Impaired Folate Metabolism Research Articles

Association of Increased Homocysteine Level with Impaired Folate Metabolism and Vitamins B Deficiency in Early Onset of Multiple Sclerosis

Association of Increased Homocysteine Level with Impaired Folate Metabolism and Vitamins B Deficiency in Early Onset of Multiple Sclerosis

Navigating thrombotic terrain: unveiling a novel homocystinuria mutation associated with thrombophilia in a 16 year old

BackgroundThrombophilia is characterised by an abnormality of blood coagulation that increases thrombosis. Homocystinuria encompasses a group of disorders marked by increased levels of homocysteine and other amino acids detectable in the bloodstream and urine. Conversely, homocystinuria due to methylenetetrahydrofolatereductase (MTHFR) deficiency, a rarer disorder, stems from impaired folate metabolism due to deficient MTHFR enzyme.Case presentationA 16-year-old boy presented with walking difficulties, headaches, and thrombotic events, thrombophilia workup led to a diagnosis of homocystinuria due to a novel mutation in MTHFR gene. Anticoagulant therapy was initiated which showed clinical improvement, but financial constraints hindered follow-up.ConclusionsThis case highlights the complexities of diagnosing and treating paediatric thrombophilia, particularly in resource-limited settings. Notably, the identified homozygous autosomal recessive (AR) missense variation in the MTHFR gene (Exon 4—c582C>G) represents a novel mutation, suggesting the ongoing significance of genetic research in elucidating the underlying mechanisms of thrombotic disorders.

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

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

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

Fetal hyperhomocysteinemia is associated with placental inflammation and early breakdown of maternal-fetal tolerance in pre-term birth.

Hyperhomocysteinemia (hypHcy) due to impaired folate metabolism is shown to be a risk factor for preterm birth (PTB) and low birth weight (LBW) in mothers. However, the relationship of fetal hypHcy with adverse pregnancy outcomes is under-represented. The present study aims to investigate the association of fetal hypHcy with oxidative stress and placental inflammation that can contribute to an early breakdown of maternal-fetal tolerance in pre-term birth (PTB). Cord blood and placenta tissue were collected from PTB and term infant group. Levels of homocysteine, folic acid, vitamin B12 and oxidative stress markers (MDA, T-AOC, 8-OHdG) were measured in cord blood serum using ELISA and respective standard assay kits. Relative expression of candidate genes (TNF-α, IL-6, IL1-β, VEGF-A, MMP2 and MMP9) was also checked using RT-PCR and immunoblotting/immunohistochemistry. PTB infants showed significantly higher levels of homocysteine (P=.02) and lower levels of vitamin B12 (P=.005) as compared to term infants. We also found that PTB infants with hypHcy had lower T-AOC (P=.003) and higher MDA (P=.04) levels as compared to term infants with normal homocysteine levels. The mRNA and protein levels of TNF-α, VEGF-A, MMP2 and MMP9 were significantly higher in hypHcy PTB infants. Our results show that fetal hypHcy is associated with oxidative stress and an increase in inflammatory markers in the placenta. Thus, in conclusion, our study demonstrates that fetal hypHcy during pregnancy is a potential risk factor that may initiate an early breakdown of uterine quiescence due to activation of inflammatory processes leading to PTB.

Demographic and biochemical risk factors of Down Syndrome in pregnant women: A pilot study in Sylhet division of Bangladesh

