Rare LRP6 variants identified in spina bifida patients.
ABSTRACT: Several single-nucleotide variants (SNVs) in low-density lipoprotein receptor-related protein 6 (Lrp6) cause neural tube defects (NTDs) in mice. We therefore examined LRP6 in 192 unrelated infants from California with the NTD, spina bifida, and found four heterozygous missense SNVs, three of which were predicted to be deleterious, among NTD cases and not in 190 ethnically matched nonmalformed controls. Parents and siblings could not be tested because of the study design. Like Crooked tail and Ringleschwanz mouse variants, the p.Tyr544Cys Lrp6 protein failed to bind the chaperone protein mesoderm development and impaired Lrp6 subcellular localization to the plasma membrane of MDCK II cells. Only the p.Tyr544Cys Lrp6 variant downregulated canonical Wnt signaling in a TopFlash luciferase reporter in vitro assay. In contrast, three Lrp6 mutants (p.Ala3Val, p.Tyr544Cys, and p.Arg1574Leu) increased noncanonical Wnt/planar cell polarity (PCP) signaling in an Ap1-luciferase assay. Thus, LRP6 variants outside of YWTD repeats could potentially predispose embryos to NTDs, whereas Lrp6 modulation of Wnt/PCP signaling would be more essential than its canonical pathway role in neural tube closure.
Project description:<h4>Background</h4>Mouse 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.<h4>Methods</h4>We 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.<h4>Results</h4>CELSR1, 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.<h4>Conclusions</h4>These 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.
Project description:Wnt signaling has been classified as canonical Wnt/β-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/β-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.
Project description:Fractures are a common comorbidity in children with the neural tube defect (NTD) spina bifida. Mutations in the Wnt/planar cell polarity (PCP) pathway contribute to NTDs in humans and mice, but whether this pathway independently determines bone mass is poorly understood. Here, we first confirmed that core Wnt/PCP components are expressed in osteoblasts and osteoclasts in vitro. In vivo, we performed detailed µCT comparisons of bone structure in tibiae from young male mice heterozygous for NTD-associated mutations versus WT littermates. PCP signalling disruption caused by Vangl2 (Vangl2Lp/+) or Celsr1 (Celsr1Crsh/+) mutations significantly reduced trabecular bone mass and distal tibial cortical thickness. NTD-associated mutations in non-PCP transcription factors were also investigated. Pax3 mutation (Pax3Sp2H/+) had minimal effects on bone mass. Zic2 mutation (Zic2Ku/+) significantly altered the position of the tibia/fibula junction and diminished cortical bone in the proximal tibia. Beyond these genes, we bioinformatically documented the known extent of shared genetic networks between NTDs and bone properties. 46 genes involved in neural tube closure are annotated with bone-related ontologies. These findings document shared genetic networks between spina bifida risk and bone structure, including PCP components and Zic2. Genetic variants which predispose to spina bifida may therefore independently diminish bone mass.
Project description: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.
Project description:Purpose: Identify genes and pathways affected in tuft embryos with NTDs Results: Expression of genes associated with neural tube closure and components of non-canonical WNT signaling/PCP pathways were affected Conclusions: TET1 regulates genes associated with neural tube closure Overall design: RNA pooled from the rostrums of E9 (18-22 somites) tuft/tuft embryos with NTD compared with respective wildtype background strain
Project description:A cranial neural tube defect in Crooked tail (Cd) mice is prevented with prenatal dietary folic acid Cd positional cloning reveals a missense mutation of a highly conserved amino acid in the low density lipoprotein receptor-related protein 6 (Lrp6), a coreceptor required for Wnt canonical signaling. Molecular modeling predicts that Lrp6(Cd) alters a hinge region of the second YWTD beta-propeller domain. Mutant LRP6 binds to Wnt and Dickkopf1 (Dkk1) but not Mesd1, and Dkk1 cannot antagonize Wnt in Cd/Cd cells, resulting in hyperactivity. NIH 3T3 cells transfected with a mutant Lrp6 plasmid resist Dkk1 antagonism much like Cd/+ cells, confirming the significance of the mutation. The Lrp6 mutation in Cd mice provides evidence for a functional connection between Wnt signaling and folate rescue of neural tube defects.
Project description: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.
Project description: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.
Project description:Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical-basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD-specific variants were significantly enriched in the aPKC-binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over-expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC-binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.
Project description:Extensive studies that have sought causative mutation(s) for neural tube defects (NTDs) have yielded limited positive findings to date. One possible reason for this is that many studies have been confined to analyses of germline mutations and so may have missed other, non-germline mutations in NTD cases. We hypothesize that somatic mutations of planar polarity pathway (PCP) genes may play a role in the development of NTDs. Torrent™ Personal Genome Machine™ (PGM) sequencing was designed for selected PCP genes in paired DNA samples extracted from the tissues of lesion sites and umbilical cord from 48 cases. Sanger sequencing was used to validate the detected mutations. The source and distribution of the validated mutations in tissues from different germ layers were investigated. Subcellular location, western blotting, and luciferase assays were performed to better understand the effects of the mutations on protein localization, protein level, and pathway signaling. ix somatic mutations were identified and validated, which showed diverse distributions in different tissues. Three somatic mutations were novel/rare: CELSR1 p.Gln2125His, FZD6 p.Gln88Glu, and VANGL1 p.Arg374His. FZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus, and disrupted the colocalization of CELSR1 and FZD6. This mutation affected non-canonical WNT signaling in luciferase assays. VANGL1 p.Arg374His impaired the co-localization of CELSR1 and VANGL1, increased the protein levels of VANGL1, and influenced cell migration. In all, 7/48 (14.5%) of the studied NTD cases contained somatic PCP mutations. Somatic mutations in PCP genes (e.g., FZD6 and VANGL1) are associated with human NTDs, and they may occur in different stages and regions during embryonic development, resulting in a varied distribution in fetal tissues/organs.