Jianxin Wu

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.

Maternal serum vitamin B12, folate and homocysteine and the risk of neural tube defects in the offspring in a high-risk area of China.

OBJECTIVE To examine the association between the risk of neural tube defects (NTD) and maternal serum vitamin B12, folate and homocysteine in a high-risk area of China. DESIGN A case-control study was carried out in Luliang mountain area of Shanxi Province. SUBJECTS/SETTING A total of eighty-four NTD pregnancies and 110 matched controls were included in the study; their serum vitamin B12 and folate concentrations were measured by chemiluminescent immunoenzyme assay and total homocysteine concentrations by fluorescent polarisation immunoassay. RESULTS Serum vitamin B12 and folate concentrations were lower in NTD-affected pregnant women than in controls (P < 0.01). Serum total homocysteine was higher in the NTD group than in controls at less than 21 weeks of gestation (P < 0.01). Adjusted odds ratios revealed that women with lower vitamin B12 (adjusted OR=4.96; 95 % CI 1.94, 12.67) and folate (adjusted OR=3.23; 95 % CI 1.33, 7.85) concentrations had a higher risk of NTD compared to controls. Based on dietary analysis, less consumption of meat, egg or milk, fresh vegetables and fruit intake would increase the risk of NTD. CONCLUSIONS Lower serum concentrations of folate and vitamin B12 are related to the increased risk of NTD in high-risk populations. Both folate and vitamin B12 intake insufficiency could contribute to the increased risk of NTD. A dietary supplement, combining folate and vitamin B12, might be an effective measure to decrease the NTD incidence in these areas.

Neural tube defects and disturbed maternal folate- and homocysteine-mediated one-carbon metabolism

Disturbances in maternal folate and homocysteine metabolism are associated with neural tube defects (NTDs). However, the role played by specific components in the one-carbon metabolic pathways leading to NTDs remains unclear. Here, we conducted a case-control study to investigate the relationship between the disturbed one-carbon metabolism and the risk of NTD-affected pregnancies. Major components were examined in population-based samples of women who had NTD-affected pregnancies (case subjects, n=46) or unaffected by any birth defects (control subjects, n=44). We used the newly developed high-performance liquid chromatography tandem mass spectrometry along with a routine chemiluminescent assay, to measure serum concentrations of 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), folic acid, serine, histidine, homocysteine, cystathionine, methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), total folate and vitamin B12 in both groups adjusting for lifestyle and sociodemographic variables. We found significantly lower serum concentrations of 5-MeTHF (P<0.001), 5-FoTHF (P=0.004), total folate (P<0.001) and vitamin B12 (P=0.005) and remarkably higher concentrations of SAH (P=0.016) in cases than in controls. Therefore, these compounds could be identified as potential risk factors for NTDs early diagnosis. Further analysis of relevant genetic and epidemiologic investigations may provide more insights into the pathogenesis of NTDs and enhance current nutritional intervention strategies to reduce the risk of NTD-affected pregnancies.

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.

MicroRNAs: potential regulators involved in human anencephaly.

MicroRNAs (miRNAs) are posttranscriptional regulators of messenger RNA activity. Neural tube defects (NTDs) are severe congenital anomalies that substantially impact an infants morbidity and mortality. The miRNAs are known to be dynamically regulated during neurodevelopment; their role in human NTDs, however, is still unknown. In this study, we show the presence of a specific miRNA expression profile from tissues of fetuses with anencephaly, one of the most severe forms of NTDs. Furthermore, we map the target genes of these miRNAs in the human genome. In comparison to healthy human fetal brain tissues, tissues from fetuses with anencephaly exhibited 97 down-regulated and 116 up-regulated miRNAs. The microarray findings were extended using real-time qRT-PCR for nine miRNAs. Specifically, of these validated miRNAs, miR-126, miR-198, and miR-451 were up-regulated, while miR-9, miR-212, miR-124, miR-138, and miR-103/107 were down-regulated in the tissues of fetuses with anencephaly. A bioinformatic analysis showed 881 potential target genes that are regulated by the validated miRNAs. Seventy-nine of these potential genes are involved in a protein interaction network. There were 6 co-occurrence annotations within the GOSlim process and 7 co-occurrence annotations within the GOSlim function found by GeneCodis 2.0. Our results suggest that miRNA dysregulation is possibly involved in the pathogenesis of anencephaly.

Association between MTR A2756G and MTRR A66G polymorphisms and maternal risk for neural tube defects: A meta-analysis

BACKGROUND Methionine synthase (MTR) and methionine synthase reductase (MTRR) genes have been considered to be implicated in the development of neural tube defects (NTDs). However, the results are inconsistent. Accordingly, we conducted a meta-analysis to further investigate such an association. METHODS Published literature from PubMed and Embase databases was retrieved. All studies evaluating the association between MTR A2756G or MTRR A66G polymorphism and maternal risk for NTDs were included. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using the fixed- or random-effects model. RESULTS A total of 11 studies (1005 cases and 2098 controls) on MTR A2756G polymorphism and 10 studies (1211 cases and 2003 controls) on MTRR A66G polymorphism were included. Overall, this meta-analysis revealed no significant association between maternal MTR A2756G polymorphism and NTD susceptibility in either genetic model. A significant association between MTRR A66G polymorphism and maternal risk for NTDs was observed for GG vs. AA (OR=1.31, 95% CI 1.03-1.67) among Caucasians. CONCLUSION The present meta-analysis indicated that MTRR A66G polymorphism, but not MTR A2756G, is significantly associated with maternal risk for NTDs in Caucasians.

