Scribble1 plays an important role in the pathogenesis of neural tube defects through its mediating effect of Par-3 and Vangl1/2 localization.

Scribble1 (Scrib1) is a tumor suppressor gene that has long been established as an essential component of apicobasal polarity (ABP). In mouse models, mutations in Scrib1 cause a severe form of neural tube defects (NTDs) as a result of a defective planar cell polarity (PCP) signaling. In this study, we dissected the role of Scrib1 in the pathogenesis of NTDs in its mouse mutant Circletail (Crc), in cell lines and in a human NTD cohort. While there were no obvious defects in ABP in the Scrib1Crc/Crc neuroepihelial cells, we identified an abnormal localization of the apical protein Par-3 and of the PCP protein Vangl2. These results were concordant with those obtained following a partial knockdown of Scrib1 in MDCK II cells. Par-3 was able to rescue the localization defect of Vangl1 (paralog of Vangl2) caused by partial knockdown of Scrib1 suggesting that Scrib1 exerts its effect on Vangl1 localization indirectly through Par-3. This conclusion is supported by our findings of an apical enrichment of Vangl1 following a partial knockdown of Par-3. Re-sequencing analysis of SCRIB1 in 473 NTD patients led to the identification of 5 rare heterozygous missense mutations that were predicted to be pathogenic. Two of these mutations, p.Gly263Ser and p.Gln808His, and 2 mouse NTD mutations, p.Ile285Lys and p.Glu814Gly, affected Scrib1 membrane localization and its modulating role of Par-3 and Vangl1 localization. Our study demonstrates an important role of Scrib1 in the pathogenesis of NTDs through its mediating effect of Par-3 and Vangl1/2 localization and most likely independently of ABP.

Rare deleterious variants in GRHL3 are associated with human spina bifida.

Neural tube defects, including spina bifida, are among the most common birth defects caused by failure of neural tube closure during development. They have a complex etiology involving largely undetermined environmental and genetic factors. Previous studies in mouse models have implicated the transcription factor Grhl3 as an important factor in the pathogenesis of spina bifida. In the present study, we conducted a resequencing analysis of GRHL3 in a cohort of 233 familial and sporadic cases of spina bifida. We identified two novel truncating variants: one homozygous frameshift variant, p.Asp16Aspfs*10, in two affected siblings and one heterozygous intronic splicing variant, p.Ala318Glyfs*26. We also identified five missense variants, one of which was demonstrated to reduce the activation of gene targets in a luciferase reporter assay. With the previously identified p.Arg391Cys variant, eight variants were found in GRHL3. Comparison of the variant rate between our cohort and the ExAC database identified a significant enrichment of deleterious variants in GRHL3 in the whole gene and the transactivation region in spina bifida patients. These data provide strong evidence for a role of GRHL3 as a predisposing factor to spina bifida and will help dissect the complex etiology and pathogenic mechanisms of these malformations.

Novel mutations in Lrp6 orthologs in mouse and human neural tube defects affect a highly dosage-sensitive Wnt non-canonical planar cell polarity pathway.

Wnt signaling has been classified as canonical Wnt/ß-catenin-dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs), respectively. Both pathways seem to antagonize each other, and recent studies have implicated a number of molecular switches that activate one pathway while simultaneously inhibiting the other thereby partially mediating this antagonism. The lipoprotein receptor-related protein Lrp6 is crucial for the activation of the Wnt/ß-catenin pathway, but its function in Wnt/PCP signaling remains largely unknown. In this study, we investigate the role of Lrp6 as a molecular switch between both Wnt pathways in a novel ENU mouse mutant of Lrp6 (Skax26(m1Jus)) and in human NTDs. We demonstrate that Skax26(m1Jus) represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6(Skax26-Jus) genetically interacts with a PCP mutant (Vangl2(Lp)) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells' polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate the importance of a tightly regulated and highly dosage-sensitive antagonism between both Wnt pathways in this process.

Sacral agenesis: a pilot whole exome sequencing and copy number study.

