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RESEARCHOpenAccessArrayCGHimprovesdetectionofmutationsintheGALCgeneassociatedwithKrabbediseaseAliceKTanner1,EphremLHChin1,PatriciaKDuffner2andMadhuriHegde1*AbstractBackground:KrabbediseaseisanautosomalrecessivelysosomalstoragedisordercausedbymutationsintheGALCgene.
ThemostcommonmutationintheCaucasianpopulationisa30-kbdeletionofexons11through17.
TherearefewotherreportsofintragenicGALCdeletionsorduplications,dueinparttodifficultiesdetectingthem.
Methodsandresults:Weusedgene-targetedarraycomparativegenomichybridization(CGH)toanalyzetheGALCgeneinindividualswithKrabbediseaseinwhomsequenceanalysiswith30-kbdeletionanalysisidentifiedonlyonemutation.
Inoursampleof33cases,traditionalapproachesfailedtoidentifytwopathogenicmutationsinfive(15.
2%)individualswithconfirmedKrabbedisease.
TheadditionofarrayCGHdeletion/duplicationanalysistothegenetictestingstrategyledtotheidentificationofasecondpathogenicmutationinthree(9.
1%)ofthesefiveindividuals.
Inallthreecases,thedeletionorduplicationidentifiedthrougharrayCGHwasanovelGALCmutation,includingtheonlyreportedduplicationintheGALCgene,whichwouldhavebeenmissedbytraditionaltestingmethodologies.
Wereportthesethreecasesindetail.
Thesecondmutationremainsunknownintheremainingtwoindividuals(6.
1%),despiteourfullbatteryoftesting.
Conclusions:AnalysisoftheGALCgeneusingarrayCGHdeletion/duplicationtestingincreasedthetwo-mutationdetectionratefrom84.
8%to93.
9%inaffectedindividuals.
BettermutationdetectionratesareimportantforimprovingmoleculardiagnosisofKrabbedisease,aswellasforprovidingprenatalandcarriertestinginfamilymembers.
Keywords:Krabbedisease,GALC,Deletion,Duplication,ArrayCGHBackgroundKrabbedisease,alsocalledgloboidcellleukodystrophy,isanautosomalrecessivelysosomalstoragedisorderin-volvingprogressivedamagetothewhitematterofthecentralandperipheralnervoussystems(reviewedin[1]).
Thediseaseiscausedbydeficiencyoftheenzymegalac-tocerebrosidase(GALC),whichleadstoaninabilitytodegradegalactolipidsfoundmainlyinthemyelinsheath[2].
Symptomsincludespasticity,irritability,anddevel-opmentaldelayandregression,whichprogresstoaseveredecerebrateconditionwithnovoluntarymove-ments[3].
Whiletheageofonsetandprogressionofthediseaseisvariable,85-90%ofaffectedindividualsde-velopsymptomsinthefirstsixmonthsoflife,withamediansurvivalof17months[1,4].
Deathisoftenduetorespiratoryinfectionsorcerebralhyperpyrexia.
Lateinfantile-andjuvenile-onsetformswithlongersurvivalperiodsarealsoseen,andadultsmaypresentwithweak-ness,lossofmanualdexterity,andparesthesiaintheirextremities[1].
TheprevalenceofKrabbediseaseisap-proximatelyonein100,000intheUSandEurope[5]withhigherfrequenciesintheDruzeandMuslimArabsinIsrael[6].
MutationsintheGALCgene(14q31)causeKrabbedisease[7],andnumerousnonsense,missense,smallin-sertion,andsmalldeletionmutationsspanningtheen-tirelengthoftheGALCgenehavebeendescribed[3].
Themostcommonmutation,consistingofapproxi-mately40%ofallelesfromaffectedindividualswithEuropeanancestryand35%ofallelesfromthosewithMexicanancestry,isa30-kbdeletionbeginninginin-tron10andextendingninekbbeyondthepolyadenyla-tionsignal[3,8,9].
