Nde Greene

Analysis of the planar cell polarity gene Vangl2 and its co-expressed paralogue Vangl1 in neural tube defect patients

To the Editor:Neural tube defects (NTD) are a heterogeneous group ofcongenitalmalformationsthataffectthebrainandspinalcord.The most severe phenotype is craniorachischisis, where thehindbrain and entire neural tube remain open. In human, itmay account for up to 10–20% of all human NTD, particularlywhen taking into account early fetal loss and therapeutictermination [Seller, 1987; Kirillova et al., 2000].Studies in Drosophila, mouse, Xenopus, and zebrafish haveindependently identified a number of genes whose productsfunction in a pathway to establish planar cell polarity (PCP).Mutationofanyofthesegenesinvertebratesresultsinanopenneuraltubeequivalenttothehumandefectcraniorachischisis.Consequently, PCP is emerging as an important biochemicalpathway for vertebrate neurulation. Through studying theLoop-tail mouse, the causitive gene Vangl2 was the first ofthe PCP genes to be identified as the cause of severe NTD[Kibar et al., 2001; Murdoch et al., 2001]. Initiation of neuraltube closure at the hindbrain–cervical boundary (Closure 1)requires the floor plate to narrow by the process of convergentextension (CE) during late gastrulation. If CE fails, the neuralfolds remain too far apart to meet and fuse [reviewed in Coppet al., 2003]. Genetic and biochemical studies have shown thatestablishment of PCP involves crucial interactions betweenmolecules such as frizzled (Fz), dishevelled (Dsh/Dvl), fla-mingo/starry night (fmi/stan/Celsr1), prickle (pk), rhoA, andstrabismus/Vangogh(Stbm/Vang)[reviewedinStrutt,2003].An added level of complexity is demonstrated by the interac-tion of the apical-basal polarity gene scribble(Scrb1), which inthe mouse mutant circletail (Crc) demonstrates a highlysimilarphenotypetobothLpandScy/Crshmutants[Murdochet al., 2003].In this study, we set out to investigate VANGL2 for a role inhuman NTD. We were also aware of a highly conservedparalogue of Vangl2 called Vangl1, which is present on mousechromosome 3qF2.2/human 1p13.1. This locus represents aduplicated block similar to the Vangl2/VANGL2 locus onmouse 1qH3/human 1q23.2. To investigate a potentiallysimilar or overlapping role for Vangl1/VANGL1 in neurula-tion, expression was first investigated using reverse tran-scriptase-PCR (RT-PCR) in both human and mouse fetaltissues (as previously described in Doudney et al., 2002).Vangl1isdetectedinwholemouseembryocDNAsspanningatleast the developmental period between E7.5 and E16.5(Fig. 1A). Human VANGL1 is widely expressed at both eightand 14 weeks gestation in all tissues investigated with theexception of placenta (Fig. 1B,C). To establish the spatial andtemporal expression of Vangl1, we performed wholemount insitu hybridization analysis of normal (þ/þ or Lp/þ) and Lp/Lpneurulation-stagemouseembryos.AtE9,Vangl1exhibiteda pattern of expression confined to the developing neural tubefrom the level of the hindbrain to the posterior neuropore(Fig. 1D,E,F, and H), with expression declining in a cranial tocaudal direction. Lp/Lp mutant embryos showed no obviousdifference in Vangl1 expression, compared to Lp/þ and wildtypeembryos(Fig.1D,E,andF).Vangl1transcriptsareabsentin all three embryos at the cardiac region, whereas Vangl2 ispresent. In comparison to Vangl2 expression, Vangl1 tran-scripts are more confined to the midline at the initiation ofClosure 1 at E8.5, and extended more caudally along the bodyaxis (compare Fig. 1G,H). Transverse sections along the bodyaxis of E9 embryos (Fig. 1D and E) showed that expression ofVangl1 is restricted to the ventral portion of the neural tube(Fig. 1I,J). In contrast to Vangl2, which at the same stage isexpressed in the flanking neuroepithelium but absent fromcells in the floor plate region [Murdoch et al., 2001], Vangl1transcripts are confined strictly to the differentiating floorplate in the cranial region (Fig. 1I–1 and 2). Interestingly, atthis level, Lp/Lp embryos showed a more diffuse expressionpattern,withVangl1expressionextendinglaterallywithinthepersistently open neural tube. More caudally, beyond the gapin expression at the cardiac level (Fig. 1I–3, J-3), expressionwas high and confined to the ventral half of the neural tube.Hence,cellsofboththefuturefloorplateandtheventro-lateralneuroepithelium express Vangl1 at these caudal levels of thebody axis. There did not appear to be any major differences inthe pattern of Vangl1 expression between Lp/þ and Lp/Lpembryos in the caudal region at E9, although the radicallydifferentneuraltubemorphology (closedinLp/þ,openinLp/Lp) might obscure subtle differences.Fromthisanalysis,weconcludethatVangl2andVangl1areco-expressed in the developing neural tube but with someimportantdifferences.MostnotablyVangl1becomesrestrictedtothefloorplateregionofthemorematureneuraltubewhichisquite different from Vangl2, which becomes specificallyexcludedfromthefloorplate,althoughitsexpressioncontinuesin non-floor plate cells of the ventral neural tube [Murdochet al.,2001].These differences may underlie theapparent lackof redundancy between the two genes, with functional Vangl1not compensating for the Vangl2 missense mutations identi-fiedintheLpmouse.ThegenerationofamouseVangl1mutantwill therefore be helpful in understanding their separate rolesin neural tube development. In contrast, comparative studiesin zebrafish show that trilobite (vangl2) and vangl1 havelargely non-overlapping expression patterns [Jessen andSolnica-Krezel, 2004]. Nevertheless, vangl1 over-expressionThis article contains supplementary material, which may beviewed at the American Journal of Medical Genetics websiteat http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html.Grant sponsor: SPARKS; Grant sponsor: The Institute ofObstetrics and Gynaecology Trust; Grant sponsor: The Hammer-smith Hospital Trust; Grant sponsor: The Wellcome Trust.*Correspondence to: K. Doudney, Institute of Reproductive andDevelopmental Biology, Imperial College London, HammersmithHospital, Du Cane Road, London, W12 0NN, UK.E-mail: kdoudney@imperial.ac.uk; p.stanier@imperial.ac.ukReceived 8 October 2004; Accepted 17 March 2005DOI 10.1002/ajmg.a.30766 2005 Wiley-Liss, Inc.

