Expanding the Mutational Spectrum Associated to Neural Tube Defects: Literature Revision and Description of Novel VANGL1 Mutations E. Merello1, S. Mascelli1, A. Raso1, G. Piatelli1, A. Consales1, A. Cama1, Z. Kibar2, V. Capra1, and Patrizia De Marco*1

Background: Neural Tube Defects (NTD) are a common class of birth defects that occur in approximately 1 in 1000 live births. Both genetic and nongenetic factors are involved in the etiology of NTD. Planar cell polarity (PCP) genes plays a critical role in neural tube closure in model organisms. Studies in humans have identified nonsynonymous mutations in PCP pathway genes, including the VANGL genes, that may play a role as risk factors for NTD. Methods: Here, we present the results of VANGL1 and VANGL2 mutational screening in a series of 53 NTD patients and 27 couples with a previous NTD affected pregnancy. Results: We identified three heterozygous missense variants in VANGL1, p.Ala187Val, p.Asp389His, and p.Arg517His, that are absent in controls and predicted to be detrimental on the protein function and, thus, we expanded the mutational spectrum of VANGL1 in NTD cases. We did not identify any new variants having an evident pathogenic effect on protein function in VANGL2. Moreover, we reviewed all the rare

nonsynonymous or synonymous variants of VANGL1 and VANGL2 found in patients and controls so far published and re-evaluated them for their pathogenic role by in silico prediction tools. Association tests were performed to demonstrate the enrichment of deleterious variants in reviewed cases versus controls from Exome Variant Server (EVS). Conclusion: We showed a significant (p 5 7.0E-5) association between VANGL1 rare genetic variants, especially missense mutations, and NTDs risk.

Introduction

it controls the uniform orientation of hairs and bristles on the body (Adler et al., 2000; Adler, 2002). There are two evolutionarily conserved sets of PCP factors that act together to coordinate PCP establishment: the Frizzled (Fz)/Flamingo (Fmi) core genes and the Fat/Dachsous (Ds) PCP system (Matakatsu and Blair, 2004; Casal et al., 2006). The components of the Fz/Fmi system include the transmembrane proteins Van Gogh/Strabismus (Vang, Vangl1/2 in vertebrates) (Taylor et al., 1998; Wolff and Rubin, 1998), Frizzled (Fz) (Vinson and Adler, 1987), and Flamingo (Fmi, or Starry Night, Celsr in vertebrates) (Usui et al., 1999), and the cytoplasmic proteins Prickle (Pk) (Gubb et al., 1999), Dishevelled (Dsh/Dvl) (Theisen et al., 1994) and Diego (Dgo, Ankrd6 in vertebrates) (Feiguin et al., 2001). PCP signaling uses intra- and inter-cellular feedback interactions between its core components to establish their characteristic asymmetric intracellular distributions and coordinate planar polarity of cell populations. Downstream of the PCP system are so-called “PCP effectors”, which are the novel proteins, Inturned, Fuzzy and Fritz that mediate the PCP signaling in different tissues (Collier and Gubb, 1997; Adler, 2002; Adler et al., 2004). In vertebrate gastrula, PCP pathway has been implicated in the regulation of convergent extension (CE), a medially directed movement of the cells with intercalation in the midline which leads to narrowing and lengthening of the neural plate (Wallingford et al., 2002; Wallingford, 2005). Evidence for the involvement of the PCP pathway in CE process has emerged from studies of a wide range of mutants of orthologs of Drosophila PCP genes in several

Neural Tube Defects (NTD; MIM #182940) constitute one of the most common congenital abnormalities in humans, occurring in 1 per 1000 live births (De Marco et al., 2006). They arise when the neural tube, the embryonic precursor of the brain and spinal cord, fails to close during neurulation (Copp et al., 2003). NTD range across a spectrum of pathologies, from very benign closed spinal dysraphisms, to the severe open forms, such as myelomeningocele and craniorachischisis (Tortori-Donati et al., 2000). Despite many advances in the understanding of NTD and the identification of many genes related to NTD, the fundamental etiology for the majority of NTD cases remains unclear (De Marco et al., 2006). Planar cell polarity (PCP) signaling pathway, also referred to as the noncanonical Wnt pathway which is important for polarized cell movement through the activation of cytoskeletal pathways, has been shown to play multiple roles during neural tube closure. The PCP pathway was first identified in the fruit-fly Drosophila, where

This research has been supported by Ricerca Corrente Ministero Salute- Italy, Fondazione San Paolo and private funding resources. 1 Istituto Giannina Gaslini, Genova, Italy 2 Department of Neurosciences, CHU Sainte Justine Research Center and University of Montreal, Montreal, Qu ebec, Canada. *Correspondence to: Patrizia De Marco, UOC Neurochirurgia, Istituto Giannina Gaslini, Genova, Italy. E-mail: [email protected] Published online 10 September 2014 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdra.23305

