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RESEARCHARTICLEDeletionsofNRXN1(Neurexin-1)PredisposetoaWideSpectrumofDevelopmentalDisordersMichaelS.
L.
Ching,1,2YipingShen,2,3,7Wen-HannTan,2,4ShafaliS.
Jeste,2,5EricM.
Morrow,6XiaoliChen,7,8NahitM.
Mukaddes,9Seung-YunYoo,4EllenHanson,1,2RachelHundley,1,2ChristinaAustin,4RonaldE.
Becker,1,2GerardT.
Berry,2,4KatherineDriscoll,1,2ElizabethC.
Engle,2,5,10,11,12SandraFriedman,1,2JamesF.
Gusella,2,3,13FukiM.
Hisama,2,4MiraB.
Irons,2,4TinaLaosca,1,2ElaineLeClair,1,2DavidT.
Miller,2,4,7MichaelNeessen,1,2JonathanD.
Picker,2,4LeonardRappaport,1,2CynthiaM.
Rooney,2,5DeanP.
Sarco,2,5JoanM.
Stoler,2,4ChristopherA.
Walsh,2,4,11,14RobertR.
Wolff,2,5TingZhang,8RamziH.
Nasir,1,2*Bai-LinWu2,7,15**onbehalfoftheChildren'sHospitalBostonGenotypePhenotypeStudyGroup1DivisionofDevelopmentalMedicine,Children'sHospitalBoston,Boston,Massachusetts2HarvardMedicalSchool,Boston,Massachusetts3CenterforHumanGeneticResearch,MassachusettsGeneralHospital,Boston,Massachusetts4DivisionofGenetics,Children'sHospitalBoston,Boston,Massachusetts5DepartmentofNeurology,Children'sHospitalBoston,Boston,Massachusetts6DepartmentofMolecularBiology,CellBiologyandBiochemistry,BrownUniversity,Providence,RhodeIsland7DepartmentofLaboratoryMedicine,Children'sHospitalBoston,Boston,Massachusetts8DepartmentofMolecularImmunology,CapitalInstituteofPediatrics,Beijing,China9IstanbulFacultyofMedicine,DepartmentofChildPsychiatry,IstanbulUniversity,Istanbul,Turkey10Children'sHospitalBoston,HowardHughesMedicalInstitute,Boston,Massachusetts11MantonCenterforOrphanDiseaseResearch,Children'sHospitalBoston,Boston,Massachusetts12DepartmentofOphthalmology,Children'sHospitalBoston,Boston,Massachusetts13DepartmentofGenetics,HarvardMedicalSchool,Boston,Massachusetts14HowardHughesMedicalInstitute,BethIsraelDeaconessMedicalCenter,Boston,Massachusetts15Children'sHospitalandInstitutesofBiomedicalScience,FudanUniversity,Shanghai,ChinaReceived1July2009;Accepted15December2009AdditionalSupportingInformationmaybefoundintheonlineversionofthisarticle.
TheChildren'sHospitalBostonGenotypePhenotypeStudyGroupalsoincludesOmarS.
Khwaja,AnnapurnaPoduri,MustafaSahinandMagdiSobeih,DepartmentofNeurology,Children'sHospitalBoston,HarvardMedicalSchool,Boston,Massachusetts.
Grantsponsors:NancyLurieMarksFamilyFoundation;SimonsFoundation;AutismSpeaks;NIH;Grantnumbers:5K23MH080954-02,1R01MH083565.
M.
S.
L.
ChingandY.
Shencontributedequallytothiswork.
*Correspondenceto:RamziH.
Nasir,DivisionofDevelopmentalMedicine,Children'sHospitalBoston,HarvardMedicalSchool.
300LongwoodAve.
,Boston,MA02115.
E-mail:ramzi.
nasir@childrens.
harvard.
edu**Correspondenceto:Bai-LinWu,DepartmentsofLaboratoryMedicineandPathology,Children'sHospitalBoston,HarvardMedicalSchool.
300LongwoodAve.
,Boston,MA02115.
E-mail:bai-lin.
wu@childrens.
harvard.
eduPublishedonline7April2010inWileyInterScience(www.
interscience.
wiley.
com)DOI10.
1002/ajmg.
b.
310632010Wiley-Liss,Inc.
937NeuropsychiatricGeneticsResearchhasimplicatedmutationsinthegeneforneurexin-1(NRXN1)inavarietyofconditionsincludingautism,schizo-phrenia,andnicotinedependence.
Toourknowledge,therehavebeennopublishedreportsdescribingthebreadthofthepheno-typeassociatedwithmutationsinNRXN1.
WepresentamedicalrecordreviewofsubjectswithdeletionsinvolvingexonicsequencesofNRXN1.
Weascertainedcasesfrom3,540individ-ualsreferredclinicallyforcomparativegenomichybridizationtestingfromMarch2007toJanuary2009.
Twelvesubjectswereidentiedwithexonicdeletions.
ThephenotypeofindividualswithNRXN1deletionisvariableandincludesautismspectrumdisorders,mentalretardation,languagedelays,andhypotonia.
TherewasastatisticallysignicantincreaseinNRXN1deletioninourclinicalsamplecomparedtocontrolpopulationsdescribedintheliterature(P8.
9107).
ThreeadditionalsubjectswithNRXN1deletionsandautismwereidentiedthroughtheHomozygosityMappingCollaborativeforAutism,andthisdeletionsegregatedwiththephenotype.
OurstudyindicatesthatdeletionsofNRXN1predisposetoawidespectrumofdevelopmentaldisorders.
2010Wiley-Liss,Inc.
Keywords:NRXN1(neurexin-1);developmentaldisorders;arrayCGH;NRXN1exonicdeletions;CNVINTRODUCTIONNeurexinsareagroupofhighlypolymorphiccellsurfaceproteinsinvolvedinsynapseformationandsignaling[Ushkaryovetal.
,1992;MisslerandSudhof,1998;Missleretal.
,2003;Grafetal.
,2004;NamandChen,2005].
Therearethreehumanneurexingenes(NRXN1,NRXN2,andNRXN3),eachofwhichhastwoindepen-dentpromotersresultinginanaandabneurexinforeachgene[Ushkaryovetal.
,1992;Ichtchenkoetal.
,1996].
Multiplealterna-tivesplicingleadstothepossibilityofgreaterthanathousanddistinctneurexinisoforms[Ullrichetal.
,1995].
Theirexpressionisbelievedtobespatiallyandtemporallyregulatedthroughoutdevelopment[PuschelandBetz,1995;Zengetal.
,2006].
StructureandFunctionofNRXN1NRXN1,locatedonchromosome2p16.
3,isoneofthelargestknownhumangenes(1.
1Mbwith24exons)[TabuchiandSudhof,2002].
Itissubjecttorelativelyfrequentdisruptionincludingmissensechanges,translocation,wholegenedeletion,andintra-geniccopynumberalterations[Fengetal.
,2006;Szatmarietal.
,2007;InternationalSchizophreniaConsortium,2008;Kimetal.
,2008;Kirovetal.
,2008;Marshalletal.
,2008;Morrowetal.
,2008;Yanetal.
,2008;Zahiretal.
,2008;Glessneretal.
,2009;Rujescuetal.
,2009].
Thelongertranscript,NRXN1-a,encodesanN-terminalsignalpeptidewiththreerepeatsoftwolaminin/neurexin/sexhormone-bindingglobulin(LNS)domainsseparatedbyanEGF-likesequence(Fig.
1).
Followingtheserepeats,thereisanO-glycosyla-tionsequence,atransmembranedomain,andacytoplasmictailof55aminoacids.
Neurexin-1-ahasbeenshowntointeractwithcertainneuroliginisoformsandneurexin-bindingproteinsknownasneurexophilins.
Thispresynapticmoleculeisalsorequiredforcalcium-triggeredneurotransmitterreleaseandthefunctionofvoltage-gatedcalciumchannelsinthesynapsesofthebrainstemandneocortex[Missleretal.
,2003;Zhangetal.
,2005;Dudanovaetal.
,2006].
Mouseknockoutsofallthreea-neurexingenesdonotdemonstratemajorabnormalitiesofaxonalpathndingduringdevelopment[Dudanovaetal.
,2007],althoughsynapticfunctionisseverelyimpaired.
Micewithknockoutsofindividuala-neurexingeneshavemodestlydecreasedpostnatalviability,whiledoubleknockoutmicehavegreatlydecreasedpostnatalsurvival.
Tripleknockoutmicedonotsurvivepasttherstdayoflife[Missleretal.
,2003].
Neurexin-1-bismuchshorterthanNeurexin-1-a,asveofthesixLNSdomainsandtheinterveningEGFsequencesarereplacedwithashortb-neurexin-specicsequence(Fig.
1)[MisslerandSudhof,1998].
Neurexin-1-bhasbeenshowntointeractwiththepostsynapticneuroliginfamilyofcelladhesionmoleculesanddystroglycans[Ichtchenkoetal.
,1995;Sugitaetal.
,2001;Aracetal.
,2007;Comolettietal.
,2007;Chenetal.
,2008].
NomousemodelswithknockoutsofNRXN1-b,aloneorincombinationwithNRXN1-a,haveyetbeenanalyzed[Sudhof,2008].
ForeachofNeurexin-1-aandNeurexin-1-b,multipleproteincodingisoformsofNRXN1havebeenidentied,whosestructureandfunctionsarenotwellunderstood.
NRXN1MutationsinHumansThereisincreasingevidencethatNRXN1disruptions[Kimetal.
,2008],pointmutations[Fengetal.
,2006;Yanetal.
,2008],anddeletions[Glessneretal.
,2009;Marshalletal.
,2008;Morrowetal.
,2008;Szatmarietal.
,2007]areassociatedwithautismspectrumdisorders.
NRXN1hasalsobeenfoundtobeassociatedwithautisminalargegenome-widesinglenucleotidepolymorphismassocia-tionstudy[Wangetal.
,2009].
NRXN1deletionshavealsobeenassociatedwithavarietyofotherconditionsincludingschizophrenia[InternationalSchizophreniaConsortium,2008;Kirovetal.
,2008;Vrijenhoeketal.
,2008;Walshetal.
,2008;Needetal.
,2009;Rujescuetal.
,2009],nicotinedependence[Bierutetal.
,2007;Nussbaumetal.
,2008],HowtoCitethisArticle:ChingMSL,ShenY,TanW-H,JesteSS,MorrowEM,ChenX,MukaddessNM,YooS-Y,HansonE,HundleyR,AustinC,BeckerRE,BerryGT,DriscollK,EngleEC,FriedmanS,GusellaJF,HisamaFM,IronsMB,LaoscaT,LeClairE,MillerDT,NeessenM,PickerJD,RappaportL,RooneyCM,SarcoDP,StolerJM,WalshCA,WolffRR,ZhangT,NasirRH,WuB-L,onbehalfoftheChildren'sHospitalBostonGenotypePhenotypeStudyGroup.
