Rozprawy doktorskie na temat „Folic acid Metabolism”

Orientador: Ricardo Della Coletta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A fissura labial e/ou palatina (FL/P) não-sindrômica é uma malformação congênita do lábio e/ou palato com alta frequência na população brasileira. A etiologia das fissuras é complexa e conta com a participação de fatores genéticos e ambientais. Inúmeros estudos demonstraram que variantes polimórficas das enzimas relacionadas ao metabolismo do ácido fólico podem ser importantes fatores de risco materno para o nascimento de uma criança FL/P não-sindrômica. O objetivo deste estudo foi estudar a influência do consumo de suplementos vitamínicos durante o primeiro trimestre de gravidez e comparar a frequência alélica e genotípica de 4 genes (MTHFR, MTHFD1, MTR e RFC1) que codificam enzimas da via metabólica do ácido fólico entre mães de indivíduos portadores de FL/P não-sindrômicas (grupo experimental) e mães de indivíduos clinicamente normais (grupo controle). Amostras de DNA de 184 mães do grupo controle e de 106 mães do grupo experimental foram genotipadas por reação em cadeia da polimerase associada à análise de polimorfismo de fragmentos de restrição enzimática (PCR-RFLP). A ausência de suplemento vitamínico durante o primeiro trimestre de gravidez aumentou de forma discreta (aproximadamente em 0,4 vezes) o risco de uma mulher ter um filho com FL/P não-sindrômica. Dos 15 polimorfismos analisados neste estudo, 2 apresentaram diferenças entre os grupos. No polimorfismo rs2274976 do gene MTHFR, o alelo A e o genótipo GA ocorreram em uma frequência significantemente maior no grupo experimental que no grupo controle (p<0,000001), aumentando em aproximadamente 6 vezes o risco de uma mãe ter um filho com FL/P não-sindrômica. O genótipo AA no lócus polimórfico rs2236225 do gene MTHFD1 foi significantemente mais prevalente no grupo experimental comparado com o grupo controle (p=0,02). A presença deste genótipo aumentou em aproximadamente 2 vezes o risco de uma mãe ter um filho com FL/P não-sindrômica. Análise multivariada demonstrou que estes fatores contribuíram de maneira independente para a etiologia das FL/P não-sindrômicas. O presente estudo demonstra que os polimorfismos rs2274976 do gene MTHFR e rs2236225 do gene MTHFD1 e a suplementação vitamínica durante o primeiro trimestre de gravidez estão associados ao desenvolvimento de FL/P não-sindrômicas na população brasileira. Este estudo corrobora com evidências prévias que demonstraram a influência de fatores ambientais e genéticos na etiopatogenia das FL/P não-sindrômicas.
Abstract: Nonsyndromic cleft lip with or without cleft palate (CL/P) is a congenital malformation of the lip and/or palate with elevating frequency in the Brazilian population. The etiology of the nonsyndromic CL/P is complex and both environmental and genetic factors play important roles. Several studies demonstrated that polymorphisms in the folic acid metabolic enzymes may be important maternal risk factor for the birth of a child with nonsyndromic CL/P. The aim of this study was to determine the influence of the multivitamin supplements during the first trimester of pregnancy and to compare the allele and genotypic frequencies of 4 genes (MTHFR, MTHFD1, MTR and RFC1) that encode enzymes of the acid folic metabolic pathway between mothers of nonsyndromic CL/P patients (experimental group) and mothers of clinically normal children (control group). DNA samples from 184 mothers of the control group and from 106 mothers of the experimental group were genotyped by polymerase chain reaction associated with reaction fragment length polymorphism (PCR-RFLP). The lack of multivitamin supplementation during the pregnancy first trimester increased in approximately 0.4-fold the maternal risk of a nonsyndromic CL/P child. Two out of 15 polymorphisms showed differences between groups. In rs2274976 MTHFR polymorphism, allele A and genotype GA occurred in a significantly higher frequency on experimental group when compared to control group (p<0.000001), rising in approximately 6 times the risk of a mother giving birth to a nonsyndromic CL/P child. Genotype AA in the rs2236225 MTHFD1 polymorphic locus was significantly more prevalent in experimental group than in control group (p=0.02). This genotype raised in approximately twice the risk of a mother giving birth to a nonsyndromic CL/P child. Multivariate analysis demonstrated that those factors contributed in an independent manner to nonsyndromic CL/P etiology. The present study shows that rs2274976 MTHFR and rs2236225 MTHFD1 polymorphisms, as well as the multivitamin supplementation during the first trimester of pregnancy, are associated with the development of nonsyndromic CL/P in the Brazilian population. This study corroborates with previous evidences demonstrating the influence of environmental and genetic factor on etiopathogenesis of the nonsyndromic CL/P.
Mestrado
Patologia
Mestre em Estomatopatologia

Reprints of the author's previously published articles included as an appendix. Bibliography: leaves 165-188. "This thesis describes a series of experiments that aimed to investigate the effects of folic acid deficiency and defects in folate metabolism on chromosome damage rates in human lymphocytes. The accumulation of chromosome damage over time is an important issue because it is thought to contribute to the mechanism of ageing and the aetiology of diseases of age such as cancer and Alzheimer's disease."

Folate deficiency, a prevalent vitamin deficiency in America, can stem from environmental and/or genetic causes. The most common inborn error of folate metabolism is deficiency of methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Severe MTHFR deficiency results in hyperhomocysteinemia and homocystinuria; patients present with developmental delay, and various neurological and vascular disorders. This thesis describes three mutations identified in the MTHFR locus in patients with severe deficiency: 1025T→C (M→T), 1027T→G (W→G), and 1768G→A (E→K). Genotype-phenotype correlations are described, along with biochemical characterization of three mutations (983A→G (N→S), 1025T→C, 1027T→G). All three mutations exert their effect by decreasing Vmax without changing the enzyme's affinity for its substrate, 5-methyltetrahydrofolate. The 983A→G variant also conferred decreased affinity for FAD, a cofactor.
The more common and mild deficiency observed in the general healthy population is probably due in part to insufficient dietary intake of folate. Folate deficiency has been associated with increased risk for colon cancer. In a pilot study presented here, the impact of altered folate intake on tumor multiplicity in the Min mouse, a model for multiple intestinal neoplasia, was assessed. Folate deficient diets did not produce a consistent change in tumor numbers. However, a linear correlation between S-adenosylmethionine and S-adenosylhomocysteine content of preneoplastic tissue and tumor multiplicity was identified.
This thesis contributes to our understanding of the impact of genetic- and/or dietary-induced folate deficiency on cellular and organismal functions.

