Association between interleukin-6 polymorphism in the-174 G/C region and hearing loss in the elderly with a history of occupational noise exposure

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

www.bjorl.org

ORIGINAL

ARTICLE

Association

between

interleukin-6

polymorphism

in

the

₋

174

G/C

region

and

hearing

loss

in

the

elderly

with

a

history

of

occupational

noise

exposure

夽

,

夽夽

Miula

Portelinha

Braga

_,

_Sandra

_Mara

_Maciel

,

Luciana

Lozza

de

Moraes

Marchiori

,

Regina

Célia

Poli-Frederico

a_{UniversidadeNortedoParaná(UNOPAR),Londrina,PR,Brazil} b_{UniversityofLondon,London,UnitedKingdom}

c_{DepartmentofPediatricDentistry,UniversidadeEstadualdeMaringá(UEM),Maringá,PR,Brazil} d_{UniversidadeEstadualdeLondrina(UEL),Londrina,PR,Brazil}

e_{UniversidadeEstadualPaulista‘‘JúliodeMesquitaFilho’’(UNESP),Botucatu,PR,Brazil}

Received29July2013;accepted1February2014 Availableonline22July2014

KEYWORDS Occupationalnoise; Sensorineuralhearing loss;

Cytokines

Abstract

Introduction:The biologicalprocesses involvedinnoise-inducedhearing loss(NIHL)arestill unclear.Theinvolvementofinflammationinthisconditionhasbeensuggested.

Objective: Toinvestigate the associationbetween interleukin --- 6 (IL-6)polymorphism and susceptibilitytoNIHL.

Methods:Thiswasacross-sectionalstudywithasampleof191independentelderly individ-uals aged>60yearsofage.Informationonexposure tooccupationalnoisewas obtainedby interviews.Audiologicalevaluationwasperformedusingpuretoneaudiometryandgenotyped throughPCRbyrestrictionfragmentlengthpolymorphism---PCR-RFLP.Datawereanalyzedusing thechi-squaretestandtheoddsratio(OR),withthesignificancelevelsetat5%.

Results:Amongelderlywithhearingloss(78.0%),18.8%hadahistoryofexposureto occupa-tionalnoise.Therewasastatisticallysignificantassociationbetweenthegenotypefrequencies oftheIL-6−174andNIHL.TheelderlywiththeCCgenotypewerelesslikelytohavehearing lossduetooccupational noiseexposurewhen comparedtothosecarrying theGGgenotype (OR=0.0124;95%CI0.0023---0.0671;p<0.001).

Conclusion: ThisstudysuggeststhereisanassociationofpolymorphismsintheIL-6 geneat position---G174Cwithsusceptibilitytonoise-inducedhearingloss.

© 2014Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.

夽 _{Pleasecitethisarticleas:BragaMP,MacielSM,MarchioriLL,Poli-FredericoRC.Associationbetweeninterleukin-6polymorphisminthe}

−174G/Cregionandhearinglossintheelderlywithahistoryofoccupationalnoiseexposure.BrazJOtorhinolaryngol.2014;80:373---8.

夽夽

Institution:UniversidadeNortedoParaná(UNOPAR),Londrina,PR,Brazil.

∗_{Correspondingauthorat:UniversidadeNortedoParaná(UNOPAR),Londrina,PR,Brazil.}

E-mail:luciana.marchiori@unopar.br(L.L.M.Marchiori).

http://dx.doi.org/10.1016/j.bjorl.2014.07.001

PALAVRAS-CHAVE Ruídoocupacional; Perdaauditiva neurossensorial; Citocinas

Associac¸ãoentreinterleucina-6polimorfismonaregiãode-174G/Ceperdaauditiva emidososcomhistóriadeexposic¸ãoaoruídoocupacional

Resumo

Introduc¸ão:Osprocessosbiológicosenvolvidosnaperdaauditivainduzidaporruído(PAIR)ainda nãoestãoclaros.Oenvolvimentodeprocessoinflamatórionestacondic¸ãotemsidosugerido.

Objetivo:Investigar a associac¸ãoentre opolimorfismo nogene dainterleucina-6 (IL-6)ea suscetibilidadeàPAIR.

Método: Trata-sedeestudotransversalcomamostrade191idososindependentesacimade60 anosdeidade.Informac¸õessobreaexposic¸ãoaoruídoocupacionalforamobtidaspor entrevis-tas.Aavaliac¸ãoaudiológicafoirealizadapormeiodeaudiometriatonalliminareagenotipagem pelatécnicadaPCR-RFLP.Osdadosforamanalisadosusando-seotesteQui-quadradoearazão dechances(OR),comoníveldesignificânciafixadoem5%.

