Association of MTHFR (C677T, A1298C) and MTRR A66G polymorphisms with fatty acids profile and risk of neural tube defects.

OBJECTIVE: This study aims to determine if 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and methionine synthase reductase (MTRR A66G) gene polymorphisms were associated with fatty acid (FA) levels in mothers of fetuses with neural tube defects (NTDs) and whether these associations were modified by environmental factors. METHODS: Plasma FA composition was assessed using capillary gas chromatography. Concentrations of studied FA were compared between 42 mothers of NTDs fetuses and 30 controls as a function of each polymorphism by the Kruskal-Wallis nonparametric test. RESULTS: In MTHFR gene C677T polymorphism, cases with (CT + TT) genotype had lower monounsaturated FAs (MUFA) and omega-3 polyunsaturated FA (n-3 PUFA) levels, but higher omega-6 polyunsaturated FAs (n-6 PUFA) and omega-6 polyunsaturated FAs: omega-3 polyunsaturated FAs (n-6:n-3) ratio levels. In MTRR gene A66G polymorphism, cases with (AG + GG) genotype had lower MUFA levels, but higher PUFA and n-6 PUFA levels. Controls with (AG + GG) genotype had lower n-6 PUFA levels. In MTHFR gene C677T polymorphism, cases with smoking spouses and (CT + TT) genotype had lower MUFA and n-3 PUFA levels, but higher PUFA, n-6 PUFA, and n-6:n-3 ratio levels. Cases with (CT + TT) genotype and who used sauna during pregnancy had lower n-3 PUFA levels. In MTRR gene A66G polymorphism, cases with (AG + GG) genotype and who used sauna during pregnancy had higher PUFA and n-6 PUFA levels. CONCLUSIONS: Further research is required to clarify the association of FA metabolism and (MTHFR, MTRR) polymorphisms with NTDs.

Mutational and structural studies of (ßα)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism.

Methylene-tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one-carbon (C1) metabolisms. F420-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H4F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H4F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer-F420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride-transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl-tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5-iminium cation of methylene-tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Association of Increased Homocysteine Levels with Impaired Folate Metabolism and Vitamin B Deficiency in Early-Onset Multiple Sclerosis.

The contents of homocysteine (HCy), cyanocobalamin (vitamin B12), folic acid (vitamin B9), and pyridoxine (vitamin B6) were analyzed and the genotypes of the main gene polymorphisms associated with folate metabolism (C677T and A1298C of the MTHFR gene, A2756G of the MTR gene and A66G of the MTRR gene) were determined in children at the onset of multiple sclerosis (MS) (with disease duration of no more than six months), healthy children under 18 years (control group), healthy adults without neurological pathology, adult patients with MS at the onset of disease, and adult patients with long-term MS. A significant increase in the HCy levels was found in children at the MS onset compared to healthy children of the corresponding age. It was established that the content of HCy in children has a high predictive value. At the same time, an increase in the HCy levels was not accompanied by the deficiency of vitamins B6, B9, and B12 in the blood. The lack of correlation between the laboratory signs of vitamin deficiency and HCy levels may be due to the polymorphic variants of folate cycle genes. An increased HCy level should be considered as a marker of functional disorders of folate metabolism accompanying the development of pathological process in pediatric MS. Our finding can be used to develop new approaches to the prevention of demyelination in children and treatment of pediatric MS.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

Congenital septal defects in Karachi, Pakistan: an update of mutational screening by high-resolution melting (HRM) analysis of MTHFR C677T.

BACKGROUND: Congenital heart defects (CHDs) are the heart structural malformations present at birth. Septal defects account for 40% of CHD, including atrial, ventricular and atrioventricular septal defects. In Pakistan, the prevalence of CHD is 3.4 in 1000, and a study estimated that 60,000 babies are born with CHD annually. Methylenetetrahydrofolate reductase (MTHFR), a chief enzyme, involved in the folate metabolism. The missense mutation, C677T (rs1801133), exists in MTHFR gene, results in a MTHFR thermolabile variant having low enzymatic activity. The study is aim to identify the MTHFR C677T variant association with septal defects. METHODS: Samples of 194 CHD patients (age [Formula: see text]= 5.8 ± 5.1) and 50 normal echo controls (age [Formula: see text]= 6.0 ± 4.9), confirmed by pediatric consultant, were collected. Extracted DNA, quantified by agarose gel electrophoresis and nanodrop, was screened for SNP by high-resolution melting (HRM). Further, HRM results were confirmed using restriction analysis and sequencing. HRM was simply and precisely genotyped the samples within 3 h at low cost. RESULTS: Genotypic data suggested that heterozygous mutant (CT) was frequent in congenital septal defect patients (0.26) which was higher than controls (0.143), p > 0.05. Mutant (TT) genotype was not found in this study. CONCLUSIONS: rs1801133 has lack of significant association with congenital septal defects. The absence of TT genotype in this study suggesting the role of natural selection in targeted population. HRM is an easy, fast and next generation of PCR, which may be used for applied genomics.

