www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Association
of
glutamate
metabotropic
receptor
polymorphisms
and
sensorineural
hearing
loss
in
adults
of
different
age
groups
夽
Sanlin
Xie,
Jianzhong
Li,
Wentao
Wang,
Xianming
Chen
∗FuzhouGeneralHospital,DepartmentofOtolaryngology-HeadandNeckSurgery,FujianSheng,China
Received25February2018;accepted17April2018 Availableonline18May2018
KEYWORDS Sensorineuralhearing loss; Glutamate metabotropic receptor7; Singlenucleotide polymorphism Abstract
Introduction:Sensorineuralhearing lossisacommonchallengeallovertheworld,including asectionoftheyoungpopulation.Whiletherehavebeenmanypublishedreportsassociating glutamatemetabotropicreceptor7withsensorineuralhearingloss,thereisnoreport,tilldate, abouttheassociationofglutamatemetabotropicreceptor7polymorphismswithsensorineural hearinglossatdifferentages.
Objective:Totestthe associationbetweenthesinglenucleotide polymorphismsrs11928865 andrs11920109oftheglutamatemetabotropicreceptor7withsensorineuralhearing lossin adultsofdifferentagegroups.
Methods:A total of 1661subjects were studied. The individuals agedbetween 30 and50, andbetween51and70yearswithsensorineuralhearinglosscomprisedgroupAandgroupB, respectively.Individualsagedbetween30and50;andbetween51and70yearswithouthearing losscomprisedcontrolgroupsCandD,respectively.TheMassARRAYmethodwasusedtoanalyze thegenotypes.
Results:Thedifferenceingenotypesfortheglutamatemetabotropicreceptor7rs11928865 sin-glenucleotidepolymorphismbetweenpatientsinthegroupsBandDwasstatisticallysignificant (p=0.018).Thedistributionfrequenciesofgenotypesinpatientsthatwereagedbetween30 and50yearswerenotsignificantlydifferent.Thedifferenceingenotypesforthers11920109 sin-glenucleotidepolymorphismbetweenthesensorineuralhearinglossgroupsandcontrolgroups showednostatisticalsignificance.
夽 Pleasecitethisarticleas:XieS,LiJ,WangW,ChenX.Associationofglutamatemetabotropicreceptorpolymorphismsandsensorineural
hearinglossinadultsofdifferentagegroups.BrazJOtorhinolaryngol.2019;85:560---4.
∗Correspondingauthor.
E-mail:3327747924@qq.com(X.Chen).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2018.04.007
1808-8694/©2018PublishedbyElsevierEditoraLtda.onbehalfofAssociac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial. ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Conclusion: Thers11928865singlenucleotidepolymorphismwasassociatedwiththe suscepti-bilitytohearinglossinpatientsingroupBbutnotwiththoseingroupA.
© 2018 Publishedby Elsevier Editora Ltda. onbehalf of Associac¸˜ao Brasileira de Otorrino-laringologiaeCirurgiaC´ervico-Facial.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/). PALAVRAS-CHAVE Perdaauditiva neurossensorial; Receptor metabotrópicode glutamato7; Polimorfismode nucleotídeoúnico
Associac¸ãoentrepolimorfismosdogenedoreceptormetabotrópicodeglutamato7e perdaauditivaneurossensorialemadultosdediferentesfaixasetárias
Resumo
Introduc¸ão: Aperdaauditivaneurossensorialéumdesafiocomumnomundotodo,incluiuma parte da populac¸ão jovem. Embora haja muitos relatos que associem o gene do receptor metabotrópicodeglutamato7comperdaauditivaneurossensorial,nãohárelato,atéa pre-sentedata,sobreaassociac¸ãodepolimorfismosdoreceptormetabotrópicodeglutamato7com perdaauditivaneurossensorialemdiferentesfaixasetárias.
Objetivo: Testar a associac¸ão entre os polimorfismos de nucleotídeo único, rs11928865 e rs11920109 doreceptor metabotrópicodeglutamato7e perdaauditivaneurossensorialem adultosdediferentesfaixasetárias.