Worldwide research in down syndrome have shown that advanced maternal age (˃30 years) at conception, lower folate status and/or impaired folate metabolism, maternal weight gain during pregnancy, low socioeconomic conditions, radiation exposure, use of contraceptive pills, and taking tobacco/smoking are major maternal risk factors for giving birth child with DS. However, the occurrence of those factors in pregnant women at advanced age has not yet been investigated in Bangladesh. Therefore, a large scale study was needed to explore the overall scenario in Bangladesh. This pilot cross sectional study was aimed to determine the serum folate and homocysteine status at advanced age pregnant women. We measured serum folate and homocysteine concentrations using the Enzyme-Linked Immunosorbent Assay (ELISA). Out of 70 pregnant women, serumfolate level was low (<5 ng/ml) in 13 (18.57%), intermediate (5-6.9 ng/ml) in 8 (11.43%) and normal in 49 (70%) whereas homocysteine concentrations were found low (<5 ng/ml) in all participants. Finally, questionnaire data indicates, low socioeconomic status, overweight during pregnancy, taking contraceptive pills and taking tobacco/smoking was higher percentage in folate-deficient pregnant women than normal serum folate level women. Additionally, women (n=13) with low folate level belongs to low socioeconomic status, overweight, taking contraceptive pills and tobacco/smoking did not give birth child with DS phenotypes. Together, our preliminary findings give an overview of DS associated risk factors in Bangladeshi pregnant women. Large scale study is going on. In next study, we might able to investigate DS risk factors, their associations with serum folate and homocysteine levels and finally with DS phenotypes in child throughout the country. Keywords: Down syndrome; risk factors; folic acid; homocysteine; pregnant women; Bangladesh.

Addressing optimal folate and related B-vitamin status through the lifecycle: health impacts and challenges.

The functional effects of folate within C1 metabolism involve interrelationships with vitamin B12, vitamin B6 and riboflavin, and related gene-nutrient interactions. These B vitamins have important roles throughout life, from pregnancy, through childhood, to middle and older age. Achieving optimal nutritional status for preventing folate-related disease is challenging, however, primarily as a result of the poor stability and incomplete bioavailability of folate from natural food sources when compared with the synthetic vitamin form, folic acid. Thus, in European countries, measures to prevent neural tube defects (NTD) have been largely ineffective because of the generally poor compliance of women with folic acid supplementation as recommended before and in early pregnancy. In contrast, countries worldwide with mandatory folic acid fortification policies have experienced marked reductions in NTD. Low vitamin B12 status is associated with increased risk of cognitive dysfunction, CVD and osteoporosis. Achieving optimal B12 status can be problematic for older people, however, primarily owing to food-bound B12 malabsorption which leads to sub-clinical deficiency even with high dietary B12 intakes. Optimising B-vitamin intake may be particularly important for sub-populations with impaired folate metabolism owing to genetic characteristics, most notably the 677C→T variant in the gene encoding the enzyme methylenetetrahydrofolate reductase (MTHFR). This common folate polymorphism is linked with several adverse health outcomes, including stroke, however, recent evidence has identified its novel interaction with riboflavin (the MTHFR cofactor) in relation to blood pressure and risk of developing hypertension. This review addresses why and how the optimal status of folate-related B vitamins should be achieved through the lifecycle.

The thymidylate synthase enhancer region (TSER) polymorphism increases the risk of thymic lymphoid hyperplasia in patients with Myasthenia Gravis

BackgroundMyasthenia Gravis (MG) is caused, in approximately 80% of the patients, by autoantibodies against the nicotinic acetylcholine receptor (AChR). The disease is often associated with pathological changes of the thymus: thymic epithelial tumours are present in about 10–20% of the patients, while up to 80% of the patients with early disease onset have thymic hyperplasia. Folate metabolism is required for the production of DNA precursors and for proper DNA methylation reactions, and impaired folate metabolism has been often associated with cellular growth and cancer. MethodsWe investigated if major polymorphisms of folate-related genes, namely MTHFR c.677C>T, MTR c.2756A>G, MTRR c.66A>G and TYMS TSER (a 28-bp tandem repeat in the 5′ promoter enhancer region of TYMS) increase the risk of pathological changes of the thymus in AChR+ MG patients. A total of 526 AChR+ MG patients, including 132 patients with normal (involuted) thymus, 146 patients with thymic hyperplasia, and 248 patients with a thymoma were included in the study. Allele and genotype comparisons were performed among the three study groups, after correcting for multiple testing. ResultsThe frequency of the TYMS TSER 3R allele was significantly higher in MG patients with thymic hyperplasia (P=0.004), and the TYMS TSER 3R3R genotype was significantly associated with increased risk of thymic hyperplasia [OR 2.71 (95% CI: 1.34–5.47)]. ConclusionsThe 3R allele in the thymidylate synthase promoter enhancer region results in increased protein production, required for the synthesis of DNA precursors. The present study suggests that the TYMS TSER 3R allele increases the risk of thymic lymphoid hyperplasia in AChR+ MG patients.