The relationship between colorectal cancer (CRC) and folate metabolism is complex. Dietary folate, depending on the timing and dose, may either prevent or enhance tumour initiation and/or growth, and polymorphisms in the genes encoding folate-metabolising enzymes may also modulate risk. In this thesis, the Apcmin/+ mouse model of CRC was used to investigate the effect of nutritional and genetic disturbances in folate metabolism on tumourigenesis and to examine various mechanisms.
The reduced folate carrier I (RFC1) is responsible for the cellular uptake and intestinal absorption of folate, primarily the 5-methyltetrahydrofolate (5-methylTHF) derivative. Methionine synthase (MTR) uses 5-methylTHF to remethylate homocysteine to methionine, which may be activated and used to methylate substrates such as DNA. 5-MethylTHF is also the product of the methylenetetrahydrofolate reductase (MTHFR)-catalysed reduction of 5,10-methyleneTHF, which is also used to convert dUMP to dTMP.
Adenoma number and load were reduced in Rfc1+/-Apc min/+ mice, compared with Rfc1+/+Apc min/+ mice, but were similar in Mtr+/-Apc min/+ and Mtr+/+ Apcmin/+ mice. Neither Rfc1 nor Mtr genotype affected global DNA methylation, apoptosis or plasma homocysteine (tHcy) levels. In the experiments involving Mtr mice, dietary folate deficiency increased adenoma number, plasma tHcy, and apoptosis, and decreased global DNA methylation. Neither Mtr nor Rfc1 genotype affected the dUTP/dTTP ratio in the intestine of mice not predisposed to adenoma formation.
Adenoma number was decreased in Mthfr+/-Apc min/+ mice (compared with Mthfr+/+Apc min/+ mice) and in Mthfr+/+Apc min/+ offspring of Mthfr+/- mothers (compared with Mthfr+/+Apcmin/+ offspring of Mthfr+/+ mothers). A folate-deficient diet, when initiated prior to conception, significantly decreased adenoma number and decreased global DNA methylation. Overall, adenoma number was inversely correlated with plasma tHcy, dUTP/dTTP ratio and apoptosis. When initiated at three weeks of age, a folate-enriched diet significantly increased adenoma number in Apcmin/+ mice. In the intestines of mice not predisposed to adenoma formation, Mthfr deficiency decreased, and folic acid deficiency increased, the dUTP/dTTP ratio.
These results support the evidence that MTHFR polymorphisms are protective in CRC tumourigenesis and that depending on stage or predisposition, folate may inhibit or enhance tumour growth.

Glutamate formiminotransferase deficiency (OMIM 229100) is an autosomal recessive disorder marked by clinical heterogeneity. The severe phenotype, first identified in patients of Japanese descent, includes high levels of formiminoglutamate (FIGLU) in the urine in response to histidine loading, megaloblastic anemia, and mental retardation. The mild phenotype is marked by high levels of FIGLU in the urine in the absence of histidine loading, mild developmental delay and no hematological abnormalities. The gene for human glutamate formiminotransferase-cyclodeaminase consists of 15 exons and is located at 21q22.3. The protein consists of a tetramer of dimers, with dimerization essential for both formiminotransferase and cyclodeaminase activity.
Genomic DNA extracted from cell lines from three patients with suspected glutamate formiminotransferase deficiency was analyzed by PCR and sequencing of individual exons. Cell lines WG 1758 and WG 1759 are from two siblings of Germanic descent. Both siblings are heterozygous for the mutations c457 C → T and c940 G → C. The c457 C → T changes a conserved arginine to a cysteine in a loop involved in the binding of formiminotetrahydrofolate to the enzyme. The c940 G → C mutation converts an arginine to a proline in an alpha-helix essential for the dimerization of the formiminotransferase domain. Cell line WG 1795 is from a patient of Danish descent. The patient appears to be hemizygous for a c1033 insG mutation. Quantitative PCR suggests the presence of a deletion on the other chromosome, which minimally encompasses exon 9. All of the FTCD gene changes were absent in 100 control individuals (200 alleles).

Orientadores: Carmen Silvia Passos Lima, Maria de Lourdes Setsuko Ayrizono
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: O desenvolvimento de câncer colorretal (CCR) é resultado de uma complexa interação de variáveis, incluindo elementos externos, como a exposição a agentes ambientais e dietéticos, e fatores internos, de natureza somática ou hereditária. Não está estabelecido se genótipos de polimorfismos de baixa penetrância em genes relacionados com o metabolismo do ácido fólico, como o metilenotetrahidrofolato redutase (MTHFR C677T e MTHFR A1298C), o metionina sintase (MTR A2756G), o metionina sintase redutase (MTRR A66G) e o timidilato sintase (TS 2R3R), estão associados com o risco de ocorrência da doença ou com suas manifestações clínicas. Portanto, o objetivo deste estudo foi verificar se esses polimorfismos gênicos influenciam o risco de ocorrência do adenocarcinoma colorretal esporádico (ACRE) e suas manifestações clínicas e biológicas em pacientes da região sudeste do Brasil. Foram avaliados 113 pacientes com ACRE e 188 controles, considerando os aspectos clínicos como a idade, o sexo, a raça, a localização, o grau de diferenciação do tumor, o estágio e os genótipos de cada gene. Os genótipos dos polimorfismos dos genes MTHFR, MTR, MTRR e TS foram avaliados por meio da reação em cadeia da polimerase (PCR) seguida ou não por digestão enzimática. O significado estatístico das diferenças entre grupos foi calculado por meio do teste da probabilidade exata de Fisher ou qui-quadrado. As determinações dos riscos de ocorrência do ACRE, a que pacientes e controles foram submetidos, foram obtidas por meio das razões das chances (ORs) e calculadas considerando um intervalo de confiança de 95%. Portadores dos genótipos MTRR 66AG+GG, do MTHFR 1298AC+CC+677CT+TT, do MTHFR 677CT+TT+MTR 2756AG+GG, do MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG e MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG apresentaram riscos 1,99, 3,26, 2,22, 10,92 e 14,88 vezes maiores, respectivamente, de desenvolver ACRE do que os outros. Além disso, os indivíduos com o genótipo MTHFR 677CT+TT e os genótipos MTR 2756AG+GG tiveram um risco de 2,12 e 1,42 vezes maior de desenvolver ACRE com idade menor do que 50 anos. Afro-Brasileiros com o genótipo GG do polimorfismo MTRR A66G tiveram risco 1,98 vezes maior de desenvolver ACRE, e indivíduos com o genótipo MTR 2756AG+GG e os genótipos MTHFR 677CT+TT estiveram sob risco 2,11 e risco 1,62 vezes maiores de ocorrência de tumores indiferenciados e avançados, respectivamente, do que os demais. Portadores dos genótipos MTHFR 1298AC+CC e MTHFR 1298AC+CC + MTRR 66AG+GG estiveram sob riscos 1,42 e 3,07 vezes maiores de tumor no reto, respectivamente, enquanto que portadores dos genótipos MTHFR 677CT+TT e MTHFR 677CT+TT + TS 2R3R+3R3R estiveram sob riscos 1,55 e 5,39 vezes maiores de tumor de cólon, respectivamente, do que portadores dos genótipos selvagens. Estes dados sugerem que polimorfismos dos genes MTHFR, MTR, MTRR e TS, que codificam enzimas que participam do metabolismo do ácido fólico, especialmente em combinação, têm papéis consistentes para o risco de desenvolver ACRE em indivíduos da região sudeste do Brasil
Abstract: The development of colorectal cancer (CRC) is the result of a complex interaction of variables, including external factors such as exposure to environmental agents and dietary factors and internal factors, whether somatic or hereditary. Is not been established genotypes with low penetrance polymorphisms in genes related to metabolism of folic acid such as methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G) and thymidylate synthase (TS 2R3R), are associated with the risk of the disease or its clinical manifestations. Therefore, the aim of this study was to determine whether these genetic polymorphisms influence the risk of sporadic colorectal adenocarcinoma (SCA) and their clinical and biological manifestations in patients from southeast Brazil. For this, we analyzed 113 patients with SCA and 188 controls, considering the clinical aspects such as age, sex, race, location, stage, degree of tumor differentiation and the genotypes of each gene described above. The genotypes of the polymorphisms of the MTHFR, MTR, MTRR and TS were assessed by polymerase chain reaction (PCR) and enzyme digestion. The statistical significance of differences between groups was calculated using the probability test of Fisher's exact or chi-square. Determination of the risks of SCA, the patients and controls were submitted, was obtained through the odds ratios (ORs) and calculated assuming a range of 95%. Carriers of the MTRR 66AG + GG, the MTHFR 1298AC+CC + 677CT+TT, the MTHFR 677CT+TT + MTR 2756AG+GG, the MTHFR 1298AC+CC + 677CT+TT + MTR 2756AG+GG, and the MTHFR 1298AC+CC + 677CT+TT + MTRR 66AG+GG genotypes had a 1.99, a 3.26, a 2.22, a 10.92 and a 14.88-fold increased risks for SCA than others, respectively. In addition, individuals with the MTHFR 677CT+TT and the MTR 2756AG+GG genotypes had a 2.12 and a 1.42-fold increased risks for SCA diagnosed under 50 years. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk for SCA, and individuals with the MTR 2756AG+GG and the MTHFR 677CT+TT genotypes were under a 2.11 and a 1.62-fold increased risks for undifferentiated and advanced tumors, respectively, than others. Carriers of the MTHFR 1298AC+CC and the MTHFR 1298AC+CC + MTRR 66AG+GG genotypes had a 1.42 and a 3.07-fold increased risks for rectal tumor, respectively, while carriers of the MTHFR 677CT+TT and the MTHFR 677CT+TT + TS 2R3R+3R3R genotypes had a 1.55 and a 5.39-fold increased risks for colon tumor, respectively, than carriers of the wild genotypes. This data suggest that polymorphisms of genes MTHFR, MTR, MTRR and TS, which encode folate-dependent enzymes, particularly in combination, have consistent roles for SCA risk in southeastern Brazil
Doutorado
Fisiopatologia Cirúrgica
Doutor em Ciências

Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a methyl donor for conversion of homocysteine to methionine. A common thermolabile variant causes mild MTHFR deficiency, induces mild hyperhomocysteinemia when plasma folate levels are low and increases risk for neural tube defects (NTD) and pregnancy loss. To increase our understanding of Mthfr regulation, the 5' and 3' regions of the mouse cDNA and gene were characterized. These studies revealed two major promoters, an internal coding exon in the 5'UTR, alternative transcriptional and translational start sites and alternative splicing and polyadenylation. These data suggest that Mthfr regulation is likely to be complex. To investigate the role of Mthfr in NTD, several approaches were taken. First, folate and MTHFR co-factor, flavin adenine dinucleotide, were shown to stabilize normal and thermolabile MTHFR during heat inactivation, suggesting that folate might prevent hyperhomocysteinemia in individuals with thermolabile enzyme through protein stabilization. Next, in situ hybridization of neurulating mouse embryos showed that Mthfr is expressed in the forebrain, hindbrain, branchial arches, blood vessels, gut, and importantly, in the ventral part of the neural tube. Mthfr+/- mice were then used as a model of mild deficiency to address the effects of maternal and embryonic Mthfr deficiency on development. When combined with inadequate dietary folate, Mthfr +/- pregnant females showed a two-fold higher rate of pregnancy loss than Mthfr+/+ pregnant females. As well, a percentage of day 10.5 embryos from only the Mthfr+/- pregnant females were underdeveloped by 2 days. These effects were not apparent when dietary folate was sufficient, consistent with a genetic-nutritional interactive effect. Finally, folate metabolism was investigated in an NTD model, the curly-tail (ct) mouse, since the ct defect and Mthfr were mapped in close proximity. However, Mthfr sequence in ct mice was simila

Hyperhomocysteinemia is a risk factor for cardiovascular disease and stroke. Nutritional and/or genetic disruptions in homocysteine metabolism can cause hyperhomocysteinemia. Mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the 677C → T mutation in the MTHFR gene is the most common genetic cause of hyperhomocysteinemia. The 677C → T variant is associated with an increased risk for neural tube defects, pregnancy complications, schizophrenia and Down syndrome, and with a decreased risk for colon cancer and leukemia. This variant is also a potential risk factor for vascular disease. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. We have generated mice with a knockout of the Mthfr gene. The Mthfr-deficient mice exhibit hyperhomocysteinemia and decreased methylation capacity. The Mthfr+/- mice appear normal, whereas the Mthfr-/- mice are smaller and have reduced survival. Abnormal external granule neuron development associated with increased cell death in the cerebellum was observed in the Mthfr-/- mice.
Evidence for cardiovascular pathology was obtained in several ways. Impaired aortic relaxation response to acetylcholine was seen in the Mthfr +/- mice fed a high methionine diet. Both Mthfr+/- and Mthfr-/- mice fed a low folate high methionine diet developed myocardial fibrosis in the left ventricle. Abnormal lipid deposition in the proximal portion of the aorta was observed in older Mthfr+/- and Mthfr-/- mice. After crossing Mthfr -deficient mice with apoE-null mice, we demonstrated that MTHFR deficiency promoted atherogenesis and its progression in the apoE-null mice.
Gene expression in brain of Mthfr-deficient mice was investigated via microarray analysis. Five genes with altered expression in the brain of Mthfr-/- mouse were validated by RT-PCR. In biochemical studies of human MTHFR, both FAD and folate were shown to stabilize the purified recombinant wild type and mutant MTHFRs from the baculovirus expression system against heat inactivation. The effect of folate appeared to be secondary to that of FAD, and S-adenosylmethionine (SAM) inhibited purified wild type and mutant MTHFRs with similar efficiency.
This dissertation will significantly contribute to our understanding of the role of MTHFR in human disease.

The principle aim of this study was to investigate biological predictors of response and resistance to multiple myeloma treatment. Two hypothesis had been proposed as responsible of responsiveness: SNPs in DNA repair and Folate pathway, and P-gp dependent efflux. As a first objective, panel of SNPs in DNA repair and Folate pathway genes, were analyzed. It was a retrospective study in a group of 454, previously untreated, MM patients enrolled in a randomized phase III open-label study. Results show that some SNPs in Folate pathway are correlated with response to MM treatment. MTR genotype was associated with favorable response in the overall population of MM patients. However, this relation, disappear after adjustment for treatment response. When poor responder includes very good partial response, partial response and stable/progressive disease MTFHR rs1801131 genotype was associated with poor response to therapy. This relation - unlike in MTR – was still significant after adjustment for treatment response. Identification of this genetic variant in MM patients could be used as an independent prognostic factor for therapeutic outcome in the clinical practice. In the second objective, basic disposition characteristics of bortezomib was investigated. We demonstrated that bortezomib is a P-gp substrate in a bi-directional transport study. We obtain apparent permeability rate values that together with solubility values can have a crucial implication in better understanding of bortezomib pharmacokinetics with respect to the importance of membrane transporters. Subsequently, in view of the importance of P-gp for bortezomib responsiveness a panel of SNPs in ABCB1 gene - coding for P-gp - were analyzed. In particular we analyzed five SNPs, none of them however correlated with treatment responsiveness. However, we found a significant association between ABCB1 variants and cytogenetic abnormalities. In particular, deletion of chromosome 17 and t(4;14) translocation were present in patients harboring rs60023214 and rs2038502 variants respectively.