Resultados: Entre os idosos com perda auditiva (78,0%), 18,8% apresentavam histórico de exposic¸ãoao ruídoocupacional.Houveassociac¸ãoestatisticamentesignificanteentreas fre-quênciasgenotípicasdaIL-6-174eaPAIR.OsidososportadoresdogenótipoCCforammenos propensosaapresentarperdaauditivaporexposic¸ãoaoruídoocupacionalquandocomparados aaquelesportadoresdogenótipoGG(OR=0,0124;95%IC0,0023-0,0671;p<0,001).

Conclusão:Opresenteestudosugereaassociac¸ãodopolimorfismonogenedaIL-6naposic¸ão -G174Ccomasuscetibilidadeàperdaauditivainduzidaporruído.

©2014Associac¸ãoBrasileira deOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicadopor ElsevierEditoraLtda.Todososdireitosreservados.

Introduction

Elderly health has increasingly aroused the interest of researchers,asthepopulationinbothdevelopedand devel-oping countries is aging.1 _{It is estimated that by 2040} developingcountrieswillhaveabillionpeopleaged60years orolder.2_{Giventherapidityandmagnitudeofthisincrease,} careforthisspecificgroupisessential,sotheycanagein goodhealthandwithgoodqualityoflife.3

Themostcommontypesofhearinglossarepresbycusis (duetoaging)andnoise-inducedhearingloss(NIHL).4_NIHL isduetocontinuedexposuretoloudnoiselevels,resulting in gradualloss of auditory acuity andis usually bilateral, symmetric,sensorineuralandirreversible.Itusuallyaffects the high audiometric frequencies of 3000---6000Hz.5 _The etiology of hearing loss also has genetic components, in additiontoenvironmental ones, and morerecently, inner earinflammatory responseinduction andup-regulation of proinflammatorycytokineswereevaluatedinthepresence ofexacerbatednoise.5---7_{Severalresearchershavereported} thepresenceofinflammatorycellsinthesteadystateand theirincreaseafterlesionsintheinnerear.6,8---10

Noise exposure induces the expression of pro-inflammatorycytokines,including tumor necrosisfactor-␣ (TNF-␣), interleukin 1␤ (IL-1␤) and interleukin-6 (IL-6).11 So et al.12 _{observed a transient up-regulation of IL-6 in} cisplatin-treated animal models. Wakabayashi et al.13 investigatedtheeffectofIL-6inhibitionusingananti-IL-6 (MR16-1) antibody in mice. These authors found that MR16-1 showed a protective effect against noise-induced cochlear injury, mainly due to neuronal loss suppression and presumably by relieving the inflammatory response, similar data were found by Nakamoto et al.14 _These authorssuggestedthatthesuppressionofproinflammatory

cytokineHSF-1inthecochleabytheadministrationofGGA (geranyl---geranyl---acetone) could be an important way to protecttheinnerear.

Theexpressionofcytokinesmaybeinfluencedbygenetic variation, resultingin pathogenic conditions15 _{and several} studies haveinvestigatedsinglenucleotidepolymorphisms (SNPs) as risk factors for inflammatory diseases.16 _These SNPsmayaffecttheexpression,secretionandcellular trans-portof interleukins17,18 _{andcanalsodecreasethelevel of} the IL-1receptorantagonist(IL-1Ra), which increasesthe productionandactivityofIL-1␤.19

The SNP in the region −174 of the interleukin-6 gene contains thereplacement of G byC, andthe presence of theCallelerepresentsaprotectivefunctionduetoreduced productionofIL-6.20

ConsideringthatNIHLisinvolvedintheinflammatory pro-cess,thisstudyaimedtoevaluatetheassociationbetween the proinflammatory cytokine-6 (IL-6) gene polymorphism andsusceptibilitytoNIHLinphysicallyindependentelderly Brazilians.

