offspringwww.hhh258

GeneticAnalysesoftheMouseDeafnessMutationsVaritint-Waddler(Va)andJerker(Espnje)HUNGJ.
KIM,TORRANCEJACKSON,ANDKONRADNOBEN–TRAUTHSectiononNeurogenetics,LaboratoryofMolecularBiology,NationalInstituteonDeafnessandOtherCommunicationDisorders,NationalInstitutesofHealth,Rockville,MD20850,USAReceived:26March2002;Accepted:3July2002;Onlinepublication:18September2002ABSTRACTGeneticstudiesonspontaneousmousemutantswithhearingdefectshaveprovidedimportantinsightsintothefunctionofgenesexpressedininnerearhaircells.
Herewereportonourgeneticanalysesofthedeafmutantsvaritint-waddler(Va)andjerker(Espnje).
Ahigh-resolutiongeneticmaplocalizesVaJtoa0.
14±0.
08cMregionbetweenD3Mit85andD3Mit259ondistalchromosome3.
Bycomparativemapping,thehumanorthologresidesat1p22.
3be-tweenmarkersD1S3449andD1S2252.
Tostudytheeffectofdifferentgeneticbackgroundsonthehear-ingphenotype,EspnjeandVaJwerecrossedtovariousinbredstrains.
Auditory-evokedbrainstemresponsetestsonF2progenydemonstratethatexpression,in-heritance,andpenetranceofthehearingphenotypearesolelycontrolledbythemutantallele.
TotestforageneticinteractionbetweenEspnjeandCdh23v,audi-toryfunctionwasanalyzedindoubleheterozygotes;nosignicantincreasesofthresholdsofsoundpres-surelevelswereobserved.
TheresultsestablishtheframeworkforcloningtheVageneandprovidevaluableinsightsintothegeneticsofdeafnessmuta-tionsinthemouse.
Keywords:deafness,modifyingalleles,Cdh23,Espin,jerker,varitint-waddlerINTRODUCTIONGeneticcharacteristicsoftraitssuchaspenetrance,inheritance,andexpressivityareoftencoregulated(modied)byallelessegregatinginthebackgroundstrain(forreviewseeNadeau2001).
Inthemouse,deafnessisgenerallyinheritedasasingle,autosomal,Mendeliantraitwithonlyverylittlevariationinpen-etranceandexpressivity.
However,afewcasesofmultifactorialinheritancewerereportedinwhichthepresenceofasecondgeneticfactorhasamodifyingeffectonpenetranceandinheritanceofthediseaseallele(Ikedaetal.
1999;Johnsonetal.
2001,Ngetal.
2001;Noben–Trauthetal.
1997).
Forinstance,thedominantalleleofmodier-of-tubby-hearing1(moth1),segregatinginAKR/J,rescueshearinglossinasignicantfractionoftubbyhomozygotes(Ikedaetal.
1999).
Recently,nonsynonymoussequencepoly-morphismsinthemicrotubule-associatedprotein1agene(Mtap1a)wereassociatedwithmoth1supportingpreviousevidencethattubbyplaysaroleatsynapses(Ikedaetal.
2002).
Themolecularmechanismsun-derlyinggeneticmodicationarecurrentlynotwellunderstood,butfurtherstudieswillundoubtedlyshowhowmodiersexerttheireffectsonthemutantallele.
Inaddition,identifyingandstudyingthemechanismsofmodiersmayalsohelpunderstandthegeneticcomplexityofage-related,noise-induced,oraminoglycoside-inducedhearingloss.
Varitint-waddler(Va)andjerker(je,Espnje)aretwospontaneousmutationsexhibitingdefectsinthesensoryepitheliumoftheorganofCortiandvestibuleleadingtodeafnessandcirclingbehavior.
Vaisasemidominantmutationandaroseinacrossbetween(C57black/C57brown)F1andC57black(CloudmanandBunker1945).
Vahomozygotesandheterozy-Presentaddress(HungJ.
