Identification and characterization of novel rare mutations in the planar cell polarity gene PRICKLE1 in human neural tube defects

The planar cell polarity (PCP) pathway controls the process of convergent extension (CE) during gastrulation and neural tube closure, and has been implicated in the pathogenesis of neural tube defects (NTDs) in animal models and human cohorts. In this study, we analyzed the role of one core PCP gene PRICKLE1 in these malformations. We screened this gene in 810 unrelated NTD patients and identified seven rare missense heterozygous mutations that were absent in all controls analyzed and predicted to be functionally deleterious using bioinformatics. Functional validation of five PRICKLE1 variants in a zebrafish model demonstrated that one variant, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a (zpk1a) in a dominant fashion. Our study demonstrates that PRICKLE1 could act as a predisposing factor to human NTDs and further expands our knowledge of the role of PCP genes in the pathogenesis of these malformations.     

Novel mutations in VANGL1 in neural tube defects

Neural tube defects (NTDs) are severe congenital malformations caused by failure of the neural tube to close during neurulation. Their etiology is complex involving both environmental and genetic factors. We have recently reported three mutations in the planar cell polarity gene VANGL1 associated with NTDs. The aim of the present study was to define the role of VANGL1 genetic variants in the development of NTDs in a large cohort of various ethnic origins. We identified five novel missense variants in VANGL1, p.Ser83Leu, p.Phe153Ser, p.Arg181Gln, p.Leu202Phe and p.Ala404Ser, occurring in sporadic and familial cases of spinal dysraphisms. All five variants affect evolutionary conserved residues and are absent from all controls analyzed. This study provides further evidence supporting the role of VANGL1 as a risk factor in the development of spinal NTDs. © 2009 Wiley‐Liss, Inc.     

Identification of novel rare mutations of DACT1 in human neural tube defects

Neural tube defects (NTDs) constitute the second most frequent cause of human congenital abnormalities. Complex multigenetic causes have been suggested to contribute to NTDs. The planar cell polarity (PCP) pathway plays a critical role in neural tube closure in model organisms and in human. Knockout of Dact1 (Dapper, Frodo) leads to deregulated PCP signaling with defective neural tube in mice. Here, we report that five missense heterozygote mutations of the DACT1 gene are specifically identified in 167 stillborn or miscarried Han Chinese fetuses with neural tube defects. Our biochemical analyses revealed that among the five mutations, N356K and R45W show loss‐of‐function or reduced activities in inducing Dishevelled2 (DVL2) degradation and inhibiting jun‐N‐terminal kinase (JNK) phosphorylation, implicating mutated DACT1 as a risk factor for human NTDs. Our findings, together with early reports, suggest that rare mutations of the PCP‐related genes may constitute a great contribution to human NTDs. Hum Mutat 33:1450–1455, 2012. © 2012 Wiley Periodicals, Inc.     

Contribution of VANGL2 mutations to isolated neural tube defects

Kibar Z, Salem S, Bosoi CM, Pauwels E, De Marco P, Merello E, Bassuk AG, Capra V, Gros P. Contribution of VANGL2 mutations to isolated neural tube defects. Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non‐canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi‐ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.     

Exploring gene—gene interactions in the etiology of neural tube defects

The role of susceptibility genes in the etiology of birth defects is unclear, but may involve in some cases multiple alleles at multiple loci. We suggest a simple epidemiologic approach to explore gene-gene interactions, and use it to reevaluate data from a recent case-control study on the possible association of neural tube defects (NTDs) with specific mutations of two genes, 5,10-methylene-tetrahydrofolate reductase (MTHFR) and cystathionine-β synthase (CBS). We found that, compared with the common genotype, homozygosity for the MTHFR mutation alone was associated with a two-fold increased risk for NTDs, while homozygosity for the CBS mutation alone was not a risk factor. However, homozygous individuals for the mutations at both loci had a five-fold greater risk for NTDs than those with the reference genotype. Though the original study was too small to detect statistically significant differences among most of the risk estimates, these results, if confirmed by independent and larger studies, suggest that gene-gene interaction may play a role in modulating the susceptibility to NTDs in a proportion of affected individuals. This approach, moreover, could be a valuable adjunct to the study of gene-gene interactions in the etiology of human disease.     

