Effect of epigenetic activating of Dlk1-Dio3 imprinted cluster on miR-370 expression due to folate deficiency during nerve development.

Proper Dlk1-Dio3 imprinting plays a critical role in embryogenesis, and folic acid deficiency may affect the imprinting of this locus through epigenetic regulation. However, whether and how folic acid directly impacts the imprinting status of Dlk1-Dio3 to affect neural development remain unclear. Here, we found decreased IG-DMR (intergenic -differentially methylated regions) methylation in the folate-deficient encephalocele in humans, suggesting that abnormal Dlk1-Dio3 imprinting status is related to neural tube defects (NTDs) caused by folate deficiency. Similar results were obtained with folate-deficient embryonic stem cells. By miRNA chip analysis, folic acid deficiency led to changes in multiple miRNAs, including the upregulation of 15 miRNAs located in the Dlk1-Dio3 locus. Real-time PCR confirmed that seven of these miRNAs were upregulated, especially miR-370. In contrast to normal embryonic development, in which expression of miR-370 is highest at E9.5, the abnormally high and sustained expression of miRNA-370 in folate-deficient E13.5 embryos may contribute to NTDs. In addition, we found that DNMT3A (de novo DNA methyltransferases 3A) is a direct target gene of miR-370 in neural cells, and DNMT3A participates in the role of miR-370 in inhibiting cell migration. Finally, in the folate-deficient mouse model, Dlk1-Dio3 epigenetic activation was found in fetal brain tissue, along with the upregulation of miR-370 and the downregulation of DNMT3A. Collectively, our findings demonstrate a pivotal role of folate in the epigenetic regulation of Dlk1-Dio3 imprinting during neurogenesis, revealing an elegant mechanism for the activation of Dlk1-Dio3 locus miRNAs in folic acid deficiency.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Folate deficiency disturbs PEG10 methylation modifications in human spina bifida.

BACKGROUND: Paternally expressed gene 10 (PEG10) is believed to be a key imprinted gene involved in placenta formation. However, its role in human folate-related spina bifida (SB) remains unclear. METHODS: The methylation status of the germline differentially methylated region (gDMR) in the PEG10/sarcoglycan epsilon (SGCE) imprinted cluster was compared between SB patients and control samples. Moreover, the influence of ectopic PEG10 expression on apoptosis was assessed to explore the underlying mechanisms related to folate deficiency-induced aberrant gDMR methylation in SB. RESULTS: The case group exhibited a significant increase in the methylation level of the gDMR and a marked reduction in the mRNA and protein expression of PEG10 compared with the control group. A prominent negative correlation was found between the folate level in brain tissue and gDMR methylation status (r = -0.62, P = 0.001). A cell model treated with a demethylating agent showed a significant elevation of PEG10 transcription level, as well as other imprinted genes in this cluster. In addition, the inhibition of PEG10 was found to be accompanied by aberrant activation of apoptosis in SB. CONCLUSIONS: Our findings suggest that disturbed gDMR methylation of the PEG10/SGCE cluster due to folate deficiency is involved in SB through aberrant activation of apoptosis. IMPACT: Disturbed genomic imprinting has been verified to be involved in neural tube defects (NTDs). However, little is known about the effect of ectopic expression of imprinted gene PEG10 on human NTDs. Aberrant methylation status of the germline differentially methylated region (gDMR) of PEG10/SGCE cluster due to folate deficiency has been found to result in the inhibition of PEG10 and has a marked association with an increased occurrence of spina bifida. Inhibited expression of PEG10 partly is found to be related to the abnormal activation of apoptosis in spina bifida.

DNA methylation abnormalities of imprinted genes in congenital heart disease: a pilot study.