Kim):DepartmentofOtolaryngologyandHeadandNeckSurgery,GeorgetownUniversityMedicalCenter,3800ReservoirRoadNW,Washington,CE20007-2197Correspondenceto:KonradNoben–TrauthNationalInstitutesofHealthResearchCourtRockville,Md20850.
Telephone:(301)402-4223;fax:(301)402-5354;email:nobentk@nidcd.
nih.
gov83JARO04:83–90(2002)DOI:10.
1007/s10162-002-3011-0JAROJournaloftheAssociationforResearchinOtolaryngologygotesaredeaf,circle,andexpressavariegatedanddilutecoatcolor.
Asecondallele,VaJ,aroseinacrosssegregatingforVaandexhibitsalessseverepheno-type(Lane1969,1972).
VaJhomozygotesandhet-erozygotesshownormalvestibularbehavior,arelessvariegated,butarebothdeaf.
Light-microscopic,electrophysiological,andultrastructuralstudiesoftheinnerearofVaandVaJidentieddefectsinorganofCortiandstriavascularis(CableandSteel1998;Deol1954).
Correlatingwiththecoatcolorphenotype,cochleaphysiologyandmorphologyaremorepro-nouncedinhomozygotes.
Jerkerisarecessivemutationwhichwasrstrec-ognizedbyamousefancierin1938andlaterde-scribedbyGru¨nebergasa''dancingadultfemalemouse''showingerraticcirclingbehavior,jerkingheadmovements,hyperactivity,anddeafness(Gru¨nebergetal.
1941).
Recently,aframe-shiftmu-tationintheEspingene(Espn),encodinganactinbundlingprotein,wasidentiedastheunderlyingmolecularcause(Zhengetal.
2000).
Espnlocalizestostereocilia,suggestinganorganizingroleduringfor-mationoftheactinlamentnetwork(Zhengetal.
2000).
Inhomozygotes,auditory-evokedbrainstemresponses(ABR),compoundactionpotentials,andcochleamicrophonicsareabsent(Sjo¨stromandAn-niko1992;SteelandBock1983).
Interestingly,al-thoughjerkerisinheritedasrecessiveallele,heterozygotesdevelopalateonsetABRthresholdshift(Sjo¨stromandAnniko1990).
Thispartialhear-inglossisaccompaniedbyahistopathologysimilartodefectsobservedinhomozygotes(Sjo¨stromandAn-niko1990),whichcouldbetheresultofadelayeddominanteffectofjerkerortheresultofinheritancemodicationcausedbythegeneticbackground.
Identicationandcharacterizationofgenesun-derlyingdeafnessinmicecontinuetoinuenceourunderstandingofhowhaircellsdevelopandfunction(forreviewseeSteelandKros2001).
Manyofthesegenesarebeingidentiedthroughageneticap-proachusingspontaneousmousemutantsincombi-nationwithgeneticmappingandmolecularcloningstrategies.
Inthisarticle,wereportontherststeptowardscloningtheValocusandonourre-sultsfromasystematicscreenforVaandEspnjemodiergenes.
METHODSANDMATERIALSMiceandcrossesAllmousestrainswereobtainedfromTheJacksonLaboratory,BarHarbor,ME.
AnimalcareandusewasinaccordancewithNIHguidelinesandproce-dureswereapprovedunderanimalstudyprotocol971/97.
Varitint-waddlercrossesForgeneticne-mapping,VaJheterozygouswerecrossedagainstCAST/EiandCZECHII/Ei.
Hetero-zygousF1progenywerephenotypicallyscoredat3–4weeksofagebytheabsenceofapreyer'sreexandtheirvariegatedcoatcolor.
ExpressionofthecoatcolorphenotypeintheF2intercrossvariedfromalargetosmallwhitebellyspot.
F2progenywithare-combinantchromosomeoverthecriticalregionweretestedforhearingbyABRanalyses.
Toidentifymodiers,heterozygousVaJmicewereoutcrossedandF1progenywithawhitebellyspotwereassumedtobe+/VaJandintercrossed.
VaJ/VaJand+/VaJwereclassiedaccordingtotheirvariegatedcoatcolorandtestedforhearingat12weeksofage.
