FZD6 is a novel gene for human neural tube defects.

2011·0.32 MB·English

by  De Marco, Patrizia

R A
ESEARCH RTICLE
OFFICIALJOURNAL
FZD6 is a Novel Gene for Human Neural Tube Defects
PatriziaDeMarco,1†ElisaMerello,1†AndreaRossi,2GianlucaPiatelli,1ArmandoCama,1ZohaKibar,3and www.hgvs.org
ValeriaCapra1∗
1NeurosurgeryDepartment,G.GasliniInstitute,Genova,Italy;2NeuroradiologyDepartment,G.Gaslini,Institute,Genova,Italy;3Departmentof
ObstetricsandGynecology,CHUSainteJustineResearchCenterandUniversityofMontreal,Montreal,Que´bec,Canada
Communicatedby AndrewO.M. Wilkie
Received14June2011;acceptedrevisedmanuscript7October2011.
Publishedonline1November2011inWileyOnlineLibrary(www.wiley.com/humanmutation).DOI:10.1002/humu.21643
monNTDsareanencephaly,whichresultsfromfailureoffusionof
thecranialneuraltube,andmyelomeningocele(commonlycalled
ABSTRACT: Neuraltubedefects(NTDs)areseveremal-
spinabifida),whichresultsfromfailureoffusionofneuraltubein
formations of the central nervous system, affecting 1 of
thespinalregion.ArelativelyrareformofNTDs,knownascran-
1,000 live births. Mouse models were instrumental in
iorachischisis,resultsfromfailureofneuraltubeclosurealongthe
definingthesignalingpathwaysdefectiveinNTDs,includ-
entirebodyaxis.Anencephalyandmyelomeningocelearereferred
ingtheplanarcellpolarity(PCP),alsocallednoncanonical
as “open” NTDs because the affected regions are exposed to the
Frizzled/Disheveled pathway. Based on the highly pene-
trant occurrence of NTDs in double Fzd3/Fzd6−/− mu- body surface. There are also a number of closed or skin-covered
conditions that involve the neural tube, including encephalocele,
tant mice, we investigated the role of the human ortho-
andmeningocele,lipomyelomeningocele,alsoreferredtoasspina
logues, FZD3 and FZD6, by resequencing a cohort of
bifidaocculta,andcaudalregression[Rossietal.,2004].
473NTDspatientsand639ethnicallymatchedcontrols.
NTDshaveacomplexetiologyinvolvingbothenvironmentaland
While we could not demonstrate a significant contribu-
genetic factors. Although folic acid, supplemented in the mother
tionofFZD3gene,weidentifiedfiverareFZD6variants
periconceptionally, appears to have dramatically reduced the fre-
that were absent in all controls and predicted to have a
quency of NTDs [Czeizel and Duda´s, 1992; MRC Vitamin Study
functionaleffectbycomputational analysis:onedenovo
ResearchGroup,1991],themechanismbywhichfolatedeficiency
frameshift mutation (c.1843_1844insA), three missense
predisposestoNTDsremainsunclear.
changes(p.Arg405Gln,p.Arg511Cysp.Arg511His),and
onesubstitution(c.∗20C>T)affectingthe3(cid:3)-untranslated Amajorcellulareventoccurringduringneurulationisconver-
gentextension,amorphogeneticprocessbywhichthepresumptive
region (UTR) of the gene. The overall rate of predicted
notochordandneuralplatelengthenandnarrowduetothemedi-
deleteriousvariantsofFZD6was5.1-foldhigherincases
olateralintercalationofcells[UenoandGreene,2003;Wallingford
comparedtocontrols,resultinginasignificantlyincreased
etal.,2002].Duringthisprocess,cellselongatemediolaterallyand
NTDs mutation burden. This study demonstrates that
produce polarized cellular protrusions that enable them to move
rarenonsynonymousvariantsinFZD6maycontributeto
directionally and to intercalate to other neighboring cells [Keller
NTDsinhumansandenlargesthespectrumofmutations
etal.,2000;WallingfordandHarland,2002].Convergentextension
thatlinkPCPpathwaytoNTDs.