BACKGROUND: Caudal regression syndrome (CRS) or sacral agenesis is a rare congenital disorder characterized by a constellation of congenital caudal anomalies affecting the caudal spine and spinal cord, the hindgut, the urogenital system, and the lower limbs. CRS is a complex condition, attributed to an abnormal development of the caudal mesoderm, likely caused by the effect of interacting genetic and environmental factors. A well-known risk factor is maternal type 1 diabetes. METHOD: Whole exome sequencing and copy number variation (CNV) analyses were conducted on 4 Caucasian trios to identify de novo and inherited rare mutations. RESULTS: In this pilot study, exome sequencing and copy number variation (CNV) analyses implicate a number of candidate genes, including SPTBN5, MORN1, ZNF330, CLTCL1 and PDZD2. De novo mutations were found in SPTBN5, MORN1 and ZNF330 and inherited predicted damaging mutations in PDZD2 (homozygous) and CLTCL1 (compound heterozygous). Importantly, predicted damaging mutations in PTEN (heterozygous), in its direct regulator GLTSCR2 (compound heterozygous) and in VANGL1 (heterozygous) were identified. These genes had previously been linked with the CRS phenotype. Two CNV deletions, one de novo (chr3q13.13) and one homozygous (chr8p23.2), were detected in one of our CRS patients. These deletions overlapped with CNVs previously reported in patients with similar phenotype. CONCLUSION: Despite the genetic diversity and the complexity of the phenotype, this pilot study identified genetic features common across CRS patients.

Spinal lipoma as a dysembryogenetic anomaly: Four unusual cases of ectopic iliac rib within the spinal lipoma.

BACKGROUND: Congenital spinal lipomas are closed spinal dysraphisms belonging to the neural tube defects (NTDs) group. They include a broad spectrum of lesions ranging from simple lipomas of the filum terminale to complex malformations. On histological evaluation, various tissue components of ectodermal, mesodermal or endodermal origin are found within the lipomas, with prevalence for nerves and striated muscle and, more rarely, cartilage and bone. Overall, rib malformations have been occasionally observed in patients with NTDs and in NTD mouse models. However, an ectopic rib arising within the spinal lipoma and articulating with the iliac crest has not been reported in either animal models or in humans. CASES: We describe four patients affected by lipomyeloschisis or lipomyelomeningocele, with an unusual fibrocartilaginous protuberance arising within the lipoma and connecting to one iliac crest, strongly resembling an ectopic rib. Histological evaluation confirmed the presence of cartilaginous tissue. CONCLUSION: We expand the clinical spectrum of fibrocartilaginous anomalies associated with spinal lipoma, suggesting the presence of an ectopic rib as a new possible phenotype in NTDs. A careful analysis by neuroradiologists and pathologists should be performed in spinal lipomas to assess the presence of an ectopic rib or other uncommon developmental anomalies. Furthermore, molecular studies are required to detect the genetic cause of this unusual phenotype. Birth Defects Research (Part A) 106:530-535, 2016. © 2016 Wiley Periodicals, Inc.

Loss-of-function de novo mutations play an important role in severe human neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are very common and severe birth defects that are caused by failure of neural tube closure and that have a complex aetiology. Anencephaly and spina bifida are severe NTDs that affect reproductive fitness and suggest a role for de novo mutations (DNMs) in their aetiology. METHODS: We used whole-exome sequencing in 43 sporadic cases affected with myelomeningocele or anencephaly and their unaffected parents to identify DNMs in their exomes. RESULTS: We identified 42 coding DNMs in 25 cases, of which 6 were loss of function (LoF) showing a higher rate of LoF DNM in our cohort compared with control cohorts. Notably, we identified two protein-truncating DNMs in two independent cases in SHROOM3, previously associated with NTDs only in animal models. We have demonstrated a significant enrichment of LoF DNMs in this gene in NTDs compared with the gene specific DNM rate and to the DNM rate estimated from control cohorts. We also identified one nonsense DNM in PAX3 and two potentially causative missense DNMs in GRHL3 and PTPRS. CONCLUSIONS: Our study demonstrates an important role of LoF DNMs in the development of NTDs and strongly implicates SHROOM3 in its aetiology.