Association of C677T and A1298C polymorphisms of the MTHFR gene with maternal risk for Down syndrome: A meta-analysis of case-control studies.

BACKGROUND: Several studies around the world support the hypothesis that genetic polymorphisms involved in folate metabolism could be related to the maternal risk for Down syndrome (DS). Most of them investigated the role of MTHFR C677T and/or A1298C polymorphisms as maternal risk factors for DS, but their results are often conflicting and still inconclusive. METHODS: We conducted a systematic review and meta-analysis to clarify the association of MTHFR C677T and/or A1298C polymorphisms with the maternal risk of DS. Our search strategy selected 42 eligible case control studies for a total of 4131 case mothers and 5452 control mothers. The Newcastle-Ottawa Scale was used to assess the methodological quality of the selected studies. To assess the confidence of statistically significant associations we applied false positive report probability test, and we performed the trial sequential analysis to minimize the type I error and random error. RESULTS: We observed significant associations between the MTHFR C677T polymorphism and maternal risk for DS for each of the genetic models investigated (dominant, recessive, codominant, and allelic contrast). Subgroup analysis by region revelated significant association in the Asian population for all the genetic models investigated. Significant associations were also found for certain genetic models in North American, South American, and Middle Eastern populations, while no association was observed in Europeans. The MTHFR A1298C polymorphism did not show any association with the maternal risk of DS, either alone or in combination with the C677T one. The results of false positive report probability to verify the confidence of a significant association suggest that the association between the MTHFR C677T polymorphism and the maternal risk for DS is noteworthy, with high confidence in Asians. CONCLUSION: The results of this meta-analysis support that the MTHFR C677T polymorphism, but not the A1298C one, is associated with the maternal risk for DS. Further studies are required to better characterize the contribution of gene-gene and gene-nutrient interactions as well as those of other regional or ethnic factors that could explain the observed different effect size in different populations.

Structural and functional characterization of a mycobacterial methylenetetrahydrofolate reductase utilizing NADH as the exclusive cofactor.

5,10-Methylenetetraydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. MSMEG_6649, a non-canonical MTHFR from Mycobacterium smegmatis, was previously reported as a monomeric protein lacking the flavin coenzyme. However, the structural basis for its unique flavin-independent catalytic mechanism remains poorly understood. Here, we determined the crystal structures of apo MTHFR MSMEG_6649 and its complex with NADH from M. smegmatis. Structural analysis revealed that the groove formed by the loops 4 and 5 of non-canonical MSMEG_6649 interacting with FAD was significantly larger than that of canonical MTHFR. Meanwhile, the NADH-binding site in MSMEG_6649 is highly similar to the FAD binding site in canonical MTHFR, suggesting that NADH plays the same role (immediate hydride donor for methylenetetraydrofolate) as FAD in the catalytic reaction. Using biochemical analysis, molecular modeling, and site-directed mutagenesis, the critical residues participating in the binding of NADH and the substrate 5,10-methylenetetrahydrofolate as well as the product 5-methyltetrahydrofolate were identified and validated. Taken together, this work not only provides a good starting point for understanding the potential catalytic mechanism for MSMEG_6649, but also identifies an exploitable target for the development of anti-mycobacterial drugs.

Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH2 -H4 folate substrate. NmMTHFR utilized NADH (Km = 44 µM) as an electron donor in the NAD(P)H-CH2 -H4 folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH2 -H4 folate substrate to bind. The unique topology of its NADH and CH2 -H4 folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.

Gender-specific association of SLC19A1 and MTHFR genetic polymorphism with oxidative stress biomarkers and plasma folate levels in older adults.

