Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

Exome sequencing identifies variants in FKBP4 that are associated with recurrent fetal loss in humans.

Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages and affects an estimated 1.5% of couples trying to conceive. RPL has been attributed to genetic, endocrine, immune and thrombophilic disorders, but many cases remain unexplained. We investigated a Bangladeshi family where the proband experienced 29 consecutive pregnancy losses with no successful pregnancies from three different marriages. Whole exome sequencing identified rare genetic variants in several candidate genes. These were further investigated in Asian and white European RPL cohorts, and in Bangladeshi controls. FKBP4, encoding the immunophilin FK506-binding protein 4, was identified as a plausible candidate, with three further novel variants identified in Asian patients. None were found in European patients or controls. In silico structural studies predicted damaging effects of the variants in the structure-function properties of the FKBP52 protein. These were located within domains reported to be involved in Hsp90 binding and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Profound effects on PPIase activity were demonstrated in transiently transfected HEK293 cells comparing wild-type and mutant FKBP4 constructs. Mice lacking FKBP4 have been previously reported as infertile through implantation failure. This study therefore strongly implicates FKBP4 as associated with fetal losses in humans, particularly in the Asian population.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

Overexpression of Grainyhead-like 3 causes spina bifida and interacts genetically with mutant alleles of Grhl2 and Vangl2 in mice.

The genetic basis of human neural tube defects (NTDs), such as anencephaly and spina bifida (SB), is complex and heterogeneous. Grainyhead-like genes represent candidates for involvement in NTDs based on the presence of SB and exencephaly in mice carrying loss-of-function alleles of Grhl2 or Grhl3. We found that reinstatement of Grhl3 expression, by bacterial artificial chromosome (BAC)-mediated transgenesis, prevents SB in Grhl3-null embryos, as in the Grhl3 hypomorphic curly tail strain. Notably, however, further increase in expression of Grhl3 causes highly penetrant SB. Grhl3 overexpression recapitulates the spinal NTD phenotype of loss-of-function embryos, although the underlying mechanism differs. However, it does not phenocopy other defects of Grhl3-null embryos such as abnormal axial curvature, cranial NTDs (exencephaly) or skin barrier defects, the latter being rescued by the Grhl3-transgene. Grhl2 and Grhl3 can form homodimers and heterodimers, suggesting a possible model in which defects arising from overexpression of Grhl3 result from sequestration of Grhl2 in heterodimers, mimicking Grhl2 loss of function. This hypothesis predicts that increased abundance of Grhl2 would have an ameliorating effect in Grhl3 overexpressing embryo. Instead, we observed a striking additive genetic interaction between Grhl2 and Grhl3 gain-of-function alleles. Severe SB arose in embryos in which both genes were expressed at moderately elevated levels that individually do not cause NTDs. Furthermore, moderate Grhl3 overexpression also interacted with the Vangl2Lp allele to cause SB, demonstrating genetic interaction with the planar cell polarity signalling pathway that is implicated in mouse and human NTDs.

SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20.

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.

Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice.

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development.

Familial Absent Uvula With Velopharyngeal Incompetence-A New Syndrome?

We present a family with a previously undescribed abnormality of the palate and oropharynx which involved the absence of the uvula and the anterior pillar of the fauces, rudimentary posterior pillar of the fauces, and hypernasality. Eight family members over 4 generations are affected in a pattern consistent with autosomal dominant inheritance. A causal role for the FOXF2 gene has been identified and previously reported. We describe the management of the proband, which involved attempting to lengthen the palate and to retroposition the abnormally anteriorly directed velar musculature, along with speech therapy.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Enrichment of Clinically Relevant Organisms in Spontaneous Preterm-Delivered Placentas and Reagent Contamination across All Clinical Groups in a Large Pregnancy Cohort in the United Kingdom.