HumMutat33:384–390,2012.(cid:4)C 2011WileyPeriodicals,Inc. ismediatedbythehighlyconservedplanarcellpolarity(PCP)path-
way,alsocalledthenoncanonicalFrizzled-Dishevelledsignalingcas-
KEYWORDS: neuraltubedefects(NTD);planarcellpo- cade.CorePCPgenesinclude:Strabismus/VanGogh(Stbm/Vang),
laritypathway;FZD6 Frizzled(Fz),Dishevelled(Dsh),Flamingo(Fmi),Prickle(Pk),and
Diego(Dgo)[SimonsandMlodzik,2008].Evidencefortheinvolve-
ment of the PCP pathway in convergent extension in vertebrates
hasemergedfromstudiesofawiderangeofmutantsoforthologs
Introduction
ofPCPgenesinseveralanimalmodelssuchaszebrafish,Xenopus,
andmouse[Barrow,2006;Heisenbergetal.,2000;Montcouquiol
Neuraltubedefects(NTDs;MIM#182940)arecongenitalmal-
etal.,2006;TadaandSmith,2000;Wallingfordetal.,2000;Walling-
formationsduetotheincompleteclosureoftheneuraltubeduring
ford,2005].Recently,weidentifiedmutationsintwohumanPCP
the early development. NTDs are known to occur in 1 of every
genes,VANGL1andVANGL2,providingevidenceforapathogenic
1,000 pregnancies, with varying rates reported among the world
role of PCP signaling in human NTDs [Kibar et al., 2007, 2009,
populations[Bottoetal.,1999;Coppetal.,2003].Themostcom-
2011].OtherPCPgeneshaveapotentialroleinconvergentexten-
sionmovementsandneuraltubeclosureinnormalandabnormal
neurulation,andhenceneedtobestudiedinlargehumanNTDs
AdditionalSupportingInformationmaybefoundintheonlineversionofthisarticle. cohorts.
†Theseauthorscontributedequallytothiswork. Frizzleds are seven-pass transmembrane (7TM) receptors that
∗Correspondenceto: ValeriaCapra,NeurosurgeryDepartment,G.GasliniInstitute, transduce critical cellular signals during development. Frizzleds
Genova,16148,Italy.E-mail:[email protected] shareacysteine-richdomain(CRD)intheN-terminalextracellu-
Contractgrantsponsors:ItalianTelethonFoundation(Grantno.GGP08051);theCana- larregion,whichbindsseveralsecretedproteins,andamongthem,
dianInstitutesforHealthResearch;theSickKidsFoundation;theFondsdelaRecherche proteinsoftheWntfamily[SchulteandBryja,2007].Aminoacid
enSante´duQue´bec. hydropathy analysis predicts aconventional 7TMstructure,three
(cid:3)C 2011WILEYPERIODICALS,INC.
extra-andthreeintracellularloops,andanintracellularcarboxy- ResequencingandGenotyping
terminalPDZ(Postsynapticdensity95[PSD-85];Discslarge[Dlg];
GenomicDNAwasisolatedfromEDTAperipheralbloodsam-
Zonulaoccludens-1 [ZO-1]) domain involved inprotein interac-
ples,byusingtheQIAampDNAbloodKit(Qiagen,Milan,Italy),
tion[SchulteandBryja,2007].Threemainsignalingpathwaysare
accordingtothemanufacturer’sprotocol.Thegenomicstructuresof
activated by Frizzleds: the PCP pathway, the canonical Wnt/β-
humanFZD3andFZD6weredeterminedusingtheNCBIGenBank
cateninpathway,andtheWnt/calciumpathway[SchulteandBryja,
(FZD3:NG_029723.1andNM_017412.3;FZD6:NG_028909.1and
2007].Wntsignalingisimportantforcelldivision(proliferation),
NM_001164615.1)(Fig.1;Supp.Fig.S1).Thewhole-codingregion
attachmentofcellstooneanother(adhesion),cellmovement(mi-
ofFZD3(3,933bplong)andFZD6(3,806bplong)withabout180bp
gration),andmanyotherprocessesbeforeandafterbirth[Umb-
ofthe5(cid:4)-untranslatedregion(UTR)and150bpofthe3(cid:4)UTRwere
haueretal.,2000;Wangetal.,1996].Itiswidelyacceptedthatbind-
amplified.Theportionsoftheintronsthatweresequencedranged
ingofthephosphoproteinDisheveled(Dvl/Dsh)anditsmembrane
from120to160bpwithanaverageofalmost140bp.Polymerase
recruitmentbyFrizzledisthecriticaleventinFrizzled-inducedsig-
chain reaction (PCR) was carried out using the AmpliTaqGold
nal transduction for all three Frizzled signaling pathways [Cong
(Applied Biosystems, Monze, Italy) as per manufacturer’s in-
etal.,2004].Therearecurrently11mammalianFrizzledproteins
structions. Direct dye terminator sequencing of PCR products
[SchulteandBryja,2007],whichhavebeenimplicatedinavarietyof
