RESUMO
Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.
Assuntos
Metilação de DNA/efeitos dos fármacos , DNA/metabolismo , Ácido Fólico/farmacologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espermatozoides/metabolismo , Testículo/metabolismo , Animais , DNA/genética , Metilação de DNA/genética , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/citologia , Testículo/citologiaRESUMO
Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5' to the upstream translational start site or in another region 3' to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.
Assuntos
Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica , Metilação de DNA , Dieta , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Transformação Celular Neoplásica/genética , Ilhas de CpG , Modelos Animais de Doenças , Epigênese Genética , Feminino , Expressão Gênica , Loci Gênicos , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/metabolismoRESUMO
Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.
Assuntos
Proteínas de Transporte/genética , Homocistinúria/genética , Erros Inatos do Metabolismo/genética , Ácido Metilmalônico/urina , Mutação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Sequência Conservada , Fibroblastos/metabolismo , Haplótipos/genética , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Oxirredutases , Dobramento de Proteína , Homologia Estrutural de Proteína , Vitamina B 12/metabolismoRESUMO
Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low-folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr(+/+) mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr(+/-) animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS-related proteins, the protein synthetic machinery, and fatty acid-related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy.
Assuntos
Carcinogênese/metabolismo , Ácido Fólico/metabolismo , Homocistinúria/complicações , Neoplasias Intestinais/etiologia , Neoplasias Intestinais/metabolismo , Intestinos/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/complicações , Proteoma/metabolismo , Animais , Carcinogênese/patologia , Dieta , Modelos Animais de Doenças , Feminino , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteômica , Transtornos Psicóticos/complicaçõesRESUMO
SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.
Assuntos
Deficiência de Ácido Fólico , Metilenotetra-Hidrofolato Redutase (NADPH2) , Hepatopatia Gordurosa não Alcoólica , Animais , Feminino , Humanos , Masculino , Camundongos , Betaína , Colina/metabolismo , Ácido Fólico , Deficiência de Ácido Fólico/metabolismo , Genótipo , Homocisteína , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/etiologia , S-AdenosilmetioninaRESUMO
The brain is particularly sensitive to folate metabolic disturbances, since methyl groups are critical for its functions. Methylenetetrahydrofolate reductase (MTHFR) generates the primary circulatory form of folate required for homocysteine remethylation to methionine. Neurological disturbances have been described in homocystinuria caused by severe MTHFR deficiency. The goal of this study was to determine if behavioral anomalies are present in severe Mthfr-deficient (Mthfr(-/-)) mice and to identify neurobiological changes that could contribute to these anomalies. Adult male mice of 3 Mthfr genotypes (+/+, +/-, -/-) were tested on motor, anxiety, exploratory and cognitive tasks. Volumes (whole brain and hippocampus) and morphology, global DNA methylation, apoptosis, expression of choline acetyltransferase (ChAT) and glucocorticoid receptor (GR), and concentrations of choline metabolites were assessed in hippocampus. Mthfr(-/-) mice had impairments in motor function and in short- and long-term memory, increased exploratory behavior and decreased anxiety. They showed decreased whole brain and hippocampal volumes, reduced thickness of the pyramidal cell layer of CA1 and CA3, and increased apoptosis in hippocampus. There was a disturbance in choline metabolism as manifested by differences in acetylcholine, betaine or glycerophosphocholine concentrations, and by increased ChAT levels. Mthfr(-/-) mice also had increased GR mRNA and protein. Our study has revealed significant anomalies in affective behavior and impairments in memory of Mthfr(-/-) mice. We identified structural changes, increased apoptosis, altered choline metabolism and GR dysregulation in hippocampus. These findings, as well as some similar observations in cerebellum, could contribute to the behavioral changes and suggest that choline is a critical metabolite in homocystinuria.
Assuntos
Hipocampo/metabolismo , Hipocampo/patologia , Homocistinúria/diagnóstico , Espasticidade Muscular/diagnóstico , Animais , Apoptose/genética , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Colina/metabolismo , Colina O-Acetiltransferase/metabolismo , Metilação de DNA , Homocisteína/sangue , Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Camundongos Knockout , Espasticidade Muscular/genética , Tamanho do Órgão , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/genética , Receptores de Glucocorticoides/metabolismoRESUMO
Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in a P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.
Assuntos
Ácido Fólico , Placenta , Animais , Suplementos Nutricionais , Feminino , Ácido Fólico/farmacologia , Expressão Gênica , Hipocampo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C â T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues.