Role of the planar cell polarity gene Protein tyrosine kinase 7 in neural tube defects in humans.

BACKGROUND: Neural tube defects (NTDs) are among the most common congenital defects affecting approximately 1 in 1000 live births in North America. Their etiology is complex including environmental and genetic factors. Defects in the planar cell polarity (PCP) signaling pathway have been strongly associated with NTDs in animal models and human cohorts. Protein tyrosine kinase 7 (Ptk7) was shown to cause a very severe form of NTDs called craniorachischisis in a mouse model and genetically interacts with a core PCP member Vangl2 where double heterozygotes suffer from spina bifida. In this study, we examined the role of PTK7 in human NTDs to determine whether variants at this gene predispose to these defects. METHODS: We sequenced the coding region and the exon-intron junctions of PTK7 in a cohort of 473 patients affected with various forms of open and closed NTDs. Novel and rare variants(<1%) were genotyped in a cohort of 473 individuals. Their pathogenic effect was predicted in silico and functionally in an overexpression assay in a well-established zebrafish model. RESULTS: We identified in our cohort 6 rare variants, 3 of which were absent in public databases. One variant, p.Gly348Ser, acted as a hypermorph when overexpressed in the zebrafish model. CONCLUSION: We detected potentially pathogenic PTK7 variants in 1.1% of our NTD cohort. Our findings implicate PTK7 as a risk factor for NTDs and provide additional evidence for a pathogenic role of PCP signaling in these malformations.

Genetic studies of ANKRD6 as a molecular switch between Wnt signaling pathways in human neural tube defects.

BACKGROUND: Planar cell polarity (PCP) is a major branch of Wnt signaling that controls the process of convergent extension in gastrulation and neurulation. PCP defects were associated with neural tube defects (NTDs) that are the most common central nervous system anomalies. PCP signaling is highly dosage sensitive and exhibits an antagonistic relationship with the canonical Wnt/ß-catenin pathway. Diversin, encoded by Ankrd6, is an ankyrin repeat protein that activates the non canonical PCP signaling and simultaneously inhibits the canonical pathway. METHODS: In this study, we analyzed this dual role of ANKRD6 in NTDs. We sequenced its coding region in 473 NTD patients and 150 controls, and we validated the effect of the identified variants on Wnt signaling using reporter assays in mammalian cells. RESULTS: We identified four rare missense mutations in 0.8% of the NTD patients and two rare missense mutations in 1.3% of the controls. Notably, when all six mutations were validated, only two mutations identified in NTD patients, p.Pro548Leu, p.Arg632His, significantly altered DIVERSIN activity in Wnt signaling assays in a hypomorphic manner. CONCLUSION: Rare missense mutations in ANKRD6 could affect a balanced reciprocal antagonism between both Wnt pathways in neurulation and act as predisposing factors to NTDs in a subset of patients.

Planar cell polarity gene mutations contribute to the etiology of human neural tube defects in our population.

Neural Tube Defects (NTDs) are congenital malformations that involve failure of the neural tube closure during the early phases of development at any level of the rostro-caudal axis. The planar cell polarity (PCP) pathway is a highly conserved, noncanonical Wnt-Frizzled-Dishevelled signaling cascade, that was first identified in the fruit fly Drosophila. We are here reviewing the role of the PCP pathway genes in the etiology of human NTDs, updating the list of the rare and deleterious mutations identified so far. We report 50 rare nonsynonymous mutations of PCP genes in 54 patients having a pathogenic effect on the protein function. Thirteen mutations that have previously been reported as novel are now reported in public databases, although at very low frequencies. The mutations were private, mostly missense, and transmitted by a healthy parent. To date, no clear genotype-phenotype correlation has been possible to create. Even if PCP pathway genes are involved in the pathogenesis of neural tube defects, future studies will be necessary to better dissect the genetic causes underlying these complex malformations.

Mutations in the planar cell polarity gene, Fuzzy, are associated with neural tube defects in humans.

