Lihua Wu

Relation between hypomethylation of long interspersed nucleotide elements and risk of neural tube defects

BACKGROUND Impaired one-carbon metabolism is thought to be associated with the risk of neural tube defects (NTDs); however, the role of methylation in NTDs remains unclear. Long interspersed nucleotide element-1 (LINE-1) constitutes 17-25% of the human genome. LINE-1 hypomethylation correlates with global DNA methylation levels in cancerous cells, but limited information is available on LINE-1 methylation in NTDs. OBJECTIVE We determined whether LINE-1 methylation patterns were associated with neural tube development and the possible relations between DNA methylation and key maternal metabolites involved in folate-mediated one-carbon metabolism. DESIGN Global methylation, maternal plasma folic acid, vitamin B-12, and total homocysteine (tHcy) concentrations were assessed in 48 NTD and 49 control samples by immunoassay, and LINE-1 methylation levels were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS Methylation levels of genomic DNA and LINE-1 decreased significantly in the neural tissue of NTD samples. The risk of NTDs increased with decreasing levels of LINE-1 methylation, with an odds ratio of 5.246 (95% CI: 1.519, 18.124; P = 0.009) for the lowest quartile (methylation level < or = 57.94%) compared with the highest quartile (methylation level > or = 60.94%). Compared with control subjects, case subjects had lower maternal plasma concentrations of vitamin B-12. CONCLUSIONS Hypomethylation of LINE-1 and genomic DNA was associated with an increased risk of NTDs. Functional insufficiency of maternal plasma vitamin B-12 was associated with NTDs, although no significant correlation could be established between maternal folic acid, vitamin B, tHcy, and LINE-1 methylation.

Tissue-specific distribution of aberrant DNA methylation associated with maternal low-folate status in human neural tube defects

This study compares the density and tissue-specific distribution of 5-methyl cytosine (5mC) in genomic DNA from human fetuses with or without neural tube defects (NTD) and examines whether low maternal serum folate is a possible correlate and/or risk factor for NTD. The results demonstrate significant hypomethylation of brain genomic DNA in NTD fetuses relative to controls (P<.01), as well as relative hypermethylation of skin and heart in NTD fetuses. In normal fetuses, the level of 5mC in liver genomic DNA decreased from fetal week 18 to 28 and increased over the same developmental period in kidney genomic DNA, but these trends were absent in genomic DNA from NTD fetuses. Mean maternal serum folate was significantly lower in NTD fetuses than in controls (P<.01), and maternal serum folate correlated with density of 5mC in genomic brain DNA from NTD fetuses (r=0.610). The results indicate that aberrant DNA methylation in NTD may be due to maternal folate deficiency and may be involved in the pathogenesis of NTD in humans.

Global DNA hypomethylation is associated with NTD‐affected pregnancy: A case‐control study

BACKGROUND Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD-affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS A hospital-based case-control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS Sixty-five NTD-affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736-fold increased risk for NTDs (95% confidence interval, 1.731-19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS Global DNA hypomethylation in fetal brain tissue was associated with NTD-affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. (c) 2010 Wiley-Liss, Inc.

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.

Glutamate carboxypeptidase II gene polymorphisms and neural tube defects in a high-risk Chinese population

Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate into N-acetylaspartate and glutamate in the brain. Animal experiments suggested that GCPII plays an essential role in early embryonic development. Previous studies provided conflicting results on the effect of the GCPII rs61886492 C>T (or 1561C>T) polymorphism on NTDs. In the Lvliang area of Shanxi province, where the incidence of NTDs is the highest in China, a case–control study was conducted to investigate possible association between the GCPII rs61886492 and rs202676 polymorphisms and NTD risk. Results indicated all the case and control samples displayed the rs61886492 GG genotype. Although no significant differences in rs202676 genotype or allele frequencies were found between the NTD and control groups, the combined AG+GG genotype group was significantly associated with anencephaly (p = 0.03, OR = 2.11, 95% CI, 1.11–4.01), but not with spina bifida or encephalocele. Overall, the rs202676 A>G polymorphism is a potential risk factor for anencephaly. The results of this study suggest that phenotypic heterogeneity may exist among NTDs in this Chinese population.

