Bo Niu

Genomic DNA Hypomethylation Is Associated with Neural Tube Defects Induced by Methotrexate Inhibition of Folate Metabolism

DNA methylation is thought to be involved in the etiology of neural tube defects (NTDs). However, the exact mechanism between DNA methylation and NTDs remains unclear. Herein, we investigated the change of methylation in mouse model of NTDs associated with folate dysmetabolism by use of ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS), liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS), microarray, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Real time quantitative PCR. Results showed that NTD neural tube tissues had lower concentrations of 5-methyltetrahydrofolate (5-MeTHF, P = 0.005), 5-formyltetrahydrofolate (5-FoTHF, P = 0.040), S-adenosylmethionine (SAM, P = 0.004) and higher concentrations of folic acid (P = 0.041), homocysteine (Hcy, P = 0.006) and S-adenosylhomocysteine (SAH, P = 0.045) compared to control. Methylation levels of genomic DNA decreased significantly in the embryonic neural tube tissue of NTD samples. 132 differentially methylated regions (35 low methylated regions and 97 high methylated regions) were selected by microarray. Two genes (Siah1b, Prkx) in Wnt signal pathway demonstrated lower methylated regions (peak) and higher expression in NTDs (P<0.05; P<0.05). Results suggest that DNA hypomethylation was one of the possible epigenetic variations correlated with the occurrence of NTDs induced by folate dysmetabolism and that Siah1b, Prkx in Wnt pathway may be candidate genes for NTDs.

Detection of Copy Number Variants Reveals Association of Cilia Genes with Neural Tube Defects

Background Neural tube defects (NTDs) are one of the most common birth defects caused by a combination of genetic and environmental factors. Currently, little is known about the genetic basis of NTDs although up to 70% of human NTDs were reported to be attributed to genetic factors. Here we performed genome-wide copy number variants (CNVs) detection in a cohort of Chinese NTD patients in order to exam the potential role of CNVs in the pathogenesis of NTDs. Methods The genomic DNA from eighty-five NTD cases and seventy-five matched normal controls were subjected for whole genome CNVs analysis. Non-DGV (the Database of Genomic Variants) CNVs from each group were further analyzed for their associations with NTDs. Gene content in non-DGV CNVs as well as participating pathways were examined. Results Fifty-five and twenty-six non-DGV CNVs were detected in cases and controls respectively. Among them, forty and nineteen CNVs involve genes (genic CNV). Significantly more non-DGV CNVs and non-DGV genic CNVs were detected in NTD patients than in control (41.2% vs. 25.3%, p<0.05 and 37.6% vs. 20%, p<0.05). Non-DGV genic CNVs are associated with a 2.65-fold increased risk for NTDs (95% CI: 1.24–5.87). Interestingly, there are 41 cilia genes involved in non-DGV CNVs from NTD patients which is significantly enriched in cases compared with that in controls (24.7% vs. 9.3%, p<0.05), corresponding with a 3.19-fold increased risk for NTDs (95% CI: 1.27–8.01). Pathway analyses further suggested that two ciliogenesis pathways, tight junction and protein kinase A signaling, are top canonical pathways implicated in NTD-specific CNVs, and these two novel pathways interact with known NTD pathways. Conclusions Evidence from the genome-wide CNV study suggests that genic CNVs, particularly ciliogenic CNVs are associated with NTDs and two ciliogenesis pathways, tight junction and protein kinase A signaling, are potential pathways involved in NTD pathogenesis.

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.

Association Between a 45-bp 3′Untranslated Insertion/Deletion Polymorphism in Exon 8 of UCP2 Gene and Neural Tube Defects in a High-Risk Area of China

Uncoupling protein 2(UCP2) is an attractive candidate gene for screening neural tube defects (NTDs) risk. In this study, polymerase chain reaction and agarose gel electrophoresis were used to determine the distribution of the polymorphism in a case group of 140 deliveries with NTDs, and a control group of 251 normal newborns. We found that the frequencies of allele I and genotypes ID + II were higher in the case group than in the control group (P = .167, OR = 1.4, 95% CI, 0.9-2.1; P = .132, OR = 1.44, 95% CI, 0.89-2.33, respectively); and at low maternal educational level, the frequency of ID + II genotypes was significantly higher in the NTD case group (P < .05, OR = 1.7, 95% CI, 1.01-2.79). The result suggested that the polymorphism in UCP2 may be a potential genetic risk factor for NTDs in a high-risk area of China, and the association was influenced by maternal education.

Rare Deleterious PARD3 Variants in the aPKC-Binding Region are Implicated in the Pathogenesis of Human Cranial Neural Tube Defects Via Disrupting Apical Tight Junction Formation

Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical–basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD‐specific variants were significantly enriched in the aPKC‐binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over‐expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC‐binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.