was carried out using the ABI Prism Big Dye Systems (Applied
developmentalprocesses,includingseveralthatinvolvethenervous
Biosystems).SampleswererunonABI3700automatedsequencer
system.Fzd3isrequiredforaxonaloutgrowthandguidanceinthe
(Applied Biosystems) and analyzed using the PhredPhrap soft-
centralnervoussystem(CNS)[Wangetal.2002,2006b].Targeted
ware 5.04 (http://droog.gs.washington.edu/polyphred). Nonsyn-
deletionofthemouseFzd3geneleadstoacurledtailphenotype
onymouschangesthatweremostlikelyexpectedtohaveadetrimen-
andflexedhindlimbsinnewborns,andcephalicneuraltubeclosure
taleffectwerefurthergenotypedusingtheiPLEXTM Goldassayfor
inasmallpercentageofembryos[Wangetal.2002,2006a].Double
mutantsFzd3–/–/Fzd6–/– embryosexhibitcraniorachischisis(afully SNPGenotyping(Sequenom,SanDiego,CA)[Ehrichetal.,2005].
ForvariantsidentifiedinNTDscases,wetestedthecosegregation
openneuraltube)andcurled-tailwitha100%penetrance,apartially
bysequencingthecorrespondingfragmentinavailableadditional
penetrantfailureofeyelidclosure,andmisorientatedauditoryand
familymembers.
vestibularsensoryhaircells,providingthemostdirectevidencefor
afunctionalconnectionbetweenPCPcomponentsandmammalian
tissuefusionprocesses[Wangetal.,2006a].Expressionstudiesin Bioinformatics
humanshaveshownthatFZD3(MIM#606143)andFZD6(MIM#
603409)arewidelyexpressedinbothembryonicandadulttissues, Mutations were annotated according to the HGVS nomencla-
includingbrainandCNS[Salaetal.,2000;Tokuharaetal.,1998]. ture(http://www.hgvs.org/mutnomen).Nucleotidenumberingre-
TheroleofPCPsignalinginthepathogenesisofNTDsinanimal flects cDNA numbering with +1 corresponding to the A of the
modelsandhumansaswellastheroleofFzd3andFzd6inneural ATG translation initiation codon 1 in the reference sequence,
tube closure in mouse models prompted us to investigate if the according to journal guidelines. A variant was designated as
human orthologs FZD3 and FZD6 genes could play a role in the novel if it was not found in either dbSNP Build 133 or in
pathogenesis of human NTDs, by resequencing these genes in a the 1,000 Genome Project (release of May 2011). The poten-
largecohortofpatientsandcontrols. tialpathogeniceffectofthemissensemutationsonproteinfunc-
tionwaspredictedusingtwosoftwareprograms:PolyPhen(Poly-
morphism Phenotyping) (http://genetics.bwh.harvard.edu/pph/)
SubjectsandMethods andPANTHER(ProteinAnalysisThroughEvolutionaryRelation-
ships) (http://www.pantherdb.org/). Possible effects on the sec-
ondary structure of the protein were assessed by Dompred soft-
PatientsandControls
ware (http://bioinf.cs.ucl.ac.uk/dompred/) that is designated to
Thepatientscohortconsistedof(1)391Italianpatientsrecruited predict putative protein domains and their boundaries. We used
attheSpinaBifidaCenteroftheGasliniHospitalinGenova,Italy; an input E-value cutoff default of 0.01 and the number of PSI-
amongthem,284havealreadybeenincludedinourpreviousstudies BLAST interactions at the default of 5. Multiple alignments of
[Kibaretal.,2007,2009,2011];(2)82patientsrecruitedatSainte the FZD3 and FZD6 proteins were done using the CLUSTAL W
JustineHospitalinMontreal,Canada.Detailedclinicalinformation program,freelyavailableonline(http://npsa-pbil.ibcp.fr).Localiza-
on both cohorts is presented as Supp. Table S1. All patients had tion of the variants in protein domains was assessed by Uniprot
isolatednonsyndromicNTDs.Around19%ofpatientshadaposi- (http://www.uniprot.org/).Potentialeffectofthe3(cid:4) UTRvariants
tivefamilyhistorydocumentedbyclinicalrecords(MRIandX-ray byalteringMicroRNAs(miRNA)bindingsiteswasevaluatedbyMi-
images). croRNA.orgsite(http://www.microrna.org)thatusesthemirSVR
Thecontrolgroupcomprised433Italianindividualsconsisting regressionmodelfortargetsitepredictions(mirSVRscore≤–0.1).
