Role of amniotic fluid homocysteine level and of fetal 5,10‐methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects

A mutation in the gene 5,10‐methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C→T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C→T MTHFR mutation (44% vs 17%, P ≤ 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 μmol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex. Am. J. Med. Genet. 90:12–16, 2000. © 2000 Wiley‐Liss, Inc.     

Amniotic fluid homocysteine levels, 5,10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites

A specific gene mutation leading to altered homocysteine metabolism has been identified in parents and fetuses with neural tube defects (NTDs). In addition, current animal and human data indicate that spine closure occurs simultaneously in five separate sites that then fuse. We sought to determine whether either this mutation or abnormal amniotic fluid homocysteine levels are associated with all five neural tube closure sites. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80) and from normal controls matched for race, month and year of amniocentesis, and maternal age. Cases were categorized according to defect site by using all available medical records. The presence or absence of the 677C-->T mutation of 5, 10-methylenetetrahydrafolate reductase (MTHFR) gene was determined, and homocysteine levels were measured; case and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs. 17%, P < or = 0. 001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 micromol/L), 27% vs. 10%, P = 0.02. Most (83%) of control cases had both normal MTHFR alleles and normal amniotic fluid homocysteine levels (normal/normal), whereas only 56% of NTD case were normal/normal (P = 0.001). When evaluated by defect site, only defects involving the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele were significantly less likely to be normal/normal than controls (P = 0.007, 0.0003, and 0.007, respectively), suggesting a strong association with the 677C-->T allele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum included many cases that had both normal MTHFR alleles and normal homocysteine and were not significantly different from controls. The 677C-->T MTHFR mutation and elevated homocysteine levels appear to be disproportionately associated with defects spanning the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum may not be related to altered homocysteine metabolism.     

Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase,folate levels in red blood cells,and risk of neural tube defects

Folic acid administration to women in the periconceptional period reduces the occurrence of neural tube defects (NTDs) in their offspring. A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), 677C→12;T, is the first genetic risk factor for NTDs in man identified at the molecular level. The gene encoding another folate-dependent enzyme, methionine synthase (MTR), has recently been cloned and a common variant, 2756A→12;G, has been identified. We assessed genotypes and folate status in 56 patients with spina bifida, 62 mothers of patients, 97 children without NTDs (controls), and 90 mothers of controls, to determine the impact of these factors on NTD risk. Twenty percent of cases and 18% of case mothers were homozygous for the MTHFR polymorphism, compared to 11% of controls and 11% of control mothers, indicating that the mutant genotype conferred an increased risk for NTDs. The risk was further increased if both mother and child had this genotype. The MTR polymorphism was associated with a decreased O.R. (O.R.); none of the cases and only 10% of controls were homozygous for this variant. Red blood cell (RBC) folate was lower in cases and in case mothers, compared to their respective controls. Having a RBC folate in the lowest quartile of the control distribution was associated with an O.R. of 2.56 (95% CI 1.28–5.13) for being a case and of 3.05 (95% CI 1.54–6.03) for being a case mother. The combination of homozygous mutant MTHFR genotype and RBC folate in the lowest quartile conferred an O.R. for being a NTD case of 13.43 (CI 2.49–72.33) and an O.R. for having a child with NTD of 3.28 (CI 0.84–12.85). We propose that the genetic-nutrient interaction—MTHFR polymorphism and low folate status—is associated with a greater risk for NTDs than either variable alone. Am. J. Med. Genet. 84:151–157, 1999. © 1999 Wiley-Liss, Inc.     

Single‐site neural tube closure in human embryos revisited

Since the multi‐site closure theory was first proposed in 1991 as explanation for the preferential localizations of neural tube defects, the closure of the neural tube has been debated. Although the multi‐site closure theory is much cited in clinical literature, single‐site closure is most apparent in literature concerning embryology. Inspired by Victor Hamburgers (1900–2001) statement that “our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right”, we decided to critically review both theories of neural tube closure. To verify the theories of closure, we studied serial histological sections of 10 mouse embryos between 8.5 and 9.5 days of gestation and 18 human embryos of the Carnegie collection between Carnegie stage 9 (19–21 days) and 13 (28–32 days). Neural tube closure was histologically defined by the neuroepithelial remodeling of the two adjoining neural fold tips in the midline. We did not observe multiple fusion sites in neither mouse nor human embryos. A meta‐analysis of case reports on neural tube defects showed that defects can occur at any level of the neural axis. Our data indicate that the human neural tube fuses at a single site and, therefore, we propose to reinstate the single‐site closure theory for neural tube closure. We showed that neural tube defects are not restricted to a specific location, thereby refuting the reasoning underlying the multi‐site closure theory. Clin. Anat. 30:988–999, 2017. © 2017 Wiley Periodicals, Inc.     