C 2014 Wiley Periodicals, Inc. V

Birth Defects Research (Part A) 103:51–61, 2015. C 2014 Wiley Periodicals, Inc. V

Key words: Neural Tube Defects (NTD); Planar Cell Polarity (PCP) pathway; VANGL1; VANGL2

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animal models such as zebrafish, Xenopus and mouse (Tada and Smith, 2000; Heisenberg et al., 2000; Barrow, 2006; Montcouquiol et al., 2006). PCP mutant mice presenting severe NTD forms (mainly craniorachischisis, and exencephaly) at high penetrance (95–100%) are: Loop-tail (Lp) (Kibar et al., 2001), Celsr1 (a mouse Fmi ortholog) (Curtin et al., 2003), and double mutants Dvl1/Dvl2 (Hamblet et al., 2002), Dvl2/Dvl3 (Etheridge et al., 2008), Dvl2/Vangl2 (Wang et al., 2006a), Fzd3/Fzd6 (Wang et al., 2006b). Also, mouse mutants of non-core vertebrate-specific PCP genes including Ptk7 (Lu et al., 2004; Paudyal et al., 2010), Scrib1 (homolog of Drosophila Scribble) (Murdoch et al., 2003), Dact-1 (Suriben et al., 2009), and the tissue-specific PCP effectors mutants, Fuz (homolog of Drosophila Fuzzy) and Intu (homolog of Drosophila Inturned), develop severe forms of NTD (Gray et al., 2009; Heydeck et al., 2009; Zeng et al., 2010). PCP mutants are characterized by other types of tissue dysmorphogenesis, including defective orientation of coat hair shafts and follicles (Devenport and Fuchs, 2008), disruption of stereocilia polarity in the inner ear and irregularities in left–right determination (Wang et al., 2006a). Looptail (Lp) is a semidominant mutation in which Lp/ 1 heterozygotes display uro-genital defects and a characteristic “looped” tail. Lp/Lp homozygotes die in utero of a severe NTD called craniorachischisis (Strong and Hollander, 1949; Smith and Stein, 1962; Gerelli and Copp, 1997). Additional defects in Lp/Lp embryos are noticed in the inner ear (organization of hair cells of the cochlea) and the heart (outflow tract defects) (Murdoch et al., 2001). Lp is caused by mutations in Vangl2 a gene that codes for a 521-amino acid transmembrane (TM) protein composed of four putative TM domains in the N-terminal half (Curtin et al., 2003). The C-terminal half is predicted to be cytoplasmic and is possibly involved in intra-cellular signaling and/or interaction with other proteins. During development, Vangl2 is expressed along the neural tube at the time of closure, from the midbrain/hindbrain boundary to its most caudal extent, marking the entire neuroepithelial dorsal–ventral axis, including floor plate cells. Lpassociated Vangl2 mutations (Vangl2 D255E, Vangl2 S464N) map to the cytoplasmic domain of the protein and abrogate interaction of Vangl2 with Dishevelled (Kibar et al., 2001; Murdoch et al., 2001). The second vertebrate homolog of the fly Stbm found in zebrafish, frogs, mice, and humans is Vangl1, sharing 68% identity with Vangl2 (Katoh, 2002). Tissue cell distribution of Vangl1 mRNA shows a nonoverlapping expression pattern with that of Vangl2. During neurulation, Vangl1 mRNA expression is limited to the narrow strip of cells along the midline of the floor plate, in an area that lacks Vangl2 mRNA expression (Torban et al., 2004). Heterozygote (gt/1) and homozygote (gt/gt) Vangl1 mutant mice are viable and fertile, although Vangl1 homozygotes display subtle alterations in

