Does prenatal screening for 5,10-methylenetetrahydrofolate reductase (MTHFR) mutations in high-risk neural tube defect pregnancies make sense?

Despite the fact that neural tube defects (NTDs) are the most common congenital malformations of the central nervous system, investigators have yet to identify responsible gene(s). Research efforts have been productive in the identification of environmental factors, such as periconceptional folic acid supplementation, that modulate risk for the development of NTDs. Studies of the folic acid biosynthetic pathway led to the discovery of an association between elevated levels of homocysteine and NTD risk. Researchers subsequently identified single nucleotide polymorphisms in the gene coding for the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR). Association studies suggested it was a potential risk factor for NTDs, because the thermolabile form of the enzyme led to elevated homocysteine concentrations when folic acid intake is low. Numerous studies analyzing MTHFR variants have resulted in positive associations with increased NTD risk only in certain populations, suggesting that these variants are not large contributors to the etiology of NTDs. With our limited understanding of the genes involved in regulating NTD susceptibility, the paucity of data on how folic acid protects the developing embryo, as well as the observed decrease in birth prevalence of NTDs following folic acid supplementation and food fortification, it makes little sense for prospective parents to be tested for MTHFR variants, or for variants of other known folate pathway genes.     

Folate status and neural tube defects

Periconceptional folic acid supplementation prevents approximately 70% of neural tube defects (NTDs). While most women carrying affected fetuses do not have deficient blood folate levels, the risk of having an NTD affected child is inversely correlated with pregnancy red cell folate levels. Current research is focused on the discovery of genetic abnormalities in folate related enzymes which might explain the role of folate in NTD prevention. The first candidate gene to emerge was the C677T variant of 5,10-methylenetetrahydrofolate reductase. Normal subjects who are homozygous for the mutation (TT) have red cell folate status some 20% lower than expected. It is now established that the prevalence of the TT genotype is significantly higher among spina bifida cases and their parents. Nevertheless, our studies show that the variant does not account for the reduced blood folate levels in many NTD affected mothers. We conclude that low maternal folate status may in itself be the most important risk factor for NTDs and that food fortification may be the only population strategy of benefit in the effort to eliminate NTDs.     

Folate-mediated one-carbon metabolism and neural tube defects: balancing genome synthesis and gene expression

Neural tube defects (NTDs) refer to a cluster of neurodevelopmental conditions associated with failure of neural tube closure during embryonic development. Worldwide prevalence of NTDs ranges from approximately 0.5 to 60 per 10,000 births, with regional and population-specific variation in prevalence. Numerous environmental and genetic influences contribute to NTD etiology; accumulating evidence from population-based studies has demonstrated that folate status is a significant determinant of NTD risk. Folate-mediated one-carbon metabolism (OCM) is essential for de novo nucleotide biosynthesis, methionine biosynthesis, and cellular methylation reactions. Periconceptional maternal supplementation with folic acid can prevent occurrence of NTDs in the general population by up to 70%; currently several countries fortify their food supply with folic acid for the prevention of NTDs. Despite the unambiguous impact of folate status on NTD risk, the mechanism by which folic acid protects against NTDs remains unknown. Identification of the mechanism by which folate status affects neural tube closure will assist in developing more efficacious and better targeted preventative measures. In this review, we summarize current research on the relationship between folate status and NTDs, with an emphasis on linking genetic variation, folate nutriture, and specific metabolic and/or genomic pathways that intersect to determine NTD outcomes.     

Metabolic effects and the methylenetetrahydrofolate reductase (MTHFR) polymorphism associated with neural tube defects in southern Brazil