Bmp2 is required for cephalic neural tube closure in the mouse

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2 chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2 mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose‐dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and normal cephalic neural tube closure in a dose‐dependent manner. Developmental Dynamics 238:110–122, 2009. Published 2008 Wiley‐Liss, Inc.     

5,10 Methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects

Persons with a thermolabile form of the enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) have reduced enzyme activity and increased plasma homocysteine which can be lowered by supplemental folic acid. Thermolability of the enzyme has recently been shown to be caused by a common mutation (677C^→T) in the MTHFR gene. We studied 41 fibroblast cultures from NTD-affected fetuses and compared their genotypes with those of 109 blood specimens from individuals in the general population. 677C^→T homozygosity was associated with a 7.2 fold increased risk for NTDs (95% confidence interval: 1.8–30.3; p value: 0.001). These preliminary data suggest that the 677C^→T polymorphism of the MTHFR gene is a risk factor for spina bifida and anencephaly that may provide a partial biologic explanation for why folic acid prevents these types of NTD.     

Possible interaction of genotypes at cystathionine β-synthase and methylenetetrahydrofolate reductase (MTHFR) in neural tube defects

Neural tube defects are a common, complex disorder with genetic and environmental components to risk. We investigated the previously reported interaction between homozygosity for the thermolabile variant at the methylenetetrahydrofolate reductase and heterozygosity for the 844ins68 allele at the cystathionine beta-synthase loci in cases with lumbosacral myelomeningocele and their parents. Using control allele frequencies from our sample pooled with those published in the literature, we confirm a marginally significant interaction at these two loci. This finding suggests that additional, larger studies are warranted to investigate this possible interaction in more detail.     

Sex,neural tube defects,and multisite closure of the human neural tube

While neural tube defects (NTD) overall have a female sex bias, this does not apply to all sites along the neuraxis. The findings regarding sex and NTD in a series of midtrimester fetuses are reviewed, and then analysed in terms of the recent hypothesis that during embryogenesis of the human neural tube there are multiple closure sites, rather than a single zipping up process. Females more often than males tend to have craniorachischisis, spina bifida involving the thorax, the holoacrania form of anencephaly, anencephaly and cervical spina bifida and encephalocoeles, while males more often than females have spina bifida affecting the lower spine. Meroacrania occurs equally in both sexes. Other sources indicate that there is a male bias in frontoethmoidal encephalocoeles. Since sex seems to be a factor that is differentially associated with lack of closure of specific areas of the neural tube, it would seem to support the notion that there are multiple closure sites in the human neural tube. However, no association was found between a particular sex and either the type of NTD which have an isolated abnormality or those NTD associated with developmental abnormalities of other body systems. © 1995 Wiley-Liss, Inc.     

Amniotic fluid folate, vitamin B12 and transcobalamins in neural tube defects

Levels of folate, vitamin B12, the vitamin B12 binding proteins, apotranscobalamin I, II and III (TC I, II and III) and the unsaturated vitamin B12 binding capacity (UBBC) were measured in mid-trimester amniotic fluids from normal pregnancies, and from those where the fetus had open spina bifida, anencephaly or omphalocoele, and where the fetus was normal but the mother had had a previous neural tube defect pregnancy. At 15-19 weeks' gestation, vitamin B12 levels were low in the fluids of all the types of abnormal fetuses, and also of normal fetuses where there had been a previous NTD sib. In contradistinction, TC I, II and III and UBBC levels were generally abnormally high in all these groups. Low vitamin B12 levels in the face of high carrier protein levels suggest deranged vitamin B12 production or transport. Since these abnormalities are present in fluids from normal sibs of NTD individuals as well as from those with midline lesions, an inherited defect is implied. We propose that at least part of the genetic predisposition to NTD, and possibly other midline defects, could reside in an abnormality connected with vitamin B12 production, transport or metabolism, and a mechanism is suggested.     