Au cours du développement, les modifications du métabolisme des monocarbones liées à une malnutrition peuvent être délétères autant pour la mère que pour le nouveau-né. De plus, les conséquences à long terme d'une carence en période gestationnelle et périnatale sont mal connues, notamment en ce qui concerne les pathologies cardiaques et hépatiques. Nous avons mis en oeuvre un modèle nutritionnel de rates adultes carencées en donneurs de groupements méthyles (vitamines B12, folates et choline) avant la gestation. Ces micronutriments participent à la régulation de différentes enzymes impliquées dans le métabolisme de l'homocystéine. Afin de se placer dans un contexte de physiopathologie, proche de la situation clinique évaluée, nous avons choisi d'alimenter les rates avec un régime carencé un mois avant la mise en accouplement et de poursuivre ce régime pendant la période d'allaitement. Nous avons évalué les répercussions métaboliques et fonctionnelles du régime sur les tissus myocardique et hépatique, chez le nouveau né à 21 jours. Nous avons étudié l'effet de cette carence en groupements méthyles sur le métabolisme énergétique lipidique et sur la carnitine. Conséquences de la carence au niveau myocardique : Le régime carencé en donneurs de méthyles induit une hypertrophie cardiaque avec une augmentation de l'épaisseur du myocarde et un agrandissement des cardiomyocytes. L'étude protéomique du myocarde et l'analyse des données par bioinformatique identifient PGC-1[alpha], PPAR[alpha] et ERR[alpha] comme principaux déterminants des variations d'expression des protéines du métabolisme oxydatif mitochondrial. Nous avons observé une diminution d'expression de PPAR[alpha] et ERR[alpha] et une inactivation de PGC1[alpha] par hypométhylation et hyperacétylation, en lien avec une diminution d'expression de PRMT1 et de SIRT1 et une augmentation de SAH. Conséquences de la carence au niveau du foie : La carence s'accompagne de l'apparition d'une stéatose hépatique microvésiculaire, avec une élévation des taux tissulaires de lipides et de triglycérides. De plus, nous avons observé une augmentation des marqueurs pro inflammatoires sans augmentation des marqueurs de fibrose. A cet égard, nos résultats ont montré qu'un déficit de synthèse de carnitine, impliquée dans la bêta-oxydation et le stockage des acides gras, jouerait un rôle déterminant dans la pathogenèse de la stéatose chez le nouveau-né. Il existe également une dérégulation du métabolisme oxydatif des acides gras, avec diminution d'activité des complexes I et II de la chaîne respiratoire, qui résulte d'une hypométhylation de PGC1 et d'une diminution d'expression de PPAR[alpha], ER[alpha] et ERR[alpha]. En conclusion, nos résultats montrent que la carence maternelle en donneurs de méthyles, induit des modifications sur la fonction de PGC-1[alpha]. Ces modifications sont associées à des altérations de l'oxydation des acides gras et sur la fonction mitochondriale pendant la période néonatale, ce qui entraîne l'accumulation de lipides dans les tissus myocardique et hépatique. Le lien entre la carence en donneurs de méthyles et l'altération de la méthylation de PGC-1[alpha] modifie les activités des enzymes impliquées dans la méthylation et l'acétylation de PGC-1. Ces enzymes sont aussi liées à des modifications épigénomiques qui modulent la fonction et l'expression des protéines. Nos résultats sont en accord avec les études de population de Barker et al, qui suggèrent que la nutrition maternelle pendant les étapes précoces de la vie est corrélée avec le risque de maladies cardio-vasculaires dans la vie adulte indépendamment des autres facteurs de risque
During development, changes in carbon metabolism related to malnutrition may be deleterious for both the mother and the newborn. In addition, long-term consequences of a methyl deficiency gestational and prenatal are poorly understood. We are particularly interested in studying these effects on the heart and liver. We have used a nutritional model of adult rats deficient in methyl donors (vitamin B12, folate and choline) before pregnancy. To be placed in a context of pathophysiology, close to the clinical situation, we chose to feed the rats with a methyl deficient diet one month before mating and continue this diet during the suckling period. We evaluated the metabolic and functional effects of this diet on myocardial and hepatic tissues, in the newborn pups in 21 days old. We studied the effects of methyl deficient diet on lipid and energy metabolism and carnitine. Consequences of the deficiency at the myocardial: The diet deficient in methyl donors induces cardiac hypertrophy with an increase in myocardial thickness and enlargement of cardiomyocytes. The proteomics analysis of the bioinformatics data identifies PGC-1[alpha], ERR[alpha] and PPAR[alpha] as major determinants of changes in protein expression of mitochondrial oxidative metabolism. We observed a decreased expression of PPAR[alpha] and ERR[alpha] and an inactivation of PGC1[alpha] by hypomethylation and hyperacetylation, in conjunction with a decrease in the expression of PRMT1 and SIRT1 and increased the level of SAH. Consequences of the deficiency at liver: Deficiency is accompanied by the appearance of microvesicular hepatic steatosis, with elevated tissue levels of lipids and triglycerides. Increase of inflammation was observed in this model with no changes in fibrosis score. In this respect, our results showed a deficiency of carnitine synthesis, involved in the beta-oxidation and storage of fatty acids, play a role in the pathogenesis of hepatic steatosis in the newborn. There is also a deregulation of the oxidative metabolism of fatty acids, with decreased activity of complex I and II of the respiratory chain, resulting in hypomethylation of PGC1 and decreased expression of PPAR[alpha], ER[alpha] and ERR[alpha]. In conclusion, our results show that maternal deprivation in methyl donors impaired the function of PGC-1[alpha]. These changes are associated with alterations in fatty acid oxidation and mitochondrial function during the neonatal period, with lipid accumulation in myocardial tissue and liver. The link between the methyl donor deficiency and impaired methylation of PGC-1[alpha] alters the activities of enzymes involved in methylation and acetylation of PGC-1[alpha]. These enzymes are also associated with epigenetic changes and the function and gene expression. Our results are consistent with population studies by Barker and colleagues, who suggest that maternal nutrition during early stages of life is correlated with the risk of cardiovascular disease in adult, independent of other risk factors

