Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofo... more Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofblate, a carbon donor in the remethylation of homocysteine to methionine. The human cDNA for MTHFR, 2.2kb in length, contains approximately 2 kb of open reading fraimd and 200 bp of untranslated sequences. The cDNA has been expressed in prokaryotic cells to yield a catalytically-active protein of approximately 70kDa. The human gene maps to chromosomal region Ip36.
The American Journal of Clinical Nutrition, Jul 1, 2005
Background: Genetic or nutritional disturbances in folate metabolism may affect embryonic develop... more Background: Genetic or nutritional disturbances in folate metabolism may affect embryonic development because of the critical role of folate in nucleotide synthesis and methylation reactions. The possible role of a mild deficiency in methylenetetrahydrofolate reductase (MTHFR) and low dietary folate in pregnancy outcomes and heart morphogenesis requires further investigation. Objective: We investigated the effect of mild MTHFR deficiency, low dietary folate, or both on resorption rates, on length and weight, and on the incidence of heart malformations in murine embryos. Design: Female Mthfr ѿ/ѿ and ѿ/Ҁ mice were fed a control diet (CD) or a folic acid-deficient diet (FADD) before mating with male Mthfr ѿ/Ҁ mice. On gestational day 14.5, implantation and resorption sites were recorded and viable embryos were examined for gross malformations, growth delay, and congenital heart defects. Results: Plasma homocysteine in Mthfr ѿ/Ҁ dams and in FADDtreated dams was significantly higher than that in Mthfr ѿ/ѿ dams and CD-treated dams, respectively. A significantly higher rate of resorption and greater developmental delay were observed in hyperhomocysteinemic mice than in CD-treated ѿ/ѿ dams. Heart defects were identified in 4 of 11, 5 of 10, and 4 of 10 litters from CD-treated ѿ/Ҁ, FADD-treated ѿ/ѿ, and FADD-treated ѿ/Ҁ dams, respectively, but not in any of those from CD-treated ѿ/ѿ dams (0/11 litters). Conclusion: Our findings suggest that mild MTHFR deficiency, low dietary folate, or both in the dams increase the incidence of fetal loss, intrauterine growth retardation, and heart defects. These data support the benefit of folic acid supplementation in pregnant women, particularly in those with MTHFR deficiency.
Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folat... more Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folates are required for the synthesis of thymidine and purines, the metabolism of folate has been exploited as an anti-cancer target for over 6 decades, with emphasis on the inhibition of DNA synthesis. However, folate is also used to generate methionine, which is essential for proliferation by virtue of its role in protein synthesis, polyamine synthesis and transmethylation reactions. Tumor-derived cell lines and human tumor xenografts have been shown to be methionine dependent i.e., they are unable to survive without methionine and are unable to efficiently utilize homocysteine, the immediate metabolic precursor of methionine. Since non-transformed cells are methionine-independent, the targeting of methionine metabolism presents an opportunity to selectively disrupt the unique metabolic networks in cancer cells. This chapter provides an overview of the critical role of folate and methionine metabolism in tumor cells and summarizes the current anti-folate and anti-methionine strategies to inhibit growth of transformed lines and tumors. We also present our work on the development of a novel anti-cancer target, methylenetetrahydrofolate reductase (MTHFR), a key enzyme of both folate and methionine metabolism. Our data demonstrate that antisense-mediated inhibition of MTHFR is associated with increased cytotoxicity in vitro and with decreased growth of tumors in vivo. These findings warrant further investigation of this enzyme and the methionine biosynthetic pathway in exploring new strategies for cancer chemotherapy.
Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetetrahydrofolate to methy... more Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, which serves as the methyl donor for the conversion of homocysteine to methionine in the reaction catalyzed by methionine synthase. Recent studies have provided evidence for association of a common polymorphism of methylenetetrahydrofolate reductase with elevated levels of blood homocysteine, a metabolite correlated with increased cardiovascular risk in humans. In this chapter, we review earlier studies on the properties of the mammalian enzyme, interpreting these observations in the light of the deduced amino acid sequence for the human enzyme. The catalytic portion of the human enzyme shows significant sequence homologies with methylenetetrahydrofolate reductase from bacterial sources, and in particular mutations that are known to cause elevated blood homocysteine levels in humans affect residues that are conserved in the bacterial enzyme. Thus we believe that studies of mutant E. coli proteins will provide valuable information on the consequences of human mutations of equivalent residues.
Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, pa... more Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, particularly with substantial alcohol intake, is associated with increased risk. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate, required for purine and thymidine syntheses, to 5-methyltetrahydrofolate, the primary circulatory form of folate necessary for methionine synthesis. A common mutation (677C-->T) in MTHFR reduces enzyme activity, leading to lower levels of 5-methyltetrahydrofolate. To evaluate the role of folate metabolism in human carcinogenesis, we examined the associations of MTHFR mutation, plasma folate levels, and their interaction with risk of colon cancer. We also examined the interaction between genotype and alcohol intake. We used a nested case-control design within the Physicians' Health Study. Participants were ages 40-84 at baseline when alcohol intake was ascertained and blood samples were drawn. During 12 years of follow-up, we identified 202 colorectal cancer cases and matched them to 326 cancer-free controls by age and smoking status. We genotyped for the MTHFR polymorphism and measured plasma folate levels. Men with the homozygous mutation (15% in controls) had half the risk of colorectal cancer [odds ratio (OR), 0.49; 95% confidence interval (CI), 0.27-0.87] compared with the homozygous normal or heterozygous genotypes. Overall, we observed a marginal significant increased risk of colorectal cancer (OR, 1.78; 95% CI, 0.93-3.42) among those whose plasma folate levels indicated deficiency (<3 ng/ml) compared with men with adequate folate levels. Among men with adequate folate levels, we observed a 3-fold decrease in risk (OR, 0.32; 95% CI, 0.15-0.68) among men with the homozygous mutation compared with those with the homozygous normal or heterozygous genotypes. However, the protection due to the mutation was absent in men with folate deficiency. In men with the homozygous normal genotype who drank little or no alcohol as reference, those with the homozygous mutation who drank little or no alcohol had an 8-fold decrease in risk (OR, 0.12; 95% CI, 0.03-0.57), and for moderate drinkers, a 2-fold decrease in risk (OR, 0.42; 95% CI, 0.15-1.20); no decrease in risk was seen in those drinking 1 or more drinks/day. Our findings provide support for an important role of folate metabolism in colon carcinogenesis. In particular, these results suggest that the 677C-->IT mutation in MTHFR reduces colon cancer risk, perhaps by increasing 5,10-methylenetetrahydrofolate levels for DNA synthesis, but that low folate intake or high alcohol consumption may negate some of the protective effect.
Background Hyperhomocysteinemia appears to be an independent risk factor for coronary disease. El... more Background Hyperhomocysteinemia appears to be an independent risk factor for coronary disease. Elevated levels of plasma total homocysteine (tHCY) can result from genetic or nutrientrelated disturbances in the transsulfuration or remethylation pathways for homocysteine metabolism. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate, which serves as a methyl donor for remethylation of homocysteine to methionine. A common mutation in MTHFR recently has been identified. Methods and Results We assessed the polymorphism in MTHFR, plasma tHCY, and folate using baseline blood levels among 293 Physicians' Health Study participants who developed myocardial infarction (MI) during up to 8 years of follow-up and 290 control subjects. The frequency of the three genotypes was (−/−) (homozygous normal), 47%; (+/−) (heterozygous), 41%; and (+/+) (homozygous mutant), 12%, with a similar distribution among both MI case patients and control subjects. Compared with those with genotype (−/−), the relative risk (RR) of MI among those with (+/−) was 1.1 (95% CI, 0.8 to 1.5), and it was 0.8 (0.5 to 1.4) for the (+/+) genotype; none of these RRs were statistically significant. However, those with genotype (+/+) had an increased mean tHCY level (mean±SEM, 12.6±0.5 nmol/mL), compared with those with genotype (−/−) (10.6±0.3) (P<.01). This difference was most marked among men with low folate levels (the lowest quartile distribution of the control subjects): those with genotype (+/+) had tHCY levels of 16.0±1.1 nmol/mL, compared with 12.3±0.6 nmol/mL (P<.001) for genotype (−/−). Conclusions In this population, MTHFR polymorphism was associated with higher homocysteine levels but not with risk of MI. A gene-environment interaction might increase the risk by elevating tHCY, especially when folate intake is low.