Multi-site neural tube closure in humans and maternal folate supplementation

A group of 13 individuals with neural tube defects (NTD) despite maternal periconceptional folate supplementation was examined to determine precisely which closure sites along the neural tube had failed during embryogenesis. All the common forms of NTD, and thus all the usual closure sites, were represented. This suggests that folate deficiency does not act specifically on one region of the neural tube; rather, it may be more generally involved in the cause of human NTD. © 1995 Wiley-Liss, Inc.     

生育过神经管缺陷儿的妇女MTHFR基因C677T多态性与血浆Hcy水平测定

目的探讨MTHFR基因C677T多态性及血浆Hcy水平与神经管缺陷的关系。方法采用PCR-RFLP方法对30例生育过NTD患儿的母亲（NTD组）和34例生育过正常儿的母亲（对照组）进行MTHFR基因C677T等位基因检测,同时用高效液相色谱结合荧光检测法对两组母亲血浆中Hcy水平进行测定。结果NTD母亲组中VV基因型的频率为（36.7%）,V等位基因频率为（65.0%）,正常对照母亲组中VV基因型的频率为（17.6%）,V等位基因频率为（44.1%）,两组比较有显著性差异（P〈0.05）。NTD母亲组Hcy水平为（11.24±3.2）μmol/L,正常对照母亲组Hcy水平为（8.96±3.3）μmol/L,两组比较也有显著性差异（P〈0.01）。结论MTHFR基因C677T多态是神经管缺陷发病的遗传危险因素,它通过血浆中同型半胱氨酸水平升高而引起神经管缺陷。     

Mammalian embryos show metabolic plasticity toward the surrounding environment during neural tube closure

Developing embryos rewire energy metabolism for developmental processes. However, little is known about how metabolic rewiring is coupled with development in a spatiotemporal manner. Here, we show that mammalian embryos display plasticity of glucose metabolism in response to the extracellular environment at the neural tube closure (NTC) stage, when the intrauterine environment changes upon placentation. To study how embryos modulate their metabolic state upon environmental change, we analyzed the steady‐state level of ATP upon exposure to extrauterine environments using both an enzymatic assay and a genetically encoded ATP sensor. Upon environmental changes, NTC‐stage embryos exhibited increased ATP content, whereas embryos before and after NTC did not. The increased ATP in the NTC‐stage embryos seemed to depend on glycolysis. Intriguingly, an increase in mitochondrial membrane potential (ΔΨm) was also observed in the neural ectoderm (NE) and the neural plate border of the non‐neural ectoderm (NNE) region. This implies that glycolysis can be coupled with the TCA cycle in the NE and the neural plate border depending on environmental context. Disrupting ΔΨm inhibited folding of the cranial neural plate. Thus, we propose that embryos tune metabolic plasticity to enable coupling of glucose metabolism with the extracellular environment at the NTC stage.     

Generation and characterization of a novel neural crest marker allele,Inka1‐LacZ,reveals a role for Inka1 in mouse neural tube closure

Previous studies identified Inka1 as a gene regulated by AP‐2α in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock‐in allele. Inka1‐LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1^?/? mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP‐2α is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not. Developmental Dynamics 239:1188–1196, 2010. © 2010 Wiley‐Liss, Inc.     

Increased levels of apo-transcobalamins I and II in amniotic fluid from pregnant women with previous neural tube defect offspring

In an attempt to identify biochemical components of the genetic predisposition to neural tube defects (NTDs), levels of folate, cobalamin, apo-transcobalamins I and II and alpha-fetoprotein were studied in midtrimester amniotic fluid from 24 pregnant women who had previously had a child with NTD. The control group consisted of 76 mothers, subjected to amniocentesis for reasons other than risk of NTD in offspring. Only pregnancies with normal outcome were included. No differences were found between groups for levels of folate, cobalamin or alpha-fetoprotein. Folate intake or metabolism did not appear to differ between groups. In contrast, the level of apo-transcobalamin I was doubled and the level of apo-transcobalamin II tripled in amniotic fluid from women who had had a child with NTD compared with the control group. Since the variation in apo-transcobalamin II in adults is to a high degree genetically determined, the present results may suggest that the genetic predisposition to NTD is associated with variation in this protein. Further studies are needed to substantiate or reject this possibility.     