Epidemiological evidence linking dietary folate deficiency and risk for colorectal cancer is conflicting. Studies using animal models indicate that timing, dose and presence of pre-malignant lesions will influence whether folate deficiency prevents or promotes tumor formation. In this thesis a new model of spontaneous tumor formation due to long-term dietary folate deficiency alone, in non-transgenic mice and without carcinogen induction, is developed. The mechanisms by which folate deficiency might influence cancer risk are also examined.
BALB/c mice, with or without a null allele in a key folate-metabolizing enzyme, Methylenetetrahydrofolate reductase (Mthfr ), develop intestinal tumors due to dietary folate deficiency alone. On folate-deficient (FD) diets, 12.5% of Mthfr+/+ mice and 28.1% of Mthfr+/- mice developed tumors; mice on control diet (CD) did not. C57B1/6 mice (a strain resistant to other methods of tumor induction) placed on the same diets for the same amount of time did not develop any tumors. To investigate possible mechanisms the levels of DNA damage (dUTP/dTTP ratio and p-H2AX staining) and DNA methylation (thin layer chromatography) were examined. FD BALB/c, but not C57B1/6 mice, had a trend towards increased dUTP/dTTP and DNA double-strand breaks and decreased global DNA methylation compared to CD mice. To determine why the FD diet affects the BALB/c and not the C57Bl/6 strain, the expression of genes involved in folate metabolism was examined. Several changes in gene expression were observed. In particular, BALB/c mice had increased Mthfr expression and MTHFR activity compared to C57Bl/6 mice. Increased MTHFR activity may deplete 5,10-methylenetetrahydrofolate supplies for the dTMP synthesis, increasing the dUMP levels and, possibly, DNA damage. The levels of several DNA repair genes were also examined. Two genes involved in base excision repair, Thymine DNA glycosylase (Tdg) and Apurinic/apyrimidinic endonuclease 1 (Apex1), were increased in FD C57B1/6 compared to FD BALB/c mice suggesting increased DNA repair capacity.
These results support the evidence that dietary folate deficiency promotes intestinal tumor formation possibly through increased DNA damage, with subsequent defects in DNA repair.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Introduction: Breast cancer is the second most common cancer in the world, being the most common among women. This disease is multifactorial involving lifestyle, hormonal, environmental and genetic factors. The folate metabolism may be associated with the development of breast cancer, since folate plays a crucial role in the synthesis, regulation and DNA methylation. Polymorphisms in genes involved in metabolism, such as MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G modify the efficiency of the enzymes, causing abnormal changes in gene expression, inactivation of tumor suppressor genes and activation of oncogenesis. Objectives: To investigate the frequency of polymorphisms in MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), MTR A2756G (rs1805087) and MTRR A66G (rs1801394) genes in patients with breast cancer comparing to individuals with no history neoplasia; to evaluate the association between polymorphisms and risk factors (age, smoking habits, alcohol consumption, number gestations, body mass index and hormone therapy) and the clinical histopathological parameters (tumor size, node involvement, metastasis and cancer subtypes) of breast cancer. Materials and methods: The present case-control study involved 606 Brazilian women, 128 case group and 478 control group. For molecular analysis, genomic DNA was extracted from peripheral blood leukocytes. Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) was used in the genotyping of polymorphisms in the genes MTHFR and MTR and PCR real time for polymorphsm in gene MTRR. The clinical and pathological data were obtained from medical records. For statistical analysis, program used were MINITAB 14.0 (multiple logistic regression), and SNPstats (inheritance models and Hardy-Weinberg Equilibrium) program. Results: Women aged 50 and over (OR: 2.65; 95% IC: 1.65-4.26; p<0.001) and alcohol consumption (OR: 1.76; 95% IC: 1.09-2.85; p=0.021) are associated increased risk for breast cancer. Smoking habits (OR:1.07; 95%CI:0.65-1.79; p=0.782), number of pregnancies (OR:0.86; 95%CI:0.54- 1.38; p=0.536), BMI ≥25 Kg/m2 (OR:1.24; 95%CI:0.75-2.06; p=0.405), and hormone therapy (OR: 1.41; 95%CI:0.86-2.33; p=0.174) are not associated with the risk of breast cancer. For MTHFR A1298C (rs1801131), we observed reduced risk of developing disease in codominant model (genotype CC – OR: 0.22; 95%CI: 0.06-0.74; p=0.014), recessive model (OR: 0.22; 95%CI: 0.07- 0.76; p=0.004), and log-additive model (OR: 0.70; 95%CI: 0.49-0.98; p=0.035), however no significant associations was found between MTHFR C677T (rs1801133), MTR A2756G (rs1805087), and MTRR A66G (rs1801394) polymorphisms and breast cancer risk. In relation to clinical histopathological parameters, we not found significant association between polymorphisms studies and breast tumors. Conclusions: Women aged 50 and over and who drink alcohol have a higher risk of developing breast cancer. The MTHFR A1298C polymorphism was associated with decreased risk in breast cancer. This is the first study the association between the genotypic these polymorphisms, and clinical histopathological parameters involving women population from the Northwest region of Sao Paulo State, Brazil.
Introdução: O câncer de mama é o segundo tipo de câncer mais frequente no mundo, sendo o mais comum entre as mulheres. Esta doença é multifatorial envolvendo o estilo de vida, fatores hormonais, ambientais e genéticos. O metabolismo do folato pode estar associada ao desenvolvimento do câncer de mama, já que o folato desempenha papel crucial na síntese, regulação e metilação do DNA. Polimorfismos nos genes participantes desse metabolismo, como MTHFR C677T, MTHFR A1298C, MTR A2756G e MTRR A66G modificam a eficiência das enzimas, causando alterações anormais na expressão gênica, inativação dos genes supressores de tumor e ativação da oncogênese. Objetivos: Investigar a frequência dos polimorfismos nos genes MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), MTR A2756G (rs1805087) e MTRR A66G (rs1801394) em pacientes com câncer de mama, comparando-a com aquela observada em indivíduos sem história de neoplasia; avaliar a associação entre os polimorfismos e os fatores de risco (idade, tabagismo, consumo de álcool, número de gestações, IMC e terapia hormonal) e as características clinico-patológicas (classificação TNM e fenotípica) no desenvolvimento do câncer de mama. Métodos: O presente estudo casocontrole envolveu 606 mulheres, sendo 128 no grupo caso e 478 no grupo controle. Para a análise molecular, o DNA genômico foi extraído a partir de sangue periférico. A técnica de Reação em Cadeia da Polimerase e digestão enzimática (PCR-RFLP) foi utilizada na genotipagem dos polimorfismos nos genes MTHFR e MTR e a técnica de PCR em tempo real para o polimorfismo no gene MTRR. Os dados clínico-histopatológicos foram obtidos por meio de prontuário médico. Para a análise estatística foram utilizados os programas MINITAB 14.0 (Regressão Logística Múltipla) e SNPstats (modelos de herança e Equilíbrio de Hardy-Weinberg). Resultados: Mulheres com 50 anos ou mais (OR: 2,65; 95% IC: 1,65-4,26; p<0,001) e que ingerem bebida alcoólica (OR: 1,76; 95% IC: 1,09-2,85; p=0,021) possuem risco aumentado para desenvolver câncer de mama. O hábito tabagista (OR: 1,07; 95% IC: 0,65-1,79; p=0,782), número de gestações (OR: 0,86 95% IC: 0,54-1,38; p=0,536), índice de massa corpórea (OR: 1,24 95% IC: 0,75-2,06; p=0,405) e terapia hormonal (OR: 1,41 95% IC: 0,86-2,33; p=0,174) não foram associados com o risco de câncer de mama. Quanto ao polimorfismo MTHFR A1298C (rs1801131), foi observado uma redução no risco de desenvolver a doença no modelo codominante (genótipo CC - OR: 0,22; 95% IC: 0,06-0,74; p=0,01), modelo recessivo (OR: 0,22; 95% IC: 0,07-0,76; p=0,004) e modelo log-aditivo (OR: 0,70; 95% IC: 0,49-0,98; p=0,03), enquanto que os polimorfismos MTHFR C677T (rs1801133), MTR A2756G (rs1805087) e MTRR A66G (rs1801394) não foram associados ao risco de câncer de mama. Com relação aos parâmetros clínicospatológicos, não foi encontrado associação entre os polimorfismos estudados e o os tumores de mama. Conclusões: Mulheres com 50 anos e que ingerem bebida alcoólica possuem risco aumentado para desenvolver câncer de mama. O polimorfismo MTHFR A1298C está associado com a diminuição do risco no desses polimorfismos e clínico-patológico das mulheres brasileiras do noroeste do estado de São Paulo, Brasil.