of randomly selected children admitted to the Gaslini Children’s
Hospitalformiscellaneousillnessesandhealthyyoungadultswho AssociationAnalysis
contributedsamplestothebloodbankoftheGasliniInstitute.The
samples were anonymous and information associated with these Rare variants were defined as those having a minor allele fre-
samples included only sex, region of birth, and age. The control quency(MAF)ofatmost1%.Chi-squareanalysisandFisher’sexact
group also included 206 Caucasian controls of French–Canadian test(two-tailed)wasusedtotestforassociationwiththevariants.
ancestry.Allcontrolindividuals(N=639)wereseenbyaneurologist
andconfirmedtobehealthy.Samplesfrompatientsandcontrols Results
were collected with the approval of the Local Ethics Committees
andwritteninformedconsentwasobtainedfromallparticipating Theresequencinganalysisofall1,112subjectsidentifiedatotalof
patients,parents,andcontrols. 12commonvariants(MAF≥1%)and28rarevariants(MAF<1%),
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Figure1. RarenonsynonymousFZD6variants.A:SchematicrepresentationsofFZD6(RefSeq:NG_028909.1;NM_001164615.1)genewiththe
approximatelocationsoftheidentifiedrarenonsynonymousvariants.FZD6mutationsabsentincontrolsandpredictedtohaveafunctionaleffect
arecircledinred.TheDNAmutationnumberingisaccordingtocDNAnumberingwithnucleotide+1astheAoftheATGtranslationinitiation
codoninthereferencesequence.Ex,exon.B:TopologicalmodeloftheFZ6protein.Theapproximatepositionsofthemutationsidentifiedare
shown.NonsynonymousFZD6mutationsabsentincontrolsandpredictedtohaveafunctionaleffectarecircledinred.CRD,cysteine-richdomain.
C:ClustalWproteinsequencealignmentofhumanFZD6withhumanFZD3andorthologuesfromotherspecies.ResiduesconservedbetweenFZD
andotherfamilymembersaregrayhighlighted.EnsembleaccessionnumbersofhumanFZD6(hFZD6):ENSP000004290551;humanFZD3(hFZD3):
ENSP000002400931,PantroglodytesFZD6 (pFZD6): ENSPTRP000000437521,gorillaFZD6 (gFZD6): ENSGOP000000153811;mouseFZD6 (mFZD6):
ENSMUSP000000229061,gallusFZD6(gaFZD6):ENSGALP000000258431,XenopusFZD6(xFZD6):ENSXETP00000088811,DanioRerioFZD6(dFZD6):
ENSDARP000000627021,andDrosophilaFZD6(drFZD6):FBpp00754851.
