www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Molecular
study
of
hearing
loss
in
Minas
Gerais,
Brazil
夽
Raíssa
de
Oliveira
Aquino
Schüffner,
Karla
Lima
Nascimento,
Fábio
André
Dias,
Pedro
Henrique
Teodoro
da
Silva,
Wrgelles
Godinho
Bordone
Pires,
Nilson
Moreira
Cipriano
Junior,
Luciana
Lara
dos
Santos
∗UniversidadeFederaldeSãoJoãodel-Rei,Divinópolis,MG,Brazil
Received6June2018;accepted15December2018 Availableonline20February2019
KEYWORDS Hearingloss; GJB2; GJB6; Brazil Abstract
Introduction:Deafnessisthemostfrequentsensorydeficitinhumans.Incidenceisestimated at4:1000birthsinBrazil.Specificprogramsforclinicalcareofpatientswithhearinglossare stillscarceinBrazilandtheissueisanimportantpublichealthproblem.
Objective: Todeterminethefrequencyof35delGandD13S1830mutationsinGJB2andGJB6 genesrespectivelyinpatientswithnon-syndromicsensorineuralhearinglossfromMinasGerais, Brazil.
Methods:Thisresearchinvolved53individuals,whowereassessedbyaquestionnairefor pre-dictingthepossibilityofnon-syndromicdeafnessandfordatacollecting.Samplesweretested forthepresenceofthe35delGmutationinGJB2geneandD13S1830inGJB6genebypolymerase chainreactionandrestrictionenzymedigestion.
Results:Epidemiologicalresearchhasshownthatthemajorityofthesubjectsareunawareof theetiologyandthepathogenesisofhearingloss.In9patients(16.98%),35delGmutationwas foundinheterozygosisandtheallelefrequencywasestimatetobearound8.5%.Although9.61% ofthepatientsreportedhavingsomedegreeofconsanguinitybetweentheparentsand12.08% reportedothercasesofdeafnessintheirfamilies,thismutationwasnotfoundinhomozygosis. TheD13S1830mutationwasnotfoundinthisstudy.
Conclusion: Thisresearchdescribesforthefirsttimethefrequencyofthe35delGandD13S1830 mutation inhearing-impairedindividuals from Minas Gerais,Brazil, and thecollected data reinforcetheneedforfurtherstudiesinthispopulationduetoheterogeneityofhearingloss. © 2019 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-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 Pleasecitethisarticleas:SchüffnerRO,NascimentoKL,DiasFA,SilvaPH,PiresWG,CiprianoJuniorNM,etal.Molecularstudyofhearing
lossinMinasGerais,Brazil.BrazJOtorhinolaryngol.2020;86:327---31.
∗Correspondingauthor.
E-mail:llaramg@hotmail.com(L.L.Santos).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2018.12.005
1808-8694/©2019Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
PALAVRAS-CHAVE
Perdaauditiva; GJB2;
GJB6; Brasil
EstudomoleculardaperdaauditivaemMinasGerais,Brasil Resumo
Introduc¸ão:Asurdezéodéficitsensorialmaisfrequenteemhumanos.Estima-sequea incidên-ciasejade4:1.000nascimentosnoBrasil.Programasespecíficosparaatendimentoclínicode pacientescomperdaauditivasãoescassosnoBrasileaquestãoéumimportanteproblemade saúdepública.
Objetivo:Determinarafrequênciadasmutac¸ões35delGnogeneGJB2eD13S1830noGJB6em pacientesdeficientesauditivosdeorigemneurossensorialenão sindrômicadeMinasGerais, Brasil.
Método: Apesquisaenvolveu53indivíduosselecionadospormeiodequestionáriooqualavaliou apossibilidadedesurdeznãosindrômicaentreoutrosdados.Asamostrasforamtestadasquanto àpresenc¸adamutac¸ão35delGnogeneGJB2eD13S1830nogeneGJB6porreac¸ãoemcadeia dapolimeraseedigestãocomenzimaderestric¸ão.