Method

Studypopulation

Ofapopulation of43,610elderlyindividualstreatedin 38basic healthunits(BHUs) intheurban areaofthecity, the sample size wasset at 343 individuals, considering a confidenceintervalof95%andamarginoferrorof5%.21

Aimingatobtainingsample representativeness,random stratificationwasperformedconsideringgenderandregions ofthecity.Thestudyincludedindividualsaged60yearsor older, ofboth genders,living independentlyand classified at level 3or 4, asproposedby Spirduso.22 _This classifica-tionassesses the level of independence of theelderly, in whichlevel1indicatesalackofself-mobilityandlevelfive indicatesathletes.Theelderlywhohadsomediseaseor lim-itationsthatpreventedthetests,suchasphysicalormental impairment, were excluded fromthe sample. All partici-pantssignedaninformedconsentform.

Audiologicalassessment

Theaudiologicalassessmentwasperformedbyconventional pure toneaudiometry,in aninteracoustics audiometer.To determine the severity of hearing loss, the criteria pro-posedby BIAP--- Bureau Internacional d’AudioPhonologie --- aninstitutionformedbyseveralassociationsofEuropean countries with the main objective of guiding the activity ofprofessionalsintheseregions,wereused.The meansof hearingthresholdsindBinairconduction,at500Hz,1kHz, 2kHzand4kHzandthe02/11997recommendationswere analyzed, boththe right andleftears wereevaluatedfor agroupof individualswithahistoryofoccupationalnoise andthegroupofindividualswithnohistoryofoccupational noise.23---25

Evaluationofoccupationalnoiseexposure

The assessment of occupational noise exposure was per-formed through interviews with the elderly participants, using a semi-structured questionnaire. Information was collectedonwhetherworkwasperformedinanoisy environ-mentornot,howmanyyearsofworkinanoisyenvironment andwhether theindividual worea hearingaid. Moreover, demographic characteristics (gender, age and ethnicity) werecollected.

GenotypingforIL-6G-174C(rs1800795)gene polymorphism

Peripheralblood samples werecollected in vacuum tubes containing6%ethylenediaminetetraaceticacid(EDTA)and DNAwasextractedfromperipheralbloodleukocytes,using theprotocoldescribedbyOlerupandZeterquist.26

Basedontheoriginalsample, theDNAwasdilutedtoa concentrationof100ng/mL.Theconcentrationwas deter-minedbyspectrophotometerat260nmand280nm(Biomate 3,ThermoFisherScientific,Madison,USA).

The C to T polymorphic site located at position −174 of theIL-6(rs1800795)gene wasamplifiedby polymerase chain reaction (PCR) resulting in a 99bp fragment. The PCR mixture contained a mixture of a buffer solution pH 8.0 1×, 1.5mM of MgCl2, 8mM each of

deoxyribonu-cleotidetriphosphates,1␮Mofeachprimerand1UTaqDNA

polymerase (Invitrogen, Carlsbad, CA, USA). The primers usedwere F:5′_{-TTGTCAAGACATGCCAAGTGCT-3}′ _andR:5′ -GCCTCAGAGACATCTCCAGTCC-3′ _{(Invitrogen,Carlsbad,CA,} USA).27

PCRamplificationwasperformedinathermocycler (TC-512 Techne resistance, Burlington, NJ, USA) under the followingconditions:initialdenaturationat95◦_{Cfor 5min} followedby30cyclesof95◦_Cfor1min,56◦_Cfor1minand 72◦_{Cfor1min,andfinalextensionat72}◦_Cfor5min.

The PCR product was digested with 2U of restriction enzymeNlaIII(Invitrogen,Carlsbad,CA,USA) overnightat 65◦_{C.Thedigestedfragmentswereseparatedon2%agarose} gel(InvitrogenLifeTechnologies,SãoPaulo,Brazil).A sam-plewithknowngenotype wasusedaspositivecontroland ultra-purewaterwasusedasnegativePCRcontrol.

The100-bpDNAmolecularweightmarker(Ladder, Invi-trogen) was included on each gel stained with SybrSafe (InvitrogenLifeTechnologies,SãoPaulo,Brazil)and visual-izedunderUVillumination.Thereadingandinterpretation ofagarosegelsweremadewithLabImageL-PIX(HE)1D-L340 (Loccus Biotechnology, São Paulo, Brazil) program. Frag-mentsof13,227and59bp(alleleG),andfragmentsof13, 118,109and59bp(alleleC)wereobserved.

Statisticalanalysis

Thestatisticalanalysiswasperformedusingthestatistical packageSPSS17.0(SPSSInc.,Chicago,IL,USA).Totestthe association between genotype frequency and hearing loss related to a history of occupational noise exposure, the chi-squaretestwasperformed.TheHardy---Weinberg equi-libriumwastestedineachgroupusingthechi-squaretest. Theoddsratio(OR)withaconfidenceintervalof95%(95% CI)wascalculated.Thesignificancelevelwassetat5%.