Neural tube defects (NTDs) are a heterogeneous group of common severe congenital anomalies which affect 1-2 infants per 1000 births. Most genetic and/or environmental factors that contribute to the pathogenesis of human NTDs are unknown. Recently, however, pathogenic mutations of VANGL1 and VANGL2 genes have been associated with some cases of human NTDs. Vangl genes encode proteins of the planar cell polarity (PCP) pathway that regulates cell behavior during early stages of neural tube formation. Homozygous disruption of PCP genes in mice results in a spectrum of NTDs, including defects that affect the entire neural axis (craniorachischisis), cranial NTDs (exencephaly) and spina bifida. In this paper, we report the dynamic expression of another PCP gene, Fuzzy, during neural tube formation in mice. We also identify non-synonymous Fuzzy amino acid substitutions in some patients with NTDs and demonstrate that several of these Fuzzy mutations affect formation of primary cilia and ciliary length or affect directional cell movement. Since Fuzzy knockout mice exhibit both NTDs and defective primary cilia and Fuzzy is expressed in the emerging neural tube, we propose that mutations in Fuzzy may account for a subset of NTDs in humans.

Rare missense variants in DVL1, one of the human counterparts of the Drosophila dishevelled gene, do not confer increased risk for neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are severe malformations that arise when the neural tube fails to close during embryogenesis. The planar cell polarity pathway is involved in neural tube closure and has been implicated in the pathogenesis of NTDs both in animal models and human cohorts. Dishevelled (Dvl/Dsh) is a multi-module protein and a key regulator of both the canonical Wnt and the PCP pathway. In mouse, all Dvl1(-/-) ; Dvl2(-/-) double mutants display craniorachischisis, a severe form of open NTDs. Recently, we have reported a possible role for rare variants of DVL2 as risk factors for NTDs. METHODS: In view of these data, we hypothesized that DVL1 mutations might increase the risk for NTDs in some cases. Resequencing of the DVL1 gene in a cohort of 473 NTDs patients and 150 ethnically matched controls was performed. Prediction of the downstream effects of the nonsynonymous variants was done using computational methods. RESULTS: We identified six missense variants that were absent in our ethnically matched controls group, and four of them (p.Arg153Cys; p.Glu544Arg; p.Arg568Trp; p.Val644Phe) were predicted to have a functional effect on protein structure by one or more bioinformatic programs. However, there was no difference in the overall rate of deleterious variants between the patients and controls (four in patients and three in controls; p=0.36). CONCLUSION: Our findings did not provide evidence for the implication of DVL1 in the pathogenesis of human NTDs.

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.

The tumor suppressor hamartin enhances Dbl protein transforming activity through interaction with ezrin.

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between ß1 and ß2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.

Role of the planar cell polarity gene CELSR1 in neural tube defects and caudal agenesis.

BACKGROUND: Neural tube defects (NTDs), including anencephaly and spina bifida, have a complex etiology. Defects in the planar cell polarity (PCP) signaling pathway have been strongly associated with NTDs in animal models and human cohorts. In this genetic study, we examined the core PCP gene CELSR1 in NTDs and caudal agenesis cases to determine whether mutations at this gene predispose to these defects. METHODS: We sequenced the coding region and the exon-intron junctions of CELSR1 in a cohort of 473 patients affected with various forms of open and closed NTDs (412) or caudal agenesis (61). Novel and rare variants (<1%) were genotyped in a cohort of 639 ethnically-matched individuals. The effect of novel missense mutations absent in controls and in public databases on protein function was predicted in silico. RESULTS: We identified in our cohort one nonsense mutation in exon 1 of CELSR1 that truncates the majority of the protein in one patient with NTD and one in-frame 12 bp deletion that removes a putative PKC phosphorylation "SSR" motif in one caudal agenesis patient. We also detected a total of 13 novel missense variants in 12 patients (11 NTDs and 1 caudal agenesis) that were predicted to be pathogenic in silico. CONCLUSIONS: We detected novel CELSR1 mutations predicted to be pathogenic in 2.9% of our NTD cohort and 3.3% of our caudal agenesis cohort. Our findings implicate CELSR1 as a risk factor for NTDs or caudal agenesis and provide additional evidence for a pathogenic role of PCP signaling in these malformations.