2010.
DeletionsofNRXN1(Neurexin-1)PredisposetoaWideSpectrumofDevelopmentalDisorders.
AmJMedGenetPartB153B:937–947.
938AMERICANJOURNALOFMEDICALGENETICSPARTBandotherphysicalmanifestationssuchasvertebralanomalies[Zahiretal.
,2008].
PriorreportsofabnormalitiesinNRXN1havefocusedonpopulationswithspecicdiagnoses(e.
g.
,autism,schizophrenia).
However,theclinicalsignicanceofcopynumbervariants(CNV),suchasdeletioninvolvingoneormoreexonsofNRXN1,andtherangeofphenotypicmanifestationsofsubjectswithNRXN1dele-tionCNVremainsunclear.
WedescribehereagroupofsubjectswithNRXN1deletionswhodemonstrateawiderangeofphysicalanddevelopmentalphenotypes.
MATERIALSANDMETHODSClinicalCohortRecordReviewFromMarch2007toJanuary2009,atotalof3,540subjectsatChildren'sHospitalBostonwereevaluatedforgenomicimbalance(deletionandduplication)usingtheAgilent244Khumangenomeoligonucleotidecomparativegenomichybridization(CGH)mi-croarrays(G4411B,AgilentTechnologies,PaloAlto,CA)accordingtothemanufacturer'sinstructions[OligonucleotideArray-BasedCGHforGenomicDNAAnalysisprotocolversion3(AgilentTechnologies)].
Themajorityofthereferralswereforclinicalfeaturesofdevelopmentaldisorders(developmentaldelay,autismspectrumdisorders,mentalretardation)ormultiplecongenitalmalformationsasdeterminedbyspecialistsinClinicalGenetics,Neurology,andDevelopmentalMedicine.
Onehundredthirtyprobescoverthe1.
12MbregionoftheNRXN1geneontheAgilent244KCGHarray.
Theaverageinterp-robespacewithintheNRXN1geneis8.
6kb.
Thispermitsthereliabledetectionofsmallintragenicdeletionsdownto43kbinsize.
ImageswerecapturedbyAgilentscannerandquantiedusingFeatureExtractionsoftwarev9.
0(AgilentTechnologies).
CGHAnalyticsSoftwarev3.
4(AgilentTechnologies)wassubsequentlyusedfordatanormalization,qualityevaluationanddatavisualiza-tion.
CopynumberaberrationwasindicatedusingtheAberrationDetectionMethod2(ADM-2)algorithm.
DeletionsinvolvingveormoreconsecutiveprobeswereconsideredastrueCNV.
Fortwolargerdeletions,uorescentinsituhybridization(FISH)testingusingprobeRP11-800C7wascarriedoutfordeletionconrmationandparentaltesting.
ThesmallerdeletionswereconrmedbyPCR-basedbreakpointmappingmethods.
Thepri-mersusedforeachcasearelistedintheSupplementaryMaterial.
SubjectswithdeletionsinvolvingexonicsequenceofNRXN1wereincludedinourreview.
Twodevelopmentalbehavioralpedia-tricians(RHN,MSLC),aclinicalgeneticist(WHT),andapediatricneurologist(SSJ)reviewedeachofthemedicalrecords.
Theclinicalhistory,physicalexamination,laboratorydata,andradiologicalreportsofeachsubjectwerereviewed.
AdditionalReportofCasesWithNRXN1DeletionandAutismCaseswithexonicandintragenicNRXN1deletionswerealsocontributedfromtheHomozygosityMappingCollaborativeforAutism(HMCA)whichutilizedtheAffymetrixGeneChipHumanMapping500KArraySetusingCNVdetectionmethodspreviouslydescribed[Morrowetal.
,2008].
ThisworkwasapprovedbytheInstitutionalReviewBoardsatthecorrespondinghospitals.
RESULTSClinicalCohortRecordReviewWeidentied12subjectsthroughChildren'sHospitalBostonwithdeletionsinvolvingexonicsequencesofNRXN1(TableIandFig.
1).
Thedeletionsreportedhererangefrom65kbto5Mbandmostofthesecasesarepredictedtoaffecttheinitialstructuraldomainsoftheprotein(Fig.
1).
FIG.
1.
Illustratesthesizeandrangeofthe12deletionCNVsinrelationtotheexonsandproteindomainsofNRXN1-aand-bintheUCSCGenomeBrowser(http://genome.
ucsc.
edu)[Kentetal.
,2002].
Thetoptrackshowsthegenomicpositionandsizeofthe12deletionCNVs.
ThemiddletracksshowthegeneannotationsinRefSeqandEnsembl.
TheRefseqGenesshowtheaandbisoformsoftheNRXN1gene;theEnsemblgenepredictionshowsseveralotherminorisoformsoftheNRXN1gene.
ThebottompanelshowstheproteindomainsoftheNRXN1-ageneproduct.
SP,signalpeptide;LNS,laminin/neurexin/sexhormone-bindingglobulindomain;EGF,epitheliumgrowthfactorlikedomain;OS,O-glycosylationsequence;TM,transmembranedomain;CT,cytoplasmictail.
tail.
[Colorgurecanbeviewedintheonlineissue,whichisavailableatwww.
interscience.
wiley.
com.
]CHINGETAL.
939TABLEI.
DeletionsWithinNRXN1inOurSamplePatientDeletionlocation(hg18build)Sizeofdeletion(kb)InheritanceExons–intronsdeletedOthergenetictestsandresults(additionalimbalance)IndicationfortestingConrmationmethod146,938,685–52,015,8855,077MaternalFISHnormal;paternalstudyunavailableAllKaryotypingandFragileXtest:normal(contiguousdeletionincludingFSHR,LHCGR,STN1)ModeratementalretardationFISH250,128,256–54,050,7133,923DenovoAllexceptthelasttwoexonsNoneGlobaldevelopmentaldelays,suspectedautismFISH350,897,002–51,212,385315PaternalExon1–5;partialintron5Karyotypingandchromosome15methylation:normalGrossmotordelay,hypotoniaPCR450,936,914–51,167,934231PaternalExon1–5;partialintron5Karyotyping,fragileXtest,SALL1,andCHD7mutationtest:normalPDD-NOS,hypotoniaPCR550,920,082–51,059,469139DenovoExon3,4,5;partialintrons2,5NoneVACTERLNotdone651,059,410–51,316,396257MaternalExon1,2;partialintron2KaryotypingandfragileXtest:normalPDD-NOS,motorcoordinationdelaysPCR751,090,504–51,212,385122PaternalExon1–3;partialintron3Karyotyping,FragileXtest,andPTENmutationtest:normalAutism,moderatementalretardationPCR850,522,892–50,827,767305DenovoExon6–17;partialintrons5,17aFragileXtest:normal(deletionat3p24.
3from21492764to21806824,maternallyinherited)MildmentalretardationPCR950,689,280–50,853,329164Unknown(fosterfamily)Exon6–8;partialintrons5,8aKaryotyping:normalLanguagedelay,prenatalsubstanceexposurePCR1050,714,297–50,853,329139DenovoIntron5aKaryotypingandfragileXtest:normalPDD-NOSPCR1150,735,499–50,811,01876MaternalIntron5aKaryotyping,PTEN,andNSD1mutationtests:normal(duplicationsat5p13.
2from37241141to37758854,paternallyinherited;at15q26.
3from98059710to98842423,maternallyinherited;at17p11.
2from21147675to21442522maternallyinherited)Hypotonia,muscleweakness,largebirthweightPCR1250,735,499–50,801,23366MaternalIntron5aNonePoorweightgain,mildcraniofacialdysmorphismPCRFSHR,follicle-stimulatinghormonereceptor;LHCGR,luteinizinghormone/choriogonadotropinreceptor;STN1,StonedB-likefactor;PDD-NOS,pervasivedevelopmentaldisorder,nototherwisespecied;VACTERL,vertebralanomalies,analatresia,cardiacmalformations,tracheoesophagealstula,renalanomalies,andlimbanomalies;SALL1,sal-like1(Drosophila);CHD7,chromodomainhelicaseDNA-bindingprotein7;PTEN,phosphataseandtensinhomolog;NSD1,nuclearreceptor-bindingSETdomainprotein1.
aDeletionsofintron5inthesepatientsinvolveanexonofaminorisoformofNRXN1.
940AMERICANJOURNALOFMEDICALGENETICSPARTBOfthese12deletions,4weredenovoCNVnotidentiedineitherparent,3werematernallyinherited,3werepaternallyinherited,andtheparentalsamplesfor1(subject9)werenotavailable.
Insubjects1,paternalsampleswerenotavailablebutthedeletionwasnotidentiedinmaternaltesting.
Insubjects1–9,thedeletionsinvolvedatleasttwoexonsofNRXN1-a,whileinsubjects10–12,thedeletionsinvolvedonlyanexonofaminorexpressedNRXN1isoform.
Thegenomicimbal-ancesinvolvingNRXN1aresummarizedinTableIandtheclinicalmanifestationsaresummarizedinTablesIIandIII.
FurtherclinicaldataareavailableintheSupplementaryMaterial.
Detailedclinicalrecordswereavailablefromgeneticistsin9outof12subjects,developmental-behavioralpediatriciansin6/12,psychologistsin6/12,andneurologistsin4/12.
Fourofthe12subjects(4,6,7,and10)werediagnosedwithautismspectrumdisorders;ineachpositivecase,thisdiagnosiswassupportedbytheAutismDiagnosticObservationSchedule.
Anothersubject(2)wassuspectedofhavingautismbuttheevaluationwasnotavailableforreview;healsohadglobaldevelopmentaldelays.
Twosubjectshadmentalretardationwithoutadiagnosisofanautismspectrumdisorder(1and8).
Subject1,inaddition,hadabsenceseizuresandanEEGconsistentwithaprimarygeneralizedepilepsy.
Onesubject(3)wastooyoungtoascertainforanautismspectrumdisorderorcognitivedelays.
Ninesubjectshadclinicaldocumentationofexpressiveorreceptivelanguagedelays.
Milddysmorphicfeatureswerepresentinsevensubjects(2,3,4,7,8,9,and12);threesubjectshadhemangiomas(2,6,and10).
Hypotoniawaspresentinfoursubjects(3,4,9,and11).
Twosubjects(5and12)hadventricularseptaldefects.
MedicalrecordreviewalsorevealedthefollowingcharacteristicsinthesixparentsfromwhomtheNRXN1deletionwasinherited.
