文献-identification of gene copy number variations in patients with mental retardation using array-cgh novel syndromes in a large fre

EuropeanJournalofMedicalGenetics53(2010)66e75ContentslistsavailableatScienceDirectEuropeanJournalofMedicalGeneticsjournalhomepage:/locate/ejmgOriginalarticleIdenticationofgenecopynumbervariationsinpatientswithmentalretardationusingarray-CGH:NovelsyndromesinalargeFrenchseriesSylvieJaillarda,b,*,SeverineDrunatc,ClaudeBendavidb,AzzedineAbourac,AmandineEtcheverryb,HubertJourneld,AndreeDelahayee,LaurentPasquierb,f,DominiqueBonneaug,AnnickToutainh,LydieBurgleni,AgnesGuichetg,EvaPipirase,BrigitteGilbert-Dussardierj,BrigitteBenzackenc,e,DominiqueMartin-Coignardk,CatherineHenrya,AlbertDavidl,JosetteLucasa,JeanMosserb,VeroniqueDavidb,m,SylvieOdentb,f,AlainVerloesc,ChristeleDubourgb,mabcdeLaboratoiredeCytogenetiqueetBiologieCellulaire,CHUPontchaillou,Rennes,FranceCNRSUMR6061,IFR140GFAS,FacultedeMedecine,UniversitedeRennes1,FranceDepartementdeGenetique,INSERMU676,CHURobertDebre,APHP,Paris,FranceServicedeGenetiqueMedicale,CHBA,Vannes,FranceLaboratoiredeCytogenetique,CHUJeanVerdier,SMBH,ParisXIII,U676Gressens,Bondy,APHP,FrancefgServicedeGenetiqueMedicale,CHUHpitalSud,Rennes,FranceServicedeGenetique,CHUAngers,Angers,FrancehServicedeGenetique,CHUBretonneau,Tours,FranceijServicedeGenetiqueetdEmbryologieMedicales,CHUArmandTrousseau,Paris,APHP,FranceServicedeGenetiqueMedicale,CHUPoitiers,Poitiers,FrancekServicedeGenetique,CHLeMans,LeMans,FrancelServicedeGenetiqueMedicale,InstitutdeBiologie,CHUHtelDieu,Nantes,FrancemLaboratoiredeGenetiqueMoleculaire,CHUPontchaillou,Rennes,FrancearticleinfoArticlehistory:Received14April2009Accepted17October2009Availableonline28October2009Keywords:Array-CGHNovelsyndromesMicrodeletionMentalretardation1.IntroductionabstractArray-CGHhasrevealedalargenumberofcopynumbervariations(CNVs)inpatientswithmultiplecongenitalanomaliesand/ormentalretardation(MCA/MR).Accordingtocriteriarecentlylisted,pathogenicitywasclearlysuspectedforsomeCNVsbutbenignCNVs,consideredaspolymorphisms,havecomplicatedtheinterpretationoftheresults.Inthisstudy,genomicDNAsfrom132Frenchpatientswithunexplainedmentalretardationwereanalysedbygenomewidehigh-resolutionAgilent44Koligonu-cleotidearrays.Theresultswereinaccordancewiththoseobservedinpreviousstudies:thedetectionrateofpathogenicCNVswas14.4%.Anon-randominvolvementofseveralchromosomalregionswasobserved.Someofthemicroimbalancesrecurrentlyinvolvedregions(1q21.1,2q23.1,2q32q33,7p13,17p13.3,17p11.2,17q21.31)correspondingtoknownornovelsyndromes.ForallthepathogenicCNVs,furthercasesareneededtoallowmoreaccurategenotypeephenotypecorrelationsunderscoringtheimportanceofdatabasestogrouppatientswithsimilarmoleculardata.2009ElsevierMassonSAS.Allrightsreserved.ofmicroimbalancesandhaveidentiedsuchaberrationsin10e15%ofMCA/MRpatientswithnormalkaryotype17,20,21,23,24,29,40,Fromtheobservationthatsubtelomericanomaliescanbefoundinabout5%ofMCA/MRpatients22,itwassuspectedthatsmallinterstitialimbalancesmayalsobeanimportantcauseofMCA/MR46,65thatisunderestimatedbycurrentcytogeneticsmethods(conventionalandmolecularcytogenetics).Whole-genomearray-basedtechnologieshavebecameanessentialtoolforthedetection*Correspondingauthor.LaboratoiredeCytogenetiqueetBiologieCellulaire,CHUPontchaillou,2rueHenriLeGuilloux,Rennes,France.Tel.:33299284389.E-mailaddress:sylvie.jaillardchu-rennes.fr(S.Jaillard).41,45,46,54,59,65,66.Thechallengenowistodescribenovelsyndromeswhichcanbeachievedbycomparingpatientscarryingthesamesubtlerearrangement.Array-CGHcangiveanaccuratedelineationoftheimbalanceswhichraisesthepossibilityofmakinggenotypeephenotypecorrelations,inordertoidentifyminimalcriticalregionsandcandidategenesforapatternofclinicalfeaturesincludinglearningdisability.Finemappingofgenomicimbalancesmayindeedbeusefultonarrowdownregionssus-pectedtocontaindosagesensitivegenescriticalfornormaldevelopment35.1769-7212/$eseefrontmatterdoi:10.1016/j.ejmg.2009.10.0022009ElsevierMassonSAS.Allrightsreserved.S.Jaillardetal./EuropeanJournalofMedicalGenetics53(2010)66e75672.Materialsandmethods2.1.PatientsSubjectswithunexplainedmentalretardationwereselectedbyclinicalgeneticistsaccordingtocriteriaderivedfromthechecklistofdeVriesetal.18,whichislinkedwithahigherlikelihoodofmanifestingsubmicroscopiccopynumberchanges,particularly,subtelomericimbalances.Criteriaforthepresentstudycorres-pondedtomentalretardationwithatleastoneassociatedcriterion(familialhistoryofmentalretardation,growthabnormalities,facialdysmorphicfeatures,congenitalmalformations,seizuresandbehavorialorsleepingdisabilities).Allpatientshadnormalstandardand/orhigh-resolutionkaryotypingandnormalfragileXtesting.Patientswereexcludedfromthestudyincaseofidentiedaetiologyofmentalretardation,abnormalkaryotypeandmicrodeletionsyndrome.SubtelomericimbalanceswereexcludedbysubtelomericFISHusingtheToTelVysionprobes(AbbottMolecularInc.,DownersGrove,Illinois,USA)orbyMultiplexLigation-DependantProbeAmplication(MLPA)usingtheSalsaMLPAP036BandP070kits(MRC-Hollandb.v,Amsterdam,TheNetherlands).Ofthe132patients,64wererecruitedatgeneticsclinicalcentersintheWestofFrance(HUGO,HpitauxUniversitairesduGrandOuest)and68wererecruitedatgeneticsclinicalcentersinParis.Informedconsentforgeneticinvestigationsonmentalretar-dationwasobtainedfromparticipatingfamilies.Detailedclinicalhistorywasobtainedandphysicalexaminationwascarriedoutforthe132patients.2.2.Array-CGHOligonucleotidearray-CGHwasperformedusingtheAgilentHumanGenomeCGHmicroarray44K(AgilentTechnologies,SantaClara,CA,USA).Thesemicroarraysareconstitutedofmorethan44,00060-meroligonucleotideprobesthatspanbothcodingandnon-codingregions,withemphasisonwell-characterizedgenesrepresentedbyatleast1probeandcancer-relevantgenesbyatleast2probes(morethan30,000genesrepresented).Coverageofthehumangenomeismadewithanaveragespatialresolutionof35kb.Theexperimentswereperformedaccordingtoversion4.0(June2006)oftheprotocolprovidedbyAgilent(AgilentOligo-nucleotideArray-BasedCGHforGenomicDNAAnalysis).ReferencegDNAwasfromsinglemaleorfemale,inordertobettercharacterizethecopynumberpolymorphisms(CNPs).MicroarrayswerescannedusingtheAgilentscannerG2565BA.ImageswereanalysedusingAgilentFeatureExtractionSoftwareversion9.1(CGH-v4_91protocol),permittingcreationofQCreportsforeachpatientwherethevalueoftheDerivativeLogRatioSpread(DLRS),whichisthespreadofthederivativelogratiovalues,wasusedasaqualitycriterion.AgraphicaloverviewandanalysisofthedatawereobtainedbyusingtheAgilentCGHanalyticssoftwareversion3.4.27(statisticalalgorithm:z-score,sensitivitythreshold:2.5,movingaveragewindow:5points).Onlyimbalancesinvolvingthreeormoreadjacentprobeswereretainedandfurtherevaluated,basedonpreviousassessmentsthatwiththisplatform,themajorityofthesingleanddouble-probealterationsobservedarelikelytobefalse-positivecalls21.Identicationofprobeswithasignicantgainorlosswasbasedonthelog2ratioplotdeviationfrom0withcut-offvaluesof0.5e1and0.5to1respectively.Thevalidationmethodwaschosendependingontheimba-lancetype(deletionorduplication)andsize,andonpracticalreasons(typeofsampleavailable:DNAand/ormetaphasespreads).2.3.MultiplexPCR/liquidchromatography(MPLC)DuplexPCRwasdesigned,associatingunlabeledprimersforanendogenouscontrolgene,HMBS,andfortheregionshowingimbalanceinthetestedpatient.Thisenablestheirsimultaneousamplicationundersemi-quantitativeconditions.PrimersweredesignedusingPrimerPremierSoftware(PremierBiosoftInterna-tional,PaloAlto,CA,USA).DuplexPCRfornormalcontrol,eachpatientandparentsgDNAswasperformedasdescribedelsewhere5,19,26.DataanalysiswasperformedusingtheNavigatorSoft-ware(Transgenomic,Omaha,NE,USA),normalisationwasachievedwithHMBSpeakandrelativepeakintensitiesforeachamplicondirectlyreectedgenomiccopynumber.2.4.QuantitativePCRReal-timequantitativePCR(qPCR)wascarriedoutonanABIprism7700(AppliedBiosystems,FosterCity,CA,USA)withtheuseofuorescentSYBRGreendye.Gene-specicprimersfortargetgenesandendogenouscontrolgenes(PLAGL1andSDC4)weredesignedusingPrimerExpressSoftware(AppliedBiosystems,FosterCity,CA,USA).Eachsamplewasruninduplicateforthequanticationofthetargetgenesascomparedtotheendogenouscontrolgenes.DataevaluationwascarriedoutusingtheABIPrismSequenceDetectionSystem(AppliedBiosystems,FosterCity,CA,USA)andMicrosoftExcel,usingthecomparativeDDthresholdcyclenumber(Ct)method.2.5.Fluorescenceinsituhybridization(FISH)ClonesmappingtotheunbalancedregionswereselectedonpublicdatabasesUCSCGenomeBrowserandEnsemblHumanGenomeBrowser.CloneswereobtainedfortheRP11libraryfromtheBACPACResourceCenter(ChildrensHospitalOaklandResearchInstitute,Oakland,CA,USA),fromDrMarianoRocchi(CytogeneticsDepartment,InstituteofGenetics,BariUniversity,Italia),fromDrJorisAndrieux(GeneticsDepartment,LilleUniversityHospital,France),fromtheWellcomeTrustSangerInstituteandfortheCTDlibraryfromInvitrogenCorporation(Invitrogen,Carlsbad,USA).TheclonesavailableforthisstudyarelistedinTable1.DNAswerelabelledbyrandomprimingusingtheBioprimeArrayCGHGenomicLabelingSystem(Invitrogen,Carlsbad,USA)orbynicktranslationusingtheNickTranslationKitVysis(AbbottMolecularInc.,DownersGrove,IL,USA).Probeswerehybridizedtometaphaseslidesobtainedfromphytohaemaglutinin-stimulatedperipheralbloodlymphocyteculturesforpatientsandparents.SlideswereanalysedwithanepiuorescentmicroscopeOlympusBX61andimageswerecapturedusingIsissoftware(MetaSystems,Altlus-sheim,Germany).FISHsignalswereexaminedbothonmetaphasechromosomesandinterphasenuclei.2.6.Multiplexligation-dependantprobeamplication(MLPA)TheSalsaMLPAkitsP036BandP070“humantelomeres”fromMRCHollandwereusedconcomitantlytodetectdeletionsorgainsinsubtelomericregionsandfortheacrocentricchromosomesinthesubcentromericregion,accordingtotheMLPADNAdetectionandquanticationprotocolfromMRC-Holland.3.ResultsCriteriaofpathogenicityestablishedbyLeeetal.31wereusedtodetermineclinicalrelevanceoftheCNVsobserved.AccordingtoLeeetal.CNVsaremorelikelytobepathogenicifcorrespondtooneofthefollowingcriteriai.e.theyaredenovo,S.Jaillardetal./EuropeanJournalofMedicalGenetics53(2010)66e7568Table1ClinicalandmolecularfeaturesofpatientswithpathogenicCNVs.PatientChromosomalClinicalfeaturesImbalanceImbalanceImbalanceValidationMethodOriginKnownsyndromeorP1imbalancedel(1)(q21.1q21.1)Mentalretardation,facialdysmorphism,post-natalgrowthretardation,minimalsize(bp)983,507start(bp)143,866,417end(bp)144,849,923FISH(RP11-337C18,candidategeneassociatedmaternal1q21.1microdeletionand12genesextremityabnormalities,cerebellarvermishypoplasia,atrialseptaldefect,multi-cysticrenaldysplasiaRP11e533N14,RP11-102F23)microduplication11,38P2dup(2)(p16.1p13.2)Severementalretardation,post-natalgrowthretardation,14,879,51057,443,10872,322,617FISH(RP11-81L7,denovo86genesmacrocephaly,epilepsy,aorticcoarctation,facialdysmorphism,PierreRobinsequenceRP11-728F15,RP11-564H1)P3del(2)(q23.1q23.1)Severementalretardation,languageimpairment,inappropriate257,300149,061,980149,319,279FISH(RP11-659J19)denovo2q23.1microdeletion262geneslaughter,post-natalgrowthretardation,microcephaly,epilepsy,largemouth,ataxia,valgusfeet,micropenisP4del(2)(q22.3q23.1)Severementalretardation,languageimpairment,inappropriate2,520,605146,841,970149,362,575FISH(RP11-95O9,denovo2q23.1microdeletion264geneslaughter,autisticbehaviour,aggressivity,sleepingdifculties,post-natalgrowthretardation,relativemicrocephaly,epilepsy,ataxia,feedingdifculties,facialdysmorphism,clinodactylyofthefthnger,hypogenitalismRP11-107E5,RP11-548K13,RP11-515K12,RP11-341F20,RP11-72H23)P5del(2)(q31.2q33.1)Severementalretardation,post-natalgrowthretardation,ectodermal26,090,717176,637,788202,728,505FISH(RP11e91M5)denovo2q33.1deletionsyndrome128genesdysplasia(hairandteethinvolvement)61P6dup(3)(p14.3p14.3)Mildtoseverementalretardation,cerebellarvermishypoplasia,1,056,17756,305,07357,361,250qPCRdenovo10genesanteriorpachygyria,hypoplasiaofthecorpuscallosum,facialdysmorphism,bilateralinguinalhernia,feetandlegsdistortionP7del(3)(q13.31q21.3)Severementalretardation,facialdysmorphism,hypotonia,agenesisof13,007,667115,508,275128,515,941FISH(RP11-153K19,NS87genesthecorpuscallosum,colicstenosis,genitaliaabnormalities,jointstiffnessRP11-324H4,RP11-322C21,RP11-689D3)P8del(3)(q26.33q27.1