NEW VANGL1 MUTATIONS IN NTD

polarity of inner hair cells of the cochlea. Remarkably, Vangl1(gt/1);Vangl2(Lp/1) double heterozygotes show profound developmental defects that include severe craniorachischisis, inner ear defects and cardiac abnormality (Torban et al., 2008). These results show that genetic interaction between Vangl1 and Vangl2 genes may causes NTD and raise the possibility that interaction between individual Vangl genes and other genetic loci and/or environmental factors may additionally contribute to the etiology of NTD. In the last years also on the basis of findings on animal models, seven human core PCP genes, VANGL1, VANGL2, PRICKLE1, FZD6, CELSR1, DVL2, ANKRD6 (Kibar et al., 2007, 2009, 2011; Lei et al. 2010; Bosoi et al. 2011; De Marco et al., 2012; Allache et al., 2012; Robinson et al., 2012; De Marco et al., 2013; Lei et al., 2014; Allache et al., 2014a), one PCP-effector gene, FUZ (Seo et al., 2011) and three non-core PCP-related genes, SCRIB, DACT1, and LRP6 (Robinson et al., 2012; Shi et al., 2012; Allache et al., 2014b) have been implicated as predisposing factors of isolated nonsyndromic NTD by mutational screening or case-control studies. Rare nonsynonymous variants absent in controls and predicted to be functionally damaging have been found in various NTD series. The putative mutations were located in different domains of their respective proteins. All were in a heterozygous form and nearly all were private mutations. Here, we report the results of VANGL1 and VANGL2 mutational screening in a series of 80 individuals including NTDs patients and couples with a personal history of NTD-affected pregnancy. We identified three new rare heterozygous VANGL1 mutations. We reviewed the reported rare nonsynonymous or synonymous variants of VANGL1 and VANGL2 found in patients and controls and published in the last 9 years and re-evaluated them for their pathogenic role by in silico prediction tools. Test associations were performed to draw conclusions on the overall role of rare genetic variants in VANGL1 and VANGL2 on NTD risk.

Subjects and Methods PATIENTS AND CONTROLS

We evaluated three different groups all recruited between the period of 2011 to 2014: Group 1 included 48 affected newborns or children (mean age 7.5 years); 19% of these patients had a diagnosis of open isolated NTD (MMC, myelomeningocele) and 81% was affected by closed isolated NTD, including both cranial (meningocele) and spinal forms (lipomyeloschisis, tethered cord, complex dysraphisms); Group 2 included 5 adults affected by closed forms of NTD (mean age, 42 years) including a women presenting occult spinal dysraphism who interrupted a pregnancy complicated by an open form of NTD (anencephaly); Group 3 included 27 healthy parents (mean age 34 years) who have had one (N 5 23) or two (N 5 4)

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pregnancies complicated by open forms of NTD (anencephaly and MMC) and gave contents for mutational screening given their increased risk following genetic counseling. The majority of the individuals were recruited at the Spina Bifida Center of the Gaslini Hospital in Genoa, Italy. Nineteen individuals were sent from other national hospitals (Torino, Milano, Bologna, Bari, Palermo, Trento, Firenze, Verbania). All individuals were Italian with the exception of one Romanian and one Eritrean patient. All individuals from the three groups were re-evaluated by a clinical geneticist and diagnosis was made on the basis of MRI, Xray images and clinical records, according to Rossi et al. (2004). All individuals were analyzed for VANGL1 mutations and only 43 of them (31 from Group 1; 1 from Group 2; 11 from Group 3) were analyzed for VANGL2. For variants identified in NTD cases, we tested their cosegregation by sequencing the corresponding fragment in available additional family members. The control group comprised 200 Italian individuals consisting of randomly selected children admitted to the Gaslini Children’s Hospital for miscellaneous illnesses and healthy young adults who contributed samples to the blood bank of the Gaslini Institute. The samples were anonymous, and information associated with these samples included only sex, region of birth, and age. Normal individuals had participated in our previous screening studies (Kibar et al., 2007, 2009, 2011; Bosoi et al., 2011; De Marco et al., 2013). Samples from patients and controls were collected with the approval of the Local Ethics Committees and written informed consent was obtained from all participating patients, parents and controls. RESEQUENCING AND GENOTYPING

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the reference sequence. A variant was designated as novel if it was not found in either the NCBI dbSNP Build 129 or 132, 1000 Genome Project and National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project databases. Prediction and understanding of the downstream effects of the variants was done using freely available online computational methods. Deleterious effects of nonsynonymous SNPs were assessed using Mutation Taster (http://www.mutationtaster.org/) and/or PolyPhen-2 (Polymorphism Phenotyping v2) (http://genetics.bwh.harvard.edu/pph2/) and SIFT (Sorting Intolerant from Tolerant) (http://sift.jcvi.org/). Effects of synonymous variants were evaluated only by Mutation Taster prediction tool. Localization of the variants in protein domains were assessed by Uniprot (http://www.uniprot.org/). The level of conservation of a nucleotide was evaluated by the phyloP program (values between 214 and 16) based on the multiple alignment of genome sequences of 46 different species. Sites predicted to be conserved are assigned positive scores, while sites predicted to be fast-evolving are assigned negative scores. To biophysically validate the proposed impact of mutation on protein structure and function, Align-GVGD (http://agvgd.iarc.fr/agvgd_input.php), was used. This software combines the biophysical characteristics such as side chain composition, polarity and volume of amino acids and protein multiple sequence alignments. Grantham Variation (GV) and Grantham (GD) scores predict where amino acid substitutions fall in a spectrum from deleterious to neutral. The outputs of Align GVGD are combined to provide a seven-tiered genetic risk classifier: C0, C15, C25, C35, C45, C55, and C65 where C0 describes the category of variants least likely to be deleterious and C65 describes the category of variants most likely to be deleterious.

