Braz. j. . vol.82 número4

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www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Prevalence

of

mitochondrial

DNA

mutations

in

sporadic

patients

with

nonsyndromic

sensorineural

hearing

loss

夽

Hua

Jiang,

Jia

Chen,

Ying

Li,

Peng-Fang

Lin,

Jian-Guo

He,

Bei-Bei

Yang

∗

DepartmentofOtolaryngology,2ndAffiliatedHospital,SchoolofMedicine,ZhejiangUniversity,Zhejiang,China

Received29March2015;accepted6June2015 Availableonline5November2015

KEYWORDS

Mitochondria; rRNA; tRNA; Hearingloss

Abstract

Introduction:SeveralmitochondrialDNAmutationshavebeenreportedtobeassociatedwith nonsyndromichearinglossinseveralfamilies.However,littleisknownabouttheprevalenceof thesemutationsinsporadicpatientswithnonsyndromicsensorineuralhearingloss.

Objective: Thepurposeofourstudywastoinvestigatetheincidenceofthesemitochondrial DNAmutationsinsuchpopulation.

Methods:Atotalof178sporadicpatientswithnonsyndromicsensorineuralhearinglosswere enrolled in this study.Genomic DNA was extracted from the peripheral bloodsample. We employed the SNaPshot® _sequencing _method _to _detect _five _{mitochondrial} _DNA _mutations,

includingA1555GandA827Gin12SrRNAgeneandA7445G,7472insC,andT7511CintRNASer(UCN)

gene.Meanwhile,weusedpolymerasechainreactionandsequencedtheproducts toscreen GJB2genemutationsinpatientscarryingmitochondrialDNAmutations.

Results:WefailedtodetectthepresenceofA1555Gmutationin12SrRNAgene,andofA7445G, 7472insC,T7511CmutationsintRNASer(UCN)_gene_in_our_population._However,_we_found_that₆

patients(3.37%)werecarriersofahomozygousA827Gmutationandoneofthemalsocarried homozygousGJB2235delCmutation.

Conclusion: Ourfindingsinthepresentstudyindicatethateveninsporadicpatientswith non-syndromicsensorineuralhearingloss,mitochondrialDNAmutationsmightalsocontributetothe clinicalphenotype.

© 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

夽 _Please_cite_this_article_as:_Jiang_H,_Chen_J,_Li_Y,_Lin_P-F,_He_J-G,_Yang_B-B._Prevalence_of_{mitochondrial}_DNA_mutations_in_sporadic_patients

withnonsyndromicsensorineuralhearingloss.BrazJOtorhinolaryngol.2016;82:391---6.

∗_{Corresponding}_author.

E-mail:yangbb1959@sina.com(B.-B.Yang).

http://dx.doi.org/10.1016/j.bjorl.2015.06.006

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PALAVRAS-CHAVE

Mitocôndrias; rRNA; tRNA;

Deficiênciaauditiva

Prevalênciademutac¸õesnoDNAmitocondrialempacientesesporádicoscom deficiênciaauditivasensorioneuralnãosindrômica

Resumo

Introdução:DiversasmutaçõesdoDNAmitocondrialtemsidodescritas,emdiferentesfamílias, associadasàdeficiênciaauditivanãosindrômica.Noentanto,poucosesabesobrea prevalên-ciadessas mutações em pacientesesporádicos comdeficiência auditivasensorioneural não sindrômica.

Objetivo:A finalidade donosso estudo foi investigar aincidência dessas mutac¸ões no DNA mitocondrialnessapopulac¸ão.

Método: Nototal,178pacientesesporádicoscomdeficiênciaauditivasensorioneuralnão sin-drômicaforamrecrutadosparaparticipac¸ãonoestudo.ODNAgenômicofoiextraídodeamostra desangueperiférico.Utilizamos ométodode sequenciamentoSNaPshot® _para _detecc_¸ão_de

cincomutac¸õesdoDNAmitocondrial:A1555GeA827Gnogene12SrRNAeA7445G,7472insC eT7511CnogenetRNASer(UCN)_._{Paralelamente,}_utilizamos_a_reac_¸ão_de_polimerase_em_cadeia_e

sequenciamososprodutosparatriagemdasmutac¸õesnogeneGJB2nospacientesportadores demutac¸õesnoDNAmitocondrial.