Método: Umtotalde1661indivíduosforamestudados.Osindivíduoscomidadeentre30e50 anoseentre51e70anoscomperdaauditivaneurossensorialconstituíramogrupoAeogrupo B,respectivamente.Indivíduoscomidadeentre30e50anos;eentre51e70anossemperda auditivaforamosgruposcontroleCeD,respectivamente.OmétodoMassARRAYfoiutilizado paraanalisarosgenótipos.
Resultados: Adiferenc¸anosgenótiposparaopolimorfismodenucleotídeoúnicors11928865do genereceptormetabotrópicodeglutamato7entreospacientesdosGruposBeDfoi estatistica-mentesignificante(p=0,018).Asfrequênciasdedistribuic¸ãodosgenótiposnospacientesentre 30e50anosnãoforamsignificantementediferentes.Adiferenc¸anosgenótiposparao polimor-fismodenucleotídeoúnicors11920109entreosgruposcomperdaauditivaneurossensorialeos gruposcontrolenãomostrousignificânciaestatística.
Conclusão:Opolimorfismodenucleotídeoúnicors11928865foiassociadoàsuscetibilidadepara perdaauditivaempacientesdogrupoB,masnãoàquelesdogrupoA.
© 2018Publicadopor ElsevierEditora Ltda.em nomede Associac¸˜ao Brasileira de Otorrino-laringologia eCirurgiaC´ervico-Facial.Este ´eumartigo Open Accesssob umalicenc¸a CCBY (http://creativecommons.org/licenses/by/4.0/).
Introduction
Singlenucleotidepolymorphisms(SNPs)maycausechanges inthequalityorquantityofproteinexpression,whichmay be related to the occurrence of certain diseases or the susceptibilitytodiseases. Glutamateis animportant neu-rotransmitterofthe auditorysystem.The proteinproduct of the glutamate metabotropic receptor 7 gene (GRM7), mGluR7, is widely distributed in the inner and outer hair cells andspiral ganglion nervecell. Sensorineural hearing loss(SHL)isacommonchallengeallovertheworld, includ-ing a section of the young population. There have been manypublishedreports associatingGRM7withSHL. Fried-manetal.haveassociatedthegeneGRM7withsusceptibility toSHLforindividualsfromEuropeancountries.1The
pres-enceofGRM7SNPcorrelatessignificantlywithSHLinelderly ChineseHanmen,anddifferenttypesofhearinglosspresent differentgenepolymorphisms.2However,theSHLsubjects
inthesestudieswereallover50yearsofage.Interestingly, thereisnoreport,tilldate,abouttheassociationofGRM7 polymorphismswithSHLatdifferentages.Whetheryounger
subjectswithSHLalsofeatureanassociationGRM7SNPsis unclear.
Here,weselectedagroupofSHLpatientsbetween30---50 and51---70years-of-ageandcorrespondingcontrolgroupsfor ageneticanalysisofGRM7.Wecomparedthesusceptibility toSHL in thepatients of these differentage groups with GRM7SNPs.
Methods
Subjects
Atotalof1661HanChinesevolunteersparticipatedinthis study.Theywere all30---70 years-of-age.They visitedour hospitalbetweenJanuary2013andJanuary2017.Thestudy wasapprovedbytheEthicsCommitteeofHospitaland writ-teninformedconsentwasobtainedfromeachsubject;the ethicscommittee’sapprovalnumberwas2015003.Atotalof 467subjects,agedbetween30and50years,including236 menand231womenwithameanageof40.54±5.32years, with SHL comprised group A. A total of 278 individuals,
agedbetween30and50years,including147menand131 womenwithameanageof40.23±4.99years,withoutSHL comprisedcontrol group C. A total of 439 subjects, aged between51and70years,including208menand231women withameanageof62.02±3.44years,withSHLcomprised groupB.Finally,477 individuals,agedbetween 51and70 years, including 257 men and 220 women with a mean age of 61.08±4.31 years,without SHL comprisedcontrol groupD.