BACKGROUND: Congenital heart disease (CHD) is resulted from the interaction of genetic aberration and environmental factors. Imprinted genes, which are regulated by epigenetic modifications, are essential for the normal embryonic development. However, the role of imprinted genes in the etiology of CHD remains unclear. METHODS: After the samples were treated with bisulfate salt, imprinted genes methylation were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. T test and One-way ANOVA were performed to evaluate the differences among groups. Odds ratios (ORs) were performed to evaluate the incidence risk of CHD in relation to methylation levels. RESULTS: We investigated the alterations of imprinted gene germline differential methylation regions (gDMRs) methylation in patients with CHD. Eighteen imprinted genes that are known to affect early embryonic development were selected and the methylation modification genes were detected by massarray in 27 CHD children and 28 healthy children. Altered gDMR methylation level of 8 imprinted genes was found, including 2 imprinted genes with hypermethylation of GRB10 and MEST and 6 genes with hypomethylation of PEG10, NAP1L5, INPP5F, PLAGL1, NESP and MEG3. Stratified analysis showed that the methylation degree of imprinted genes was different in different types of CHD. Risk analysis showed that 6 imprinted genes, except MEST and NAP1L5, within a specific methylation level range were the risk factors for CHD CONCLUSION: Altered methylation of imprinted genes is associated with CHD and varies in different types of CHD. Further experiments are warranted to identify the methylation characteristics of imprinted genes in different types of CHD and clarify the etiologies of imprinted genes in CHD.

The effect of folic acid deficiency on Mest/Peg1 in neural tube defects.

OBJECTIVE: Neural tube defects (NTDs) are one of the most common and serious birth defects in human beings caused by genetic and environmental factors. Folate insufficiency is involved in the occurrence of NTDs and folic acid supplementation can prevent NTDs occurrence, however, the underlying mechanism remains poorly understood. METHODS: We established cell and animal models of folic acid deficiency to detect the methylation modification and expression levels of genes by MassARRAY and real-time PCR, respectively. Results and conclusion: In the present study, we found firstly that in human folic acid-insufficient NTDs, the methylation level of imprinted gene Mest/Peg1 was decreased. By using a folic acid-deficient cell model, we demonstrated that Mest/Peg1 methylation was descended. Meanwhile, the mRNA level of Mest/Peg1 was up-regulated via hypomethylation modification under low folic acid conditions. Consistent with the results in cell models, Mest/Peg1 expression was elevated through hypomethylation regulation in folate-deficient animal models. Furthermore, the up-regulation of Mest/Peg1 inhibited the expression of Lrp6 gene, a crucial component of Wnt pathway. Similar results with Lrp6 down-regulation of fetal brain were verified in animal models under folic acid-deficient condition. Taken together, our findings indicated folic acid increased the expression of Mest/Peg1 via hypomethylation modification, and then inhibited Lrp6 expression, which may ultimately impact on the development of nervous system through the inactivation of Wnt pathway.

Imprinting aberrations of SNRPN, ZAC1 and INPP5F genes involved in the pathogenesis of congenital heart disease with extracardiac malformations.

Congenital heart disease (CHD) with extracardiac malformations (EM) is the most common multiple malformation, resulting from the interaction between genetic abnormalities and environmental factors. Most studies have attributed the causes of CHD with EM to chromosomal abnormalities. However, multi-system dysplasia is usually caused by both genetic mutations and epigenetic dysregulation. The epigenetic mechanisms underlying the pathogenesis of CHD with EM remain unclear. In this study, we investigated the mechanisms of imprinting alterations, including those of the Small nuclear ribonucleoprotein polypeptide N (SNRPN), PLAG1 like zinc finger 1 (ZAC1) and inositol polyphosphate-5-phosphatase F (INPP5F) genes, in the pathogenesis of CHD with EM. The methylation levels of SNRPN, ZAC1, and INPP5F genes were analysed by the MassARRAY platform in 24 children with CHD with EM and 20 healthy controls. The expression levels of these genes were detected by real-time polymerase chain reaction (PCR). The correlation between methylation regulation and gene expression was confirmed using 5-azacytidine (5-Aza) treated cells. The methylation levels of SNRPN and ZAC1 genes were significantly increased in CHD with EM, while that of INPP5F was decreased. The methylation alterations of these genes were negatively correlated with expression. Risk analysis showed that abnormal hypermethylation of SNRPN and ZAC1 resulted in 5.545 and 7.438 times higher risks of CHD with EM, respectively, and the abnormal hypomethylation of INPP5F was 8.38 times higher than that of the control group. We concluded that abnormally high methylation levels of SNRPN and ZAC1 and decreased levels of INPP5F imply an increased risk of CHD with EM by altering their gene functions. This study provides evidence of imprinted regulation in the pathogenesis of multiple malformations.