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.

The maternal ITPK1 gene polymorphism is associated with neural tube defects in a high-risk Chinese population.

Background Epidemiological surveys and animal studies have revealed that inositol metabolism is associated with NTDs, but the mechanisms are not clear. Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a pivotal regulatory enzyme in inositol metabolic pathway. The objective was to assess the potential impact of the maternal ITPK1 genotypes on the inositol parameter and on the NTD risk in a NTD high-risk area in China. Methodology/Results A case-control study of pregnant women affected with NTDs (n = 200) and controls (n = 320) was carried out. 13 tag SNPs of ITPK1 were selected and genotyped by the Sequenom MassArray system. We found that 4 tag SNPs were statistically significant in spina bifida group (P<0.05). MACH was used to impute the un-genotyped SNPs in ITPK1 locus and showed that 3 meaningful SNPs in the non-coding regions were significant. We also predicted the binding capacity of transcription factors in the positive SNPs using the bioinformatics method and found that only rs3783903 was located in the conserved sequence of activator protein-1 (AP-1). To further study the association between biochemical values and genotypes, maternal plasma inositol hexakisphosphate (IP6) levels were also assessed using LC-MS. The maternal plasma IP6 concentrations in the spina bifida subgroup were 7.1% lower than control (136.67 vs. 147.05 ng mL−1, P<0.05), and significantly lower in rs3783903 GG genotype than others (P<0.05). EMSA showed a different allelic binding capacity of AP-1 in rs3783903, which was affected by an A→G exchange. The RT-PCR suggested the ITPK1 expression was decreased significantly in mutant-type of rs3783903 compared with wild-type in the 60 healthy pregnancies (P<0.05). Conclusions/Significance These results suggested that the maternal rs3783903 of ITPK1 might be associated with spina bifida, and the allele G of rs3783903 might affect the binding of AP-1 and the decrease of maternal plasma IP6 concentration in this Chinese population.

Maternal PCMT1 gene polymorphisms and the risk of neural tube defects in a Chinese population of Lvliang high-risk area

Protein-L-isoaspartate (D-aspartate) O-methyltransferase 1 (PCMT1) gene encodes for the protein repair enzyme L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), which is known to protect certain neural cells from Bax-induced apoptosis. Previous study has shown that PCMT1 polymorphisms rs4552 and rs4816 of infant are associated with spina bifida in the Californian population. The association between maternal polymorphism and neural tube defects is still uncovered. A case-control study was conducted to investigate a possible association between maternal PCMT1 and NTDs in Lvliang high-risk area of Shanxi Province in China, using a high-resolution DNA melting analysis genotyping method. We found that increased risk for anencephaly in isolated NTDs compared with the normal control group was observed for the G (vs. A) allele (p=0.034, OR=1.896, 95% CI, 1.04-3.45) and genotypes GG+GA (p=0.025, OR=2.237, 95% CI, 1.09-4.57). Although the significance was lost after multiple comparison correction, the results implied that maternal polymorphisms in PCMT1 might be a potential genetic risk factor for isolated anencephaly in this Chinese population.

dNTP deficiency induced by HU via inhibiting ribonucleotide reductase affects neural tube development.

Exposure to environmental toxic chemicals in utero during the neural tube development period can cause developmental disorders. To evaluate the disruption of neural tube development programming, the murine neural tube defects (NTDs) model was induced by interrupting folate metabolism using methotrexate in our previous study. The present study aimed to examine the effects of dNTP deficiency induced by hydroxyurea (HU), a specific ribonucleotide reductase (RNR) inhibitor, during murine neural tube development. Pregnant C57BL/6J mice were intraperitoneally injected with various doses of HU on gestation day (GD) 7.5, and the embryos were checked on GD 11.5. RNR activity and deoxynucleoside triphosphate (dNTP) levels were measured in the optimal dose. Additionally, DNA damage was examined by comet analysis and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. Cellular behaviors in NTDs embryos were evaluated with phosphorylation of histone H3 (PH-3) and caspase-3 using immunohistochemistry and western blot analysis. The results showed that NTDs were observed mostly with HU treatment at an optimal dose of 225 mg/kg b/w. RNR activity was inhibited and dNTP levels were decreased in HU-treated embryos with NTDs. Additionally, increased DNA damage, decreased proliferation, and increased caspase-3 were significant in NTDs embryos compared to the controls. Results indicated that HU induced murine NTDs model by disturbing dNTP metabolism and further led to the abnormal cell balance between proliferation and apoptosis.

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.