Resultados: A pesquisaepidemiológica mostrou que a maioriados indivíduos desconhece a etiologiadaperdaauditiva.Em 9pacientes(16,98%),amutac¸ão35delGfoiencontrada em heterozigoseeafrequênciaalélicafoiestimadaem8,5%.Embora9,61%daspessoastenham relatadoalgum graude consanguinidadeentreos paise12,08%relatassemoutros casosde surdezemsuasfamílias,essamutac¸ãonãofoiencontradaemhomozigose.Amutac¸ãoD13S1830 nãofoiencontradanesteestudo.
Conclusão:Este trabalho descreve pela primeira vez a frequência da mutac¸ão 35delG e D13S1830em deficientesauditivosdeMinas Gerais,Brasil,eosdadoscoletadosreforc¸ama necessidadedemaisestudosnessapopulac¸ãodevidoàheterogeneidadedaperdaauditiva. © 2019 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Deafness is the most frequent sensory deficit in humans, withan incidence that ranges from1:300 to1:1000 chil-dren. Accordingto theWorld HealthOrganization (WHO), hearinglossaffectsabout10%oftheworldpopulation.1The
frequencyofdeafnessinBrazilisestimatedat4:1000births. InBrazilenvironmentalfactorsaccountfor80%ofcases,and geneticcausesareresponsiblebytheother20%.2,3
Non-syndromic hereditaryforms, which arenot associ-atedwithotherclinical manifestations,accountfor about 70%ofcasesandcanbeautossomalrecessive(DFNB), autos-somal dominant (DFNA), X-linked or mitochondrial. The recessiveformsaremoreprevalent(75%---80%ofcases)and tendtobemoresevereandoftenassociatedwithdefects inthecochlea,leadingtotheneurosensorialdisability.The dominant forms (15%---20% of cases) are milder and con-tributeinmostcasestopost-lingualdeafnessprogressive. TheX-linked formsarerarer, aswell asthe mitochondrial form.4---8 The early identification of deafness with
etiolog-icaldiagnosis alsoenablesearly treatment, improvingthe prognosis.In Brazil,thespecificprogramsfor clinicalcare of patients with hearing loss are still scarce and this is animportantpublichealth problem.Theresearchof non-syndromic Congenital Deafness with Guthrie test is not availableattheBrazilianpublichealthsystem(SUS,Unified HealthSystem) and just Otoacoustic EmissionsTest (OAE) arerequired,bylaw,inthenewborns.9Besides,molecular
diagnosisismorerestrictedincientificresearches,andthe highgeneticheterogeneityofnon-syndromichearinglossis acomplicatingfactor.10
Morethan150locihavebeenmappedand93geneshave beenidentifiedfornon-syndromichearingloss,11thoughthe
involved genes and frequencies of mutations are strongly influenced bythe ethniccomposition of thepopulation.12
Mutationsinthreegenesencodingconnexin,GJB2(Cx26),
GJB6(Cx30),andGJB3(Cx31),werefoundtocausehearing impairment,andtheoccurrenceinthefirsttwoaremost fre-quentlydescribed.2VariationsinGJB2geneaccountforup
to50%ofallgeneticallycausednon-syndromichearingloss casesinCaucasians.13Therearenoreportsonthefrequency
ofthemutationsinthesegenesindeafnesspopulationfrom MinasGerais,Brazilandhereweestimatedthefrequencyof twofrequentlydescribed mutations, 35delGinGJB2 gene andD13S1830 inGJB6genein unrelated hearing-impaired patientsfromMinasGerais,Brazil.