Low folate concentration impacts mismatch repair deficiency in neural tube defects.

Aim: To know the cause of sequence variants in neural tube defect (NTD). Materials & methods: We sequenced genes implicated in neural tube closure (NTC) in a Chinese cohort and elucidated the molecular mechanism-driving mutations. Results: In NTD cases, an increase in specific variants was identified, potentially deleterious rare variants harbored in H3K36me3 occupancy regions that recruits mismatch repair (MMR) machinery. Lower folate concentrations in local brain tissues were also observed. In neuroectoderm cells, folic acid insufficiency attenuated association of Msh6 to H3K36me3, and reduced bindings to NTC genes. Rare variants in human NTDs were featured by MMR deficiency and more severe microsatellite instability. Conclusion: Our work suggests a mechanistic link between folate insufficiency and MMR deficiency that correlates with an increase of rare variants in NTC genes.

Relationship between HLA-DPA1 genetic polymorphism and anembryonic pregnancy.

BACKGROUND: Human leukocyte antigen (HLA)-DP is an HLA class II molecule. Overexpression of HLA class II molecules in placental trophoblast cells may induce pregnancy loss. However, the association between HLA-DP and pregnancy loss remains unclear. HLA-DPA1 is an HLA-DP peptide chain. The objective of this study was to assess the association between HLA-DPA1 genetic polymorphism and anembryonic pregnancy, a type of early pregnancy loss, in the Chinese population. METHODS: A case-control study was designed to compare the frequencies of HLA-DPA1 gene polymorphisms in an anembryonic pregnancy group and a control group. Sixty-eight cases and 122 controls were recruited. Statistical analysis was performed to assess the correlation between single-nucleotide polymorphisms (SNPs) and anembryonic pregnancy susceptibility. MassARRAY high-throughput DNA analysis was used to analyze 19 HLA-DPA1 SNPs. To explore how HLA-DPA1 polymorphism could affect anembryonic pregnancy, HLA-DPA1 serum levels were analyzed by ELISA. RESULTS: Homozygous typing of rs1431403 (CC and TT) significantly increased the risk of anembryonic pregnancy in the case group (ORCC  = 3.13, 95% CI: 1.50-6.53; ORTT  = 2.96, 95% CI: 1.31-6.66; ORCC+TT  = 3.06, 95% CI: 1.62-5.78). In samples with high HLA-DPA1 levels (≥1,500 pg/ml), the homozygous rs1431403 genotypes (nCC  = 21, 43.8%; nTT  = 20, 57.1%) were observed more frequently than were heterozygous genotypes. CONCLUSION: HLA-DPA1 rs1431403 may be a risk factor for anembryonic pregnancy in the Chinese population. Homozygous rs1431403 genotypes (CC and TT) may increase the risk of anembryonic pregnancy by aberrantly increasing the HLA-DPA1 levels.

Low Maternal Dietary Folate Alters Retrotranspose by Methylation Regulation in Intrauterine Growth Retardation (IUGR) Fetuses in a Mouse Model.