Resultados: Emnossapopulação,nãoconseguimosdetectarapresençadamutaçãoA1555Gno gene12SrRNAenemasmutaçõesA7445G,7472insCeT7511CnogenetRNASer(UCN)_._Entretanto,

constatamosqueseispacientes(3,37%)eramportadoresdamutac¸ãohomozigotaA827G;eum delestambémportavaamutac¸ãohomozigotaGJB2235delC.

Conclusão:Nossosachadosnopresenteestudoindicamque,mesmoempacientes esporádi-coscomdeficiênciaauditivasensorioneuralnãosindrômica,asmutac¸õesdoDNAmitocondrial tambémpodemcontribuirparaofenótipoclínico.

© 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Publicado por Elsevier Editora Ltda. Este é um artigo Open Access sob uma licença CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Sensorineuralhearingloss (SNHL)is oneof themost

com-moncongenital disorders.The incidence is approximately

onein1000newbornsworldwide.1---3_Over_half_of_them_have

ageneticcausewithautosomaldominant,autosomal

reces-sive,X-linkedormitochondrialpatternofinheritance.4,5_Up

to now, more than 200 point mutations in mitochondrial

DNA(mtDNA) have been reportedin the mtDNA mutation

database MITOMAP.6 _Among _them,_several _mutations _have

beenfoundtobeassociatedwithsyndromic,nonsyndromic

andaminoglycoside-induced hearing loss,especially those

in 12SrRNAand tRNASer(UCN) _genes. _The _A1555G, _which _is

locatedinthehighlyconservedA-siteofmitochondrial12S

rRNA,wasthefirstonetobedescribedtobeassociatedwith

SNHLandfoundtoberesponsibleforboth

aminoglycoside-inducedand nonsyndromichearingloss inseveral families

worldwide.7---10 _The _A827G, _a _homoplasmic _mutation _in

mtDNA12SrRNA,wasrecentlyfoundtoberesponsiblefor

nonsyndromichearinglossinallmaternally relatedfamily

membersinaChinesefamily.11_It_also_has_been_reported_to

beassociatedwithbothaminoglycoside-ototoxicityand

non-syndromichearingloss insporadic individuals.12,13 _Besides

the12S rRNAgene, themitochondrial tRNASer(UCN) _gene _is

also associated with SNHL,as well as withnonsyndromic

deafness,asseveralmutationshavebeenidentified,

includ-ingA7445G,14,15_7472insC,16 _T7511C.17,18

Although, asmentioned above,mtDNA mutations have

been reported to be associated with nonsyndromic SNHL

in severalfamilies,little is knownabout theincidence of

these mtDNA mutations in sporadic patients with

nonsyn-dromicSNHL.Ourprevious studywasfocusedontheGJB2

and SLC26A4 mutations in patients withautosomal

reces-sivenonsyndromichearingloss(ARNHL).Consequently,we

findthatalthoughtheGJB2andSLC26A4genearethemost

commondeafnessgenes,onlyasmallpartofthesepatients

carry homozygous or compound heterozygous pathogenic

mutations.19 _That_indicates_that_other_deafness_genes_may

contributetotheclinicalphenotype.Inthisstudyweuseda

rapidmethod,SNaPshot®_sequencing,_to_screen₁₇₈_sporadic

patients with nonsyndromic SNHL to estimate the

preva-lenceofmtDNAmutationsinsuchpopulation.Moreover,to

identify the role of the GJB2 gene in the deafness

phe-notype, we also screened the GJB2 gene in the patients

carryingmtDNAmutations.

Methods

Patientsandsamples

A total of 178 sporadic patients withnonsyndromic SNHL

participatedinthisinvestigation.Theyoriginatedfrom

var-iousregionsofourprovince.Eachpatientreceivedcareful

physicalexamination,andacomprehensiveclinicalhistory

wasrecorded.Patientswithdominantfamilyhistory,a

his-tory of aminoglycoside exposure, and syndromic hearing

loss were excluded from the study. All subjects

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Table1 PCRprimersequences.

