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.

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.

A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA. In this study, we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations, and increased mortality. After genetic mapping and whole-exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ animals had a profound delay in erythroid maturation and increased mortality at embryonic day (E) 12.5, which improved by E14.5. Surviving mutant animals had macrocytic anemia at birth, as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus/+ mutant mouse will be useful in studying the factors influencing the variable penetrance that is observed in DBA.

Severe Congenital Hypothyroidism Due to a Novel Deep Intronic Mutation in the TSH Receptor Gene Causing Intron Retention.

In 3 Somalian siblings with severe nongoitrous congenital hypothyroidism, exome sequencing identified a variant in TSHR predicted to be benign in isoform 3 but leading to an intronic mutation in isoform 1 (NM_00369:c.692 + 130C>A), which is the isoform expressed in the thyroid. This mutation creates a pseudoexon that results in a protein that, if transcribed, would lack the transmembrane domain, thereby hampering its expression at the cell surface. Our findings illustrate that the interpretation of exome analysis requires knowledge of the relevant isoform expression and of the biology of the disease. This is the first description of a deep intronic mutation creating a pseudoexon and inactivating the thyroid stimulating hormone (TSH) receptor.

Update on the Role of the Non-Canonical Wnt/Planar Cell Polarity Pathway in Neural Tube Defects.

Neural tube defects (NTDs), including spina bifida and anencephaly, represent the most severe and common malformations of the central nervous system affecting 0.7-3 per 1000 live births. They result from the failure of neural tube closure during the first few weeks of pregnancy. They have a complex etiology that implicate a large number of genetic and environmental factors that remain largely undetermined. Extensive studies in vertebrate models have strongly implicated the non-canonical Wnt/planar cell polarity (PCP) signaling pathway in the pathogenesis of NTDs. The defects in this pathway lead to a defective convergent extension that is a major morphogenetic process essential for neural tube elongation and subsequent closure. A large number of genetic studies in human NTDs have demonstrated an important role of PCP signaling in their etiology. However, the relative contribution of this pathway to this complex etiology awaits a better picture of the complete genetic architecture of these defects. The emergence of new genome technologies and bioinformatics pipelines, complemented with the powerful tool of animal models for variant interpretation as well as significant collaborative efforts, will help to dissect the complex genetics of NTDs. The ultimate goal is to develop better preventive and counseling strategies for families affected by these devastating conditions.

Whole exome sequencing identifies novel predisposing genes in neural tube defects.

BACKGROUND: Neural tube defects (NTD) are among the most common defects affecting 1:1000 births. They are caused by a failure of neural tube closure during development. Their clinical presentation is diverse and dependent on the site and severity of the original defect on the embryonic axis. The etiology of NTD is multifactorial involving environmental factors and genetic variants that remain largely unknown. METHODS: We have conducted a whole exome sequencing (WES) study in five new NTD families and pooled the results with WES data from three NTD families and 43 trios that were previously investigated by our group. We analyzed the data using biased candidate gene and unbiased gene burden approaches. RESULTS: We identified four novel loss-of-function variants in three genes, MTHFR, DLC1, and ITGB1, previously associated with NTD. Notably, DLC1 carried two protein truncating variants in two independent cases. We also demonstrated an enrichment of variants in MYO1E involved in cytoskeletal remodeling. This enrichment reached borderline significance in a replication cohort supporting the association of this new candidate gene to NTD. CONCLUSION: These data provide some key insights into the pathogenic mechanisms of human NTD and demonstrate the power of next-generation sequencing in deciphering the genetics of this complex trait.

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.

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.

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.

Genetic Testing in a Cohort of Complex Esophageal Atresia.

The objective of the present study is to describe a cohort of complex esophageal atresia and the yield of genetic tests performed for such patients. We selected 45 patients with complex esophageal atresia (EA), namely those having at least one associated anomaly. We reviewed their medical records to assess clinical features, other diagnoses, and genetic investigations. Most of the patients had a diagnosis of VACTERL association (56%) with no genetic variant identified. Interestingly, 5 patients in the cohort (11%) had a right pulmonary hypoplasia or agenesis. A majority of our cohort (73%) had genetic testing; 60% were karyotyped (abnormal in 4 of the 27 patients tested), 31% had aCGH (abnormal in 1 of the 14 patients tested), and 31% had diepoxybutane (DEB) testing for Fanconi anemia (abnormal in 2 of the 14 patients tested). One patient had exome sequencing studies, but no candidate gene was identified. Various anomalies were associated with EA, and overall a genetic variant could be identified in 7 of the 33 patients tested. Chromosomal studies such as aCGH and chromosomal breakage studies should be considered, and their yield varied between 7 and 14%. Other genetic investigations such as exome sequencing could possibly have even higher yields but will need to be assessed in a large cohort. Improved genetic diagnoses in EA may improve the management of these patients by directing specific surveillance and management schemes.

Exome sequencing of two Italian pedigrees with non-isolated Chiari malformation type I reveals candidate genes for cranio-facial development.