Subject4,whohadpervasivedevelopmentaldisorder,notother-wisespecied(PDD-NOS)andhypotonia,inheritedhisdeletionfromhisfatherwhoisalsoreportedtobesociallyawkward.
Subject6,whohadPDD-NOSandcoordinationissues,inheritedthedeletionfromamotherwithahistoryoflanguagedelayandsocialskilldifculties.
Subject11,whohashypotonia,weakness,andPolandanomaly,inheritedthedeletionfromamotherwhohasahistoryofjointhypermobility,osteoarthritis,mitralvalveprolapse,severemigraines,andseverebreastasymmetry.
Thefatherofsubject3(hypotonia,grossmotordelay),thefatherofsubject7(autism,mentalretardation)andthemotherofsubject12(poorweightgain,craniofacialdysmorphism)arereportedtobehealthywithoutdevelopmentalormedicalconcerns.
AdditionalReportofCasesWithNRXN1DeletionandAutismInadditiontotheChildren'sHospitalBostoncases,wereportherethreecasesfromtwofamiliesascertainedthroughtheHMCA[Morrowetal.
,2008].
TheNRXN1deletionsineachwerediscov-eredtosegregatewithIQbelow70inthesepedigrees(Fig.
2).
Allthreeaffectedchildrenwerecarriersandunaffectedchildrenwerenot.
ThedeletionswereinheritedfromfatherswhowerefoundtohaveASDsymptomsandIQbetween60and70,whilenon-carriermotherswerenotontheautismspectrumandwithIQsinthenormalrange.
Thedeletionforthesubjectintherstfamilyisexonicandintragenic,whilethedeletionforthetwosiblingsinthesecondfamilyisupstreamandmayaffectgeneexpression.
Furtherinvestigationisnecessarytosubstantiatethisasafunctionaldele-tion,eventhoughitsegregateswithdisease.
SignicanceTestToestablishtherelevanceoftheseCNV,wecomparedthefrequencyofdeletionsinvolvingNRXN1-aexonsinourChildren'sHospitalBostonpopulation,inwhomCGHtestingwasconsideredtobeclinicallyindicated,tothefrequencyofsimilardeletionsdetectedbyarraygenomicprolingofequivalentresolutioninnormalpop-ulations.
Itsaraetal.
[2009]detectedthreedeletionsinvolvingNRXN1-aexonsin2,493normalindividuals.
TheInternationalSchizophreniaConsortium[2008]reportedtwoexonicdeletioncasesin3,181normalcontrols.
Anotherlarge-scaleschizophreniastudyidentiedvedeletioncasesamong33,746normalcontrols[Rujescuetal.
,2009].
Recently,Glessneretal.
[2009]reportednodeletionCNVinvolvingNRXN1-aamong1,409AutismCase–Control(ACC)controlsamplesand1,110AutismGeneticResourceExchange(AGRE)controls.
Collectively,thefrequencyofexonicdeletionofNRXN1-aincontrolpopulationsis10/51,939(0.
019%);thisdifferssignicantlyfromthefrequencyofexonicdeletionCNVweobservedinourclinicallyreferredpopulation(9/3,540)(0.
25%;P8.
9107,two-tailedFisher'sexacttest).
Therearenoavailabledataonthefrequencyofminorisoformexonicdeletionsincontrolpopulationsandthusthesesubjects(n3)wereexcludedfromthesignicancetest.
DISCUSSIONTherecentrecognitionofgenomicimbalanceinmanychromo-somalregionsthatareassociatedwithautism,mentalretardation,andschizophreniaisduetotheincreasinguseofwholegenomehigh-resolutionarrayCGHintheevaluationofindividualswiththesedisorders.
OurclinicalsubjectswithNRXN1deletionwereascertainedthroughapatientpopulationpresentingwithabroadrangeofreferringdiagnoses.
Throughacarefulreviewofmedicalrecords,weidentiedinoursubjectsanumberofclinicalfeaturesthathadnotbeenpreviouslyassociatedwithNRXN1deletions.
Theseincludelanguagedelays,mentalretardationwithoutautism,hypotonia,andhemangiomas.
Inaddition,twoofoursubjects(5and12)hadventricularseptaldefects.
Interestingly,thehumancDNAhomologoustoratNRXN1-ahasbeenisolatedinbothbrainandhearttissuessuggest-ingapotentialroleforNeurexin-1inbothbrainandheartdevel-opment[Nagaseetal.
,1998].
Oneofthesesubjects(5)alsohadevidenceofmultiplecongenitalanomaliesincludingvertebralanomaliesintheformofaVACTERLassociation.
Vertebralanomalieshavealsobeenreportedinoneothercaseintheliterature[Zahiretal.
,2008].
Apreviousreportshowedthepresenceofaseizuredisorderintwounrelatedindividualssharingthesamemissensevariantinexon1ofNRXN1-b[Fengetal.
,2006].
Inourcohort,onlyonesubjecthadaseizuredisorder(subject1),althoughhis5MbdeletionencompassedtheentireNRXN1geneaswellasthegenesforfollicle-stimulatinghormonereceptor(FSHR),luteinizingCHINGETAL.
941TABLEII.
NeurologicalandDevelopmentalCharacteristicsSubjectSexAgeatascertainmentAutismspectrumdisorderCognitive-developmentalndingsLanguagedelayMotorinvolvementHistoryofseizures/EEGresultsMRI-brainBehavioralfeatures1M16yNoMR;SB5:FSIQ44;VIQ44;NVIQ48;(CA14y)ExpressiveandreceptiveWalkedat18monthsHistoryofseizures;abnormalEEGNormalInattention,impulsivity,hyperactivity2M2yAutismsuspected,noformalevaluationavailableGlobaldevelopmentaldelaysExpressiveandreceptiveNotdocumentedNotdocumentedNotperformedornotdocumentedNotdocumented3F10moNotsuspectedNoconcernsreported.
TestingnotdocumentedNoMildgrossmotordelay,hypotoniaNoneNotperformedornotdocumentedNotdocumented4M4yPDD-NOS(ADOS)WPPSI-IIIVIQ77,PIQ98(CA4y)ExpressiveHypotoniaEEGNormalNotperformedornotdocumentedAttentionconcerns5F6yNoNoconcernsreported.
Testingnotdocumented6monthreceptivedelayNormalNotdocumentedNotperformedornotdocumentedNotdocumented6F7yPDD-NOS(ADOS)BayleyIImentalscale91,29mo(CA31mo)ExpressiveMotorcoordinationdisorderNoneNotperformedornotdocumentedNotdocumented7M14yAutism(ADOS)MR:SB5:FSIQ47;VIQ46;NVIQ53ExpressiveandreceptiveNormalEEGnormalNotperformedornotdocumentedHyperactivity8F11yNoMR:WISC-IV:VCI67,PRI63,WMI59,PSI75,FSIQ58(CA11y)ExpressiveandreceptiveNormalNoneNormalInattention,dgety,disorganized9F4yNoAcademicdelaysreported.
TestingnotdocumentedExpressiveandreceptiveHypotoniaNoneNormalImpulsivityandinattention10M2yPDD-NOS(ADOS)BayleyIIIcognitivescore95(average)ExpressiveandreceptiveNormalNotdocumentedNotperformedornotdocumentedNotdocumented11M8yNoNoconcernsreported.
TestingnotdocumentedNoProximalanddistalweakness,hypotoniaNoneNotperformedornotdocumentedNotdocumented12F19moNotdocumentedNotdocumentedNotdocumentedNormalNoneNotperformedornotdocumentedNotdocumentedADOS,autismdiagnosticobservationschedule;BayleyII,BayleyScalesofInfantDevelopment,secondedition;BayleyIII,Bayleyscalesofinfantandtoddlerdevelopment,thirdedition;CA,chronologicalageattesting;MR,mentalretardation;SB5,Stanford-Binetintelligencescales,fthedition;FSIQ,fullscaleIQ;VIQ,verbalIQ;NVIQ,nonverbalIQ;PIQ,performanceIQ;WPPSI-III,Wechslerpreschoolandprimaryscaleofintelligence,thirdedition;WISC-IV,Wechslerintelligencescaleforchildren,fourthedition;VCI,verbalcomprehensionindex,PRI,perceptualreasoningindex;WMI,workingmemoryindex;PSI,processingspeedindex;y,years;mo,months.
942AMERICANJOURNALOFMEDICALGENETICSPARTBTABLEIII.
RelevantPhysicalCharacteristicsSubjectDysmorphicfeaturesVertebral/skeletalCardiacSkin1NoneNotdocumentedNormalNotdocumented2FrontalbossingHistoryofplagiocephalyResolvedheartmurmurHemangiomaonneck3Epicanthalfolds;hypertelorismsmallerbifrontalregionProminentcoronalsutures,feet:higharchesandsomewhatsmalllengthNormalLighterthanparents4Down-slantingpalpebralssures;antevertednares;mildretrognathia,pointedchinNotdocumentedNormalNormal5NoneCurved2ndtoes,incompletefusionofringofrstcervicalvertebraNarrowedaorticarch,2VSDsNotdocumented6NoneBilateralhipdysplasiaProlongedQTc(457msec)Hemangiomaonneck7Slightlydeepseteyes,largeearsNormalNormalNormal8Longface,malarhypoplasia,prominenttubularnosewithpointednasaltip,hypoplasticalaenase,longatphiltrum,thinvermilion,prominentchin,longslenderngers,thintoesNotdocumentedNormalNormal9Lownasalbridge,smalljaw,verysmoothphiltrum.
Slightlyatmid-faceandprominentcheeksMildclinodactylyandunevendigitlengthsNormalNotdocumented10Dolichocephaly(32-weekprematureinfant)NotdocumentedNormalHemangiomaonback11NoneChest-rightmildPolandanomalyNormalEczema12Relativemacrocephaly(headcircumference90%),cuppingofleftear,frontalbossingOpenanteriorfontanelleat19monthsSmallmuscularVSD,fenestrationinatrialseptum,smallPDANotdocumentedVSD,ventricularseptaldefect;PDA,patentductusarteriosus;QTc,correctedQTinterval(normal<440msec).
CHINGETAL.
943hormone/choriogonadotropinreceptor(LHCGR),andStonedB-likefactor(STN1).
Toourknowledge,noneofthesegeneshasbeenassociatedwithseizuresormentalretardationintheliterature.
AlthoughwecannotbecertainthatthesefeaturesareadirectconsequenceofNRXN1deletion,ourobservationssuggestthatthephenotypiccharacteristicsofNRXN1deletionmaybewiderthanpreviouslyreported.
