACS, lista de referências bibliográficas dos artigos selecionados, busca manual em anais de congressos e comunicação pessoal. Foram incluídos estudos caso-controle que avaliaram a presença dos polimorfismos genéticos em mães de crianças com SD por trissomia livre do cromossomo 21 (mães-caso) e em mães de crianças sem histórico de anormalidades cromossômicas, síndromes ou malformações (mães-controle). Os critérios de exclusão consistiram em estudos que incluíram mães de crianças com SD por translocação ou mosaicismo, relatos de caso, editoriais e artigos de revisão. A extração dos dados e a avaliação da qualidade dos estudos foram feitas por dois investigadores. A metanálise avaliou a associação entre cada polimorfismo e o risco materno para a SD por meio dos modelos genéticos dominante, recessivo, codominante e alélico. Medidas de desfecho dicotômicas foram sumarizadas utilizando-se modelos de efeito fixo e randômico e os resultados foram expressos em odds ratio (OR) com intervalo de confiança de 95% (IC 95%). A heterogeneidade entre estudos foi calculada pelo teste Q e pela estatística I2 e suas potenciais fontes foram investigadas pelas análises de sensibilidade e subgrupo. O viés de publicação foi estimado pelos funnel plot e teste de regressão linear. Resultados Coletivamente, 30 estudos caso-controle preencheram os critérios de elegibilidade, o que totalizou 3.101 mães-caso e 3.967 mães-controle. Foi verificada associação significativa entre os polimorfismos MTHFR C677T e MTRR A66G e o risco materno para SD. As análises de subgrupo de acordo com a etnia revelaram associações significativas para o polimorfismo MTHFR C677T e o risco materno para a SD em caucasianos, brasileiros e asiáticos e para o polimorfismo MTRR A66G em caucasianos. Adicionalmente, foi encontrada associação significativa para o polimorfismo RFC1 A80G e o risco materno para a SD e também nas análises de subgrupo de asiáticos e de mães com idade materna inferior a 35 anos no momento da concepção. Finalmente, o genótipo MTHFD1 1958GA revelou-se fator de risco materno para o nascimento de indivíduos com SD quando a análise foi restringida aos estudos cujo grupo controle estava em equilíbrio de Hardy-Weinberg. Nenhuma associação foi verificada para os polimorfismos MTHFR A1298C, MTR A2756G, CβS 844ins68 e TC2 C776G. Conclusões Os polimorfismos MTHFR C677T, MTRR A66G, RFC1 A80G e MTHFD1 G1958A são fatores de risco materno para a SD.

A cobalamina (vitamina B12) e o ácido fólico são nutrientes essenciais para síntese de DNA, metionina e S-adenosilmetionina (SAM). A SAM é uma potente doadora de grupo metil necessário nas reações de metilação do DNA. Deficiências dessas vitaminas podem comprometer as concentrações da SAM e, indiretamente, a metilação do DNA. O objetivo deste estudo foi avaliar o efeito das concentrações de vitaminas (no sangue e na dieta), metabólitos (no soro) e do polimorfismo MTHFR C677T na taxa de metilação global do DNA. Participaram do estudo 103 gestantes, das quais foram colhidas amostras de sangue nas idades gestacionais de 16, 28 e 36 semanas. Foram realizadas as determinações dos valores de folato eritrocitário, e das concentrações séricas de folato, cobalamina, vitamina B6, homocisteína (tHcy), metionina, ácido metilmalônico (MMA), SAM e S-adenosilhomocisteína (SAH), além genotipagem para o polimorfismo MTHFR C677T por PCR-RFLP. A taxa de metilação global do DNA foi avaliada indiretamente por reação de extensão usando [3H]dCTP. Para avaliação nutricional foi aplicado um inquérito recordatório de 24 horas no dia de cada coleta. Devido ao uso de suplementação com ácido fólico e/ou polivitamínicos, as participantes foram classificadas em quatro