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Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP
Introduction: Congenital heart defects (CHD) are present in approximately 40 to 60% of individuals with Down syndrome (DS). It is the leading cause of death in the first years of life in individuals with the syndrome. Polymorphisms in maternal and fetal genes encoding enzymes involved in folate metabolism have been associated with the development of congenital heart defects. Objectives: To assess if the presence of polymorphism (MTHFR rs4846048, MTHFR rs4846049, hsa-mir-149 rs2292832, MTHFR C677T, MTHFR A1298C, MTHFR T1317C, MTR A2756G, MTRR A66G, SLC19A1 A80G, TC2 A67G, TC2 C776G, CßS 844ins68, CßS T833C, MTHFD1 G1958A, BHMT G742A, DHFR del 19 pb, and SHMT C1420T) in individuals with DS is associated with the occurrence of CHD in these individuals. We also evaluated the association between maternal genetic polymorphisms MTHFR rs4846048, MTHFR rs4846049 and hsa-mir-149 rs2292832, and the presence of CHD in offspring with DS. Methods: This study included 139 individuals (80 individuals with DS and CHD, and 59 control subjects with DS without congenital heart disease). Molecular analysis of MTHFR rs4846048, MTHFR rs4846049 and hsa-mir-149 rs2292832 was carried out by real time polymerase chain reaction allelic discrimination. Genotyping data of MTHFR C677T, MTHFR A1298C, MTHFR T1317C, MTR A2756G, MTRR A66G, SLC19A1 A80G, TC2 A67G, TC2 C776G, CßS 844ins68, CßS T833C, MTHFD1 G1958A, BHMT G742A, DHFR del 19 pb, and SHMT C1420T were obtained from database of previous studies of the research group and also used to assess the risk for the occurrence of CHD in this study. Multiple logistic regression analyzes were performed to assess the risk of CHD in the presence of 17 polymorphisms in dominant and recessive genetic models. The median number of mutant alleles between groups was assessed by the Mann-Whitney test. Genotypic combination analysis was performed for the polymorphisms MTHFR rs4846048, MTHFR rs4846049, and hsa-mir-149 rs2292832, using Fisher's exact test, dominant model. Results: Multiple logistic regression analysis involving individuals with DS showed no association between 17 polymorphisms and the risk for CHD. The median number of polymorphic alleles did not differ among individuals with DS with and without CHD. On the other hand, the maternal genotypes hsa-mir-149 rs2292832 CT or TT were associated with reduced risk for isolated heart disease in the offspring (OR = 0,31; 95% CI = 0,13 to 0,72; P = 0,0063). The analysis of genotypic combinations of MTHFR rs4846048, MTHFR rs4846049, and hsa-mir-149 rs2292832 in individuals with DS, and their mothers showed no association between the different combinations and the risk for congenital heart disease. Conclusions: There is no evidence of association between the polymorphisms analyzed in individuals with DS and the occurrence of CHD. However, a lower risk of isolated congenital heart disease for individuals with DS is observed in the presence of maternal genotypes hsa-mir-149 rs2292832 CT or TT.
Introdução: Defeitos cardíacos congênitos (DCC) estão presentes em aproximadamente 40 a 60% dos indivíduos com a síndrome de Down (SD) e representam a principal causa de morte nos primeiros anos de vida em indivíduos com a síndrome. Polimorfismos em genes maternos e fetais, que codificam enzimas envolvidas no metabolismo do folato, têm sido associados com o desenvolvimento de cardiopatias congênitas. Objetivos: Avaliar se a presença dos polimorfismos MTHFR rs4846048, MTHFR rs4846049, hsa-mir-149 rs2292832, MTHFR C677T, MTHFR A1298C, MTHFR T1317C, MTR A2756G, MTRR A66G, SLC19A1 A80G, TC2 A67G, TC2 C776G, CßS 844ins68, CßS T833C, MTHFD1 G1958A, BHMT G742A, DHFR del 19 pb, SHMT C1420T em indivíduos com SD está associada com a ocorrência de DCC nesses indivíduos. Também foi avaliada a associação entre os polimorfismos genéticos maternos MTHFR rs4846048, MTHFR rs4846049 e hsa-mir-149 rs2292832 e a presença de DCC na prole com SD. Casuística e Método: Este estudo incluiu 139 indivíduos (80 indivíduos com SD e DCC e 59 indivíduos controles com SD, sem cardiopatia congênita). A análise molecular dos polimorfismos MTHFR rs4846048, MTHFR rs4846049 e hsa-mir-149 rs2292832 foi realizada pelo método discriminação alélica por meio de reação em cadeia da polimerase em tempo real. Os dados da genotipagem dos polimorfismos MTHFR C677T, MTHFR A1298C, MTHFR T1317C, MTR A2756G, MTRR A66G, SLC19A1 A80G, TC2 A67G, TC2 C776G, CßS 844ins68, CßS T833C, MTHFD1 G1958A, BHMT G742A, DHFR del 19 pb, SHMT C1420T foram obtidos de banco de dados de trabalhos previamente publicados pelo grupo de pesquisa e utilizados para avaliar o risco para a ocorrência de DCC no presente estudo. Análises de regressão logística múltipla foram realizadas para avaliar o risco de DCC na presença dos 17 polimorfismos nos modelos genéticos dominante e recessivo. A mediana do número de alelos mutantes entre os grupos foi avaliada pelo teste de Mann-Whitney. Análise de combinação genotípica foi realizada para os polimorfismos MTHFR rs4846048, MTHFR rs4846049 e hsa-mir-149 rs2292832, utilizando o teste exato de Fisher, no modelo dominante. Resultados: As análises de regressão logística múltipla, envolvendo os indivíduos com SD, não evidenciaram associação entre os 17 polimorfismos e o risco para DCC. A mediana do número de alelos polimórficos também não diferiu entre os indivíduos com SD com e sem DCC. Por outro lado, os genótipos maternos hsa-mir-149 rs2292832 CT ou TT foram associados ao risco reduzido para cardiopatia isolada na prole com SD (OR = 0,31; IC 95% = 0,13-0,72; P = 0,0063). A análise das combinações genotípicas dos polimorfismos MTHFR rs4846048, MTHFR rs4846049 e hsa-mir-149 rs2292832, nos indivíduos com SD e nas suas mães, não mostrou associação entre as diferentes combinações e o risco para cardiopatia congênita. Conclusões: Na casuística avaliada não há evidências de associação entre os polimorfismos analisados em indivíduos com SD e a ocorrência de DCC; entretanto um menor risco de cardiopatia congênita isolada para os indivíduos com SD é observado na presença dos genótipos maternos hsa-mir-149 rs2292832 CT ou TT.