The30-kbdeletionmutationresultsintheclassicinfantileformofthediseaseinthe*Correspondence:mhegde@emory.
edu1EmoryGeneticsLaboratory,DepartmentofHumanGenetics,EmoryUniversity,Atlanta,GA,USAFulllistofauthorinformationisavailableattheendofthearticle2012Tanneretal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Tanneretal.
OrphanetJournalofRareDiseases2012,7:38http://www.
ojrd.
com/content/7/1/38homozygousstateorwhenintrans(onoppositechro-mosomes)withanothermutationassociatedwithseveredisease[3].
TherearefewreportsofanyotherlargedeletionsorlargeduplicationsencompassingtheGALCgene.
Thetechniquescurrentlyavailablefordetectingsingle-andmulti-exondeletionsandduplicationsincludemultiplexPCR,quantitativePCR,Southernblotting,multiplexligation-dependentprobeamplification(MLPA),detec-tionofvirtuallyallmutations-SSCP(DOVAM-S),andsingleconditionamplification/internalprimersequen-cing(SCAIP).
Duetothedifficultyandcomplexityoftestdevelopmenttoroutinelyandreliablydetectdosagedifferences,clinicallaboratorieshavenotofferedthistesting,andthereforedataonthepresenceofthesetypesofmutationsarelacking.
Tofurtherexplorethedeletion/duplicationmutationspectrumoftheGALCgene,wehavedevelopedacom-prehensivemutationdetectionstrategywhichbeginswithsequenceanalysisoftheGALCgeneincombinationwithmutation-specifictestingforthe30-kbdeletion.
Iftwomutationsarenotfoundusingthisapproach,des-piteanestablishedbiochemicaldiagnosisforthepatient,thistestingisfollowedbytargetedarraycomparativegenomichybridization(CGH)tolookforcopynumberchangeswithintheGALCgene.
Herewepresentmuta-tionidentificationstatisticsforGALCanalysisatEmoryGeneticsLaboratory(EGL);wealsoidentifytwonovelGALCdeletionsanddescribetheonlylargeGALCdupli-cationreportedinanindividualwithKrabbedisease.
ResultsEmoryGeneticsLaboratory(EGL)startedofferingarrayCGH-baseddeletion/duplicationtestingfortheGALCgeneinJanuaryof2008.
FromJanuaryof2008throughAugustof2011,approximately100samplesweresub-mittedtoEGLforfullGALCgeneanalysis.
Therewere33casesinwhichweperformedcompleteGALCgeneanalysis(sequenceanalysiswith30-kbdeletionanalysisfollowedbyarrayCGHdeletion/duplicationanalysis,ifnecessary).
Thesecasesincludedsamplesfromindivi-dualsreportedtobeenzymaticallyorclinicallyaffectedbyKrabbedisease,andalsopairedparentalsamplesfromcasesinwhichtheaffectedindividualwasdeceased.
Theremainderofthesampleswerenotin-formativeforourstudyandincludedsamplesforwhichnoclinicalinformationwassubmitted,samplessentforcarriertestinginindividualswithafamilyhistoryofKrabbediseaseinwhichthefamilialmutationwasnotknown,andsamplessentforcomprehensivecarriertest-ingfromindividualswithpartnersknowntobecarriersforKrabbedisease.
Analysisbeganwithsequencingofthe17exonsandflankingintronicregionsoftheGALCgene,alongwithallele-specificPCRanalysisforthecommon30-kbdeletion.
Ifamutation(s)wasnotfound,theorderingphysicianhadtheoptionofreflextestingwitharrayCGHtodetectsingleandmultipleexondele-tionsandduplications.
ResultsofGALCanalysisforthe33casesofconfirmedKrabbediseasearegivenintable1.
In28ofthe33cases(84.
8%),bothmutationswereidentifiedviasequenceanalysisincombinationwithanalysisforthe30-kbdele-tion.
