branchesya

HUMANMUTATIONMutationinBrief#845(2005)OnlineMUTATIONINBRIEF2005WILEY-LISS,INC.
Received12April2005;acceptedrevisedmanuscript13July2005.
NovelSequenceVariantsintheTMC1GeneinPakistaniFamilieswithAutosomalRecessiveHearingImpairmentRegieLynP.
Santos1,4,MuhammadWajid2,MohammadNasimKhan2,NathanMcArthur1,ThanhL.
Pham1,AttyaBhatti2,KwanghyukLee1,SabaIrshad2,AsifMir2,KaiYan1,MariaH.
Chahrour1,MuhammadAnsar3,WasimAhmad2andSuzanneM.
Leal1*1DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine,Houston,Texas;2DepartmentofBiologicalSciences,Quaid-I-AzamUniversity,Islamabad,Pakistan;3DepartmentofBiosciences,COMSATSInstituteofInformationTechnology,Islamabad,Pakistan;4GeneticEpidemiologyUnit,DepartmentofEpidemiologyandBiostatistics,ErasmusMedicalCentre,Rotterdam,theNetherlands*Correspondenceto:Dr.
SuzanneM.
Leal,DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine,OneBaylorPlaza,AlkekN1619.
01,Houston,Texas77030;Tel.
:1(713)798-4011;Fax:1(713)798-4373;E-mail:sleal@bcm.
tmc.
eduGrantsponsor:NationalInstitutesofHealth-NationalInstituteofDeafnessandOtherCommunicationDisorders(NIH-NIDCD)andHigherEducationCommission,Islamabad,Pakistan;Grantnumber:DC03594(NIH-NIDCD).
CommunicatedbyPaoloM.
FortinaThoughmanyhearingimpairmentgeneshavebeenidentified,onlyafewofthesegeneshavebeenscreenedinpopulationstudies.
Forthisstudy,168PakistanifamilieswithautosomalrecessivehearingimpairmentnotduetomutationsintheGJB2(Cx26)geneunderwentagenomescan.
Two-pointandmultipointparametriclinkageanalyseswerecarriedout.
Twelvefamilieshadtwo-pointormultipointLODscoresof1.
4orgreaterwithinthetransmembranecochlearexpressedgene1(TMC1)regionandweresubjectedtofurtherscreeningwithdirectDNAsequencing.
Fivenovelputativelyfunctionalnon-synonymoussequencevariants,c.
830A>G(p.
Y277C),c.
1114G>A(p.
V372M),c.
1334G>A(p.
R445H),c.
2004T>G(p.
S668R)andc.
2035G>A(p.
E679K),werefoundtosegregatewithinsevenfamilies,butwerenotobservedin234Pakistanicontrolchromosomes.
Thevariantsc.
830A>G(p.
Y277C),c.
1114G>A(p.
V372M)andc.
1334G>A(p.
R445H)occurredathighlyconservedregionsandwerepredictedtoliewithinhydrophobictransmembranedomains,whilenon-synonymousvariantsc.
2004T>G(p.
S668R)andc.
2035G>A(p.
E679K)occurredinextracellularregionsthatwerenothighlyconserved.
Thereisevidencethatthec.
2004T>G(p.
S668R)variantmayhaveoccurredataphosphorylationsite.
Onefamilyhastheknownsplicesitemutationc.
536-8T>A.
Theprevalenceofnon-syndromichearingimpairmentduetoTMC1inthisPakistanipopulationis4.
4%(95%CI:1.
9,8.
6%).
TheTMC1proteinmighthaveanimportantfunctioninK+channelsofinnerhaircells,whichwouldbeconsistentwiththehypotheticalstructureofproteindomainsinwhichsequencevariantswereidentified.
2005Wiley-Liss,Inc.
KEYWORDS:TMC1;autosomalrecessivenon-syndromichearingimpairment;Pakistan;prevalenceINTRODUCTIONDuetothehighlycomplexstructureandfunctionofthehumaninnerear,itisnotsurprisingthatsensorineuralhearingimpairment(HI)isgeneticallyheterogeneous.
Currently,37non-syndromichearingimpairment(NSHI)DOI:10.
1002/humu.
93742Santosetal.
geneshavebeenidentified,ofwhich21areassociatedwithautosomalrecessive(AR)NSHI(VanCampG,SmithRJH.
HereditaryHearingLossHomepage.
URL:http://webhost.
ua.
ac.
be/hhh/Accessed1March2005).
However,oftheARNSHIgenes,onlyafew[e.
g.
GJB2(MIM#121011),CDH23(MIM#605516)andWFS1(MIM#606201)]havebeenwellcharacterizedintermsofprevalenceandsequencevariantsfoundindifferentpopulations.
AdditionalstudiesofgenesthatareinvolvedinNSHIwithinvariouspopulationsarenecessarytobetterunderstandtheirprevalence,spectrumofsequencevariantsandpublichealthsignificance.
Inthisstudy,TMC1(MIM#606706)wasstudiedwithinthePakistanipopulation.
Atotalof168PakistanifamilieswithARNSHIthatwerenegativeforGJB2mutationsunderwenta10cMgenomescan.
Twelvefamilieshadtwo-pointormultipointLODscoresof>1.
4withinthe9q21.
13regionwhereTMC1isphysicallymapped.
ThesefamilieswereselectedforsequencingoftheTMC1gene.
ThereaftertheprevalenceofTMC1variantsinthispopulationwasestimated.
Also,bylocatingtheresiduesatpredictedtransmembranedomainsandstudyingevolutionaryconservationinmultiplesequencealignment,thepossibleeffectsontheTMC1proteinproductofbothnovelandpreviouslyreportedsequencevariantswereexamined.
MATERIALSANDMETHODSAscertainmentofstudysubjectsThestudywasapprovedbytheQuaid-I-AzamUniversityInstitutionalReviewBoardandbytheInstitutionalReviewBoardforHumanSubjectResearchforBaylorCollegeofMedicineandAffiliatedHospitals.
Informedconsentwasobtainedfromallfamilymemberswhoparticipatedinthestudy.
Forthisstudy,192unrelatedPakistanifamilieswithatleasttwoARNSHIindividualswereascertainedfromvariousregionsofPakistan.
Medicalandfamilyhistoryandinformationonpedigreestructurewasobtainedfrommultiplefamilymembers.
Puretoneaudiometryat250-8000Hzwasperformedforselectedsubjects.
Allhearing-impairedfamilymembersunderwentphysicalexamination.
Withinthesefamilies,noclinicalfeaturesthatwouldindicatethattheHIwaspartofasyndromewereobserved.
Inaddition,nogrossvestibularinvolvementwasnoted.
TheHIphenotypewasprelingual,severetoprofound,andwasnotknowntobecausedbyinflammatorymiddleeardiseaseorspecificenvironmentalfactors.
Additionally,117unrelatedhearingindividualswithoutafamilyhistoryofhearingimpairmentwereascertainedfromPakistan.
GenomescanDNAwasisolatedfromvenousbloodsamplesfollowingastandardprotocol(Grimbergetal.
,1989),quantifiedbyspectrophotometryatopticaldensity260,andstoredat-20°C.
Ofthe192families,twelvefamilieswerepositiveforGJB2(GenBankaccession#NM_004004.
3)mutations.
TwelveotherfamilieswerenotincludedforgenomescanduetoaninsufficientnumberofDNAsamplesinordertocarryoutalinkageanalysis.
DNAsamplesfrom126familiesweredilutedto40ηg/landsenttotheCenterforInheritedDiseaseResearch(CIDR;URL:http://www.
cidr.
jhmi.
edu/)forgenomescan,whiledilutedDNAsamplesfrom42familiesweresenttotheNationalHeart,LungandBloodInstitute(NHLBI)MammalianGenotypingService(CenterforMedicalGenetics,Marshfield,WI,USA;URL:http://research.
marshfieldclinic.
org/genetics/)forgenotyping.
From2002to2004,samplesweresentinsixbatches,withanaverageof395shorttandemrepeat(STR)markersspacedat~10cMapartforeachgenomescanthatwasdoneonall22autosomesandtheXandYchromosomes.
LinkageanalysisLinkageanalyseswereperformedunderafullypenetrantautosomalrecessivemodelwithadiseaseallelefrequencyof0.
001.
TheMLINKprogramoftheFASTLINKcomputerpackagewasutilizedfortwo-pointlinkageanalysis(Cottinghametal.
,1993),whilemultipointanalysiswasperformedusingALLEGRO(Gudbjartssonetal.
,2002)withmapdistancesfromtheMarshfieldgeneticmap(Bromanetal.
,1998).
SomeofthefamiliesweretoolargetoanalyzeintheirentiretyusingALLEGROandwerethereforebrokenintotwoormorebranchesfortheanalysis,thentheLODscoresfromeachbranchofthefamilyweresummed.
Markerallelefrequencieswereestimatedfromthedatabymeansofbothobservedandreconstructedgenotypesoffoundersfromeachpedigreeandtheotherpedigreesinthesamegenomescan.
TMC1VariantsinPakistaniFamilies3SequencingTMC1Polymerasechainreaction(PCR)primersweredesignedfor24exonsplus1000basepairsofthepromoterregionofTMC1(GenBankaccession#NM_138691.
2)viaPrimer3software(RozenandSkaletsky,2000).
DNAfromoneunaffectedandtwoaffectedindividualsfromeachfamilyweredilutedto5ηg/l,amplified,andpurifiedwithExoSAP-IT(USBCorp.
,Cleveland,Ohio,USA;URL:http://www.
usbweb.
com/).
SequencingwiththeappropriateprimerswasperformedwiththeBigDyeTerminatorv3.
1CycleSequencingKittogetherwithanAppliedBiosystems3700DNAAnalyzer(AppleraCorp.
,FosterCity,CA;URL:http://www.
appliedbiosystems.
com/).
SequencevariantswereidentifiedviaSequencherTMVersion4.
1.
4software(GeneCodesCorp.
,AnnArbor,MI,USA;URL:http://www.
genecodes.
com/sequencher/).
Whenasequencevariantwasfound,DNAsamplesfromtherestofthefamilyweresequencedfortheexoninwhichthevariantwasidentified.
Similarly,117controlindividualsfromPakistanwerescreenedforthesameexon.
ProteinsequenceanalysisTodeterminetheevolutionaryconservationofidentifiedsubstitutions,theExPASy(ExpertProteinAnalysisSystem;URL:http://us.
expasy.
org/)proteomicsserveroftheSwissInstituteofBioinformatics(SIB)wasusedtolookforhomologuesoftheTMC1protein.
ExPASyusestheNCBIBLASTP1.
5.
4-Paracelprogram(Altschuletal.
,1997)tosearchtheExPASy/UniProtdatabase.
ToperformtheBLASTPsearch,thedefaultsettingswereused,exceptthatthethresholdforexpectedrandommatches(E)wassettoamoreconservativevalueof10-4inordertominimizefalsepositiveresults.
Ofthe81querymatches(scorerange58-1196),onematchperspecieswaschosenforfurtheralignment.
Nineproteins,includingthehumanTMC1sequence,werethensubmittedformultiplesequencealignmentviaClustalW(Thompsonetal.
,1994)attheEuropeanBioinformaticsInstitute(EBI;URL:http://www.
ebi.
ac.
uk/clustalw/)usingdefaultsettings.
Membrane-spanningdomainsoftheTMC1proteinwerepredictedthroughtheTMHMMServerv.
2.
0(Kroghetal.
,2001).
AnindependentstudydemonstratedthatTMHMMv.
2.
0had92%truepositivepredictions,whichwerehigherthanallothertransmembraneproteinpredictionprograms(Molleretal.
,2001).
Tocheckformotifsorpatternswithintheproteinsequence,thePROSITEdatabaseofSIBandtheEuropeanMolecularBiologyLaboratory(EMBL)-EBIwasscanned(Sigristetal.
,2002).
ThewebserverPolyPhen,alsofromEMBL,wasusedtoassessthefunctionaleffectofidentifiedsubstitutions(Ramenskyetal.
,2002).
RESULTSOf168familiesthatunderwenttwo-pointand/ormultipointlinkageanalysis,twelvefamilieshadtwo-pointormultipointLODscoresof1.
4orgreaterbetweenmarkersflankingtheTMC1gene.
Table1showstheLODscoresperfamily,alongwithethno-linguisticinformationandsequencevariantsthatwerefoundtosegregatewithinfamilies.
Thesesequencevariantswerefoundinthehomozygousstateinthehearing-impairedindividualsbutnotamongunaffectedfamilymembers.
4Santosetal.
Table1.
FamiliesscreenedforTMC1mutations*FamilyPlaceoforiginLanguage2-pointLODMultipointLODSequencevariant4008DGKhan,PunjabSairiki3.
14.
0c.
2035G>A(p.
E679K)4027MianWali,NWFP-PunjabborderPunjabi2.
5;2.
6atchr.
111.
1;2.
0atchr.
11None4033Sarghoda,PunjabPunjabi2.
2;1.
2atchr.
22.
9;2.
1atchr.
2c.
1334G>A(p.
R445H)4049Bahwalpur,PunjabSairiki4.
74.
8c.
830A>G(p.
Y277C)4070Kashmir,AJKKashmiri2.
43.
0c.
2004T>G(p.
S668R)4090Chistian,PunjabPunjabi1.
4;2.
1atchr.
101.
8;-0.
6atchr.
10c.
536-8T>A4119Sadiqabad,PunjabSairiki2.
42.
6c.
1114G>A(p.
V372M)4138KotliKshmir,AJKKashmiri2.
63.
3c.
2004T>G(p.
S668R)4156Larkhana,SindSindi2.
2;2.
3atchr.
83.
1;0atchr.
8None4160Dhandi,SindSindi3.
03.
6c.
1114G>A(p.
V372M)4165TajGhar,PunjabSairiki1.
5;2.
0atchr.
172.
7;2.
8atchr.
17None4173Patoki,PunjabPunjabi1.
6;1.
7atchr.
11.
6;1.
9atchr.
1None*TMC1(GenBankaccession#NM_138691.
2).
NWFP=NorthwesternFrontierProvince;AJK=AzadJammunandKashmir.
Thetraditionalnomenclatureforc.
536-8T>AwasIVS10-8T>A.
Table2.
TMC1sequencevariantsfoundtosegregatewithinPakistanifamiliesExonNucleotidechangeAminoacidchangeDomain*EvolutionaryconservationPredictedfunctionaleffectAllelefrequenciesamongcontrolchromosomes11c.
536-8T>ASpliceacceptorN/AN/AExon11skipped0/23413c.
830A>Gp.
Y277CTM2IdenticalProbablydamaging0/23415c.
1114G>Ap.
V372MTM3ConservedBenign0/23416c.
1334G>Ap.
R445HTM4IdenticalBenign0/23421c.
2004T>Gp.
S668RECNon-conservedBenign;PKCphosphorylationsite§0/23421c.
2035G>Ap.
E679KECNon-conservedBenign0/234*TMHMMv.
2.
0predictedsixmembrane-spanningdomainsfortheTMC1protein(Kroghetal.
,2001).
TM=transmembrane;EC=extracellular;N/A=notapplicable.
HumanTMC1sequencecomparedwithTMC1-likeproteinsequencesof8specieswhichwereidentifiedviatheNCBIBLASTP(Altschuletal.
,1997)programandtheExPASy/UniProtdatabases.
Speciesinclude1mammal,1amphibian,2bonyfishes,3insectsand1nematode.
ComparisondoneviaClustalW(Thompsonetal.
,1994).
Theserineresidueatposition668andglutamicacidat679areconservedinM.
musculusTmc1andF.
rubripesTmc2-relatedproteins.
Additionally,S668isalsoconservedinA.
gambiaeTmcA-likeproteinandD.
melanogasterCG3280-PA.
PossibleeffectofaminoacidsubstitutionsontheproteinstructureandfunctionwerepredictedviathewebserverPolyPhen(Ramenskyetal.
,2002).
§Thep.
S668RsequencechangewasfoundtooccuratapossibleproteinkinaseC(PKC)phosphorylationsiteaccordingtothePROSITEdatabase(Sigristetal.
,2002).
TMC1VariantsinPakistaniFamilies5Eightfamilieswerepositiveforputativelyfunctionalsequencevariants.
Oftheidentifiedvariants,c.
830A>G(p.
Y277C),whichoccurredatahighlyconservedresidue,wasdeemedtobedamagingbecauseofachangefromahydroxyltoasulfhydrylsidechainintheaminoacidthatwaslocatedwithinthesecondtransmembrane(TM)domain(Table2).
Thec.
1114G>A(p.
V372M)andc.
1334G>A(p.
R445H)variantswerealsoathighlyconservedTMresidues,buttheaminoacidwaschangedtoanotherofthesamesubclass.
Residuesforvariantsc.
2004T>G(p.
S668R)andc.
2035G>A(p.
E679K)werelessconservedandoccurredwithintheextracellularregion.
TheselastfoursequencechangeswereconsideredbenignbythePolyphenwebsite,thoughscanningthroughPROSITEshowedthattheserineresidueatposition668maybelongtoaproteinkinaseCphosphorylationsite.
Thenucleotidechangec.
2004T>Cremovesthisputativephosphorylationsite.
Alsoc.
1114G>A(p.
V372M)andc.
2004T>G(p.
S668R)wereeachfoundtosegregatewiththeNSHIstatusintwofamilies.
Onefamilyhastheknownsplicesitemutationc.
536-8T>A(Kurimaetal.
,2002).
Allthereportedsequencevariantswerenotfoundincontrolsubjects,whowerelinguisticallymatchedforthePunjabi-andSairiki-speakingfamilies.
DuetothelimitedsamplesfromtheremoteSindiandKashmiriregions,mostofthecontrolsforthec.
2004T>GvariantarePunjabi,whileforthec.
1114G>AvariantmostofthecontrolsspeakSairiki.
Severalpolymorphismswerealsoobservedinthetwelvefamilies(Table3).
Noneofthesevariantssegregatedwithhearingimpairmentstatuswithinthefamilies.
Twoofthevariantswerenon-synonymoussubstitutions,butwerealsoconsideredbenignpolymorphismsbecausetheydidnotsegregatewithHIstatus.
Thesesubstitutionsalsooccurredatnon-conservednon-transmembraneresidues.
Asidefromc.
536-8T>A,otherTMC1sequencevariantsthatwereidentifiedinpreviousresearchwerenotobservedinthefamiliesinthisstudy,butwerealsoanalyzedintermsofevolutionaryconservationandoccurrenceattransmembranedomainsandknownmotifs(Table4).
Table3.
TMC1polymorphismsthatdonotsegregatewithinfamiliesExonNucleotideChange1g.
74G>A(-467fromtheATG)3g.
[236-67G>A(+)322A>G(-219fromtheATG)]4g.
346-11delT6c.
45C>T(synonymous)8c.
237-44G>A8c.
241G>A(p.
E81K)*10c.
[535+101A>G(+)535+106_113delCAAACAAA]14c.
1029+85T>C16c.
1404+32A>G17c.
1457T>C(p.
M486T)22c.
2208+22A>G*p.
E81KdoesnotsegregatewiththeHIstatusinthreefamilies.
Additionallyp.
E81Kisattheintracellularglutamicacid(E)-richregionandisnon-conserved.
p.
M486Twasfoundintheheterozygousstateintwohearingindividualsfromtwodifferentfamiliesbutnotamongthehearing-impaired.
Theresidueispredictedtobeintracellularandisnon-conserved.
6Santosetal.
Table4.
PreviouslypublishedsequencevariantsintheTMC1gene*ExonNucleotideChangeProteinChangeDomainEvolutionaryConservationPredictedFunctionalEffectIntron3-5IVS3_IVS5del27kbTranscriptioninitiationsiteremovedN/AN/AExons4and5deleted7c.
100C>Tp.
R34XICNon-conservedTruncatedprotein;Occursatglutamic-acidrichN-terminusregion;cAMP-andcGMP-dependentproteinkinasephosphorylationsite8c.
295delAFrameshiftN/AN/ATruncatedprotein13c.
884+1G>ASplicedonorN/AN/AExon13skipped15c.
1165C>Tp.
R389XECNon-conservedTruncatedprotein17c.
1534C>Tp.
R512XICNon-conservedTruncatedprotein19c.
1714G>A§p.
D572NICNon-conservedBenign;CaseinkinaseIIphosphorylationsite20c.
1960A>Gp.
M654VTM5Non-conservedBenign*TMC1sequencechangeswerereportedbyKurimaetal.
(2002)exceptforp.
R389Xatexon15(Meyeretal.
,2005).
Membrane-spanningdomains,evolutionaryconservationandfunctionaleffectweredeterminedasdescribedinTable2.
Becausethearticledidnotspecifytheexactpositionatwhichthisdeletionoccurred,itwasnotpossibletorenamethissequencevariantaccordingtothecurrentnomenclaturerecommendations.
Thetraditionalnomenclatureforc.
884+1G>AwasIVS13+1G>A.
§c.
1714G>A(p.
D572N)wasobservedinaNorthAmericanfamilywithdominanthearingimpairment.
TMC1VariantsinPakistaniFamilies7DISCUSSIONPutativelyfunctionalTMC1variantswereobservedineightofthe168Pakistanifamiliesthatunderwentagenomescan.
TwelveadditionalfamiliesinthestudydidnotundergoagenomescanbutareknowntohavefunctionalvariantsintheGJB2gene(Santosetal.
,2005).
Thus,whenGJB2-affectedfamilieswereincluded,theprevalenceofTMC1mutationsinthisPakistanipopulationwas4.
4%(95%CI:1.
9,8.
6).
Thisisalowerprevalenceratethanthepreviouslyreportedprevalenceof5.
4+3.
0%amongconsanguineousIndianandPakistanifamilies(Kurimaetal.
,2002).
However,thedifferenceintheseprevalenceratesisnotstatisticallysignificant.
Among12familiesinwhichtheTMC1genewassequenced,apotentiallyfunctionalvariantwasnotidentifiedinfourfamilies(Table1).
Threeofthesefamilies(4027,4165and4173)hadLODscoresofsuggestivelinkage(1.
9-2.
8)inotherchromosomalregions,whichmaycontainthegenethatcausestheHIinthesefamilies.
Infamily4156,thetwo-pointLODscorewasrelativelyhighforchromosome2,butthemultipointLODscoreatchromosome2waszero,whileamultipointLODscoreof3.
1wasobtainedintheTMC1region.
Forthisfamily,itispossiblethatthefunctionalvariant(s)arewithintheintronicregionsofTMC1,orinanothergenethatliesclosetotheTMC1gene.
ThepredictedstructureofTMC1bearssimilaritytothatoftheα-subunitofvoltage-dependentK+channels,whichhassixα-helicalTMsegmentsandintracellularNandCtermini(HanlonandWallace,2002).
ThefirstfourTMdomainsoftheK+channelα-subunitactasvoltagesensorsforactivationgating(Li-Smerinetal.
,2000),whereastheinterveningsegmentbetweenTM5andTM6appearstoconferchannelselectivity(HanlonandWallace,2002).
ThreehighlyconservedTMC1sequencevariantsinthisstudy–c.
830A>G(p.
Y277C),c.
1114G>A(p.
V372M)andc.
1334G>A(p.
R445H)–liewithinthecentralportionofhydrophobicTMsegments.
Meanwhile,theTMC1variantsc.
2004T>G(p.
S668R)andc.
2035G>A(p.
E679K)werefoundbetweenTM5andTM6amongaclusteroflessconservedresidues.
Thoughtheseaminoacidresidueswerenon-conservedwhenhumanTMC1wasalignedwitheightproteinsfromotherspecies,alignmentwithotherhumanandmurineTMCproteinsshowsthattheseresiduesareconservedformembersoftheTMCsubfamilyA,TMC1,TMC2andTMC3(Keresztesetal.
,2003;Kurimaetal.
,2003).
Additionally,theserineresidueatposition668isaputativeproteinkinaseCphosphorylationsite.
Therefore,eventhoughthePolyphenserverlabeledfouroffivenovelsubstitutionsasbenign,webelievethatallfivevariantsarefunctional.
ItshouldbenotedthatthePolyphenserversuccessfullypredictsonly82%ofmutationsintheSwissProtproteindatabase(Ramenskyetal.
,2002).
Thedeafness(dn)mouseisthehomologousmodelforrecessiveTMC1mutations,whiletheBeethoven(Bth)mouseisidentifiedwithdominantTMC1mutationsthatcausepostlingualhearingimpairment(Kurimaetal.
,2002;Vreugdeetal.
,2002).
InthemouseTmc1expressionhasbeenfoundtolocalizetocochlearhaircells(Kurimaetal.
,2002).
Inmicroscopicsectionsofdnmousecochlea,Tmc1mutationscausedthegreatestdamagetoinnerhaircells(IHC),withprogressivedegenerationthatbeginsatpostnatalday13,whichisthetimeofonsetofhearingfunctioninmice(Vreugdeetal.
,2002;Pujoletal.
,1983).
Infunctionalstudies,thecochlearmicrophonic,whichisameasureoftheelectricpotentialthatisgeneratedbytheorganofCortiafteracousticstimulation,wasneverdetectedindnmicefrompostnatalday12onwards(SteelandBock,1980;BockandSteel,1983).
ThesestudiesfurthersupportthefindingsofbilateralprofoundprelingualhearingimpairmentduetorecessivehomozygousTMC1mutations,andimplicatetheIHCasthemainsiteofhearingdysfunction.
ItwassuggestedthatTMC1mightbeanionchannelortransporterwhichmediatesK+homeostasisintheinnerear(Keresztesetal.
,2003).
BecauseK+transportacrosshaircellmembranesrequiresveryrapidresponse,theuseofsecondmessengerorcascademechanismshasbeendeemedhighlyunlikely,andthegating-springmodelformechanoelectricaltransductionislargelyfavored(CoreyandHudspeth,1983).
Thismodelproposedthatmechanicalforcesbroughtaboutbybendingofstereociliaandtensiononthetiplinksdirectlyactivateionchannels.
IfitistruethatTMC1isanionchannelthatismainlylocalizedintheIHC,thenitmightbeinvolvedinthemostbasicauditoryprocessofhair-celltransduction.
Inconclusion,fivenovelsequencevariantsoftheTMC1geneforhearingimpairmentwereidentifiedinthisstudy.
ThesevariantswerefoundinTMC1regionsthatmightbecriticaltofunctionofK+channelsinhaircells.
InthehighlyconsanguineousPakistanipopulation,theprevalenceofputativelyfunctionalTMC1variantswasfoundtobelow.
However,thetrueimpactofgeneticdefectsinTMC1cannotbeknownuntilmorestudiesaredoneinotherpopulations.
ACKNOWLEDGMENTSWewishtothankthefamilymembersfortheirinvaluableparticipationandcooperation.
WearealsogratefultoRAHarrisforhisusefulcommentsandsuggestions.
ThisworkwasmadepossiblethroughtheNationalInstitutes8Santosetal.
ofHealth-NationalInstituteofDeafnessandOtherCommunicationDisordersgrantR01-DC03594.
GenotypingserviceswereprovidedbytheCenterforInheritedDiseaseResearch(CIDR)andtheNationalHeart,LungandBloodInstituteMammalianGenotypingService.
CIDRisfullyfundedthroughafederalcontractfromtheNationalInstitutesofHealthtoTheJohnsHopkinsUniversity,ContractNumberN01-HG-65403.
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