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.

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2.

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 S464N ) in neural tube development and inner ear hair cell polarity. The Vangl2I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.

Genomic study of severe fetal anomalies and discovery of GREB1L mutations in renal agenesis.

PURPOSE: Fetal anomalies represent a poorly studied group of developmental disorders. Our objective was to assess the impact of whole-exome sequencing (WES) on the investigation of these anomalies. METHODS: We performed WES in 101 fetuses or stillborns who presented prenatally with severe anomalies, including renal a/dysgenesis, VACTERL association (vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal anomalies, and limb abnormalities), brain anomalies, suspected ciliopathies, multiple major malformations, and akinesia. RESULTS: A molecular diagnosis was obtained in 19 cases (19%). In 13 of these cases, the diagnosis was not initially suspected by the clinicians because the phenotype was nonspecific or atypical, corresponding in some cases to the severe end of the spectrum of a known disease (e.g., MNX1-, RYR1-, or TUBB-related disorders). In addition, we identified likely pathogenic variants in genes (DSTYK, ACTB, and HIVEP2) previously associated with phenotypes that were substantially different from those found in our cases. Finally, we identified variants in novel candidate genes that were associated with perinatal lethality, including de novo mutations in GREB1L in two cases with bilateral renal agenesis, which represents a significant enrichment of such mutations in our cohort. CONCLUSION: Our study opens a window on the distinctive genetic landscape associated with fetal anomalies and highlights the power-but also the challenges-of WES in prenatal diagnosis.

A genome-wide association study identifies candidate loci associated to syringomyelia secondary to Chiari-like malformation in Cavalier King Charles Spaniels.

BACKGROUND: Syringomyelia (SM) is a common condition affecting brachycephalic toy breed dogs and is characterized by the development of fluid-filled cavities within the spinal cord. It is often concurrent with a complex developmental malformation of the skull and craniocervical vertebrae called Chiari-like malformation (CM) characterized by a conformational change and overcrowding of the brain and cervical spinal cord particularly at the craniocervical junction. CM and SM have a polygenic mode of inheritance with variable penetrance. RESULTS: We identified six cranial T1-weighted sagittal MRI measurements that were associated to maximum transverse diameter of the syrinx cavity. Increased syrinx transverse diameter has been correlated previously with increased likelihood of behavioral signs of pain. We next conducted a whole genome association study of these traits in 65 Cavalier King Charles Spaniel (CKCS) dogs (33 controls, 32 with extreme phenotypes). Two loci on CFA22 and CFA26 were found to be significantly associated to two traits associated with a reduced volume and altered orientation of the caudal cranial fossa. Their reconstructed haplotypes defined two associated regions that harbor only two genes: PCDH17 on CFA22 and ZWINT on CFA26. PCDH17 codes for a cell adhesion molecule expressed specifically in the brain and spinal cord. ZWINT plays a role in chromosome segregation and its expression is increased with the onset of neuropathic pain. Targeted genomic sequencing of these regions identified respectively 37 and 339 SNPs with significantly associated P values. Genotyping of tagSNPs selected from these 2 candidate loci in an extended cohort of 461 CKCS (187 unaffected, 274 SM affected) identified 2 SNPs on CFA22 that were significantly associated to SM strengthening the candidacy of this locus in SM development. CONCLUSIONS: We identified 2 loci on CFA22 and CFA26 that contained only 2 genes, PCDH17 and ZWINT, significantly associated to two traits associated with syrinx transverse diameter. The locus on CFA22 was significantly associated to SM secondary to CM in the CKCS dog breed strengthening its candidacy for this disease. This study will provide an entry point for identification of the genetic factors predisposing to this condition and its underlying pathogenic mechanisms.

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.

The impairment of MAGMAS function in human is responsible for a severe skeletal dysplasia.

Impairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process.

Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment.

Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans.

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.

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.

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.

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.

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.

Risk of meningomyelocele mediated by the common 22q11.2 deletion.