Sixteenofthesecaseshadtwomutationsidentifiedthroughsequenceanalysis,eighthadonemutationiden-tifiedthroughsequenceanalysisandonecopyofthe30-kbdeletion,andfourhadahomozygous30-kbdeletion.
Inanadditionalthreeofthe33cases(9.
1%),sequenceanalysiswithanalysisofthe30-kbdeletionidentifiedonemutation,whilereflexdeletion/duplicationtestingidentifiedasecondmutation;thesecasesarediscussedinmoredetailbelowascases1-3.
Theoveralldetectionratefortwomutationsusingacombinationofsequenceanalysisandarray-CGHdeletion/duplicationtestingwastherefore31/33(93.
9%).
Intwoofthe33cases(6.
1%),comprehensiveGALCmutationanalysis,includingsequenceanalysis,30-kbde-letionanalysis,anddeletion/duplicationanalysis,failedtoidentifytwoknownmutations.
Whenonlyonemuta-tionisidentifiedinindividualswithabiochemicaldiag-nosisofarecessivedisease,itisEGL'scustomarypracticetodesignasecondsetofPCRprimersfortheentiregeneinvolved.
ResequencingoftheGALCgenewithanalternativeprimersetalsofailedtoidentifyasecondmutationintheseindividuals,reducingtheprob-abilityofalleledropout.
Inoneofthetwocases,theprobandwasdeceasedbutreportedlyhadclinicalfea-turesconsistentwithKrabbediseaseandanenzymaticdiagnosisofKrabbediseasefromanotherlaboratory(la-boratoryreportsoftheenzymeanalysiswerenotpro-videdtoEGL).
Sequenceanalysiswith30-kbdeletionanalysisoftheparentsidentifiedonecopyofthe30-kbdeletioninthemother,butwasnegativeinthefather.
Reflexdeletion/duplicationtestinginthefatherwasalsoTable1GALCMutationsIdentifiedin33BiochemicallyConfirmedKrabbeDiseasePatientsatEmoryGeneticsLaboratoryMutationCategory#ofPatients%ofPatientsTwopointmutations1616/33=48.
5%Onepointmutationandone30-kbdeletion88/33=24.
2%Two30-kbdeletions44/33=12.
1%Oneknownmutationandonelargedeletionorduplication33/33=9.
1%OneknownmutationandnegativearrayCGH22/33=6.
1%Tanneretal.
OrphanetJournalofRareDiseases2012,7:38Page2of9http://www.
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com/content/7/1/38negative.
Inthesecondcase,theprobandhadabio-chemicaldiagnosisofKrabbediseasefromanotherla-boratory;nootherinformationontheprobandwasprovided.
Sequenceanalysiswith30-kbdeletionanalysisidentifiedonemissensemutationandoneintronicvari-antofunknownclinicalsignificance(c.
909-10A>G;IVS8-10A>G)intheaffectedindividual.
Reflexdele-tion/duplicationtestingwasnegative.
Testingthepar-entsofthesecondindividualwasrecommendedtoaidininterpretationoftheunknownvariant,buthasnotbeenorderedatEGLtodate.
Case1Case1wasaneight-month-oldCaucasianmalereportedtohavedeficientGALCenzymeactivity.
Sequenceana-lysiswith30-kbdeletionanalysisidentifiedonecopyofafive-bpdeletioninexon16oftheGALCgene.
Asecondmutationwasnotidentified.
Aftertheaffectedindividualpassedaway,samplesfromtheparentsweresenttoEGLfortesting.
Theindividual'smotherwasfoundtocarrythefive-bpdeletion.
Array-CGHdeletion/duplicationtestingoftheindividual'sfatherdetecteda6.
9-kbdele-tionofexon8(seefigure1).
Case2Case2wasaone-year-oldCaucasianfemalereportedtohavedeficientGALCenzymeactivitybybloodassayandbyfibroblastassay,andclinicalfeaturesconsistentwithKrabbedisease.
Sequenceanalysiswith30-kbdeletionanalysisidentifiedonecopyofthe30-kbdeletioninthisindividual.
