Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

OBJECTIVE: Circular RNAs (circRNAs) participate in several important pathological processes and have been used in the diagnosis and treatment of various diseases. This study aimed to investigate the role of circRNAs in neural tube defects (NTDs). METHOD: We characterized circRNA-associated competitive endogenous RNA (ceRNA) networks in brain tissue of low folate -induced NTDs mouse at embryonic day 13.5 by high-throughput sequencing. The expression levels of Circzfp644, miR-20-5p and Gas7 were detected by RT-PCR. Gas7 and Circzfp644 functions were determined by miRNA-mimics and inhibitors in mouse teratocarcinoma cells (F9 cells), and luciferase gene reporter assay was assessed in the F9 cells. In addition, the expression levels of Circzfp644, miR-20-5p and Gas7 were determined by Nanostring in human NTDs tissues. RESULTS: We detected 57 circRNA transcripts, 16 miRNAs, and 148 mRNAs that were significantly dysregulated in NTDs brain tissues compared with their expression levels in control (normal) tissues. Circzfp644 shared miRNA response elements with the growth arrest specific 7 ( Gas7) gene and competitively bound with miR-20-5p to increase the expression of Gas7. Downregulation of Circzfp644 and Gas7 and upregulation of miR-20-5p were found in human NTD tissue. CONCLUSION This study provides new perspectives on the role of circRNAs in nervous system development and the pathogenesis of NTDs.
Humans ; Animals ; Mice ; RNA, Circular/genetics* ; MicroRNAs/metabolism* ; Down-Regulation ; Neural Tube Defects/genetics* ; Folic Acid

Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 μg/d) has been recommended for adults in the European Union, and its increased intake (600 μg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).
Adult ; Animals ; Female ; Flour ; Folic Acid/metabolism* ; Folic Acid Deficiency/prevention & control* ; Food, Fortified ; Humans ; Infant, Newborn ; Mammals/metabolism* ; Pregnancy ; Triticum/metabolism*

Objective: This study aimed to investigate the effects of Methods: In this study, 0.1% DMG was supplemented in 20% casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system. Results: Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 μmol/L to 28.49 ± 0.50 μmol/L; Conclusion DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.
Animals ; Biomarkers/metabolism* ; Chromatography, Liquid ; Diet ; Dietary Supplements ; Folic Acid Deficiency/metabolism* ; Homocysteine/metabolism* ; Liver/metabolism* ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sarcosine/metabolism*

Hyperhomocysteinemia (Hhcy) is an independent risk factor for Alzheimer's disease (AD). Visual dysfunction is commonly found and is positively correlated with the severity of cognitive defects in AD patients. Our previous study demonstrated that Hhcy induces memory deficits with AD-like tau and amyloid-β (Aβ) pathologies in the hippocampus, and supplementation with folate and vitamin B12 (FB) prevents the Hhcy-induced AD-like pathologies in the hippocampus. Here, we investigated whether Hhcy also induces AD-like pathologies in the retina and the effects of FB. An Hhcy rat model was produced by vena caudalis injection of homocysteine for 14 days, and the effects of FB were assessed by simultaneous supplementation with FB in drinking water. We found that Hhcy induced vessel damage with Aβ and tau pathologies in the retina, while simultaneous supplementation with FB remarkably attenuated the Hhcy-induced tau hyperphosphorylation at multiple AD-related sites and Aβ accumulation in the retina. The mechanisms involved downregulation of amyloid precursor protein (APP), presenilin-1, beta-site APP-cleaving enzyme 1, and protein phosphatase-2A. Our data suggest that the retina may serve as a window for evaluating the effects of FB on hyperhomocysteinemia-induced Alzheimer-like pathologies.
Alzheimer Disease ; etiology ; metabolism ; pathology ; therapy ; Amyloid beta-Peptides ; metabolism ; Animals ; Dietary Supplements ; Disease Models, Animal ; Folic Acid ; therapeutic use ; Homocysteine ; Hyperhomocysteinemia ; complications ; metabolism ; pathology ; therapy ; Male ; Rats, Sprague-Dawley ; Retina ; metabolism ; pathology ; Retinal Vessels ; metabolism ; pathology ; Vitamin B 12 ; therapeutic use ; tau Proteins ; metabolism