)Severementalretardation,anophtalmia,agenesisofthecorpus3,788,389180,966,165184,754,554FISH(RP11-43F17)denovoSOX2deletion3614genescallosum,post-natalgrowthretardation,microcephaly,coarsefacies,epilepsy,hypotoniaP9del(5)(q14.3q14.3)Severementalretardation,hypotonia,epilepsy,craniofacialanomalies,216,43288,051,97088,268,402MPLCandFISH(CTD-denovo1genestereotypicmovements2328P23,RP11-1147F22,RP11-110I3)P10del(7)(p14.1p13)Severementalretardation,hypotonia,bidthumbs,feedingdifculties,6,858,38837,663,44544,521,833FISH(RP11e52M17)denovoGreigsyndrome7,4750genesrenalabnormalities(bilateralrenalhypoplasia,megaureter,vesicoureteralreux,renalinsufciency),cutislaxa,facialdysmorphism,cerebralatrophyP11del(10)(q24.32q24.32)Severementalretardation,autisticbehaviour,hypotonia,macrocephaly,417,162104,251,636104,668,797MPLCdenovo10genescerebellarvermisagenesis,partialsightP12dup(17)(p13.3p13.3)Severementalretardation,languageimpairment,epilepsy,facial438,6181,059,8521498,470FISH(RP11-818O24)NSEmbeddedinthe9genesdysmorphism,vaginalduplicationMillereDiekersyndromecriticalregion13P13del(17)(p11.2p11.2)Moderatementalretardation,clinicalfeaturesofMyhresyndrome1,452,78318,709,50420,162,287FISH(RP11-369K5)NSEmbeddedintheSmith-18genesMagenissyndromecriticalregionP14del(17)(q21.31q21.31)Moderatementalretardation,lowbirthweight,feedingproblems,493,11341,073,48641,566,599FISH(RP11-669E14)denovo17q21.31microdeletion6genesepilepsy,microcephaly,facialdysmorphism,atrialseptaldefect,ventricularseptaldefect,deafness,hypotonia,jointlaxity,bilateralinguinalhernia28,49,53P15del(17)(q21.31q21.31)Mentalretardation,neonatalhypotonia,epilepsy,hypoplasiaofthe442,19541,073,48641,515,681FISH(RP11-669E14)denovo17q21.31microdeletion6genescorpuscallosum,neuronalheterotopia,facialdysmorphism,cryptorchidism,hypermetropia28,49,53P16dup(18)(q21.2pter)andMentalretardation,hypoplasiaofthecorpuscallosum,epilepsy,auralBorderlineFISH(D18Z1)denovodel(18)(q21.2qter)atresia,genitaliaabnormalities,hypotonia,facialdysmorphism,ptosis,trigonocephalyprobe:51,501,43251,501,491dup(22)(q11.1q11.21)P175genesretardation,hypoplasiaofthecorpuscallosum,epilepsy,hypotonia,jointlaxitySeverementalretardation,behaviouraldifculties,post-natalgrowth267,450FISH(RP11-150P15)16,591,96416,324,514MLPA(P036Bkit)maternalEmbeddedintheCateyesyndromecriticalregion37P18dup(X)(p22.11p22.11)2genescryptorchidism,shortngerswithsyndactylies,shortandhollowfeetModerate-mildmentalretardation,microcephaly,facialdysmorphism,468,009qPCR22,278,56021,810,551maternalP19dup(X)(p22.11p22.11)2genesweight2SD,shortngersandfeet,clubfeet,leftfootsyndactylyII-IIIModerate-mildmentalretardation,microcephaly,facialdysmorphism,468,00921,810,55122,278,560qPCRmaternalMappositionsrefertotheGenomeAssemblyMay2004hg17.ThevalidationmethodusedwaseithermultiplexPCR/liquidchromatography(MPLC),uorescenceinsituhybridization(FISH),multiplexligation-dependantprobeamplication(MLPA)orquantitativePCR(qPCR).Themethodchosendependingontheimbalancetypeandsize,andonpracticalreasons.Thenumberofg