Clinicalevaluationandaudiologicalmeasurement
Allparticipantsunderwenta routineannual health check-upthatincludedashortquestionnaireofdemographicdata, systemicdiseasehistory,personallifehistoryforhabitssuch assmokingand drinking,chest X-ray electrocardiography, andbiochemicalprofilingofbloodsamples.
All subjects were analyzed by pure tone audiometry, tympanometry, and examination of the auditory brain-stemresponse(ABR). Audiometrywasdone usingamodel GSI-61 pure tone audiometer (Grason-Stadler, Inc., Madi-son WI, USA). Tympanometry used the GSI-Tymp star II (Grason-Stadler,Inc.).Theauditorybrainstemresponsewas measuredusingtheSmartEP(IntelligentHearing Systems, Miami,FL,USA).Thepuretonethresholdaverage(PTA)of the patients’ air frequency at 0.5kHz, 1kHz, 2kHz, and 4kHz were calculated for each individual,and then indi-viduals were grouped according to the PTA of the ears. Thedecreaseinhearingintheexperimentalgroupreached beyondthe 26dB hearing level (HL) of two or more con-secutivedrops in frequency.The subjectswere examined with ABR, and the peak latency of I, III, and V wave, andthe interpeak latency of I---V wave weredetermined, andinterlaterallatencydifferenceofthepeaklatencyand interpeaklatencywerecompared.WhenpeaklatencyofV waveextended longerthan6.1ms;orwaveIIIandwaveV couldnot be induced; or the I---Vwave interpeak latency lengthening was greater than or equal to 4.0ms; or the interlateral latency difference with V wave was greater than 0.4ms, and those who were considered positive for postcochlearlesions,thosewhomeettheseconditionswill not be included in the research. Subjects were excluded if theyhad conductive deafness, noise-induced deafness, anddeafnesscausedbyotherdiseasessuchasdrug-induced deafness,suddendeafness,Meniere’sdisease,otosclerosis, adhesive otitis,acoustic neuroma,and autoimmune deaf-ness.Additionally,subjectswerealsoexcludediftheyhad systemic diseases including diabetes, hypertension, heart disease, hyperlipidemia, chronic kidney disease, chronic obstructivepulmonary disease,and alcohol abuse.Among thecandidateswhomettheseconditions,subjectswhohad progressivelossof hearingaftertheage of50yearswere categorizedinGroupB,whereassubjectswhohad progres-sive hearing loss after 29 years of age were allotted to GroupA.
SequenomMassARRAYSNPanalysistests
Based on prior studies, rs11928865 and rs11920109 were selectedastheGRM7SNPsrelevanttoage-relatedSHL.1,2
Genotypingwasperformedusingpolymerasechainreaction
(PCR) amplification and MassARRAY-specific SNP analyses. GenomicDNAwasextractedfromwholebloodsamplesusing theQIAampDNABloodminikit(Qiagen,Valencia,CA,USA) accordingtothemanufacturer’sinstructions.Primerswere designed using the AssayDesigner 3.1 softwareand were synthesizedby theBeijingGenomics Institute,China.The MassARRAYmethodwasusedtoanalyzethegenotypeofthe twoselectedSNPloci.PCRamplificationwasperformedon the384apertureconfigurationPCR(AppliedBiosystems, Bei-jing,China).ThePCRcyclingconditionsincludedaninitial denaturationstepat94◦Cfor5min,followedby45cycles ofdenaturationat94◦Cfor20s,annealingat56◦Cfor30s, and an extensionat 72◦C for 1min, and afinal extension stepat72◦Cfor3min.SequencingofthePCRproductwas performed with the MassARRAY analyzer (Applied Beijing GenomicsInstitute,Beijing,China).