Meningomyelocele is one of the most severe forms of neural tube defects (NTDs) and the most frequent structural birth defect of the central nervous system. We assembled the Spina Bifida Sequencing Consortium to identify causes. Exome and genome sequencing of 715 parent-offspring trios identified six patients with chromosomal 22q11.2 deletions, suggesting a 23-fold increased risk compared with the general population. Furthermore, analysis of a separate 22q11.2 deletion cohort suggested a 12- to 15-fold increased NTD risk of meningomyelocele. The loss of Crkl, one of several neural tube-expressed genes within the minimal deletion interval, was sufficient to replicate NTDs in mice, where both penetrance and expressivity were exacerbated by maternal folate deficiency. Thus, the common 22q11.2 deletion confers substantial meningomyelocele risk, which is partially alleviated by folate supplementation.

Identification and biochemical characterization of a novel mutation in DDX11 causing Warsaw breakage syndrome.

Mutations in the gene encoding the iron-sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity.

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.

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.

A novel hypomorphic Looptail allele at the planar cell polarity Vangl2 gene.

Vangl2 forms part of the planar cell polarity signalling pathway and is the gene defective in the Looptail (Lp) mouse mutant. Two previously described alleles, Lp and Lp(m1Jus) , segregate in a semi-dominant fashion, with heterozygotes displaying the looped-tail appearance, while homozygotes show the neural tube defect called craniorachischisis. Here, we report a novel experimentally induced allele, Lp(m2Jus) , that carries a missense mutation, R259L, in Vangl2. This mutation was specific to the Lp phenotype and absent from both parental strains and 28 other inbred strains. Notably, this mutation segregates in a recessive manner with all heterozygotes appearing normal and 47% of homozygotes showing a looped-tail. Homozygous Lp(m2Jus) embryos showed spina bifida in 12%. Lp(m2Jus) genetically interacts with Lp with 77% of compound heterozygotes displaying craniorachischisis. Vangl2(R259L) behaved like the wild-type allele in overexpression and morpholino knockdown/rescue assays in zebrafish embryos. These data suggest that Lp(m2Jus) represents a new hypomorphic allele of Lp.

Whole-Exome Sequencing in Congenital Hypothyroidism Due to Thyroid Dysgenesis.

Context: Congenital hypothyroidism due to thyroid dysgenesis (CHTD) is a predominantly sporadic and nonsyndromic (NS) condition of unknown etiology. NS-CHTD shows a 40-fold increase in relative risk among first-degree relatives (1 in 100 compared with a birth prevalence of 1 in 4000 in the general population), but a discordance rate between monozygotic (MZ) twins of 92%. This suggests a two-hit mechanism, combining a genetic predisposition (incomplete penetrance of inherited variants) with postzygotic events (accounting for MZ twin discordance). Objective: To evaluate whether whole-exome sequencing (WES) allows to identify new predisposing genes in NS-CHTD. Methods: We performed a case-control study by comparing the whole exome of 36 nonconsanguineous cases of NS-CHTD (33 with lingual thyroid ectopy and 3 with athyreosis, based on technetium pertechnetate scintigraphy at diagnosis) with that of 301 unaffected controls to assess for enrichment in rare protein-altering variants. We performed an unbiased approach using a gene-based burden with a false discovery rate correction. Moreover, we identified all rare pathogenic and likely pathogenic variants, based on in silico prediction tools, in 27 genes previously associated with congenital hypothyroidism (CH) (thyroid dysgenesis [TD] and dyshormonogenesis). Results: After correction for multiple testing, no enrichment in rare protein-altering variants was observed in NS-CHTD. Pathogenic or likely pathogenic variants (21 variants in 12 CH genes) were identified in 42% of cases. Eight percent of cases had variants in more than one gene (oligogenic group); these were not more severely affected than monogenic cases. Moreover, cases with protein-altering variants in dyshormonogenesis-related genes were not more severely affected than those without. Conclusions: No new predisposing genes were identified following an unbiased analysis of WES data in a well-characterized NS-CHTD cohort. Nonetheless, the discovery rate of rare pathogenic or likely pathogenic variants was 42%. Eight percent of the cases harbored multiple variants in genes associated with TD or dyshormonogenesis, but these variants did not explain the variability of hypothyroidism observed in dysgenesis. WES did not identify a genetic cause in NS-CHTD cases, confirming the complex etiology of this disease. Additional studies in larger cohorts and/or novel discovery approaches are required.

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.