BACKGROUND: The importance of metabolic factors in neural tube defects (NTDs) has been the focus of many investigations. Several authors have suggested that abnormalities in homocysteine metabolism, such as hyperhomocysteinemia, folate deficiency, and low vitamin B12, may be responsible for these malformations and that both nutritional factors and genetic abnormalities are associated with them. METHODS: We conducted a case-control study to investigate the influence of biochemical and genetic factors in NTDs in infants in southern Brazil. Levels of folate, vitamin B12, total homocysteine (t-Hcy) and the 677C>T and 1298A>C polymorphisms of the MTHFR gene were analyzed in 41 NTD child-mother pairs and 44 normal child-mother control pairs. RESULTS: Subjects in the case group had a higher mean blood folate level than those in the control group. The level of vitamin B12 was lower in mothers in the NTD group than in control mothers (p = 0.004). The level of t-Hcy was not different in the two groups, but t-Hcy and vitamin B12 were correlated (p = 0.002). There was no difference in the genotype distribution for 677C>T and 1298A>C polymorphisms of MTHFR in the case and control pairs. The level of t-Hcy was correlated with 677TT. CONCLUSIONS: Despite the small sample in this study, we suggest that low vitamin B12 and, consequently, hyperhomocysteinemia are important risk factors for NTDs in our population.     

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. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 877–893, 2014     

Genetic basis of neural tube defects. II. Genes correlated with folate and methionine metabolism

Effective supplementation with folate, which prevents neural tube defect (NTD) occurrence, and high homocysteine levels in the blood of NTD children's mothers suggest that genes involved in folate and homocysteine metabolism can be involved in NTD aetiology. Genes encoding methylenetetrahydrofolate reductase (MTHFR) or methylenetetrahydrofolate dehydrogenase (MTHFD) belong to the first group. Genes encoding methionine synthase (MTR), its regulator - methionine synthase reductase (MTRR) and also cystathionine synthase (CBS) can be included in the second group. We present a current list of the folate and homocysteine metabolism genes that are known to be involved in NTD and pay special attention to primary and secondary NTD prevention.     

Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice

BACKGROUND: The etiology of neural tube defects (NTDs) is multifactorial, with environmental and genetic determinants. Folate supplementation prevents the majority of NTDs, and a polymorphism in methylenetetrahydrofolate reductase (MTHFR) has become recognized as a genetic risk factor. The mechanisms by which folate affects NTD development are unclear. The Splotch (Sp) mouse is a well-characterized mouse model for studying spontaneous NTDs. To assess the potential interaction between folate metabolism and the Sp mutant in NTD development, we studied mice with both Sp and Mthfr mutations, as well as the interaction between Sp and low dietary folate. METHODS: Wild-type, single Mthfr+/-mutant, single Sp/+mutant, and double mutant (Mthfr+/-, Sp/+) female mice were mated with males of the same genotype. Embryos were examined for NTDs on gestational day (GD) 13.5. To investigate the effects of folate deficiency on Sp mice, Sp/+female mice were fed a control diet (CD), a moderately folic acid-deficient diet (MFADD), or a severely folic acid-deficient diet (SFADD). They were mated with Sp/+males and the embryos were examined. RESULTS: There were no differences in the incidence or severity of NTDs in embryos from double-mutant mating pairs compared to those from single Sp mutants. Embryos from Mthfr+/-dams did not exhibit NTDs. Diets deficient in folate did not influence the incidence or severity of NTDs in embryos from Sp/+mice. CONCLUSIONS: We did not observe an interaction between Sp and Mthfr mutations, or between the Sp mutation and low dietary folate, in NTD development in Splotch mice.     

Proportion of neural tube defects attributable to known risk factors

BACKGROUND

Recognized risk factors for neural tube defects (NTDs) poorly predict population‐level NTD risk. However, the proportion of NTDs that can be attributed to these risk factors is uncertain.

METHODS

To determine the proportion of NTD cases that is attributable to known or suspected risk factors (i.e., female infant sex, family history of NTDs, and maternal Hispanic ethnicity, obesity, pregestational diabetes, gestational diabetes, low dietary folate intake, lack of folic acid supplementation, anticonvulsant use, and hot tub or sauna use), we estimated the adjusted population attributable fraction (aAF) for each factor, using the method of Eide and Geffler and data from the National Birth Defects Prevention Study.

RESULTS

Our analyses of these data indicate that the proportion of cases of spina bifida and anencephaly that can be attributed to known risk factors is 28% and 44%, respectively. For spina bifida, the factor with the greatest attributable fraction was maternal obesity (aAF, 10%), whereas for anencephaly it was Hispanic ethnicity (aAF, 15%).