Statisticalanalyses
Statistical analyses of thedata wereperformed using the SPSS20.0software(SPSSInc.,Chicago,IL,USA).By apply-ingtheHardy---Weinbergprinciple,weexaminedthegroup representationofthegenefrequenciesineachgroup.The measurement data were assessed using the t-test, and thecategoricaldatawereassessedusingamultilayer Chi-squaretestorFisher’sexacttest.Theintergroupalleleand genotypefrequencycomparisonswereperformedusingthe chi-squaretest.Therelativerisksofthegenotypesare indi-catedbythedominanceratio(OddsRatio---OR)andthe95% ConfidenceInterval(95%CI).Thedifferencewasstatistically significantatp<0.05.
Results
ThespeechrecognitiontestrangeofGroupAwas26---120dB and the average hearing threshold was 50.91±19.34dB. The respective values for Group B were 26---120dB and 53.53±18.44dB.The respectivevalues for Group C were −5dB to 25dB and 9.22±6.39dB. The respective values for GroupD were−5dBto25dB and11.33±6.24dB.The patientswereage-andgender-matchedbetweenthe hear-inglossandcontrolgroupsfortheanalyses.Therewereno significantdifferencesinage andgender betweentheSHL andcontrolgroups.
Genotyping
The genotype and allele frequency distributions of rs11928865 and rs11920109 were in accordance with the Hardy-Weinbergequilibrium.
Table 1 shows the differences in the rs11928865 (Pfreq=0.937, ORunadjusted=1.035, 95% CI=0.787---1.360)
and rs11920109 (Pfreq=0.815, ORunadjusted=1.052, 95%
CI=0.815---1.300) genotypeandallelefrequenciesfor indi-vidualsin the30---50 yearsofage SHLandcontrolgroups, whichdidnotdiffersignificantlyfromeachother(Table1).
Table2shows the distributionofthe rs11928865 geno-types and allele frequencies for individuals in Group B and control groups. The genotype distributions of rs11928865weresignificantlydifferentbetweenthegroups
Table1 Associationofthers11928865andrs11920109SNPs inthe30---50year-oldsensorineuralhearinglossgroupandthe 30---50year-oldcontrolgroup.
SNP Genotypedistribution Allelefrequencies ORUnadjusted(95%CI)
Pfreq
Control(n=278) % Case(n=467) % Control % Case %
rs11928865 TT 188 67.60 321 68.70 T 455 81.83 769 82.33 1.035(0.787---1.360) AT 79 28.40 127 27.20 A 101 18.17 165 17.67 Pfreq=0.937 AA 11 4.00 19 4.10 rs11920109 CC 90 32.40 154 33.00 C 314 56.47 539 57.71 1.052(0.815---1.300) CT 134 48.20 231 49.50 T 242 43.53 395 42.29 Pfreq=0.815 TT 54 19.40 82 17.60
Table2 Associationofthers11928865andrs11920109SNPs inthe51---70year-oldsensorineuralhearinglossgroupandthe 51---70year-oldcontrolgroup.
SNP Genotypedistribution Allelefrequencies ORUnadjusted(95%CI)
Pfreq
Controln=477 % Casen=467 % Control % Case %
rs11928865 TT 311 65.20 332 75.60 T 776 81.34 759 86.45 1.463(1.136---1.884) AT 154 32.30 95 21.60 A 178 18.66 119 13.55 Pfreq=0.001 AA 12 2.50 12 2.70 rs11920109 CC 220 46.10 185 42.10 C 638 66.88 558 63.55 0.864(0.712---1.047) CT 198 41.50 188 42.80 T 316 33.12 320 36.45 Pfreq=0.349 TT 59 12.40 66 15.00 (Pfreq=0.001, ORunadjusted=1.463, 95% CI=1.136---1.884). However,the genotype distributions of rs11920109SNPin thesegroups werenotsignificantlydifferent(Pfreq=0.349, ORunadjusted=0.846,95%CI=0.712---1.047)(Table2).
Table3showslogisticanalysesresultsafterthedatawere adjustedforageandgender.Logisticregressionmodelswere constructedtocontrolforpotentialconfoundingeffectson theGRM7genotypes,whichshowedasignificantdifference inthers11928865TTversusATallelesinthe51---70year-old SHL and control groups (p=0.000, ORunadjusted=0.578, 95%
CI=0.429---0.779),whereasthedifferenceintheTTvs.AA alleleswasnotstatisticallysignificant(p=0.260,OR=0.617, 95%CI=0.266---1.429)(Table3).
The genotype distributions were significantly different betweenindividualsagedbetween51and70yearswithor without SHL, indicating that the rs11928865 SNPin GRM7 isassociatedwiththesusceptibilitytoSHLinindividualsin this agerange. However,rs11928865 wasnot significantly differentbetweenindividualsagedbetween30and50years withandwithoutSHL,whichsuggeststhatrs11928865isnot relatedtothesusceptibilitytoSHLinthisagegroup. Fur-thermore,noassociationwasidentifiedbetweenrs11920109 andSHLinthisstudy.
Discussion
The incidence of SHL is closely related to age, and the incidenceofSHLgraduallyincreaseswithage. Environmen-talandgeneticfactorsarethetwoleadingcausesof SHL.
Genetic factors account for 35%---55% of the age-related hearing impairment.3 Gene abnormalities such as the
encoding connexin 26 gene mutation,4 grainyhead-like 2
frameshift mutation,5 and cadherin genemutations have
beenassociatedwithSHL.6 However,mostadultSHLlacks
atypicalfamilygenetichistory.
This study shows that rs11928865 in GRM7 was associ-atedwithsusceptibilitytoSHL,whichisconsistentwiththe resultsofprevious studies.1,2However,thesubjectsinthe
previousstudiesdifferedwithrespecttotheinclusionand exclusioncriteria usedin this study as well asother fac-torssuchasage,gender,andgeneralcondition.Luoetal. selectedChinesemaleswhowereagedbetween70and100 yearsastheirstudysubjects,andthestudyofsystemic dis-easewasalsoincludedinthestudy.2rs11928865showeda
significantassociationwithSHLinanotherstudywith Euro-pean subjects aged 53 years or older.1 In another study,
theAmerican subjects hadan average age of 71.3 years; asignificant correlation betweenGRM7SNPs andauditory perceptionwasreported.7
Furthermore, previous studies have also demonstrated that gender and age may affect SHL.8,9 The risk for SHL
directlycorrelateswithage;men aremore susceptibleto SHLthanwomen,andhearinglosstendstobemoresevere inmenthaninwomen.10
In the results of this study, no association between the GRM7 rs11928865 locus and SHL in group A, and no positive results for the rs11920109 locus were observed, reflecting the complexity of SHL due to the involve-ment of multiple genes, environmental factors, ototoxic
Table3 Logisticregressionmodel:analysisoftheeffectsofGRM7SNPrs11928865genotypesinthe51---70year-oldsensorineural hearinglossgroupsandthe51---70year-oldcontrolgroups.
SNPs Genotype Control Case p OR 95%CI
rs11928865 TT 311 332
AT 154 95 0.000 0.578 0.492---0.779
AA 12 12 0.260 0.617 0.266---1.429
drugs,systemicdiseases,andlifestylehabits.Anumberof pathogenic candidategenes includingthe genes encoding connexin26(GJB2),4,11,12 grainyhead-like2(GRHL2),5
cad-herin(CDH23),6 potassium voltage-gated channel member
4(KCNQ4),13 N-acetyltransferase(NAT),14---16apolipoprotein
E(APOE),17anduncouplingprotein2(UCP2)18,19havebeen
relatedtothegeneticsusceptibilityofSHL,anditislikely thatthesegenesinteractinthepathogenesisofSHL.
Conclusion
Inconclusion,rs11928865wasassociatedwithsusceptibility toSHLforindividualsingroupB,butnotforindividualsin groupA.Thissuggeststhatdifferencesintheageofonset of SHL in the two age groups, as measured by the time andnatureofhearingloss,mightbeexplainedbydifferent geneticetiologies.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Wesincerelythankthepatientsfortheirparticipation.This workwassupported bythe NaturalScienceFoundation of FuJianProvinceunderGrant(no.2015J01405).
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