analysisinpatientswithOFCDandLenzmicrophthalmiasyndromesmentalretarda - PDF document

analysisinpatientswithOFCDandLenzmicrophthalmiasyndromes,mentalretardationwithocularanomalies,andcardiaclateralitydefectsEmmaHilton,JenniferJohnston,SandraWhalen,NobuhikoOkamotoYoshikazuHatsukawa,JuntaroNishio,HiroshiKohara,YoshikoHirano,SeijiMizunoChiharuTorii,KenjiroKosaki,SylvieManouvrier,OdileBoute,RahatPerveenCarolineLaw *Correspondence:ProfessorLGBiesecker,NationalHumanGenomeResearchInstitute,NIH,49ConventDrive,MSC4472,Bethesda,MD20892-4472,USA.Tel:13014022041;Fax:13014022170;E-mail:leslieb@helix.nih.govTheseauthorscontributedequallytothiswork. EuropeanJournalofHumanGenetics(2009)17,1325–13352009MacmillanPublishersLimitedAllrightsreserved1018-4813/09$32.00www.nature.com/ejhg OFCDsyndrome,(2)putativeX-linked(‘Lenz’)microphthalmiasyndrome,(3)isolatedoculardefectsand(4)lateralityphenotypes.WepresentanewcohortoffemaleswithOFCDsyndromeandnullmutationsinsupportingthehypothesisthatBCORisthesolemolecularcauseofthissyndrome.WeidentifyforthefirsttimemutationsintwofemaleswithOFCDsyndromeandoneapparentlyasymptomaticfemale.WepresentafemalediagnosedwithisolatedoculardefectsandidentifyminorfeaturesofOFCDsyndrome,suggestingthatOFCDsyndromemaybemildandunderdiagnosed.WehavesequencedacohortofmalesdiagnosedwithputativeX-linkedmicrophthalmiaandfoundamutation,p.P85L,inasinglecase,suggestingthatBCORmutationsarenotamajorcauseofX-linkedmicrophthalmiainmales.Theabsenceofmutationsinapanelofpatientswithnon-specificlateralitydefectssuggeststhatmutationsinarenotamajorcauseofisolatedheartandlateralitydefects.PhenotypicanalysisofOFCDandLenzmicrophthalmiasyndromesshowsthatinadditiontothestandarddiagnosticcriteriaofcongenitalcataract,microphthalmiaandradiculomegaly,patientsshouldbeexaminedforskeletaldefects,particularlyradioulnarsynostosis,andcardiac/lateralitydefects.EuropeanJournalofHumanGenetics(2009)17,1325–1335;doi:10.1038/ejhg.2009.52;publishedonline15April2009BCL-6corepressor;oculofaciocardiodentalsyndrome;Lenzmicrophthalmiasyndrome;mentalretardation;oculardefectsIntroductionTheX-linkedmicrophthalmiasyndromes,11ofwhicharecurrentlydefined(Table1),compriseaclinicallyandmolecularlydiversegroupofdisorders,anumberofwhichoverlapwithX-linkedmentalretardationsyndromes.Lenzmicrophthalmiasyndromehasbeenrecognizedformorethan50yearsandwaspreviouslyassumedtobeaunitaryentity.However,clinicalandlinkageanalysessuggestthatitmaybeaetiologicallyheterogeneous,linkedtobothXq27-q28(MCOPS1;MIM309800)orXp11.4(MCOPS2;MIM300166).Specifically,theMCOPS2formofLenzmicrophthalmiasyndromehasbeenshowntobecausedbymutationoftheBCL-6corepressorgene(thegeneticaetiologyofMCOPS1remainsunknown.TheoriginalreportofLenzdescribedafamilywithvariableocularmanifestations,includinganophthalmia,micro-phthalmiaandcoloboma.Therewerenumerousextra-ocularanomaliesincludingmentalretardation,palatalanddentalanomalies,congenitalheartdefects,skeletaldefects(affectingthefingersandclavicles),unilateralrenalaplasiaandcryptorchidism;thisphenotypesubstantiallyoverlapswithotherX-linkedmicrophthalmiasyndromes(Table1).AnotherX-linkedmicrophthalmia-associatedcondition,oculofaciocardiodental(OFCD)syndrome,ischaracterizedbyoculardefects(congenitalcataracts,microphthalmia),facialanomalies(septatenasaltip,highnasalbridge,midfacehypoplasia,palatalanomalies),congenitalcardiacdefects(atrial/ventricularseptaldefects,othercomplexheartdefects),dentalirregularities(canineradic

ulomegaly,delayedandpersistentdentition,hypodontia)andskeletalanomalies(syndactyly,hammer-typeflexiondeformi-5–10Allaffectedindividualsarefemale,withseveralincidencesofmother-daughtertransmission.In2004,asequencevariantwasidentifiedwithinthegeneinasinglefamilywithLenzmicrophthalmiaThemissensemutation(c.254CT,p.P85L)cosegregatedwiththediseasephenotypeandwasnotidentifiedinmorethan450controlchromosomes.ItrepresentsthesolemolecularcauseofLenzmicrophthal-miasyndromeidentifiedtodate.Inthesamestudy,mutationswerefoundinalltestedpatientswithOFCDsyndrome,suggestingthetwoconditionswereallelic.Themutationtypesincludednonsense,frameshift,deletionandsplicingmutations,afindingsubsequentlyreplicatedbyothers.Importantly,OFCDsyndromehasbeenshowntoencompassdefectsoflaterality,includingtheheartandotherviscera,suggestingthatisnecessaryforleft–rightasymmetricdevelopmentandthatmutationsinmayrepresentasourceofthereportedmaleexcessoflateralitydefects.Thisstudyaimstoexpandourunderstandingofthephenotypesassociatedwithmutationsin,support-ingthehypothesisthatOFCDsyndromeissolelyasso-ciatedwithnullmutationsinandtoidentifyadditionalpatientswithLenzmicrophthalmiasyndromewhocarrymutationsin.Wehavedemonstratedthatmutationsinmayberesponsibleforapparentlyisolatedocularanomaliesandtestedthehypothesisthatmutationscausenon-syndromiccardiac/lateralitydefects.Thecontinuousaccumulationofpatientdataallowsthefrequencyofnon-cardinalphenotypestobeestimatedandimprovediagnosisandtreatmentofMaterialsandmethodsDirectsequencingoftheThecodingexonsandflankingintronicsequencesofthegenewereamplifiedbyPCRanddirectlysequencedaspreviouslyreported. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics Semi-quantitativemultiplexfluorescentPCRanalysisoftheDeletionswithinthegeneweredetectedbysemi-quantitativemultiplexfluorescentPCR(QMF-PCR).Allexonsofthegeneandthreecontrolgenes(–chromosome21,–chromosome7andFactorIXchromosomeX)wereamplifiedintwoduplicatemultiplexreactions(primersequencesinSupplementaryTable1).Oneofeachprimerpairwaslabelledwiththefluorescentphosphoramidite6-FAMdye.Amplificationswereper-formedin25lreactionsusingtheQIAGENMultiplexPCRkit(Qiagen,France)with75ngofgenomicDNAandamixofprimers(concentrationrange1.3–8).Thereactionstartedwithaninitialdenaturationof15minat95Cfollowedby23cyclesat95Cfor30s/60Cfor30s/72Cfor45swithanincrementof3spercycle.Finalextensionwasat72Cfor10min.PCRproductswerepurifiedandprocessedaspreviouslydescribed.TwocontrolDNAs(maleandfemale)wereincludedineachexperiment.Resultswereanalysedbysuperimposingfluorescentprofilesoftestedpatientsandcontrols.insituhybridizationanalysisoftheTodeterminetheproportionofcellswithadeletioninthegene,fluorescentinsituhybridization(FISH)wasperformedaspreviouslydescribed.Metaphasespreadsofperipheralleucocyteswereobtainedaccordingtostandardtechniques.BACclonesusedinFISHexperimentswereprovidedbyTheWellcomeTrustSangerInstitute(Cambridge,UK).CloneslocalizedonchromosomeXp11.4(RP11-320G24,RP11-330L22andRP11-429N5)weredirectlylabelledwithCy3.Chromosomeswerecounter-stainedwithDAPI.ThespecificsignalintensityanditssublocalizationalongthechromosomeaxiswereanalysedusingaLeicafluorescencemicroscopeequippedwiththeVisilog-6program(Noesis,LesUlis,France).EthicsapprovalThehumansubjectresearchdescribedherewasreviewedandapprovedbyethicscommitteesatStMary’sHospital(Manchester,UK),theNationalIn

stitutesofHealth(Bethesda, Table1DefinedX-linkedmicrophthalmiasyndromesSyndromenomenclatureandalternativenamesMIMDescriptionLocusGeneMCOPS1Lenzmicrophthalmiasyndrome(MAA)309800Micro/anophthalmia,dentalanomalies,skeletal/digitalanomalies,mentalretardation,facialdysmorphiaOculofaciocardiodentalsyndrome300166Micro/anophthalmia,congenitalcataracts,radiculomegaly,cardiacdefects,skeletal/digitalanomalies,facialdysmorphiaMCOPS4301590Micro/anophthalmia,ankyloblepharon,mentalMidassyndrome309810Microphthalmia,linearskinpigmentationdefectsXp22.2MCOPCT3302300Microphthalmia,congenitalcataractMCOPCB1300345Microphthalmia,coloboma(Focaldermalhypoplasia)Goltzsyndrome305600Microphthalmia,coloboma,sclerocornea,linearskinlesions,digitalanomalies,mentalretardationBRESHECKsyndrome300404Microphthalmia,CNSanomalies,skeletalanomalies,mentalretardation,renalhypoplasia(Nance–Horansyndrome)302350Microcornea,cataracts,dentalanomaliesXp22.13(Norriedisease)310600Microphthalmia,neuroretinaldegeneration,mentalretardation,sensorineuraldeafnessNotassigned;proposedaspartoftheRenpenningsyndrome300463Microphthalmia,coloboma,skeletal/digitalanomalies,mentalretardation,facialdysmorphiaMCOPS1,microphthalmia,syndromic1;MAA,microphthalmiaandassociatedanomalies;MCOPS2,microphthalmia,syndromic2;MAA2,microphthalmiaandassociatedanomalies2;MCOPS4,microphthalmia,syndromic4;MCOPS7,microphthalmia,syndromic7;MCOPCT3,microphthalmia,isolated,withcataract3;MCOPCB1,microphthalmia,isolated,withcoloboma1;BRESHECK,brainanomalies,retardation,ectodermaldysplasia,skeletalmalformations,Hirschsprungdisease,ear/eyeanomalies,cleftpalate/cryptorchidism,andkidneydysplasia/hypoplasia;CNS,centralnervoussystem.Notethelargephenotypicoverlapofclinicalfeatureswithinthisgroupofdisorders. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics MD,USA),BaylorCollegeofMedicine(Houston,TX,USA),andHopitalHenriMondor(Creteil,France).ResultsSequenceandcopynumberanalysisofpatientswithOFCDsyndromeFromexistingliteratureandpreviouswork,wehaveidentified33femaleswithOFCDsyndrome.3,5–11,16–25thisstudy,twoofthesepatientsaredescribedinfurtherclinicaldetailandmutationalanalysisofpletedinafamily(motheranddaughter).Inadditiontothesefourpreviouslyreportedpatients,wehaveidentifiedafurther31femalesputativelydiagnosedwithOFCDsyndromeandfoundmutationsininallfamilies,bringingthecohortofpatientsdescribedhereto35cases.Asummaryofthephenotypesandmutat-ionsidentifiedinpatientswithOFCDsyndromeisgiveninTable2.ThepositionsofmutationsinrelationtotheexonsequencesareshowninFigure1.ThereferencesequenceusedformutationnumberingisgiveninSupplementaryData1.The31novelpatientsdescribedherebringsthetotalnumberofreportedcasesofOFCDsyndrometo64.WehavealsosequencedthreefemaleswithfeaturesofOFCDsyndromewhoprovednegativeformutations;however,thephenotypeswereatypicalforOFCDsyndrome,withanotablelackofradiculomegalyinthedentalphenotypeinallThemutationsidentifiedhereandinotherstudiesinvolvedeletionsofsignificantportionsofthecodingsequence,alterationstoconservedspliceacceptorsites(predictedtoleadtoexonskippingwithconcomitantframeshifts)orsmallinsertions/deletionswhichcauseframeshiftsandgenerateprematurestopcodons;itispredictedthatsuchprematurestopcodonswilltriggernonsense-mediateddecayofthemRNAalthoughthegenerationofC-terminallytruncatedproteinspeciesisalsopossible.Theobservationthat,inallfemalestes

tedtodate,X-inactivationisgrosslyskewedinfavourofthewild-typeallele(datanotshown)suggeststhatlossofwild-typeBCORproteinconferssignificantselectivedisadvantage,certainlyinhaemato-logicallineages.FemaleswithOFCDsyndromearethere-forefunctionalmosaics,withcellpopulationsandtissuesexpressingeitherwild-typeBCOR(wherelackofBCORfunctionispresumedlethal)ornoBCOR/truncatedBCOR(wherelackofBCORfunctioncanbesupported).Amongstthepathogenicvariantsaretwofurtherinstancesofapreviouslyreportedc.2926CTp.R976XnonsenseInthisstudy,wereportforthefirsttimetwofamilieswithindividualsmosaicforamutationinXVIandOFCDXVII).InOFCDXVI,theindividualsmosaicforamutationhavethecardinalphenotypeassociatedwithOFCDsyndrome.Incontrast,themosaicindividualfromOFCDXVIIisasymptomatic.Inbothfamilies,offspringpresentwiththecardinalOFCDsyn-dromephenotypeandarenon-mosaicforheterozygousOFCDXVIIndividualsXVI.1andXVI.2aremonozygotictwinsisters,bothpresentingwithaclassicalOFCDsyndromephenotype.IndividualXVI.3(thedaughterofindividualXVI.1)alsopresentedwithanoverlappingarrayoffeatures.UsingaquantitativefluorescentPCRmethod,adeletionofatleastexons4–15wasdetectedinpatientsXVI.1andXVI.2.However,thedecreaseinPCRproductamountderivedfromthegenewasestimatedataround75%ofcontrolpeakheight,ratherthanthe50%expectedforanon-mosaicdeletion(Figure2aandb).Thisresultwasrecapitulatedforallfragmentsamplifiedandstronglysuggestedsomaticmo-saicismofthisdeletion.Bycontrast,amplificationwasreducedto50%ofcontrolpeakheightinpatientXVI.3,suggestingnon-mosaicdistributionofthe(Figure2c).FISHanalysiswasundertakentoconfirmsomaticmosaicisminindividualsXVI.1andXVI.2.FISHanalysisonmetaphasechromosomesfrombothpatientsdemonstratedadeletioncorrespondingtoBACcloneRP11-330L22in52/100metaphasepreparationsforeachpatient,withonlyonesignalfromtheXchromosome(Figure2g;datafrompatientXVI.1notshown).Twodistinctsignalsweredetectedintheremaining48leucocytepreparations(Figure2h;datafrompatientXVI.1notshown).XchromosomeswereidentifiedusinganXcentromeremarkerandbybandingpattern(notshown).Theseresultssupportedthehypothesisthatthesetwoindividualsaremosaicforthedeletedregion.ForpatientXVI.3,100/100metaphasepreparationsgeneratedasinglesignal,confirmingthenon-mosaicnatureofthedeletioninthispatient(Figure2i).TwoBACclonesflankingRP11-330L22,RP11-429N5(proximal)encompassingexons4–15ofBCORandRP11-320G24(distal)weretestedinpatientXVI.3.Twospecificsignalsweredetectedineachmetaphasepreparation,suggestingthattheseregionsarenotdeletedinthisfamily(theproximalRP11-429N5probecontainssufficientsequenceoutsideofthegenetopermitOFCDXVIITheproband(XVII.2),a14–month-oldgirl,presentedwithaclassicalOFCDsyndromephenotype.ThemotherwasasymptomaticandhadanormalpanoramicdentalX-ray.QuantitativefluorescentPCRdetectedareducedamountofproductcorrespondingtoexons13and14,suggestingadeletionoftheseexonsinbothfemales.ComparisonofamountsofPCRproductobtainedsuggestedthatthemother(XVII.1)wasmosaicforthisdeletion,withtheamountofPCRproductat75%ofthecontrolpeakheight MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics Table2ClinicaldataandidentifiedmutationsinforthepatientcohortpresentedhereFamilyCaseAgeOcularFacial/cranialCardiacDentalSkeletalOtherMutationOFCDI134years8daysCongenitalcataractMicrophthalmia(2)Ptosis(1)Lensdislocation(2)Opticdiscdysplasia(2)SeptatenasalcartilageHighnasalbridge(2)Heartmurmur(1)ASD(2)VSD