Open neural tube defects: Immunocytochemical demonstration of neuroepithelial cells in amniotic fluid

Cytologic evaluation of second trimester amniotic fluid (AF) is a rapid, inexpensive adjunct to prenatal diagnosis of open neural tube defects (ONTDs). Our goal was to determine whether the neural-appearing cells and/or large foamy macrophages in the AF of anencephalics are indeed of neural and/or glial origin. In two second trimester patients with elevated serum alpha-fetoprotein (AFP) and polyhydramnios, fetal sonogram studies showed anencephaly; amniocentesis was performed for AF-AFP, cytogenetic, and cytologic studies. AF sediment smears were initially Papanicolaou-stained; next, the same smears were immunoperoxidase (IP)-stained for glial fibrillary acidic protein (GFAP). If GFAP negative, slides were restained for synaptophysin (SYN) and neuron-specific enolase (NSE). Both AFs contained small neural-appearing cells (5–10 μm) singly and in clusters, with dense, round, homogeneous nuclei, an occasional nucleolus, and scant cytoplasmic rim. These were GFAP negative and SYN and NSE positive; the large vacuolated, lipid-laden macrophages (20–40 μm) were negative for all three IP stains. In conclusion, positive IP staining for SYN and NSE supports the morphologic impression that small dark cells in AF are of neural origin, while negative IP staining of large foamy macrophages suggests nonneural, nonglial origin. Diagn. Cytopathol. 16:143–144, 1997. © 1997 Wiley-Liss, Inc.     

The thymic findings in stillborns with neural tube defects

Neural tube defects are among the most common congenital anomalies causing perinatal deaths. Other organ system anomalies may be associated with neural tube defects: for instance, various types of thymus pathology have been reported in these patients. In this study thymic changes were investigated in 30 stillborns with neural tube defects seen between January 1988 and June 1989. Thymic weights were significantly reduced in 14 cases and increased in 7. One patient had a double thymus, and in two cases no thymus tissue could be found. These findings suggest a primary developmental defect of the neural crest, affecting the orderly development of the thymus gland.     

Amniotic fluid analysis in prenatal diagnosis of neural tube defects: a comparison between six biochemical tests supplementary to the measurement of amniotic fluid alpha-fetoprotein

Concanavalin A (con A) and lens culinaris agglutinin (LCA) microheterogeneity pattern of AFP (crossed affinity immunoelectrophoresis), alpha-2-macroglobulin and synaptic membrane protein D-2 (rocket immunoelectrophoresis) and qualitative (polyacrylamide gel electrophoresis) and quantitative (enzyme kinetic reaction rate) acetylcholinesterase were analysed in 87 consequtive samples from normal pregnancies and 37 abnormal samples (fetal neural tube defect or abdominal wall defect). Very few false positive results were obtained in normal pregnancies with any of the tests. In all cases of neural tube defects the correct result was obtained with qualitative acetylcholinesterase analysis, whereas only 2/3 of the abdominal wall defects were correctly predicted. Testing with con A or LCA was less optimal in neural tube defects, whereas all abdominal wall defects could be predicted correctly. Acetylcholinesterase in the quantitative test and protein D-2 did not decrease the rate of false results. Determination of the alpha-2-macroglobulin concentration performed well in the present study, but is not recommended because of the very high susceptibility to contamination of amniotic fluid with fetal or maternal blood.     

Updated investigations of the role of methylenetetrahydrofolate reductase in human neural tube defects

Folate supplementation appears to reduce the risk for neural tube defects (NTDs). Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene in the folate metabolism pathway that has been extensively studied in different human populations. We examined the risk associated with having the thermolabile variant (TT) of MTHFR in a study of 175 American Caucasians with NTDs and their families. We found a significant association in patients compared with 195 unrelated controls [odds ratio (OR) = 2.13, 95% confidence interval (95% CI) = 1.11-4.09)], but not in mothers (OR = 1.29, 95% CI = 0.622-2.67) or in fathers (OR = 1.45, 95% CI = 0.681-3.09). We found no evidence for unequal transmission from parents to an affected child (p > 0.10). We failed to find a previously reported association for a combined haplotype for MTHFR and cystathionine beta-synthase, except in subjects with NTDs compared with 559 pooled controls (OR = 2.87, 95% CI = 1.03-8.03). We found no evidence for an association for a novel CA-repeat polymorphism identified in a gene closely linked to MTHFR (p > 0.10). Our studies continue to suggest that additional candidate genes other than MTHFR may be responsible for an increased risk to NTD in some American Caucasian families.