JerkercrossesIngeneral,homozygousEspnjewereoutcrossedandmatingpairsofobligateheterozygoteswereestab-lishedtoproducetheF2offspring.
TogenerateCdh23v-2J/Espnjedoubleheterozygotes,Cdh23v-2Jho-mozygoteswerecrossedtoEspnjeheterozygotesandviceversa.
GenotypingbyPCRDNAwasisolatedfromtailbiopsiesthroughdigestionin50mMTris/Cl,pH8.
0,100mMEDTA,pH8.
0,100mMNaCl,1%SDS,and500lgproteinaseKat55°Covernight,followedbyphenol/chloroformex-tractionandprecipitationin0.
7volisopropanol,0.
3Msodiumacetate,pH4.
5.
DNAwasresuspendedinTE,pH8.
5,andadjustedtoaconcentrationof100ng/lL.
ForPCRamplication,100ngDNAwasmixedin500mMKCl,100mMTris/Cl,pH8.
3,2.
5mMMgCl2,0.
2lMoligonucleotides,20lMdNTP(AmershamPharmacia),and0.
02UthermostableDNApolymerase.
The10-lLreactionwasrunthroughthefollowingprogram:initialdenatur-ationfor2minat95°C,20sat94°C,20sat52°C,30sat72°Cfor49cycles;followedbya2-minexten-sionat72°C.
ForSSLPanalysesusingMITmarkers(Invitrogen),PCRproductswereseparatedbyelectro-phoresisin4%MetaPhoragarose(FMC)andvisual-izedbystaininginethidiumbromide.
ToidentifytheCdh23v-2Jallele,PCRproductswereseparatedbyelec-trophoresisin2%agarose,bandswereexcised,puri-ed(Qiagen),anddirectlysequencedusingBigDyeTMterminatorchemistry(AppliedBiosystems,FosterCity,CA)andABI377automatedsequencer(AppliedBio-systems).
Thejerkermutation(2426delG)abolishesaBsmBIrestrictionenzymerecognitionsite.
ToidentifytheEspnjeallele,weusedforwardprimerEspn-F20,5-GCACCTGCTTTGTGGGAAATC-3,andreverseprimerEspn-R19,5-GTCTGAAGTAATGGAGTGGT-84KIMETAL.
:MouseDeafnessMutationsVaandEspnjeTGACG-3,toamplifya342bpproductspanningthedeletion.
RestrictiondigestwithBsmBIcleavesthewild-typealleleina211-and131-bpproductbutleavesthemutantalleleuncut.
PCRproductswereprecipitatedin1volethanol,0.
2lLpelletpaint(Novagen),resuspendedin10lLTE,pH7.
5,di-gestedwiththeappropriaterestrictionenzyme,andseparatedona12%polyacrylamidegel,followedbystaininginethidiumbromide.
Auditory-evokedbrainstemresponseanalysesTotestmiceforauditory-evokedbrainstemresponse(ABR)thresholds,acomputer-aidedevokedpotentialsystem(IntelligentHearingSystem,Miami,FL)wasused.
TheSmart-EPv2.
21,modiedforhigh-fre-quencycapabilityandcoupledtohigh-frequencytransducers,wasusedtogeneratespecicacousticstimuliandtoamplify,measure,anddisplaytheevokedbrainstemresponsesofanesthetizedmice(concentration=0.
31mg/gbodyweight).
Subdermalneedleelectrodeswereinsertedatthevertex(active),ventrolaterallytotherightear(reference)andtheleftear(ground).
Specicacousticstimuliweredeliveredmonoaurallythrough3-cmplastictubechannelsfromthehigh-frequencytransducers.
Miceweretestedwithclickstimuliandalsowith8-,16-,and32-kHzpure-tonepipsatvaryingintensityfrom100dBsoundpressurelevels(SPL)to10dBSPL.
Acousticstimuliwerepresentedat19.
1timespersecond.
ABRthresholdsweredeterminedforeachstimulusfre-quencybyidentifyingthelowestintensityproducingareproducibleABRpatternonthecomputerscreen(atleasttwoconsistentpeaks).