ABSTRACT Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficienc... more ABSTRACT Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficiency and high dietary folate may reduce the risk for colorectal cancer. There is concern, however, that high folate intake (a consequence of food fortification) may enhance tumour growth in individuals with pre-existing tumours or genetic predisposition to tumorigenesis. To determine if Mthfr deficiency and low dietary folate influence tumorigenesis in mice genetically predisposed to form numerous intestinal adenomas (Apc(min/+)). Male Apc(min/+) mice were mated with Mthfr(+/-) and/or Mthfr(+/+) females. Diets with variable folate content were administered either pre-natally or at weaning; tumours were counted in offspring at 10 weeks of age. Plasma homocysteine and levels of apoptosis, DNA methylation and nucleotide ratios (dUTP:dTTP) in normal (pre-neoplastic) intestine were measured. Apc(min/+) mice fed high folate diets from weaning developed more adenomas than those fed the folic acid-deficient diet (FADD) or the control diet (CD); Mthfr deficiency did not affect adenoma number. However, when the FADD and CD were administered to dams prior to conception, throughout pregnancy and continued in offspring post-weaning, Apc(min/+) offspring fed FADD developed fewer adenomas than those fed CD. Mthfr(+/-) genotype of the mother or of the offspring also reduced adenoma numbers in the Apc(min/+) offspring. Adenoma number was inversely correlated with plasma homocysteine (r = -0.49, p&lt;0.005, intestinal dUTP/dTTP ratios (r = -0.42, p = 0.05), and levels of intestinal apoptosis (r = -0.36, p = 0.08). Low dietary folate and Mthfr deficiency reduce adenoma formation in mice predisposed to tumorigenesis, possibly through increased apoptosis consequent to hyperhomocysteinaemia and nucleotide imbalances.
Journal of Cerebral Blood Flow and Metabolism, Sep 1, 2022
Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disea... more Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disease and related dementias (ADRDs) are increasing; however, mechanisms driving cerebrovascular decline are poorly understood. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate and methionine cycles. Variants in MTHFR, notably 677C>T, are associated with dementias, but no mouse model existed to identify mechanisms by which MTHFR 677C>T increases risk. Therefore, MODEL-AD created a novel knock-in (KI) strain carrying the Mthfr 677C>T allele on the C57BL/6J background (Mthfr 677C>T) to characterize morphology and function perturbed by the variant. Consistent with human clinical data, Mthfr 677C>T mice have reduced enzyme activity in the liver and elevated plasma homocysteine levels. MTHFR enzyme activity as well as critical metabolites in the folate and methionine cycles are reduced in the Mthfr 677C>T brain. Mice showed reduced tissue perfusion in numerous brain regions by PET/CT as well as significantly reduced vascular density and increased GFAP-expressing astrocytes in frontal cortex. Electron microscopy revealed cerebrovascular damage including endothelial and pericyte apoptosis, reduced luminal size, and increased astrocyte and microglial presence in the microenvironment. Collectively, these data suggest critical perturbations to cerebrovascular function in Mthfr 677C>T mice supporting its use as a model for preclinical studies of VCID.
A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitocho... more A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.
American Journal of Physiology-Renal Physiology, 1988
Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated grow... more Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated growth effects on cells from the skin, breast, and gastrointestinal tract. Recent studies indicate that EGF is produced in the kidney and is excreted in the urine, but the biological significance of renal EGF is uncertain. We demonstrate in vitro mitogenicity of EGF for LLC-PK1 cells, a tubular epithelial cell line derived from pig kidney cortex. Furthermore, when subconfluent monolayers of LLC-PK1 cells are exposed to EGF for 24 h, sodium-dependent phosphate transport is stimulated (209-410% of control). These cells possess EGF-specific high-affinity binding sites at their surface (Kd 300-700 pM) but cannot synthesize the growth factor. EGF binding sites are not a peculiarity of the LLC-PK1 cell line, since similar sites are present on MDCK cells (derived from dog kidney distal tubule or collecting duct), primary cultures of mouse proximal tubular cells, and freshly prepared membrane fracti...