Assuntos
Embrião de Mamíferos/metabolismo , Homocistinúria/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Espasticidade Muscular/metabolismo , Regiões Promotoras Genéticas , Animais , Vasos Sanguíneos/metabolismo , Sistema Nervoso Central/metabolismo , Feminino , Genótipo , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Placenta/metabolismo , Polimorfismo de Nucleotídeo Único , Gravidez , Transtornos Psicóticos/metabolismo , Testículo/metabolismoRESUMO
Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/- mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.
Assuntos
Colesterol/metabolismo , Metilação de DNA/efeitos dos fármacos , Ácido Fólico/farmacologia , Fígado/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Colina/metabolismo , Dieta , Ácido Fólico/administração & dosagem , Alimentos Fortificados , Homeostase/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína 2 Associada à Farmacorresistência Múltipla , Receptores de Lipoproteínas/genéticaRESUMO
SCOPE: Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development. METHODS AND RESULTS: Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene. CONCLUSION: Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.
Assuntos
Ácido Fólico/farmacologia , Homocistinúria/induzido quimicamente , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/induzido quimicamente , Placenta/metabolismo , Fatores Sexuais , Animais , Metilação de DNA , Suplementos Nutricionais , Feminino , Ácido Fólico/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Ftálicos/sangue , Gravidez , Transtornos Psicóticos , S-Adenosilmetionina/sangue , Transcriptoma/efeitos dos fármacosRESUMO
Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Ácido Fólico/efeitos adversos , Fígado/metabolismo , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Recomendações Nutricionais , Caracteres Sexuais , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Troca Materno-Fetal , Transtornos da Memória/induzido quimicamente , Camundongos Endogâmicos C57BL , Fosfatidilcolinas/metabolismo , Gravidez , Esfingomielinas/metabolismoRESUMO
Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer's disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/- mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/- genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.
Assuntos
Envelhecimento/patologia , Encéfalo/patologia , Dieta , Ácido Fólico/metabolismo , Homocistinúria/complicações , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/complicações , Peptídeos beta-Amiloides/metabolismo , Animais , Ansiedade/complicações , Ansiedade/fisiopatologia , Encéfalo/fisiopatologia , Sobrevivência Celular , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Colina/metabolismo , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética , Ácido Glutâmico/metabolismo , Homocistinúria/fisiopatologia , Fígado/metabolismo , Masculino , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Metilação , Camundongos Endogâmicos BALB C , Espasticidade Muscular/fisiopatologia , Fatores de Crescimento Neural/metabolismo , Neurônios/patologia , Fosfolipídeos/metabolismo , Transtornos Psicóticos/complicações , Transtornos Psicóticos/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , S-Adenosilmetionina/metabolismo , Transmissão SinápticaRESUMO
SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677CâT) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.
Assuntos
Homocistinúria/metabolismo , Inflamação/etiologia , Metabolismo dos Lipídeos , Fígado/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/metabolismo , Tecido Adiposo/metabolismo , Animais , Glicemia/análise , Fígado Gorduroso/etiologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Transtornos Psicóticos/metabolismoRESUMO
Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr(+/+) and Mthfr(+/-) mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr(+/+) and Mthfr(+/-) pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. We also examined the effects of VPA on MTHFR expression in HepG2 cells and on MTHFR activity and homocysteine levels in mice. Mthfr(+/+) mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar in Mthfr(+/-) mice with the two treatments (25-27%). NTDs were only observed in one group (VPA-treated Mthfr(+/+)). In HepG2 cells, VPA increased MTHFR promoter activity and MTHFR mRNA and protein (2.5- and 3.7-fold, respectively). Consistent with cellular MTHFR upregulation by VPA, brain MTHFR enzyme activity was increased and plasma homocysteine was decreased in VPA-treated pregnant mice compared to saline-treated animals. These results underscore the importance of folate interconversion in VPA-induced teratogenicity, since VPA increases MTHFR expression and has lower teratogenic potential in MTHFR deficiency.