In this study, differences in the placental microbiota from term and preterm deliveries in a large pregnancy cohort in the United Kingdom were studied by using 16S-targeted amplicon sequencing. The impacts of contamination from DNA extraction, PCR reagents, and the delivery itself were also examined. A total of 400 placental samples from 256 singleton pregnancies were analyzed, and differences between spontaneous preterm-, nonspontaneous preterm-, and term-delivered placentas were investigated. DNA from recently delivered placentas was extracted, and screening for bacterial DNA was carried out by using targeted sequencing of the 16S rRNA gene on the Illumina MiSeq platform. Sequenced reads were analyzed for the presence of contaminating operational taxonomic units (OTUs) identified via sequencing of negative extraction and PCR-blank samples. Differential abundances and between-sample (beta) diversity metrics were then compared. A large proportion of the reads sequenced from the extracted placental samples mapped to OTUs that were also found for negative extractions. Striking differences in the compositions of samples were also observed, according to whether the placenta was delivered abdominally or vaginally, providing strong circumstantial evidence for delivery contamination as an important contributor to observed microbial profiles. When OTU- and genus-level abundances were compared between the groups of interest, a number of organisms were enriched in the spontaneous preterm-delivery cohort, including organisms that have been associated previously with adverse pregnancy outcomes, specifically Mycoplasma spp. and Ureaplasma spp. However, analyses of the overall community structure did not reveal convincing evidence for the existence of a reproducible "preterm placental microbiome."IMPORTANCE Preterm birth is associated with both psychological and physical disabilities and is the leading cause of infant morbidity and mortality worldwide. Infection is known to be an important cause of spontaneous preterm birth, and recent research has implicated variation in the "placental microbiome" in the risk of preterm birth. Consistent with data from previous studies, the abundances of certain clinically relevant species differed between spontaneous preterm- and nonspontaneous preterm- or term-delivered placentas. These results support the view that a proportion of spontaneous preterm births have an intrauterine-infection component. However, an additional observation from this study was that a substantial proportion of sequenced reads were contaminating reads rather than DNA from endogenous, clinically relevant species. This observation warrants caution in the interpretation of sequencing outputs from low-biomass samples such as the placenta.

Cutis laxa and excessive bone growth due to de novo mutations in PTDSS1.

The cutis laxa syndromes are multisystem disorders that share loose redundant inelastic and wrinkled skin as a common hallmark clinical feature. The underlying molecular defects are heterogeneous and 13 different genes have been involved until now, all of them being implicated in elastic fiber assembly. We provide here molecular and clinical characterization of three unrelated patients with a very rare phenotype associating cutis laxa, facial dysmorphism, severe growth retardation, hyperostotic skeletal dysplasia, and intellectual disability. This disorder called Lenz-Majewski syndrome (LMS) is associated with gain of function mutations in PTDSS1, encoding an enzyme involved in phospholipid biosynthesis. This report illustrates that LMS is an unequivocal cutis laxa syndrome and expands the clinical and molecular spectrum of this group of disorders. In the neonatal period, brachydactyly and facial dysmorphism are two early distinctive signs, later followed by intellectual disability and hyperostotic skeletal dysplasia with severe dwarfism allowing differentiation of this condition from other cutis laxa phenotypes. Further studies are needed to understand the link between PTDSS1 and extra cellular matrix assembly.

Mutations in SNX14 cause a distinctive autosomal-recessive cerebellar ataxia and intellectual disability syndrome.

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.

Sumoylation in Craniofacial Disorders.

Craniofacial development requires a complex series of coordinated and finely tuned events to take place, during a relatively short time frame. These events are set in motion by switching on and off transcriptional cascades that involve the use of numerous signalling pathways and a multitude of factors that act at the site of gene transcription. It is now well known that amidst the subtlety of this process lies the intricate world of protein modification, and the posttranslational addition of the small ubiquitin -like modifier, SUMO, is an example that has been implicated in this process. Many proteins that are required for formation of various structures in the embryonic head and face adapt specific functions with SUMO modification. Interestingly, the main clinical phenotype reported for a disruption of the SUMO1 locus is the common birth defect cleft lip and palate. In this chapter therefore, we discuss the role of SUMO1 in craniofacial development, with emphasis on orofacial clefts. We suggest that these defects can be a sensitive indication of down regulated SUMO modification at a critical stage during embryogenesis. As well as specific mutations affecting the ability of particular proteins to be sumoylated, non-genetic events may have the effect of down-regulating the SUMO pathway to give the same result. Enzymes regulating the SUMO pathway may become important therapeutic targets in the preventative and treatment therapies for craniofacial defects in the future.

Maternal inheritance of a promoter variant in the imprinted PHLDA2 gene significantly increases birth weight.

Birth weight is an important indicator of both perinatal and adult health, but little is known about the genetic factors contributing to its variability. Intrauterine growth restriction is a leading cause of perinatal morbidity and mortality and is also associated with adult disease. A significant correlation has been reported between lower birth weight and increased expression of the maternal PHLDA2 allele in term placenta (the normal imprinting pattern was maintained). However, a mechanism that explains the transcriptional regulation of PHLDA2 on in utero growth has yet to be described. In this study, we sequenced the PHLDA2 promoter region in 263 fetal DNA samples to identify polymorphic variants. We used a luciferase reporter assay to identify in the PHLDA2 promoter a 15 bp repeat sequence (RS1) variant that significantly reduces PHLDA2-promoter efficiency. RS1 genotyping was then performed in three independent white European normal birth cohorts. Meta-analysis of all three (total n = 9,433) showed that maternal inheritance of RS1 resulted in a significant 93 g increase in birth weight (p = 0.01; 95% confidence interval [CI] = 22-163). Moreover, when the mother was homozygous for RS1, the influence on birth weight was 155 g (p = 0.04; 95% CI = 9-300), which is a similar magnitude to the reduction in birth weight caused by maternal smoking.

Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion.

Annexin A5 is a placental anti-coagulant protein that contains four nucleotide substitutions (M2 haplotype) in its promoter. This haplotype is a risk factor for recurrent spontaneous abortion (RSA). The influence of the M2 haplotype in the gestational timing of spontaneous abortions, paternal risk and relationships with known risk factors were investigated. European couples (n = 500) who had experienced three or more consecutive spontaneous abortions, and two fertile control groups, were selected for this study. The allele frequency of M2 was significantly higher among patients who had experienced early RSA than among controls (P = 0.002). No difference was found between controls and patients who had undergone late spontaneous abortions. No difference was found between patients who had experienced RSA who had a live birth or no live births, or between patients who were positive or negative for known risk factors. Male and female partners in each group had similar allele frequencies of M2. The M2 haplotype is a risk factor for early spontaneous abortions, before the 12th week of gestation, and confers about the same relative risk to carriers of both sexes. Having one or more M2 allele(s) in combination with other risk factors further increases the RSA risk.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Fat dads must not be blamed for their children's health problems.

The relationship between the parental genomes in terms of the future growth and development of their offspring is not critical. For the majority of the genome the tissue-specific gene expression and epigenetic status is shared between the parents equally, with both alleles contributing without parental bias. For a very small number of genes the rules change and control of expression is restricted to a specific, parentally derived allele, a phenomenon known as genomic imprinting. The insulin-like growth factor 2 (Igf2/IGF2) is a robustly imprinted gene, important for fetal growth in both mice and humans. In utero IGF2 exhibits paternal expression, which is controlled by several mechanisms, including the maternally expressing untranslated H19 gene. In the study by Soubry et al., a correlation is drawn between the IGF2 methylation status in fetal cord blood leucocytes, and the obesity status of the father from whom the active IGF2 allele is derived through his sperm. These data imply that paternal obesity affects the normal IGF2 methylation in the sperm and this in turn alters the expression of IGF2 in the baby.

Is LMNB1 a susceptibility gene for neural tube defects in humans?

BACKGROUND: Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds. METHODS: Mutation analysis by DNA sequencing was performed on all exons of LMNB1 in 239 samples from patients with NTDs from the United Kingdom, Sweden, and United States. Possible functional effects of missense variants were analyzed by bioinformatics prediction and fluorescence in photobleaching. RESULTS: In NTD patients, we identified two unique missense variants that were predicted to disrupt protein structure/function and represent putative contributory mutations. Fluorescence loss in photobleaching analysis showed that the A436T variant compromised stability of lamin B1 interaction within the lamina. CONCLUSION: The genetic basis of human NTDs appears highly heterogenous with possible involvement of multiple predisposing genes. We hypothesize that rare variants of LMNB1 may contribute to susceptibility to NTDs.

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.

Investigation of SUMO pathway genes in the etiology of nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: SUMO1 has been implicated as having a role in the causation of cleft lip with or without cleft palate (CLP), both directly and through association studies in humans and, perhaps more controversially, in transgenic mouse studies. METHODS: To screen for sequence variants that might be responsible for human CLP, we performed direct DNA sequence analysis in a well-characterized white European cohort of 192 patients. We screened the genes encoding SUMO1, SUMO2, and SUMO3, as well as the E3 ligases PIAS1 and PIAS2, which are required for sumoylation. Variants were analyzed in a cohort of 192 unaffected white European controls. RESULTS: Only two missense variants were identified, both within SUMO3, however, these were both present in multiple affected individuals and a similar number of controls. Other variants identified, apart from a single synonymous change in PIAS1, were all present within flanking intronic regions distant from splice consensus sites. Moreover, most other variants were previously reported in dbSNP and were shown to be present at a similar frequency in cases and controls. CONCLUSIONS: Our findings indicate that mutations identified in the SUMO-related genes tested, including three novel coding SNPs, do not directly contribute to the incidence of CLP.

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension.

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesized that this glycosaminoglycan-PCP interaction may regulate CE, but, surprisingly, DiO labelling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.