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The congenital facial clefts are characterized by incomplete formation of the structures that separate the oral and nasal cavity. It is known that several environmental and genetic factors are involved in its development, among these, polymorphisms associated with folic acid metabolism have been investigated. In this sense, the objective was to observe the frequency of polymorphisms C677T and A1298C methylenetetrahydrofolate reductase gene (MTHFR), methionine synthase A2756G of (MTR), A66G of methionine synthase reductase (MTRR) A80G and the reduced folate carrier (RFC1) in patients with non-syndromic oral clefts, trying to match them with their development. Methods: We studied 140 patients with non-syndromic oral clefts and their mothers and 175 control subjects with their mothers, who underwent a questionnaire to obtain family information. Were collecting blood for DNA extraction from patients and their mothers to identify the genotypes of both by PCRRFLP, in addition to carrying out the determination of glucose, AST, ALT and serum creatinine, folic acid and vitamin B12 Serum and plasma homocysteine, and the hemogram. Results: Most patients have cleft lip and palate (55.8%), followed by isolated cleft palate (24.2%) and cleft lip (20%). Regarding gender, 62% of patients were male and 48% female and, after subdivision of the type of screwdriver according to sex was found a prevalence of males in the cracks of the type lip and palate (69 %) and lip (69.2%) and in the case of cleft palate was a female predominance (59%). The average concentration of serum folate in the group of mothers of cleft patients was significantly lower (13.8 ? 2.4 ng / mL) compared with the group of mothers of control subjects (18.8 ? 3.4 ng / mL) This was also observed for the group of cleft children as compared to controls, the dosage of folic acid had a significant difference with values of 15.6 ? 0.6 (ng / mL) and 17.9 ? 0.6 (ng / mL), respectively. For the biochemical measurements of glucose, AST, ALT and creatinine were not statistically different, nor was observed for haematological parameters performed. In assessing the frequency of polymorphisms C677T and A1298C MTHFR, A2756G MTR, MTRR A66G and A80G of the RFC1 there was no statistically significant difference in genotype distribution between cases and controls both for mothers and in the cleft. Conclusion: Although not observed association of polymorphisms with the development of cracks, the decrease in serum folate in the group of cleft patients and their mothers may reflect a disturbance in the metabolism of this metabolite, necessitating further studies such as studies methylation and expression to further elucidate the involvement of folate in the development of oral clefts
As fendas orais s?o malforma??es caracterizadas pela forma??o incompleta das estruturas que separam a cavidade nasal e oral. Sabe-se que v?rios fatores ambientais e gen?ticos est?o envolvidos no seu desenvolvimento, dentre esses, polimorfismos associados ao metabolismo do ?cido f?lico t?m sido alvo de estudos. Neste sentido, o objetivo deste trabalho foi observar a freq??ncia dos polimorfismos C677T e o A1298C do gene da Metilenotetrahidrofolato redutase (MTHFR), A2756G da Metionina Sintase (MTR), A66G da Metionina Sintase Redutase (MTRR) e A80G do Transportador de folato reduzido (RFC1) em pacientes portadores de fendas orais n?o-sindr?micas, buscando associ?-los ao desenvolvimento das mesmas. Casu?stica e M?todos: Foram avaliados 140 portadores de fendas orais n?o-sindr?micas e suas m?es e 175 indiv?duos controles com suas m?es, que foram submetidos a um question?rio familiar para obten??o de informa??es. Foi realizada a coleta de sangue para extra??o do DNA dos pacientes e de suas m?es para identifica??o dos gen?tipos de ambos atrav?s de PCR-RFLP, al?m da realiza??o das dosagens de glicose, AST, ALT e creatinina s?ricos, dosagens de ?cido f?lico e vitamina B12 s?ricos e homociste?na plasm?tica, al?m da realiza??o de hemograma. Resultados: A maioria dos pacientes s?o portadores de fenda l?bio-palatina (55,8%), seguida da fenda palatina isolada (24,2%) e da fenda labial (20%). Em rela??o ao sexo, 62% dos pacientes e s?o do sexo masculino e 48% do sexo feminino e, ap?s subdivis?o do tipo de fenda de acordo com o sexo constatou-se uma preval?ncia do sexo masculino nas fendas do tipo l?bio-palatina (69%) e labial (69,2%) e no caso das fendas palatinas isoladas houve uma predomin?ncia do sexo feminino (59%). A concentra??o m?dia de ?cido f?lico s?rico no grupo das m?es de pacientes fissurados foi significativamente inferior (13,8?2,4ng/mL) quando comparada com o grupo das m?es dos indiv?duos controles (18,8?3,4ng/mL), o que tamb?m foi observado para o grupo dos pacientes fissurados quando comparado aos filhos controles, a dosagem de ?cido f?lico apresentou diferen?a significante com valores de 15,6?0,6(ng/mL) e 17,9?0,6(ng/mL), respectivamente. Para as dosagens bioqu?micas de glicose, AST, ALT e creatinina n?o foram observadas diferen?as estat?sticas, assim como n?o foi observado para os par?metros hematol?gicos realizados. Na avalia??o da freq??ncia dos polimorfismos C677T e A1298C da MTHFR, A2756G da MTR, A66G da MTRR e A80G do RFC1 n?o foi observada diferen?a estatisticamente significante na distribui??o dos gen?tipos entre caso e controle tanto em rela??o as m?es quanto nos pacientes fissurados. Conclus?o: Apesar de n?o ter sido observada associa??o dos polimorfismos estudados com o desenvolvimento das fendas, a diminui??o na concentra??o s?rica de ?cido f?lico no grupo dos pacientes fissurados e de suas m?es pode refletir algum dist?rbio no metabolismo desse metab?lito, sendo necess?rio mais estudos como estudos de metila??o e express?o para melhor elucidar o envolvimento do ?cido f?lico no desenvolvimento das fendas orais

Os defeitos de fechamento de tubo neural constituem uma das malformações mais freqüentes na espécie humana, apresentando alta morbi-mortalidade. Sua etiologia é considerada multifatorial, estando envolvidos fatores genéticos e ambientais. Estes fatores estão relacionados principalmente com o metabolismo da homocisteína. Realizamos um estudo de caso-controle com o objetivo de estudar os fatores bioquímicos e genéticos relacionados ao DTN na nossa população. Em pares de afetados com DTN e suas mães e pares de pacientes normais e suas mães foram avaliados dosagem de folato, vitamina B12, homocisteína e polimorfismos da enzima metileno tetraidrofolato redutase (MTHFR), C677T e A1298C. A dosagem de folato nos casos foi 11,37 ng/mL(±6,72) e nos controles 5,64 ng/mL(±4,16) (p<0,001). O folato sérico das mães foi 7,27 ng/mL (±4,48) e 3,90 ng/mL (±1,77) nas mães controles (p<0,001). A média de dosagem de vitamina B12 foi de 641,88 pg/mL ((±262,21) nos casos e 743,27 pg/mL (±433,52) nos controles (p= 0,205). A média de dosagem de vitamina B12 nas mães dos casos foi 354,75 pg/mL (±142,06) e 465,25 pg/mL (±194,91) nas mães controles (p=0,004). O nível de homocisteína plasmático médio foi 6,89 μmol/L(±4,48) para os casos e 5,41 μmol/L (±2,55) para os controles (p=0,099). Nas mães dos casos a dosagem média de homocisteína foi 7,23 μmol/L (±2,64) e 7,00 μmol/L (±2,24) nas mães controles (p=0,666). Não houve diferença entre a freqüência dos genótipos C677T e A1298C da MTHFR nos casos e controles e suas mães. Para o polimorfismo C677T as freqüências dos alelo C e T foram respectivamente 0,6585 e 0,3414 nos pacientes com DTN; 0,6590 e 0,3410 nos controles; 0,6460 e 0,3540 nas mães dos casos e 0,6136 e 0,3860 nas mães controles. Para o polimorfismo A1298C as freqüências dos alelos A e C foram respectivamente 0,7436 e 0,2564 nos pacientes com DTN; 0,7610 e 0,2390 nos controles; 0,8055 e 0,1945 nas mães dos casos e 0,8065 e 0,1935 nas mães controles. Identificamos que indivíduos homozigotos 677TT apresentam um maior nível de homocisteína e este é inversamente relacionado com os níveis de vitamina B12. Estes achados sugerem que uma alteração metabólica relacionada ao metabolismo da homocisteína e principalmente devido à diminuição da vitamina B12 seja um fator de risco para DTN na nossa população.
Neural tube defects (NTD) are among the most common birth defect leading to great disabilities. The etiology is multifactorial, involving the combined action of both genetic and environmental factors. Those factors have been related to homocysteine metabolism. We performed a case control study in order to evaluate the biochemical and genetic factors related to NTD in the South of Brazil. A mother- NTD children pair and mother normal children were evaluated for folate, vitamin B12, homocysteine and two polymorphism of the methylene tetrahydrofolate reductase (MTHFR), C677T and A1298C. The folate level was 11,37 ng/mL (±6,72) in the NTD patients and 5,64 ng/mL(±4,16) in the controls (p<0,001). The folate was 7,27 ng/mL (±4,48) in the NTD mothers and 3,90 ng/mL (±1,77) in the control mothers (p<0,001). The level of vitamin B12 was 641,88 pg/mL ((±262,21) in the NTD case and 743,27 pg/mL (±433,52) in the controls (p= 0,205). The levels of vitamin B12 in the NTD mothers was 354,75 pg/mL (±142,06) and 465,25 pg/mL (±194,91) in the control mothers (p=0,004). The pasmatic homocysteine level was 6,89 μmol/L(±4,48) for the NTD cases and 5,41 μmol/L (±2,55) for the controls (p=0,099). The NTD mothers showed homocysteine level of 7,23 μmol/L (±2,64) and the controls mothers demonstrated 7,00 μmol/L (±2,24) (p=0,666). We could not observed a difference between the frequency of the genotypes C677T and A1298C in case and controls. The frequency of the alele C and T for the polymorphism C677T were respectively 0,6585 and 0,3414 for the NTD patients; 0,6590 and 0,3410 for the controls; 0,6460 and 0,3540 for NTD mothers; 0,6136 and 0,3860 for the control mothers. The frequencies of the allele A and C for the polymorphism A1298C were respectively 0,7436 and 0,2564 for NTD patients, 0,7610 and 0,2390 for controls; 0,8055 and 0,1945 for NTD mothers; 0,8065 and 0,1935 for controls mothers. We could demonstrated that the homozygous 677TT had a higher level of homocysteine and this was related to low level of vitamin B12. Those findings suggest that biochemical and genetic factors related to homocysteine metabolism and vitamin B12 deficiency are a risk factor to NTD in our population.