ThemutationswehaveobservedinourclinicalcohortareprimarilyinNRXN1-a.
Subjectswithsmalldeletions(under3-Mb)clusteredintotwogroups(Fig.
1).
Onegroup(subjects3–7)haddeletionsinvolvingpartoftheinitialLNSandEGFdomains-encodingregionsofNRXN1-a.
Oftheseveindividuals,threehadautismspectrumdisorders.
OneadditionalcasefromtheHMCAwasalsofoundtohaveadeletioninthisregion,whichissimilartothedeletioninsubject7fromtheclinicallyreferredcohort.
Asecondgroup(subjects8–12)haddeletionsthatclusteredaroundaregionfurtherfromtheapromotorofthegene(Fig.
1).
Allveofthesesubjects'deletionsencompassedanexonofanisoformwhosefunctionisnotwellunderstood.
Furthermore,whiletwosubjects(8and9)haddeletionsinvolvingotherexonsofNRXN1-aaswellasthisminorisoform,threesubjects'deletions(10–12)containonlytheexonofthisminorisoform.
ThisminorisoformisanEnsemblannotatedtranscript,namedENST00000406859(Fig.
1).
Itcontains13exonswith2,590bptranscriptionand856residuesoftranslationlength.
Thecodedprotein(ENSP00000385681)consistsofoneLNSandEGFdomain.
Itsfunctioniscurrentlyunknown.
Onesuchsubject(10)withadenovodeletioninthisregionhasbeendiagnosedwithPDD-NOS,suggestingpotentialclinicalrele-vanceforthisisoform.
Thisdeletioninintron5hasnottoourknowledgebeenpreviouslyreportedasbeingassociatedwithabnormaldevelopment.
Neurexin-1-bmutationswerelesscommon.
TwoofthesubjectsinourcohorthadlargedeletionsencompassingexonsforNRXN1-aand-b.
MissensevariantsinNRXN1-b(R8P,L13F,S14L,andT40S)havepreviouslybeenidentiedinindividualswithautism[Fengetal.
,2006;Kimetal.
,2008].
Relativesoftheseindividualswithautismwhosharedthesemissensemutationsdemonstratedsomedegreeoflearningorbehavioralissuesbutdidnotappeartomeetfullautismspectrumdisordercriteria[Fengetal.
,2006;Kimetal.
,2008].
Thisisconsistentwithourndingsofamixedphenotypeassociatedwithdeletionsinthisregionrangingfromautismspectrumdisorderstohypotoniawithcarrierrelativeswhoarenotasaffected.
InadditiontotheirNRXN1deletions,subjects8and11hadadditionalgenomicimbalancesasdescribedinTableI.
Thesegenomicimbalanceswereallinheritedfromunaffectedparents.
Thetwoduplicationson15q26.
3and17p11.
2insubject11overlapFIG.
2.
A:NRXN1-adeletionssegregatewithautismspectrumdisorder(ASD)andmildmentalretardation.
Pedigree1showsco-segregationofahemizygousCNVbetweenrs17041500andrs17512199whichdeletestherstthreecodingexons(DelEx1-3)ofNRXN1-a.
TheCNViscarriedbyallsubjectswithASDanddiminishedintelligencequotient(IQ),butnotbyatypicallydevelopingsibling.
Pedigree2showsco-segregationofahemizygousCNVwhichdeleteslikelyregulatory,genomicDNAupstream(Del50Reg)ofNRXN1-a.
PDD-NOS,pervasivedevelopmentdisorder,nototherwisespecied.
,wild-type,non-deletedDNA.
B:MappingofinferredCNdataSNP-by-SNPontheUCSCgenomebrowserdemonstratestheextentacrosstheNRXN1locus.
VerticalredlinesindicateeachSNPwithcopynumberof1or2.
Horizontalgreenlinesdemarcatetheextentofeachdeletion.
AlignmentofannotatedgenesintheRefSeqdatabaseareshownaswellasarepresentationofvertebrateconservationusingmultizandrelatedtoolsintheUCSC/PennStateBioinformaticscomparativegenomicalignmentpipeline.
Ofnote,Del50Regdeletesthelastfourexonsofanuncharacterized,splicedmRNAAK127244thatisexpressedinbrain.
ThegeneistranscribedintheoppositedirectionasNRXN1-ayetthetranscriptionstartsiteiswithin3.
5kbsuggestingthatthismRNAmaybetranscribedcoordinatelywithNRXN1-a.
[Colorgurecanbeviewedintheonlineissue,whichisavailableatwww.
interscience.
wiley.
com.
]944AMERICANJOURNALOFMEDICALGENETICSPARTBwithknownbenignCNVsandareunlikelycontributoryfactorstothepatient'scondition.
Theduplicationat5p13.
2insubject11anddeletionat3p24.
3insubject8arenotpreviouslyreportedCNVbutcontainnoknowngenesassociatedwithdevelopmentaldisorders,thusareconsideredasCNVofunknownsignicance.
Nevertheless,itisunclearwhethertheseCNVsmodiedtheobservedphenotype.
NRXN1andSynapseFunctionPriorstudieshavefunctionallylinkedothermoleculesthatareassociatedwithNRXN1toarangeofneuropsychiatricdisordersincludingautism.
Theseincludeneuroligins3and4(NLGN3,NLGN4)andSH3andmultipleankyrinrepeatdomains3(SHANK3)[Jamainetal.
,2002;Laumonnieretal.
,2004;Durandetal.
,2007;Moessneretal.
,2007;Lawson-Yuenetal.
,2008].
Inaddition,CNTNAP2(contactinassociatedprotein-like2)[Alarconetal.
,2008;Arkingetal.
,2008;Bakkalogluetal.
,2008]andcadherin10(CDH10)and9(CDH9)havebeenalsoassociatedwithautismspectrumdisorders[Wangetal.
,2009].
OurndingthatNRXN1isalsoassociatedwithautismanddevelopmentaldisordersaddsfurtherevidencetotheimportanceofthismolecularfamilytothedevelopmentofneurodevelopmentaldisorders.
ThefunctionofNRXN1infacilitatingsynaptictransmissionsuggeststhatmutationsinthisgenemaypredisposetoaneurologicdisconnectionsyndrome.
Long-rangedisconnectionsbetweenneuralnetworkshavebeenhypothesizedtobecausativeinsomepopulationswithautism[Barnea-Goralyetal.
,2004;Frith,2004;Justetal.
,2004;GeschwindandLevitt,2007].
TheeffectsofNRXN1onlanguagedevelopmentandhypotoniamaylikewiseberelatedtolong-rangeconnectivitywithinthebrain.
PhenotypicVariationPhenotypicvariationsmayreectthehighlypleiotropiceffectsobservedforspecicCNVssuchasthoseassociatedwithNRXN1.
Inaddition,anumberofoursubjectsinheritedNRXN1deletionsfromtheirparents.
Thedetailedphenotypeoftheseparentswerenotdescribedinthemedicalrecordsexceptinthefamilyhistory,buttheparentswereostensiblylessaffectedthantheirchildren.
ThissuggeststhatdeletionintheNRXN1genemaynotbefullypenetrant,orinteractswithothergenesresultinginthevariablephenotype.
Furtherresearcheffortstoinvestigatesuchvariablephenotypesassociatedwiththisunstablegenomicregionwillpro-videfurtherinsightintotheroleofNRXN1inthedevelopmentoflanguagedelays,autismspectrumdisorders,andphysicalfeatures.
LimitationsTheaccuracyandcompletenessoftheclinicalphenotypeidentiedinthisstudyisentirelydependentontheclinicalinformationthatwasdocumentedinthemedicalrecordsofthesesubjects,oftenbeforetheNRXN1deletionswereidentiedinthem.
Becauseoftheclinicalvariabilityexhibitedinourcohort,thesubjectswereseenbyavarietyofspecialists,whichaffectedthecompletenessofdata.
Inaddition,theparentswerenotformallyassessedtoascertaintheircognitive,physical,andbehavioralphenotypes.
Asnotedabove,reviewoffamilyhistorysuggeststhatsomeparentsmayhavesharedsimilarphenotypestotheirchildren.
Weareconduct-ingfurthertestingonboththesubjectsandtheirparentstobetterclarifydevelopmentaland/orsocialcognitionissuesinsubjectsandtheirparents.
ForthedeletionCNVsignicancetest,weusedthenormalcontroldatageneratedbydifferentgenomicprolingarrayplat-formsasreference.
Knowingthatthesensitivityandspecicitydifferfromonearrayplatformtoanother,thismaynotbeanoptimalcomparison.
However,theeffortwasmadetominimizethedetectionbiasbetweendifferentarrayplatforms.
Herewehaveonlychosenrecentstudiesusingarrayplatformofsimilarresolutionasours.
AllthesepublishedarticlesreportedthedetectionofsmallerCNV,suggestingthattechnicallyallthesearrayplatformswereabletodetectanyCNVidentiedinthisstudy.
Thusthiscomparison,althoughanapproximation,isontheconservativeside.
Finallyweacknowledgethatwhileourclinicallyascertainedsubjectswerenotdrawnfromacohortwithasinglediagnosissuchasautismorschizophrenia,theywereascertainedfromaheteroge-neouslyaffectedgroupinwhomgenetictestingwasconsideredclinicallyrelevant.
Asaresult,thereisascertainmentbiasandourndingsmaynotreectthetruedistributionofphysicalanddevelopmentalndingsintheNRXN1deletionphenotype.
Never-theless,wehavedemonstratedthatthereareanumberofotherphenotypicfeaturespresentinthisclinicalpopulationbeyondwhathaspreviouslybeenidentiedintheliterature.
CONCLUSIONWefoundawiderangeofphenotypicfeaturesinagroupofsubjectswithNRXN1deletionswhowereclinicallyreferredforgenetictesting.
Theseincludesubjectswithautismspectrumdisorders,mentalretardation,languagedelays,hypotonia,hemangiomas,andtheVACTERLassociation.
ACKNOWLEDGMENTSTheauthorsgratefullyacknowledgetheassistancebyourcolleaguesfromtheDNADiagnosticsLab:VaLip,XiaomingSheng,AnnReinhard,HongFang,SivTang,HongShao,HaitaoZhu,SamTang,andAndrewChengfortechnicalsupportofarrayCGH;ChristopherA.
WalshLab:DanielleGleasonandDanielRakiecfortechnicalsupportandRobertSeanHillforbioinformaticssupport.
WearefurthergratefulforthesupportfromtheNancyLurieMarksFamilyFoundation(C.
A.
W.
),theSimonsFoundation(C.
A.
W.
andJ.
F.
G.
),AutismSpeaks(J.
F.
G.
),andtheNIH(5K23MH080954-02toE.
