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.     

A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein,causes neural tube closure defects in mice

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155^msp3, a unique ENU‐induced allele in mice. Homozygous Baf155^mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA‐Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014     

Genetic basis of neural tube defects. I. Regulatory genes for the neurulation process

Neural tube defects (NTD) together with cardiovascular system defects are the most common malformations in the Polish population (2.05-2.68/1000 newborns). They arise during early embryogenesis and are caused by an improper neural groove closure during the neurulation process. NTD can arise from the influence of specific environmental factors on the foetus. The genetic factor is also very important, because NTDs have multigenetic conditioning. It was suggested that genes connected with the regulation of neurulation could also be involved in NTD aetiology, especially when their deletion or modification leads to neural tube defects in the mouse model. Examples are genes from the PAX family, T (Brachyury), BRCA1 and PDGFRA genes.     

Reduction in Valproic Acid–Induced Neural Tube Defects by Maternal Immune Stimulation: Role of Apoptosis

Teratogenic deregulation of apoptosis during development is a possible mechanism for birth defects. Administration of valproic acid (VA) during first trimester of pregnancy causes neural tube defects (NTDs). Nonspecific stimulation of the mother's immune system has been shown to reduce various teratogen‐induced fetal malformations including NTDs in rodents. This present study investigated the role of reduced apoptosis by maternal immune stimulation in prevention of VA‐induced NTDs in CD‐1 mice. Prevention of VA‐induced NTDs by nonspecific maternal immune stimulation using IFNγ was employed to evaluate the role of reduced apoptosis by IFNγ in this protective mechanism. Apoptosis was quantified using flow cytometry. Terminal Transferase dUTP Nick End Labeling assay was used to localize the apoptosis. Increased apoptosis, suggesting involvement in VA teratogenicity, was observed along the neural tube in both normal and abnormal embryos from VA‐exposed dams. Increased apoptosis in normal VA‐exposed embryos suggests that VA may alter other cellular processes such as cell proliferation and differentiation in addition to apoptosis. Apoptotic levels in embryos with closed neural tubes from IFNγ + VA dams were similar to controls indicating resistance to VA‐induced apoptosis and protection against teratogenicity of VA. In IFNγ + VA exposed embryos with open neural tubes, maternal immune stimulation failed to regulate apoptosis resulting in an NTD. Overall, these results suggest that VA alters several biological processes including apoptosis in the developing embryos to induce fetal malformations. Resistance to VA‐induced apoptosis in embryos resulting from maternal immune stimulation may be involved in protective mechanism.     

Ribonucleotide reductase subunit R1: a gene conferring sensitivity to valproic acid-induced neural tube defects in mice

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly.     

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.     

<Emphasis Type="Italic">Pax1/E2a</Emphasis> Double-Mutant Mice Develop Non-Lethal Neural Tube Defects that Resemble Human Malformations

Many mouse models exist for neural tube defects (NTDs), but only few of them are relevant for human patients that are born alive with spina bifida aperta. NTDs in humans show a complex inheritance, which most likely result from the involvement of a variety of predisposing genetic and environmental factors. Hints toward the identity of predisposing genetic factors for human NTDs could come from mouse studies on the development of the neural tube and spinal cord, as well as from studies on associated features of this type of diseases. Among such features is the observation that pregnancies affected by a neural tube defect frequently show changes in thymus morphology, and in both neonatal and maternal T-cell repertoire. The genes for E2a and Pax1 have both been implicated in not only paraxial mesodermal development, but also in that of the immune system. Moreover, Pax1 mutant mice have been shown to display NTDs in digenic mouse models. In the present study we have investigated the phenotype of E2a null mutant mice that are also heterozygous for the so-called undulated mutation in Pax1. Here we report that such double-mutant mice develop a non-lethal NTD that strongly resembles the classic human NTD: spina bifida aperta, associated with defects of the axial skeleton, immune system and urinary tract.     

Describing the Prevalence of Neural Tube Defects Worldwide: A Systematic Literature Review

BackgroundFolate-sensitive neural tube defects (NTDs) are an important, preventable cause of morbidity and mortality worldwide. There is a need to describe the current global burden of NTDs and identify gaps in available NTD data.ConclusionsMany WHO member states (120/194) did not have any data on NTD prevalence. Where data are collected, prevalence estimates vary widely. These findings highlight the need for greater NTD surveillance efforts, especially in lower-income countries. NTDs are an important public health problem that can be prevented with folic acid supplementation and fortification of staple foods.     

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell–cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.Other members of NTD Collaborative Group involved in this study are listed in the appendix     

The emerging role of epigenetic mechanisms in the etiology of neural tube defects

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1,000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the etiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.Key words: neural tube defects, diet, folic acid, epigenome, epigenetic regulation, methylation, chromatin, histones, acetylation     

The involvement of cell death and survival in neural tube defects: a distinct role for apoptosis and autophagy?