Simultaneous quantification of 11 pivotal metabolites in neural tube defects by HPLC–electrospray tandem mass spectrometry

One-carbon metabolism that involves folate metabolism and homocysteine metabolism plays a powerful role in embryonic development. Any impairment to this metabolism during the neurulation process would trigger the occurrence of neural tube defects (NTDs). The great importance of one-carbon metabolism necessitates the establishment of methodology to determine the relative compounds involved in the metabolic cycles. We have developed a sensitive method for measurement of 11 pivotal compounds by using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS) in sera of pregnant women. Use of an aqueous chromatography column increased retention time and separation of the polar compounds in the system, resulting in fewer co-elution and interference from the other compounds that can lead to ion suppression. Calibration curves suitable for the analysis of maternal serum were linear (r(2)>0.997) with limits of detection from 0.05 to 1ng/mL. Intra-day coefficients of variation (CVs) and inter-day CVs were both lower than 11%. With the developed method, 96 serum samples including 46 cases and 50 controls were analyzed. The established method provided a reliable method for quantifying most of the compounds involved in the one-carbon metabolism simultaneously, thus made it possible to elucidate NTDs with multiple factors instead of one single and provided a solid foundation for the diagnosis and prevention of NTDs as well as some other one-carbon metabolism related diseases.

Association of genomic instability, and the methylation status of imprinted genes and mismatch-repair genes, with neural tube defects

We studied the genomic instability and methylation status of the mismatch-repair (MMR) genes hMLH1 and hMSH2, and the imprinted genes H19/IGF2, in fetuses with neural tube defects (NTDs) to explore the pathogenesis of the disease. Microsatellite instability (MSI) was observed in 23 of 50 NTD patients. Five NTD patients showed high-degree MSI (MSI-H) and 18 showed low-degree MSI (MSI-L). The frequencies of mutated microsatellite loci were 3/50 (6%) for BatT-25, 10/50 (20%) for Bat-26, 3/50 (6%) for Bat34C4, 6/50 (12%) for D2S123, 4/50 (8%) for D2S119, and 3/50 (6%) for D3S1611. The promoter regions of the hMLH1 and hMSH2 genes were unmethylated in NTD patients, as determined by methylation-specific PCR. The hMLH1 and hMSH2 promoter methylation patterns, the methylation levels of H19 DMR1, and IGF2 DMR0 were detected by bisulfite sequencing PCR, sub-cloning, and sequencing. The hMSH2 promoter sequence was unmethylated, and the hMLH1 promoter showed a specific methylation pattern at two CpG sites. The methylation levels of H19 DMR1 in the NTD and control groups are 73.3%±15.9 and 58.3%±11.2, respectively. The methylation level of the NTD group was higher than that of the control group (Students t-test, P<0.05). There is no significant difference in IGF2 DMR0 methylation level between the two groups. All of the results presented here suggest that genomic instability, the MMR system, and hyper-methylation of the H19 DMR1 may be correlated with the occurrence of NTDs.

Unusual Patterns of Neural Tube Defects in a High Risk Region of Northern China

OBJECTIVE To study the prevalence of different types of neural tube defects (NTDs) in Luliang Prefecture, Shanxi province, where the prevalence of NTDs is unusually high and the correlation between NTDs prevalence and patterns. METHODS A surveillance population-based birth defects was performed in Luliang Prefecture, Shanxi province. RESULTS The results of our study showed that the prevalence of NTDs was 2-fold higher in Luliang Prefecture than in other areas of Shanxi province. Unusual patterns of NTDs were found, however, multiple NTDs were relatively common in Luliang Prefecture, accounting for over 13% of all NTDs cases in China. CONCLUSION The prevalence of NTDs is associated with its patterns.

Cystathionine beta-synthase 844ins68 polymorphism is unrelated to susceptibility to neural tube defects.

OBJECTIVE Cystathionine beta-synthase (CBS) 844ins68 polymorphism has been implicated in the development of neural tube defects (NTDs). However, the results of different studies are inconsistent. Thus, we conducted a meta-analysis to further investigate this association. METHODS Published studies were retrieved from PubMed, Embase, China National Knowledge Infrastructure, and Wanfang Data. Studies that evaluated the association between CBS 844ins68 polymorphism and NTD risk among mothers, children, or fathers were included. The pooled odds ratios with 95% confidence interval were calculated using a fixed effects model or a random effects model. RESULTS A total of eight studies on mothers (641 cases and 1145 controls), eight studies on children (852 cases and 1912 controls), and five studies (263 cases and 1562 controls) on fathers were included. The meta-analysis revealed no significant association between CBS 844ins68 polymorphism and NTD risk among mothers, children, and fathers under either genetic model. CONCLUSION The present meta-analysis indicates that CBS 844ins68 polymorphism is not a good predictor of risk for NTDs.