M.
M.
and1R01MH083565toC.
A.
W).
E.
C.
E.
andC.
A.
W.
areInvestigatorsoftheHowardHughesMedicalInstitute.
Y.
S.
holdsaYoungInvestigatorAwardfromtheChildren'sTumorFoundationandCatalystAwardfromHarvardMedicalSchool,E.
M.
M.
holdsaCareerAwardforMedicalScientistsfromtheBurroughsWellcomeFund,B.
L.
W.
holdsaFudanScholarResearchAwardfromFudanUniversity.
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Walsh,2,4,11,14RobertR.
Wolff,2,5TingZhang,8RamziH.
Nasir,1,2*Bai-LinWu2,7,15**onbehalfoftheChildren'sHospitalBostonGenotypePhenotypeStudyGroup1DivisionofDevelopmentalMedicine,Children'sHospitalBoston,Boston,Massachusetts2HarvardMedicalSchool,Boston,Massachusetts3CenterforHumanGeneticResearch,MassachusettsGeneralHospital,Boston,Massachusetts4DivisionofGenetics,Children'sHospitalBoston,Boston,Massachusetts5DepartmentofNeurology,Children'sHospitalBoston,Boston,Massachusetts6DepartmentofMolecularBiology,CellBiologyandBiochemistry,BrownUniversity,Providence,RhodeIsland7DepartmentofLaboratoryMedicine,Children'sHospitalBoston,Boston,Massachusetts8DepartmentofMolecularImmunology,CapitalInstituteofPediatrics,Beijing,China9IstanbulFacultyofMedicine,DepartmentofChildPsychiatry,IstanbulUniversity,Istanbul,Turkey10Children'sHospitalBoston,HowardHughesMedicalInstitute,Boston,Massachusetts11MantonCenterforOrphanDiseaseResearch,Children'sHospitalBoston,Boston,Massachusetts12DepartmentofOphthalmology,Children'sHospitalBoston,Boston,Massachusetts13DepartmentofGenetics,HarvardMedicalSchool,Boston,Massachusetts14HowardHughesMedicalInstitute,BethIsraelDeaconessMedicalCenter,Boston,Massachusetts15Children'sHospitalandInstitutesofBiomedicalScience,FudanUniversity,Shanghai,ChinaReceived1July2009;Accepted15December2009AdditionalSupportingInformationmaybefoundintheonlineversionofthisarticle.
TheChildren'sHospitalBostonGenotypePhenotypeStudyGroupalsoincludesOmarS.
Khwaja,AnnapurnaPoduri,MustafaSahinandMagdiSobeih,DepartmentofNeurology,Children'sHospitalBoston,HarvardMedicalSchool,Boston,Massachusetts.
Grantsponsors:NancyLurieMarksFamilyFoundation;SimonsFoundation;AutismSpeaks;NIH;Grantnumbers:5K23MH080954-02,1R01MH083565.
M.
S.
L.
ChingandY.
Shencontributedequallytothiswork.
*Correspondenceto:RamziH.
Nasir,DivisionofDevelopmentalMedicine,Children'sHospitalBoston,HarvardMedicalSchool.
300LongwoodAve.
,Boston,MA02115.
E-mail:ramzi.
nasir@childrens.
harvard.
edu**Correspondenceto:Bai-LinWu,DepartmentsofLaboratoryMedicineandPathology,Children'sHospitalBoston,HarvardMedicalSchool.
300LongwoodAve.
,Boston,MA02115.
E-mail:bai-lin.
wu@childrens.
harvard.
eduPublishedonline7April2010inWileyInterScience(www.
interscience.
wiley.
com)DOI10.
1002/ajmg.
b.
310632010Wiley-Liss,Inc.
937NeuropsychiatricGeneticsResearchhasimplicatedmutationsinthegeneforneurexin-1(NRXN1)inavarietyofconditionsincludingautism,schizo-phrenia,andnicotinedependence.
Toourknowledge,therehavebeennopublishedreportsdescribingthebreadthofthepheno-typeassociatedwithmutationsinNRXN1.
WepresentamedicalrecordreviewofsubjectswithdeletionsinvolvingexonicsequencesofNRXN1.
Weascertainedcasesfrom3,540individ-ualsreferredclinicallyforcomparativegenomichybridizationtestingfromMarch2007toJanuary2009.
Twelvesubjectswereidentiedwithexonicdeletions.
ThephenotypeofindividualswithNRXN1deletionisvariableandincludesautismspectrumdisorders,mentalretardation,languagedelays,andhypotonia.
TherewasastatisticallysignicantincreaseinNRXN1deletioninourclinicalsamplecomparedtocontrolpopulationsdescribedintheliterature(P8.
9107).
ThreeadditionalsubjectswithNRXN1deletionsandautismwereidentiedthroughtheHomozygosityMappingCollaborativeforAutism,andthisdeletionsegregatedwiththephenotype.
OurstudyindicatesthatdeletionsofNRXN1predisposetoawidespectrumofdevelopmentaldisorders.
2010Wiley-Liss,Inc.
Keywords:NRXN1(neurexin-1);developmentaldisorders;arrayCGH;NRXN1exonicdeletions;CNVINTRODUCTIONNeurexinsareagroupofhighlypolymorphiccellsurfaceproteinsinvolvedinsynapseformationandsignaling[Ushkaryovetal.
,1992;MisslerandSudhof,1998;Missleretal.
,2003;Grafetal.
,2004;NamandChen,2005].
Therearethreehumanneurexingenes(NRXN1,NRXN2,andNRXN3),eachofwhichhastwoindepen-dentpromotersresultinginanaandabneurexinforeachgene[Ushkaryovetal.
,1992;Ichtchenkoetal.
,1996].
Multiplealterna-tivesplicingleadstothepossibilityofgreaterthanathousanddistinctneurexinisoforms[Ullrichetal.
,1995].
Theirexpressionisbelievedtobespatiallyandtemporallyregulatedthroughoutdevelopment[PuschelandBetz,1995;Zengetal.
,2006].
StructureandFunctionofNRXN1NRXN1,locatedonchromosome2p16.
3,isoneofthelargestknownhumangenes(1.
1Mbwith24exons)[TabuchiandSudhof,2002].
Itissubjecttorelativelyfrequentdisruptionincludingmissensechanges,translocation,wholegenedeletion,andintra-geniccopynumberalterations[Fengetal.
,2006;Szatmarietal.
,2007;InternationalSchizophreniaConsortium,2008;Kimetal.
,2008;Kirovetal.
,2008;Marshalletal.
,2008;Morrowetal.
,2008;Yanetal.
,2008;Zahiretal.
,2008;Glessneretal.
,2009;Rujescuetal.
,2009].
Thelongertranscript,NRXN1-a,encodesanN-terminalsignalpeptidewiththreerepeatsoftwolaminin/neurexin/sexhormone-bindingglobulin(LNS)domainsseparatedbyanEGF-likesequence(Fig.
1).
Followingtheserepeats,thereisanO-glycosyla-tionsequence,atransmembranedomain,andacytoplasmictailof55aminoacids.
Neurexin-1-ahasbeenshowntointeractwithcertainneuroliginisoformsandneurexin-bindingproteinsknownasneurexophilins.
Thispresynapticmoleculeisalsorequiredforcalcium-triggeredneurotransmitterreleaseandthefunctionofvoltage-gatedcalciumchannelsinthesynapsesofthebrainstemandneocortex[Missleretal.
,2003;Zhangetal.
,2005;Dudanovaetal.
,2006].
Mouseknockoutsofallthreea-neurexingenesdonotdemonstratemajorabnormalitiesofaxonalpathndingduringdevelopment[Dudanovaetal.
,2007],althoughsynapticfunctionisseverelyimpaired.
Micewithknockoutsofindividuala-neurexingeneshavemodestlydecreasedpostnatalviability,whiledoubleknockoutmicehavegreatlydecreasedpostnatalsurvival.
Tripleknockoutmicedonotsurvivepasttherstdayoflife[Missleretal.
,2003].
Neurexin-1-bismuchshorterthanNeurexin-1-a,asveofthesixLNSdomainsandtheinterveningEGFsequencesarereplacedwithashortb-neurexin-specicsequence(Fig.
1)[MisslerandSudhof,1998].
Neurexin-1-bhasbeenshowntointeractwiththepostsynapticneuroliginfamilyofcelladhesionmoleculesanddystroglycans[Ichtchenkoetal.
,1995;Sugitaetal.
,2001;Aracetal.
,2007;Comolettietal.
,2007;Chenetal.
,2008].
NomousemodelswithknockoutsofNRXN1-b,aloneorincombinationwithNRXN1-a,haveyetbeenanalyzed[Sudhof,2008].
ForeachofNeurexin-1-aandNeurexin-1-b,multipleproteincodingisoformsofNRXN1havebeenidentied,whosestructureandfunctionsarenotwellunderstood.
NRXN1MutationsinHumansThereisincreasingevidencethatNRXN1disruptions[Kimetal.
,2008],pointmutations[Fengetal.
,2006;Yanetal.
,2008],anddeletions[Glessneretal.
,2009;Marshalletal.
,2008;Morrowetal.
,2008;Szatmarietal.
,2007]areassociatedwithautismspectrumdisorders.
NRXN1hasalsobeenfoundtobeassociatedwithautisminalargegenome-widesinglenucleotidepolymorphismassocia-tionstudy[Wangetal.
,2009].
NRXN1deletionshavealsobeenassociatedwithavarietyofotherconditionsincludingschizophrenia[InternationalSchizophreniaConsortium,2008;Kirovetal.
,2008;Vrijenhoeketal.
,2008;Walshetal.
,2008;Needetal.
,2009;Rujescuetal.
,2009],nicotinedependence[Bierutetal.
,2007;Nussbaumetal.
,2008],HowtoCitethisArticle:ChingMSL,ShenY,TanW-H,JesteSS,MorrowEM,ChenX,MukaddessNM,YooS-Y,HansonE,HundleyR,AustinC,BeckerRE,BerryGT,DriscollK,EngleEC,FriedmanS,GusellaJF,HisamaFM,IronsMB,LaoscaT,LeClairE,MillerDT,NeessenM,PickerJD,RappaportL,RooneyCM,SarcoDP,StolerJM,WalshCA,WolffRR,ZhangT,NasirRH,WuB-L,onbehalfoftheChildren'sHospitalBostonGenotypePhenotypeStudyGroup.
2010.
DeletionsofNRXN1(Neurexin-1)PredisposetoaWideSpectrumofDevelopmentalDisorders.
AmJMedGenetPartB153B:937–947.
938AMERICANJOURNALOFMEDICALGENETICSPARTBandotherphysicalmanifestationssuchasvertebralanomalies[Zahiretal.