BACKGROUND Maternal folate deficiency-mediated metabolic disruption is considered to be associated with the risk of intrauterine growth retardation (IUGR), but the exact mechanism remains unclear. The retrotransposon long interspersed nucleotide element-1 (LINE-1), which can induce birth defects via RNA intermediates, plays crucial roles during embryonic development. We investigated potential relationships between maternal folate and DNA methylation, and possible roles of LINE-1 in IUGR. MATERIAL AND METHODS The IUGR model was established by feeding female mice 1 of 3 diets - control diet (CD), folate-deficient diet for 2 weeks (FD2w), and folate-deficient diet for 4 weeks (FD4w) - prior to mating. Maternal serum folate, 5-methyltetrahydrofolate (5-MeTHF), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) concentrations and global DNA methylation were assessed by LC/MS/MS method. LINE-1 methylation levels in fetuses were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LINE-1 expression levels were validated by real-time PCR. RESULTS Maternal folate deficiency caused plasma folate and 5-MeTHF levels to decrease and SAH level to increase in the FD4w group. Compared with the CD group, methylation levels of genomic DNA and LINE-1 decreased significantly in placenta and fetal tissues from the FD4w group. Expression of LINE-1 open reading frame 1 (ORF1) protein was elevated in fetal liver tissues. Furthermore, a strong correlation was found between methylation and disrupted one-carbon metabolism, implying that dietary folate plays important roles during embryogenesis. CONCLUSIONS Maternal dietary folate deficiency impaired one-carbon metabolism, leading to global DNA and LINE-1 hypomethylation, and then increased retrotransposition in fetuses, which can lead to IUGR.

The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Folate deficiency in early development leads to disturbance in multiple processes, including neurogenesis during which fibroblast growth factor (FGF) pathway is one of the crucial pathways. Whether folic acid (FA) directly affects FGF pathways to influence neurodevelopment and the possible mechanism remains unclear. In this study, we presented evidence that in human FA-insufficient encephalocele, the FGF pathway was interfered. Furthermore, in Brachyury knockout mice devoid of such T-box transcription factors regulating embryonic neuromesodermal bipotency and a key component of FGF pathway, change in expression of Brachyury downstream targets, activator Fgf8 and suppressor dual specificity phosphatase 6 was detected, along with the reduction in expression of other key FGF pathway genes. By using a FA-deficient cell model, we further demonstrated that decrease in Brachyury expression was through alteration in hypermethylation at the Brachyury promoter region under FA deficiency conditions, and suppression of Brachyury promoted the inactivation of the FGF pathway. Correspondingly, FA supplementation partially reverses the effects seen in FA-deficient embryoid bodies. Lastly, in mice with maternal folate-deficient diets, aberrant FGF pathway activity was found in fetal brain dysplasia. Taken together, our findings highlight the effect of FA on FGF pathways during neurogenesis, and the mechanism may be due to the low expression of Brachyury gene via hypermethylation under FA-insufficient conditions.-Chang, S., Lu, X., Wang, S., Wang, Z., Huo, J., Huang, J., Shangguan, S., Li, S., Zou, J., Bao, Y., Guo, J., Wang, F., Niu, B., Zhang, T., Qiu, Z., Wu, J., Wang, L. The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Five microRNAs in serum as potential biomarkers for prostate cancer risk assessment and therapeutic intervention.

BACKGROUND: Prostate cancer (PCa) is a common malignant human tumor and one of the main causes of cancer-related deaths in men. At present, prostate-specific antigen levels are widely used to diagnose PCa in the clinic, but they are not sufficient for an accurate early diagnosis or prognosis. METHODS: To identify potential molecular markers for PCa, we used real-time PCR to measure the expression levels of various microRNAs, including miR-1825, miR-484, miR-205, miR-141, and let-7b, in the serum of 72 PCa patients and 34 healthy controls. RESULTS: miR-1825, miR-484, miR-205, miR-141, and let-7b were shown to be highly specific for PCa, suggesting that they could be used as PCa tumor screening biomarkers. miR-205 may also be used as a biomarker for indicating bone metastasis in PCa patients, miR-1825 levels may help indicate tumor-node-metastasis classification, the evaluation of treatment effects, and determining prognosis, while let-7b levels may indicate potential tumor malignancy and the hormone resistance status and could be used as a basis to adjust individual treatments for the high-risk, early diagnosis of refractory PCa. CONCLUSION: This study identified possible PCa tumor markers to more accurately predict the occurrence, progression, and prognosis of PCa, and which could be used in the development of tumor drug therapy.