OBJECTIVE: Autism spectrum disorder (ASD) is a complex neurodevelopmental syndrome with an increasingly prevalent etiology, yet not fully understood. It has been thought that vitamin D, complex B vitamin levels and homocysteine are associated with environmental factors and are important in ASD. The aim of this study was to examine serum vitamin D, vitamin D receptor (VDR), homocysteine, vitamin B6, vitamin B12 and folate levels in ASD. METHODS: In this study, serum vitamin D and VDR, homocysteine, vitamins B6, B12 and folate levels were determined in 60 patients with ASD (aged 3 to 12 years) and in 45 age-gender matched healthy controls. In addition, calcium, phosphorus and alkaline phosphatase, which are associated with vitamin D metabolism, were measured from serum in both groups. ASD severity was evaluted by the Childhood Autism Rating Scale (CARS). RESULTS: Serum vitamin D and VDR were substantially reduced in patients with ASD in comparision to control group. However, homocysteine level was significantly higher and vitamin B6, vitamin B12 and folate were also reduced in patients with ASD. Total CARS score showed a positive association with homocysteine and a negative correlation with vitamins D, B6, B12, folate and VDR. CONCLUSION: This comprehensive study, which examines many parameters has shown that low serum levels of vitamins D, B6, B12, folate and VDR as well as high homocysteine are important in the etiopathogenesis of ASD. However, further studies are required to define the precise mechanism(s) of these parameters and their contributions to the etiology and treatment of ASD.
Alkaline Phosphatase ; Autism Spectrum Disorder* ; Autistic Disorder* ; Calcium ; Child* ; Folic Acid ; Homocysteine ; Humans ; Metabolism ; Phosphorus ; Receptors, Calcitriol* ; Vitamin B 12 ; Vitamin B 6 ; Vitamin D* ; Vitamins*

OBJECTIVE: To identify the associations between polymorphisms of the 3′-untranslated region (UTR) of methylenetetrahydrofolate reductase (MTHFR) gene, which codes for an important regulatory enzyme primarily involved in folate metabolism, and idiopathic recurrent pregnancy loss (RPL) in Korean women. METHODS: The study population comprised 369 RPL patients and 228 controls. MTHFR 2572C>A, 4869C>G, 5488C>T, and 6685T>C 3′-UTR polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis or by TaqMan allelic discrimination assays. Natural killer cell proportions were determined by flow cytometry. RESULTS: The MTHFR 2572-5488-6685 (A-C-T) haplotype had an adjusted odds ratio of 0.420 (95% confidence interval, 0.178–0.994; p=0.048) for RPL. Analysis of variance revealed that MTHFR 4869C>G was associated with altered CD56⁺ natural killer cell percentages (CC, 17.91%±8.04%; CG, 12.67%±4.64%; p=0.024) and folate levels (CC, 12.01±7.18 mg/mL; CG, 22.15±26.25 mg/mL; p=0.006). CONCLUSION: Variants in the 3′-UTR of MTHFR are potential biomarkers for RPL. However, these results should be validated in additional studies of ethnically diverse groups of patients.
Biomarkers ; Discrimination (Psychology) ; Female ; Flow Cytometry ; Folic Acid ; Haplotypes ; Humans ; Killer Cells, Natural* ; Metabolism ; Methylenetetrahydrofolate Reductase (NADPH2) ; Odds Ratio ; Pregnancy*

OBJECTIVETo study the effects of maternal folate deficiency on fetal growth and development and the methylation profiles of insulin-like growth factor system in the offspring rats.

METHODSTwenty-two Sprague-Dawley female rats were randomly assigned to two groups: a folate deficient group (n=12) and a control group (n=10). They were fed with folate deficient and normal diet respectively. Dams were mated after 2 weeks of feeding. Eight female rats from each group were pregnant. On the 20th day of gestation, the fetuses were delivered by caesarean section. Thirty-two fetal rats from each group were randomly selected and the body length and weight were measured. Eight fetal rats from each group were randomly selected and ELISA was used to measure the level of folate content, IGF-1 and IGFBP-3 in the fetal brain and liver. Three fetal rats from each group were randomly selected and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was used to detect the methylation level of insulin-like growth factor system in the fetal brain and liver. ELISA was used to measure the level of IGF-1 and IGFBP-3 in the maternal serum from both groups.