of which 22 were never reported before. No variants that affect RareVariantsinFZD6Gene
highlyconservedconsensussplicesiteswereidentified(Supp.Ta-
bleS2).Allofthecommonvariantswereknownsinglenucleotide We identified five rare FZD6 variants that were absent in all
polymorphisms (SNPs) and none were associated with NTDs (P controls and predicted to have a functional effect. One of them
> 0.05;) (Supp. Table S3). Rare variants were overrepresented in is a de novo frameshift mutation that introduces a premature
patientscomparedtocontrols(34patients/14controls;P=0.0001; stop codon (p.Cys615X [c.1843_1844insA]), three are missense
Table1andSupp.TableS4).NoNTDpatientorcontrolwasacarrier changes (p.Arg405Gln [c.1214G>A], p.Arg511Cys [c.1531C>T],
ofraremissensemutationsinbothFZD3andFZD6. p.Arg511His[c.1532G>A]),andlastoneisasinglenucleotidesub-
ToidentifymutationsthatcauseorpredisposetoNTDs,wefo- stitution(c.∗20C>T),affectingthe3(cid:4)UTRoftheFZD6gene.(Fig.1;
cused our analysis on 13 (three of FZD3 and 10 of FZD6; Supp. Table1).Nevertheless,theoverallrateofpredicteddeleteriousvari-
TableS2)rarenonsynonymous(missense,frameshift,and3(cid:4)UTR) antswas5.1-foldhigherincasescomparedtocontrols,forsignifi-
variants as these were more likely to have a substantial effect on cantlyincreasedmutationburdenwithNTDs(ninepatients/three
proteinfunction.Amultifacetedcomputationalapproachbasedon controls;P=0.036).
thefunctionalpredictionofnonsynonymousvariants,theconser- The FZD6 p.Cys615X (c.1843_1844insA) was a de novo muta-
vationofthealteredaminoacid,theeffectonthesecondaryprotein tion (absent in the parents), deriving from the insertion of an A
structure,andalterationofmiRNAs-bindingsitefornoncoding3(cid:4) nucleotideatposition1,843ofthecodingregionthatintroducesa
UTRvariantswasusedtohighlightvariationspotentiallyinvolved prematurestopcodonatposition615(Figs.1A,1B,and2).Itwas
inNTDs. identified in an Italian patient affected by a complex dysraphism
ResequencingoftheFZD3geneidentifiedonlyonepossiblydam- consistingofanteriorthoracicmeningocele,hydromyelia,intradu-
agingmutation,suggestingthatthisgenedoesnotplayamajorrole ral lipoma, scoliosis, and multiple vertebral and costal anomalies
inthecausationofNTDs(Supp.TableS4andSupp.Fig.S1). (Table2).Maternityandpaternitywereconfirmedbymicrosatellite
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Table1. NovelandKnownRareVariantsintheCodingandFlankingIntronicSequenceofFZD6Gene
mirSVR
Nucleotidechangea Aminoacid Resequencing Aminoacid PANTHERprediction PolyPhenprediction regressionmodel
(rsID) change 473pt/639ct conservation DompredPredictionb (subPSECscore)c (PSICscore)d (mirSVRscore)e
c.374+7A>C - 1/0 - - - - -
c.418C>T(rs80216383) p.His140Tyr 2/1f No Randomcoiled,alteration Deleterious(–4.05) Possiblydamaging(2.11) -
c.765C>T p.Gly255Gly 3/0 Yes Randomcoiled,- - - -
c.1125G>A p.Leu374Leu 1/0 Yes Alpha-helix,- - - -
c.1163C>A p.Ala388Asp 1/2 Yes Alpha-helix,alteration Deleterious(–5.52) Possiblydamaging(1.92) -
c.1214G>A p.Arg405Gln 2/0 Yes Randomcoiled,alteration NA Benign(0.38) -
c.1392+67A>T - 1/0 - - - -
c.1392+115C>T - 1/0 - - - -
c.1393-109G>T - 1/0 - - - -
c.1531C>T p.Arg511Cys 1/0 Yes Randomcoiled,alteration Notdeleterious(–2.47) Possiblydamaging(2.45) -
c.1532G>A p.Arg511His 1/0 Yes Randomcoiled,alteration Deleterious(–3.65) Benign(0.15) -
c.1809C>T p.Asp603Asp 1/0 No Randomcoiled,- - - -
c.1810G>A(rs79408516) p.Gly604Arg 2/1f No Randomcoiled,no Notdeleterious(–1.99) Benign(0.004)
alteration
c.1843_1844insA p.Cys615X 1/0 Yes - - - -
c.1858T>A(rs116195528) p.Ser620Thr 2/1f No Randomcoiled,no Notdeleterious(–1.02) Benign(0.01) -
alteration
c.1991C>A(rs12549394) p.Ala664Glu 2/2 No Randomcoiled,no NA Benign(0.35) -
alteration
c.∗20C>T - 1/0 - - - - Probably
alterating
miRNAbinding
site(–0.6)
Rarevariants 24/7
Rarefunctionaldeleteriousvariants 9/3(P=0.036)
aFZD6GenBankRefSeqnumberNM_001164615.1andNG_028909.1.NucleotidenumberingreflectscDNAnumberingwith+1correspondingtotheAoftheATGtranslation
initiationcodon1inthereferencesequence.
