Neural Tube Defects Phenotype Research Articles

The interaction of endorepellin and neurexin triggers neuroepithelial autophagy and maintains neural tube development

Heparan sulfate proteoglycan 2 (HSPG2) gene encodes the matrix protein Perlecan, and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects (NTDs). We discovered rare genetic variants of HSPG2 in 10% cases compared to only 4% in controls among a cohort of 369 NTDs. Endorepellin, a peptide cleaved from the domain V of Perlecan, is known to promote angiogenesis and autophagy in endothelial cells. The roles of enderepellin in neurodevelopment remain unclear so far. Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo. Through the endocytic pathway, the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker, which is necessary for normal neural tube closure. We created knock-in (KI) mouse models with human-derived HSPG2 variants, using sperm-like stem cells that had been genetically edited by CRISPR/Cas9. We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos. Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases. Furthermore, we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation. Therefore, autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants.

Neural tube defects and epigenetics: role of histone post-translational histone modifications.

Neural tube defects (NTDs) are the most common congenital anomalies of the CNS. It is widely appreciated that both genetic and environmental factors contribute to their etiology. The inability to ascribe clear genetic patterns of inheritance to various NTD phenotypes suggests it is possible that epigenetic mechanisms are involved in the etiology of NTDs. In this context, the contribution of DNA methylation as an underlying contributing factor to the etiology of NTDs has been extensively reviewed. Here, an updated accounting of the evidence linking post-translational histone modifications to these birth defects, relying heavily upon studies in humans, and the possible molecular implications inferred from reports based on cellular and animal models, are presented.

Transcriptional Signature of Valproic Acid-Induced Neural Tube Defects in Human Spinal Cord Organoids.

In vertebrates, the entire central nervous system is derived from the neural tube, which is formed through a conserved early developmental morphogenetic process called neurulation. Although the perturbations in neurulation caused by genetic or environmental factors lead to neural tube defects (NTDs), the most common congenital malformation and the precise molecular pathological cascades mediating NTDs are not well understood. Recently, we have developed human spinal cord organoids (hSCOs) that recapitulate some aspects of human neurulation and observed that valproic acid (VPA) could cause neurulation defects in an organoid model. In this study, we identified and verified the significant changes in cell-cell junctional genes/proteins in VPA-treated organoids using transcriptomic and immunostaining analysis. Furthermore, VPA-treated mouse embryos exhibited impaired gene expression and NTD phenotypes, similar to those observed in the hSCO model. Collectively, our data demonstrate that hSCOs provide a valuable biological resource for dissecting the molecular pathways underlying the currently unknown human neurulation process using destructive biological analysis tools.

Gene-environment interactions underlying the etiology of neural tube defects.

Neural tube defects (NTDs) consist of severe structural malformations of the brain and spinal cord and are the second most common structural birth defect in humans, accounting for approximately 2700 affected pregnancies every year in the United States. These numbers are highly significant, considering that birth defects remain a leading cause of infant mortality in the United States, affecting approximately 120,000 babies born annually. Survivors of these congenital malformations face long-term disability and lifelong challenges imposed by severe physical burdens compromising the afflicted individual's overall quality of life. Clearly, birth defects, and especially NTDs remain a global public health challenge, and the source of significant financial repercussions for healthcare systems worldwide. In order to better understand the role gene-environment interactions play in the etiology of NTDs, this chapter provides an overview of NTD phenotypes and their embryonic origins, discusses the genetic landscape of NTDs as it is currently understood, with a focus on experimental models that best illustrate how environmental factors modulate individual susceptibility to these birth defects. As folic acid interventions have proven to be effective in reducing the prevalence of NTDs, the chapter ends with a discussion on the impact that maternal dietary status has on NTD prevalence from a population perspective.

Comparison of inherited neural tube defects in companion animals and livestock.

Neural tube defects (NTDs) are congenital malformations resulting from the improper or incomplete closure of the neural tube during embryonic development. A number of similar malformations of the protective coverings surrounding the central nervous system are also often included under this umbrella term, which may not strictly fit this definition. A range of NTD phenotypes exist and have been reported in humans and a wide range of domestic and livestock species. In the veterinary literature, these include cases of anencephaly, encephalocele, dermoid sinus, spina bifida, and craniorachischisis. While environmental factors have a role, genetic predisposition may account for a significant part of the risk of NTDs in these animal cases. Studies of laboratory model species (fish, birds, amphibians, and rodents) have been instrumental in improving our understanding of the neurulation process. In mice, over 200 genes that may be involved in this process have been identified and variant phenotypes investigated. Like laboratory mouse models, domestic animals and livestock species display a wide range of NTD phenotypes. They remain, however, a largely underutilized population and could complement already established laboratory models. Here we review reports of NTDs in companion animals and livestock, and compare these to other animal species and human cases. We aim to highlight the potential of nonlaboratory animal models for mutation discovery as well as general insights into the mechanisms of neurulation and the development of NTDs.