,2008].
PriorreportsofabnormalitiesinNRXN1havefocusedonpopulationswithspecicdiagnoses(e.
g.
,autism,schizophrenia).
However,theclinicalsignicanceofcopynumbervariants(CNV),suchasdeletioninvolvingoneormoreexonsofNRXN1,andtherangeofphenotypicmanifestationsofsubjectswithNRXN1dele-tionCNVremainsunclear.
WedescribehereagroupofsubjectswithNRXN1deletionswhodemonstrateawiderangeofphysicalanddevelopmentalphenotypes.
MATERIALSANDMETHODSClinicalCohortRecordReviewFromMarch2007toJanuary2009,atotalof3,540subjectsatChildren'sHospitalBostonwereevaluatedforgenomicimbalance(deletionandduplication)usingtheAgilent244Khumangenomeoligonucleotidecomparativegenomichybridization(CGH)mi-croarrays(G4411B,AgilentTechnologies,PaloAlto,CA)accordingtothemanufacturer'sinstructions[OligonucleotideArray-BasedCGHforGenomicDNAAnalysisprotocolversion3(AgilentTechnologies)].
Themajorityofthereferralswereforclinicalfeaturesofdevelopmentaldisorders(developmentaldelay,autismspectrumdisorders,mentalretardation)ormultiplecongenitalmalformationsasdeterminedbyspecialistsinClinicalGenetics,Neurology,andDevelopmentalMedicine.
Onehundredthirtyprobescoverthe1.
12MbregionoftheNRXN1geneontheAgilent244KCGHarray.
Theaverageinterp-robespacewithintheNRXN1geneis8.
6kb.
Thispermitsthereliabledetectionofsmallintragenicdeletionsdownto43kbinsize.
ImageswerecapturedbyAgilentscannerandquantiedusingFeatureExtractionsoftwarev9.
0(AgilentTechnologies).
CGHAnalyticsSoftwarev3.
4(AgilentTechnologies)wassubsequentlyusedfordatanormalization,qualityevaluationanddatavisualiza-tion.
CopynumberaberrationwasindicatedusingtheAberrationDetectionMethod2(ADM-2)algorithm.
DeletionsinvolvingveormoreconsecutiveprobeswereconsideredastrueCNV.
Fortwolargerdeletions,uorescentinsituhybridization(FISH)testingusingprobeRP11-800C7wascarriedoutfordeletionconrmationandparentaltesting.
ThesmallerdeletionswereconrmedbyPCR-basedbreakpointmappingmethods.
Thepri-mersusedforeachcasearelistedintheSupplementaryMaterial.
SubjectswithdeletionsinvolvingexonicsequenceofNRXN1wereincludedinourreview.
Twodevelopmentalbehavioralpedia-tricians(RHN,MSLC),aclinicalgeneticist(WHT),andapediatricneurologist(SSJ)reviewedeachofthemedicalrecords.
Theclinicalhistory,physicalexamination,laboratorydata,andradiologicalreportsofeachsubjectwerereviewed.
AdditionalReportofCasesWithNRXN1DeletionandAutismCaseswithexonicandintragenicNRXN1deletionswerealsocontributedfromtheHomozygosityMappingCollaborativeforAutism(HMCA)whichutilizedtheAffymetrixGeneChipHumanMapping500KArraySetusingCNVdetectionmethodspreviouslydescribed[Morrowetal.
,2008].
ThisworkwasapprovedbytheInstitutionalReviewBoardsatthecorrespondinghospitals.
RESULTSClinicalCohortRecordReviewWeidentied12subjectsthroughChildren'sHospitalBostonwithdeletionsinvolvingexonicsequencesofNRXN1(TableIandFig.
1).
Thedeletionsreportedhererangefrom65kbto5Mbandmostofthesecasesarepredictedtoaffecttheinitialstructuraldomainsoftheprotein(Fig.
1).
FIG.
1.
Illustratesthesizeandrangeofthe12deletionCNVsinrelationtotheexonsandproteindomainsofNRXN1-aand-bintheUCSCGenomeBrowser(http://genome.
ucsc.
edu)[Kentetal.
,2002].
Thetoptrackshowsthegenomicpositionandsizeofthe12deletionCNVs.
ThemiddletracksshowthegeneannotationsinRefSeqandEnsembl.
TheRefseqGenesshowtheaandbisoformsoftheNRXN1gene;theEnsemblgenepredictionshowsseveralotherminorisoformsoftheNRXN1gene.
ThebottompanelshowstheproteindomainsoftheNRXN1-ageneproduct.
SP,signalpeptide;LNS,laminin/neurexin/sexhormone-bindingglobulindomain;EGF,epitheliumgrowthfactorlikedomain;OS,O-glycosylationsequence;TM,transmembranedomain;CT,cytoplasmictail.
tail.
[Colorgurecanbeviewedintheonlineissue,whichisavailableatwww.
interscience.
wiley.
com.
]CHINGETAL.
939TABLEI.
DeletionsWithinNRXN1inOurSamplePatientDeletionlocation(hg18build)Sizeofdeletion(kb)InheritanceExons–intronsdeletedOthergenetictestsandresults(additionalimbalance)IndicationfortestingConrmationmethod146,938,685–52,015,8855,077MaternalFISHnormal;paternalstudyunavailableAllKaryotypingandFragileXtest:normal(contiguousdeletionincludingFSHR,LHCGR,STN1)ModeratementalretardationFISH250,128,256–54,050,7133,923DenovoAllexceptthelasttwoexonsNoneGlobaldevelopmentaldelays,suspectedautismFISH350,897,002–51,212,385315PaternalExon1–5;partialintron5Karyotypingandchromosome15methylation:normalGrossmotordelay,hypotoniaPCR450,936,914–51,167,934231PaternalExon1–5;partialintron5Karyotyping,fragileXtest,SALL1,andCHD7mutationtest:normalPDD-NOS,hypotoniaPCR550,920,082–51,059,469139DenovoExon3,4,5;partialintrons2,5NoneVACTERLNotdone651,059,410–51,316,396257MaternalExon1,2;partialintron2KaryotypingandfragileXtest:normalPDD-NOS,motorcoordinationdelaysPCR751,090,504–51,212,385122PaternalExon1–3;partialintron3Karyotyping,FragileXtest,andPTENmutationtest:normalAutism,moderatementalretardationPCR850,522,892–50,827,767305DenovoExon6–17;partialintrons5,17aFragileXtest:normal(deletionat3p24.
3from21492764to21806824,maternallyinherited)MildmentalretardationPCR950,689,280–50,853,329164Unknown(fosterfamily)Exon6–8;partialintrons5,8aKaryotyping:normalLanguagedelay,prenatalsubstanceexposurePCR1050,714,297–50,853,329139DenovoIntron5aKaryotypingandfragileXtest:normalPDD-NOSPCR1150,735,499–50,811,01876MaternalIntron5aKaryotyping,PTEN,andNSD1mutationtests:normal(duplicationsat5p13.
2from37241141to37758854,paternallyinherited;at15q26.
3from98059710to98842423,maternallyinherited;at17p11.
2from21147675to21442522maternallyinherited)Hypotonia,muscleweakness,largebirthweightPCR1250,735,499–50,801,23366MaternalIntron5aNonePoorweightgain,mildcraniofacialdysmorphismPCRFSHR,follicle-stimulatinghormonereceptor;LHCGR,luteinizinghormone/choriogonadotropinreceptor;STN1,StonedB-likefactor;PDD-NOS,pervasivedevelopmentaldisorder,nototherwisespecied;VACTERL,vertebralanomalies,analatresia,cardiacmalformations,tracheoesophagealstula,renalanomalies,andlimbanomalies;SALL1,sal-like1(Drosophila);CHD7,chromodomainhelicaseDNA-bindingprotein7;PTEN,phosphataseandtensinhomolog;NSD1,nuclearreceptor-bindingSETdomainprotein1.
aDeletionsofintron5inthesepatientsinvolveanexonofaminorisoformofNRXN1.
940AMERICANJOURNALOFMEDICALGENETICSPARTBOfthese12deletions,4weredenovoCNVnotidentiedineitherparent,3werematernallyinherited,3werepaternallyinherited,andtheparentalsamplesfor1(subject9)werenotavailable.
Insubjects1,paternalsampleswerenotavailablebutthedeletionwasnotidentiedinmaternaltesting.
Insubjects1–9,thedeletionsinvolvedatleasttwoexonsofNRXN1-a,whileinsubjects10–12,thedeletionsinvolvedonlyanexonofaminorexpressedNRXN1isoform.
Thegenomicimbal-ancesinvolvingNRXN1aresummarizedinTableIandtheclinicalmanifestationsaresummarizedinTablesIIandIII.
FurtherclinicaldataareavailableintheSupplementaryMaterial.
Detailedclinicalrecordswereavailablefromgeneticistsin9outof12subjects,developmental-behavioralpediatriciansin6/12,psychologistsin6/12,andneurologistsin4/12.
Fourofthe12subjects(4,6,7,and10)werediagnosedwithautismspectrumdisorders;ineachpositivecase,thisdiagnosiswassupportedbytheAutismDiagnosticObservationSchedule.
Anothersubject(2)wassuspectedofhavingautismbuttheevaluationwasnotavailableforreview;healsohadglobaldevelopmentaldelays.
Twosubjectshadmentalretardationwithoutadiagnosisofanautismspectrumdisorder(1and8).
Subject1,inaddition,hadabsenceseizuresandanEEGconsistentwithaprimarygeneralizedepilepsy.
Onesubject(3)wastooyoungtoascertainforanautismspectrumdisorderorcognitivedelays.
Ninesubjectshadclinicaldocumentationofexpressiveorreceptivelanguagedelays.
Milddysmorphicfeatureswerepresentinsevensubjects(2,3,4,7,8,9,and12);threesubjectshadhemangiomas(2,6,and10).
Hypotoniawaspresentinfoursubjects(3,4,9,and11).
Twosubjects(5and12)hadventricularseptaldefects.
MedicalrecordreviewalsorevealedthefollowingcharacteristicsinthesixparentsfromwhomtheNRXN1deletionwasinherited.
Subject4,whohadpervasivedevelopmentaldisorder,notother-wisespecied(PDD-NOS)andhypotonia,inheritedhisdeletionfromhisfatherwhoisalsoreportedtobesociallyawkward.
Subject6,whohadPDD-NOSandcoordinationissues,inheritedthedeletionfromamotherwithahistoryoflanguagedelayandsocialskilldifculties.