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz
The interaction between folate and methylenetetrahydrofolate reductase (MTHFR) gene is an example of a strong gene-nutrient interaction. MTHFR 677C→T polymorphism may be associated with homocysteine in the modulation of the cardiovascular disease (CVD). Additionally, the interaction between the polymorphism and folate has been linked to a protective effect in individuals with colorectal cancer (CRC). The aim of this work is to assess the possible association between MTHFR 677C→T polymorphism, CVD and CRC, modified by folate and homocysteine. The predictive response of CRC patients carrying MTHFR 677C→T polymorphism, treated with 5-fluorouracil, is also briefly examined, along with the current strategies to inhibit tumours, involving MTHFR gene as a target. Studies were found by searches of electronic literature for papers up to October 2014 using the terms “MTHFR 677C→T polymorphism” “folate,” “cardiovascular disease,” “homocysteine,” “colorectal cancer” and “chemotherapy”. Collected studies were mainly observational, randomised controlled trials, meta-analyses and systematic reviews, approximately from the last 15 years. The association between MTHFR 677C→T polymorphism and CVD was found, although results from folate supplementation trials demonstrated no benefit in CVD. High supply of folate and TT genotype carriers may have a lower risk to develop colon cancer in some populations. The type and amount of folate, along with its supplementation when carcinogenesis is already stablished may increase the risk for CRC. MTHFR 677C→T polymorphism seems to be associated with better prognosis and less toxicity in 5-fluorouracil monotherapy. MTHFR inhibition technique shows promising results as an anti-cancer therapy. Findings are inconsistent to recommend folate supplementation in TT genotype carriers with CVD and CRC. A genetic and environmental risk assessment for CRC risk in primary care, regarding folate and MTHFR 677C→T polymorphism is worth considering. Further research on combinatory MTHFR polymorphisms and riboflavin effect, could help clarify the association between MTHFR 677C→T polymorphism, folate and disease.
Pensa-se que a variante do enzima codificado pelo polimorfismo MTHFR 677C→T possa estar associado à homocisteína e consequentemente, associado ao risco de doenças cardiovasculares (CVD); assim como a associação entre o folato e a mesma variante polimórfica possa proteger contra o cancro colorretal (CRC). O objetivo deste trabalho é correlacionar o polimorfismo MTHFR 677C→T com CVD e CRC, associado ao folato e à homocisteína. A resposta terapêutica ao 5-fluorouracil em pacientes com CRC e com o polimorfismo é também, brevemente analisada, juntamente com as atuais estratégias de inibição de tumores, envolvendo MTHFR. Os estudos foram identificados por pesquisa eletrónica de referências bibliográficas publicadas até outubro de 2014, usando as terminologias: “polimorfismo MTHFR 677C→T”, “folato”, “doença cardiovascular”, “homocisteína”, “cancro colorretal” e “quimioterapia”. Compreenderam principalmente estudos observacionais, clínicos randomizados, metaanálises e revisões sistemáticas dos últimos 15 anos. A associação entre o polimorfismo e CVD foi encontrada, embora os resultados de estudos com suplementação de folato não demonstrem benefício, em CVD. O elevado consumo de folato e o genótipo TT parecem estar associados a menor risco de cancro do cólon. O tipo e quantidade de folato, juntamente com a fase da carcinogénese na qual se inicia a suplementação do mesmo podem estar relacionados com o aumento do risco de CRC. O polimorfismo parece estar associado a um melhor prognóstico e menor toxicidade em tratamentos com 5-fluorouracil. A técnica de inibição de MTHFR mostra resultados promissores como terapia anticancerígena. Os resultados são inconsistentes para recomendar suplementos de folato em indivíduos TT. Testes de avaliação da interação gene-ambiente, em relação ao folato e ao polimorfismo referido podem ser justificáveis, em indivíduos em risco de CRC. Mais estudos que combinem os polimorfismos do MTHFR e que analisem o efeito da riboflavina, ajudariam a compreender a associação entre o polimorfismo MTHFR 677C→T, o folato e a doença.

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Folic acid can effectively reduce blood homocysteine levels. Recent studies have shown that hyperhomocysteinemia is also associated with liver disorders. However, the underlying mechanisms remain unclear. The general objective of my study was to investigate the biochemical and molecular mechanisms of homocysteine-induced liver injury and abnormal lipid metabolism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 weeks. An elevation of serum aminotransferases activities (indicator for liver injury) and an increase in hepatic lipid peroxidation were observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production led to oxidative stress in the liver. Reduction of oxidative stress by inhibiting superoxide anion production ameliorated hyperhomocysteinemia-induced liver injury. A significant elevation of hepatic and serum cholesterol concentrations in hyperhomocysteinemic rats was observed, exclusively due to increased expression of HMG-CoA reductase in hepatocytes. The molecular mechanisms of homocysteine-induced adverse effects were further investigated in isolated rat hepatocytes and in human hepatoma cells (HepG2). Hcy stimulated HMG-CoA reductase expression in hepatocytes via activation of transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element binding protein (CREB) and nuclear factor Y (NF-Y). Activation of these 3 transcription factors was detected in hyperhomocysteinemic rat liver and in homocysteine-treated hepatocytes. Pretreatment of hepatocytes with inhibitors for individual transcription factors effectively attenuated Hcy-induced HMG-CoA reductase mRNA expression. Supplementation of folic acid in diet significantly reduced serum homocysteine level and effectively inhibited hyperhomocysteinemia-induced superoxide anion production, resulting in amelioration of oxidative stress-mediated liver injury in hyperhomocysteinemic rats. These results reflected a protective role of folic acid in hyperhomocysteinemia-induced liver injury. In conclusion, the present study demonstrates that (1) hyperhomocysteinemia can cause oxidative stress and liver injury; (2) homocysteine stimulates cholesterol biosynthesis in hepatocytes via transcriptional regulation of HMG-CoA reductase expression; (3) supplementation of folic acid offers a hepatoprotective effect during hyperhomocysteinemia. Oxidative stress and accumulation of cholesterol in the liver contribute to liver injury associated with hyperhomocysteinemia. The role of folic acid in maintaining good health may extend beyond the cardiovascular system to encompass hyperhomocysteinemia-associated liver disorders.
October 2007

Reprints of the author's previously published articles included as an appendix.
Bibliography: leaves 165-188.
xiv, 189 leaves : ill. (some col.) ; 30 cm.
"This thesis describes a series of experiments that aimed to investigate the effects of folic acid deficiency and defects in folate metabolism on chromosome damage rates in human lymphocytes. The accumulation of chromosome damage over time is an important issue because it is thought to contribute to the mechanism of ageing and the aetiology of diseases of age such as cancer and Alzheimer's disease."
Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2002?