Gene PCRprimers Predictedsize

12SrRNA

A1555G F:5′_{-GCATCAAGCACGCAGCAATG-3}′ ₉₂₆_bp

A827G R:5′_{-TAGGTTTAGCTCAGAGCGGTCAAGTTA-3}′

tRNASer(UCN)

A7445G F:5′_{-CCCCCACCCTACCACACATTC-3}′ ₅₂₄_bp

7472insC R:5′_{-GGTGTACTCGTAGGTTCAGTACCATTGG-3}′

T7511C

GJB2

Exon1 F:5′_{-TGGGGAACTCATGGGGGCTCAAAG-3}′ ₄₂₅_bp

R:5′_{-AGGTTCCTGGCCGGGCAGTCC-3}′

Exon2 F:5′_{-TCAGAGAAGTCTCCCTGTTCTGTCC-3}′ ₉₁₆_bp

R:5′_{-TGAGGCCTACAGGGGTTTCAA-3}′

Table2 Extensionprimersequences.

Gene Extensionprimers

12SrRNA

A1555G R:5′_{-TTTTTTTTTTTTTAACCCCTACGCATTTATATAGAGGAG-3}′

A827G R:5′_{-TTTTTTTTTTCACGGGAAACAGCAGTGATTA-3}′

tRNASer(UCN)

A7445G 5′_{-TTTTTTTTTTTTTTTTTTTTTTTTATTCGAAGAACCCGTATACATAAAATCTAG-3}′

7472insC F:5′_{-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAGGAATCGAACCCCCCA-3}′

T7511C F:5′_{-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCCATGGCCTCCATGACTT-3}′

response (ABR), otoacoustic emissions (OAE) and tympa-nometry.The severityof hearing impairmentwas defined asnormal(<26dB),mild (26---40dB),moderate(41---70dB), severe(71---90dB)andprofound (>90dB).Peripheralblood sampleswereobtainedfromthepatientsforgenomicDNA isolation.

Informedconsentwasobtainedfromadultpatientsand fromtheparentsofchildren,andthisstudywasapprovedby localEthicsCommittee(Ethical Committeeapproval num-ber:2013---007).

SNaPshot® sequencing

Afinal20␮LPCRreactionmixturecontained1×GCBuffer

I,3.0mMMg2+_;_0.3_mM_dNTPs,₁_U_HotStarTaq_polymerase,

10ngtemplateDNA,and1␮Mprimermixture,includingtwo

pairsofprimerstoamplifyfivemutationregions(Table1).

ThePCRconditionswereasfollows:95◦_C_for₂_min;₁₁_cycles

of94◦_C_for₂₀_s,_65---0.5◦_C_per_cycle_for₄₀_s,₇₂◦_C_for₉₀_s;₂₄

cyclesof94◦_C_for₂₀_s,₅₉◦_C_for₃₀_s,₇₂◦_C_for₉₀_s;₇₂◦_C_for

2min.ForpurificationofPCRproducts,5Ushrimpalkaline

phosphatase(SAP)(Promega)and2UExonucleaseI

(Epicen-tre)wereaddedinto15␮LPCRproducts.Themixturewas

incubatedat37◦_C_for₆₀_min,_followed_by_incubation_at₇₅◦_C

for15min.

Thesinglebaseextension(SBE)wasperformedinafinal

10␮Lreactionmixture,containing5␮LSNaPshot®Multiplex

Kit(AppliedBiosystems),2␮LpurifiedmultiplexPCR

prod-uct, and 0.8␮M extension primer mixture (Table 2). The

reactionprogramwas96◦_C_for₁_min;₂₈_cycles_of₉₆◦_C_for

10s,52◦_C_for₅_s,₆₀◦_C_for₃₀_s;₄◦_C_forever._Then,_the_SBE

products were purifiedby using SAP. For sequence

analy-sis,0.5␮LpurifiedmultiplexSBEproductsweremixedwith

0.5␮LLiz120SIZESTANDARD(AppliedBiosystems)and9␮L

Hi-Di(AppliedBiosystems),anddenaturedat95◦_C_for₅_min.

Then,theproductsweresequencedbytheABI3730XLDNA

sequencer.ThedatawasanalyzedbytheGeneMapperv4.1

software(AppliedBiosystems).

GJB2mutationdetectionbypolymerasechain reaction

Two exons of GJB2 were amplified from DNA samples by

polymerasechainreaction (PCR).The primerswere listed

in Table 1. PCR amplification and subsequent purification

wereperformed aswe describedbefore.ThepurifiedPCR

productsweresequencedusingthedideoxychainterminator

methodonan ABI 3730XL DNAsequencer(Applied

Biosys-tems)withtheABIBigDyeTerminatorv3.1CycleSequencing

Kit (Applied Biosystems), according tothe manufacturer’s

protocols.TheprimerswerethesameasthoseforthePCR

amplification.

Results

The studysamples consisted of82 males and96 females.

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Table3 ClinicalandgeneticcharacteristicsofsixpatientscarryingA827GinmtDNA12SrRNA.

Patient Sex Ageofonset(years) Levelofhearingimpairment A827GinmtDNA12SrRNA MutationsinGJB2

Right Left

#61 Male 0 Profound Profound +/+ 235delC/235delC

#105 Female 0 Profound Profound +/+ WT/WT

#210 Male 0 Profound Profound +/+ WT/WT

years,with a mean age of 5.53±4.44 years.The results of audiometric testing revealed cochlear involvement in allparticipantsand thatallof them sufferedfromsevere (71---90dBtoprofound(>90dB))bilateralsensorineural hear-ingimpairment.

In all 178 sporadic patients with nonsyndromic SNHL, wefailedtodetectthepresenceoftheA7445G,7472insC, T7511CmutationsinthetRNASer(UCN)_gene._Moreover,_we_did

notdetect A1555G mutationin the 12SrRNAgene inthis population. However, the A827G variant in the 12S rRNA genewasfoundin 6patients,whoallcarriedhomozygous mutation.To illuminate the role of the GJB2 genein the phenotypicexpressionintheindividualswithmtDNA muta-tions,wescreenedtheGJB2genemutationsinthesubjects carryingA827Gmutation.Consequently, onlyone patient, whohadhomozygous235delC,carriedpathogenicmutation

intheGJB2gene(Table3).

Discussion

Most of the previously reported studies focused on the

association between the mtDNA mutations and the

pop-ulationwithaminoglycoside-induced and/ornonsyndromic

hearingloss.12,19_Several_mtDNA_mutations,_such_as_A1555G

and A827G in the 12S rRNA gene, A7445G, 7472insC and

T7511CinthetRNASer(UCN)_gene_have _been_identified_to_be

associatedwithsuchpopulations.10,13,14,19,20_However,_these

mutationsare often notsufficient to producethe clinical

phenotype.14,19 _In_patients _with_ARNHL,_similar_with_other

studies,ourpreviousstudyfoundthatabout30---40%patients

carriedpathogenicmutationsinthemostcommondeafness

genes,butnearly40%ofthesepatientscarriedheterozygous

mutation.Thus, other genemutations may together

con-tributetothephenotype.Althoughtheassociationbetween

the mtDNA mutations and nonsyndromic hearing loss has

beenidentifiedinseveralfamilies,littleisknownaboutthe

prevalence of mtDNA mutations in sporadic patients with

nonsyndromicSNHL.