Micewerekeptonaheatingpadinthesoundproofchamberduringtest-ing.
SamplesofCBA/CaJmiceweretestedperiodi-callyasreferencefornormalhearing.
RESULTSANDDISCUSSIONGeneticmapofVaJPreviouslinkageanalysesplacedVatothedistalendofchromosome3(74.
8cM)(Laneetal.
1992;Mo-FIG.
1.
GeneticmapofVaJ.
A.
Haplotypeanalysis.
Markersareindicatedontheleftandnumbersofrecombinationspertotalmeiosesaregivenontheright.
BlackboxesindicatetheVaJderivedalleleandthewhiteboxesindicatethewildtypederived(CAST/EiorCZECHII/Ei)allele.
Thefrequencyoccurrenceofeachhaplotypeisshownonthebottom.
B.
Chromosomallocation.
MarkerorderontheleftanddistancesincentiMorgan(cM)±SEontherightareshownalongthedistalendofchromosome3(cMposition71.
8–76.
2).
TheVaJcriticalintervalishighlighted.
Thehumanhomologousregionisshownontheleft.
KIMETAL.
:MouseDeafnessMutationsVaandEspnje8586KIMETAL.
:MouseDeafnessMutationsVaandEspnjebraatenetal.
1984).
WiththeultimategoalofcloningVa,weconstructedahigh-resolutiongeneticmap.
Twointersubspecicintercrosses[(B6C3Fe-a/a-Hox-a13HdVaJ/+·CAST/Ei)F1·F1]and[(B6C3Fe-a/a-Hoxa13HdVaJ/+·CZECHII/Ei)F1·F1]werees-tablishedwhichproduced445and618F2progeny,respectively.
Initially,these2126meiosesweregeno-typedwithSSLPmarkersD3Mit320andD3Mit200,whichidentied124meioticrecombinations.
TomapVaJmoreprecisely,theserecombinantswerefurtheranalyzedwithfourinterveningSSLPmarkers(Fig.
1).
MarkersD3Mit260andD3Mit85arerecombinantwithVaJanddenetheproximalendoftheintervalwitharecombinationfrequencyof2/2126(0.
09±0.
07cM).
Onthedistalend,D3Mit259recombineswithVaJwithafrequencyof1/2126(0.
05cM).
Therefore,VaJlocalizestoa0.
14±0.
08-cMminimalregionatdistalchromosome3(Fig.
1).
Givenanestimatedsizeofthemousegenomeof1400cM(Copelandetal.
1993)andaphysicaldistanceof2.
7·109bp(www.
ensembl.
org),thecriticalintervalspansacalculateddistanceofap-proximately270kb.
TheinsertsizeoftheRPCl-23bacterialarticialchromosomelibraryaverages197kb(Osoegawaetal.
2000).
Thus,the2000-meiosesVaJcrossprovidestheresolutiontodelineatethecriticalintervaltonotmorethantwooverlappingBACclonescontainingapproximatelyonetothreegenes.
Theseestimatesareingoodagreementwithdistancesobservedinpreviousgeneticandphysicalmapsinvolvinglociondifferentchromosomes(Brydaetal.
2001).
Withthegoalofidentifyingpotentialdiseaseho-mologsinhumans,theVaintervalwaslinkedtothehumanphysicalmap.
Todevelopgeneticmarkers,geneslocatednearVaJwereanalyzedforpolymor-phisms.
Onthe5-untranslatedregion(UTR)ofthePrkacbgeneencodingthebeta-catalyticsubunitofthecAMP-dependentproteinkinase(NP_035230),ase-quencelengthpolymorphismbetweentheC57BL/6Jstrainandthewild-derivedparentalstrainsCAST/EiandCZECHII/Eiwasidentied.
TypingthepanelofrecombinantswiththismarkerplacedPrkacb0.
32cMdistaltoVaJ(Fig.
1).
Onthehumanmap,PRKACBisfoundon1p22.
3(Landeretal.
2001).
Onthecom-positemouselinkagemap,thegeneencodingCys-teine-rich-protein61(Cyr61,formerlyIgfbp10)isconcordantwithD3Mit113andD3Mit85(MGD2002).