Subject11,whohashypotonia,weakness,andPolandanomaly,inheritedthedeletionfromamotherwhohasahistoryofjointhypermobility,osteoarthritis,mitralvalveprolapse,severemigraines,andseverebreastasymmetry.
Thefatherofsubject3(hypotonia,grossmotordelay),thefatherofsubject7(autism,mentalretardation)andthemotherofsubject12(poorweightgain,craniofacialdysmorphism)arereportedtobehealthywithoutdevelopmentalormedicalconcerns.
AdditionalReportofCasesWithNRXN1DeletionandAutismInadditiontotheChildren'sHospitalBostoncases,wereportherethreecasesfromtwofamiliesascertainedthroughtheHMCA[Morrowetal.
,2008].
TheNRXN1deletionsineachwerediscov-eredtosegregatewithIQbelow70inthesepedigrees(Fig.
2).
Allthreeaffectedchildrenwerecarriersandunaffectedchildrenwerenot.
ThedeletionswereinheritedfromfatherswhowerefoundtohaveASDsymptomsandIQbetween60and70,whilenon-carriermotherswerenotontheautismspectrumandwithIQsinthenormalrange.
Thedeletionforthesubjectintherstfamilyisexonicandintragenic,whilethedeletionforthetwosiblingsinthesecondfamilyisupstreamandmayaffectgeneexpression.
Furtherinvestigationisnecessarytosubstantiatethisasafunctionaldele-tion,eventhoughitsegregateswithdisease.
SignicanceTestToestablishtherelevanceoftheseCNV,wecomparedthefrequencyofdeletionsinvolvingNRXN1-aexonsinourChildren'sHospitalBostonpopulation,inwhomCGHtestingwasconsideredtobeclinicallyindicated,tothefrequencyofsimilardeletionsdetectedbyarraygenomicprolingofequivalentresolutioninnormalpop-ulations.
Itsaraetal.
[2009]detectedthreedeletionsinvolvingNRXN1-aexonsin2,493normalindividuals.
TheInternationalSchizophreniaConsortium[2008]reportedtwoexonicdeletioncasesin3,181normalcontrols.
Anotherlarge-scaleschizophreniastudyidentiedvedeletioncasesamong33,746normalcontrols[Rujescuetal.
,2009].
Recently,Glessneretal.
[2009]reportednodeletionCNVinvolvingNRXN1-aamong1,409AutismCase–Control(ACC)controlsamplesand1,110AutismGeneticResourceExchange(AGRE)controls.
Collectively,thefrequencyofexonicdeletionofNRXN1-aincontrolpopulationsis10/51,939(0.
019%);thisdifferssignicantlyfromthefrequencyofexonicdeletionCNVweobservedinourclinicallyreferredpopulation(9/3,540)(0.
25%;P8.
9107,two-tailedFisher'sexacttest).
Therearenoavailabledataonthefrequencyofminorisoformexonicdeletionsincontrolpopulationsandthusthesesubjects(n3)wereexcludedfromthesignicancetest.
DISCUSSIONTherecentrecognitionofgenomicimbalanceinmanychromo-somalregionsthatareassociatedwithautism,mentalretardation,andschizophreniaisduetotheincreasinguseofwholegenomehigh-resolutionarrayCGHintheevaluationofindividualswiththesedisorders.
OurclinicalsubjectswithNRXN1deletionwereascertainedthroughapatientpopulationpresentingwithabroadrangeofreferringdiagnoses.
Throughacarefulreviewofmedicalrecords,weidentiedinoursubjectsanumberofclinicalfeaturesthathadnotbeenpreviouslyassociatedwithNRXN1deletions.
Theseincludelanguagedelays,mentalretardationwithoutautism,hypotonia,andhemangiomas.
Inaddition,twoofoursubjects(5and12)hadventricularseptaldefects.
Interestingly,thehumancDNAhomologoustoratNRXN1-ahasbeenisolatedinbothbrainandhearttissuessuggest-ingapotentialroleforNeurexin-1inbothbrainandheartdevel-opment[Nagaseetal.
,1998].
Oneofthesesubjects(5)alsohadevidenceofmultiplecongenitalanomaliesincludingvertebralanomaliesintheformofaVACTERLassociation.
Vertebralanomalieshavealsobeenreportedinoneothercaseintheliterature[Zahiretal.
,2008].
Apreviousreportshowedthepresenceofaseizuredisorderintwounrelatedindividualssharingthesamemissensevariantinexon1ofNRXN1-b[Fengetal.
,2006].
Inourcohort,onlyonesubjecthadaseizuredisorder(subject1),althoughhis5MbdeletionencompassedtheentireNRXN1geneaswellasthegenesforfollicle-stimulatinghormonereceptor(FSHR),luteinizingCHINGETAL.
941TABLEII.
NeurologicalandDevelopmentalCharacteristicsSubjectSexAgeatascertainmentAutismspectrumdisorderCognitive-developmentalndingsLanguagedelayMotorinvolvementHistoryofseizures/EEGresultsMRI-brainBehavioralfeatures1M16yNoMR;SB5:FSIQ44;VIQ44;NVIQ48;(CA14y)ExpressiveandreceptiveWalkedat18monthsHistoryofseizures;abnormalEEGNormalInattention,impulsivity,hyperactivity2M2yAutismsuspected,noformalevaluationavailableGlobaldevelopmentaldelaysExpressiveandreceptiveNotdocumentedNotdocumentedNotperformedornotdocumentedNotdocumented3F10moNotsuspectedNoconcernsreported.
TestingnotdocumentedNoMildgrossmotordelay,hypotoniaNoneNotperformedornotdocumentedNotdocumented4M4yPDD-NOS(ADOS)WPPSI-IIIVIQ77,PIQ98(CA4y)ExpressiveHypotoniaEEGNormalNotperformedornotdocumentedAttentionconcerns5F6yNoNoconcernsreported.
Testingnotdocumented6monthreceptivedelayNormalNotdocumentedNotperformedornotdocumentedNotdocumented6F7yPDD-NOS(ADOS)BayleyIImentalscale91,29mo(CA31mo)ExpressiveMotorcoordinationdisorderNoneNotperformedornotdocumentedNotdocumented7M14yAutism(ADOS)MR:SB5:FSIQ47;VIQ46;NVIQ53ExpressiveandreceptiveNormalEEGnormalNotperformedornotdocumentedHyperactivity8F11yNoMR:WISC-IV:VCI67,PRI63,WMI59,PSI75,FSIQ58(CA11y)ExpressiveandreceptiveNormalNoneNormalInattention,dgety,disorganized9F4yNoAcademicdelaysreported.
TestingnotdocumentedExpressiveandreceptiveHypotoniaNoneNormalImpulsivityandinattention10M2yPDD-NOS(ADOS)BayleyIIIcognitivescore95(average)ExpressiveandreceptiveNormalNotdocumentedNotperformedornotdocumentedNotdocumented11M8yNoNoconcernsreported.
TestingnotdocumentedNoProximalanddistalweakness,hypotoniaNoneNotperformedornotdocumentedNotdocumented12F19moNotdocumentedNotdocumentedNotdocumentedNormalNoneNotperformedornotdocumentedNotdocumentedADOS,autismdiagnosticobservationschedule;BayleyII,BayleyScalesofInfantDevelopment,secondedition;BayleyIII,Bayleyscalesofinfantandtoddlerdevelopment,thirdedition;CA,chronologicalageattesting;MR,mentalretardation;SB5,Stanford-Binetintelligencescales,fthedition;FSIQ,fullscaleIQ;VIQ,verbalIQ;NVIQ,nonverbalIQ;PIQ,performanceIQ;WPPSI-III,Wechslerpreschoolandprimaryscaleofintelligence,thirdedition;WISC-IV,Wechslerintelligencescaleforchildren,fourthedition;VCI,verbalcomprehensionindex,PRI,perceptualreasoningindex;WMI,workingmemoryindex;PSI,processingspeedindex;y,years;mo,months.
942AMERICANJOURNALOFMEDICALGENETICSPARTBTABLEIII.
RelevantPhysicalCharacteristicsSubjectDysmorphicfeaturesVertebral/skeletalCardiacSkin1NoneNotdocumentedNormalNotdocumented2FrontalbossingHistoryofplagiocephalyResolvedheartmurmurHemangiomaonneck3Epicanthalfolds;hypertelorismsmallerbifrontalregionProminentcoronalsutures,feet:higharchesandsomewhatsmalllengthNormalLighterthanparents4Down-slantingpalpebralssures;antevertednares;mildretrognathia,pointedchinNotdocumentedNormalNormal5NoneCurved2ndtoes,incompletefusionofringofrstcervicalvertebraNarrowedaorticarch,2VSDsNotdocumented6NoneBilateralhipdysplasiaProlongedQTc(457msec)Hemangiomaonneck7Slightlydeepseteyes,largeearsNormalNormalNormal8Longface,malarhypoplasia,prominenttubularnosewithpointednasaltip,hypoplasticalaenase,longatphiltrum,thinvermilion,prominentchin,longslenderngers,thintoesNotdocumentedNormalNormal9Lownasalbridge,smalljaw,verysmoothphiltrum.
Slightlyatmid-faceandprominentcheeksMildclinodactylyandunevendigitlengthsNormalNotdocumented10Dolichocephaly(32-weekprematureinfant)NotdocumentedNormalHemangiomaonback11NoneChest-rightmildPolandanomalyNormalEczema12Relativemacrocephaly(headcircumference90%),cuppingofleftear,frontalbossingOpenanteriorfontanelleat19monthsSmallmuscularVSD,fenestrationinatrialseptum,smallPDANotdocumentedVSD,ventricularseptaldefect;PDA,patentductusarteriosus;QTc,correctedQTinterval(normal<440msec).
CHINGETAL.
943hormone/choriogonadotropinreceptor(LHCGR),andStonedB-likefactor(STN1).
Toourknowledge,noneofthesegeneshasbeenassociatedwithseizuresormentalretardationintheliterature.
AlthoughwecannotbecertainthatthesefeaturesareadirectconsequenceofNRXN1deletion,ourobservationssuggestthatthephenotypiccharacteristicsofNRXN1deletionmaybewiderthanpreviouslyreported.
ThemutationswehaveobservedinourclinicalcohortareprimarilyinNRXN1-a.
Subjectswithsmalldeletions(under3-Mb)clusteredintotwogroups(Fig.
1).
Onegroup(subjects3–7)haddeletionsinvolvingpartoftheinitialLNSandEGFdomains-encodingregionsofNRXN1-a.
Oftheseveindividuals,threehadautismspectrumdisorders.
OneadditionalcasefromtheHMCAwasalsofoundtohaveadeletioninthisregion,whichissimilartothedeletioninsubject7fromtheclinicallyreferredcohort.
