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

BACKGROUNDNeural tube defects are the most common human birth defects. The causes are multifactorial with complex genetic and environmental factors, although the exact genetic causes are unknown. This research was conducted to study the frequency of Msx2 gene polymorphisms in 59 women with a history of pregnancy with a neural tube defect and in 73 healthy controls. We aimed to determine the effect of this genetic polymorphism on the incidence of neural tube defects in the Han Chinese population.

METHODSWe studied 59 mothers with at least one previous child with a neural tube defect (the case group) and 73 case-control subjects during the same period, from Shanxi Province, China. We analyzed the genotypic distributions and allele frequencies of Msx2 C386T polymorphisms in DNA samples from the case and control groups. A three-dimensional protein model was predicted using Swiss-Pdb Viewer software version 4.0. Disease association was analyzed using chi-square tests.

RESULTSSignificant differences were observed in the genotypes and allele frequencies of the Msx2 C386T allele between the case and control groups (CT: 32% vs. 15%, P = 0.0073 and TT 15% vs. 4%, P = 0.013, respectively). Logistic regression analysis showed that the C386T mutation is a potential risk factor for neural tube defects (P < 0.05; OR: 3.466; 95%CI: 1.831 - 6.560). Three-dimensional structure prediction revealed that the Msx2 C386T mutation results in a threonine substitution for methionine at position 129 of exon 2, which might lead to structural mutations or dysfunctions in the protein encoded by Msx2.

CONCLUSIONMaternal Msx2 C386T gene polymorphisms were associated with fetal neural tube defects in Han Chinese women in Shanxi Province.

Adult ; Asian Continental Ancestry Group ; genetics ; China ; Female ; Homeodomain Proteins ; chemistry ; genetics ; metabolism ; Humans ; Neural Tube Defects ; epidemiology ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Pregnancy ; Protein Structure, Secondary ; Young Adult

OBJECTIVETo explore the relationship between multi-trace elements levels in hair and human neural tube defects as well as other risk factors.

METHODSUsing 88 paired cases and controls, an 1:1 matched case control study was carried out. The study subjects were collected from the China-U. S. Collaborative Project on Neural Tube Defects Prevention and Birth Defects Surveillance System. Risk factors were obtained by field investigation with standardized questionnaires and hair trace elements levels were determined by AAS and ICP-MS methods. Microwave digestion was used to digest hair samples. The detected elements would include three groups, namely nutritional elements: Cr, Mn, Cu, Zn, Co, Mo; toxic elements: Pb, As, Cd, Hg; and Lanthanons: Y, La, Pr, Nd. Cox Proportional Hazard Regression Model was used to perform risk factors analysis.

RESULTSPregnancy fever appeared to be a risk factor of neural tube defects (OR = 6.525, P = 0.034) while hair zinc level (OR = 0.541 microg/100 g, P = 0.02) and times of prenatal physical examination (OR = 0.634, P < 0.001) served as two protective factors appeared in the last model.

CONCLUSIONZinc deficiency might serve as a risk factor for human neural tube defects, suggesting that the avoidance of pregnancy infection together with more periodical prenatal physical examination might reduce the incidence of neural tube defects.

Adult ; Analysis of Variance ; Case-Control Studies ; Diet ; Female ; Hair ; metabolism ; Humans ; Infant, Newborn ; Logistic Models ; Male ; Neural Tube Defects ; etiology ; metabolism ; Pregnancy ; Pregnancy Complications ; metabolism ; Prenatal Care ; Risk Factors ; Surveys and Questionnaires ; Trace Elements ; metabolism