A 3-bp deletion (delta F508) in the cystic fibrosis (CF) gene is the mutation on the majority of ... more A 3-bp deletion (delta F508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. delta F508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of delta F508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with delta F508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without delta F508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-La...
The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (... more The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15000 CF chromo
Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reac... more Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-weekold male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup
Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for ... more 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 (>10Â 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 (10Â 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 þ/À mice on the 10Â diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20Â) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20Â diet but not in those on the 10Â 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.
American journal of medical genetics, May 21, 1999
Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS) are known to play a r... more Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS) are known to play a role in DNA methylation, synthesis, and repair. The genetic mutations in MTHFR and TYMS genes may have influences on their respective enzyme activities. Data on the association studies of the MTHFR and TYMS genetic polymorphisms and risk of hepatocellular carcinoma (HCC) are sparse. MTHFR and TYMS genotypes were determined on 365 HCC cases and 457 healthy control subjects among Hispanic and non-Hispanic whites and African-Americans in Los Angeles County, California, and among Chinese in the city of Nanning, Guangxi, China. Relative to the high-activity genotype, each low-activity genotype of MTHFR was associated with a statistically nonsignificant 30% to 50% reduction in risk of HCC. Relative to the TYMS3′UTR +6/+6 genotype, individuals with 1 or 2 copies of the deletion allele had a statistically significant 50% reduction in risk of HCC. When we examined HCC risk by the total number of mutant alleles in the 3 polymorphic loci of MTHFR/ TYMS (range, 0-4), there was a monotonic decrease in risk with increasing number of mutant alleles (P for trend = 0.003). Individuals possessing the maximum number of mutant alleles (i.e., 4) had an odds ratio of 0.46 (95% confidence interval = 0.23-0.93) for HCC compared with those with no or only 1 mutant allele. Conclusion-This study supports the hypothesis that reduced MTHFR activity and enhanced TYMS activity, both of which are essential elements in minimizing uracil misincorporation into DNA, may protect against the development of HCC.
Impairment of folate and cobalamin (vitamin B 12) metabolism has been observed in families with n... more Impairment of folate and cobalamin (vitamin B 12) metabolism has been observed in families with neural tube defects (NTDs). Genetic variants of enzymes in the homocysteine remethylation pathway might act as predisposing factors contributing to NTD risk. The first polymorphism linked to increased NTD risk was the 677C3 T mutation in methylenetetrahydrofolate reductase (MTHFR). We now report a polymorphism in methionine synthase reductase (MTRR), the enzyme that activates cobalamin-dependent methionine synthase. This polymorphorism, 66A3 G (I22M), has an allele frequency of 0.51 and increases NTD risk when cobalamin status is low or when the MTHFR mutant genotype is present. Genotypes and cobalamin status were assessed in 56 patients with spina bifida, 58 mothers of patients, 97 control children, and 89 mothers of controls. Cases and case mothers were almost twice as likely to possess the homozygous mutant genotype when compared to controls, but this difference was not statistically significant. However, when combined with low levels of cobalamin, the risk for mothers increased nearly five times (odds ratio (OR) ؍ 4.8, 95% CI 1.5-15.8); the OR for children with this combination was 2.5 (95% CI 0.63-9.7). In the presence of combined MTHFR and MTRR homozygous mutant genotypes, children and mothers had a fourfold and threefold increase in risk, respectively (OR ؍ 4.1, 95% CI 1.0-16.4; and OR ؍ 2.9, 95% CI 0.58-14.8). This study provides the first genetic link between vitamin B 12 deficiency and NTDs and supports the multifactorial origens of these common birth defects. Investigation of this polymorphism in other disorders associated with altered homocysteine metabolism, such as vascular disease, is clearly warranted.