Asecondmutationwasnotidentified.
Test-ingoftheparentsindicatedthatthechild'smotherwasthecarrierofthe30-kbdeletion.
Array-CGHdeletion/duplicationanalysisoftheindividual'sfatherrevealedanovel11-kbduplicationencompassingexons11through14.
Subsequentdeletion/duplicationtestingoftheaffectedindividualrevealedacomplexcopynumberpat-ternintheGALCgeneresultingfromtheoverlappingcombinationofthe30-kbdeletion,whichextendsfromexons11throughtheendofthegene,andaduplicationofexons11through14(seefigure2).
Inthisindividual,thecombinationofadeletionandaduplicationresultedinanarrayresultwithnormalcopynumberforexons11through14,whileexons15throughtheendofthegeneweredeleted.
Intheabsenceofparentaltesting,thiscombinedsetofcopynumberchangeswouldhavebeendifficulttodetectandinterpret.
Thisindividualwasconfirmedtobethesameindividualreportedin[10]andistheonlyreportofalargeduplicationintheGALCgenetodate.
Case3Case3wasa17-month-oldEastIndianfemale.
TestinginanotherlaboratoryindicateddeficientGALCactivityandahomozygoussinglebasepairdeletioninexon1oftheGALCgene.
Testingofthisindividual'sparentsindi-catedthatthemotherwasacarrierofthesinglebasepairdeletion,buttestingforthemutationinthefatherwasnegative.
SamplesweresubmittedtoEGLforarray-CGHdeletion/duplicationtestingfortheaffectedindi-vidualandherfather.
Thistestingrevealedadeletionofexons1through6inbothindividuals(seefigure3).
Thedeletionthattheaffectedindividualinheritedfromherfathercausedthemutationinheritedfromhermothertoappearhomozygousbysequenceanalysis.
DiscussionArrayCGHiscurrentlybeingusedsuccessfullyinmanymolecularcytogeneticlaboratoriestodetectgrossaltera-tionsinthehumangenome.
Mostcytogeneticarrays,however,arenotdesignedtodetectsmall(intragenic)deletionsandduplications.
Thedevelopmentofaclinicaltestfordetectingintrageniccopynumberchangeshasthepotentialtoraisethemutationdetectionratefortesteddiseases,therebyimprovingmoleculardiagnosisofaffectedindividuals.
Gene-targetedarrayCGHoffersapowerfulalternativetothecurrentmethodsusedfordetectingthesemutations.
WehaveadaptedarrayCGHtechnologyandsuccess-fullyshownthatitcandetectintragenicdeletionsandduplicationsinalargesetofgenes,includingGALC,usingasinglearray[11].
AstudybyAradhyaetal.
demonstratedthatgene-targetedarrayCGHwasabletoidentifypartialorwholegenedeletionsandduplicationsinapproximately5%ofaclinicalcohortsenttoadiag-nosticlaboratoryfortesting[12].
Whenbrokendownbymodeofinheritance,thepositiverateapproximately5%forautosomaldominantgenes,approximately10%forautosomalrecessivegenes,andapproximately3.
5%forX-linkedgenes.
TheyconcludethatintrageniccopynumbermutationsaremoreprevalentthatpreviouslysuspectedinMendeliandisordersandshouldbepartoftheirroutinediagnosticworkup.
Inaddition,astudybyWangetal.
reporteda3%positiveratewhenusinggenetargetedarrayCGHtoidentifydeletionsandduplicationinamitochondrialandmetabolicpatientcohort,andconcludethatgenetargetedarrayCGHisusefulasacomplementarydiagnostictestforgenesequenceana-lysis[13].
Thecasereportspresentedheredemonstratetheutil-ityofgene-targetedarrayCGHinclinicalmoleculardiagnosticstoimproveandclarifymutationdetection,aswellastoidentifycopynumbermutationsthatother-wisewouldhavebeenmissedbyconventionalanalysis.