(2)Pulmonaryvalvestenosis(2)Uneruptedsecondaryteeth(1)Rootradiculomegaly(1)Hypodontia(1)Notrecorded(2)Hammertoes(1)Radioulnarsynostosis(1)Poorfeeding(2)c.2926COFCDII123yearsCongenitalcataractSeptatenasalcartilageVSDPersistantprimaryteethUneruptedsecondaryteethRootradiculomegalyHammertoesHearingimpairmentc.1539_1540insGOFCDIII169years65years43years40years21years4yearsCongenitalcataractGlaucoma(2,4)Phthisisbulbi(1)Septatenasalcartilage(1,3,4,5)Cleftpalate(6)Notaffected(2)ASD(6)VSD(6)DORV(6)Notrecorded(1,2,3,4,5)Delayedprimarydentition(6)Persistentprimaryteeth(1,3,5)Uneruptedsecondaryteeth(1,2,3,4,5)Rootradiculomegaly(1,2,3,5)Hypodontia(5)Hammertoes(1,2,4,5)Second-thirdtoesyndactyly(3,6)Radioulnarsynostosis(4,6)Mentalretardation(6)Asplenia(6)OFCDIV137years11yearsCongenitalcataractMicrophthalmiaMicrocorneaIrissynechia(1)SeptatenasalcartilageHighnasalbridgeLong/narrowface(1)Higharchedpalate(2)Aorticvalvestenosis(1)Notaffected(2)PersistantprimaryteethUneruptedsecondaryteethRootradiculomegalyFusionofteeth(1)Duplicatedteeth(2)Hammertoes(1)Second-thirdtoesyndactylyMildmentalretardation(2)HearingimpairmentVomiting/refluxOFCDV131yearsCongenitalcataractSeptatenasalcartilageHighnasalbridgeASDPersistantprimaryteethUneruptedsecondaryteethRootradiculomegalyHammertoesRadioulnarsynostosisLumbarscoliosisc.4512_4514delTTGinsAOFCDVI170years41years40years10yearsCongenitalcataractMicrophthalmia(2,4)Irissynechia(2)Septatenasalcartilage(2,4)Submucosalcleftpalate(4)Bifiduvula(4)Notrecorded(1,3)ASD(4)Notaffected(2,3)Notrecorded(1)Persistentprimaryteeth(2,4)Uneruptedsecondaryteeth(2,4)Rootradiculomegaly(4)Hypodontia(2,4)Fusionofteeth(4)Duplicatedteeth(2,4)Notrecorded(1,3)Notrecorded(1,3)Hammertoes(2)Second-thirdtoesyndactyly(2)Scoliosis(4)Hearingimpairment(1)c.3621delAOFCDVII15yearsCongenitalcataractNotaffectedNotrecordedPersistentprimaryteethSecond-thirdtoesyndactylyc.4303_4307delCCATGOFCDVIII112yearsCongenitalcataractSeptatenasalcartilageHighnasalbridgeSubmucosalcleftpalatePentalogyofFallotDelayeddentitionRootradiculomegalySecond-thirdtoesyndactylyRadioulnarsynostosisMildmentalretardationVesicoureteralrefluxOFCDIX17yearsCongenitalcataractMicrophthalmiaMicrocorneaSeptatenasalcartilageCleftpalateASDPersistentprimaryteethUneruptedsecondaryteethRootradiculomegalyHammertoesBilateralradioulnarsynostosisAltersconservedspliceacceptorsiteandpredicts:p.delexon9fsX18OFCDX12yearsCongenitalcataractMicrophthalmiaMicrocorneaSeptatenasalcartilageHighnasalbridgeSubmucosalcleftpalateASDDelayedprimarydentitionHammertoesMildmentalretardationc.2428COFCDXI142years13yearsCongenitalcataractGlaucoma(1)Retinaldetachment(1)SeptatenasalcartilageLong/narrowface(1)Simpleears(2)Tricuspidvalveinsufficiency(2)RootradiculomegalyHammertoes(2)Shortfingers(1)Cerebralatrophy(2)ADHD(2)OFCDXII117yearsCongenitalcataractSeptatenasalcartilageHigharchedpalateASDRootradiculomegalyHammertoesSecond-thirdtoesyndactylyMildmentalretardationLargedeletionencompassingatleastexons2-15OFCDXIII15yearsCongenitalcataractNotaffectedASDDelayedprimarydentitionHammertoesSecond-thirdtoesyndactylyHearingimpairmentc.3649_3667dup19OFCDXIV12yearsCongenitalcataractSeptatenasalcartilageSimpleearsPrimarydentitionunaffectedSecond-thirdtoesyndactylyLimitedsupinationatwristPartialfusionofvertebraec.3427_3428insAOFCDXV13yearsCongenitalcataractNotaffectedASDPrimarydentitionunaff

ectedNotrecordedc.3848-1Gp.delexon9fsX18OFCDXVI127years27years18monthsCongenitalcataractSeptatenasalcartilage(1,2)Highnasalbridge(1,2)Long/narrowface(1,2)Notaffected(3)ASD(3)VSD(1)Notaffected(2)Delayedprimarydentition(3)Delayeddentition(1,2)Persistentprimaryteeth(1,2)Rootradiculomegaly(1,2)Second-thirdtoesyndactylyRadioulnarsynostosis(2)Scoliosis(2)Largedeletionencompassingatleastexons4-15Somaticmosaicism(1,2)Heterozygous(3)OFCDXVII133years14monthsCongenitalcataract(2)Microcornea(2)Notaffected(1)Septatenasalcartilage(2)Long/narrowface(2)Notaffected(1)Notaffected(1,2)Delayedprimarydentition(2)Notaffected(1)Second-thirdtoesyndactyly(2)Notaffected(1)Smalldeletionencompassingexons13and14Somaticmosaicism(1)Heterozygous(2)OFCDXVIII120yearsCongenitalcataractSeptatenasalcartilageHighnasalbridgeLong/narrowfaceCleftpalateASDRootradiculomegalySecond-thirdtoesyndactylyLimitedsupinationTp.R1514X MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics (Figure2d).Amplificationfromexons8and9wereatthecontrolintensity.AmplificationfromprobandXVII.2wasreducedto50%forexons13and14,suggestinganon-mosaicdistributionofthisdeletion(Figure2e).PCRamplificationandsequencingofaregionspanningtheproposeddeletionregionwasperformedtorefinethedeletionto1410bpfromintron12–14(Figure2f).ClinicalfeaturesofOFCDsyndromeThecohortpresentedherecomprises31newfemalesandfourpreviouslydescribedfemaleswithmutationsin(35patients).However,onefemale(XVII.1)isasymptomatic,despiteprovingmosaicforation,andthusisexcludedfromthephenotypicevaluation,bringingthecohortofsymptomaticfemalesto34.Asummaryofthefrequencyofphenotypeswithinthe34symptomaticfemalesisgiveninTable3.OFCDsyndromeisassociatedwithoculardefectsandthecardinalmanifestationofcongenitalcataractispresentineachindividualinthenewcohortpresentedhere(34/34).Inadditiontocongenitalcataract,patientsdisplayedanarrayofeyeanomalies,mostfrequentlymicrophthalmiaand/ormicrocorneain82%ofpatients(28/34;Figure3aandb).Inourcohort,31patientswereexaminedforfacialanomaliesandafacialphenotypewasrecordedin26patients.Themostspecificfacialmanifestationisseptatenasalcartilage,observedin96%(25/26)ofaffectedpatients(Figure3aandb),oftenassociatedwithahighnasalbridgeandlongand/ornarrowface.Manyofthepatientshadpalatalanomalies,includingcleftpalate,high-archedpalateandbifiduvula(31%;8/26).Whereexamined,themajorityofpatientsinthecohortpresentedherehaveacongenitalheartdefect(74%;20/27),with85%(17/20)presentingwithaseptaldefect,mostcommonlyanatrialseptaldefect.FurthercardiacanomaliessuchaspentalogyofFallot,double-outlet-right-ventricle,valveinsufficienciesandpatentductusarteriosusareobserved,suggestingaroleforinmultiplecardiacInthiscohort,30patientswereexaminedfordentaldefects.Owingtoage,datacorrespondingonlytoprimarydentitionwereavailableforeightpatients.Ofthese,75%(6/8)haveadentalphenotypeofdelayedprimarydentitionand/orpersistentprimaryteeth.Twopatientshavethusfarunaffectedprimarydentitionbutitisanticipatedthatsecondarydentitionwillbeaffected.Dataregardingbothprimaryandsecondarydentitionareavailablefor22patientsinthiscohortandallhaveavarietyofdentalanomaliesassociatedwithOFCDsyn-drome,thatis,delayed/persistentdentitionwithmultiple Table2(FamilyCaseAgeOcularFacial/cranialCardiacDentalSkeletalOtherMutationOFCDXIX114yearsCongenitalcataractSeptatenasalcartilageHighnasalbridgeLong/narrowfaceSimpleearsNota

ffectedDelayeddentitionRootradiculomegalyHypodontiaFusionofteethNotrecordedMildmentalretardationc.570delCp.W191GfsX25OFCDXX112yearsCongenitalcataractSeptatenasalcartilageHighnasalbridgeLong/narrowfaceNotaffectedDelayeddentitionPersistentprimaryteethRootradiculomegalyHammertoesSecond-thirdtoesyndactylyc.863delCp.P288RfsX90OFCDXXI110monthsCongenitalcataractNotrecordedASDNotrecordedNotrecordedc.2926CTp.R976XLenzI17yearsMicrophthalmiaNarrowforeheadSimpleearsASDNotaffectedMultiplepartialfingerFifthfingerclinodactylyRadioulnarsynostosisMentalretardationPatientsarerepresentedbytwonumbers:thefirstindicatesthefamily,thesecondindicatestheindividualcase.Featuresspecifictoindividualswithinafamilyareindicatedinparentheses().Agesareatthetimeofexamination.ASD,atrialseptaldefect;VSD,ventricularseptaldefect;MI,mitralvalveinsufficiency;PDA,patentductusarteriosus;DORV,doubleoutletrightventricle;ADHD,attentiondeficithyperactivitydisorder.FullphenotypesinMcGovernetalmutationidentifiedinthisstudy.BriefphenotypeandmutationinHiltonetalFullphenotypeofindividualandfamilymembersdescribedinthisstudy. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics uneruptedteeth,rootradiculomegalyandabsent/dupli-cated/fusedteeth(100%;22/22;Figure3c).Rootradiculo-megaly,auniqueandcardinaldiagnosticfeatureofOFCDsyndrome,ispresentin91%(20/22)ofpatients,withdiagnosislimitedintheremainingtwopatientsbytheunavailabilityofadentalX-ray. Figure1Locationofreportedmutationswithinthegene.Arrowheadsabovethediagramindicatemutationsdescribedinpreviousreports.Mutationsindicatedbelowthediagramarethoseidentifiedinthisstudy(seeTable2).ArrowheadsinwhiteindicatemutationscorrespondingtofemaleswithOFCDsyndrome.ArrowheadsingreyindicatemutationscorrespondingtomaleswithLenzmicrophthalmiasyndrome.Arrowheadinblackindicatesanin-framedeletionoccurringinafemalewithOFCDsyndrome.Deletionsareindicatedbysolidhorizontallines. Figure2EvidenceforsomaticmosaicisminindividualsXVI.1,XVI.2andXVII.1.()QMF-PCRfluorescentspectraobtainedforfragmentsofthegeneandcontrolgenes,withpatientpeaksinredandcontrolsubjectpeaksinblue.PatientXVI.1(),patientXVI.2()andpatientXVII.1)displaydiminishedpeakintensitytoapproximately75%ofthecontrolpeakintensity,suggestingmosaicismofthedeletedregion.PatientXVI.3)andpatientXVII.2()displaydiminishedpeakintensitytoapproximately50%ofthecontrolpeakintensity,suggestinganon-mosaicdeletion.(SequencechromatogramofpatientXVII.2,confirmingadeletionbetweenintrons12and14,resultinginlossofexons13and14.(g–i)FISHanalysisusingBACcloneRP11-330L22,encompassingthegene,withsignalsindicatedbywhitearrows.DataforpatientXVI.1areequivalenttopatientXVI.2.PatientXVI.2()displaysasinglesignalinapproximately50%ofmetaphasepreparations()whereastheremainingpreparationsshowtwospecificsignals(),confirmingthattheseindividualsaremosaicforadeletionofthegene.ForpatientXVI.3(),aspecificsinglesignalwasobservedin100%ofmetaphasepreparations,confirmingthatthedeletionofthegeneisnon-mosaicandpresentinallcells. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics AnumberofabnormalitiesoftheskeletonhavebeenreportedtobeassociatedwithOFCDsyndromeandin29patientsexamined,weobserveskeletalanomaliesin28cases.Hammertoesarepresentin54%(15/28)ofaffectedpatients(Figure3e)andsecond-thirdtoesyndac-tylyin57%(16/28;Figure3d);93%(26/28)ofpatientshaveatleastoneofthesephenotypes.Rad