ProvidedCyr61localizescentromerictoVaJ,wepre-dictthehumanorthologofVatomapto1p22.
3be-tweenmarkersD1S3449andD1S2252.
Severalinheriteddiseaselocihavebeenlocalizedtoornear1p22,includingthedominantformofhearinglossDFNA37(Talebizadehetal.
2000;VanCampandSmith2002).
AlthoughdeafindividualsofthisfamilywerenotreportedtoexpressadditionalsymptomshomologoustotheVaphenotype,Vaisagoodcan-didategeneforDFNA37asitiswellestablishedthatdifferentmutationsinthesamelocuscanproducedifferentdiseaseoutcomes(Liuetal.
1997;Weiletal.
1995).
Inarecentreport,VawasdiscussedasacandidategeneforthemissinggeneintheDFNA2locus(GoldsteinandLalwani2002).
However,mark-ersD1S255andD1S2713,whichdeterminetheDFNA2criticalintervalinthisfamily,mapapproxi-mately50MbtelomerictothepredictedhumanhomologofVa.
Varitint-waddlermodierscreenVaJheterozygotesexpressaslightlydilutecoatcolor,asmallwhiteventralspot,andsometimesawhiteforeheadpatch.
VaJhomozygotesarealmostentirelyvariegatedonboththeventralandthedorsalside(Lane1972).
CoatcolorinVaheterozygotesismoredilutewithanentirelywhiteventralsideandalargewhiteforeheadspot(Fig.
2).
ThephenotypicbaselinewasestablishedthroughABRmeasurements.
Attwoweeksofage,VaJheterozygotesrespondedtothe8-and16-kHzstimulionlyatsoundpressurelevels(SPL)of88±2dBSPL(n=12)and94±1dBSPL(n=12),respectively.
Vaheterozygotesreturnedpo-sitivewaveformstoa8-kHzstimulusatintensitiesof98±2dBSPL(n=9).
OnlyoneofvetestedVaJhomozygotesrespondedtoa8-kHzstimulusat95dBSPL.
Noresponsecouldbeelicitedwithclickand32-kHzstimulifrom+/VaJ,+/Va,andVaJ/VaJ.
Atthreeweeksofage,thresholdsinVaJheterozygotesin-creasedto>100dBSPL(datanotshown).
Severityofhearinglossanddegreeofvariegationcorrelateswithalleletype(+/VaJ100;817±3;8>100;8>100;5VaJ/C3H27±4;922±8;6>100;818±3;8>100;29>100;5VaJ/DBA65±7;235±3;7>100;519±2;1299±1;31>100;10VaJ/BALB41±3;337±8;6>100;323±8;8>100;14>100;7VaJ/CZECH21±5;726±4;8>100;1226±6;999±5;32>100;4VaJ/B627±3;328±6;6>100;817±5;14>100;18>100;1aThreshold±standarddeviationfora16-kHzstimulusisgivenindBSPLfollowedbythenumberofanimalstested.
TABLE2JerkerphenotypeoninbredandvariousmixedbackgroundsAuditoryphenotypeaVestibularphenotypebF1F2Strain/crossAffectedTotal%affectedp>0.
001cNormalNormalAffectedJE/CZECH167674250.
0922±3;530±4;33>100;22JE/CAST171631270.
2528±3;529±5;45>100;20Je/DBA35127280.
532±6;1225±6;24>100;13Je/BALB3097310.
1817±3;526±5;25>100;12Je/A35108320.
0835±9;728±6;24>100;15Je/12923124190.
0133±3;624±4;35>100;9JE/Led44108410.
0630±6;5>100;8aThreshold±standarddeviationfora16-kHzstimulusisgivenindBSPLfollowedbythenumberofanimalstested.
bF2progeny.
cSignicancelevelofchi-squaredtest.
dN2progeny.
88KIMETAL.
:MouseDeafnessMutationsVaandEspnjeInanaveragesamplesizeof100F2progenyperoutcross,segregationofthevestibularphenotypedidnotsignicantlydeviatefromtheratioexpectedforarecessivetrait(Table2).