Methods
Sample
Weconductedacross-sectionalstudy,inwhich53disabled, 32malesand21 females,allunrelated,werestudied. We includedinthestudypatientswithoutmedicalconfirmation aboutdisability’scause.Therefore,wereincludedpatients withhearing impairmentclassifiedasunknown case, with no diagnostic hypothesis about the cause of hearing loss, absenceofconfirmationthroughspecifictestsand/or medi-calconfirmationdescribedinthereport.Therewasnoage restrictionforparticipationinthisstudy.Allindividualsare residents of the Midwest region of Minas Gerais. Patients
wererecruitedfromschoolAudio-VisualAssistancefor Hear-ingImpaired(AAVIDA)andtheDeafAssociation,bothfrom Divinópolis,MinasGerais,Brazil.
This study was approved by the Ethics Committee on Human Research from Universidade Federal de São João Del Rei, Minas Gerais, with favourable opinion number 009/2011. All steps for clarification of the study were performed using the Brazilian Sign Language (Libras) and Portugueselanguage,soallparticipantshavefullautonomy in deciding their participationin research.Those individ-uals whohave decidedto participatesigned an informed consent.
We usedaquestionnairetocollectdata relatedtoage of onset of hearing loss, presence of other cases in the family,consanguineousrelationshipsinthefamily, manifes-tationofsyndromesassociatedtohearinglosstoconfirming non-syndromic deafness and exclusion of environmental causeswithmedicalconfirmation:prenatalinfectionssuch asrubellaortoxoplasmosis,postnatalinfections,and expo-suretoototoxicdrugs.
Molecularanalysis
Bloodsamples(5mL)werecollectedfromthepatientsand genomicDNAisolatedaccordingtostandard procedures.14
Theamplificationofthegenomicregionforanalysisofthe 35delG mutation in the GJB2 gene was performed with primerspreviouslydescribedandtheannealingtemperature usedwas65◦C.2ThePCRproducesan89bpfragmentwhich
isdigestedbyrestrictionenzymeBstNI.Individualswithout the35delGmutationhavetherestrictionsitefortheenzyme BstNIand producetwofragmentsof 69bp and20bp.The 35delGdeletionabolishestherestrictionsiteoftheenzyme andconsequentlyhomozygousindividualsforthismutation produceonlyafragmentof89bpandheterozygous individ-ualsproducethethreefragmentsdescribedabove.2
The D13S1830mutationintheGJB6genewasdetected bystandardPCRtechniqueusingthreeprimers,F:(TTTAGG GCATGATTGGGGTGATTT),R1:(CACCATGCGTAGCCT TAACCATTTT)andR2:(TCATCGGGGGTGTCAACAAACA).2
Theannealingtemperature was62◦C.TheprimersR1and R2arelocated inthe geneencodingconnexin30 protein, andprimerFis locatedbeforethe342kbregion,whichis deletedinmutantindividuals.The reverseprimer (R2),in turn, is located close tothe breakpoint DNA in the pres-enceofthemutation.Normalindividualswhodonothave themutationwillform681pbproductsamplifiedbyprimers R1andR2.Whenthedeletionoccurs,theprimerannealing regionR2is lostapproaching theregions annealingof the primersFandR1andamplifyafragmentof460pb. There-fore,theheterozygousindividualshaveproductswith681pb and460pbandhomozygousmutatedonly460pbfragment. Twomutations(35delGandD13S1830)werescreenedinall participants.
ThePCRproductswerevisualizedon8%polyacrylamide gelstainedwithsilvernitrate.TheDNAextractionproduct wasstoredinthefreezer(mean−6◦C)andinasimilar man-nerasthePCRproducts.Allanalyzeswerecarriedoutina specializedlaboratory inmolecular biologyof theFederal UniversityofSãoJoãodel-Rei,Divinópolis,MG,Brazil.
Results
Inthisstudy,atotalof53individualswithhearing impair-mentwereevaluated.Theageofpatientsrangedfrom5to 42yearsold,withanaverageof23.5years.
Regardingthestageofhearingloss,32.07%ofthe indi-vidualspresentedmanifestationatbirth,26.41%perceived thedisabilitybefore2yearsold,and11.32%after2years old.This information wasnot assessed for 30.18% of the individuals.