Genomic DNA was isolated from EDTA peripheral blood samples, by using the QIAamp DNA blood Kit (Qiagen Inc., Valencia, CA), according to the manufacturer’s protocol. The genomic structures of human VANGL1 and VANGL2 were determined using the NCBI GenBank (VANGL1: NM_138959.2; VANGL2: NM_020335.2). VANGL gene exons and exon-intron boundaries were PCR amplified using specific primers as previously reported (Kibar et al., 2007, 2011). Direct dye terminator sequencing of PCR products was carried out using the ABI Prism Big Dye Systems (Applied Biosystems; Foster City, CA). Samples were run on ABI 3700 automated sequencer and analyzed using the PhredPhrap software 5.04 (http://droog.gs.washington. edu/polyphred). The putative mutation was confirmed by sequencing in both directions of the PCR products.

Rare variants were defined as those having a minor allele frequency (MAF) of 1%. All the stop-gain/loss, splice, Indel, synonymous and missense mutations with MAFT (p.Ala187Val), c.1165G>C (p.Asp389His), and c.1550G>A (p.Arg517His), that were not present in 200 ethnically

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NEW VANGL1 MUTATIONS IN NTD

TABLE 1. Rare (MAFC (p.His350His; rs147220426) and the c.1068G>A (p.Lys356Lys), both predicted to cause splice site changes by Mutation Taster. These two changes were found in a child with a cranial form of NTD, cephalocele, and in a father of a fetus affected by MMC, respectively. Two common mutations were also identified one missense change, the c.346G>A (p.Ala116Thr; rs4839469) and a synonymous change the c.330C>T (pTyr110Tyr; rs41275546) that were predicted to be benign polymorphisms. MUTATIONAL ANALYSIS OF VANGL2

We failed to identify any new variants having an evident pathogenic effect on protein function in 43 individuals

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NEW VANGL1 MUTATIONS IN NTD

FIGURE 1. VANGL1 new missense variants. A: Electrophoregrams showing the nucleotide changes. B: Partial alignment of human VANGL1 sequence with orthologues from other species. Residues conserved between VANGL and other family members are black highlighted. Ensemble accession numbers of human VANGL1 ENST00000355485; Ptroglodyte ENSPTRG00000001147; Mmulatta ENSMMUG00000008967; Fcatus ENSFCAG00000010493; Mus Musculus ENSMUSG00000027860; Ggallus ENSGALG00000015025; Trubripes ENSTRUG00000003698; Drerio ENSDARG00000004305; Celegans B0410.2.WT: wildtype sequence; MT: mutant sequence. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(Table 2). Three common synonymous variants were identified, the p.Lys379Lys (rs12086448); the p.Gly445Gly (rs17380127), and the p.Pro467Pro (rs17380141), that did not introduce splice site changes. Eight of 43 patients were carriers of all three synonymous changes demonstrating that they constitute a relatively common haplotype among Italians. No NTD patient or control was a carrier of rare missense mutations in both VANGL1 and VANGL2. REVIEW OF LITERATURE AND ASSOCIATION TESTS

We revised all the published rare (MAF< 1%) nonsynonymous or synonymous variants of VANGL1or VANGL2 found in patients and controls and re-evaluated them for their pathogenic role by in silico predictive tools (Tables 1 and 2; Fig. 2). Mutational reports of VANGL1 were published in four previous studies, two of them were published by our group (Kibar et al., 2007, 2009; Bartsch et al., 2012; Doudney et al., 2005). In a recent additional study, Cai et al. (2014) performed a case-control study of two

common VANGL1 polymorphisms, p.Glu347Ala and p.Ala116Thr; thus, we excluded this report because it was not pertinent to the aims of our review. Few published studies reported results of VANGL2 re-sequencing (Lei et al., 2010; Kibar et al., 2011). Including this study, a total of 1007 NTDs patients have been so far re-sequenced for VANGL1 and 501 were of Italic origin. Overall, 60 rare VANGL1 mutations were identified, 48 in NTD patients and 12 in controls; thirteen were not present in public database, while all others were reported with very low frequencies. They were mostly missense (40/60; 67%) mutations and private. Thirty-four variants of patients and 11 of controls were predicted to be detrimental (missense variants were considered detrimental by at least two of three prediction tools). A group of 200 Italian controls individuals was re-sequenced identifying four VANGL1 variants that Mutation Taster predicted to be deleterious (Kibar et al., 2009) This control group was inadequate for

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TABLE 2. Rare (MAF

Expanding the mutational spectrum associated to neural tube defects: literature revision and description of novel VANGL1 mutations.

Neural Tube Defects (NTD) are a common class of birth defects that occur in approximately 1 in 1000 live births. Both genetic and nongenetic factors a...
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