Kit Doudney

The T-box transcription factor gene TBX22 is mutated in X-linked cleft palate and ankyloglossia.

Formation of the secondary palate is a complex step during craniofacial development. Disturbance of the events affecting palatogenesis results in a failure of the palate to close. As a consequence of deformity, an affected child will have problems with feeding, speech, hearing, dentition and psychological development. Cleft palate occurs frequently, affecting approximately 1 in 1,500 births; it is usually considered a sporadic occurrence resulting from an interaction between genetic and environmental factors. Although several susceptibility loci have been implicated, attempts to link genetic variation to functional effects have met with little success. Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a semidominant X-linked disorder previously described in several large families of different ethnic origins and has been the subject of several studies that localized the causative gene to Xq21 (refs. 10–13). Here we show that CPX is caused by mutations in the gene encoding the recently described T-box transcription factor TBX22 (ref. 14). Members of the T-box gene family are known to play essential roles in early vertebrate development, especially in mesoderm specification. We demonstrate that TBX22 is a major gene determinant crucial to human palatogenesis. The spectrum of nonsense, splice-site, frameshift and missense mutations we have identified in this study indicates that the cleft phenotype results from a complete loss of TBX22 function.

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. Hum Mutat 33:440–447, 2012.

Epithelial cell polarity genes are required for neural tube closure

Human neural tube defects (NTD) are a heterogeneous group that exhibit complex inheritance, making it difficult to identify the underlying cause. Due to the uniform genetic background, inbred mouse strains are a more amenable target for genetic studies. We investigated the loop‐tail (Lp) mouse as a model for the severe NTD, craniorachischisis. A homozygous point mutation was identified in the transmembrane protein Vangl2, which in Drosophila has been shown to function in the planar cell polarity (PCP) pathway. Morphological analysis of the Lp mice shows that the defect results from an abnormally broad floor plate, most likely through a failure in convergent extension. The elevated neural folds remain too far apart to contact, inhibiting neural tube closure. Recently, two other mouse mutants (crash and circletail) were described with a similar phenotype to Lp and were investigated as potentially new alleles. Mapping studies, however, showed that both mutants segregated to distinct loci. In the crash (Crsh) mouse, a mutation was identified in Celsr1, a seven pass transmembrane receptor that encodes a protein orthologous to Drosophila Flamingo. Like Vangl2, this gene also functions in the PCP pathway. While in circletail, a point mutation was identified introducing a premature stop codon into the apical‐basal cell polarity gene scribble (Scrb1). We subsequently demonstrated a genetic interaction between all three genes, where double heterozygotes exhibit the same homozygous NTD phenotype. This strongly suggests both a candidate gene pathway and that interaction between independent recessive alleles may be a possible explanation for the complex inheritance in severe human NTD.

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.

Genomic organization and embryonic expression of Igsf8, an immunoglobulin superfamily member implicated in development of the nervous system and organ epithelia.

Igsf8 is an immunoglobulin protein that binds to the tetraspanin molecules, CD81 and CD9. We describe the genomic organization of mouse and human Igsf8, and reveal a dynamic expression pattern during embryonic and fetal development. Igsf8 is first expressed at E9.5 in a ventral domain of the neural tube, with dorsal expression apparent at E10.5. We show that the ventral, but not the dorsal, domain of neural tube expression is dependent on Shh signaling. From E11.5, Igsf8 is expressed at the lateral edge of the ventricular zone, in early postmitotic neuroblasts, and in dorsal root and cranial ganglia. Igsf8 is also expressed in the branchial arches, dorsal pancreatic primordium, neural retina, olfactory epithelium, gut, kidney, and lung.

A family with pseudodominant Friedreich’s ataxia showing marked variation of phenotype between affected siblings

A family with pseudodominant Friedreich’s ataxia is described showing marked variation of phenotype between affected siblings. The mother of this family (III-3) developed a spastic ataxic tetraplegia with neuropathy at 34 years of age; her husband, who was unrelated, was clinically normal. Of their nine children, two (IV-2, IV-3), including one with multiple sclerosis (IV-3), developed a mild spinocerebellar degeneration in the third decade. Three in their late 20s had an asymptomatic spinocerebellar degeneration (IV-4, IV-5, IV-6) and one was confined to a wheelchair at 15 years with typical Friedreich’s ataxia (IV-9). Three other siblings (IV-1, IV-7, IV-8) were clinically normal. The father proved to be heterozygous for the triplet repeat expansion at the Friedreich’s ataxia locus and all clinically affected members were homozygous for alleles in the expanded size range. This family confirms that homozygote-heterozygote mating is the genetic basis for some families with apparent autosomal dominant Friedreich’s ataxia.

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

Physical evidence for the position of the Friedreich's ataxia locus FRDA proximal to D9S5

Orientation of the Friedreichs ataxia locus (FRDA) with respect to D9S15 and D9S5 has proved critical to the design of subsequent cloning strategies. The rarity of recombination events between FRDA and these markers, originally used to determine assignment to human chromosome region 9q13-->q21.1, has necessitated the instigation of physical mapping studies to determine order and, hence, the precise location of the disease gene. Simultaneous fluorescence in situ hybridisation using cosmid clones located in close proximity to the ends of a 1.2-Mb yeast artificial chromosome clone extending into the FRDA candidate region provides physical evidence for the order of the marker loci to be cen-D9S202-D9S5-D9S15-qter. The possibility that a pericentric inversion, occurring naturally in approximately 1% of the normal population, may affect the order of markers within this region has been eliminated. Considered in association with the interpretation of a recombination event detected in a single affected individual, these data indicate that the FRDA locus is located proximal to D9S5.