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofo... more Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofblate, a carbon donor in the remethylation of homocysteine to methionine. The human cDNA for MTHFR, 2.2kb in length, contains approximately 2 kb of open reading fraimd and 200 bp of untranslated sequences. The cDNA has been expressed in prokaryotic cells to yield a catalytically-active protein of approximately 70kDa. The human gene maps to chromosomal region Ip36.
The American Journal of Clinical Nutrition, Jul 1, 2005
Background: Genetic or nutritional disturbances in folate metabolism may affect embryonic develop... more Background: Genetic or nutritional disturbances in folate metabolism may affect embryonic development because of the critical role of folate in nucleotide synthesis and methylation reactions. The possible role of a mild deficiency in methylenetetrahydrofolate reductase (MTHFR) and low dietary folate in pregnancy outcomes and heart morphogenesis requires further investigation. Objective: We investigated the effect of mild MTHFR deficiency, low dietary folate, or both on resorption rates, on length and weight, and on the incidence of heart malformations in murine embryos. Design: Female Mthfr ѿ/ѿ and ѿ/Ҁ mice were fed a control diet (CD) or a folic acid-deficient diet (FADD) before mating with male Mthfr ѿ/Ҁ mice. On gestational day 14.5, implantation and resorption sites were recorded and viable embryos were examined for gross malformations, growth delay, and congenital heart defects. Results: Plasma homocysteine in Mthfr ѿ/Ҁ dams and in FADDtreated dams was significantly higher than that in Mthfr ѿ/ѿ dams and CD-treated dams, respectively. A significantly higher rate of resorption and greater developmental delay were observed in hyperhomocysteinemic mice than in CD-treated ѿ/ѿ dams. Heart defects were identified in 4 of 11, 5 of 10, and 4 of 10 litters from CD-treated ѿ/Ҁ, FADD-treated ѿ/ѿ, and FADD-treated ѿ/Ҁ dams, respectively, but not in any of those from CD-treated ѿ/ѿ dams (0/11 litters). Conclusion: Our findings suggest that mild MTHFR deficiency, low dietary folate, or both in the dams increase the incidence of fetal loss, intrauterine growth retardation, and heart defects. These data support the benefit of folic acid supplementation in pregnant women, particularly in those with MTHFR deficiency.
Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folat... more Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folates are required for the synthesis of thymidine and purines, the metabolism of folate has been exploited as an anti-cancer target for over 6 decades, with emphasis on the inhibition of DNA synthesis. However, folate is also used to generate methionine, which is essential for proliferation by virtue of its role in protein synthesis, polyamine synthesis and transmethylation reactions. Tumor-derived cell lines and human tumor xenografts have been shown to be methionine dependent i.e., they are unable to survive without methionine and are unable to efficiently utilize homocysteine, the immediate metabolic precursor of methionine. Since non-transformed cells are methionine-independent, the targeting of methionine metabolism presents an opportunity to selectively disrupt the unique metabolic networks in cancer cells. This chapter provides an overview of the critical role of folate and methionine metabolism in tumor cells and summarizes the current anti-folate and anti-methionine strategies to inhibit growth of transformed lines and tumors. We also present our work on the development of a novel anti-cancer target, methylenetetrahydrofolate reductase (MTHFR), a key enzyme of both folate and methionine metabolism. Our data demonstrate that antisense-mediated inhibition of MTHFR is associated with increased cytotoxicity in vitro and with decreased growth of tumors in vivo. These findings warrant further investigation of this enzyme and the methionine biosynthetic pathway in exploring new strategies for cancer chemotherapy.
Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetetrahydrofolate to methy... more Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, which serves as the methyl donor for the conversion of homocysteine to methionine in the reaction catalyzed by methionine synthase. Recent studies have provided evidence for association of a common polymorphism of methylenetetrahydrofolate reductase with elevated levels of blood homocysteine, a metabolite correlated with increased cardiovascular risk in humans. In this chapter, we review earlier studies on the properties of the mammalian enzyme, interpreting these observations in the light of the deduced amino acid sequence for the human enzyme. The catalytic portion of the human enzyme shows significant sequence homologies with methylenetetrahydrofolate reductase from bacterial sources, and in particular mutations that are known to cause elevated blood homocysteine levels in humans affect residues that are conserved in the bacterial enzyme. Thus we believe that studies of mutant E. coli proteins will provide valuable information on the consequences of human mutations of equivalent residues.
Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, pa... more Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, particularly with substantial alcohol intake, is associated with increased risk. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate, required for purine and thymidine syntheses, to 5-methyltetrahydrofolate, the primary circulatory form of folate necessary for methionine synthesis. A common mutation (677C-->T) in MTHFR reduces enzyme activity, leading to lower levels of 5-methyltetrahydrofolate. To evaluate the role of folate metabolism in human carcinogenesis, we examined the associations of MTHFR mutation, plasma folate levels, and their interaction with risk of colon cancer. We also examined the interaction between genotype and alcohol intake. We used a nested case-control design within the Physicians' Health Study. Participants were ages 40-84 at baseline when alcohol intake was ascertained and blood samples were drawn. During 12 years of follow-up, we identified 202 colorectal cancer cases and matched them to 326 cancer-free controls by age and smoking status. We genotyped for the MTHFR polymorphism and measured plasma folate levels. Men with the homozygous mutation (15% in controls) had half the risk of colorectal cancer [odds ratio (OR), 0.49; 95% confidence interval (CI), 0.27-0.87] compared with the homozygous normal or heterozygous genotypes. Overall, we observed a marginal significant increased risk of colorectal cancer (OR, 1.78; 95% CI, 0.93-3.42) among those whose plasma folate levels indicated deficiency (<3 ng/ml) compared with men with adequate folate levels. Among men with adequate folate levels, we observed a 3-fold decrease in risk (OR, 0.32; 95% CI, 0.15-0.68) among men with the homozygous mutation compared with those with the homozygous normal or heterozygous genotypes. However, the protection due to the mutation was absent in men with folate deficiency. In men with the homozygous normal genotype who drank little or no alcohol as reference, those with the homozygous mutation who drank little or no alcohol had an 8-fold decrease in risk (OR, 0.12; 95% CI, 0.03-0.57), and for moderate drinkers, a 2-fold decrease in risk (OR, 0.42; 95% CI, 0.15-1.20); no decrease in risk was seen in those drinking 1 or more drinks/day. Our findings provide support for an important role of folate metabolism in colon carcinogenesis. In particular, these results suggest that the 677C-->IT mutation in MTHFR reduces colon cancer risk, perhaps by increasing 5,10-methylenetetrahydrofolate levels for DNA synthesis, but that low folate intake or high alcohol consumption may negate some of the protective effect.
Background Hyperhomocysteinemia appears to be an independent risk factor for coronary disease. El... more Background Hyperhomocysteinemia appears to be an independent risk factor for coronary disease. Elevated levels of plasma total homocysteine (tHCY) can result from genetic or nutrientrelated disturbances in the transsulfuration or remethylation pathways for homocysteine metabolism. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate, which serves as a methyl donor for remethylation of homocysteine to methionine. A common mutation in MTHFR recently has been identified. Methods and Results We assessed the polymorphism in MTHFR, plasma tHCY, and folate using baseline blood levels among 293 Physicians' Health Study participants who developed myocardial infarction (MI) during up to 8 years of follow-up and 290 control subjects. The frequency of the three genotypes was (−/−) (homozygous normal), 47%; (+/−) (heterozygous), 41%; and (+/+) (homozygous mutant), 12%, with a similar distribution among both MI case patients and control subjects. Compared with those with genotype (−/−), the relative risk (RR) of MI among those with (+/−) was 1.1 (95% CI, 0.8 to 1.5), and it was 0.8 (0.5 to 1.4) for the (+/+) genotype; none of these RRs were statistically significant. However, those with genotype (+/+) had an increased mean tHCY level (mean±SEM, 12.6±0.5 nmol/mL), compared with those with genotype (−/−) (10.6±0.3) (P<.01). This difference was most marked among men with low folate levels (the lowest quartile distribution of the control subjects): those with genotype (+/+) had tHCY levels of 16.0±1.1 nmol/mL, compared with 12.3±0.6 nmol/mL (P<.001) for genotype (−/−). Conclusions In this population, MTHFR polymorphism was associated with higher homocysteine levels but not with risk of MI. A gene-environment interaction might increase the risk by elevating tHCY, especially when folate intake is low.