Results

Ofa totalof 343,the moleculargeneticprocedureswere performedin191elderly.Ofthese,31.9%reportedahistory of noise exposure, with 18.8% exhibiting hearing impair-ment.Ofthe68.1%withnohistoryofnoiseexposure,59.2% hadhearingloss.Themeanagewas67.8±5.3years,with ahigherproportion(58.6%)ofelderlywomen(Table1).

Of the assessed elderlyindividuals, 56% were homozy-gous for the G allele, 7.8% were homozygous for the C alleleand 36.2% were heterozygous (Table 1). The geno-typedistributionfor IL-6gene wasin agreementwiththe Hardy---Weinbergequilibrium(p>0.05).

Table1 Generalcharacteristics,allelicandgenotypic fre-quenciesamongBrazilianelderly(n=191).

Characteristics n %

Gender

Male 79 41.4

Female 112 58.6

Age(years)

60---64 63 33.0

65---74 104 54.5

75or+ 24 12.6

Mean67.8(SD=5.3)

Historyofnoiseexposure

Nohistoryofnoiseexposure

Nohearingloss 17 8.9

Withhearingloss 113 59.2

Withhistoryofnoiseexposure

Nohearingloss 25 13.1

Withhearingloss 36 18.8

Genotypicfrequency

GG 107 56.0

CC 15 7.8

GC 69 36.2

Allelicfrequency

G 283 74.1

C 99 25.9

Discussion

Although the mechanism and role of proinflammatory cytokines in NIHL are not well understood, it is known

that the structure and expression of cytokines may be affected by genetic variation, such as single nucleotide polymorphisms(SNPs),whichresultsinevidentpathological consequences.15 _{SNP functionality regarding gene} expres-sionisanimportantsubjectindisease-associatedstudies.16 Inthepresentstudy,thefrequenciesofallelesGandC inthestudypopulationwere74.1%and25.9%,respectively. Otherstudieshavealsodemonstratedapredominanceofthe ancestralallele(G)rangingfrom60%to70%.28_Furthermore, weobservedahigherfrequencyofGGgenotype(56%), fol-lowedbyGC(36.2%)andCC(7.8%).Thisisconsistentwith theaforementionedstudies.20,28

We observed an association between the GG genotype of IL-6and hearingloss withhistoryof exposure to occu-pational noise (2=40.201; p<0.001). The elderly with

the CC genotype had lower susceptibility to hearing loss due tooccupational noise exposure (OR=0.0124; 95% CI: 0.0023---0.0671;p<0.01)comparedtoelderlypatientswith the GG genotype. The presence of theC allele results in lower expression of IL-6 after an inflammatory stimulus, whencomparedwiththeGallele.29_{Itissuggestedthatthe} CCgenotypeconfersaprotectiveeffectagainstthe develop-mentofcomorbidities,whichcanbeverifiedinthisanalysis, inwhichthefrequencytheCalleleinthehearinglossgroup withoutexposuretonoisewashigherthanintheNIHLgroup. ThestudiesbyHiroseetal.6_{andFujiokaetal.}11_pointed outthepossibilityofinflammatorychangesinthe cochlea after noise stimuli. Fujioka et al.11 _{demonstratedfor the} first time the induction of pro-inflammatory cytokines in the cochlea exposed tothe noise andobserved increased expression of interleukins TNF-␣, IL-1␤ and IL-63h after noiseexposure.Theseauthorspointedoutthatthisisa self-protection mechanism against exposure to large amounts ofnoiseandthattheconsequentover-expressionof inter-leukinsforlongperiodsmustworsencochlearfunction.

Table2 Associationbetweengenotypefrequencyfortheinterleukin-6G-174Cgenepolymorphismandhearinglossrelatedto historyofoccupationalnoiseexposure(n=191).