Amongpatientswhoansweredquestionsaboutthe etiol-ogyofdeafness,nobodyhadmedicalreportconfirmingthe etiologyofhearingloss.Patientswithsuspicionsaboutthe possiblecauseof disabilitybut without medical confirma-tioncorrespondedto60.37%ofthesample;datawerenot availablefor39.62%.Thelargenumberofmissingdatamay berelatedtothelack ofinformationof individualsonthe probablecauseofhearingdisability.
BrazilianSignLanguage(Libras)isaformof communica-tionusedbymostparticipants,86.79%.Only9.43%useoral communicationanddatawerenotaccessedfor3.77%ofthe sample.
Inconsanguinityanalysis,9.61%ofpeoplereported hav-ing some degree of consanguinity between the parents (confirmedonlybysimplegenedogram)and12.08%reported othercasesofdeafnessintheirfamilies.
About molecular analysis, individuals with deafness of unknown etiology and who lived in the Midwest state of MinasGerais,Brazil,wereinvestigatedforthepresenceof 35delGandD13S1830mutationsin GJB2andGJB6 respec-tively.Ofthe106allelespresentinthe53individualswho underwentmolecularanalysis,ninewere35delGmutation. So, the allele frequency of 35delG mutation in the sam-plestudiedwas8.5%.Allsubjectswiththismutationwere heterozygous(16.98%). Eventhough patientsreportedthe presence of consanguinity relationships in some families, homozygousindividuals forthis mutationwerenot found. TheD13S1830mutationwasnotfoundinanyofthe individ-ualstested.
Discussion
Casesofpre-lingualdeafnessmaybeassociatedwith envi-ronmental factors or genetic inheritance. Environmental factorsinBrazilareamongthemajorcausesofhearingloss, andcongenital rubella syndromeand neonatal anoxia are themostprevalentassociatedriskfactors(33.5%),followed bythoseofunknownetiology(18.5%).Inpostlingual deaf-nesscasesthelossmaybeaconsequenceoftheuseototoxic drugs,familyhistoryofhearinglossorinsomecasescanbe consideredidiopathic.15
The35delGmutationintheGJB2geneisthemaincause of genetic deafness in caucasian populations and it has beendescribed in allstudies conductedin other Brazilian regions.13,15,16Thismutationwasfoundinheterozygosityin
16.98%of the sample studied. Recessive formsof hearing loss are the most prevalent in non-syndromic hereditary forms. When patients had the 35delG mutation in both alleles(homozygous), it is possible toaffirm that hearing impairmentmanifestedasaresultofthis genotype. How-ever, when the mutation is in heterozygosis, as in most
Table1 Frequencyof35delGmutationinpreviousstudiesperformedinBrazilianpopulations.
Region Samplepopulation Allele35delG References
MinasGeraisState 53patientswithnonsyndromichearingloss 8.5% Presentstudy SãoPauloState 300patientswithnonsyndromichearingloss 9.8% 17
SãoPauloState 180patientswithnonsyndromichearingloss 13.30% 18
SãoPauloState 33patientswithcongenitalnonsyndromicsensorineuralhearingloss 21.21% 8
EspiritoSantoState 77patientswithnonsyndromichearingloss 7.8% 2
NorthBrazil 77patientswithnonsyndromichearingloss 5.19% 19
cases, it is necessary to do further analysis in the GJB2
gene,toinvestigateapossiblecompoundheterozygosisas ajustificationofthemanifestationofdeafness.