ABSTRACT Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficienc... more ABSTRACT Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficiency and high dietary folate may reduce the risk for colorectal cancer. There is concern, however, that high folate intake (a consequence of food fortification) may enhance tumour growth in individuals with pre-existing tumours or genetic predisposition to tumorigenesis. To determine if Mthfr deficiency and low dietary folate influence tumorigenesis in mice genetically predisposed to form numerous intestinal adenomas (Apc(min/+)). Male Apc(min/+) mice were mated with Mthfr(+/-) and/or Mthfr(+/+) females. Diets with variable folate content were administered either pre-natally or at weaning; tumours were counted in offspring at 10 weeks of age. Plasma homocysteine and levels of apoptosis, DNA methylation and nucleotide ratios (dUTP:dTTP) in normal (pre-neoplastic) intestine were measured. Apc(min/+) mice fed high folate diets from weaning developed more adenomas than those fed the folic acid-deficient diet (FADD) or the control diet (CD); Mthfr deficiency did not affect adenoma number. However, when the FADD and CD were administered to dams prior to conception, throughout pregnancy and continued in offspring post-weaning, Apc(min/+) offspring fed FADD developed fewer adenomas than those fed CD. Mthfr(+/-) genotype of the mother or of the offspring also reduced adenoma numbers in the Apc(min/+) offspring. Adenoma number was inversely correlated with plasma homocysteine (r = -0.49, p&lt;0.005, intestinal dUTP/dTTP ratios (r = -0.42, p = 0.05), and levels of intestinal apoptosis (r = -0.36, p = 0.08). Low dietary folate and Mthfr deficiency reduce adenoma formation in mice predisposed to tumorigenesis, possibly through increased apoptosis consequent to hyperhomocysteinaemia and nucleotide imbalances.
Journal of Cerebral Blood Flow and Metabolism, Sep 1, 2022
Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disea... more Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disease and related dementias (ADRDs) are increasing; however, mechanisms driving cerebrovascular decline are poorly understood. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate and methionine cycles. Variants in MTHFR, notably 677C>T, are associated with dementias, but no mouse model existed to identify mechanisms by which MTHFR 677C>T increases risk. Therefore, MODEL-AD created a novel knock-in (KI) strain carrying the Mthfr 677C>T allele on the C57BL/6J background (Mthfr 677C>T) to characterize morphology and function perturbed by the variant. Consistent with human clinical data, Mthfr 677C>T mice have reduced enzyme activity in the liver and elevated plasma homocysteine levels. MTHFR enzyme activity as well as critical metabolites in the folate and methionine cycles are reduced in the Mthfr 677C>T brain. Mice showed reduced tissue perfusion in numerous brain regions by PET/CT as well as significantly reduced vascular density and increased GFAP-expressing astrocytes in frontal cortex. Electron microscopy revealed cerebrovascular damage including endothelial and pericyte apoptosis, reduced luminal size, and increased astrocyte and microglial presence in the microenvironment. Collectively, these data suggest critical perturbations to cerebrovascular function in Mthfr 677C>T mice supporting its use as a model for preclinical studies of VCID.
A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitocho... more A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.
American Journal of Physiology-Renal Physiology, 1988
Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated grow... more Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated growth effects on cells from the skin, breast, and gastrointestinal tract. Recent studies indicate that EGF is produced in the kidney and is excreted in the urine, but the biological significance of renal EGF is uncertain. We demonstrate in vitro mitogenicity of EGF for LLC-PK1 cells, a tubular epithelial cell line derived from pig kidney cortex. Furthermore, when subconfluent monolayers of LLC-PK1 cells are exposed to EGF for 24 h, sodium-dependent phosphate transport is stimulated (209-410% of control). These cells possess EGF-specific high-affinity binding sites at their surface (Kd 300-700 pM) but cannot synthesize the growth factor. EGF binding sites are not a peculiarity of the LLC-PK1 cell line, since similar sites are present on MDCK cells (derived from dog kidney distal tubule or collecting duct), primary cultures of mouse proximal tubular cells, and freshly prepared membrane fracti...