Ascase3illustrates,apparentlyhomozygousmutationsinaffectedindividualsshouldbeverifiedbytestingparentsforthemutationand/orperformingdeletion/duplicationanalysisontheaffectedindividualtoruleTanneretal.
OrphanetJournalofRareDiseases2012,7:38Page3of9http://www.
ojrd.
com/content/7/1/38outthepresenceofonecopyofthemutationononealleleandadeletionontheotherallele.
Ofthe11individualsinthisstudywithapparentlyhomozygousmutationsidenti-fiedbysequenceanalysiswith30-kbdeletionanalysis(sevenpointmutationsandfour30-kbdeletions),parentalsamplesweresubmittedononlythree;inallthreecases,theparentswereshowntoeachcarryonecopyofthenowverifiedhomozygousmutationidentifiedinthechild.
Withoutparentaltestingontheothereightcases,itisnotpossibletoruleoutthepresenceofadeletion,possiblyleadingtoanunderrepresentationofthefrequencyofnoveldeletionsintheGALCgene.
Inaddition,thenovel11-kbduplicationofexons11-14incase2wouldnothavebeendetectedbytheuseoftraditionalmethodologies.
Thisduplicationnormalizesthecopynumberintheprobandfromexons11-14,inspiteofthefactthattheprobandcarriesthecommon30-kbdeletionontheotherallele.
Thedetectionofade-letionandduplicationintransinthesamegenewithpartiallyoverlappingexonsclearlyprovedthepowerofaCGHtodetectgene-targeteddeletionsandduplica-tions.
Thebetterablewearetodetectmutations,themoreoptionstherewillbeformolecularprenataltestingandcarriertestinginfamilymembers.
ConclusionsAnalgorithmformoleculartestingforKrabbediseaseisgiveninfigure4.
First,geneticcounsellingandmolecu-lartestingforKrabbediseasecanbeofferedtoindivi-dualswithclinicalsymptoms,lowlevelsofGALCFigure1Case1.
A.
Pedigreeoftheprobandandparents.
Afilledsymbolindicatestheaffectedindividual;symbolswithadotinthemiddleindicatecarriers.
GALCgenotypesaregivenforeachindividualundertherespectivesymbol.
Sequenceanalysiswith30-kbdeletionanalysisidentifiedonecopyofafive-bpdeletioninexon16intheprobandandhismother.
Asecondmutationwasnotidentified.
ArrayCGHanalysiswasperformedontheproband'sfathertoidentifythesecondmutation.
B.
GALCarrayCGHresultsforthefather.
AdiagramoftheGALCgeneisgivenabovetheresultswithexonnumbersindicated.
Thefathercarriesadeletionofexon8oftheGALCgene.
(Thepresenceofthedeletionofexon8intheprobandisinferred,astheprobandpassedawaybeforedeletion/duplicationanalysiswasperformed).
Tanneretal.
OrphanetJournalofRareDiseases2012,7:38Page4of9http://www.
ojrd.
com/content/7/1/38enzyme,orpositivenewbornscreenresults.
InitialtestingshouldbeginwithGALCgenesequenceanalysis,alongwithallele-specificPCRforthe30-kbdeletion,asthesemethodshavethehighestdetectionrateandarecosteffective.
Ifthesemethodsdonotidentifytwoknownmutations,GALCarrayCGHdeletion/duplicationanalysisshouldbeoffered.
Iftwomutationsareidentified,prenataltestingcanbeofferedafterparentaltestingtoconfirmthatthetwomutationsareintrans.
Sincesequenceana-lysisalonecannotdistinguishahomozygousmutationfromasequencemutationthatliesintranswithadele-tioninthesamegene,mutation-specifictestingofbothparentsofindividualswithanapparentlyhomozygousmutationisespeciallyimportant.
Parentaltestingshouldbefollowedbydeletion/duplicationtestinginthosecasesinwhichoneparenttestsnegativeforthemuta-tion.