Concepts in the neurosurgical care of patients with spinal neural tube defects: An embryologic approach.

The neural tube defects (NTDs) are a heterogeneous group of structural birth defects that arise from a complex array of multiple genetic and environmental factors and adversely affect the structure and function of the brain and spinal cord. Spinal NTDs are clinically more common than cranial NTDs. There remains a significant gap in linking the multiple NTD phenotypes to current genomic understanding. This article summarizes the neurosurgical clinical approach to spinal NTDs by correlating each step of embryonic development of the human nervous system with key management concepts for defects that arise at that step. The NTDs are broadly classified as open or closed. Open defects include myelomeningocele (MMC), encephalocele, and anencephaly. Closed defects are also known as occult spinal dysraphism and are characterized by intact skin over the spinal defect. They are more common and often cause neurologic decline from tethered cord syndrome. Failure of primary neurulation gives rise to open myelomeningocele (MMC). Surgical closure of an open MMC focuses on realigning the tissue layers that failed to separate during neurulation. In utero closure is a promising recent technique. Chronic neurosurgical management largely focuses treating hydrocephalus. The Chiari II malformation is uniformly present in MMC patients and may cause brainstem dysfunction. Tethered spinal cord may progressively impair normal neurologic function but typically responds well to surgical untethering. Surgical closure of MMC centers on approximated realignment of embryologically disordered neural tissue. Clinical surgical management decisions in the spinal NTDs remains challenging but standardized principles have emerged.

Inositol Deficiency Increases Neural Tube Defect Susceptibility by PI3K/AKT Signaling Pathway (P11-009-19)

ObjectivesTo explore the possible mechanisms of inositol deficiency in neural tube defects (NTDs). MethodsThe gas chromatography–mass spectrometry (GC-MS) analysis was used to detect the plasma inositol levels of NTD pregnancies and non-NTD controls from a high-prevalence area of Shanxi Province in northern China. A maternal MI deficient mouse model was established by using intraperitoneal injection of Li2CO3, which act as an inhibitor of inositol 1-phosphatase (IMPase) and a key enzyme for the synthesis and recycling of inositol in vivo. Using the phospho-protein array to find the possible differential phosphorylated proteins between the NTD and control group in MI deficiency human and mouse respectively. We performed pathway analysis on the differential phosphorylated proteins using KEGG databases of human NTDs with MI deficiency. ResultsWe found that maternal MI deficiency was associated with an increased risk of NTDs (Pearson correlation coefficient r = –0.19, P < 0.05). The MI levels of the pregnant mouse decreased significantly after Li2CO3 injection and reach to the lowest level at 8h after the injection (23.72mg/L vs 44.15 mg/L P < 0.05). Repeat trials showed that the average incidence of NTDs was 26.7%. The NTD phenotypes included encephalocele (85.8%), anencephaly (5.7%) and spina bifida (8.5%). The result of phospho-protein array displayed that PI3K-Akt signaling pathway (hsa04151) was the most enriching pathway. We then validated the proteins involved in the PI3K-Akt signaling pathway using a mouse phospho-protein array. A total of 21 proteins were found to be differentially phosphorylated (Fold change > 1.5), and 14 of them were differentially phosphorylated in both the human and mouse, including YWHAZ, AKT1, AKT2, BCL2, CCND1, NOS3, PTK2, IRS1, MTOR (mTOR), TP53, RPS6KB1 (p70S6K), PDPK1, PPP2CA and PTEN. Further pathway analysis on the 14 common differentially phosphorylated protein found that PI3K/Akt/mTOR/p70S6K pathway was the most disturbed signaling pathway induced by MI deficiency. ConclusionsOur study indicated that PI3K-Akt signaling pathway might be one of the important regulatory mechanisms of inositol deficiency-induced NTDs. Funding SourcesThis study was supported by The National Key Basic Research Program (2018YFC1002500), National Nature Science Foundation of China (81571443 to JW, 81801451 to JG).

Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice.

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

Genetic analysis of Wnt/PCP genes in neural tube defects

BackgroundMouse homozygous mutants in Wnt/planar cell polarity (PCP) pathway genes have been shown to cause neural tube defects (NTDs) through the disruption of normal morphogenetic processes critical to neural tube closure (NTC). Knockout mice that are heterozygotes of single PCP genes likely fail to produce NTD phenotypes, yet damaging variants detected in human NTDs are almost always heterozygous, suggesting that other deleterious interacting variants are likely to be present. Nonetheless, the Wnt/PCP pathway remains a genetic hotspot. Addressing these issues is essential for understanding the genetic etiology of human NTDs.MethodsWe performed targeted next-generation sequencing (NGS) on 30 NTD-predisposing Wnt/PCP pathway genes in 184 Chinese NTD cases. We subsequently replicated our findings for the CELSR1 gene in an independent cohort of 292 Caucasian NTD samples from the USA. Functional validations were confirmed using in vitro assays.ResultsCELSR1, CELSR2 and CELSR3 genes were significantly clustered with rare driver coding mutations (q-value< 0.05) demonstrated by OncodriveCLUST. During the validation stage, the number of rare loss of function (LoF) variants in CELSR1 was significantly enriched in NTDs compared with the LoF counts in the ExAC database (p < 0.001). Functional studies indicated compound heterozygote variants of CELSR2 p.Thr2026Met and DVL3 p.Asp403Asn result in down regulation of PCP signals.ConclusionsThese data indicate rare damaging variants of the CELSR genes, identified in ~ 14% of NTD cases, are expected to be driver genes in the Wnt/PCP pathway. Compound damaging variants of CELSR genes and other Wnt/PCP genes, which were observed in 3.3% of the studied NTD cohort, are also expected to amplify these effects at the pathway level.

Digenic variants of planar cell polarity genes in human neural tube defect patients

Neural tube defects (NTDs) are considered to be a complex genetic disorder, although the identity of the genetic factors remains largely unknown. Mouse model studies suggest a multifactorial oligogenic pattern of inheritance for NTDs, yet evidence from published human studies is surprisingly absent. In the present study, targeted next-generation sequencing was performed to screen for DNA variants in the entire coding regions and intron-exon boundaries of targeted genes using DNA samples from 510 NTD cases. These candidate genes were PCP genes, including VANGL1, VANGL2, CELSR1, SCRIB, DVL2, DVL3 and PTK7. Candidate variants were validated using Sanger sequencing. A total of 397 single nucleotide variants(SNVs) were identified with a mean depth of approximately 570×. Of these identified SNVs, 74 were predicted to affect protein function and had a minor allele frequency of <0.01 or unknown. Among these 74 missense SNVs, 10 were identified from six NTD cases that carried two mutated genes. Of the six NTD cases, three spina bifida cases and one anencephaly case carried digenic variants in the CELSR1 and SCRIB gene; one anencephaly case carried variants in the CELSR1 and DVL3 gene; and one spina bifida case carried variants in the PTK7 and SCRIB genes. Three cases that parental samples were available were confirmed to be compound heterozygous. None of the digenic variants were found in the 1000 genome database. The findings imply that genetic variation might interact in a digenic fashion to generate the visible NTD phenotypes and emphasize the importance of these genetic interactions in the development of NTDs in humans.

Genetic backgrounds and modifier genes of NTD mouse models: An opportunity for greater understanding of the multifactorial etiology of neural tube defects

Neurulation, the early embryonic process of forming the presumptive brain and spinal cord, is highly complex and involves hundreds of genes in multiple genetic pathways. Mice have long served as a genetic model for studying human neurulation, and the resulting neural tube defects (NTDs) that arise when neurulation is disrupted. Because mice appear to show mostly single gene inheritance for NTDs and humans show multifactorial inheritance, mice sometimes have been characterized as a simpler model for the identification and study of NTD genes. But are they a simple model? When viewed on different genetic backgrounds, many genes show significant variation in the penetrance and expressivity of NTD phenotypes, suggesting the presence of modifier loci that interact with the target gene to affect the phenotypic expression. Looking at mutations on different genetic backgrounds provides us with an opportunity to explore these complex genetic interactions, which are likely to better emulate similar processes in human neurulation. Here, we review NTD genes known to show strain-specific phenotypic variation. We focus particularly on the gene Cecr2, which is studied using both a hypomorphic and a presumptive null mutation on two different backgrounds: one susceptible (BALB/c) and one resistant (FVB/N) to NTDs. This strain difference has led to a search for genetic modifiers within a region on murine chromosome 19. Understanding how genetic variants alter the phenotypic outcome in NTD mouse models will help to direct future studies in humans, particularly now that more genome wide sequencing approaches are being used. Birth Defects Research 109:140-152, 2017. © 2016 Wiley Periodicals, Inc.