Neural tube defects (NTDs), such as spina bifida (SB) or exencephaly, are common congenital malformations leading to infant mortality or severe disability. The etiology of NTDs is multifactorial with a strong genetic component. More than 70 NTD mouse models have been reported, suggesting the involvement of distinct pathogenetic mechanisms, including faulty cell death regulation. In this review, we focus on the contribution of functional genomics in elucidating the role of apoptosis and autophagy genes in neurodevelopment. On the basis of compared phenotypical analysis, here we discuss the relative importance of a tuned control of both apoptosome-mediated cell death and basal autophagy for regulating the correct morphogenesis and cell number in developing central nervous system (CNS). The pharmacological modulation of genes involved in these processes may thus represent a novel strategy for interfering with the occurrence of NTDs.     

Quantitative Measurement of <Emphasis Type="Italic">PARD3</Emphasis> Copy Number Variations in Human Neural Tube Defects

Although more than 200 genes are known to be related to neural tube defects (NTDs), the exact molecular basis is still unclear. Evaluating the contribution of copy number variation (CNV) might be a priority because CNV involves changes in the copy number of large segments of DNA, leading to phenotypic traits and disease susceptibility. Recent studies have documented that the polarity protein partitioning defective 3 homolog (Pard3) plays an essential role in the process of neural tube closure. The aim of this study was to assess the role of PARD3 CNVs in the etiology of human NTDs. Relative quantitative PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to quantitative measurement of CNVs in 25 PARD3 exons in 202 NTD cases and 231 controls from a region of China with a high prevalence of NTDs. The results showed that microduplications ranging from 3 to 4 were evident in coding Exon 21 and Exon 25 in both case and control groups. A novel heterozygous microdeletion spanning 444 bp of Exon 14 was identified in two cases of anencephaly and is absent from all controls analyzed. Expression analyses indicated that this heterozygotic microdeletion showed no tissue specificity and led to defective expression of PARD3. Our study provides further evidence implicating PARD3 in the etiology of NTDs.     

Fetal DNA hypermethylation in tight junction pathway is associated with neural tube defects: A genome-wide DNA methylation analysis

Neural tube defects (NTDs) are a spectrum of severe congenital malformations of fusion failure of the neural tube during early embryogenesis. Evidence on aberrant DNA methylation in NTD development remains scarce, especially when exposure to environmental pollutant is taken into consideration. DNA methylation profiling was quantified using the Infinium HumanMethylation450 array in neural tissues from 10 NTD cases and 8 non-malformed controls (stage 1). Subsequent validation was performed using a Sequenom MassARRAY system in neural tissues from 20 NTD cases and 20 non-malformed controls (stage 2). Correlation analysis of differentially methylated CpG sites in fetal neural tissues and polycyclic aromatic hydrocarbons concentrations in fetal neural tissues and maternal serum was conducted. Differentially methylated CpG sites of neural tissues were further validated in fetal mice with NTDs induced by benzo(a)pyrene given to pregnant mice. Differentially hypermethylated CpG sites in neural tissues from 17 genes and 6 pathways were identified in stage 1. Subsequently, differentially hypermethylated CpG sites in neural tissues from 6 genes (BDKRB2, CTNNA1, CYFIP2, MMP7, MYH2, and TIAM2) were confirmed in stage 2. Correlation analysis showed that methylated CpG sites in CTNNA1 and MYH2 from NTD cases were positively correlated to polycyclic aromatic hydrocarbon level in fetal neural tissues and maternal serum. The correlation was confirmed in NTD-affected fetal mice that were exposed to benzo(a)pyrene in utero. In conclusion, hypermethylation of the CTNNA1 and MYH2 genes in tight junction pathway is associated with the risk for NTDs, and the DNA methylation aberration may be caused by exposure to benzo(a)pyrene.     

Using genomewide mutagenesis screens to identify the genes required for neural tube closure in the mouse

BACKGROUND: Neural tube closure is a critical embryological process that requires the coordination of many molecular and cellular events. Only recently has the molecular basis of the cell movements that drive neural tube closure begun to be elucidated. This has been accomplished in part due to the analysis of a growing number of genetically targeted and naturally occurring mouse mutant strains that have neural tube defects (NTDs). Currently there are more than 100 genes that when mutated result in NTDs in the mouse. Yet only approximately 10% of genes in the mouse genome have been mutated and their gross phenotype analyzed, suggesting that only a small percentage of the genes that can cause NTDs have been identified. METHODS: In order to more systematically and fully understand the genetic basis of neural tube closure and to begin to define the molecular pathways that direct this key embryonic event, our laboratories have undertaken a forward genetic screen in mice. From this we hope to gain a better understanding of the regulation of this complex morphogenic processes. CONCLUSIONS: The mouse provides a good model for human neural tube closure, and therefore the information gained from generating novel mouse models of NTDs will help to predict the genes responsible for human NTDs and provide experimental evidence for how they function.     

The emerging role of epigenetic mechanisms in the etiology of neural tube defects

The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the aetiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements, and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.