Mitochondrial DNA mutations in tRNA genes can cause

tRNAmodificationand afailurein tRNAmetabolism, thus

impairingtheproteinsynthesis andreducing theATP

syn-thesis, which are considered to be the main pathogenic

factors.21---25 _The_A7445G _mutation_was_first_identified_in_a

familywithnonsyndromicdeafness.26_This_mutation_leads_to

the3′_end_{endonucleolytic}_processing_defect_in_the_L-strand

polycistronic RNA precursor.27 _{Consequently,} _the _A7445G

mutation can cause a reduction of more than 50% in the

tRNASer(UCN)_level_and_a_decrease_in_protein_synthesis,

play-ingadeterminantroleintherespiratoryphenotypeofthe

mutantcelllines.28,29 _The_7472insC_was_originally_reported

byTirantietal.,whichislikelytoalterthestructureofthe

TpsiCloopinthetRNASer(UCN)_clover_leaf_secondary

struc-ture,andhasbeenprovedtoimpairboth5′_and₃′_processing

of the tRNASer(UCN) _and _cause _a _drop _in _the _steady-state

level of the tRNASer(UCN)_.16,24,30 _The _T7511C _mutation _has

beenidentifiedtobeassociatedwithnonsyndromichearing

loss in several differentethnic families.17,18,31 _This

muta-tion can affect the processing of L-strandRNA precursor,

spanning tRNASer(UCN) _as_well _as_ND6 _mRNA, _specifically _in

the 5′ _end _of _tRNA.23,32 _As _a _result, _the_T7511C _mutation

causesareductionoftheleveloftRNASer(UCN)_and_an

impair-mentinmitochondrialproteinsynthesis.22,28_Although_these

mutationsinthetRNASer(UCN)_have_been_found_to_be

associ-atedwiththepatientswithaminoglycoside-inducedand/or

nonsyndromic hearing loss, many studies failed todetect

such mutations in those patients. In the study reported

by Xing etal., theyanalyzed the molecular

characteriza-tionof a Chinesefamilywithaminoglycoside-induced and

nonsyndromic hearing loss, but did not find the A7445G,

7472insC,andT7511CmutationsinthetRNASer(UCN) _gene.33

InanArgentineanfamilywithaminoglycoside-induced

hear-ing loss,such mutations werealsonot found.34 _Moreover,

Liet al. performed amolecular analysis in 164unrelated

Caucasian individuals with nonsyndromic hearing

impair-ment and failed to detect the presence of the A7445G,

7472insC,andT7511CmutationsinthetRNASer(UCN) _gene.12

Abreu-Silva et al. also did not find these mutations in

203unselectedBrazilianhearing-impairedpatients.35_Up_to

now, little is known about the incidence of these

muta-tions in the tRNASer(UCN) _gene _in _sporadic _patients _with

nonsyndromic SNHL. In the present study, we explored

thesemutationsinsuchpopulation,butnoneofthese

sub-jectswasfoundtocarrysuchmutationsinthetRNASer(UCN)

gene.

The A1555G mutation creates a specific GC base pair,

whichmakesthesecondarystructureofmitochondrial12S

rRNAmoreclosely resemblingthecorrespondingregionof

E. coli 16S rRNA and binding aminoglycosides with high

affinities.36 _Thus, _the _patients _carrying _this _mutation _can

suffer from SNHLafter using aminoglycosides.13 _However,

previous studies have identified that A1555G mutation in

12S rRNA is also associated with nonsyndromic hearing

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muchlowerthaninaminoglycoside-inducedhearing

impair-ment.Luetal.reportedthattheincidencesoftheA1555G

mutation were 1.43% and 10.41% in a Chinese pediatric

populationwithnonsyndromicandaminoglycoside-induced

hearing loss, respectively.19 _Similarly, _in _a _Polish

popula-tion,Rydzaniczetal.observedthattheincidencewas5.5%

and 1.6% in the cohorts withaminoglycoside-induced and

nonsyndromichearingloss.37 _In_a_Caucasian_pediatric

non-syndromic hearing loss population, the frequency of the

A1555G mutationwas0.6%.12 _In _this_study,_we _found _that

none of the subjects carriedthe A1555G mutation.

Com-paredwithprevious reports, theincidenceof theA1555G

mutationinourpopulationwithnonsyndromicSNHLis

rela-tivelylower.