Oncetwomutationshavebeenidentifiedandconfirmedinparentalsamples,testingbyknownmu-tationanalysiswouldalsobepossibleforotheraffectedfamilymembersorascarriertestinginadultfamilymembers.
Thisstudyof33KrabbecasesrevealsthatarrayCGHdeletion/duplicationanalysisoftheGALCgeneincreasedtherateofdetectionoftwomutationsfrom84.
8%,forsequenceanalysiswith30-kbdeletionanalysis,upto93.
9%,andenabledustouncoverthreepreviouslyun-reportedcopynumbermutations.
Inadditiontothe33casespresentedhere,therewerealsothreeotherFigure2Case2.
A.
Pedigreeoftheprobandandparents.
Afilledsymbolindicatestheaffectedindividual;symbolswithadotinthemiddleindicatecarriers.
GALCgenotypesaregivenforeachindividualundertherespectivesymbol.
Sequenceanalysiswith30-kbdeletionanalysisidentifiedonecopyofthe30-kbdeletionintheprobandandhermother.
Asecondmutationwasnotidentified.
ArrayCGHanalysiswasperformedontheproband'sfathertoidentifythesecondmutation,whichwasconfirmedintheproband.
B.
GALCarrayCGHresultsforthefather(top),mother(middle),andproband(bottom).
AdiagramoftheGALCgeneisgivenabovetheresultswithexonnumbersindicated.
Thefathercarriesaduplicationofexons11through14,whilethemothercarriesthe30-kbdeletionofexons11through17.
Thecombinationoftheduplicationanddeletionintheprobandyieldsaneutralcopynumberforexons11through14(boxedregion),sinceshehastwocopiesofthoseexons(bothfromherfatherandnonefromhermother),whileexons15through17aredeleted(presentinonlyonecopyfromherfather).
Tanneretal.
OrphanetJournalofRareDiseases2012,7:38Page5of9http://www.
ojrd.
com/content/7/1/38casesforwhichsequenceanalysiswith30-kbdeletionanalysisfailedtoidentifytwoknownmutations,butforwhichreflexdeletion/duplicationtestingwasnotordered.
Inonecase,onlyonemutationwasdetectedthoughsequenceanalysis,whereasintheothertwocases,oneknownmutationandonevariantofunknownclinicalsignificanceweredetectedthroughsequenceanalysis.
Inallthreecases,deletion/duplicationanalysisandparentaltestingwererecommendedinthehopethatasecondmutationcouldbeidentifiedorthesignifi-canceofthevariantsmightbeclarified;noneofthistest-ing,however,hasbeenorderedatEGLtodate.
Ultimately,furtherGALCarrayCGHdeletion/duplicationtestinginclinicallaboratorieswilllikelyrefinethesestatisticsandidentifyothernovelmutations.
MethodsSequencingOligonucleotideprimersweredesignedtoamplifythe17codingexonsoftheGALCgenein16fragments.
(Oligo-nucleotidesequencesareavailablefromtheauthorsuponrequest.
)PCRproductswereanalyzedona2%agarosegel,afterwhichtheremainderofthePCRprod-uctwaspurifiedusingaMilliporeUltrafiltrationPCRpurificationkit(Millipore,Billerica,MA).
Sequencingreactionswerepreparedina10-ulreactionvolumeusingFigure3Case3.
A.
Pedigreeoftheprobandandparents.
Afilledsymbolindicatestheaffectedindividual;symbolswithadotinthemiddleindicatecarriers.
GALCgenotypesaregivenforeachindividualundertherespectivesymbol.
Sequenceanalysisperformedinanotherlaboratoryidentifiedanapparentlyhomozygousonenucleotidedeletionmutationintheproband.
Onecopyofthemutationwasidentifiedintheproband'smother,butherfatherwasnegative.
ArrayCGHanalysiswasperformedontheproband'sfathertoidentifyasuspecteddeletion,whichwasconfirmedintheproband.
B.
GALCarrayCGHresultsforthefather(top)andproband(bottom).