ABRmeasurementswerecarriedouton37–65randomlyselected12-week-oldF2progenyfromeachcross.
AllcirclingF2offspringshowedanABRpatternindistinguishablefromho-mozygotesoftheJE/Leinbredstrain,andallF2micewithanormalvestibularphenotypealsoreturnednormalthresholds(Table2).
Conversely,allanimalswithabnormalthresholdsdisplayedthecirclingbe-havior.
ThegeneticdiversitybetweentheparentalstrainsJE/Le,CAST/Ei,andCZECHII/Eiallowedustodirectlycorrelatephenotypewithgenotype.
AllF2progenyfromtheJE/CAST-jeandJE/CZECH-jein-tercrosswithvestibulardefects(n=159andn=170,respectively)werehomozygousjeandviceversa;outof20ABR-testeddeafF2progeny,allwerehomozy-gousjeandviceversa.
Insummary,vestibularandauditoryphenotypesshowednovariationintheirexpressivity,werecoexpressed,andsegregatedasre-cessivetraitsindependentofthegeneticbackgroundstested.
ThestereociliadefectinjerkerisreminiscentofthestereociliadisorganizationobservedinCdh23v-2J.
Theactin-bundlingactivityofEspnandtheassocia-tionofclassiccadherinswiththeactincytoskeletonsuggestadirectand/orindirectinteractionbetweenEspnandCdh23.
Totestsuchapossibleinteractiononthegeneticlevel,wedeterminedhearingfunctionindoubleheterozygotes(Cdh23v-2J/Espnje).
AllF1offspring(n=24)showednormalvestibularbehaviorandtherewasnostatisticallysignicantdifferenceintheABRthresholdsbetweenCdh23v-2J/+/Espnje/+andCdh23v-2J/+/Espn+/+(Table3).
Partofthisstudywasundertakentoidentifypos-siblegeneticinteractionsbetweenmutantandnor-malallelessegregatingindifferentinbredstrainsusingABRanalysesasphenotypicendpoint.
Expres-sion,inheritance,andpenetranceofhearingpheno-typeinjeandVaJarecontrolledbythemutantalleleonly.
However,ourdatadonotruleoutmodifyingeffectsonlevelsnotdifferentiatedbyABR.
Theseincludesubtleaccelerationsordelaysinpathology,pleiotropicauditoryeffects,oreffectscontrollingsecondarydownstreamevents.
Alternatively,allelesintheselectedbackgroundstrainscauseweak,nonde-tectableeffects.
Someoftheselimitationsinidenti-fyingmodiersmightbeovercomebyperforming''sensitizedscreens;''intheseassaysgeneticsofadeafnessmutationisstudiedonthebackgroundofarandomlymutagenizedgenome(Balling2001;Na-deauandFrankel2000).
Thisapproachhasthepo-tentialtoproducestrongeralleles,toallowforselectionofpronouncedeffects,toscreentowardsaturation,andtoincreaseprobabilityofpositivelyidentifyingthemodifyinglocus.
CONCLUSIONSFromourstudythefollowingconclusionscanbemade:(1)VaJmapstothedistalendofchromosome3withina0.
14-cMintervalankedbyD3Mit85andD3Mit259.
(2)ComparativemappinglocalizesthehumanorthologofVato1p22.
3betweenmarkersD1S3449andD1S2252.
(3)Expression,penetrance,andinheritanceofjeandVaJondifferentgeneticbackgroundsarecontrolledbythemutantallele.
(4)Somelimitationsinidentifyingandcharacterizingmodiersmightbeovercomebyperformingsensi-tizedscreens.
ACKNOWLEDGMENTSWewouldliketothankElizabethBryda,DorisWu,andMattKelleyfortheirhelpfulcommentsonthemanuscriptandmembersofourlaboratoryforstimulatingdiscussions.
WethankRichardPellegrinofortechnicalassistanceandMe-lissaIrby,KeithBlack,andOlgaRivasformaintainingthemousecolony.
ThisprojectwasfundedbyDIR,NIDCD,DC00056-01.
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