CONCLUSION

Our analyses indicate that known risk factors account for <50% of NTD cases. Hence, the majority of NTD cases are attributable to, as yet, unidentified factors. These findings highlight the need for continued research to identify genetic and additional nongenetic risk factors for NTDs. Further, these findings suggest that strategies that aim to reduce the risk of NTDs associated with maternal Hispanic ethnicity and obesity may have the greatest impact on the population prevalence of these conditions. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.     

Evaluation of transcobalamin II polymorphisms as neural tube defect risk factors in an Irish population

BACKGROUND: Decreased maternal folate levels are associated with having a child with a neural tube defect (NTD), and periconceptual folic acid supplementation reduces this risk by >50%. Vitamin B(12) (as methylcobalamin) is a cofactor for methionine synthase, an enzyme that plays a key role in folate metabolism. Alterations in vitamin B(12) metabolism may influence the development of NTDs. Low levels of maternal plasma vitamin B(12) and reduced binding of vitamin B(12) by transcobalamin II (TCII) are independent risk factors for NTDs. TCII levels are altered in the amniotic fluid of pregnancies affected by NTDs. Given this evidence, inherited variants in genes involved in vitamin B(12) trafficking such as TCII are candidate NTD risk factors. METHODS: We used case/control and family-based association methods to investigate whether six common polymorphisms in the TCII gene influence NTD risk. TCII genotypes were determined for more than 300 Irish NTD families and a comparable number of Irish controls. RESULTS: Allele and genotype frequencies for each polymorphism did not differ between family members and controls. CONCLUSIONS: These six TCII polymorphisms do not strongly influence NTD risk in the Irish population. The Supplementary Material for this article can be found on the Birth Defects Research (Part A) website: http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2005/73/v73.4.swanson.html     

The genetic background of the curly tail strain confers susceptibility to folate‐deficiency‐induced exencephaly

BACKGROUND : Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD‐causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects. METHODS : Folate deficiency was induced in curly tail and genetically matched wild‐type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs. RESULTS : Folate‐deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar‐sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background‐matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo‐inositol reduced the incidence of NTDs in the folate‐deficient wild‐type strain. CONCLUSIONS : Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene‐nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

The "thermolabile" variant of methylenetetrahydrofolate reductase and neural tube defects: An evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother

Recent reports have implicated the "thermolabile" (T) variant of methylenetetrahydrofolate reductase (MTHFR) in the causation of folate-dependent neural tube defects (NTDs). We report herein the largest genetic study of NTD cases (n=271) and families (n=218) to date, establishing that, in Ireland, the "TT" genotype is found in 18.8% of cases versus 8.3% of controls (odds ratio 2.57; confidence interval [CI] 1.48-4.45; P=.0005). The maternal and paternal TT genotypes have intermediate frequencies of 13.8% and 11.9%, respectively, indicating that the predominant MTHFR-related genetic effect acts via the TT genotype of the developing embryo. Analysis of the 218 family triads of mother, father, and affected child with log-linear models supports this interpretation, providing significant evidence that the case TT genotype is associated with NTDs (P=.02) but no evidence of a maternal TT genotypic effect (P=. 83). The log-linear model predicted that the risk of NTDs conferred by the case TT genotype is 1.61 (CI 1.06-2.46), consistent with the paramount importance of the case TT genotype in determining risk. There is no compelling evidence for more than a modest additional risk conferred by a maternal TT genotype. These results favor a biological model of MTHFR-related NTD pathogenesis in which suboptimal maternal folate status imposes biochemical stress on the developing embryo, a stress it is ill-equipped to tolerate if it has a TT genotype.     