PURPOSE: Recently, the role of vitamins, folate in particular, has been emphasized in the maintenance of health. Folate deficiency is known to give rise to developmental delay, pre-mature vascular disease, neural tube defects, acute leukemia, atherosclerotic vascular disease, delivery defects, breast cancers and gastrointestinal neoplasia. Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate metabolism, and influences DNA synthesis and DNA methylation. Generally, a low folate level is known to be associated with gastrointestinal neoplasms. Also, the amino- acid-changing and enzyme-activity-reducing nucleotide polymorphism (677C-->T/Ala222Val) has been described in the MTHFR polymorphism and it brings about low enzyme activity, which may reduce DNA methylation and uracil misincorporation into DNA. These processes may be critical for the oncogenic transformation of human cells. Two common single nucleotide polymorphisms (SNPs) resulting in amino-acid changes (677C T/Ala222Val and 1298A C/Glu428Ala) have been described in MTHFR. We investigated the relation between the MTHFR C677T and A1298C polymorphisms derived from colorectal cancers and from controls in the Korean population. METHODS: One hundred forty-eight (148) individuals with colorectal cancer and 288 healthy persons were analyzed. Blood sampling of each group was performed by using a PCR- RFLP analysis, and MTHFR polymorphism genotypes of 677C/C, 677C/T, 677T/T, 1298AA, 1298AC, and 1298CC were obtained. RESULTS: The genotype frequencies of MTHFR C677T polymorphisms were 25.0% (CC), 48.0% (CT), 27.0% (TT), and 75.0% (CT+TT), respectively, in case patients and 39.2% (CC), 36.8% (CT), 24.0% (TT), and 60.8% (CT+TT) in controls. The genotype frequencies of MTHFR A1298C polymorphisms were 56.1% (AA), 372% (AC), 6.8% (CC), and 43.9% (AC+CC), respectively, in case patients and 55.6% (AA), 40.3% (AC), 4.2% (CC), and 44.4% (AC+CC) in controls. The 677TT and the 677CT genotypes were associated with significantly increased risks for colorectal cancer (adjusted OR=1.77 and 95% CI=1.02~3.04 in TT; adjusted OR=2.07 and 95% CI=1.28~3.35 in CT) than was the 677CC, genotype but the the 1298CC and 1298 AC genotypes were not associated with significantly increased risks for colorectal cancer (adjusted OR=1.75 and 95% CI= 0.71~4.26 in CC; adjusted OR=0.95 and 95% CI=0.62~1.45 in AC). CONCLUSIONS: The MTHFR C677T polymorphism may be influenced by colorectal cancer, but the role of the MTHFR A1298C polymorphism needs careful interpretation and confirmation in larger studies.
Breast ; Colorectal Neoplasms* ; DNA ; DNA Methylation ; Folic Acid ; Gastrointestinal Neoplasms ; Genotype ; Humans ; Leukemia ; Metabolism ; Methylenetetrahydrofolate Reductase (NADPH2)* ; Neural Tube Defects ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Uracil ; Vascular Diseases ; Vitamins

To evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatography-mass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4 +/- 0.9 micromol/L, and 22.1 +/- 12.6 nmol/micromol creatinine) were significantly higher than that of normal pregnancies (1.0 +/- 0. 4 micromol/L, and 2.5 +/- 1.1 nmol/micromol creatinine). There was no significant difference between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.
Amniotic Fluid/*chemistry ; Biological Markers/analysis ; Biological Markers/urine ; Folic Acid/blood ; Methylmalonic Acid/analysis ; Methylmalonic Acid/*urine ; Neural Tube Defects/*diagnosis ; Neural Tube Defects/metabolism ; Pregnancy Trimester, Second ; *Prenatal Diagnosis ; Vitamin B 12/blood

To evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatography-mass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4 +/- 0.9 micromol/L, and 22.1 +/- 12.6 nmol/micromol creatinine) were significantly higher than that of normal pregnancies (1.0 +/- 0. 4 micromol/L, and 2.5 +/- 1.1 nmol/micromol creatinine). There was no significant difference between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.
Adult ; Amniotic Fluid ; chemistry ; Biomarkers ; analysis ; urine ; Female ; Folic Acid ; blood ; Humans ; Methylmalonic Acid ; analysis ; urine ; Neural Tube Defects ; diagnosis ; metabolism ; Pregnancy ; Pregnancy Trimester, Second ; Prenatal Diagnosis ; Vitamin B 12 ; blood

OBJECTIVETo describe the distribution of reduced folate carrier gene (RFC1)genotype and allele frequency between southern and northern, female and male Chinese population.