AdiagramoftheGALCgeneisgivenabovetheresultswithexonnumbersindicated.
Boththefatherandtheprobandhaveonecopyofadeletionofexons1through6.
Thedeletioninheritedfromherfathermadethemutationinexon1inheritedfromhermotherappearhomozygousintheprobandbysequenceanalysis.
Tanneretal.
OrphanetJournalofRareDiseases2012,7:38Page6of9http://www.
ojrd.
com/content/7/1/38theBDv3.
1Wsequencingkit(AppliedBiosystems,FosterCity,CA).
EachPCRproductwassequencedinbothdirectionsaccordingtoABIrecommendationsusinguni-versalM13sequencingprimers.
SequencedPCRpro-ductswerepurifiedusingSephadexWcleanupplatesbyEdgeBiosystems(Gaithersburg,MD)accordingtothemanufacturer'srecommendation.
Sampleswereheat-denaturedfor5minutesandloadedontoanABI3730xlsequencer.
SequencedatawereanalyzedusingMutationSurveyorWv3.
1(Softgenetics,PA)andSeqScapeW(Ap-pliedBiosystems,FosterCity,CA).
Allele-specificPCRTheallele-specificPCRassayforthe30-kbGALCdele-tionwasdesignedbyRafietal.
,1995.
Threeprimers,onesenseandtwoantisense,wereusedtoamplifythewild-typeandmutantalleles.
ThePCRproductswereanalyzedona2%agarosegel.
Awild-typealleleyieldsa615bpPCRproductwhereasamutantalleleyieldsa320bpPCRproduct.
ArrayCGHArraydesignThetargetedgenehigh-resolutionoligonucleotideCGHarraywascustomdesignedonOxfordGeneTechnolo-gies(OGT)180Kplatformtodetectdeletionsandduplicationsin175genesassociatedwithvariousgeneticdisorders.
Longoligonucleotides(~45–60mer)wereusedtodesignthearray,withrepeatsequencemaskingimplementedtoensuregreatersensitivityandspecificity.
TheGALCgenewascoveredby431probeswith116probescoveringthe17exonsatanaveragespacingof15bpbetweenprobes.
Theintronicregionwascoveredby315probesatanaveragespacingof25bpbetweenprobes.
Useofintronicoligonucleotideprobesallowsustodetectdosagechangeswithintheentiregenomicre-gionofthegeneanddeterminetheapproximatebreakpoints.
ExperimentalsetupDNAextractionwasperformedonpatientDNAusingaGentraPuregeneDNAextractionkitaccordingtothemanufacturer'sinstructions.
Maleandfemalewild-typecontrolDNAwasobtainedfromPromega,Inc.
Eachpa-tientandreferenceDNAsamplewassonicated,suchthattheDNAfragmentsizewasbetween200-5,000basesandverifiedona1%agarosegel.
Patientandrefer-enceDNAsampleswerelabeledusingKlenowenzyme(NEB)andCy3orCy59merwobbleprimers(TriLinkTechnologies),respectively.
Afterlabeling,eachsamplewaspurifiedbyisopropanolprecipitationandreconsti-tutedinultra-purewater.
Wecombined4ugeachofla-beledpatientandreferenceDNA,andtheproductsweredesiccatedinavacufuge(SavantDNA120),thenFigure4AlgorithmfortestingforKrabbediseaseinaffectedindividuals.
Tanneretal.
OrphanetJournalofRareDiseases2012,7:38Page7of9http://www.
ojrd.
com/content/7/1/38resuspendedinappropriatehybridizationbufferalongwithCy3andCy5controlCPK650meroligonucleotides.
ThismixturewashybridizedtoaNimbleGentargetedgeneCGHarrayfor16-20hoursat42°CinaMauiHybridizationsystem(BioMicroSystems).
Thearrayhas389,587uniquesequenceprobeswithanaveragespacingof10-bpwithincodingregionsand25-bpwithinin-tronicregions,allowingfordetectionofcopynumberchangesandbreakpointsassmallas100-bpwithintheentirecodingregion.