bLocationinproteinsecondarystructure/potentialstructurealteration.
csubPSECscore:substitutionposition-specificevolutionaryconservationscore.Continuousvaluesrangefrom0(neutral)toabout–10(mostlikelytobedeleterious).–3isthe
cutoffpoint.
dPSIC(position-specificindependentcounts)score:<1benign,>1possiblydamaging,>2probablydamaging.
emirSVRisaregressionmodelthatcomputesaweightedsumofanumberofsequenceandcontextfeaturesofthepredictedmiRNA–mRNAduplex.mirSVRdownregulation
scoresarecalibratedtocorrelatelinearlywiththeextentofdownregulationandthereforeenableaccuratescoringofgeneswithmultipletargetsitesbysimpleadditionofthe
individualtargetscores(mirSVRcutoffscore≤–0.1).ThemutationalteredthebindingsiteforMIR628.
fThesetwopatientsandonecontrolindividualarecarriersofthreeFZD6incismutations:p.His140Tyr,p.Gly604Arg,p.Ser620Thr.
pt,patients;ct,controls;NA,notaligned;3(cid:4)UTR,3(cid:4)-untranslatedregion.Deleteriousvariantsnotfoundincontrolsareinbold.
markersanalysis.Thep.Cys615Xmutationencodesforatruncated TheFZD6p.Arg511Cys(c.1531C>T)wasidentifiedinanItalian
proteinthatlacksthelast51aminoacidsinitscarboxyl-terminal patientwithmyelomeningocele(MMC)(Figs.1and2;Table2).This
tail. substitutionintroducesacysteineinanabsolutelyconservedregion
TheFZD6p.Arg405Gln(c.1214G>A)wasfoundintwounrelated andmayresultintheformationofintermoleculardisulfidebridges
patientsbothaffectedwithmyelomeningocele(Figs.1and2;Ta- leadingtoabnormalconformationalchangesoftheFZD6protein
ble2).ThisvariantwasdetectedinthefatheroftheItalianpatient (Fig.1C).Theidentificationoftwodifferentmissensemutations,
whorepresentsaNTDfamilialcasewithanaffectedthird-degree p.Arg511Hisandp.Arg511Cys,affectingthesameconservedamino
relative.Themutationaffectsaconservedresiduepredictedtolo- acidintwounrelatedNTDpatientsdemonstratethatthisresidue
calizetothethirdintracellulardomainoftheproteinandchanged could represent a mutational hot spot. Both transitions affect a
apositivelychargedresidueintoahydrophilicunchargedresidue CGdinucleotide,thereforethesequencepredisposestomutations.
(Fig. 1C). Although PANTHER and PolyPhen predicted that this Moreover, missense mutations at this site could confer a specific
variantisbenign,DomPredsoftwareshowedaneffectonthesec- pathogeniceffectassupportedbybioinformaticanalysis.
ondaryproteinstructure.Moreover,theidentificationofthisvariant TheFZD6c.∗20C>Tlocalizesatthe3(cid:4)UTRofthegene,outside
intwounrelatedpatientsaffectedwiththesamekindofopenNTDs ofproteincodingsequence.ItwasidentifiedinanItalianpatient
anditsabsenceinallcontrolsstronglysuggeststhatitismostlikely with MMC (Fig. 1A; Table 2). The C→T substitution altered the
pathogenic. predictedtargetsiteforMIR628(hsa-miR-628-3p;miRNAbinding
TheFZD6p.Arg511His(c.1532G>A)wasfoundinanItalianpa- score=–0.6;cutoffscoreis<–0.1),suggestingthatthis“apparent
tient with Caudal Regression syndrome, including vertebral and silent”variantmayhaveaneffectcreatingaberrantcis-regulatory
costalanomalies,meningocele,hydromyelia,tetheredcord,leftkid- elementformiRNA(Table1).