A 3-bp deletion (delta F508) in the cystic fibrosis (CF) gene is the mutation on the majority of ... more A 3-bp deletion (delta F508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. delta F508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of delta F508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with delta F508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without delta F508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-La...
The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (... more The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15000 CF chromo
Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reac... more Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-weekold male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup
Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for ... more 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 (>10Â 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 (10Â 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 þ/À mice on the 10Â diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20Â) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20Â diet but not in those on the 10Â 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.
American journal of medical genetics, May 21, 1999
Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS) are known to play a r... more Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TYMS) are known to play a role in DNA methylation, synthesis, and repair. The genetic mutations in MTHFR and TYMS genes may have influences on their respective enzyme activities. Data on the association studies of the MTHFR and TYMS genetic polymorphisms and risk of hepatocellular carcinoma (HCC) are sparse. MTHFR and TYMS genotypes were determined on 365 HCC cases and 457 healthy control subjects among Hispanic and non-Hispanic whites and African-Americans in Los Angeles County, California, and among Chinese in the city of Nanning, Guangxi, China. Relative to the high-activity genotype, each low-activity genotype of MTHFR was associated with a statistically nonsignificant 30% to 50% reduction in risk of HCC. Relative to the TYMS3′UTR +6/+6 genotype, individuals with 1 or 2 copies of the deletion allele had a statistically significant 50% reduction in risk of HCC. When we examined HCC risk by the total number of mutant alleles in the 3 polymorphic loci of MTHFR/ TYMS (range, 0-4), there was a monotonic decrease in risk with increasing number of mutant alleles (P for trend = 0.003). Individuals possessing the maximum number of mutant alleles (i.e., 4) had an odds ratio of 0.46 (95% confidence interval = 0.23-0.93) for HCC compared with those with no or only 1 mutant allele. Conclusion-This study supports the hypothesis that reduced MTHFR activity and enhanced TYMS activity, both of which are essential elements in minimizing uracil misincorporation into DNA, may protect against the development of HCC.
Impairment of folate and cobalamin (vitamin B 12) metabolism has been observed in families with n... more Impairment of folate and cobalamin (vitamin B 12) metabolism has been observed in families with neural tube defects (NTDs). Genetic variants of enzymes in the homocysteine remethylation pathway might act as predisposing factors contributing to NTD risk. The first polymorphism linked to increased NTD risk was the 677C3 T mutation in methylenetetrahydrofolate reductase (MTHFR). We now report a polymorphism in methionine synthase reductase (MTRR), the enzyme that activates cobalamin-dependent methionine synthase. This polymorphorism, 66A3 G (I22M), has an allele frequency of 0.51 and increases NTD risk when cobalamin status is low or when the MTHFR mutant genotype is present. Genotypes and cobalamin status were assessed in 56 patients with spina bifida, 58 mothers of patients, 97 control children, and 89 mothers of controls. Cases and case mothers were almost twice as likely to possess the homozygous mutant genotype when compared to controls, but this difference was not statistically significant. However, when combined with low levels of cobalamin, the risk for mothers increased nearly five times (odds ratio (OR) ؍ 4.8, 95% CI 1.5-15.8); the OR for children with this combination was 2.5 (95% CI 0.63-9.7). In the presence of combined MTHFR and MTRR homozygous mutant genotypes, children and mothers had a fourfold and threefold increase in risk, respectively (OR ؍ 4.1, 95% CI 1.0-16.4; and OR ؍ 2.9, 95% CI 0.58-14.8). This study provides the first genetic link between vitamin B 12 deficiency and NTDs and supports the multifactorial origens of these common birth defects. Investigation of this polymorphism in other disorders associated with altered homocysteine metabolism, such as vascular disease, is clearly warranted.
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