RESULTSThe mean fetal length and weight were lower in the folate deficient group than in the control group (P<0.05). The levels of IGF-1 and IGFBP-3 in the maternal serum, as well as folate content and IGFBP-3 in the fetal brain and liver were significantly lower in the folate deficient group than in the control group (P<0.05). The methylation levels of IGF-1R, IGF-2R, IGFBP-2, IGFBP-5, IGFBP-6 and IGFBP-7 in the fetal brain were higher in the folate deficient group than in the control group (P<0.05). The methylation levels of IGF-1R, IGF-2R, IGFBP-3 and IGFBP-5 in the fetal liver were higher in the folate deficient group than in the control group. The methylation of IGF-2 gene showed a significant reduction in the folate deficient group (P<0.05).

CONCLUSIONSMaternal folate deficiency may cause retardation of growth and development of the offspring, which is possibly associated with the changes of methylation profiles of insulin-like growth factors.

Animals ; Brain ; metabolism ; DNA Methylation ; Female ; Fetal Development ; Fetus ; metabolism ; Folic Acid Deficiency ; metabolism ; Insulin-Like Growth Factor Binding Protein 3 ; blood ; Insulin-Like Growth Factor I ; analysis ; Liver ; metabolism ; Rats ; Rats, Sprague-Dawley

BACKGROUND: Methotrexate (MTX), one of the main drugs used to treat osteosarcoma, is a representative folic acid antagonist. Polymorphisms of various enzymes involved in the metabolism of MTX could contribute to differences in response to MTX in pediatric osteosarcoma patients. METHODS: Blood and tissue samples were obtained from 37 pediatric osteosarcoma patients who were treated with high-dose MTX therapy. The following 4 single nucleotide polymorphisms (SNPs) were analyzed: ATIC 347C>G, MTHFR 677C>T, MTHFR 1298A>C and SLC19A1 80G>A. Serial plasma MTX concentrations after high-dose MTX therapy and MTX-induced toxicities were evaluated. Correlations among polymorphisms, MTX concentrations and treatment-induced toxicities were assessed. RESULTS: Plasma MTX levels at 48 hours after high-dose MTX infusion were significantly associated with SLC19A1 80G>A (P=0.031). Higher plasma levels of MTX at 48 and 72 hours were significantly associated with MTX-induced mucositis (P=0.007 and P=0.046) and renal toxicity (P=0.002), respectively. SNP of SLC19A1 gene was associated with development of severe mucositis (P=0.026). CONCLUSION: This study suggests that plasma levels of MTX are associated with GI and renal toxicities after high-dose MTX therapy, and genetic polymorphisms that affect the metabolism of MTX may influence drug concentrations and development of significant side effects in pediatric patients treated with high-dose MTX.
Folic Acid* ; Humans ; Metabolism ; Methotrexate* ; Mucositis ; Osteosarcoma* ; Plasma ; Polymorphism, Genetic* ; Polymorphism, Single Nucleotide

The folate metabolic pathway plays important roles in cellular physiology by participating in nucleotide synthesis, DNA repair and methylation, and maintenance and stability of the genome. Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme involved in folate metabolism. Polymorphisms of MTHFR may change the level of homocysteine and affect DNA synthesis and methylation, leading to an increased oxidative stress and disturbed methylation reactions and consequently affecting reproductive function. This article presents an overview on MTHFR gene polymorphisms, proposing that multicentered, large-sample and long-term prospective studies are needed to reveal the relationship between MTHFR gene polymorphisms and infertility.
DNA ; biosynthesis ; DNA Methylation ; DNA Repair ; Folic Acid ; metabolism ; Homocysteine ; metabolism ; Humans ; Infertility ; enzymology ; genetics ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Polymorphism, Genetic ; Prospective Studies