Uncoupling protein 2 polymorphisms as risk factors for NTDs

BACKGROUND: Both environmental and genetic factors are involved in the etiology of NTDs. Inadequate folate intake and obesity are important environmental risk factors. Several folate‐related genetic variants have been identified as risk factors; however, little is known about how genetic variants relate to the increased risk seen in obese women. Uncoupling Protein 2 (UCP2) is an attractive candidate to screen for NTD risk because of its possible role in obesity as well as energy metabolism, type‐2 diabetes, and the regulation of reactive oxygen species. Interestingly, a previous study found that a common UCP2 compound homozygous genotype was associated with a threefold increase in NTD risk. METHODS: We evaluated three polymorphisms, ‐866G>A, A55V, and the 3′UTR 45 bp insertion/deletion, as risk factors for NTDs in Irish NTD cases (n = 169), their mothers (n = 163), their fathers (n = 167), and normal control subjects (n = 332). RESULTS: Allele and genotype frequencies were not significantly different when comparing NTD mothers, NTD fathers, or affected children to controls. Additionally, the previously reported risk genotype (combined homozygosity of 55VV and 3′UTR 45 bp deletion/deletion) was not present at a higher frequency in any NTD group when compared to controls. CONCLUSIONS: In our Irish study population, UCP2 polymorphisms did not influence NTD risk. Moreover, the prevalence of this allele in other populations was similar to the Irish prevalence but far lower than reported in the previous NTD study, suggesting that this previous finding of an association with NTDs might have been due to an unrepresentative study sample. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Methylenetetrahydrofolate reductase mutations, a genetic cause for familial recurrent neural tube defects

Methylenetetrahydrofolate reductase (MTHFR) gene mutations have been implicated as risk factors for neural tube defects (NTDs). The best-characterized MTHFR genetic mutation 677C→T is associated with a 2-4 fold increased risk of NTD if patient is homozygous for this mutation. This risk factor is modulated by folate levels in the body. A second mutation in the MTHFR gene is an A→C transition at position 1298. The 1298A→C mutation is also a risk factor for NTD, but with a smaller relative risk than 677C→T mutation. Under conditions of low folate intake or high folate requirements, such as pregnancy, this mutation could become of clinical importance. We present a case report with MTHFR genetic mutation, who presented with recurrent familial pregnancy losses due to anencephaly/NTDs.     

Folate, homocysteine and neural tube defects: an overview

Folate administration substantially reduces the risk on neural tube detects (NTD). The interest for studying a disturbed homocysteine (Hcy) metabolism in relation to NTD was raised by the observation of elevated blood Hcy levels in mothers of a NTD child. This observation resulted in the examination of enzymes involved in the folate-dependent Hcy metabolism. Thus far, this has led to the identification of the first and likely a second genetic risk factor for NTD. The C677T and A1298C mutations in the methylenetetrahydrofolate reductase (MTHFR) gene are associated with an increased risk of NTD and cause elevated Hcy concentrations. These levels can be normalized by additional folate intake. Thus, a dysfunctional MTHFR partly explains the observed elevated Hcy levels in women with NTD pregnancies and also, in part, the protective effect of folate on NTD. Although the MTHFR polymorphisms are only moderate risk factors, population-wide they may account for an important part of the observed NTD prevalence.     

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.     

Higher serum homocysteine and lower thyroid hormone levels in pregnant women are associated with neural tube defects

BackgroundThis study tested the hypothesis that abnormal maternal metabolism of both homocysteine and thyroid hormone network in pregnant women is associated with neural tube defects (NTDs) in a part of China with high NTD prevalence.MethodsA case–control study was performed between 2007 and 2009 in Lüliang Mountains, Shanxi Province. This study included 83 pregnant women who had fetuses with NTDs (cases) and 90 pregnant women with normal fetuses (controls). In addition, a cell model to illustrate the epidemiological findings was established.ResultsFetuses of mother who had both high total homocysteine (tHcy) and inadequate free thyroxine were 3 times more at risk of developing NTDs (adjusted odds ratio = 3.5; 95 % confidence interval = 1.2–10.4; cases vs. controls) using multivariate logistic regression models. Furthermore, biological interaction between metabolisms of Hcy and thyroid hormones was demonstrated in vitro. In homocysteine thiolactone of a metabolite of Hcy-treated mouse embryonic neural stem NE4C cells, genes (Bmp7, Ctnnb1, Notch 1, Gli2, and Rxra) related to both neural tube closure and thyroid hormone network were shown to be regulated by H3K79 homocysteinylation, which increased their expression levels.ConclusionsThe effect of maternal serum high tHcy on risk of developing NTDs is depended on maternal serum level of thyroxine. Meanwhile, a higher level of tHcy might also affect both maternal metabolism of thyroid hormone and neural tube closure in embryogenesis through homocysteinylation of histones.     