Effects of MTHFR A1298C polymorphism on peripheral blood folate concentration in healthy populations: a meta-analysis of observational studies.

BACKGROUND AND OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) irreversibly converts 5,10- methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the main form of folate used in the body. Previous studies suggest that MTHFR polymorphism influences folate metabolism, but conflicting results are reported. We performed a meta-analysis to accurately characterize the association between MTHFR A1298C polymorphism and peripheral blood folate concentration in healthy populations. METHODS AND STUDY DESIGN: Studies focusing on MTHFR A1298C polymorphism and folate concentrations were identified and subjected to a metaanalysis using Review Manager 5.1. Standard mean differences (SMD) with 95% confidence intervals (95% CI) were used to assess the association between these variables. RESULTS: A total of 14 studies with 5616 healthy individuals were included in this meta-analysis. Significant differences in folate concentration were found in the MTHFR homozygote model (SMD=0.12, 95% CI=0.00-0.24, I2=17%, p=0.04) and the dominant model (SMD=0.07, 95% CI=0.01-0.14, I2=22%, p=0.02) in the general population excluding the elderly. While abnormal folate concentrations are more common in elderly, no association between MTHFR A1298C polymorphism and peripheral blood folate concentration was found in the meta-analysis when elderly were included. CONCLUSIONS: This meta-analysis indicates that, in the general population excluding the elderly, the C allele of MTHFR 1298 polymorphism is associated with the risk for an increased folate concentration.

Melatonin receptor 1B gene polymorphism rs10830963 and gestational diabetes mellitus among a Chinese population - a meta-analysis of association studies.

INTRODUCTION: Studies have been conducted to investigate the association between rs10830963 of MTNR1B and the risk of gestational diabetes mellitus (GDM), but with inconclusive results. We aimed to clarify these controversies, especially with regard to the association in the Chinese population. MATERIAL AND METHODS: A systemic literature reference search inclusive to August 12, 2016 yielded 35 articles, from which 11 studies met the inclusion criteria for the final meta-analysis, including 3889 patients with GDM and 6708 controls. RESULTS: We found statistically significant associations between rs10830963 and GDM using odds ratios (ORs) and 95% confidence intervals (CIs) [GG genotype vs. CC genotype: OR = 1.70, 95% CI: 1.38-2.10; G allele vs C allele: OR = 1.27, 95% CI: 1.20-1.36; GG+CG vs. CC (dominant model): OR = 1.31, 95% CI: 1.20-1.44; GG vs CG+CC (recessive model): OR = 1.41, 95% CI: 1.26-1.58]. In subgroup analyses stratified by ethnicity, we also observed rs10830963 to be associated with significantly increased risk of GDM in all genetic models in the Chinese population. CONCLUSIONS: Our meta-analysis indicated that the rs10830963 polymorphism might serve as a risk factor of GDM in the Chinese population.

Folate deficiency disturbs hsa-let-7 g level through methylation regulation in neural tube defects.

Folic acid deficiency during pregnancy is believed to be a high-risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid-regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa-let-7 g methylation level in NTD cases. Hypomethylation of hsa-let-7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa-let-7 g methylation directly regulates its own expression. Overexpression of hsa-let-7 g, along with its target genes, disturbed the migration and proliferation of SK-N-SH cells, implying that hsa-let-7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa-let-7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.

Altered GNAS imprinting due to folic acid deficiency contributes to poor embryo development and may lead to neural tube defects.