grupos: grupo 1 (50 gestantes que não usaram suplementação); grupo 2 (14 mulheres que fizeram uso de suplementos nas três idades gestacionais); grupo 3 (21 participantes que usaram suplementação até a 16ª semana de gravidez) e grupo 4 (10 gestantes que utilizaram suplementação nas idades gestacionais de 16 e 28 semanas apenas). A taxa de metilação global do DNA foi menor na 28ª semana de gravidez quando comparada àquelas encontradas nas 16 e 36ª semanas em todos os grupos estudados. No grupo 1, as seguintes variáveis: concentrações de folato eritrocitário, vitamina B6 sérica, MMA, SAM, creatinina, vitamina B6 da dieta, proteínas de carnes (dieta), idade gestacional de 36 semanas e o genótipo CT + TT do polimorfismo MTHFR C677T foram associadas com a taxa de metilação do DNA, bem como as interações entre os genótipos CT + TT e folato eritrocitário ou sérico ou SAM; entre folato sérico e vitamina B6; entre creatinina e MMA ou vitamina B6 da dieta, e entre SAM e proteínas de carnes da dieta. As interações entre: 1 - menores concentrações de folato sérico e baixa ingestão de ácido fólico; 2 - maiores concentrações de MMA e baixa ingestão de vitamina B6; 3 - idade gestacional de 36 semanas e genótipo CT + TT do polimorfismo C677T no gene MTHFR foram associadas ao maior risco de apresentar menores taxas de metilação global do DNA no grupo 1. / Cobalamin (B12 vitamin) and folic acid are essential nutrients to DNA, methionine and S-adenosylmethinone (SAM) synthesis. SAM, the main methyl group donator, is necessary to DNA methylation reactions. Cobalamin and folic acid deficiencies might implicate SAM concentrations and, moreover, DNA methylation. The objective of this study was to evaluate the effects of serum vitamin and metabolites, vitamin intake and MTHFR C677T genetic polymorphism on global DNA methylation. Blood samples from 103 pregnant women were collected at gestational age of 16, 28 and 36 weeks. Red blood cell folate levels (RBC fol), and serum concentrations of folate (Fol), cobalamin, B6 vitamin, homocysteine (tHcy), methionine, methylmalonic acid (MMA), SAM and S-adenosylhomocysteine (SAH) were evaluated. The MTHFR C677T polymorphism was detected by PCR-RFLP. A radiolabeled [3H]dCTP extension assay was used to assess the level of global DNA methylation. Nutrient intake was assessed by 24 hour dietary recall applied at 16, 28 and 36 weeks of pregnancy. According to folic acid and/or multivitamins supplementation, women were classified in four groups: group 1 (50 women with no supplementation); group 2 (14 subjects who took supplements at three gestational ages); group 3 (21 women which took supplements until 16 weeks of pregnancy) and group 4 (10 subjects that took supplements at gestational ages of 16 and 28 weeks, but not at 36 weeks). Global DNA methylation level was lower at 28 weeks compared with 16 or 36 weeks.of pregnancy in women from four studied groups. In the group 1, not only values of RBC fol; serum concentrations of B6 vitamin, MMA, SAM and creatinine; vitamin B6 and meat protein intake; gestational age of 36 weeks and MTHFR 677T allele (CT+TT genotypes) were related to global DNA methylation in these women, but also interactions between MTHFR 677T allele and RBC fol or serum Fol or SAM; among serum Fol and B6 vitamin; between creatinine and MMA or vitamin B6 intake; and among SAM and meat protein intake. The following interactions between: 1 - lower serum Fol concentration and lower folate intake; 2 - higher serum MMA concentration and lower B6 vitamin intake; and 3 - gestational age of 36 weeks and MTHFR 677T allele were associated with higher risk of global DNA hypomethylation in pregnant women from group 1.