Chiari malformation type I (CMI) is a congenital abnormality of the cranio-cerebral junction with an estimated incidence of 1 in 1280. CMI is characterized by underdevelopment of the occipital bone and posterior fossa (PF) and consequent cerebellar tonsil herniation. The presence for a genetic basis to CMI is supported by many lines of evidence. The cellular and molecular mechanisms leading to CM1 are poorly understood. The occipital bone formation is dependent on complex interactions between genes and molecules with pathologies resulting from disruption of this delicate process. Whole-exome sequencing of affected and not affected individuals from two Italian families with non-isolated CMI was undertaken. Single-nucleotide and short insertion-deletion variants were prioritized using KGGSeq knowledge-based platform. We identified three heterozygous missense variants: DKK1 c.121G>A (p.(A41T)) in the first family, and the LRP4 c.2552C>G (p.(T851R)) and BMP1 c.941G>A (p.(R314H)) in the second family. The variants were located at highly conserved residues, segregated with the disease, but they were not observed in 100 unaffected in-house controls. DKK1 encodes for a potent soluble WNT inhibitor that binds to LRP5 and LRP6, and is itself regulated by bone morphogenetic proteins (BMPs). DKK1 is required for embryonic head development and patterning. LRP4 is a novel osteoblast expressed receptor for DKK1 and a WNT and BMP 4 pathways integrator. Screening of DKK1 in a cohort of 65 CMI sporadic patients identified another missense variant, the c.359G>T (p.(R120L)), in two unrelated patients. These findings implicated the WNT signaling in the correct development of the cranial mesenchyme originating the PF.

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.

Use of Morphometric Mapping to Characterise Symptomatic Chiari-Like Malformation, Secondary Syringomyelia and Associated Brachycephaly in the Cavalier King Charles Spaniel.

OBJECTIVES: To characterise the symptomatic phenotype of Chiari-like malformation (CM), secondary syringomyelia (SM) and brachycephaly in the Cavalier King Charles Spaniel using morphometric measurements on mid-sagittal Magnetic Resonance images (MRI) of the brain and craniocervical junction. METHODS: This retrospective study, based on a previous quantitative analysis in the Griffon Bruxellois (GB), used 24 measurements taken on 130 T1-weighted MRI of hindbrain and cervical region. Associated brachycephaly was estimated using 26 measurements, including rostral forebrain flattening and olfactory lobe rotation, on 72 T2-weighted MRI of the whole brain. Both study cohorts were divided into three groups; Control, CM pain and SM and their morphometries compared with each other. RESULTS: Fourteen significant traits were identified in the hindbrain study and nine traits in the whole brain study, six of which were similar to the GB and suggest a common aetiology. The Control cohort had the most elliptical brain (p = 0.010), least olfactory bulb rotation (p = 0.003) and a protective angle (p = 0.004) compared to the other groups. The CM pain cohort had the greatest rostral forebrain flattening (p = 0.007), shortest basioccipital (p = 0.019), but a greater distance between the atlas and basioccipital (p = 0.002) which was protective for SM. The SM cohort had two conformation anomalies depending on the severity of craniocervical junction incongruities; i) the proximity of the dens (p <0.001) ii) increased airorhynchy with a smaller, more ventrally rotated olfactory bulb (p <0.001). Both generated 'concertina' flexures of the brain and craniocervical junction. CONCLUSION: Morphometric mapping provides a diagnostic tool for quantifying symptomatic CM, secondary SM and their relationship with brachycephaly. It is hypothesized that CM pain is associated with increased brachycephaly and SM can result from different combinations of abnormalities of the forebrain, caudal fossa and craniocervical junction which compromise the neural parenchyma and impede cerebrospinal fluid flow.

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.

Neuroblastoma Amplified Sequence (NBAS) mutation in recurrent acute liver failure: Confirmatory report in a sibship with very early onset, osteoporosis and developmental delay.

BACKGROUND: Recently, biallelic mutations in the Neuroblastoma Amplified Sequence NBAS gene have been identified in ten patients that present recurrent acute liver failure (RALF) in early infancy. In addition to severe liver dysfunction, some of these individuals also suffered from other comorbidities including cardiomyopathy, neurologic phenotypes and gastrointestinal immune defects. Here we report on a consanguineous Lebanese family with three siblings affected by RALF. Of note, neonatal spontaneous fractures, developmental delay, prominent eyes, generalized hirsutism, gum hypertrophy, and hepato-splenomegaly ​were also present. METHODS: Whole-genome SNP genotyping in all the patients, followed by exome sequencing was performed in one of the affected siblings. RESULTS: A homozygous c.409C > T (p.Arg137Trp) missense mutation in NBAS was identified in all patients. CONCLUSION: Overall, our findings confirm the involvement of NBAS in the pathogenesis of this condition characterized by severe liver dysfunction and help expand its phenotypical spectrum.

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.

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.

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.