Folate deficiency and over-supplementation causes impaired folate metabolism: Regulation and adaptation mechanisms in Caenorhabditis elegans.

Impaired folate metabolism increases the risk of birth defects, neurodegenerative and cardiovascular disease, osteoporosis and cancer. We used Caenorhabditis elegans to investigate impaired folate metabolism by RNA interference of key enzymes in the methionine synthase (MS) and thymidylate synthase (TS) cycle and by folate deficiency and over-supplementation feeding studies. Folate status is influenced by genetic variations (polymorphisms), folate deficiency and supplementation. Single RNAi of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR) and MS revealed that gene regulation is largely affected in both folate cycles. Adaptation requires a close transcriptional connection between TS and MS cycle. Coupled DHFR and MS expression is required to balance both cycles, but seems to reduce the overall rate of folate conversion. Feeding studies showed that folate over-supplementation to functioning metabolism inactivates MS and MTHFR expression and enhances TS activity, which favors DNA synthesis over methylation reactions. Folate deficiency disrupted homeostasis by favoring TS cycle and led to malformation in C. elegans offspring. Embryos show aneuploidy and are nonviable lacking DNA repair during meiotic stage of diakinesis. Single gene silencing alters gene expression in both cycles and disrupts folate homeostasis. Folate over-supplementation and deficiency favors TS over MS cycle and causes prophase DNA damage.

Maternal obesity disrupts the methionine cycle in baboon pregnancy.

Maternal intake of dietary methyl-micronutrients (e.g. folate, choline, betaine and vitamin B-12) during pregnancy is essential for normal maternal and fetal methionine metabolism, and is critical for important metabolic processes including those involved in developmental programming. Maternal obesity and nutrient excess during pregnancy influence developmental programming potentially predisposing adult offspring to a variety of chronic health problems. In the present study, we hypothesized that maternal obesity would dysregulate the maternal and fetal methionine cycle. To test this hypothesis, we developed a nulliparous baboon obesity model fed a high fat, high energy diet (HF-HED) prior to and during gestation, and examined methionine cycle biomarkers (e.g., circulating concentrations of homocysteine, methionine, choline, betaine, key amino acids, folate, and vitamin B-12). Animals were group housed allowing full physical activity and social interaction. Maternal prepregnancy percent body fat was 5% in controls and 19% in HF-HED mothers, while fetal weight was 16% lower in offspring of HF-HED mothers at term. Maternal and fetal homocysteine were higher, while maternal and fetal vitamin B-12 and betaine were lower in the HF-HED group. Elevations in circulating maternal folate were evident in the HF-HED group indicating impaired folate metabolism (methyl-trap) as a consequence of maternal vitamin B-12 depletion. Finally, fetal methionine, glycine, serine, and taurine were lower in the HF-HED fetuses. These data show that maternal obesity disturbs the methionine cycle in primate pregnancy, providing a mechanism for the epigenetic changes observed among obese pregnant women and suggesting diagnostic and therapeutic opportunities in human pregnancies complicated by obesity.

Altered folate metabolism modifies cell proliferation and progesterone secretion in human placental choriocarcinoma JEG-3 cells.