Disturbed epigenetic modifications have been linked to the pathogenesis of Neural Tube Defects (NTDs) in those with folate deficiency during pregnancy. However, evidence is lacking to delineate the critical region in epigenome regulated by parental folic acid and mechanisms by which folate deficiency affects normal embryogenesis. Our data from clinical samples revealed the presence of aberrant DNA methylation in GNAS imprinting cluster in NTD samples with low folate concentrations. Results from mouse models indicated that the establishment of GNAS imprinting was influenced by both maternal and paternal folate-deficient diets. Such aberrant GNAS imprinting was present prior to the gametogenesis period. Imprinting in Exon1A/GNAS gDMR was abolished in both spermatozoa and oocytes upon treating with a parental folate-deficient diet (3.6% in spermatozoa, 9.8% in oocytes). Interestingly, loss of imprinting in the GNAS gene cluster altered chromatin structure to an overwhelmingly open structure (58.48% in the folate-free medium group vs. 39.51% in the folate-normal medium group; P < 0.05), and led to a disturbed expression of genes in this region. Furthermore, an elevated cyclic AMP levels was observed in folate acid deficiency group. Our results imply that GNAS imprinting plays major roles in folic acid metabolism regulation during embryogenesis. Aberrant GNAS imprinting is an attribute to NTDs, providing a new perspective for explaining the molecular mechanisms by which folate supplementation in human pregnancy provides protection from NTDs.

Association between TCF7L2 polymorphisms and gestational diabetes mellitus: A meta-analysis.

AIMS/INTRODUCTION: Studies have been carried out to evaluate the correlation between TCF7L2 genetic polymorphisms and gestational diabetes mellitus (GDM) risk. However, the conclusions from these studies are incomplete, because partial single nucleotide polymorphisms (SNPs) were analyzed. We carried out a meta-analysis aimed to systematically evaluate TCF7L2 gene polymorphisms and GDM susceptibility in all population and racial/ethnic subgroups to afford a foundation for future research. MATERIALS AND METHODS: Published studies censoring TCF7L2 variants and GDM risk were captured from the EMBASE, PubMed, CNKI and Wanfang databases. The meta-analysis was processed using software of RevMan 5.2 and Stata13. The relationship between TCF7L2 polymorphism and GDM occurrence was evaluated by pooled odds ratios. Stratified analysis based on race/ethnicity was also carried out. The allele-specific odds ratios and 95% confidence intervals were counted, and based on homogeneity evaluated using the I2 -test, fixed- or random-effects pooled measures were selected. RESULTS: A total of 22 studies were covered, capturing eight TCF7L2 SNPs and involving 5,573 cases and 13,266 controls. Six of eight SNPs showed significant relationships with GDM occurrence, of which the SNPs rs7903146, rs12255372 and rs7901695 were the most powerful. Stratified analysis by race/ethnicity showed discrepant results in these three SNPs. In Caucasians and other races, all these SNPs were found to have a significant association with GDM risk, but in Asians, only SNP rs7903146 showed a significant association. CONCLUSIONS: Six of eight SNPs were found to have significant associations between TCF7L2 variants and GDM risk in the overall population, with the most powerful in SNPs being rs7903146, rs12255372 and rs7901695, but the contribution of these SNPs to GDM risk were variable among different racial/ethnic groups.

Determining the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and genomic DNA methylation level: A meta-analysis.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) polymorphism is a risk factor for neural tube defects. C677T and A1298C MTHFR polymorphisms produce an enzyme with reduced folate-related one carbon metabolism, and this has been associated with aberrant methylation modifications in DNA and protein. METHODS: A meta-analysis was conducted to assess the association between MTHFR C677T/A1298C genotypes and global genomic methylation. RESULTS: Eleven studies met the inclusion criteria. Of these, 10 were performed on C677T MTHFR genotypes and 6 were performed on A1298C MTHFR genotypes. Our results did not indicate any correlation between global methylation and MTHFR A1298C, C677T polymorphisms. CONCLUSION: The results of our study provide evidence to assess the global methylation modification alterations of MTHFR polymorphisms among individuals. However, our data did not found any conceivable proof supporting the hypothesis that common variant of MTHFR A1298C, C677T contributes to methylation modification. Birth Defects Research (Part A) 106:667-674, 2016. © 2016 Wiley Periodicals, Inc.