The A827G mutation,like A1555G mutation,is located

at the A-site of the mitochondrial 12S rRNAgene, which

is highlyevolutionarilyconservedindifferent species.34 _It

is possible that the alteration of the tertiary or

quater-nary structure of the 12S rRNA by the A827G mutation

may lead tomitochondrial dysfunction;thereby, it would

play a role in the pathogenesis of hearing loss.11,34 _The

pathogenesisofthismutationhasbeenidentifiedina

Chi-nesefamilywithnonsyndromichearingloss.11_Although_the

A827Gmutationwasalsofoundinnormalhearingcontrols,19

morestudiesconsideritasapathogenicmutation

depend-ing on its location and absence in the controls in their

studies.11---13,34,37_Meanwhile,_these_studies_also_found

incom-plete penetrance of the A827Gmutation,which indicates

thatthismutationaloneisnotsufficienttoproduceclinical

phenotype.11,34_Thus,_some_normal_hearing_subjects_carrying

theA827Gmutationmaybeattributedtoincomplete

pen-etrance.Inourstudy,wefoundthat3.37%(6/178)patients

carryhomozygousA827Gmutation.InanotherChinese

spo-radicpediatricpopulationwithnonsyndromichearingloss,

theincidenceis4.41%.13_The_incidence_of_the_A827G

muta-tion in such Chinese population seems to be higher than

inotherethnicgroups.11,37_As_previous_studies_showed_that

expressionoftheclinicalphenotypeofdeafness-associated

homoplasmicmutationsinthe12SrRNAgenerequires the

contribution of modulatingfactors, including

aminoglyco-sides or nuclear modifier genes, and the GJB2 gene is

a potential candidatemodifier gene,12,38---40 _we _performed

mutationalscreeningoftheGJB2geneinthepatients

car-rying the A827G mutation. As a result, only one patient

carried both 12S rRNA A827G mutation and homozygous

GJB2235delCmutation.ThelackofGJB2mutationinother

patients indicates that other nuclear modifier genes and

otherenvironmentalfactorsmaycontributetotheclinical

phenotypeinthesepatients.

Conclusion

InthepresentstudywescreenedmtDNAmutationsin

spo-radic patients withnonsyndromic SNHL and found a total

of 6 subjects (3.37%) carrying homozygous mtDNA

muta-tions.Ourfindingsindicatethatevensporadicpatientswith

nonsyndromicSNHLalsocarrymtDNAmutationsandthese

mutationsmightcontributetotheclinicalphenotype.

Fur-therstudiesscreeningthemutationsinmtDNAareneeded

to help us understand the prevalence and role of mtDNA

mutationsinsporadicpatientswithnonsyndromicSNHL.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This work was supported by grants from the Department

ofPublicHealth(no.2011ZDA012)andScienceTechnology

Department(no.2014C37036).

References

1.MortonNE.Geneticepidemiologyofhearingimpairment.Ann NYAcadSci.1991;630:16---31.

2.WangQJ,ZhaoYL,RaoSQ,GuoYF,He Y,LanL, etal. New-bornhearingconcurrent genescreeningcanimprovecarefor hearingloss:astudyon14,913Chinesenewborns.IntJPediatr Otorhinolaryngol.2011;75:535---42.

3.DaiP,LiuX,YuF,ZhuQ,YuanY,YangS,etal.Molecular etiol-ogyofpatientswithnonsyndromichearinglossfromdeaf-mute schoolsin18provincesofChina.ZhonghuaErKeXueZaZhi. 2006;4:1---5.

4.BayazitYA,YilmazM.Anoverviewofhereditaryhearingloss. ORLJOtorhinolaryngolRelatSpec.2006;68:57---63.

5.MortonCC.Genetics,genomicsandgenediscoveryinthe audi-torysystem.HumMolGenet.2002;11:1229---40.

6.The mitochondrial DNA mutation database. http://www.mitomap.org/[accessed20.07.14].

7.Fischel-GhodsianN,PrezantTR, BuX,OztasS.Mitochondrial ribosomalRNAgenemutationinapatientwithsporadic amino-glycosideototoxicity.AmJOtolaryngol.1993;14:399---403. 8.PandyaA,XiaX,RadnaabazarJ,BatsuuriJ,DangaansurenB,

Fischel-GhodsianN, etal. Mutationinthemitochondrial 12S rRNAgeneintwofamiliesfromMongoliawithmatrilineal amino-glycosideototoxicity.JMedGenet.1997;34:169---72.

9.delCastilloFJ,Rodríguez-BallesterosM,MartínY,ArellanoB, Gallo-TeránJ,Morales-AnguloC,etal.Heteroplasmyforthe 1555A>G mutationinthemitochondrial 12SrRNAgeneinsix Spanishfamilieswithnon-syndromichearingloss.JMedGenet. 2003;40:632---6.