Occupationalnoiseexposure Genotyping Nohearingloss

n(%)

Withhearingloss

n(%)

pvalue

Nohistory Genotypicfrequency 0.961a

GG 09(52.9) 62(54.9)

CC 01(05.9) 08(07.0)

GC 07(41.2) 43(38.1)

Allelicfrequency 0.8696b

G 25(73.5) 167(76.9)

C 09(26.5) 59(23.1)

Withhistory Genotypicfrequency <0.01c

GG 03(12.0) 33(91.7)

CC 04(16.0) 02(05.5)

GC 18(72.0) 01(02.8)

Allelicfrequency <0.01d

G 24(48.0) 67(93.0)

C 26(52.0) 05(07.0)

a

2=0.078.

b

2=0.027.

c

2=40.201.

d

ThefindingsbySoetal.12_{clearlydemonstratedthatIL-6} acts asan inducer of acutecochlear damage,considering thatatransientup-regulationofIL-6occurredafter expo-sure of animals with cisplatin. Some studies have shown that the inhibition of IL-6 production hasa cochlear pro-tectiveeffect.Wakabayashietal.21_{usedanti-IL-6(MR10-1)} antibodytoinhibitIL-6productioninmice,identifyinga pro-tectiveeffectfor noise-inducedcochlearinjury.Alongthis samelineofinvestigation,Nakamotoetal.14_foundthatthe druggeranyl---geranyl---acetone(GGA)stimulatesexpression oftheHSFgenewhich,inturn,inhibitsinflammationinthe cochlea,protectingtheinnerear.

Itisnoteworthythatthisstrongevidenceofthe involve-mentofIL-6withNIHLhasbeenverifiedbyseveralstudies in animal models.11---14 _{Evidence in human beings related} to pro-inflammatory interleukins and NIHL was recently reportedbyourgroup, whenwe investigatedthe associa-tionof polymorphismsin theIL-1␤ geneand NIHL.30 _That studydemonstrated thatthepolymorphism inthisgeneis notassociatedwithNIHLin theassessedelderly subjects. However,thefindingsofthepresentstudyshowedthat poly-morphismsintheIL-6geneshouldcontributetotheriskof NIHLintheBrazilianelderly.

Consideringthiscontext,theidentificationofthe poly-morphism in the IL-6 gene in patients with a history of hearinglossrelatedtooccupationalexposuretonoisemay helpustounderstandindividualvariabilityofinflammation resultinginhearingloss,aswell as,in thefuture,to sug-gestgenotypingindividualsforthisparticularpolymorphism (rs1800795)inordertodetermineindividualsusceptibility, providinganewstrategyforthepreventionofhearingloss relatedtonoiseexposure.

Conclusions

AnassociationbetweenthepresenceofIL-6gene polymor-phismand hearinglossassociatedwithoccupationalnoise exposurewasfound inelderlyBrazilians.Studiesbasedon large populationsand withdifferentethnicitiesshould be performedtoconfirmourfindings.

Funding

This study was funded by FUNADESP / Brazil (Fundac¸ão NacionaldeDesenvolvimentodoEnsinoSuperiorParticular).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

References

1.ShresthaLB.Populationagingindevelopingcountries.Health Affairs.2000;19:204---12.

2.KinsellaK, HeW.Anagingworld:2008.USA:CensusBureau, Washington;2009.

3.VerasR.Populationagingtoday:demands,challengesand inno-vations.RevSaudePúb.2009;43:548---54.

4.NudelmannAA,CostaEA,SeligmanJ,Iba˜nezRN.Perdaauditiva induzidapeloruído.PortoAlegre:Bagagem;1997.

5.KeithleyEM,WangX,BarkdullGC.Tumornecrosisfactoralpha caninducerecruitmentofinflammatorycellstothecochlea. OtolNeurotol.2008;29:854---9.

6.Hirose K, DiscoloCM, Keasler JR, RansohoffR. Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma.JCompNeurol.2005;489:180---94.

7.SatohH,FiresteinGS,BillingsPB,HarrisJP,KeithleyEM.Tumor necrosisfactor-alpha,aninitiator,andetanercept,aninhibitor ofcochlearinflammation.Laryngoscope.2002;112:1627---34. 8.JokayI,PappZ,SoosG,SziklaiI,DezsoB.Theeffectofchronic

otitismediaontheimmunoreactivityofhumaninnerear.Eur ArchOtorhinolaryngol.2001;258:529---32.

9.OkanoT,NakagawaT,KitaT,KadaS,YoshimotoM,Nakahata T,etal.Bonemarrow-derivedcellsexpressingIba1are consti-tutivelypresentasresidenttissuemacrophagesinthemouse cochlea.JNeurosciRes.2008;86:1758---67.

10.TanBT,LeeMM,RuanR.Bonemarrow-derivedcellsthathome to acoustic deafened cochleapreserved their hematopoietic identity.JCompNeurol.2008;509:167---79.