FZD6 is a novel gene for human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6(-/-) mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3'-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs.     

CD1+ cells in mothers of stillborn infants with neural tube defects

In healthy individuals, CD1+ cells are found in thymic tissue, but not in peripheral blood. The thymus, as a key organ of the neuroendocrine system, frequently shows gross abnormalities in infants with neural tube defects. In order to study the immuno-logic significance of fetal thymic findings, maternal T-lymphocyte subpopulations were investigated. In 10 mothers of healthy new-borns, 5 mothers of stillborn infants who had no gross abnormalities, and 5 mothers of stillborn infants with neural tube defects, CD1+, CD3+, CD4+, and CD8+ cells were studied. Only the mothers of the infants with open neural tube defects showed CD1 + cells in their peripheral blood. © 1995 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.     

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.     

Transcobalamins in the etiology of neural tube defects

In a sample of 79 pregnant women at risk offspring with neural tube defects (NTDs) and 158 controls, significantly increased median values were found for apo-transcobalamins I and II in amniotic fluid in the group at risk, thus confirming previous results. The findings may reflect a genetic disposition to NTDs associated with altered levels of apo-transcobalamins, and research on the etiology and mechanisms of NTDs should focus on these proteins.     

AMBRA1, a novel α‐synuclein‐binding protein,is implicated in the pathogenesis of multiple system atrophy

The accumulation of abnormal α‐synuclein is the major histopathological feature of Lewy body disease and multiple system atrophy (MSA), which are referred to as synucleinopathies. Cytoplasmic degradation systems, such as the autophagy‐lysosome and proteasome pathways, are involved in their pathogenesis. Autophagy is tightly regulated by several upstream proteins including UNC‐51‐like kinase 1/2, beclin1, vacuolar protein sorting‐associated protein 34 and autophagy/beclin1 regulator 1 (AMBRA1). Recently, we revealed that both cortical and brainstem‐type Lewy bodies were immunopositive for several upstream proteins of autophagy. Therefore, we conducted the present study to elucidate the role of upstream proteins of autophagy in the pathogenesis of MSA. Pathological and biochemical analyses using human brain samples revealed that AMBRA1 is a component of the pathological hallmarks of MSA and upstream proteins of autophagy are impaired in the MSA brain. In vitro and in vivo analyses revealed a ninefold stronger affinity of AMBRA1 with α‐synuclein phosphorylated at serine 129 compared with non‐phosphorylated α‐synuclein. Furthermore, a weak but significant correlation between AMBRA1 overexpression and reduction of abnormal α‐synuclein was observed. Silencing AMBRA1 function caused aggregates of α‐synuclein in the cytoplasm of mouse primary cultured neurons, which was simulated by the treatment of Bafilomycin, an autophagy inhibitor. Our results demonstrated for the first time that AMBRA1 is a novel hub binding protein of α‐synuclein and plays a central role in the pathogenesis of MSA through the degradative dynamics of α‐synuclein. These results raise the possibility that molecular modulation targeting AMBRA1 can be a promising candidate for the treatment of synucleinopathies.     

应用银染mRNA差异显示法寻找高温致神经管畸形差异表达基因

目的 :寻找高温致神经管畸形的差异表达基因。方法 :在高温致神经管畸形的动物模型上 ,分别于高温处理后 2 4、48和 72小时 ,提取鼠胚神经管组织总RNA和正常对照组鼠胚相应时间的神经管组织总RNA ,反转录合成cDNA第一链后进行差异显示PCR扩增 ,采用PAGE和银染技术显示差异条带 ,回收差异条带并经PCR二次扩增后 ,用点杂交方法筛除假阳性条带 ,再用Northern印迹杂交进一步鉴定。结果 :在高温致畸的鼠胚神经管组织中筛选到一个特别明显的差示cDNA片段N3 2 ,该片段所在基因在高温致畸的胚胎神经管组织中的表达远低于正常同龄胚的神经管组织。结论 :N3 2片段所在基因的低表达与高温致神经管畸形相关。     

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.     

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.     