ioulnarsyno-stosisismorerecentlyrecognizedasaskeletalfeatureofOFCDsyndromeandwasdetectedin25%(7/28)ofaffectedpatientsofthecohortdescribedhere(Figure3f),withafurthertwopatientsreportinglimitedDevelopmentalproblemshavebeenobservedinpatientswithOFCDsyndromeandinthecohortpresentedhere,18%(6/34)ofpatientsdemonstratealevelofmentalretardation,althoughinmostcasesthisismild.Hearingloss,bothconductiveandsensorineural,is Table3PhenotypesassociatedwithOFCDsyndromeinpatientcohortFeatureOccurrences%AffectedFemaleswithmutation35FemaleswithOFCDsyndromephenotype34/35OcularRecorded34/34Affected34/34100Congenitalcataract34/34100Microphthalmia/microcornea28/3482Coloboma1/343Ptosis3/349Secondaryglaucoma4/3412Lensdislocation1/343Opticdiscdysplasia1/343Phthisisbulbi1/343Irissynechia2/346Retinaldetachment1/343Recorded31/34Affected26/3184Septatenasalcartilage25/2696Highnasalbridge11/2642Longnarrowface8/2631Palate/uvulaanomalies8/2631Simpleears2/268CardiacRecorded27/34Affected20/2774Unresolvedheartmurmur1/205Septaldefects17/2085Patentductusarteriosus3/2015Valveincompentency4/2020PentalogyofFallot1/205Dextrocardia1/205Doubleoutletrightventricle1/205DentalRecorded30/34Primarydentitiononly8/30Primaryandsecondarydentition22/30Affected22/22100Delayed/persistent/unerupteddentition18/2282Rootradiculomegaly(secondaryteeth)20/2291Hypodontia/duplication/fusion(secondaryteeth)14/2263SkeletalRecorded29/34Affected28/2997Hammertoes15/2854Second-thirdtoesyndactyly16/2857Radioulnarsynostosis/limitedsupinationatwrist9/2832Lordosis/scoliosis/vertebralfusion7/2825Shortfingers1/284Mentalretardation6/3418Cerebralatrophy1/343ADHD1/343Hearingimpairment5/3415Poorfeeding/vomiting/reflux3/349Asplenia1/343Vesicoureteralreflux1/343ADHD,attentiondeficithyperactivitydisorder. Figure3OFCDsyndrome.(a,b)TypicalfacialfeaturesofOFCDsyndromeincludemicrophthalmia(lefteyeofeachpatient),longnarrowfaciesandseptatenasalcartilage.()ThedentalphenotypeofOFCDsyndromeincludeshypodontiaandtoothfusionandduplica-tion.(d,f)DigitanomaliesinOFCDsyndromeincludesecond-thirdtoesyndactyly()andhammertoes().()RadioulnarsynostosisinapatientwithOFCDsyndrome. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics observedin15%(5/34)ofpatients.PatientswithOFCDsyndromemaymanifestgastrointestinalproblems,possi-blyaspartofalateralityphenotype,andwereportfeedingdifficultiesandrepeatedvomitingandrefluxin9%(3/34)ofthecohortpresentedhere.Afurthermanifestationofdefectivelateralpatterning,asplenia,isobservedinoneSequenceanalysisofinmaleswithpresumedX-linked(‘Lenz’)microphthalmiasyndromeTodate,asinglefamilyinwhichmalesareaffectedbyX-linkedmicrophthalmiaremainthesolepatientswith‘Lenz’microphthalmiasyndrometohavebeenshowntocarryaWehavesequencedthegenein21maleswithaputativediagnosisofLenzmicrophthalmiasyndrome,basedonanocularphenotypepresentingwithadditionalfeaturesofLenzmicrophthal-miasyndrome,includingmentalretardation,hearingimpairmentandskeletal/digitalanomalies.Inthemajorityofcases(20/21),therewasnoevidenceofafamilyhistory.WeidentifiedamissensemutationinonepatientwithLenzmicrophthalmiasyndrome,c.254CT,predictingp.P85Lsubstitutionattheproteinlevel(LenzI;Table2).Thismissensechangeisidenticaltothemutationpre-viouslydescribedtobeassociatedwithLenzmicrophthal-miasyndrome.Thepatientpresentedwithbilateralmicrophthalmia,mildmentalretardation,atrialseptaldefectandatypicalarr

ayofskeletalanddigitalabnorm-alities.Interestingly,thepatientpresentedwithrightradioulnarsynostosis,aphenotypenotpreviouslyasso-ciatedwithLenzmicrophthalmiasyndromebutstronglylinkedtoOFCDsyndrome.Wedidnotfindamutationinin20ofthemalepatients,suggestingthat(1)thediagnosisofLenzmicro-phthalmiasyndromeisdisputedor(2)mutationsinarenotthemajorcauseofLenzmicrophthalmiasyndrome,afindingreplicatedbyothers.SequenceanalysisofinpatientswithunspecifiedoculardevelopmentalanomaliesToinvestigatethecontributionofmutationsinnon-specificformsofocularandmentalretardationsyndromes,weanalysedthegeneinapanelof96patientswithisolatedmicrophthalmia,colobomaand/ormentalretardation.Weidentifiedasinglemutationininafemalepatientwithwhathadbeenrecordedasisolatedbilateralcataractandunilateralmicrophthalmia(OFCDVII.1).Theproband’smotherhadasimilarphenotype.Themutationwasc.4303_4307delCCATGp.P1435LfsX24.Onfurtherquestioning,itwasfoundthatthepatienthadnumerousprimaryteethremovedinteenageyearsandhadsecond-thirdtoesyndactyly,sug-gestingthatthisfemalehasamildOFCDsyndromeSequenceanalysisofinpatientswithunspecifiedlateralspecificationdefectsWehavepreviouslyidentifieddefectsinlateralspecifica-tionaspartoftheclinicalspectrumofOFCDsyndrome.Wethereforehypothesizethatmutationsinaccountfornon-syndromiclateralitydefects,withheartdefectsrepresentingthemostcommonmanifestationofdefectsinlateraldetermination.Thishypothesisissupportedbytheobservationthatinpopulationstudies,thereisa2:1malepredominanceofpatientswithlateralitydefectsnotattributabletomutationsinfurtherX-linkedgenes,suchas,maythereforebeinvolvedinhumanlateralspecification.Weanalysedthegeneinapanelof96patientswithavarietyofcardiac/lateralitydefects.Weidentifiedsequencealterationsinthreepatients:afemalewithsitusinversusanddextrocardia(heterozygous,c.2288GT,p.R763L),afemalewithmesocardia,VSD,partialanomalouspulmonaryvenousreturnandintestinalmalrotation(heterozygous,c.3974AG,p.K1325R)andamalewithtranspositionofgreatarteries,pulmonaryatresia,VSDandasplenia(hemizygous,c.3974AG,p.K1325R).ThesesequencealterationsdonotappearinSNPdatabasesorwithintheavailableESTsequences.However,thesechangeswerealsoidentifiedinthreesequencesfromapanelof171ethnicallymatchedunaffectedcontrols(onefemaleheterozygousforc.2288GT,p.R763L;twofemalesheterozygousforc.3974AG,p.K1325R),suggestingthattheyarerarepolymorphismsratherthanpathogenicDiscussionInthisstudy,wehavefocusedonthepreciseclinicalfeaturesofOFCDandLenzmicrophthalmiasyndromes,allelicdisorderscausedbymutationoftheasexamplesofX-linkedsyndromicmicrophthalmia-associatedconditions.GiventheheterogeneityamongstX-linkedmicrophthalmiasyndromes,extensionofourknowledgeofthemechanismbywhichisassociatedwithOFCDandLenzmicrophthalmiasyn-dromesmaybepertinenttootherdisorders.Tothatend,wesearchedformutationsinthegeneinpatientswithOFCDsyndrome,presumedX-linkedrecessive(‘Lenz’)microphthalmiasyndrome,isolatedoculardefectsandlateralspecificationdefects.AmongstpathogenicsequencevariantsthatcauseOFCDsyndromeandLenzmicrophthalmiasyndromes,themutationspresentedhereconfirmthesharplydemarcatedgenotype/phenotypecorrelationwherebyhemizygousmalesthatcarryamissensemutationinhaveLenzmicrophthalmiasyndromeandheterozygousfemalescarryinganullallelehaveOFCDsyndrome.Thephenotypeinfemalepatientsmaybevariable,notonlyduetodifferentialX-inactivationeffects,butalsoduetosomatic Mutatio

nalspectrumofEHiltonetal EuropeanJournalofHumanGenetics mosaicism,anovelfindinginthreefemalesinthecohortpresentedhere.Todate,mutationsinarethesolemolecularcauseofOFCDsyndromeandallmutationsidentifiedarenullalleles.ItisnotablethatfemaleswithputativediagnosesofOFCDsyndromeintheabsenceofradiculomegalyprovetobenegativeformutationsin,suggestingastrongassociationofthiscardinalphenotypewithmutationsin.ThemutationwefoundinafemalepatientwithapparentlyisolatedmicrophthalmiaandcataractsuggeststhatOFCDsyndromemaybeunderdiagnosedand,atthemildendofthespectrum,mayhaverelativelyinsignificantnon-ocularfeatures.ThreepatientswithOFCDsyndromewithmanifesta-tionsconsistentwithdefectivelateralpatterning(dextrocardia,aspleniaandintestinalmalrotation)havebeendescribed.Theclinicalfeaturesoftwoofthesepatients(II.1andIII.6),whodisplaydextrocardiaandaspleniarespectively,aredescribedinmoredetailinthisreport.AsithasbeendemonstratedthatBCORisnecessaryinthefrogembryotoconfercorrectlateralorganizationofheartandintestinaltract,itishypothesizedthatdefectsoflateralpatterning,particularlyofinternalorgans,areafeatureofOFCDsyndrome.TheassociationofcardiacseptaldefectsandcorrectlateralityspecificationiswelldocumentedanditislikelythatthehighfrequencyofcardiacseptaldefectsinOFCDsyndromeisthemostcommonmanifestationofaberrantlateralityspecification.DefectsofthemidlineareaetiologicallylinkedwithlateralpatterninganditispossiblethatthefacialcleftingandseptatenasalcartilageobservedinOFCDsyndromealsoresultfromdefectivelateralpatterning.AmongsttheskeletalfeaturesassociatedwithOFCDsyndromeitisofnotethatradioulnarsynostosis,amorerecentlyrecognizedfeature,wasdetectedin25%ofthepatientswithaskeletalphenotypedescribedhere.ThiswasalsoafeatureofthepatientwithLenzmicrophthalmiasyndromewiththec.254CT,p.P85Lmissensemutation.ThisisidenticaltowhatwaspreviouslydescribedinLenzmicrophthalmiasyndrome,supportingthepathogenicityofthissequencevariantandalsosuggestingthatradioulnarsynostosisisanimportantmarkerofThegeneticcausesofLenzmicrophthalmiasyndromethusremainlargelyundefined.SubsequenttothefirstreportbyLenz(1955),therehavebeenalargenumberofreportsofX-linkedrecessivemicrophthalmia27–41Thesereportsshowbroadphenotypicvariabilityinmaleswithmultiplecongenitalanomalies.However,insomecases,theabsenceofaprovenX-linkedfamilyhistorymustcallintoquestionthevalidityofthediagnosis.Itislikelythatsomereportedcasesof‘sporadicLenzmicrophthalmiasyndrome’areinfactmisdiagnosedandrepresentotherformsof‘syndromicmicrophthalmia’.Asaresult,theexactincidenceofLenzmicrophthalmiasyndromeremainsuncertainalthoughthenumberoffamilieswithprovenX-linkedinheritanceisverysmall.Unfortunately,agenotypicdiagnosisofLenzmicrophthal-miasyndromeiscurrentlyimpossibleduetolackofgeneticinformation.Lenzmicrophthalmiasyndromehasbeenassociatedwithatleasttwogeneticloci,oneatXq27-q28andthesecondoccupiedbytheexclusionofamutationinallexceptoneofourmalecohort,weexaminedothercandidategeneswhichmightbeassociatedwithX-linkedmicrophthalmia/mentalretardation,specifically(mutationsinwhichcausemicrophthalmiaandmentalretardation)BCOR-like1(basedonhomologytoandchromosomallocationatXq26.1,intheregionoftheMCOPS1locus).Nomutationswerefoundineithergene(datanotshown),demonstratingthatneitherisamajorcauseofX-linkedmicrophthalmiasyndromes.Combinedwiththelowincidenceofmutationsininmalemicrophthalmiapatients,theseda

tasuggestthatthemajorlocusforX-linkedmicrophthalmia/mentalretardationremainsInsummary,wehaveundertakencarefulphenotypicanalysisofbothfemaleswithOFCDsyndromeandmalepatientswithMCOPS2.Apartfromoculardefects,facialdysmorphia,congenitalheartdefectsanddentalanoma-lies,wefindahighincidenceofskeletaldefects.AlthoughbothcongenitalheartdefectsanddefectsoflateralitydeterminationmaybeassociatedwithOFCD,wedidnotsequencevariantsamongstthosewithunspeci-fiedlateralspecificationdefects.Nonethelesswerecom-mendthatlateralitydefectsshouldbeinvestigated,bothascardiacmanifestationsandintestinalproblemsinpatientswithOFCDsyndrome.Finally,wesuggestthattheskeletalphenotypeofradioulnarsynostosis–whichwehaveseentobeassociatedwithbothOFCDandLenzmicrophthalmiasyndrome–asafurtherclinicalpredictorofEHisfundedbytheWellcomeTrust(UK)andBDFNewlife(UK).SWisfundedbylaFondationpourlaRechercheMedicale(France).ThisresearchwassupportedinpartbytheIntramuralResearchprogramoftheNationalHumanGenomeResearchInstitute,NIH(US).WethankPatriciaContevilleandNathalieCollotfortechnicalsupport.1ForresterS,KovachMJ,ReynoldsNM,UrbanR,KimonisV:ManifestationsinfourmaleswithandanobligatecarrieroftheLenzmicrophthalmiasyndrome.AmJMedGenet92–100.2NgD,HadleyDW,TifftCJ,BieseckerLG:GeneticheterogeneityofsyndromicX-linkedrecessivemicrophthalmia-anophthalmia:isLenzmicrophthalmiaasingledisorder?AmJMedGenet:308–314.3NgD,ThakkerN,CorcoranCMetal:OculofaciocardiodentalandLenzmicrophthalmiasyndromesresultfromdistinctclassesofmutationsinBCOR.NatGenet:411–416. MutationalspectrumofEHiltonetal EuropeanJournalofHumanGenetics 4LenzW:Recessive,sex-limitedmicrophthalmiawithmultipleabnormalities.ZKinderheilkd:384–390.5HaywardJR:Cuspidgigantism.OralSurgOralMedOralPathol:500–501.6MarashiAH,GorlinRJ:Radiculomegalyofcaninesandcongenitalcataracts–asyndrome?OralSurgOralMedOralPathol802–803.7MarashiAH,GorlinRJ:Radiculomegalyofcanineteethandcongenitalcataracts:confirmationofasyndrome.AmJMedGenet:143.8WilkieAO,TaylorD,ScamblerPJ,BaraitserM:Congenitalcataract,microphthalmiaandseptalheartdefectintwogenera-tions:anewsyndrome?ClinDysmorphol:114–119.9AalfsCM,OosterwijkJC,vanSchooneveldMJ,BegemanCJ,WabekeKB,HennekamRC:Cataracts,radiculomegaly,septalheartdefectsandhearinglossintwounrelatedadultfemaleswithnormalintelligenceandsimilarfacialappearance:confirmationofasyndrome?ClinDysmorphol:93–103.10GorlinRJ,MarashiAH,ObwegeserHL:Oculo-facio-cardio-dental(OFCD)syndrome.AmJMedGenet:290–292.11HiltonEN,MansonFD,UrquhartJEetal:Left-sidedembryonicexpressionoftheBCL-6corepressor,BCOR,isrequiredforvertebratelateralitydetermination.HumMolGenet1773–1782.12LinAE,TichoBS,HoudeK,WestgateMN,HolmesLB:Heterotaxy:Associatedconditionsandhospital-basedprevalenceinnew-GenetMed:157–172.13NielF,MartinJ,Dastot-Le-MoalFetal:RapiddetectionofCFTRgenerearrangementsimpactsongeneticcounsellingincysticJMedGenet:e118.14YauSC,BobrowM,MathewCG,AbbsSJ:AccuratediagnosisofcarriersofdeletionsandduplicationsinDuchenne/Beckermusculardystrophybyfluorescentdosageanalysis.JMedGenet:550–558.15PinkelD,StraumeT,GrayJW:Cytogeneticanalysisusingquantitative,high-sensitivityfluorescencehybridisation.NatlAcadSciUSA:2934–2938.16OpitzC,HornD,LehmannR,DimitrovaT,Fasmers-HenkeK:Oculo-facio-cardio-dental(OFCD)syndrome.JOrofacOrthop:178–185.17SchulzeBR,HornD,KobeltA,TariverdianG,StellzigA:Raredentalabnormalitiesseeninoculo-facio-cardi

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