Does dietary folic acid supplementation in mouse NTD models affect neural tube development or gamete preference at fertilization?

BackgroundNeural tube defects (NTDs) are the second most common birth defect in humans. Dietary folic acid (FA) supplementation effectively and safely reduces the incidence of these often debilitating congenital anomalies. FA plays an established role in folate and homocysteine metabolism, but the means by which it suppresses occurrence of NTDs is not understood. In addition, many cases remain resistant to the beneficial effects of folic acid supplementation. To better understand the molecular, biochemical and developmental mechanisms by which FA exerts its effect on NTDs, characterized mouse models are needed that have a defined genetic basis and known response to dietary supplementation.ResultsWe examined the effect of FA supplementation, at 5-fold the level in the control diet, on the NTD and vertebral phenotypes in Apob tm1Unc and Vangl2 Lp mice, hereafter referred to as Apob and Lp respectively. The FA supplemented diet did not reduce the incidence or severity of NTDs in Apob or Lp mutant homozygotes or the loop-tail phenotype in Lp mutant heterozygotes, suggesting that mice with these mutant alleles are resistant to FA supplementation. Folic acid supplementation also did not affect the rate of resorptions or the size of litters, but instead skewed the embryonic genotype distribution in favor of wild-type alleles.ConclusionSimilar genotypic biases have been reported for several NTD models, but were interpreted as diet-induced increases in the incidence and severity of NTDs that led to increased embryonic lethality. Absence of differences in resorption rates and litter sizes argue against induced embryonic lethality. We suggest an alternative interpretation, namely that FA supplementation led to strongly skewed allelic inheritance, perhaps from disturbances in polyamine metabolism that biases fertilization in favor of wild-type gametes.

Histone modification mapping in human brain reveals aberrant expression of histone H3 lysine 79 dimethylation in neural tube defects

Neural tube defects (NTDs) are severe, common birth defects that result from failure of neural tube closure, but their pathological mechanisms are not yet fully understood. Histone modifications have an important role in gene regulation during fetal development. We therefore hypothesized that the human NTDs may be partly caused by an imbalance in metabolism, perhaps caused by nutritional deficiencies, that leads to aberrant histone modifications. Here, we report a screen of fetal brain histone modifications using 2D nano-LC strong cation exchange reverse phase (SCX/RP) MS/MS and the identification of 61 unique post-translational modification sites on histones H1, H2a, H2b, H3, and H4. Of these, 38 sites are novel (not already found in the Uniprot database). Furthermore, we compared the histone modification patterns between normal brains and NTD brains special of which maternal folate levels were lower than of normal control. The results showed that histone H3 lysine 79 dimethylation (H3K79me2) and a novel identified site, H2bK5 monomethylation (H2bK5me1), were completely absent in individuals with NTDs. Follow-up Western blotting validated the decreased H3K79me2 expression in brains with NTDs, but the amplified samples experiments displayed that decreased H3K79me2 expression was not suitable for all samples with NTDs. Furthermore, folate-free treated mouse embryonic stem cells induced the decreased H3K79me2 level. Subsequently, our ChIP results in normal fetal brain tissues showed that H3K79me2 binds to SUFU, RARA and ITGA3 which induce NTDs phenotype after knockout in mice, and in NTDs brain tissues the bindings of H3K79me2 to these three genes were significantly altered. Taken together, our study indicated that low folate treatment might attenuate H3K79 dimethylation, further affect its regulate activation on target genes, some of which are NTDs-resulting associated, lastly interrupt early embryo developing. Our study increases the understanding of normal fetal brain histone modifications and provides a platform for investigating histone modifications in neural disease and also has an insight into a potential role of aberrant histone modification in etiology of NTDs.

Maternal exposure to ambient levels of benzene and neural tube defects among offspring: Texas, 1999-2004.