10.LuJ, QianY, Li Z,YangA, ZhuY, Li R, et al.Mitochondrial haplotypesmaymodulatethephenotypicmanifestationofthe deafness-associated 12S rRNA 1555A>G mutation. Mitochon-drion.2010;10:69---81.

11.XingG,ChenZ,WeiQ,TianH,LiX,ZhouA,etal.Maternally inheritednon-syndromichearinglossassociatedwith mitochon-drial12SrRNAA827G mutationina Chinesefamily. Biochem BiophysResCommun.2006;344:1253---7.

12.Li R, GreinwaldJHJr, YangL, ChooDI, Wenstrup RJ, Guan MX. Molecular analysis of the mitochondrial 12S rRNA and tRNASer(UCN)genesinpaediatricsubjectswithnon-syndromic hearingloss.JMedGenet.2004;41:615---20.

13.LiZ,LiR,ChenJ,LiaoZ,ZhuY,QianY,etal.Mutationalanalysis ofthemitochondrial12SrRNAgeneinChinesepediatric sub-jectswithaminoglycoside-inducedandnon-syndromichearing loss.HumGenet.2005;117:9---15.

14.Fischel-GhodsianN,PrezantTR,FournierP,StewartIA,MawM. Mitochondrialmutationassociatedwithnonsyndromicdeafness. AmJOtolaryngol.1995;16:403---8.

15.Hutchin TP, LenchNJ, Arbuzova S, Markham AF,Mueller RF. Maternally inherited hearing impairment in a family with the mitochondrial DNAA7445G mutation. EurJHum Genet. 2001;9:56---8.

(6)

associated with a novel point mutation in mitochondrial tRNASer(UCN)gene.HumMolGenet.1995;4:1421---7.

17.SueCM,TanjiK,HadjigeorgiouG,AndreuAL,NishinoI,Krishna S, et al. Maternally inherited hearing loss in a large kin-dredwithanovelT7511CmutationinthemitochondrialDNA tRNA(Ser(UCN))gene.Neurology.1999;52:1905---8.

18.IshikawaK,TamagawaY,TakahashiK,KimuraH,KusakariJ,Hara A,etal.Nonsyndromichearinglosscausedbyamitochondrial T7511Cmutation.Laryngoscope.2002;112:1494---9.

19.LuJ,LiZ,ZhuY,YangA,LiR,ZhengJ,etal.Mitochondrial 12SrRNAvariantsin1642HanChinesepediatricsubjectswith aminoglycoside-inducedand nonsyndromichearingloss. Mito-chondrion.2010;10:380---90.

20.DingY,LengJ,FanF,XiaB,XuP.TheroleofmitochondrialDNA mutationsinhearingloss.BiochemGenet.2013;51:588---602. 21.McKenzieM,LiolitsaD,HannaMG.Mitochondrialdisease:

muta-tionsandmechanisms.NeurochemRes.2004;29:589---600. 22.Li X, Fischel-Ghodsian N, Schwartz F, Yan Q, Friedman RA,

Guan MX. Biochemicalcharacterization ofthe mitochondrial tRNASer(UCN)T7511Cmutationassociatedwithnonsyndromic deafness.NucleicAcidsRes.2004;32:867---77.

23.Li X, Zhang LS, Fischel-Ghodsian N, Guan MX. Biochemi-calcharacterizationofthedeafness-associatedmitochondrial tRNASer(UCN)A7445Gmutationinosteosarcomacellcybrids. BiochemBiophysResCommun.2005;328:491---8.

24.Toompuu M, Levinger LL, Nadal A, Gomez J, Jacobs HT. The 7472 insC mtDNA mutation impairs 5′ _and ₃′

processingoftRNA(Ser(UCN)).BiochemBiophysResCommun. 2004;322:803---13.

25.TangX,LiR,ZhengJ,CaiQ,ZhangT,GongS,etal.Maternally inheritedhearinglossisassociatedwiththenovelmitochondrial tRNASer(UCN)7505T>CmutationinaHanChinesefamily.Mol GenetMetab.2010;100:57---64.