Thefrequencyof35delGmutationinasampleofhearing impairmentfromMinasGeraisstatehasbeendescribedfor thefirsttimeinthisstudy.Thisfrequencyissimilartoothers alreadydescribed inpopulations fromSoutheasternBrazil (Table1).Thosethatarenotinagreementmaybeduetothe samplesizeandtheselectioncriteriaofthesample.2,8,17---19
Despite the fact that D13S1830 is a mutation frequently describedinBrazilianpopulations,inthesamplefromMinas Geraisthisallelewasnotfound.Someotherstudiesalsodid notfoundthismutationintheir analysis.19---21 Even though
environmentalcauses of deafness may behighly frequent inBrazil,identificationofthehiddengeneticcausesbythe moleculartestsisimportant.Thegenetictestisusefulfor earlydiagnosistoachievetheobjectivesinthe rehabilita-tionfordeafpeopleandgeneticcounseling.Epidemiological research,asdescribed here, hasshown that the majority of the subjects are unaware of the etiology of deafness. Someof themsuspect ofthe cause,butnoindividualhas a medical report, confirming the lack of data regarding theetiologyandthepathogenesisofhearingloss.Allthese aspectsinvolvedintheetiologicunderstandingofdeafness is important for providing a better prognosis; stimulation ofspeechandweaknessofthelowerdyadhearing-speech, contributingtoabetterqualityoflifeforindividuals with hearinglossgeneticnon-syndromicandtheirfamilies.17
AlthoughvariationsinGJB2genealmostexclusivelycause prelingualautosomalrecessivehearingloss,thisdisorderis veryheterogeneous.13 Othermutations,orothergenesnot
studiedhere,mightbeinvolvedinthedisability pathogen-esis.The sequencingof the GJB2genecould beuseful to identifythesecondmutantalleleandinspiteofsequencing oftheGJB6genewasnotreportedinBrazilianpopulations until now, it would be interest toidentify other possible frequentmutationassociatedtothehearingdisability.
Conclusion
Thisstudydescribesforthefirsttimethemolecular inves-tigationof twogenesrelated tothehearing lossin Minas Geraispopulation.Theallelefrequencyof35delGmutation inthesamplestudiedwas8.5%andtheD13S1830mutationin GJB6genewasnotfoundinthepatientsinvestigated.These results reinforce the need for further studies about the investigationofdeafnessinBrazil,inordertoimprovepublic healthissuesandgeneticcounseling.Geneticand epidemi-ologicalinformationmakepossibleetiologicidentification,
geneticcounselingforfamiliesandbetterassessmenttothe possibilityoftreatment.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
The authors thank all patients for participating in this study,Audio-VisualAssistancefor HearingImpaired School (AAVIDA)andtheDeafAssociationfromDivinópolis.
References
1.MortonNE.Geneticepidemiologyofhearingimpairment.Ann NYAcadSci.1991;630:16---31.
2.Cordeiro-Silva Mde F, Barbosa A, Santiago M, Provetti M, Rabbi-BortoliniE.Prevalenceof35delG/GJB2and del (GJB6-D13S1830)mutationsinpatientswithnon-syndromicdeafness fromapopulationofEspiritoSanto-Brazil.BrazJ Otorhinolaryn-gol.2010;76:428---32.
3.PiattoVD,ManigliaJV. Importânciado GeneConexina26na etiologiadadeficiênciaauditivasensorioneuralnão-sindrômica. ActaAWHO.2001;20:106---12.
4.Piatto VB, Oliveira CA, Alexandrino F, Pimpinati CJ, Sar-torato EL. Prospects for genetic hearing loss screening: 35delGmutationtrackinginanewbornpopulation.JPediatr. 2005;81:139---42.
5.CarvalhoGM,RamosPZ,CastilhoAM,GuimaraesAC,Sartorato EL.Molecularstudyofpatientswithauditoryneuropathy.Mol MedRep.2016;14:481---90.
6.Shearer AE, Hildebrand MS, Smith RJHMD. Hered-itary hearing loss and deafness overview; 2017. https://www.ncbi.nlm.nih.gov/books/NBK1434/ of subor-dinatedocument[accessed08.03.17].
7.Piatto VB, Moreira OA, Silva MA, Maniglia JV, Pereira MC, SartoratoEL.Correlation between audiometric dataand the 35delG mutation in ten patients. Braz J Otorhinolaryngol. 2007;73:777---83.