Arrayswerethenwashedaccordingtothemanufacturer'srecommendationandimmediatelyscannedonaGenePix4000scanner(MolecularDevices).
Afterscanning,datawereextractedfromimages,andwithin-arraynormalizationwasaccomplishedusingmanufacturer-providedsoftware(NimbleScan).
Normal-izedlog(2)ratiodatawereanalyzedusingtwodifferentanalysisprograms:SegMNTandDNAcopyNimbleScan(NimbleGenSystems,Inc.
).
Bothsoftwareprogramsre-portbreakpointsforpredicteddeletionsorduplicationsinthepatientortestsamplerelativetothereferenceandalsodisplayresultsinabargraphwherethey-axisindicatesgainorlossofmaterial(1=gain,0=normal,-1=loss),whilethex-axisindicatesthepositionofeachfeatureonthechromosome.
Datafiles(.
gff)generatedfromdifferentaveragingwindowsusingNimbleScansoftwarewereparsedusingacustomprogram(Nimkit)thatwasdevelopedin-house.
Nimkitenablesthelaboratorytoselectandanalyzeonlythegeneofinterest.
Nimkitgeneratesagene-specificreportsummarizingbreakpointsdetectedinthegeneofinterest,therespectivelog(2)ratios,andtheexonspresentateachregion.
AllotherregionsaremaskedandnotanalyzedbyNimkit,preventinggeneticanalysisofgenesforwhichclinicaltestingwasnotrequested,incompliancewithHIPAArequirements.
Arrayqualitywasassessedbycontrolresequencingoli-gonucleotidesoneacharraythatcorrespondtosyntheticsequencesdesignedtohavenocross-hybridizationpo-tentialtoanyknownsequence.
Thissequencewasdesignedtohavethreedistinctsequencingdomainswithdifferentcharacteristics:A,B,andCdomains.
Resequen-cingwasperformedonboththeforwardandreversestrands,sothattheresequencingreporthassixdifferentscoresfortheCy3channelandsixdistinctscoresfortheCy5channel:A-forwardandA-reverse,B-forwardandB-reverse,C-forwardandC-reverse.
The"A"domaincontainedlongrunsofGnucleotidesthatcanbedifficulttosynthesize.
The"B"domaincon-tainedalargeperfecthairpinsequence.
The"C"domaincontainedastraightforwarddomainthatshouldalwaysresequence.
Failureofdomain"C"indicatedacata-strophicfailure.
ControlDNAwasspikedintoeachex-perimentforbothCGHandresequencingarrays.
Ascorefrom0-100%wasobtainedthatindicatedthesequencefidelityandcorrelatedwellwiththeoverallperformanceofamicroarrayexperiment[14].
AbbreviationsCGH:Comparativegenomichybridization;GALC:Galactocerebrosidase;MLPA:Multiplexligation-dependentprobeamplification;DOVAM-S:Detectionofvirtuallyallmutations-SSCP;SCAIP:Singleconditionamplification/internalprimersequencing;EGL:EmoryGeneticsLaboratory.
CompetinginterestsTheauthorsdeclarenocompetinginterests.
AcknowledgementsTheauthorswouldliketothankDr.
KathrynGarberforeditorialassistance.
Authordetails1EmoryGeneticsLaboratory,DepartmentofHumanGenetics,EmoryUniversity,Atlanta,GA,USA.
2HunterJamesKellyResearchInstitute,DepartmentofNeurology,SchoolofMedicine,StateUniversityofNewYorkatBuffalo,Buffalo,NY,USA.
Authors'contributionsAKTparticipatedindataanalysisanddraftedthemanuscript.
ELHCparticipatedinperformingthesequenceanalysisandarrayCGHanalysis.
PKDcollaboratedonthecaseofthelargeGALCgeneduplication.
MHconceivedofthestudy,andparticipatedinitsdesignandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
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