Reduced folate carrier gene is a risk factor for neural tube defects in a Chinese population

BACKGROUND: There is a considerable body of data demonstrating that periconceptional supplementation of folic acid can prevent a significant proportion of neural tube defects (NTDs). At present, the mechanism by which folic acid exerts its beneficial effect remains unknown. Folate transporter genes, including the reduced folate carrier gene (RFC1), have been proposed as NTD risk factors. METHODS: The study population included 104 nuclear families with NTDs and 100 nonmalformed control families. We investigated the possible association between a common RFC1 polymorphism (A80G) and NTD risk among offspring, as well as potential gene-environment interactions between the infant RFC1 genotype and maternal periconceptional use of folic acid through a population-based case-control study. RESULTS: We observed that the infants of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.04-6.36) in our study population. Among mothers who did not utilize folic acid supplements, the risk for having a child with an NTD was 3.30 (95% CI, 1.15-9.65) for offspring with the GG genotype, compared to the reference (AA) genotype. Children who had the GG genotype and whose mothers did not take folic acid had an elevated risk for NTDs (OR, 8.80; 95% CI, 2.83-28.69), compared to offspring with the AA and GA genotypes whose mothers utilized folic acid supplements. CONCLUSIONS: Our findings suggest that the RFC1 G allele is likely to be an important genetic factor in determining folate transport and subsequently may be a risk factor for NTDs in this Chinese population.     

An insertion/deletion polymorphism of the dihydrofolate reductase (DHFR) gene is associated with serum and red blood cell folate concentrations in women

A low serum folate and high homocysteine phenotype is associated with an increased risk of neural tube defects (NTDs), cardiovascular diseases and other pathologies. Thus defining both genetic and non-genetic factors that may impact folate/homocysteine metabolism will enhance our understanding of the etiologic mechanisms underlying these conditions and facilitate risk assessment. Dihydrofolate reductase catalyzes the reduction of folic acid to dihydrofolate and thereafter to tetrahydrofolate. The impact of the dihydrofolate reductase (DHFR) c.86 + 60_78 insertion/deletion (ins/del) polymorphism on folate and homocysteine concentrations was analyzed using data from healthy young adults from Northern Ireland, collected as part of visit three of the Young Hearts Project. Among men the DHFR c.86 + 60_78 polymorphism was not significantly associated with serum or red blood cell folate concentrations, or with homocysteine concentrations. Among women the DHFR c.86 + 60_78 polymorphism explained 2% of the variation in RBC folate levels and 5% of the variation in serum folate levels, but did not appear to have an independent effect on homocysteine. Relative to women with the DHFR c.86 + 60_78 ins/ins and ins/del genotypes, del/del homozygotes had increased serum and red blood cell folate concentrations and may therefore be at decreased risk of having offspring affected by NTDs and of other adverse reproductive and health outcomes attributable to low folate.     

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.     

Plasma vitamin values and antiepileptic therapy: case reports of pregnancy outcomes affected by a neural tube defect

BACKGROUND: Folic acid supplementation reduces the occurrence of neural tube defects (NTDs); however, it is not clear whether it protects against teratogenic effects of antiepileptic drugs. METHODS: We report the cases of four pregnant women receiving valproic acid therapy, who all had NTD-affected offspring, despite periconceptional 5 mg/day of folic acid supplementation (cases), and investigated homocysteine metabolism, linked with folate metabolism. Their plasma homocysteine, folates, and vitamin B6 and B12 results were compared with values of two other women, who were also receiving valproic acid and folic acid complement, but who had normal pregnancies (valproic acid controls), and values of 40 pregnant women who had normal pregnancies and were not receiving any therapy (controls without therapy). Because of the possible existence of a genetic susceptibility, polymorphisms in homocysteine metabolism were sought. RESULTS: Two cases showed a decreased phosphopyridoxal level, compared with levels in the controls not receiving therapy. The genotype TT (C677T) is an NTD genetic susceptibility, but it was observed in only one valproic acid control. Various polymorphisms were observed in the cases, but were also common in the controls. Several studies have reported that valproic acid therapy lowers vitamin B6 levels. Our case with the greatest decrease in plasma phosphopyridoxal, who was taking periconceptional folic acid plus pyridoxine therapy, had a normal second pregnancy outcome. CONCLUSIONS: In addition to folates, other vitamins, such as vitamin B6, may have played a role in NTDs in our patients taking an antiepileptic drug.