neyagenesis(Figs.1and2;Table2);itispredictedtolocalizeto In silico analysis predicted that FZD6 p.His140Tyr (c.418C>T;
the cytoplasmatic terminal domain of the protein (Fig. 1B). The rs80216383:C>T) and p.Ala388Asp (c.1163C>A) mutations, even
mutationwastransmittedfromhisapparentlyhealthymother.Al- iftheyweredetectedinoneandtwoof639controls,respectively,
though the variant represents an exchange of two basic amino could affect protein function, demonstrating that these variants
acids that is not considered to be a substantial chemical change, couldrepresentpredisposingriskfactors(Table1).Intriguingly,the
theseresiduesdoliewithinanabsolutelyconservedregionsuggest- FZD6p.His140Tyrvariantoccurredinciswithtwobenignmissense
ing intolerance for any amino acid substitution across evolution mutations,FZD6p.Gly604Arg(c.1810G>A;rs79408516:G>A)and
(Fig.1C). FZD6p.Ser620Thr(c.1858T>A;rs116195528:T>A)(Table1).
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Figure2. ElectropherogramsofnonsynonymousFZD6mutationsabsentincontrolsandpredictedtohaveafunctionaleffect.Thealteredamino
acidswiththeirpositionsandcorrespondingnucleotidechanges(capitalletters)areshownwiththesequencingtracedata.Smalllettersindicate
intronicsequence.TheFZD6p.Cys615Xmutationisshowninthe3(cid:4)→5(cid:4)directionandthecomplementnucleotideisshown.Theothermutations
areshowninsense(5(cid:4)→3(cid:4))direction.WT,wild-typesequence;M,mutatedsequence.∗,stopcodon.
Table2. ClinicalFeaturesofNTDsPatientsCarryingUniqueFZD6Mutations
Sex,ethnicgroup Mutations NTDstype Inheritance Clinicalfeatures
F,C p.Arg405Gln Spinal,open Fathercarrier Lumbo-sacralMMC,hydrocephalus.Familialcase:maternalthird-degree
relativehasopenNTD;parentsarefirst-degreecousins
M,C p.Arg405Gln Spinal,open UN(parentsNA) MMC,sphinctericincontinence,neurogenicbladder.
F,C p.Arg511Cys Spinal,open UN(parentsNA) MMC,ChiarimalformationtypeII
M,C p.Arg511His Spinal,closed,complex Mothercarrier CRS:sacralagenesis,spondilo-costaldysplasia,MC,tetheredcord,hydromyelia,
clubfoot,leftkidneyagenesis,convexdorso-lumbarscoliosis
F,C p.Cys615X Spinal,closed,complex No,denovo RightanteriorthoracicMC,hydromyelia,intradurallipoma,spondilo-costal
dysplasia,complexscoliosis
M,C c.∗20C>T Spinal,open UN(fatherUN) Lumbo-sacralMMC,ChiarimalformationtypeII,hydrocephalus
MMC,myelomeningocele;MC,meningocele;CRS,caudalregressionsyndrome;M,male;F,female;C,Caucasian;UN,unknown;NA,notavailable.
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Discussion couldcontributeinthepathogenesisofbothopenandclosedforms
ofNTDs,asreportedforotherPCPgenes,VANGL1andVANGL2
ThisisthefirststudythatsystematicallyscreenedallFZD3and [Kibaretal.,2007,2009,2010],furthersupportingacommonmech-
FZD6 coding regions and splice sites for small alterations in pa- anism involving defective PCP genes in the onset of both NTDs
tientswithNTDs.Resequencinganalysisin473NTDpatientsand forms.
639controlsidentifiedastatisticallysignificantoverrepresentation Bycontrast,wecouldnotdemonstrateasignificantcontribution
ofrarepresumedpathologicalvariantsamongthepatientsgroup. ofFZD3geneinthepathogenesisofNTDs.Infact,onlyonemuta-
Predictionandunderstandingofthedownstreameffectsofthenon- tionwasidentifiedhavinganevidentpathogeneticeffectonprotein
synonymousandnoncodingvariantswasdoneusingcomputational function.Furtherstudieswouldberequiredtodeterminewhether
methods.FivemutationsofFZD6,onedenovononsensemutation FZD3mayplayanindependentrole.