METHODRFC1 (A80G) genotype was detected, using polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR) on 720 blood spot DNA from the normal subjects.

RESULTSThe frequencies of the northern population with AA, GG and GA genotypes were 22.28%, 31.09% and 46.63%, and the frequencies of the southern population were 18.56%, 22.75% and 58.68%, respectively. Findings showed that there were significant differences between southerners and northerners in RFC1 (A80G) genotype (P < 0.01). There was no significant difference between G allele frequency of the northern (52.10%) and southern population (54.40%). The frequencies of male with RFC1 (A80G) AA, GG and GA genotype were 24.88%, 25.85% and 49.27%, and among female were 18.83%, 27.77% and 53.40%, respectively. There were no significant differences between male and female in RFC1 genotype (P > 0.05), or between G allele frequency in female (50.49%) and that in male (54.47%).

CONCLUSIONSThe distribution of RFC1 genotype seemed to be consistent with neural tube defects (NTDs) while its prevalence among the northerners was higher than that of southerners, with female having a higher NTDs prevalence. This study provided genetic epidemiological data for etiological hypothesis between RFC1 and diseases relative to folate metabolism.

Alleles ; Carrier Proteins ; genetics ; physiology ; China ; ethnology ; Female ; Folic Acid ; metabolism ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Membrane Proteins ; genetics ; Membrane Transport Proteins ; Methylenetetrahydrofolate Reductase (NADPH2) ; Mutation ; genetics ; Neural Tube Defects ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Polymorphism, Restriction Fragment Length

Folic Acid ; metabolism ; Homocysteine ; metabolism ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Neural Tube Defects ; genetics ; metabolism ; Polymorphism, Genetic

OBJECTIVE: Recent epidermiologic studies suggested that alterations in folate metabolism as a result of polymorphism in the enzyme 5,10-methylenetetrahydrofolate reductase(MTHFR) have been frequently associated with neural tube defects, vascular disease, and some cancers. A common 677C->T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of 5-methyltetrahydrofolate and increases the pool of 5,10-methylenetetrahydrofolate. A possible cause underlying altered DNA methylation could be an insufficient level of S-adenosylmethionine as a consequence of weaker alleles of MTHFR gene. Therefore, the weak MTHFR activity may underlie susceptibility to brain neoplasms. We now report the associations of MTHFR polymorphisms in three groups of adult brain tumors: gliomas, meningiomas and schwannomas. METHODS: We analyzed DNA of 71 brain tumors and 254 age- and sex-matched controls with a case-control study. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. RESULTS: The incidence of the MTHFR 677TT genotype was higher among 20 schwannoma cases compared with that of 254 controls, conferring a 5-fold increase of the risk of schwannomas(odds ratio, OR=4.75 ; 95% confidence index, CI=1.05-21.50). The homozygous mutant group had half the risk of meningioma(OR=0.42:95% CI = 0.11-1.58) compared with the homozygous normal or heterozygous genotypes. There was no significant difference in MTHFR 677TT genotype frequency between glioma group(19 cases) and control group(254 cases)(OR = 1.53 ; 95% CI = 0.30-7.73). CONCLUSION: The data indicate that the homozygous 677TT MTHFR genotype confers the significantly higher risk of schwannoma and the lower risk of meningioma. However, our study had limited a statistical power because of the small sample size, which is reflected in the wide CIs. Hence, these findings need to be confirmed in larger populations.
Adult ; Alleles ; Brain Neoplasms* ; Brain* ; Case-Control Studies ; DNA ; DNA Methylation ; Folic Acid ; Genotype ; Glioma ; Humans ; Incidence ; Meningioma ; Metabolism ; Methylenetetrahydrofolate Reductase (NADPH2) ; Neural Tube Defects ; Neurilemmoma ; Oxidoreductases* ; Risk Factors ; S-Adenosylmethionine ; Sample Size ; Vascular Diseases