BackgroundPrevious studies have reported positive associations between maternal exposure to air pollutants and several adverse birth outcomes. However, there have been no studies assessing the association between environmental levels of hazardous air pollutants, such as benzene, and neural tube defects (NTDs), a common and serious group of congenital malformations.ObjectiveOur goal was to conduct a case–control study assessing the association between ambient air levels of benzene, toluene, ethylbenzene, and xylene (BTEX) and the prevalence of NTDs among offspring.MethodsThe Texas Birth Defects Registry provided data on NTD cases (spina bifida and anencephaly) delivered between 1999 and 2004. The control group was a random sample of unaffected live births, frequency matched to cases on year of birth. Census tract–level estimates of annual BTEX levels were obtained from the U.S. Environmental Protection Agency 1999 Assessment System for Population Exposure Nationwide. Restricted cubic splines were used in mixed-effects logistic regression models to determine associations between each pollutant and NTD phenotype.ResultsMothers living in census tracts with the highest benzene levels were more likely to have offspring with spina bifida than were women living in census tracts with the lowest levels (odds ratio = 2.30; 95% confidence interval, 1.22–4.33). No significant associations were observed between anencephaly and benzene or between any of the NTD phenotypes and toluene, ethylbenzene, or xylene.ConclusionIn the first study to assess the relationship between environmental levels of BTEX and NTDs, we found an association between benzene and spina bifida. Our results contribute to the growing body of evidence regarding air pollutant exposure and adverse birth outcomes.

Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

The vacuolated lens (vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract, and occasionally a white belly spot. We previously reported that 1) the vl phenotypes are due to a mutation in an orphan G protein-coupled receptor (GPCR), Gpr161; 2) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked quantitative trait loci (QTL) to be mapped (modifiers of vacuolated lens, Modvl1-3); and 3) phenotype-based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract-modifying effect of Modvl3. We now extend this analysis in three ways. First, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined that approximately 50% of vl/vl NTD-affected embryos die during development. Second, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed, and two novel modifiers were mapped [Modvl4, logarithm of odds ratio (LOD) 4.4; Modvl5, LOD 5.0]. Third, phenotype-based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects (Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were resequenced and nonsynonymous coding variants were identified. Bioinformatics and protein modeling suggest that these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.

The orphan G protein-coupled receptor, Gpr161 , encodes the vacuolated lens locus and controls neurulation and lens development

The vacuolated lens (vl) mouse mutant causes congenital cataracts and neural tube defects (NTDs), with the NTDs being caused by abnormal neural fold apposition and fusion. Our positional cloning of vl indicates these phenotypes result from a deletion mutation in an uncharacterized orphan G protein-coupled receptor (GPCR), Gpr161. Gpr161 displays restricted expression to the lateral neural folds, developing lens, retina, limb, and CNS. Characterization of the vl mutation indicates that C-terminal tail of Gpr161 is truncated, leading to multiple effects on the protein, including reduced receptor-mediated endocytosis. We have also mapped three modifier quantitative trait loci (QTL) that affect the incidence of either the vl cataract or NTD phenotypes. Bioinformatic, sequence, genetic, and functional data have determined that Foxe3, a key regulator of lens development, is a gene responsible for the vl cataract-modifying phenotype. These studies have extended our understanding of the vl locus in three significant ways. One, the cloning of the vl locus has identified a previously uncharacterized GPCR-ligand pathway necessary for neural fold fusion and lens development, providing insight into the molecular regulation of these developmental processes. Two, our QTL analysis has established vl as a mouse model for studying the multigenic basis of NTDs and cataracts. Three, we have identified Foxe3 as a genetic modifier that interacts with Gpr161 to regulate lens development.

Maternal fumonisin exposure and risk for neural tube defects: Mechanisms in an in vivo mouse model

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, a common contaminant of corn worldwide. FB1 disrupts sphingolipid biosynthesis by inhibiting the enzyme ceramide synthase, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. A relationship between maternal ingestion of FB1-contaminated corn during early pregnancy and increased risk for neural tube defects (NTDs) has recently been proposed in human populations around the world where corn is a dietary staple. The current studies provide an in vivo mouse model of FB1 teratogenicity. Pregnant LM/Bc mice were injected with increasing doses of FB1 on GD 7.5 and 8.5, and exposed fetuses were examined for malformations. Sphingolipid profiles and (3)H-folate concentrations were measured in maternal and fetal tissues. Immunohistochemical expression of the GPI-anchored folate receptor (Folbp1) and its association with the lipid raft component, ganglioside GM1, were characterized. Rescue experiments were performed with maternal folate supplementation or administration of gangliosides. Maternal FB1 administration (20 mg/kg of body weight) during early gestation resulted in 79% NTDs in exposed fetuses. Sphingolipid profiles were significantly altered in maternal and embryonic tissues following exposure, and (3)H-folate levels and immunohistochemical expression of Folbp1 were reduced. Maternal folate supplementation partially rescued the NTD phenotype, whereas GM1 significantly restored folate concentrations and afforded almost complete protection against FB1-induced NTDs. Maternal FB1 exposure altered sphingolipid metabolism and folate concentrations in LM/Bc mice, resulting in a dose-dependent increase in NTDs that could be prevented when adequate folate levels were maintained.

Epithelial cell polarity genes are required for neural tube closure

Human neural tube defects (NTD) are a heterogeneous group that exhibit complex inheritance, making it difficult to identify the underlying cause. Due to the uniform genetic background, inbred mouse strains are a more amenable target for genetic studies. We investigated the loop-tail (Lp) mouse as a model for the severe NTD, craniorachischisis. A homozygous point mutation was identified in the transmembrane protein Vangl2, which in Drosophila has been shown to function in the planar cell polarity (PCP) pathway. Morphological analysis of the Lp mice shows that the defect results from an abnormally broad floor plate, most likely through a failure in convergent extension. The elevated neural folds remain too far apart to contact, inhibiting neural tube closure. Recently, two other mouse mutants (crash and circletail) were described with a similar phenotype to Lp and were investigated as potentially new alleles. Mapping studies, however, showed that both mutants segregated to distinct loci. In the crash (Crsh) mouse, a mutation was identified in Celsr1, a seven pass transmembrane receptor that encodes a protein orthologous to Drosophila Flamingo. Like Vangl2, this gene also functions in the PCP pathway. While in circletail, a point mutation was identified introducing a premature stop codon into the apical-basal cell polarity gene scribble (Scrb1). We subsequently demonstrated a genetic interaction between all three genes, where double heterozygotes exhibit the same homozygous NTD phenotype. This strongly suggests both a candidate gene pathway and that interaction between independent recessive alleles may be a possible explanation for the complex inheritance in severe human NTD.

TERC is not a major gene in human neural tube defects.

Neural tube defects (NTDs) are the second most common birth defects, after congenital heart defects. Telomerase, the reverse transcriptase that maintains telomere DNA, has been shown to be important for neural tube development and bilateral symmetry in the brain. In knockout mice null for the telomerase RNA component (TERC), telomere loss results in the failure of neural tube closure, primarily at the forebrain and midbrain. We investigated TERC for variants that may predispose to human NTDs in 477 NTD cases with a variety of phenotypic presentations. Two novel single nucleotide polymorphisms were identified in the human TERC sequence but showed no association with the NTD phenotype. Variants in TERC are unlikely to be a major risk factor for the most common form of human NTDs, lumbosacral myelomeningocele.

Genetic susceptibility to neural tube defect pregnancy varies with offspring phenotype

Neural tube defects (NTDs) have a well-established genetic basis, although no single genetic factor has been identified as a major risk factor in NTD susceptibility. A large number of association studies have been conducted to investigate the possibility that NTD susceptibility is linked to polymorphic variation in genes involved in early embryonic development or in the absorption or metabolism of folate, a nutrient that has been clearly associated with a reduction in the risk of NTD pregnancy. A study of three candidate gene polymorphisms at loci implicated in folate absorption and metabolism has been conducted on a population of 211 mothers of a heterogeneous mix of NTD phenotypes: 59% spina bifida aperta (SBA), 20.3% spina bifida occulta (SBO), 17% anencephaly, and 3.7% other NTD. Allele and genotype frequencies were stratified according to offspring NTD phenotype, and variation in the level of NTD risk was associated with different phenotypes. All the three variants (MTHFR 677C > T, GCPII 1561C > T, and RFC-1 80G > A) were shown to significantly influence the risk of anencephalic pregnancy. In addition, the MTHFR 677C > T variant conferred a modest protective effect in SBO mothers and the total NTD mother group, but not in SBA mothers. The RFC-1 80G > A variant elevated the risk of SBO and anencephalic pregnancy. The findings of this study suggest that NTD phenotypic heterogeneity may help explain the mixed findings of previous association studies and that different polymorphisms may hold differing degrees of significance for the various NTD phenotypes.