A cobalamina (vitamina B12) e o ácido fólico são nutrientes essenciais para síntese de DNA, metionina e S-adenosilmetionina (SAM). A SAM é uma potente doadora de grupo metil necessário nas reações de metilação do DNA. Deficiências dessas vitaminas podem comprometer as concentrações da SAM e, indiretamente, a metilação do DNA. O objetivo deste estudo foi avaliar o efeito das concentrações de vitaminas (no sangue e na dieta), metabólitos (no soro) e do polimorfismo MTHFR C677T na taxa de metilação global do DNA. Participaram do estudo 103 gestantes, das quais foram colhidas amostras de sangue nas idades gestacionais de 16, 28 e 36 semanas. Foram realizadas as determinações dos valores de folato eritrocitário, e das concentrações séricas de folato, cobalamina, vitamina B6, homocisteína (tHcy), metionina, ácido metilmalônico (MMA), SAM e S-adenosilhomocisteína (SAH), além genotipagem para o polimorfismo MTHFR C677T por PCR-RFLP. A taxa de metilação global do DNA foi avaliada indiretamente por reação de extensão usando [3H]dCTP. Para avaliação nutricional foi aplicado um inquérito recordatório de 24 horas no dia de cada coleta. Devido ao uso de suplementação com ácido fólico e/ou polivitamínicos, as participantes foram classificadas em quatro grupos: grupo 1 (50 gestantes que não usaram suplementação); grupo 2 (14 mulheres que fizeram uso de suplementos nas três idades gestacionais); grupo 3 (21 participantes que usaram suplementação até a 16ª semana de gravidez) e grupo 4 (10 gestantes que utilizaram suplementação nas idades gestacionais de 16 e 28 semanas apenas). A taxa de metilação global do DNA foi menor na 28ª semana de gravidez quando comparada àquelas encontradas nas 16 e 36ª semanas em todos os grupos estudados. No grupo 1, as seguintes variáveis: concentrações de folato eritrocitário, vitamina B6 sérica, MMA, SAM, creatinina, vitamina B6 da dieta, proteínas de carnes (dieta), idade gestacional de 36 semanas e o genótipo CT + TT do polimorfismo MTHFR C677T foram associadas com a taxa de metilação do DNA, bem como as interações entre os genótipos CT + TT e folato eritrocitário ou sérico ou SAM; entre folato sérico e vitamina B6; entre creatinina e MMA ou vitamina B6 da dieta, e entre SAM e proteínas de carnes da dieta. As interações entre: 1 - menores concentrações de folato sérico e baixa ingestão de ácido fólico; 2 - maiores concentrações de MMA e baixa ingestão de vitamina B6; 3 - idade gestacional de 36 semanas e genótipo CT + TT do polimorfismo C677T no gene MTHFR foram associadas ao maior risco de apresentar menores taxas de metilação global do DNA no grupo 1. / Cobalamin (B12 vitamin) and folic acid are essential nutrients to DNA, methionine and S-adenosylmethinone (SAM) synthesis. SAM, the main methyl group donator, is necessary to DNA methylation reactions. Cobalamin and folic acid deficiencies might implicate SAM concentrations and, moreover, DNA methylation. The objective of this study was to evaluate the effects of serum vitamin and metabolites, vitamin intake and MTHFR C677T genetic polymorphism on global DNA methylation. Blood samples from 103 pregnant women were collected at gestational age of 16, 28 and 36 weeks. Red blood cell folate levels (RBC fol), and serum concentrations of folate (Fol), cobalamin, B6 vitamin, homocysteine (tHcy), methionine, methylmalonic acid (MMA), SAM and S-adenosylhomocysteine (SAH) were evaluated. The MTHFR C677T polymorphism was detected by PCR-RFLP. A radiolabeled [3H]dCTP extension assay was used to assess the level of global DNA methylation. Nutrient intake was assessed by 24 hour dietary recall applied at 16, 28 and 36 weeks of pregnancy. According to folic acid and/or multivitamins supplementation, women were classified in four groups: group 1 (50 women with no supplementation); group 2 (14 subjects who took supplements at three gestational ages); group 3 (21 women which took supplements until 16 weeks of pregnancy) and group 4 (10 subjects that took supplements at gestational ages of 16 and 28 weeks, but not at 36 weeks). Global DNA methylation level was lower at 28 weeks compared with 16 or 36 weeks.of pregnancy in women from four studied groups. In the group 1, not only values of RBC fol; serum concentrations of B6 vitamin, MMA, SAM and creatinine; vitamin B6 and meat protein intake; gestational age of 36 weeks and MTHFR 677T allele (CT+TT genotypes) were related to global DNA methylation in these women, but also interactions between MTHFR 677T allele and RBC fol or serum Fol or SAM; among serum Fol and B6 vitamin; between creatinine and MMA or vitamin B6 intake; and among SAM and meat protein intake. The following interactions between: 1 - lower serum Fol concentration and lower folate intake; 2 - higher serum MMA concentration and lower B6 vitamin intake; and 3 - gestational age of 36 weeks and MTHFR 677T allele were associated with higher risk of global DNA hypomethylation in pregnant women from group 1.