Sonic Hedgehog Signaling Affected by Promoter Hypermethylation Induces Aberrant Gli2 Expression in Spina Bifida.

GLI2 is a key mediator of the sonic hedgehog (Shh) signaling pathway and plays an important role in neural tube development during vertebrate embryogenesis; however, the role of gli2 in human folate-related neural tube defects remains unclear. In this study, we compared methylation status and polymorphisms of gli2 between spina bifida patients and a control group to explore the underlying mechanisms related to folate deficiency in spina bifida. No single nucleotide polymorphism was found to be significantly different between the two groups, although gli2 methylation levels were significantly increased in spina bifida samples, accompanied by aberrant GLI2 expression. Moreover, a prominent negative correlation was found between the folate level in brain tissue and the gli2 methylation status (r = -0.41, P = 0.014), and gli2 hypermethylation increased the risk of spina bifida with an odds ratio of 12.45 (95 % confidence interval: 2.71-57.22, P = 0.001). In addition, we established a cell model to illustrate the effect of gli2 expression and the accessibility of chromatin affected by methylation. High gli2 and gli1 mRNA expression was detected in 5-Aza-treated cells, while gli2 hypermethylation resulted in chromatin inaccessibility and a reduced association with nuclear proteins containing transcriptional factors. More meaningful to the pathway, the effect gene of the Shh pathway, gli1, was found to have a reduced level of expression along with a decreased expression of gli2 in our cell model. Aberrant high methylation resulted in the low expression of gli2 in spina bifida, which was affected by the change in chromatin status and the capacity of transcription factor binding.

DNA methylation aberrations rather than polymorphisms of FZD3 gene increase the risk of spina bifida in a high-risk region for neural tube defects.

BACKGROUND: Animal models of neural tube defects (NTDs) have indicated roles for the Fzd3 gene and the planar cell polarity signaling pathway in convergent extension. We investigated the involvement of FZD3 in genetic and epigenetic mechanisms associated with human NTDs, especially spina bifida. We explored the effects of variants spanning the FZD3 gene in NTDs and examined the role of aberrant methylation of the FZD3 promoter on gene expression in brain tissue in spina bifida. METHODS: Six FZD3 single nucleotide polymorphisms were genotyped using a MassARRAY system in tissue from 165 NTD fetuses and 152 controls. DNA methylation aberrations in the FZD3 promoter region were detected using a MassARRAY EpiTYPER (17 CpG units from -500 to -2400 bp from the transcription start site) in brain tissue from 77 spina bifida and 74 control fetuses. RESULTS: None of the six single nucleotide polymorphisms evaluated were significantly associated with spina bifida, but the mean methylation level was significantly higher in spina bifida samples (13.70%) compared with control samples (10.91%) (p = 0.001). In terms of specific sites, DNA methylation levels were significantly higher in the spina bifida samples at 14 of the 17 CpG units, which mostly included in R2 region. FZD3 mRNA expression was negatively correlated with methylation of the FZD3 promoter region, especially the R2 region (R = 0.970; p = 0.001) in HeLa cells. CONCLUSION: The results of this study suggest that DNA methylation plays an important role in FZD3 gene expression regulation and may be associated with an increased risk of spina bifida.

Impaired methylation modifications of FZD3 alter chromatin accessibility and are involved in congenital hydrocephalus pathogenesis.

Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57 ± 3.90 vs. 7.08 ± 0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression.