Braz. j. . vol.81 número3

(1)

www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Molecular

approach

of

auditory

neuropathy

夽

Magali

Aparecida

Orate

Menezes

da

Silva

a

_,

_Vânia

_Belintani

_Piatto

b,∗

_,

_Jose

_Victor

_Maniglia

a

a_Department_of_{Otorhinolaryngology}_and_Head_and_Neck_Surgery,_Faculdade_de_Medicina_de_São_José_do_Rio_Preto_(FAMERP),

SãoJosédoRioPreto,SP,Brazil

b_Department_of_Anatomy,_Faculdade_de_Medicina_de_São_José_do_Rio_Preto_(FAMERP),_São_José_do_Rio_Preto,_SP,_Brazil

Received4July2014;accepted12August2014 Availableonline30March2015

KEYWORDS Deafness; Genes;

Molecularbiology; Mutation

Abstract

Introduction:Mutationsintheotoferlingeneareresponsibleforauditoryneuropathy. Objective: Toinvestigatetheprevalenceofmutationsinthemutationsintheotoferlingenein patientswithandwithoutauditoryneuropathy.

Methods:Thisoriginalcross-sectionalcasestudyevaluated16indexcaseswithauditory neu-ropathy,13patientswithsensorineuralhearingloss,and20normal-hearingsubjects.DNAwas extractedfromperipheralbloodleukocytes,andthemutationsintheotoferlingenesiteswere amplifiedbypolymerasechainreaction/restrictionfragmentlengthpolymorphism.

Results:The16indexcasesincludednine(56%)femalesandseven(44%)males.The13deaf patientscomprisedseven(54%)malesandsix(46%)females.Amongthe20normal-hearing sub-jects, 13 (65%) weremales andseven were (35%) females. Thirteen(81%) index cases had wild-type genotype (AA) and three (19%) hadthe heterozygous AG genotype for IVS8-2A-G (intron8)mutation.The5473C-G(exon44)mutationwasfoundinaheterozygousstate(CG) inseven (44%)indexcases andnine(56%) hadthewild-typeallele(CC). Ofthesemutants, two(25%)werecompoundheterozygotesforthemutationsfoundinintron8andexon44.All patientswithsensorineuralhearinglossandnormal-hearingindividualsdidnothavemutations (100%).

Conclusion: Therearedifferencesatthemolecularlevelinpatientswithandwithoutauditory neuropathy.

夽

Pleasecitethisarticleas:daSilvaMA,PiattoVB,ManigliaJV.Molecularapproachofauditoryneuropathy.BrazJOtorhinolaryngol. 2015;81:321---8.

∗_{Corresponding}_author.

E-mails:vbpiatto@gmail.com,vania.piatto@famerp.br(V.B.Piatto).

http://dx.doi.org/10.1016/j.bjorl.2015.03.005

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PALAVRAS-CHAVE Surdez;

Genes;

Biologiamolecular; Mutac¸ão

Abordagemmoleculardaneuropatiaauditiva

Resumo

Introdução:Mutaçõesnogenedaotoferlina(OTOF)sãoresponsáveispelaneuropatiaauditiva. Objetivo:InvestigaraprevalênciademutaçõesnogeneOTOFempacientescomesem neu-ropatiaauditiva.

Método: Estudodecasosemcortetransversalsendoavaliados16casosíndicecomneuropatia auditiva,13pacientescomdeficiênciaauditivasensorioneural(DASN)e20indivíduosouvintes. DNAfoiextraídodeleucócitosdosangueperiféricoeregiõesdogeneOTOFforamanalisadas pelatécnicaPCR-RFLP.

Resultados: Dos16casosíndice,9(56%)sãodogênerofemininoe7(44%)domasculino.Dos 13pacientescomDASN,7(54%)sãomasculinose6(46%)femininos.Dos20ouvintes,13(65%) sãomasculinose7(35%)femininos.Treze(81%)casosíndiceapresentamogenótiposelvagem (AA)e3(19%) ogenótipoheterozigoto AGparaamutaçãoIVS8-2A-G(intron8).A mutação 5473C-G(exon44)foiencontradaemheterozigose(CG)em7(44%)doscasosíndicee9(56%) apresentamogenótiposelvagem(CC).Destesmutantes,dois(25%)sãoheterozigotoscompostos paraasmutaçõesencontradasnointron8eexon44.OspacientescomDASNeosouvintesnão apresentammutações(100%).

Conclusão:Existemdiferenc¸as,aonívelmolecular,empacientescomesemneuropatia audi-tiva.

Introduction

AfterthemutationsinGJB2andGJB6gene,whichaccount forapproximately80%ofautosomal-recessivenonsyndromic hearing impairment (ARNSHI), mutations in the otoferlin gene(OTOF)arethemostfrequentgeneticcauseofdeafness inchildren.This typeofhearingloss (HL)is more compli-cated,consideringthatatanearlystageitmaypresentas auditoryneuropathy(AN)thatisnotdetectedbyneonatal screeningbasedonotoacousticemissiontesting.1

AuditoryneuropathyisauniquetypeofHLinwhich

tym-panogramsarenormal,theacousticreflexandthebrainstem

auditory evoked potentials (BAEP) are absent or severely

altered,andtheouterhaircellfunctionisnormal,asshown

bythe presenceof otoacoustic emissions (OAEs). Patients

withthisdisorder canshowvarieddegreesof HL,in

addi-tiontoseverespeechcomprehensionimpairment,whichis

disproportionaltothedegreeofhearingloss.1

In 2003, the term ‘‘autosomal-recessive nonsyndromic

auditory neuropathy’’ was defined, due not only to the

audiological characteristics, but also to genetic

discover-ies related to mutations in the OTOF gene, which were

associatedwiththistypeofhearingloss.2_The _OTOF_gene,

located on chromosome 2 (2p23-p22), contains 48 exons

and encodes the protein otoferlin, which is expressed in

cochlear inner hair cells and typeI vestibularhair cells.3

Since then, molecular studies have been published that

allowedtheidentificationofmutationsintheOTOFgeneand

associatedthem tothe autosomal-recessivenonsyndromic

AN.2---12

Therefore,thisstudyaimedtoinvestigatetheprevalence

ofmutationsintheOTOFgene:2416T-A(Y730X)inexon18,3

IVS8-2-A-Ginintron8/exon9,6_2485C-T_(Q829X)_in_exon_22,5

5473C-G(Pro1825Ala)inexon44,5_and_3032T-C_(Leu1011Pro)

inexon26,9 _in_patients_with_AN_and_in_patients_with

non-syndromicHL.

Methods

This cross-sectional study was carried out from April 1,

2010 until July 30, 2011; of 1230 cases of nonsyndromic

sensorineural hearing loss (NSHL) treated at the Hearing

LossOutpatient Clinicofthe institution,16patients were

selected(NPTgroup---neuropathy),ofbothgenders,withAN.

These weredeemedindex cases,accordingtothe

follow-ing inclusion criteria: normal otoscopy,diagnosis of NSHL

by pure tone audiometry, presence of otoacoustic

emis-sionsand/orcochlearmicrophonicinBAEP;absenceofBAEP

wavesorseverealterationintheirmorphology;normalMRI;

absenceof riskfactors (maternal---fetalinfections,

menin-gitis,ototoxicdrugs)orperipheralneuropathies.Exclusion

criteriaincludedadiagnosisofconductiveormixedhearing

loss,thepresenceofriskfactorsorperipheralneuropathies,

andanage>65years.

Atotalof13patientsofbothgenders(NSHLgroup)were

also selected, whohad received a diagnosis of NSHL and

whoseaudiologicaltestsdidnotmatchtheclinicalpicture

of ANandhad nomutationsin theconnexin 26and GJB6

genes.

FortheControlgroup(CGgroup),20individualsofboth

genders were selected, with no hearing complaints, with

normalotoscopyandwhowereunrelatedtothepatientsin

theNPTandNSHLgroups.

Theindexcases,patients,andcontrolswereofthesame

ethnicity,age ≤65years,withnoconsanguinity, andfrom

(3)

ThestudywasapprovedbytheEthicsCommitteeofthe

institution(EdictNo.44/2010).GenomicDNAwasextracted

fromperipheralbloodsamplesusingtheGEIllustra

extrac-tionkit--- BloodGenomicprepMiniSpinKitTM_(GE_Healthcare

UKLtd)accordingtothemanufacturer’sprotocol.

AllfivemutationsanalyzedintheOTOFgeneare

identi-fiedbytheirpositionsintheNCBIGeneBankRefSeqGene:

NG009937.1. Polymerase chain reaction/restriction

frag-mentlengthpolymorphism(PCR/RFLP)testswereusedfor

themolecularanalysis.

Each ofthefivePCRreactionswasprocessedina

tem-perature cycler (Bioer Technology®_, _Model _TC-XPG) _using

25uL final reaction volume,containingthe following: 1

---DNA(200---300ng);2---10pmolofeachprimer(forwardand

reverse); 3 --- Reagent Kit FideliTaqTM _PCR _MasterMix ₍₂_×₎

(GE HEALTHCARE®₎_using _protocol _according_to_the

manu-facturer’sinstructions.

The followingstandard cycleforthePCR reactionswas

used,differingonlyintheprimerannealingtime,whichis

specificforeachpairused:initialdenaturationat94◦_C_for

3min,35repeatedcyclesof60sat94◦ _C_for_{denaturation,}

60satX◦_C_for_primer_annealing_(for_the_2416T-A_mutation:

56◦_C;_IVS8-2A-G:₅₁◦_C;_2485C-T: ₅₈◦_C;_5473C-G_mutation:

59◦_C;_3032T-C_mutation:₅₂◦_C),_and_extension_of₂_minutes

at72◦

Cand10minat72◦

Cforthefinalextension.The

frag-mentsamplifiedbyPCRwererespectivelysubmittedtoRFLP

analysis,using5Uofthefollowingenzymes:DdeIenzyme,

BGlII,BfaI,FauIandMspI(NewEnglandBiolabs®_).

The products of the reactions (PCR/RFLP) were added

tobromophenol blueand submittedtoelectrophoresis on

2% agarose gel in Tris---borate---EDTA1× buffer containing

ethidium bromide(0.5ug/mL), after which the gels were

illuminatedwithUVlightingandphotographed.

Statisticalanalysis

Theresultswerepreviouslysubmittedtodescriptive

statis-ticstodeterminenormality.TheChi-squaredtestwasused

tocompareindependentvariableswithnormaldistribution

and the Kruskal---Wallis test for samples with non-normal

distribution, in addition to odds ratio with a confidence

intervalof95%(95%CI).Thelevelofsignificancewassetat

5%.StatisticaltestswereperformedusingGraphPadInStat

softwarerelease3.0(GraphPadSoftwareInc---SanDiego,

CA, United States). Consistency of genotype distribution

withtheHardy-Weinbergequilibriumwasassessedbyexact

tests.13

Results

Ofatotalof16indexcasesfromtheNPTgroup,nine(56%)

werefemalesandseven(44%)weremales.Agerangedfrom

8to65yearsforfemales(meanM24.2years,SD±20.1)and

formalesbetween1and38years(M14.1years,SD±15.7),

withnostatisticallysignificantdifference(p=0.29).

Ofthe13patientsintheNSHLgroup,seven(54%)were

malesandsix(46%)werefemales.Agevariedfrom7to37

yearsfor males(M22.7 years,SD±12.6)andbetween 12

and45yearsforfemales(M33.3years,SD±12.3),withno

significantdifference(p=0.15).

Regarding the 20 individuals in the CG, 13 (65%) were

malesandseven(35%) werefemales.Age rangedfrom19

to44yearsformales(M28.5years,SD±7.2)andbetween

20---35yearsfor females (M26.3 years,SD±6.3), withno

significantdifference(p=0.49).

Considering the demographicvariables among patients

inthethreegroups,therewasaprevalenceof femalesin

allthree,althoughitwasnotsignificant(p=0.49).Boththe

NSHLandCGgroups hadahigherprevalence intheyoung

adultagerange,totaling55%and95%,respectively.TheNPT

grouphadhigherprevalenceintheinfantgroup(38%).The

statisticalanalysisforthisdemographicvariablewasnot

sig-nificantbetweengroups(p=0.37).Similarly,themeanages

amongthethreegroups showednosignificant differences

(p=0.091;Table1).

Table 2 shows the distribution, in percentages, of the

genotypesfound in thethreegroups assessed for thefive

mutationsintheOTOFgene.IntheNPTgroup,13(81%)index

caseshadthewild genotype(AA)andthree(19%)hadthe

heterozygousgenotype AG for IVS8-2A-G mutation(intron

8).The5473C-Gmutation(exon44)wasfoundin

heterozy-gousform(CG)inseven(44%)oftheindexcasesandnine

(56%)hadthewildallele(CC).IntheNSHLandCGgroups,

100%ofpatientshadnoneoftheanalyzedmutationsinthe

OTOFgene.

Thus, in the NPT group, therewere eight (50%) index

caseswithmutationsin theOTOFgene,one(12.5%;F1.3)

heterozygousforthemutationIVS8-2A-G,five(62.5%;F1.2,

F4.1, FBT1, FBT3.2, and FBT4.2) compound heterozygous

for the mutation 5473C-G, and two (25%; F1.1 and F5GI)

compound heterozygous for the mutations found

respec-tivelyinintron8andexon44.Ofthenormal-hearingfamily

members,themotherofFamily1(F1.M)isacompound

het-erozygousforthemutationsanalyzedinintron8andexon44

andthemothersofFamiliesFBT3andFBT4areheterozygous

onlyforthe5473C-Gmutation(Table3).

The NPT group was assessed for the Hardy---Weinberg

equilibriuminrelation tomutationsfound inintron 8and

exon44. According to the calculations made, allelic

fre-quenciesA(wild-typeallele)andG(mutant allele)ofthe

IVS8-2A-Gmutation,fortheindexcasesoftheNPTgroup,

were A=0.91 and G=0.09. The observed genotypic

fre-quencies of the wild homozygous form (AA-13), mutant

heterozygous (AG-3), and mutant homozygous (GG-0) of

these index cases are consistent with the expected

fre-quencies for the Hardy-Weinberg equilibrium (2=0.171;

p=0.68).Regardingthe5473C-Gmutation,theallelic

fre-quenciesC(wild-typeallele)andG(mutantallele)forthe

indexcasesoftheNPTgroupwereC=0.78andG=0.22.For

this mutation,the genotypic frequencies observed in the

wildhomozygousform(CC-9),mutantheterozygous(GC-7),

and mutant homozygous (GG-0) of these index cases are

alsoin agreement withthe expected frequencies for the

Hardy---Weinbergequilibrium(2=0.125;p=0.26).

Regardingthedegree ofhearinglossfoundintheindex

casesoftheNPTgroup,three(19%)hadmild,five(31%)

mod-erate,five(31%)severe,andthree(19%)profoundhearing

loss.IntheNSHLgroup,four(31%)hadmoderate,two(15%)

severe,andseven(54%)profoundhearingloss.Therewere

nomildcasesinthisgroup.Statisticalanalysisforthe

vari-abledegreeofhearinglossshowednosignificantdifference

(4)

Table1 DistributionofdemographicvariablesofindexcasesintheNPTgroup,patientsfromtheNSHLgroup,andindividuals fromtheControlgroup.

Variables NPTgroup(n=16) NSHLgroup(n=13) Controlgroup(n=20) p

Gender n(%) n(%) n(%)

Male 7(44) 6(46) 7(35) 0.49a

Female 9(56) 7(54) 13(65)

Ageclassification n(%) n(%) n(%) 0.37a

Infant 1(06) 0(00) 0(00)

Child 6(38) 2(15) 0(00)

Adolescent 3(19) 2(15) 0(00)

Youngadult 4(25) 7(55) 19(95)

Adult 2(12) 2(15) 1(05)

Elderly 0(00) 0(00) 0(00)

Age M±SD M±SD M±SD p

Years 19.8±18.5 27.6±13.1 19.±18.5 0.091b

M±SD,mean±standarddeviation.

a_Chi-squared_test. b _{Kruskal---Wallis}_test.

Duetomutationidentificationonlyinintron8andexon

44intheOTOFgene,Table4showsthedegreeofhearingloss

intheNPTgroupindexcasesandgenotypesfoundinthese

cases,throughthemolecularanalysisoftheseregions.

Among the index cases with mutation, those withthe

severe(four; 25%)and profound (three; 19%) hearingloss

weremoreprevalentthanthosewithoutmutation(onlyone

severeHL,6%)ofthesame group,whichshowedahigher

prevalenceof mild(three; 19%) andmoderate(four;25%)

HL,withasignificantdifference(p=0.022).

Discussion

ANisahearingdisordercharacterizedbyseveredistortion

orabsenceofBAEPwithpreservedOAEs,thusindicatingthe

presenceofouterhaircellintegrity.14

AmongthepreviouslyidentifiedcausesofANare

hyper-bilirubinemia, neonatal hypoxia, or neurodegenerative

diseases. However,therehavebeen reportsof‘‘isolated’’

AN, i.e., without any risk factors, and no

physiopatho-logical mechanism has been proposed to explain these

Table2 Distribution,inpercentages,ofthegenotypesfoundinthethreegroupsassessedforthefivemutationsintheOTOF gene.

Genotype NPTgroup(n=16)n(%) NSHLgroup(n=13)n(%) Controlgroup(n=20)n(%)

2416T-AExon18

TT 16(100) 13(100) 20(100)

TA 0 0 0

AA 0 0 0

IVS_8-2A-G_Intron₈

AA 13(81) 13(100) 20(100)

AG 3(19) 0 0

GG 0 0 0

2485C-TExon22

CC 16(100) 13(100) 20(100)

CT 0 0 0

TT 0 0 0

5473C-GExon44

CC 9(56) 13(100) 20(100)

CG 7(44) 0 0

GG 0 0 0

3032T-CExon26

TT 16(100) 13(100) 20(100)

TC 0 0 0

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Table3 GenotypesfoundintheNPTgroupindexcasesamongthefivemutationsassessedintheOTOFgene.

Indexcases(NPTGroup) andnormal-hearing familymembers

2416T-A exon18

IVS8-2A-G intron8

2485C-T exon22

5473C-G exon44

3032T-C exon26

F1.P −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F1.M −/−(TT) −/+(AG) −/−(CC) −/+(CG) −/−(TT) F1.1 −/−(TT) −/+(AG) −/−(CC) −/+(CG) −/−(TT) F1.2 −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) F1.3 −/−(TT) −/+(AG) −/−(CC) −/−(CC) −/−(TT) F2.2M −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F2.3 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F2.6 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F2.11 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F3.P −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F3.M −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F3.2 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F3.3 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F4.1 −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) F5.GI −/−(TT) −/+(AG) −/−(CC) −/+(CG) −/−(TT) F5.GII −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) F6.1 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) FBT1 −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) FBT2 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) FBT3.M −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) FBT3.1 −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) FBT4.M −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) FBT4.1 −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) FBT4.2 −/−(TT) −/−(AA) −/−(CC) −/+(CG) −/−(TT) FBT5.GI −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT) FBT5.GII −/−(TT) −/−(AA) −/−(CC) −/−(CC) −/−(TT)

Underlined,indexcaseswithauditoryneuropathy(NPT).F,family;P,fatheroftheindexcase;M,motheroftheindexcase;GI,twin;I GII,twinII;−/−,wildhomozygousgenotypefortheanalyzedmutation;−/+,heterozygousgenotypefortheanalyzedmutation;−/+, heterozygousgenotypeforthemutationanalyzedinnormal-hearingfamilymember.

Table4 DegreeofhearinglossandthegenotypesfoundinIntron8andExon44oftheOTOFgene,inindexcasesofNPTgroup.

GroupNPT Degreeofhearingloss IVS8-2A-Gintron8 5473C-Gexon44

F1.1 Profound −/+(AG) −/+(CG)

F1.2 Severe −/−(AA) −/+(CG)

F1.3 Severe −/+(AG) −/−(CC)

F2.2M Mild −/−(AA) −/−(CC)

F2.3 Moderate −/−(AA) −/−(CC)

F2.6 Mild −/−(AA) −/−(CC)

F4.1 Severe −/−(AA) −/+(CG)

F5.GI Profound −/+(AG) −/+(CG)

F6.1 Severe −/−(AA) −/−(CC)

FBT1 Moderate −/−(AA) −/+(CG)

FBT2 Moderate −/−(AA) −/−(CC)

FBT3.1 Severe −/−(AA) −/+(CG)

FBT4.2 Profound −/−(AA) −/+(CG)

(6)

cases. Recent advances in genetic research has allowed a new approach to elucidate the physiopathology of this disease.15,16

Given the above, this study aimed to investigate the

prevalenceoffivemutationsin theOTOFgeneinpatients

withANdiagnosis,innormal-hearingsubjectsandinpatients

with NSHL. These were included in order to determine

whethermutationsintheOTOFgenecouldbeidentifiedin

normal-hearingsubjectsandinpatientswithNSHLwithout

mutationsinothergenesandwithoutAN.

Regardingthedemographicvariables,therewasa

preva-lenceoffemalesinthethreestudygroups,althoughitwas

notstatisticallysignificant.TheNSHLandCGgroups hada

higherprevalenceintheyoungadultagerangeandtheNPT

grouphadahigherprevalenceintheinfantagerange,but

withoutasignificantdifference.Thesedataareconsistent

withthefewpublishedreportsontheprevalenceofgender

andagerangerelatedtotheOTOFgene.17,18

Mostreports of mutations identifiedin the OTOF gene

have been described in homozygous form and in

con-sanguineous families.3,4,6,9,18---22 _Among _the _five _mutations

analyzed in the OTOF gene, only the IVS8-2A-G mutation

inintron 8and 5473C-Gmutationin exon44 werefound,

affecting 50% of the study index cases. In contrast to

the literature,all index cases were the offspring of

non-consanguineousmarriages,demonstratingthatmutationsin

theOTOF gene that areassociated withAN mayoccur in

thesecases.

Heterozygous cases in patients with AN have been

described,withorwithoutconsanguinity.2,5,8,10---12,23,24_In_the

present study, the IVS8-2A-G mutation was found in

het-erozygousstate(onemutantallele)in12.5%ofindexcases,

withthesameoccurringwiththe5473C-Gmutation,which

wasfoundin62.5%ofcases.In25%ofcases,thetwo

muta-tions identified occurred at the same time in the same

individual,which,therefore,arecalledcompound

heterozy-gotes. These two mutations, as well as the other three

analyzed, were not found in the NSHL group (100%) nor

in the CG group. These data are in agreement with the

literature.2,5,8,10---12,23,24

In 2000, the IVS8-2A-G mutationwasfirst described in

itshomozygousstate inthreebrothersfromIndia born to

consanguineous parents and in its heterozygous state in

theparentsandunaffectedsiblings,andwasnotidentified

in109normal-hearingindividuals fromthecontrolgroup.6

Therehavebeen nofurtherreportsonthismutation.The

5473C-Gmutationwasdescribedincompound

heterozygos-ityinaSpanishfamily,withthe2485C-Tmutationinexon

22,identifiedinanotherallele.5

Similarly,therehavebeennofurtherreportsofthe

afore-mentionedmutationinexon44.

The IVS8-2A-G mutation,by creatinga prematurestop

codon,andthe5473C-Gmutation,byalteringthebinding

capacitywith Ca+2 _in _the _wild-type _gene, _are _considered

pathogenicwhenintheirhomozygousstate.3,5_In_this_study,

bothwerefound incompound heterozygousstates onlyin

indexcaseswithAN(F1.1andF5.GI),whichcouldexplain

themolecularcauseofhearinglossintheseindividuals,as

theANphenotypehasbeenobserved inpatientswithtwo

mutationsaffectingallisoformsofotoferlinoronemutation

affectingthelong isoformandanother,theshortisoform.

Thismeans that anycombination of biallelic mutationsin

theOTOFgenemayresultinAN,astheyarepartiallyrelated

tothenature(truncatedor non-truncated)orthelocation

(longorshortisoform)ofthesemutations.23

However, the same mutations were also found in

compound heterozygosis in the normal-hearing mother

(F1.M)ofthecompoundheterozygousindexcase(F1.1).As

themotherhasnormalhearing,thecauseofHLintheindex

casemaybeduetootherexistingmutationsinotherexons

or other splicesites, ormore complexmutations,suchas

large deletions or other sequence rearrangements, which

requireamorespecificmolecularapproachthanusedhere.

Similarly,thesehypothesesmayalsobeappliedtoexplain

theothersixHLindexcases,astheyarecarriersofoneof

themutationsidentifiedinonlyoneofthealleles.

Brazilian references have not reported the mutations

identified in this study,10---12 _and _only _one _of _them

identi-fiedthe2485C-Tmutationinexon22withheterozygosityin

0.5%ofcasesofpatientswithnon-syndromicsensorineural

HL(1/200).10

Estimates of the prevalence of AN in deaf individuals

rangefrom1%to19%.25---28_In_Brazil,_the_prevalence_of_AN

amongallcasesofdeafness is1.2%.29 _This _range_of

varia-tionmaybeduetothefactthatdifferentpopulationswere

studied ordue todifferencesinthecriteriausedto

iden-tifypatients withthis disease.The prevalence of ANin a

population withoutriskfactorsis notwellestablished.9 _In

thepresentstudy,allthecriteriaforinclusionandexclusion

werestrictlyfollowed,thusdeterminingtheprevalenceof

1.3% of cases withAN in a population with sensorineural

hearinglossduringthestudyperiod.

Moreover,theBrazilianestimateofthemagnitudeofHL

in patients with ANis 29.6% mild, 55.5% moderate, 7.4%

severe, and7.5% profound HL.29 _In_the _present _study _19%

hadmild,31%moderate,31%severe,and19%profoundHL.

Among the indexcaseswithmutations in theOTOF gene,

wefoundastatisticallysignificantprevalenceofsevereand

profound HL compared to the mild and moderate HL in

indexcaseswithoutmutation.Theintensityofhearingloss

causedbyANcanrangefrommildtoprofound,witha

dis-proportionate decreaseinunderstandingspeechthatdoes

notcorrespondtothepure-toneaudiogram.

Thereissomehomogeneityinthedescriptionofthe

phe-notype ofpatients withmutations intheOTOF gene,that

ischaracterizedbyacongenitalorpre-lingualprofoundHL

notassociatedwithotherabnormalities,astheindexcases

studied.Therefore,ANistheonlyphenotypicexpressionof

mutationsintheOTOFgene,withhearinglossidentifiedby

therelativepreservationofouterhaircellfunction.9,30

ThepresenceofOAEsinthefirstmonthsoflifehas

impor-tantimplicationsforneonatalscreeningprograms,suchas

initialscreening.InthecaseofAN,thereisariskthatthis

losswillgoundiagnosed,for althoughthesepatientshave

severe toprofound degree ofhearing loss,theywould be

includedamongthecasesclassifiedas‘‘noalterationor

nor-mal’’ becauseOAEsreflect theauditory function inouter

haircellsandtheirpresencedoesnotexcludealterationsof

innerhaircells,insynapses,orintheauditorynerve.28

InthecontextofHLinvestigation,itisimportanttotake

into accountthe typeof AN loss,which is often

misdiag-nosed. Therefore, when assessing a child or patient with

deafness in the presence of OAEs, the hypothesis of AN

(7)

OTOFgeneresearchshouldbeperformed,astheywere

per-formedinthisstudy,allowinganaccurateetiologicdiagnosis

toprovide appropriate audiological treatment in atimely

manner.28

Potentiallimitationsregardingthestudyfindingsdeserve

consideration;despite thepositiveresults,theyshouldbe

interpreted with caution and should be corroborated by

independent, multicenter studies to determine the true

prevalenceofmutationsintheOTOFgeneandthe

associa-tionwithANintheBrazilianpopulation.

Conclusion

TheevidenceofassociationbetweenmutationsintheOTOF

gene and AN indicates that there are differences at the

molecular level in patients with andwithout this typeof

hearingloss;intheabsenceofriskfactors,thecombination

ofseveredistortionorabsenceofBAEPinthepresenceof

OAEsshoulddirectthediagnosisofANandthescreeningfor

mutationsin the OTOFgene. Moleculartests arevaluable

diagnosticcomplements,notonlyindividually,butalsoasa

screeningmethodforaneonatalorpopulationstudy.

Funding

ThisstudywassupportedbyResearchGrant(BAP-FAMERP).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Tothepatientsandtheirparents/guardianswho,by

agree-ingtoparticipateinthisresearch,willcontributetoabetter

futureforthechildrenofBrazil.Totheinstitutioninwhich

thisresearchwasperformed, byencouragingresearchand

teachingqualification.

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