Assuntos
Anormalidades Induzidas por Medicamentos/prevenção & controle , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Regulação para Cima/efeitos dos fármacos , Ácido Valproico/efeitos adversos , Anormalidades Induzidas por Medicamentos/etiologia , Animais , Encéfalo/enzimologia , Linhagem Celular Tumoral , Feminino , Ácido Fólico/metabolismo , Homocisteína/sangue , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Camundongos Knockout , Gravidez , TeratogênicosRESUMO
Folate deficiency and maternal smoking are strong risk factors for placental abruption. We assessed whether the reduced folate carrier [NM_194255.1: c.80A-->G (i.e., p.His27Arg)] (RFC-1) polymorphism was associated with placental abruption, and evaluated if maternal smoking modified the association between plasma folate and abruption. Data were derived from the New Jersey-Placental Abruption Study--a multicenter, case-control study of placental abruption (2002-2007). Maternal DNA was assayed for the RFC-1 c.80A-->G polymorphism using a PCR-dependent diagnostic test. Maternal folate (nmol/l) was assessed from maternal plasma, collected immediately following delivery. Due to assay limitations, folate levels at > or =60 nmol/l were truncated at 60 nmol/l. Therefore, case-control differences in folate were assessed from censored log-normal regression models following adjustment for potential confounders. Distribution of the mutant allele (G) of the RFC-1 c.80A-->G polymorphism was similar between cases (52.3%; n = 196) and controls (50.5%; n = 191), as was the homozygous mutant (G/G) genotype (OR 1.1, 95% CI 0.6-2.2). In a sub-sample of 136 cases and 140 controls, maternal plasma folate levels (mean +/- standard error) corrected for assay detection limits were similar between placental abruption cases (63.6 +/- 5.1 nmol/l) and controls (58.3 +/- 4.7 nmol/l; P = 0.270), and maternal smoking did not modify this relationship (interaction P = 0.169). We did not detect any association between the RFC-1 c.80A-->G polymorphism and placental abruption, nor was an association between plasma folate and abruption risk evident. These findings may be the consequence of high prevalence of prenatal multivitamin and folate supplementation in this population (over 80%). It is therefore not surprising that folate deficiency may be rare and that the RFC-1 c.80A-->G polymorphism is less biologically significant for placental abruption.
Assuntos
Descolamento Prematuro da Placenta/sangue , Descolamento Prematuro da Placenta/genética , Ácido Fólico/sangue , Proteínas de Membrana Transportadoras/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Gravidez , Proteína Carregadora de Folato Reduzido , Risco , Transdução de Sinais/genética , Fumar/efeitos adversos , Fumar/genéticaRESUMO
Clinical reports have suggested that low dietary folate increases risk for colorectal cancer. Animal studies for investigation of folate and tumorigenesis have used carcinogen induction or mice with germ-line mutations. We have developed a new spontaneous tumor model in which mice, with or without a null allele in a key folate-metabolizing enzyme, methylenetetrahydrofolate reductase (Mthfr), develop intestinal tumors due to low dietary folate alone. On folate-deficient diets, 12.5% of Mthfr(+/+) mice and 28.1% of Mthfr(+/-) mice developed tumors; mice on control diets were negative. Dietary and genotype effects on tumor development were significant. To investigate mechanisms of folate-dependent tumorigenesis, we examined levels of DNA damage and gene expression of two genes involved in DNA damage response and G(2)-M checkpoint regulation, polo-like kinase 1 (Plk1) and cell division cycle 25c (Cdc25c). Folate deficiency increased DNA damage and decreased expression of both genes (assessed by quantitative reverse transcription-PCR and immunofluorescence) in normal intestine compared with levels in mice on control diets. An immunofluorescence assay for CDC25c activity (phosphorylated CDC2) also found CDC25c activity to be decreased in folate-deficient normal intestine. In tumors, however, Plk1 and Cdc25c mRNA were found to be higher (11- and 3-fold, respectively) compared with normal intestine from folate-deficient mice; immunofluorescence studies of PLK1, CDC25c, and phosphorylated CDC2 supported these findings. Our data suggest that folate deficiency can initiate tumor development, that Mthfr mutation can enhance this phenomenon, and that altered expression of Plk1 and Cdc25c may contribute to folate-dependent intestinal tumorigenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Deficiência de Ácido Fólico/complicações , Neoplasias Intestinais/etiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fosfatases cdc25/genética , Animais , Divisão Celular , Dano ao DNA , Fase G2 , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fosforilação , RNA Mensageiro/análise , Quinase 1 Polo-LikeRESUMO
Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in folate and homocysteine metabolism. Research performed during the past decade has clarified our understanding of MTHFR deficiencies that cause homocystinuria or mild hyperhomocysteinemia. Our cloning of the MTHFR coding sequence was initially followed by the identification of the first deleterious mutations in MTHFR, in patients with homocystinuria and marked hyperhomocysteinemia. Shortly thereafter, we identified the 677C-->T variant and showed that it encoded a thermolabile enzyme with reduced activity. Currently, a total of 41 rare but deleterious mutations in MTHFR, as well as about 60 polymorphisms have been reported. The 677C-->T (Ala222Val) variant has been particularly noteworthy since it has become recognized as the most common genetic cause of hyperhomocysteinemia. The disruption of homocysteine metabolism by this polymorphism influences risk for several complex disorders, including cardiovascular disease, neural tube defects and some cancers. We describe here the complex structure of the MTHFR gene, summarize the current state of knowledge on rare and common mutations in MTHFR and discuss some relevant findings in a mouse model for MTHFR deficiency.