26.ReidFM,VernhamGA,JacobsHT.Anovelmitochondrialpoint mutationinamaternalpedigreewithsensorineuraldeafness. HumMutat.1994;3:243---7.

27.LevingerL,JacobsO,JamesM.Invitro3′_-end_{endonucleolytic}

processing defect in a human mitochondrial tRNA(Ser(UCN)) precursor with the U7445C substitution, which causes non-syndromicdeafness.NucleicAcidsRes.2001;29:4334---40. 28.Guan MX,Enriquez JA, Fischel-Ghodsian N,Puranam RS,Lin

CP,MawMA,etal.Thedeafness-associatedmitochondrialDNA mutationatposition7445,whichaffectstRNASer(UCN) precur-sorprocessing,haslong-rangeeffectsonNADHdehydrogenase subunitND6geneexpression.MolCellBiol.1998;18:5868---79. 29.ReidFM,RovioA,HoltIJ,JacobsHT.Molecularphenotypeof

ahumanlymphoblastoidcell-linehomoplasmicforthenp7445

deafness-associatedmitochondrialmutation.HumMolGenet. 1997;6:443---9.

30.ToompuuM,TirantiV,ZevianiM,JacobsHT.Molecular pheno-typeofthenp7472deafness-associatedmitochondrialmutation inosteosarcomacellcybrids.HumMolGenet.1999;8:2275---83. 31.Chapiro E,Feldmann D,Denoyelle F,Sternberg D,Jardel C, EliotMM,etal.TwolargeFrenchpedigreeswithnonsyndromic sensorineural deafness and the mitochondrial DNA T7511C mutation:evidenceforamodulatoryfactor.EurJHumGenet. 2002;10:851---6.

32.Puranam RS, Attardi G. The RNase P associated with HeLa cellmitochondriacontainsanessentialRNAcomponent iden-ticalinsequencetothatofthenuclearRNaseP.MolCellBiol. 2001;21:548---61.

33.XingG, Chen Z, Wei Q, TianH, Li X, Zhou A, et al. Mito-chondrial12SrRNAA827Gmutationisinvolvedinthegenetic susceptibilitytoaminoglycosideototoxicity. BiochemBiophys ResCommun.2006;346:1131---5.

34.ChaigMR,ZernottiME,SoriaNW,RomeroOF,RomeroMF,Gerez NM.Amutationinmitochondrial 12SrRNA,A827G,in Argen-tineanfamilywithhearinglossafteraminoglycosidetreatment. BiochemBiophysResCommun.2008;368:631---6.

35.Abreu-SilvaRS, Lezirovitz K,Braga MC, SpinelliM, Pirana S, Della-Rosa VA, et al. Prevalence of the A1555G (12S rRNA) andtRNASer(UCN)mitochondrialmutationsinhearing-impaired Brazilianpatients.BrazJMedBiolRes.2006;39:219---26. 36.Hamasaki K, Rando RR. Specific binding of aminoglycosides

to a human rRNA construct based on a DNA polymorphism whichcausesaminoglycoside-induceddeafness.Biochemistry. 1997;36:12323---8.

37.Rydzanicz M, Wróbel M, Pollak A, Gawecki W, Brauze D, Kostrzewska-PoczekajM,etal.Mutationanalysisof mitochon-drial 12S rRNA gene in Polish patients with non-syndromic andaminoglycoside-inducedhearingloss.BiochemBiophysRes Commun.2010;395:116---21.

38.Prezant TR, Agapian JV, Bohlman MC, Bu X, Oztas S, Qiu WQ,etal. Mitochondrialribosomal RNAmutationassociated withbothantibiotic-inducedandnon-syndromicdeafness.Nat Genet.1993;4:289---94.

39.Li R,Xing G, YanM,CaoX, LiuXZ,Bu X, etal. Cosegrega-tionofC-insertionatposition961withtheA1555Gmutation ofthemitochondrial 12S rRNAgenein alarge Chinese fam-ilywithmaternallyinheritedhearingloss.AmJMedGenetA. 2004;124A:113---7.