A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly

Neural tube defects (NTDs) affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida) may lead to lifelong neurological handicap. Collectively, NTDs rank among the most common birth defects worldwide. This study focuses on anencephaly, which despite having a similar frequency to spina bifida and being the most common type of NTD observed in mouse models, has had more limited inclusion in genetic studies. A genetic influence is strongly implicated in determining risk of NTDs and a molecular diagnosis is of fundamental importance to families both in terms of understanding the origin of the condition and for managing future pregnancies. Here we used a custom panel of 191 NTD candidate genes to screen 90 patients with cranial NTDs (n = 85 anencephaly and n = 5 craniorachischisis) with a targeted exome sequencing platform. After filtering and comparing to our in‐house control exome database (N = 509), we identified 397 rare variants (minor allele frequency, MAF < 1%), 21 of which were previously unreported and predicted damaging. This included 1 frameshift (PDGFRA), 2 stop‐gained (MAT1A; NOS2) and 18 missense variations. Together with evidence for oligogenic inheritance, this study provides new information on the possible genetic causation of anencephaly.

Analysis of the planar cell polarity gene Vangl2 and its co‐expressed paralog Vangl1 in neural tube defect patients (Am J Med Genet 136A: 90–92, 2005)

Erratum Analysis of the Planar Cell Polarity Gene Vangl2 and its Co-Expressed Paralog Vangl1 in Neural Tube Defect Patients (Am J Med Genet 136A: 90–92, 2005) K. Doudney,* G.E. Moore, P. Stanier, P. Ybot-Gonzalez, C. Paternotte, N.D.E. Greene, A.J. Copp, and R.E. Stevenson Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London, United Kingdom Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London, United Kingdom J.C. Self Research Institute, Greenwood Genetics Center, Greenwood, South Carolina

OC126: The PONTI (Prevention of neural tube defects by inositol) clinical trial

days 74 and 78 after conception. At 98 to 112 days an attempt to correct the defect was made in 11 cases and 6 fetuses remained uncorrected (control group). Using an open uterine approach the fetus was partially exposed from its tail to the lumbar spine. The skin around the defect was dissected below the dermis to permit the placement of an interface material over the exposed neural tissue and the skin. The skin was then completely closed over the defect using a separated suture. Pregnancy was allowed to continue and lambs were sacrificed between 137–145 days of gestation. The specimens were submitted to macroscopic and microscopic analysis. The correction was considered successful when more then 70% of the skin was closed. Results: The survival rate after the creation of the defect was 68% (17/25) and 90% (10/11) after correction. Macroscopically the skin was completely closed in 7 out of 10 cases in the corrected group and in 2 out of 6 cases in the control group. Neverthless 80% (8/10) were considered successful in the corrected cases. Transverse sections of fetal spine were microscopically analyzed in average 132 days of gestation (range 118–145 days) and the medulla had been reached in all fetuses in both groups. Conclusions: The new simplified technique was successful in the correction of the defect. We believe it can be more easily applied in an endoscopic approach for correction a human myelomeningocele.