11.FujiokaM,KanzakiS, OkanoHJ,MasudaM,OgawaK,Okano H.Proinflammatorycytokinesexpressioninnoise-induced dam-agedcochlea.JNeurosciRes.2006;83:575---83.

12.So H,Kim H, LeeJH, ParkC,Kim Y,Kim E,et al. Cisplatin cytotoxicityofauditorycellsrequiressecretionsof proinflam-matorycytokinesviaactivationofERKandNFkappaB.JAssoc Res.2007;8:338---55.

13.Wakabayashi K, Fujioka M, Kanzaki S, Okano HJ,Shibata S, Yamashita D, et al. Blockade of interleukin-6 signaling sup-pressedcochlearinflammatoryresponseandimprovedhearing impairment in noise-damaged mice cochlea. Neurosci Res. 2010;66:345---52.

14.Nakamoto T, Mikuriya T, Sugahara K, Hirose Y, Hashimoto T, Shimogori H, et al. Geranylgeranylacetone suppresses noise-inducedexpressionofproinflammatorycytokinesinthe cochlea.AurisNasusLarynx.2012;39:270---4.

15.Satoh H, Firestein GS, Billings PB, Harris JP, Keithley EM. Proinflammatory cytokine expression in the endolymphatic sac during innerear inflammation.J Assoc Res Otolaryngol. 2003;4:139---47.

16.DinareloCA.Interleukin-1inthepathogenesisandtreatmentof inflammatorydiseases.Blood.2011;117:3720---32.

17.HwangJH,ChenJC,YangSY,WangMF,ChanYC.Expressionof tumornecrosisfactor-␣andinterleukin-1␤genesinthecochlea andinferiorcolliculusinsalicylate-inducedtinnitus.J Neuroin-flamm.2011;8:30.

18.SanttilaS,SavinainenK,HurmeM.PresenceoftheIL-1RAallele 2 (IL1RN*2)is associated withenhanced IL-1beta production invitro.ScandJImmunol.1998;47:195---8.

19.BioqueG, CrusiusJB,KoutroubakisI,BoumaG, KostensePJ, MeuwissenSG,etal.AllelicpolymorphisminIL-1betaandIL-1 receptorantagonist(IL-1Ra)genesininflammatorybowel dis-ease.ClinExpImmunol.1995;102:379---83.

20.CostaAM,GuimarãesMCM,DeSouzaER,NóbregaOT,Bezerra ACB.Interleukin-6(G-174C)andtumournecrosisfactor-alpha (G-308A)genepolymorphismsingeriatricpatientswithchronic periodontitis.Gerodontology.2010;27:70---5.

21.Raosoft Sample Size Calculator [Internet]. Available from: http://www.raosoft.com/samplesize.html (accessed 10.06.13).

22.SpirdusoWW.Physicaldimensions ofaging.2nd ed.Barueri: Manole;2005.p.75---80.

23.NationalCommitteeonNoiseandHearingConservation. Rec-ommendationsfortheassessmentofdamagecausedbyNoise InducedHearing Loss.Acta AWHO.1996;16:45[Lettertothe Editors].

25.Manual de Procedimentos em Audiometria Tonal Limiar, LogoaudiometriaeMedidasdeImitânciaAcústica.http://www. fonoaudiologia.org.br/publicacoes/ManualdeAudiologia.pdf (accessed18.04.13).

26.OlerupO, ZetterquistH.HLA-DRtypingbyPCRamplification withsequence-specificprimers(PCR-SSP)in2hours:an alter-native to serological DR typing in clinicalpractice including donor---recipientmatchingincadaverictransplantation.Tissue Antigens.1992;39:225---35.

27.OlomolaiyeO,WoodNA,BidwellJL.AnovelNlaIIIpolymorphism inthehumanIL-6promoter.EurJImmunogenet.1998;25:267. 28.TrevilattoPC,Scarel-CaminagaRM,DeBritoJrRB, DeSouza AP, Line SRP. Polymorphism at the position −174 of IL-6 is

associated with susceptibility to chronic periodontitis in a Caucasian Brazilian population. J ClinPeriodontol. 2003;30: 438---42.

29.Fishman D, Faulds G, Jeffey R, Mohamed-Ali V, Yudkin JS, HumphriesS,etal.Theeffectofnovelpolymorphismsinthe interleukin-6(IL-6)geneonIL-6transcriptionandplasmaIL-6 levels,andanassociationwithsystemic-onsetjuvenilechronic arthritis.JClinInvestig.1998;102:1369---76.