Folate is an essential B vitamin required for de novo purine and thymidylate synthesis, and for the remethylation of homocysteine to form methionine. Folate deficiency has been associated with placenta-related pregnancy complications, as have SNP in genes of the folate-dependent enzymes, methionine synthase (MTR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1). We aimed to determine the effect of altered folate metabolism on placental cell proliferation, viability and invasive capacity and on progesterone and human chorionic gonadotropin (hCG) secretion. Human placental choriocarcinoma (JEG-3) cells cultured in low folic acid (FA) (2 nM) demonstrated 13% (P<0.001) and 26% (P<0.001) lower proliferation, 5.5% (P=0.025) and 7.5% (P=0.004) lower invasion capacity, and 5 to 7.5% (P=0.004-0.025) lower viability compared with control (20 nM) or supplemented (100 nM) cells, respectively. FA concentration had no effect on progesterone or hCG secretion. Small interfering RNA (siRNA) knockdown of MTR gene and protein expression resulted in 17.7% (P<0.0001) lower proliferation and 61% (P=0.014) higher progesterone secretion, but had no effect on cell invasion and hCG secretion. siRNA knockdown of MTHFD1 gene expression in the absence of detectable changes in protein expression resulted in 10.3% (P=0.001) lower cell proliferation, but had no effect on cell invasion and progesterone or hCG secretion. Our data indicate that impaired folate metabolism can result in lower trophoblast proliferation, and could alter viability, invasion capacity and progesterone secretion, which may explain in part the observed associations between folate and placenta-related complications.

Polymorphisms in folate pathway genes are not associated with somatic nondisjunction in turner syndrome.

Folate metabolism dysfunction can lead to DNA hypomethylation and abnormal chromosomal segregation. Previous investigations of this association have produced controversial results. Here we performed a case-control study in patients with Turner syndrome (TS) to determine the effects of genetic polymorphisms of folate pathway genes as potential risk factors for somatic chromosomal nondisjunction. TS is a useful model for this investigation because patients with TS show a high frequency of chromosome mosaicism. Here we investigated the possible association of polymorphisms of the MTHFR gene with TS risk, which has been previously investigated with controversial results. We also examined the effects of MTR, RFC1, and TYMS gene polymorphisms in TS for the first time. The risk was evaluated according to allelic and genotype (independent and combined) frequencies among 70 patients with TS and 144 age-matched healthy control subjects. Polymorphism genotyping was performed by PCR, PCR-RFLP, and PCR-ASA. The polymorphisms MTHFR 677C>T and 1298A>C, MTR 2756A>G, RFC1 80G>A, and TYMS 2R/3R-alone or in combinations-were not associated with the risk of chromosomal aneuploidy in TS. In conclusion, our present findings did not support a link between impaired folate metabolism and abnormal chromosome segregation leading to somatic nondisjunction in TS patients.

Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects

Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.

Is Folate Status a Risk Factor for Asthma or Other Allergic Diseases?

PurposeIt is controversial whether folate status is a risk factor for the development of asthma or other allergic diseases. This study was conducted to investigate whether indirect or direct exposure to folate and impaired folate metabolism, reflected as methylene-tetrahydrofolate reductase (MTHFR) C677T polymorphism, would contribute to the development of asthma and other allergic diseases.MethodsElectronic databases were searched to identify all studies assessing the association between folate status and asthma or other allergic diseases. Two reviewers independently assessed the eligibility of studies and extracted data. The relative risk (RR) or odds ratio (OR) with 95% confidence intervals (CI) was calculated and pooled.ResultsTwenty-six studies (16 cohort, 7 case-control, and 3 cross-sectional studies) were identified. Maternal folic acid supplementation was not associated with the development of asthma, atopic dermatitis (AD), eczema, and sensitization in the offspring, whereas exposure during early pregnancy was related to wheeze occurrence in the offspring (RR=1.06, 95% CI=[1.02-1.09]). The TT genotype of MTHFR C677T polymorphism was at high risk of asthma (OR=1.41, 95% CI=[1.07-1.86]).ConclusionsIt is indicated that maternal folic acid supplementation during early pregnancy may increase the risk of wheeze in early childhood and that the TT genotype of MTHFR C677T polymorphism impairing folic acid metabolism would be at high risk of asthma development. These results might provide additional information for recommendations regarding forced folate consumption or folic acid supplements during pregnancy based on its well-established benefits for the prevention of congenital malformations. However, currently available evidence is of low quality which is needed to further elucidate.