HMGB1在高温致金黄地鼠神经管畸形神经上皮细胞中的表达变化

为了探讨高温致神经管畸形(NTDs)作用的分子机制,为预防人类NTDs的发生提供理论依据,本研究在高温致金黄地鼠NTDs模型的基础上,研究HMGB1在高温致金黄地鼠NTDs神经上皮细胞中的表达变化。将孕鼠随机分为实验组和对照组,分别于水浴处理后8、16、24、40、64 h(相当于胚龄第8、8.5、9、9.5、10.5 d)处死孕鼠,剖腹取鼠胚,制备石蜡切片,应用免疫荧光染色技术检测NTDs发生过程中HMGB1在神经上皮细胞中的表达变化。结果显示:对照组和模型组孕鼠在水浴处理后8、16、24h,HMGB1免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后40、64 h,HMGB1表达部位出现了由胞浆向胞核的转移;高温处理后,HMGB1在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱。上述结果提示,HMGB1的表达变化与神经管发育密切相关,其表达降低是高温致神经管畸形发生的重要途径之一。     

Elevated rates of severe neural tube defects in a high-prevalence area in Northern China

In the northern provinces of China, the birth prevalence rate of neural tube defects (NTDs) is among the highest in the world-at about 6 per 1,000 births in rural areas. A unique population-based birth defects surveillance system in which photographs are taken of infants with selected external birth defects was implemented in two provinces in northern China and two provinces in southern China where NTD rates approximate those in the United States. In the period from March 1992 through December 1993, 660 infants with NTDs were identified by the surveillance project from a birth cohort of 251,567. We compared data from the two surveillance areas in China with data from a low-prevalence area in the United States to determine if the pattern of NTD types differs. Based on birth prevalence rates of NTDs from the Metropolitan Atlanta Congenital Defects Program, the observed to expected ratios for two types of NTDs are markedly increased at 80.8 for craniorachischisis and 25.0 for iniencephaly. Rates of these two NTDs in the southern provinces are increased to a lesser degree with observed to expected ratios of 7.1 for craniorachischisis and 2.7 for iniencephaly. The pattern of NTDs in northern China shows an increase in types that are rare in low-prevalence areas such as metropolitan Atlanta. Increased awareness of varying patterns of NTDs in different populations may have important implications for identifying etiologic and pathogenetic mechanisms of NTDs. Am. J. Med. Genet. 73:113–118, 1997. © 1997 Wiley-Liss, Inc. This article was prepared by a group consisting of both United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.     

Are common mutations of cystathionine β-synthase involved in the aetiology of neural tube defects?

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defect (NTD) pregnancies. Plasma levels of Hcy are governed by both genetic and nutritional factors and the aetiology of NTDs is also known to have both genetic and nutritional components. We therefore examined the frequency of relatively common mutations in the enzyme cystathionine β-synthase (CBS), which is one of the main enzymes that controls Hcy levels, in the NTD population. Neither the severely dysfunctional G307S CBS allele nor the recently reported 68 bp insertion/I278T CBS allele was observed at increased frequency in the cases relative to controls. We therefore conclude that loss of function CBS alleles do not account for a significant proportion of NTDs in Ireland.     

Multigeneration maternal transmission in Italian families with neural tube defects

Periconceptional vitamin supplementation with folate prevents about three-quarters of expected cases of neural tube defects (NTDs) in clinical trials. However, vitamin action may be regulated at the level of the gene, and individual susceptibility to environmental agents, including dietary components, also may be under genetic control. We investigated the presence of familial factors in a retrospective case control study of neural tube defects in Genoa, Italy. Cases included all patients treated at a single pediatric neurosurgical service. Controls matched on age and sex came from the same hospital. We found strong evidence for the contribution of genetic factors in this study. There was an excess risk of 14 for the occurrence of NTDs in first-degree relatives compared to controls (P < .0005). There was no difference in sex ratio in any group of relatives, but maternal grandparents of children with a high spinal lesion had 14% fewer offspring than paternal grandparents (P < .005), possibly because of excess miscarriages. Our study is the first to show complex patterns of inheritance in spina bifida families affecting three generations in one clinical subgroup and preferentially on the mother's side. These results support a role for genomic imprinting and highlight the value of multidisciplinary epidemiologic and clinical studies that include multiple generations. New studies incorporating dietary and genetic approaches will help clarify and extend these findings. © 1996 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 ≤ 0.001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 μmol/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. Am. J. Med. Genet. 90:6–11, 2000. © 2000 Wiley‐Liss, Inc.