Karen E. Christensen

The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects.

Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42°C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%±7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01–4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38–9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13–8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted. Hum Mutat 0,1–9, 2008.

Steatosis in Mice Is Associated with Gender, Folate Intake, and Expression of Genes of One-Carbon Metabolism

Disrupted choline metabolism may affect hepatic lipid metabolism and lead to steatosis. Because folate and the choline metabolite betaine independently serve as methyl donors for homocysteine (Hcy) remethylation to methionine, we assessed the impact of folate deficiency on steatosis, choline metabolism, and expression of 9 genes involved in folate-mediated one-carbon metabolism. Liver histology, choline metabolites, and mRNA and protein expression were examined in mice fed control (CD; 2 mg/kg folic acid) or folate-deficient diets (FD; 0.3 mg/kg folic acid) for 12 mo. Females fed CD were not steatotic (0/6), whereas males were mildly to moderately steatotic (5/6). Steatosis was observed in FD-fed males and females; it was more severe and more frequent in males (7/7) than in females (4/10) (P = 0.005). Hepatic betaine was lower in males (P = 0.014) and FD-fed mice (P < 0.001) and negatively correlated with steatosis severity in mice fed CD (r = -0.87; P = 0.001). Gender differences in the expression of 6 enzymes may contribute to increased steatosis susceptibility in males. Males relied more on betaine-dependent (folate-independent) Hcy remethylation [72% more betaine-Hcy methyltransferase (P < 0.001) and 28% less folate-dependent methionine synthase (MTR) (P < 0.001)]. FD-fed mice of both genders appeared to shift to betaine-dependent remethylation by reducing MTR expression 70% (P < 0.001) and increasing betaine demand; there was a correlation between MTR expression and betaine levels (r = 0.50; P = 0.031). Our work demonstrates that chronic folate insufficiency leads to steatosis in mice. Increased utilization of betaine for Hcy remethylation in males and in both genders during folate deficiency may lead to steatosis by disrupting choline metabolism.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice

Background: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. Objective: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. Design: Folic acid–supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr+/+ and Mthfr+/− mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. Results: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr+/− mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr+/− livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr+/− mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. Conclusions: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

Mitochondrial methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetases.

Folate-mediated metabolism involves enzyme-catalyzed reactions that occur in the cytoplasmic, mitochondrial, and nuclear compartments in mammalian cells. Which of the folate-dependent enzymes are expressed in these compartments depends on the stage of development, cell type, cell cycle, and whether or not the cell is transformed. Mitochondria become formate-generating organelles in cells and tissues expressing the MTHFD2 and MTHFD1L genes. The products of these nuclear genes were derived from trifunctional precursor proteins, expressing methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase activities. The MTHFD2 protein is a bifunctional protein with dehydrogenase and cyclohydrolase activities that arose from a trifunctional precursor through the loss of the synthetase domain and a novel adaptation to NAD rather than NADP specificity for the dehydrogenase. The MTHFD1L protein retains the size of its trifunctional precursor, but through the mutation of critical residues, both the dehydrogenase and cyclohydrolase activities have been silenced. MTHFD1L is thus a monofunctional formyltetrahydrofolate synthetase. This review discusses the properties and functions of these mitochondrial proteins and their role in supporting cytosolic purine synthesis during embryonic development and in cells undergoing rapid growth.

Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2′-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold.

Risk of congenital heart defects is influenced by genetic variation in folate metabolism

Genetic disturbances in folate metabolism may increase risk for congenital heart defects. We examined the association of heart defects with four polymorphisms in folate-related genes (methylenetetrahydrofolate reductase (MTHFR) c.677C.T, MTHFR c.1298A.C, methionine synthase reductase (MTRR) c.66A.G, and reduced folate carrier (SLC19A1) c.80A.G) in a case-control study of children (156 patients, 69 controls) and mothers of children with heart defects (181 patients, 65 controls), born before folic acid fortification. MTRR c.66A.G in children modified odds ratios for overall heart defects, specifically ventricular septal defect and aortic valve stenosis (p-value below 0.05). The 66GG and AG genotypes were associated with decreased odds ratios for heart defects (0.42, 95% confidence interval (0.18-0.97) and 0.39 (0.18-0.84), respectively). This overall association was driven by decreased risk for ventricular septal defect for 66GG and AG (odds ratio 0.32 (0.11-0.91) and 0.25 (0.09-0.65)) and decreased odds ratio for aortic valve stenosis for 66AG (0.27 (0.09-0.79)). The association of ventricular septal defect and 66AG remained significant after correction for multiple testing (p = 0.0044, multiple testing threshold p = 0.0125). Maternal MTHFR 1298AC genotype was associated with increased odds ratio for aortic valve stenosis (2.90 (1.22-6.86), p = 0.0157), but this association did not meet the higher multiple testing threshold. No association between MTHFR c.677C.T or SLC19A1 c.80A.G and heart defect risk was found. The influence of folate-related polymorphisms may be specific to certain types of heart defects; larger cohorts of mothers and children with distinct sub-classes are required to adequately address risk.