Altered Methylation of the DNA Repair Gene MGMT Is Associated with Neural Tube Defects

DNA repair is critical for proper embryogenesis, and altered expression of DNA repair genes has been associated with neural tube defects (NTDs). The expression of DNA repair enzymes may be controlled, in part, by methylation of the promoter region. To assess whether disturbed promoter methylation pattern increases the incidence of NTDs, we employed methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify the methylation levels of multiple promoter CpG sites in normal human embryos and embryos with NTDs. Of the total seven DNA repair genes, two genes (MGMT, MSH6) were examined as having disturbed methylation levels by MS-MLPA kit, while only one gene was confirmed with a significant alternated methylation pattern by MALDI-TOF MS. In our research, methylation profiling of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) showed gender difference in embryogenesis. Comparison of MGMT promoter methylation revealed that hypomethylation was associated with an increased risk for cephalic malformations, especially with female embryos (Adjusted Odds Ratio = 8.250). The majority of individual CpG units within the promoter demonstrated hypomethylation. Meanwhile, the expression of MGMT was proven to increase significant in female cases. These results underscore the role of stable promoter methylation in the DNA repair enzymes MGMT for proper embryogenesis.

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.

Hypomethylation of fetal brain genomic DNA in neural tube defects determined by a new liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) method

Neural tube defects (NTDs) are associated with significant infant morbidity and mortality. Detection of alterations in DNA methylation might provide evidence for an epigenetic mechanism in some NTDs cases. We developed a sensitive LC–MS/MS method for the quantification of global DNA methylation in low quantity samples of embryonic tissue. The limit of detection was 0.5 × 10−15 mol for 5-methyl-2′-deoxycytidine (5mdC) and the assay was linear from 1 × 10−14 mol to 1 × 10−11 mol for 5mdC with DNA input between 1 ng and 1000 ng. The calibration curve ranged from 1% to 10% with a correlation coefficient (r2) of 0.998 for methylation levels ranging from 2% to 7% of the mammalian genome. The intra-day precision was 2.19% to 7.73% and the inter-day precision was 0.87% to 4.33%, with an overall accuracy between 97.30% and 107.66%. Following method validation, we applied this method for the first time to assess global DNA methylation in fetal tissue from confirmed anencephalus and spina bifida cases. Brain tissue from these NTDs cases showed DNA hypomethylation compared to control samples, while no differences were found in skin tissue. Results from statistical analysis demonstrated that the global DNA methylation level was independent of gender. The methylation analysis suggested that hypomethylation was associated with NTDs, implicating a possible epigenetic mechanism in the pathogenesis of anencephalus and spina bifida.

Association between TCF7L2 Polymorphisms and Gestational Diabetes Mellitus: a Meta-Analysis.

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.

Polymorphisms in MTHFD1 Gene and Susceptibility to Neural Tube Defects: A Case-Control Study in a Chinese Han Population with Relatively Low Folate Levels

Background The polymorphism of methylenetetrahydrofolate dehydrogenase (MTHFD1) has been reported as a risk factor for neural tube defects (NTDs). In the present study, we aimed to investigate whether the single-nucleotide polymorphisms (SNPs) of MTHFD1 gene are associated with NTDs in a Chinese population and to determine their mechanism of action. Material/Methods MTHFD1 gene was scanned in a total of 270 NTDs cases and 192 healthy controls by using next-generation sequencing (NGS) method. After quality control procedures, 208 selected SNP sites in MTHFD1 gene were enrolled for follow-up statistical association analyses. Functional analyses were also performed for significant SNPs through bioinformatics analysis. Folic acid levels of brain tissue in available NTDs cases and healthy controls (113 and 123, respectively) were measured. Statistical and bioinformatics analyses were performed to investigate the relationship between SNPs in MTHFD1 and susceptibility to NTDs. Results Statistical analysis showed that 2 independent SNPs, rs1956545 and rs56811449, confer the risk of NTDs (P value=0.0195, OR (odds ratio)=1.41, 95% CI (confidence interval)=1.06–1.88; P value=0.0107, OR=0.56, 95% CI=0.36–0.87). The haplotype GGGG, which consists of 4 SNPs (rs2236225, rs2236224, rs1256146, and rs6573559), is also associated with risk of NTDs (P value=0.0438, OR=0.7180, 95% CI=0.5214–0.9888). The risk allele C of rs1956545 is also associated with decreased folic acid levels in the brain (P value=0.0222, standard beta=−0.2238, 95% CI=−0.4128 – −0.0349) according to analysis in the subset of NTDs cases and healthy controls. Bioinformatics analysis indicates that rs1956545 and rs56811449 are within ENCODE regulatory regions, the open chromatin regions of blastula Trophoblast cell line, and histone-marked region of brain astrocyte cell line. Conclusions The polymorphism of SNP loci rs1956545 and rs56811449 as well as a haplotype in MTHFD1 gene could serve as an indicator for the occurrence of NTDs in Chinese population and some specific genotypes of the loci may have lower risk of developing NTDs.