BACKGROUND: Plasma folate levels are closely related to antioxidant capacity and are regulated by folate pathway gene polymorphism. However, few studies have explored the gender-specific association of folate pathway gene polymorphism with oxidative stress biomarkers. The present study was designed to explore the gender-specific independent and combined impacts of solute carrier family 19 member 1 (SLC19A1) and methylenetetrahydrofolate reductase (MTHFR) genetic polymorphisms on oxidative stress biomarkers in older adults. METHODS: A total of 401 subjects were recruited, including 145 males and 256 females. Demographic characteristics of the participants were collected by using a self-administered questionnaire. Fasting venous blood samples were taken for folate pathway gene genotyping, circulating lipids parameters and erythrocyte oxidative stress biomarkers measurement. The difference of genotype distribution and the Hardy-Weinberg equilibrium was calculated by the Chi-square test. The general linear model was applied to compare the plasma folate levels and erythrocyte oxidative stress biomarkers. Multiple linear regression was used to explore the correlation between genetic risk scores and oxidative stress biomarkers. Logistic regression was used to explore the association of genetic risk scores of folate pathway gene with folate deficiency. RESULTS: The male subjects have lower plasma folate and HDL-C levels than the female ones, and the male carrying MTHFR rs1801133 (CC) or MTHFR rs2274976 (GA) genotypes have higher erythrocyte SOD activity. The plasma folate levels, erythrocyte SOD and GSH-PX activities were negatively correlated with genetic risk scores in the male subjects. A positive correlation between the genetic risk scores and folate deficiency was observed in the male subjects. CONCLUSIONS: There was association between folate pathway gene polymorphism of Solute Carrier Family 19 Member 1 (SLC19A1) and Methylenetetrahydrofolate Reductase (MTHFR) with erythrocyte SOD and GSH-PX activities, and folate levels in male but not in female aging subjects. Genetic variant of genes involved in folate metabolism has strong impact on plasma folate levels in the male aging subjects. Our data demonstrated that there was a potential interaction of gender and its genetic background in affecting the body's antioxidant capacity and the risk of folate deficiency in aging subjects.

High Folate, Perturbed One-Carbon Metabolism and Gestational Diabetes Mellitus.

Folate is a dietary micronutrient essential to one-carbon metabolism. The World Health Organisation recommends folic acid (FA) supplementation pre-conception and in early pregnancy to reduce the risk of fetal neural tube defects (NTDs). Subsequently, many countries (~92) have mandatory FA fortification policies, as well as recommendations for periconceptional FA supplementation. Mandatory fortification initiatives have been largely successful in reducing the incidence of NTDs. However, humans have limited capacity to incorporate FA into the one-carbon metabolic pathway, resulting in the increasingly ubiquitous presence of circulating unmetabolised folic acid (uFA). Excess FA intake has emerged as a risk factor in gestational diabetes mellitus (GDM). Several other one-carbon metabolism components (vitamin B12, homocysteine and choline-derived betaine) are also closely entwined with GDM risk, suggesting a role for one-carbon metabolism in GDM pathogenesis. There is growing evidence from in vitro and animal studies suggesting a role for excess FA in dysregulation of one-carbon metabolism. Specifically, high levels of FA reduce methylenetetrahydrofolate reductase (MTHFR) activity, dysregulate the balance of thymidylate synthase (TS) and methionine synthase (MTR) activity, and elevate homocysteine. High homocysteine is associated with increased oxidative stress and trophoblast apoptosis and reduced human chorionic gonadotrophin (hCG) secretion and pancreatic ß-cell function. While the relationship between high FA, perturbed one-carbon metabolism and GDM pathogenesis is not yet fully understood, here we summarise the current state of knowledge. Given rising rates of GDM, now estimated to be 14% globally, and widespread FA food fortification, further research is urgently needed to elucidate the mechanisms which underpin GDM pathogenesis.

Profiling the Influence of Gene Variants Related to Folate-Mediated One-Carbon Metabolism on the Outcome of In Vitro Fertilization (IVF) with Donor Oocytes in Recipients Receiving Folic Acid Fortification.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification.

Vitamin B-6 and riboflavin, their metabolic interaction, and relationship with MTHFR genotype in adults aged 18-102 years.

BACKGROUND: The generation of the active form of vitamin B-6, pyridoxal 5'-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear. OBJECTIVE: To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood. METHODS: Data from 5612 adults aged 18-102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined. RESULTS: Older (≥65 y) compared with younger (<65 y) adults had significantly lower PLP concentrations (P < 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27-1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P < 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P < 0.001). In participants with available dietary data (i.e., NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient ß: 2.49; 95% CI 1.75, 3.24; P < 0.001), supplement use (ß: 81.72; 95% CI: 66.01, 97.43; P < 0.001), fortified food (ß: 12.49; 95% CI: 2.08, 22.91; P = 0.019), and EGRac (ß: -65.81; 95% CI: -99.08, -32.54; P < 0.001), along with BMI (ß: -1.81; 95% CI: -3.31, -0.30; P = 0.019). CONCLUSIONS: These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively.Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017).

Vitamin D3 supplementation may attenuate morphological and molecular abnormalities of the olfactory bulb in a mouse model of Down syndrome.

Individuals with Down syndrome (DS) exhibit impaired olfactory function and are at a higher risk of developing Alzheimer's disease (AD). Olfactory dysfunction may be an early clinical symptom of AD. Recent studies have demonstrated that vitamin D3 (VD3) exerts neuroprotective effects in mouse models of AD. In this study, we investigated the effects of VD3 on the morphology, immunolocalization, and markers involved in neuropathogenic processes, apoptosis, proliferation, cell survival, and clearance of amyloid peptides, along with neuronal markers in the olfactory bulb (OB) of an adult female mouse model of DS. Morphological and molecular analyses revealed that trisomic mice exhibited a volume reduction in the external plexiform layer, a decrease in the number of mitral and granule cells, and an increase in the expression of amyloid-ß 42, caspase-3 p12, and P-glycoprotein. VD3 reversed certain morphological abnormalities in the OB of control trisomic mice (Ts(CO)) and decreased the levels of caspase-3 p12 and methylenetetrahydrofolate reductase in the treated groups. The results demonstrated that trisomy factor causes morphofunctional abnormalities in the OB of Ts(CO) mice. Moreover, VD3 could represent a therapeutic target to attenuate morphological and molecular alterations in OB.

T677T Methylenetetrahydrofolate Reductase Single Nucleotide Polymorphisms Increased Prevalence in a Subgroup of Infertile Patients with Endometriosis.

Background: Approximately 10% (190 million) of women worldwide are affected by endometriosis, ectopic deposits of endometrial tissue that create a major source of pain that affects lifestyle and reproductive function. The pathogenesis of endometriosis is an estrogen-dependent inflammatory process, influenced/catalyzed by oxidative stress and consequently defective methylation, with biochemical features centered around the folate and one-carbon cycles. We aimed to determine whether a link could be found between the two major methylenetetrahydrofolate reductase single nucleotide polymorphisms (MTHFR SNPs), c.677C>T and c.1298A>C, involved in methylation process/epigenetic marking failures, and endometriosis. Material and Methods: We studied a population of 158 patients in a group of >1500 referred for treatment of infertility. All the patients had experienced >2 failed assisted reproductive technology cycles and/or >2 miscarriages, a classical cohort for investigation in our group. Patients with endometriosis had at least stage 2+ disease confirmed by laparoscopy. Results: The prevalence of the homozygous c.677C>T isoform is doubled in the endometriosis group, 21.5% versus 10.2% in the non-endometriosis group (p > 0.01). Symmetrically, the percentage of patients in the endometriosis group with the wild type MTHFR significantly decreased by one-half (8.2%-17.2%) in the non-endometriosis group (p < 0.001). Conclusion: Determination of MTHFR c.677C>T should not be overlooked in patients with harmful endometriosis affecting their fertility. As folates metabolism is impaired in these MTHFR SNPs carrier patients, co-treatment with 5-methyl folate may constitute a successful (co)-treatment modality.

Absorption and Tissue Distribution of Folate Forms in Rats: Indications for Specific Folate Form Supplementation during Pregnancy.

Food fortification and folic acid supplementation during pregnancy have been implemented as strategies to prevent fetal malformations during pregnancy. However, with the emergence of conditions where folate metabolism and transport are disrupted, such as folate receptor alpha autoantibody (FRαAb)-induced folate deficiency, it is critical to find a folate form that is effective and safe for pharmacologic dosing for prolonged periods. Therefore, in this study, we explored the absorption and tissue distribution of folic acid (PGA), 5-methyl-tetrahydrofolate (MTHF), l-folinic acid (levofolinate), and d,l-folinic acid (Leucovorin) in adult rats. During absorption, all forms are converted to MTHF while some unconverted folate form is transported into the blood, especially PGA. The study confirms the rapid distribution of absorbed folate to the placenta and fetus. FRαAb administered, also accumulates rapidly in the placenta and blocks folate transport to the fetus and high folate concentrations are needed to circumvent or overcome the blocking of FRα. In the presence of FRαAb, both Leucovorin and levofolinate are absorbed and distributed to tissues better than the other forms. However, only 50% of the leucovorin is metabolically active whereas levofolinate is fully active and generates higher tetrahydrofolate (THF). Because levofolinate can readily incorporate into the folate cycle without needing methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) in the first pass and is relatively stable, it should be the folate form of choice during pregnancy, other disorders where large daily doses of folate are needed, and food fortification.

A Third Way of Energy Conservation in Acetogenic Bacteria.

Acetogenic bacteria are a group of strictly anaerobic bacteria that make a living from acetate formation from two molecules of CO2 via the Wood-Ljungdahl pathway (WLP). The free energy change of this reaction is very small and allows the synthesis of only a fraction of an ATP. How this pathway is coupled to energy conservation has been an enigma since its discovery ~90 years ago. Here, we describe an electron transport chain in the cytochrome- and quinone-containing acetogen Sporomusa ovata that leads from molecular hydrogen as an electron donor to an intermediate of the WLP, methylenetetrahydrofolate (methylene-tetrahydrofolate [THF]), as an electron acceptor. The catalytic site of the hydrogenase is periplasmic and likely linked cytochrome b to the membrane. We provide evidence that the MetVF-type methylenetetrahydrofolate reductase is linked proteins MvhD and HdrCBA to the cytoplasmic membrane. Membrane preparations catalyzed the H2-dependent reduction of methylene-THF to methyl-THF. In our model, a transmembrane electrochemical H+ gradient is established by both scalar and vectorial protons that leads to the synthesis of 0.5 mol ATP/mol methylene-THF by a H+-F1Fo ATP synthase. This H2- and methylene-THF-dependent electron transport chain may be present in other cytochrome-containing acetogens as well and represents a third way of chemiosmotic energy conservation in acetogens, but only in addition to the well-established respiratory enzymes Rnf and Ech. IMPORTANCE Acetogenic bacteria grow by making acetate from CO2 and are considered the first life forms on Earth since they couple CO2 reduction to the conservation of energy. How this is achieved has been an enigma ever since. Recently, two respiratory enzymes, a ferredoxin:NAD+ oxidoreductase (Rnf) and a ferredoxin:H+ oxidoreductase (Ech), have been found in cytochrome-free acetogenic model bacteria. However, some acetogens contain cytochromes in addition, and there has been a long-standing assumption of a cytochrome-containing electron transport chain in those acetogens. Here, we provide evidence for a respiratory chain in Sporomusa ovata that has a cytochrome-containing hydrogenase as the electron donor and a methylenetetrahydrofolate reductase as the terminal electron acceptor. This is the third way of chemiosmotic energy conservation found in acetogens.

5,10-Methylenetetrahydrofolate reductase becomes phosphorylated during meiotic maturation in mouse oocytes.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Mitochondrial DNA and Epigenetics: Investigating Interactions with the One-Carbon Metabolism in Obesity.

Mitochondrial DNA copy number (mtDNAcn) has been proposed for use as a surrogate biomarker of mitochondrial health, and evidence suggests that mtDNA might be methylated. Intermediates of the one-carbon cycle (1CC), which is duplicated in the cytoplasm and mitochondria, have a major role in modulating the impact of diet on the epigenome. Moreover, epigenetic pathways and the redox system are linked by the metabolism of glutathione (GSH). In a cohort of 101 normal-weight and 97 overweight/obese subjects, we evaluated mtDNAcn and methylation levels in both mitochondrial and nuclear areas to test the association of these marks with body weight, metabolic profile, and availability of 1CC intermediates associated with diet. Body composition was associated with 1CC intermediate availability. Reduced levels of GSH were measured in the overweight/obese group (p = 1.3∗10-5). A high BMI was associated with lower LINE-1 (p = 0.004) and nominally lower methylenetetrahydrofolate reductase (MTHFR) gene methylation (p = 0.047). mtDNAcn was lower in overweight/obese subjects (p = 0.004) and independently correlated with MTHFR methylation levels (p = 0.005) but not to LINE-1 methylation levels (p = 0.086). DNA methylation has been detected in the light strand but not in the heavy strand of the mtDNA. Although mtDNA methylation in the light strand did not differ between overweight/obese and normal-weight subjects, it was nominally correlated with homocysteine levels (p = 0.035) and MTHFR methylation (p = 0.033). This evidence suggests that increased body weight might perturb mitochondrial-nuclear homeostasis affecting the availability of nutrients acting as intermediates of the one-carbon cycle.