Analyses of copy number variation reveal putative susceptibility loci in MTX‐induced mouse neural tube defects

Copy number variations (CNVs) are thought to act as an important genetic mechanism underlying phenotypic heterogeneity. Impaired folate metabolism can result in neural tube defects (NTDs). However, the precise nature of the relationship between low folate status and NTDs remains unclear. Using an array-comparative genomic hybridization (aCGH) assay, we investigated whether CNVs could be detected in the NTD embryonic neural tissues of methotrexate (MTX)-induced folate dysmetabolism pregnant C57BL/6 mice and confirmed the findings with quantitative real-time PCR (qPCR). The CNVs were then comprehensively investigated using bioinformatics methods to prioritize candidate genes. We measured dihydrofolate reductase (DHFR) activity and concentrations of folate and relevant metabolites in maternal serum using enzymologic method and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three high confidence CNVs on XqA1.1, XqA1.1-qA2, and XqE3 were found in the NTD embryonic neural tissues. Twelve putative genes and three microRNAs were identified as potential susceptibility candidates in MTX-induced NTDs and possible roles in NTD pathogenesis. DHFR activity and 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), and S-adenosylmethionine (SAM) concentrations of maternal serum decreased significantly after MTX injection. These findings suggest that CNVs caused by defects in folate metabolism lead to NTD, and further support the hypothesis that folate dysmetabolism is a direct cause for CNVs in MTX-induced NTDs.

Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.

LRP2 mediates folate uptake in the developing neural tube

The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2) is a multifunctional cell-surface receptor expressed in the embryonic neuroepithelium. Loss of LRP2 in the developing murine central nervous system (CNS) causes impaired closure of the rostral neural tube at embryonic stage (E) 9.0. Similar neural tube defects (NTDs) have previously been attributed to impaired folate metabolism in mice. We therefore asked whether LRP2 might be required for the delivery of folate to neuroepithelial cells during neurulation. Uptake assays in whole-embryo cultures showed that LRP2-deficient neuroepithelial cells are unable to mediate the uptake of folate bound to soluble folate receptor 1 (sFOLR1). Consequently, folate concentrations are significantly reduced in Lrp2(-/-) embryos compared with control littermates. Moreover, the folic-acid-dependent gene Alx3 is significantly downregulated in Lrp2 mutants. In conclusion, we show that LRP2 is essential for cellular folate uptake in the developing neural tube, a crucial step for proper neural tube closure.

MTHFR677TT genotype and disease risk: is there a modulating role for B-vitamins?

Methylenetetrahydrofolate reductase (MTHFR) is a critical folate-metabolising enzyme which requires riboflavin as its co-factor. A common polymorphism (677C→T) in the MTHFR gene results in reduced MTHFR activity in vivo which in turn leads to impaired folate metabolism and elevated homocysteine concentrations. Homozygosity for this polymorphism (TT genotype) is associated with an increased risk of a number of conditions including heart disease and stroke, but there is considerable variability in the extent of excess risk in various reports. The present review will explore the evidence which supports a role for this polymorphism as a risk factor for a number of adverse health outcomes, and the potential modulating roles for B-vitamins in alleviating disease risk. The evidence is convincing in the case which links this polymorphism with hypertension and hypertensive disorders of pregnancy, particularly preeclampsia. Furthermore, elevated blood pressure was found to be highly responsive to riboflavin intervention specifically in individuals with the MTHFR 677TT genotype. Future intervention studies targeted at these genetically predisposed individuals are required to further investigate this novel gene-nutrient interaction. This polymorphism has also been associated with an increased risk of neural tube defects (NTD) and other adverse pregnancy outcomes; however, the evidence in this area has been inconsistent. Preliminary evidence has suggested that there may be a much greater need for women with the MTHFR 677TT genotype to adhere to the specific recommendation of commencing folic acid prior to conception for the prevention of NTD, but this requires further investigation.