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.

Maternal periconceptional factors affect the risk of spina bifida-affected pregnancies: an Italian case-control study.

PURPOSE: Neural tube defects, including spina bifida and anencephaly, are the second most common birth defects with an incidence in Italy of 0.4-1/1,000. Information on factors playing a role in the pathogenesis of spina bifida is based on populations with different exposures, lifestyle, social and cultural habits compared to Italian people. Our objective was to fill this gap by using data from a case-control interview study carried out at the G. Gaslini Children's Hospital, Genoa, from 2000 to 2008. METHODS: We surveyed questionnaires from 133 case mothers and 273 control women providing information on periconceptional risk factors. Univariate and multivariate logistic regression analyses were used to estimate risks by odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: Univariate results suggest that birth order, low maternal educational level, age, smoking habits, alcohol consumption, high caffeine intake, lack of folate supplementation, low and high calorie diet, occasional consumption of fruit and vegetables, high emotional stress, and environmental pollution are associated with an increased spina bifida risk. Nevertheless, high caffeine intake (OR = 10.82; 95% CI, 3.78-31), low calorie diet (OR = 5.15; 95%CI, 1.79-14), occasional consumption of fruit and vegetables (OR = 3.38; 95% CI, 1.67-6.82), alcohol consumption (OR = 3.05; 95% CI, 1.24-7.50) and, above all, lack of folate supplementation at any time of pregnancy (OR = 20.54; 95% CI, 5.41-77) mainly determined spina bifida risk in the multivariate analysis. CONCLUSION: Our findings point out that a common underlying mechanism, a disturbed folate/homocysteine metabolism, may be causative for the burden of spina bifida in the Italian population.

Mutations in VANGL1 associated with neural-tube defects.

Neural-tube defects such as anencephaly and spina bifida constitute a group of common congenital malformations caused by complex genetic and environmental factors. We have identified three mutations in the VANGL1 gene in patients with familial types (V239I and R274Q) and a sporadic type (M328T) of the disease, including a spontaneous mutation (V239I) appearing in a familial setting. In a protein-protein interaction assay V239I abolished interaction of VANGL1 protein with its binding partners, disheveled-1, -2, and -3. These findings implicate VANGL1 as a risk factor in human neural-tube defects.

Moyamoya vasculopathy shows a genetic mutational gradient decreasing from East to West.

BACKGROUND: Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease characterized by bilateral steno-occlusive changes at the terminal portion of the internal carotid arteries and an abnormal vascular network at the base of the brain determining stroke in children. Patients with a similar vasculopathy and associated conditions are affected by the moyamoya syndrome (MMS). Most of the studies focused on MMD were carried out on East-Asian population. Ring Finger 213 (RNF213) has been identified as the strongest susceptibility gene for MMD in East-Asian people. Overall, 74.5% of the East-Asian patients carry the founder variant p.Arg4810Lys of RNF213 never reported in Caucasians. A different genetic landscape among the diverse ethnic populations seems to exist. METHODS: We sequenced the coding sequence region of RNF213, TGFB1 and PDGFRB in 21 ethnically homogeneous Italian children with moyamoya; comprehensive sequencing data are available from parents of eight of them. The analyses were carried out by NGS on Thermo-fisher PGM platform. We also performed a comprehensive review of the literature about the variations of these three genes in Caucasian patients. RESULTS: Several new variants of RNF213 gene were detected, in particular, two new pathogenic mutations on RNF213 (p.Trp4677Leu and p.Cys4017Ser) were identified in one MMS case and in one MMD case, respectively. Moreover, in a MMS case a new probably causing disease mutation p.Pro1063Thr of PDGFRB was detected. CONCLUSIONS: The genetic susceptibility of Asian moyamoya vasculopathy seems to differ from the Caucasian disease. No additional differences seem to exist between MMD and MMS.

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.