8.PiattoVB,BertolloMGE,SartoratoLE,ManigliaVJ.Prevalence oftheGJB2mutationsandthedel(GJB6-D13S1830)mutationin Brazilianpatientswithdeafness.HearRes.2004;196:87---93. 9.LuzI,RibasA, KozlowskiL,WilligM,Berberian AP.Newborn
HearingScreeninginaPublicMaternityWardinCuritiba,Brazil: determiningfactorsfornotretesting.IntArchOtorhinolaryngol. 2016;20:300---4.
10.ManzoliGN,BademciG,AcostaAX,FelixTM,CengizFB,Foster J2nd,etal.Targetedresequencingofdeafnessgenesrevealsa founderMYO15AvariantinnortheasternBrazil.AnnHumGenet. 2016;80:327---31.
11.NiuZ,FengY,HuZ,LiJ,SunJ,ChenH,etal.Exomesequencing identifiesanovelmissense mutationofWFS1 asthecauseof non-syndromiclow-frequencyhearinglossinaChinesefamily. IntJPediatrOtorhinolaryngol.2017;100:1---7.
12.CampVG,Smith R.Hereditaryhearinglosshomepage;2018. http://hereditaryhearingloss.org/ of subordinate document [accessed05.03.17].
13.ParzefallT, Frohne A, KoenighoferM,Kirchnawy A, Streubel B, Schoefer C, et al. Whole-exome sequencing to identify thecause ofcongenitalsensorineuralhearinglossincarriers ofaheterozygousGJB2mutation.EurArchOtorhinolaryngol. 2017;274:3619---25.
14.MillerSA,DykesDD,PoleskyHF.Asimplesaltingoutprocedure forextractingDNAfromhumannucleatedcells.NucleicAcids Res.1988;16:1215.
15.BernardesR, BortoncelloS, ChristianiTV,SartoratoEL,Silva RC,PortoPR.Molecularinvestigationinchildrencandidatesand submittedtocochlearimplantation.Braz JOtorhinolaryngol. 2006;72:333---6.
16.OliveiraCA, Pimpinati CJ, Alexandrino F,Magna LA, Maciel-Guerra AT, Sartorato EL. Allelic frequencies of the 35delG mutationoftheGJB2geneindifferentBrazilianregions.Genet Test.2007;11:1---3.
17.BatissocoAC, Abreu-SilvaRS, Braga MC,Lezirovitz K, Della-RosaV,AlfredoTJr,etal.PrevalenceofGJB2(connexin-26) and GJB6(connexin-30)mutations ina cohortof300 Brazil-ianhearing-impairedindividuals:implicationsfordiagnosisand geneticcounseling.EarHear.2009;30:1---7.
18.SvidnickiMC,Silva-CostaSM,RamosPZ,dosSantosNZ,Martins FT,CastilhoAM,etal.Screeningofgeneticalterationsrelated tonon-syndromichearinglossusingMassARRAYiPLEX(R) tech-nology.BMCMedGenet.2015;16:85.
19.CastroLS,MarinhoAN,RodriguesEM,MarquesGC,CarvalhoTA, SilvaLC,etal.AstudyofGJB2anddelGJB6-D13S1830 muta-tionsinBraziliannon-syndromicdeafchildrenfromtheAmazon region.BrazJOtorhinolaryngol.2013;79:95---9.
20.ManzoliGN,Abe-SandesK,BittlesAH,daSilvaDS,Fernandes LdaC,PaulonRM,etal.Non-syndromichearingimpairmentin amulti-ethnicpopulationofNortheasternBrazil.IntJPediatr Otorhinolaryngol.2013;77:1077---82.
21.Melo US, SantosS, Cavalcanti HG, Andrade WT, Dantas VG, RosaMR,etal.Strategiesforgeneticstudyofhearinglossin theBraziliannortheasternregion.IntJMolEpidemiolGenet. 2014;5:11---21.