Influence of the antifolate drug Methotrexate on the development of murine neural tube defects and genomic instability

Impaired folate metabolism is considered a risk factor for neural tube defects (NTDs). However, the relationship between folate deficiency and the risk of NTDs remains unclear, because experimentally induced dietary folate deficiency is insufficient to cause NTDs in non-mutant mice. Methotrexate (MTX) is a specific folate antagonist that competitively inhibits dihydrofolate reductase (DHFR) activity. The objective of this study was to develop a folate dysmetabolism murine model, and study the development of NTDs and its mechanism. Pregnant mice were injected with different doses of MTX [0, 0.5, 1.0, 3.0, 4.5 and 6.0 mg kg(-1) body weight (b/w) intraperitoneally (i.p.)] on gestational day 7.5 and sacrificed on gestational day 11.5. DHFR activity in embryonic tissues was detected, and folate concentrations were analyzed using LC/MS/MS. Copy number variations (CNVs) in neural tube tissues were detected using array comparative genomic hybridization (aCGH). A dose of MTX 4.5 mg kg(-1) b/w, resulted in the highest incidence of NTDs (31.4%) compared with the other groups, and DHFR activities, 5-MeTHF and 5-FoTHF concentrations in embryonic tissues decreased significantly after MTX injection. Furthermore, we found three high-confidence CNVs on chromosome X using aCGH, which was confirmed by RT-PCR and MassARRAY. These results indicate that MTX could cause a folate-associated dysmetabolism, which is similar to that of dietary folate deficiency in mice. The presence of CNVs in neural tube tissues was associated with the development of NTDs.

Thrombosis and risk factors: A comment

We read with great interest the recent publication by Akar related to thrombosis and risk factors, in which he reached in conclusion that in case of need only homocysteine (Hcy) levels should be routinely analyzed and not the 5, 10-methylenetetrahydrofolate reductase (mTHFr) 677 T polymorphism [1]. The methylation of Hcy to methionine is catalyzed by the mTHFr enzyme. As far as is known, genetic deficiency of mTHFr is one cause leading to increased plasma Hcy levels [2]. But, it should not be forgotten that Hcy rises in many acquired and genetic conditions (Table) [3]. There are three main indications for determining plasma total Hcy: (a) to diagnose homocystinuria; (b) to identify individuals with or at risk of developing cobalamin or folate deficiency; (c) to assess total Hcy as a risk factor for cardiovascular and other disorders [4]. There is an involved question in this point: Is increased plasma total Hcy level related to both venous and arterial occlusive disease? And, if is it true, are polymorphism and mutation of mTHFr directly playing a role to augment the level of Hcy for cardiovascular disease (CvD)? As reviewed elsewhere, moderately increased plasma Hcy is associated with venous and arterial occlusion [5]. moreover, as it is known, the presence of mTHFr 677C→T polymorphism is a strong risk factor for increased plasma Hcy level but not for CvD [4]. In a metaanalysis including 11.162 CvD cases and 12.758 controls, with high Hcy levels in a state of low folate levels, the TT genotype was associated with a 16% increase in coronary heart disease risk [6]. Concordantly, previous studies had shown that the mTHFr 677C→T polymorphism is only associated with high Hcy levels or increased CvD risk in a setting of low folate status [6]. Hence, at higher dietary intakes of folate, the effect of the mTHFr 677C→T genotype has no adverse effect on plasma Hcy levels or on subsequent risk of CvD. The results support the hypothesis that impaired folate metabolism, resulting in high Hcy concentrations, plays a causal role in the occurrence of CvD. In view of cost effectiveness, do not investigate routinely mTHFr 677C→T polymorphism in the general or CvD population seems to be reasonable, but the other mutations of mTHFr could be still influential for high plasma Hcy levels.