Asecondgroup(subjects8–12)haddeletionsthatclusteredaroundaregionfurtherfromtheapromotorofthegene(Fig.
1).
Allveofthesesubjects'deletionsencompassedanexonofanisoformwhosefunctionisnotwellunderstood.
Furthermore,whiletwosubjects(8and9)haddeletionsinvolvingotherexonsofNRXN1-aaswellasthisminorisoform,threesubjects'deletions(10–12)containonlytheexonofthisminorisoform.
ThisminorisoformisanEnsemblannotatedtranscript,namedENST00000406859(Fig.
1).
Itcontains13exonswith2,590bptranscriptionand856residuesoftranslationlength.
Thecodedprotein(ENSP00000385681)consistsofoneLNSandEGFdomain.
Itsfunctioniscurrentlyunknown.
Onesuchsubject(10)withadenovodeletioninthisregionhasbeendiagnosedwithPDD-NOS,suggestingpotentialclinicalrele-vanceforthisisoform.
Thisdeletioninintron5hasnottoourknowledgebeenpreviouslyreportedasbeingassociatedwithabnormaldevelopment.
Neurexin-1-bmutationswerelesscommon.
TwoofthesubjectsinourcohorthadlargedeletionsencompassingexonsforNRXN1-aand-b.
MissensevariantsinNRXN1-b(R8P,L13F,S14L,andT40S)havepreviouslybeenidentiedinindividualswithautism[Fengetal.
,2006;Kimetal.
,2008].
Relativesoftheseindividualswithautismwhosharedthesemissensemutationsdemonstratedsomedegreeoflearningorbehavioralissuesbutdidnotappeartomeetfullautismspectrumdisordercriteria[Fengetal.
,2006;Kimetal.
,2008].
Thisisconsistentwithourndingsofamixedphenotypeassociatedwithdeletionsinthisregionrangingfromautismspectrumdisorderstohypotoniawithcarrierrelativeswhoarenotasaffected.
InadditiontotheirNRXN1deletions,subjects8and11hadadditionalgenomicimbalancesasdescribedinTableI.
Thesegenomicimbalanceswereallinheritedfromunaffectedparents.
Thetwoduplicationson15q26.
3and17p11.
2insubject11overlapFIG.
2.
A:NRXN1-adeletionssegregatewithautismspectrumdisorder(ASD)andmildmentalretardation.
Pedigree1showsco-segregationofahemizygousCNVbetweenrs17041500andrs17512199whichdeletestherstthreecodingexons(DelEx1-3)ofNRXN1-a.
TheCNViscarriedbyallsubjectswithASDanddiminishedintelligencequotient(IQ),butnotbyatypicallydevelopingsibling.
Pedigree2showsco-segregationofahemizygousCNVwhichdeleteslikelyregulatory,genomicDNAupstream(Del50Reg)ofNRXN1-a.
PDD-NOS,pervasivedevelopmentdisorder,nototherwisespecied.
,wild-type,non-deletedDNA.
B:MappingofinferredCNdataSNP-by-SNPontheUCSCgenomebrowserdemonstratestheextentacrosstheNRXN1locus.
VerticalredlinesindicateeachSNPwithcopynumberof1or2.
Horizontalgreenlinesdemarcatetheextentofeachdeletion.
AlignmentofannotatedgenesintheRefSeqdatabaseareshownaswellasarepresentationofvertebrateconservationusingmultizandrelatedtoolsintheUCSC/PennStateBioinformaticscomparativegenomicalignmentpipeline.
Ofnote,Del50Regdeletesthelastfourexonsofanuncharacterized,splicedmRNAAK127244thatisexpressedinbrain.
ThegeneistranscribedintheoppositedirectionasNRXN1-ayetthetranscriptionstartsiteiswithin3.
5kbsuggestingthatthismRNAmaybetranscribedcoordinatelywithNRXN1-a.
[Colorgurecanbeviewedintheonlineissue,whichisavailableatwww.
interscience.
wiley.
com.
]944AMERICANJOURNALOFMEDICALGENETICSPARTBwithknownbenignCNVsandareunlikelycontributoryfactorstothepatient'scondition.
Theduplicationat5p13.
2insubject11anddeletionat3p24.
3insubject8arenotpreviouslyreportedCNVbutcontainnoknowngenesassociatedwithdevelopmentaldisorders,thusareconsideredasCNVofunknownsignicance.
Nevertheless,itisunclearwhethertheseCNVsmodiedtheobservedphenotype.
NRXN1andSynapseFunctionPriorstudieshavefunctionallylinkedothermoleculesthatareassociatedwithNRXN1toarangeofneuropsychiatricdisordersincludingautism.
Theseincludeneuroligins3and4(NLGN3,NLGN4)andSH3andmultipleankyrinrepeatdomains3(SHANK3)[Jamainetal.
,2002;Laumonnieretal.
,2004;Durandetal.
,2007;Moessneretal.
,2007;Lawson-Yuenetal.
,2008].
Inaddition,CNTNAP2(contactinassociatedprotein-like2)[Alarconetal.
,2008;Arkingetal.
,2008;Bakkalogluetal.
,2008]andcadherin10(CDH10)and9(CDH9)havebeenalsoassociatedwithautismspectrumdisorders[Wangetal.
,2009].
OurndingthatNRXN1isalsoassociatedwithautismanddevelopmentaldisordersaddsfurtherevidencetotheimportanceofthismolecularfamilytothedevelopmentofneurodevelopmentaldisorders.
ThefunctionofNRXN1infacilitatingsynaptictransmissionsuggeststhatmutationsinthisgenemaypredisposetoaneurologicdisconnectionsyndrome.
Long-rangedisconnectionsbetweenneuralnetworkshavebeenhypothesizedtobecausativeinsomepopulationswithautism[Barnea-Goralyetal.
,2004;Frith,2004;Justetal.
,2004;GeschwindandLevitt,2007].
TheeffectsofNRXN1onlanguagedevelopmentandhypotoniamaylikewiseberelatedtolong-rangeconnectivitywithinthebrain.
PhenotypicVariationPhenotypicvariationsmayreectthehighlypleiotropiceffectsobservedforspecicCNVssuchasthoseassociatedwithNRXN1.
Inaddition,anumberofoursubjectsinheritedNRXN1deletionsfromtheirparents.
Thedetailedphenotypeoftheseparentswerenotdescribedinthemedicalrecordsexceptinthefamilyhistory,buttheparentswereostensiblylessaffectedthantheirchildren.
ThissuggeststhatdeletionintheNRXN1genemaynotbefullypenetrant,orinteractswithothergenesresultinginthevariablephenotype.
Furtherresearcheffortstoinvestigatesuchvariablephenotypesassociatedwiththisunstablegenomicregionwillpro-videfurtherinsightintotheroleofNRXN1inthedevelopmentoflanguagedelays,autismspectrumdisorders,andphysicalfeatures.
LimitationsTheaccuracyandcompletenessoftheclinicalphenotypeidentiedinthisstudyisentirelydependentontheclinicalinformationthatwasdocumentedinthemedicalrecordsofthesesubjects,oftenbeforetheNRXN1deletionswereidentiedinthem.
Becauseoftheclinicalvariabilityexhibitedinourcohort,thesubjectswereseenbyavarietyofspecialists,whichaffectedthecompletenessofdata.
Inaddition,theparentswerenotformallyassessedtoascertaintheircognitive,physical,andbehavioralphenotypes.
Asnotedabove,reviewoffamilyhistorysuggeststhatsomeparentsmayhavesharedsimilarphenotypestotheirchildren.
Weareconduct-ingfurthertestingonboththesubjectsandtheirparentstobetterclarifydevelopmentaland/orsocialcognitionissuesinsubjectsandtheirparents.
ForthedeletionCNVsignicancetest,weusedthenormalcontroldatageneratedbydifferentgenomicprolingarrayplat-formsasreference.
Knowingthatthesensitivityandspecicitydifferfromonearrayplatformtoanother,thismaynotbeanoptimalcomparison.
However,theeffortwasmadetominimizethedetectionbiasbetweendifferentarrayplatforms.
Herewehaveonlychosenrecentstudiesusingarrayplatformofsimilarresolutionasours.
AllthesepublishedarticlesreportedthedetectionofsmallerCNV,suggestingthattechnicallyallthesearrayplatformswereabletodetectanyCNVidentiedinthisstudy.
Thusthiscomparison,althoughanapproximation,isontheconservativeside.
Finallyweacknowledgethatwhileourclinicallyascertainedsubjectswerenotdrawnfromacohortwithasinglediagnosissuchasautismorschizophrenia,theywereascertainedfromaheteroge-neouslyaffectedgroupinwhomgenetictestingwasconsideredclinicallyrelevant.
Asaresult,thereisascertainmentbiasandourndingsmaynotreectthetruedistributionofphysicalanddevelopmentalndingsintheNRXN1deletionphenotype.
Never-theless,wehavedemonstratedthatthereareanumberofotherphenotypicfeaturespresentinthisclinicalpopulationbeyondwhathaspreviouslybeenidentiedintheliterature.
CONCLUSIONWefoundawiderangeofphenotypicfeaturesinagroupofsubjectswithNRXN1deletionswhowereclinicallyreferredforgenetictesting.
Theseincludesubjectswithautismspectrumdisorders,mentalretardation,languagedelays,hypotonia,hemangiomas,andtheVACTERLassociation.
ACKNOWLEDGMENTSTheauthorsgratefullyacknowledgetheassistancebyourcolleaguesfromtheDNADiagnosticsLab:VaLip,XiaomingSheng,AnnReinhard,HongFang,SivTang,HongShao,HaitaoZhu,SamTang,andAndrewChengfortechnicalsupportofarrayCGH;ChristopherA.
WalshLab:DanielleGleasonandDanielRakiecfortechnicalsupportandRobertSeanHillforbioinformaticssupport.
WearefurthergratefulforthesupportfromtheNancyLurieMarksFamilyFoundation(C.
A.
W.
),theSimonsFoundation(C.
A.
W.
andJ.
F.
G.
),AutismSpeaks(J.
F.
G.
),andtheNIH(5K23MH080954-02toE.
M.
M.
and1R01MH083565toC.
A.
W).
E.
C.
E.
andC.
A.
W.
areInvestigatorsoftheHowardHughesMedicalInstitute.
Y.
S.
holdsaYoungInvestigatorAwardfromtheChildren'sTumorFoundationandCatalystAwardfromHarvardMedicalSchool,E.
M.
M.
holdsaCareerAwardforMedicalScientistsfromtheBurroughsWellcomeFund,B.
L.
W.
holdsaFudanScholarResearchAwardfromFudanUniversity.
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