Assuntos
Genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Anormalidades Múltiplas/genética , Aborto Habitual/genética , Substituição de Aminoácidos , Animais , Apolipoproteína A-I/deficiência , HDL-Colesterol/metabolismo , Etnicidade/genética , Feminino , Retardo do Crescimento Fetal/genética , Frequência do Gene , Cardiopatias Congênitas/genética , Homocistinúria/genética , Humanos , Hiper-Homocisteinemia/epidemiologia , Hiper-Homocisteinemia/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Mutação Puntual , Gravidez , Esquizofrenia/genéticaRESUMO
Two promoters of the murine methylenetetrahydrofolate reductase gene (Mthfr), a key enzyme in folate metabolism, were characterized in Neuro-2a, NIH/3T3 and RAW 264.7 cells. Sequences of 189 bp and 273 bp were sufficient to achieve maximal activity of the upstream and downstream promoter, respectively. However, subtle differences in minimal promoter lengths and in promoter activities were observed between the cell lines. Both promoters demonstrated comparable activity in NIH/3T3 and RAW 264.7 cells, while in Neuro-2a cells, the upstream promoter was 15-fold more active than the downstream promoter. Alignment and data mining tools identified a candidate nuclear factor kappa B (NF-kappaB) binding site at the 3'end of the downstream promoter that is conserved throughout several species. NF-kappaB activation experiments in cultured cells were associated with increased Mthfr mRNA. Co-transfection of NF-kappaB and promoter constructs demonstrated Mthfr up-regulation by at least 2-fold through its downstream promoter in Neuro-2a cells; this increase was significantly reduced when the putative binding site was mutated. EMSA analysis demonstrated direct binding of NF-kappaB to this non-mutated site. This study, a first step into the elucidation of Mthfr regulation, demonstrates that two TATA-less, GC-rich promoters differentially drive transcription of Mthfr in a cell-specific manner, and provides a novel link of Mthfr to possible roles in the immune response and cell survival.
Assuntos
Regulação Enzimológica da Expressão Gênica , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , NF-kappa B/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Sequência Consenso , Sequência Conservada , Ensaio de Desvio de Mobilidade Eletroforética , Vetores Genéticos , Macrófagos/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Camundongos , Mutação , NF-kappa B/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TransfecçãoRESUMO
Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.
Assuntos
Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/genética , Proteínas Mitocondriais/genética , Mutação , Vitamina B 12/metabolismo , Pré-Escolar , Cromossomos Humanos Par 4 , Cobamidas/biossíntese , Análise Mutacional de DNA , Éxons , Feminino , Teste de Complementação Genética , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Ácido Metilmalônico/urina , Proteínas de Transporte da Membrana Mitocondrial , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to methionine. Severe MTHFR deficiency results in marked hyperhomocysteinemia and homocystinuria. Patients display developmental delay and a variety of neurological and vascular symptoms. Cloning of the human cDNA and gene has enabled the identification of 29 rare mutations in homocystinuric patients and two common variants [677C>T (A222V) and 1298A>C (E429A)] with mild enzymatic deficiency. Homozygosity for 677C>T or combined heterozygosity for both polymorphisms is associated with mild hyperhomocysteinemia. In this communication, we describe four novel mutations in patients with homocystinuria: two missense mutations (471C>G, I153M; 1025T>C, M338T), a nonsense mutation (1274G>A, W421X), and a 2-bp deletion (1553delAG). We expressed the 1025T>C mutation as well as two previously reported amino acid substitutions [983A>G (N324S) and 1027T>G (W339G)] and observed decreased enzyme activity at 10%, 36%, and 21% of control levels, respectively, with little or no effect on affinity for 5-methyltetrahydrofolate. One of these mutations, 983A>G (N324S), showed flavin adenine dinucleotide (FAD) responsiveness in vitro. Expression of these mutations in cis with the 677C>T polymorphism, as observed in the patients, resulted in an additional 50% decrease in enzyme activity. This report brings the total to 33 severe mutations identified in patients with severe MTHFR deficiency.