(FZD6 p.Cys615X), three missense variants (FZD6 p.Arg405Gln, Mouse models represent powerful tools for gene discovery in
p.Arg511Cys,p.Arg511His)affectinghighlyconservedresidues,and human disease. Fzd3–/–/Fzd6–/– mice mutants provided a success-
a3(cid:4)UTRsubstitution(FZD6c.∗20C>T)wereabsentinallcontrols fulentrypointforidentifyinghumanorthologsinvolvedinNTDs.
analyzedandhavedetrimentaleffectsonthebasisofbioinformatic However, comparison of mouse and human data provide some
tools.Thus,ourfindingssupportasignificantinvolvementofFZD6 limitedinsights,inthatdifferentlyfromdoublemutantmicethat
in the pathogenesis of a minority of NTDs patients and under- developasevereformofNTDs,mutationsofbothFZDareunlikely
scorethevalueofcandidategeneresequencingtounderstandthe tobefoundinthesameaffectedindividuals,probablyduetotheir
geneticcontributionincomplexdiseasessuchasNTDs.NTDsare lethality.Inaddition,asreportedinmice[Wangetal.,2006a],we
thesecondhumandiseasetobelinkedtomutationsofFZDgenes confirmedaredundantfunctionofFZD3andFZD6genesinneural
after familial exudative vitreoretinopathy (FEVR; MIM# 133780) tubeclosure,sincethatmutationsatonesinglegenearenotsuf-
thatarisefromlossoffunctionmutationsofFZD4,aFZDgenethat ficienttodevelopthedefect,beingthemajorityoftheminherited
hasnotfarbeenimplicatedinneuraltubeclosure[Robitailleetal., fromanhealthyparent,probablybecauseofacompensatoryeffect
2002]. ofthewild-typegene.Giventhatsingle-locusallelismisinsufficient
FZD6 mutations account only for 1% of the NTDs cases. This toexplainthevariablepenetranceandexpressivityofsuchmalfor-
mutationfrequencyiscomparabletothatdetectedinourprevious mations,geneticvariationacrossmultiplesitesofthePCPproteome
studiesinotherhumanPCPgenes[Kibaretal.,2009,2010].The mayoccurtoinfluenceclinicaloutcome.
majority of the mutations were inherited from a healthy parent In conclusion, our findings are consistent with the emerging
demonstrating incomplete penetrance. This finding suggests that model that the cumulative contributions of multiple rare alleles
mutationsatFZD6generepresentlowpenetrancevariantsthatmust withlargegeneticeffectsarefoundamongindividualswithcom-
interactwithothergenesand/orenvironmentalfactorstomodulate plextrait.IdentificationoftheroleofFZD6geneinNTDrepresents
theincidenceandtheseverityofNTDs.Alltheuniquemutations afurtherstepforwardtoourunderstandingoftheintricategenetic
wereheterozygousandprivateexceptforFZD6p.Arg405Glnfound puzzleunderlyingthesecomplexmalformations.Functionalstudies
intwounrelatedpatients:oneItalianfamilialcaseandonesporadic oftheFZD6variantswillbenecessarytoconfirmtheirimplication
Canadianpatient.Themajorityofthemutationswerelocatedinthe in NTDs risk. Finally, our promising results induce us to pursue
intracellulardomainsoftheFZDproteins.Mutagenesisstudieshave the investigation of other core PCP genes in large human NTDs
demonstratedthatseveralresiduesinthefirstandthirdintracellular cohorts.
loopsaswellasinC-terminaldomainofFZDarecrucialforsignaling
[Congetal.,2004]. Acknowledgments
Importantly,theFZD6p.Cys615XarisesfromanAinsertionin
the coding region, which introduces a premature stop codon re- We are grateful to all patients and their families. We also thank A.S.B.I.
(AssociazioneSpinaBifidaItalia),RuiuIlariaandMaraUgliettafortechnical
sulting in a truncated protein lacking the last 51 residues in its
assistance.
C-terminal tail. Genotype–phenotype correlation shows that this
de novo and severe FZD6 mutation is associated with a complex
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