Interleukin-10 gene polymorphism (-1082G/A) and allergy to efavirenz in patients infected with human immunodeficiency virus

braz j infect dis.2014;18(4):445–448

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Brief

communication

Interleukin-10

gene

polymorphism

(

−1082G/A)

and

allergy

to

efavirenz

in

patients

infected

with

human

immunodeficiency

virus

Raphael

de

Oliveira

Rodrigues

_,

_Paulo

_Germano

_de

_Carvalho

_,

Érico

Antônio

Gomes

de

Arruda

_,

_Silvia

_Helena

_Barem

_Rabenhorst

_,

Silvia

Fernandes

Ribeiro

da

Silva

_,

_Ilana

_Farias

_Ribeiro

_,

_Denise

_Girão

_Limaverde

_Lima

_,

Aparecida

Tiemi

Nagao-Dias

a_{DepartmentofClinicalAnalysisandToxicology,FacultyofPharmacy,UniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil} b_{HealthSciencesCenter,UniversidadedeFortaleza,Fortaleza,CE,Brazil}

c_{HospitalSãoJosédeDoenc¸asInfecciosas,Fortaleza,CE,Brazil}

d_{LaboratoryofMolecularGenetics,DepartmentofPathology,UniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil}

e_{LaboratoryofHistocompatibilityandTransplantationImmunology,ResearchCenterinHepatorenalDiseases,UniversidadeFederaldo} Ceará(UFC),Fortaleza,CE,Brazil

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Articlehistory:

Received11November2013 Accepted6January2014 Availableonline10May2014

Keywords: HIV Drugallergy Polymorphism IL-10

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Theaimofthepresentstudywastoinvestigatetheassociationbetweenpolymorphism intheinterleukin-10genepromoteratposition−1082inhumanimmunodeficiency virus-infectedpatientswhohadpresentedallergicreactionduetoefavirenz.Thestudyincluded 63patientstreatedattheHospitalSãoJosédeDoenc¸asInfecciosas,Fortaleza,Ceará,Brazil. Twenty-onepatientswhohadpresentedallergicreactiontoefavirenzwerecomparedto 42patientswithnoallergicreactionfollowingexposuretothisdrug.Bloodsampleswere collectedforDNAextractionandsubmittedtotherestrictionfragmentlength polymor-phism–polymerasechainreactiontechnique.The−1082AA genotypewassignificantly morefrequentinallergicpatientsascomparedtonon-allergicpatients(p=0.019;2_=5.534;

OR=3.625;95%CI=1.210–10.860).LikewisethealleleIL-10−1082Awasidentified signifi-cantlymoreoftenamongefavirenzallergicpatientsthaninthenon-allergicgroup(p=0.009; 2_{=6.787;OR=3.029;95%CI=1.290–7.111).Thesefindingssuggestthatthepolymorphism}

intheinterleukin-10genepromoter−1082G/Acanberelatedtothedevelopmentofallergic reactionstoefavirenz.

©2014ElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthor.}

E-mailaddress:raphaelolrodrigues@gmail.com(R.d.O.Rodrigues). http://dx.doi.org/10.1016/j.bjid.2014.01.009

446

Accordingto theWorld Health Organization (WHO),the term adverse drug reaction (ADR) can be defined as any noxious or unintended reaction that appears after drug administration at standard doses, for the purpose of pro-phylaxis, diagnosis and treatment of a disease.1 _Clinical manifestationsofadversereactionstoantiretroviraldrugscan affectindividualsonvariouslevelsofseverity.Themost com-monADRsoccurearlyintherapyandincludegastrointestinal effects,suchas nausea,bloating, anddiarrhea, whichmay be transient or persistent. Less common reactions include Zidovudine AZT-associated anemia and hypersensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs).2 It isknown that viral infections, such asthose caused by Epstein–Barrvirus(EBV)andhumanimmunodeficiencyvirus (HIV),constituteincreasedriskfactorsforthedevelopmentof drughypersensitivity.InthecaseofHIV,studieshaveshown thatthefrequencyofdrughypersensitivityisestimatedtobe intherangeof3–20%,andthatskinrashesrelatedtodrugs are 100 timesmorefrequent inHIV-infectedpatients than inthe generalpopulation.3 _{Themainadverseeffects} asso-ciatedwith efavirenz, a drug which belongs to the NNRTI class, are due to disorders of the central nervous system (CNS),suchasdizziness,drowsiness, headache,inability to concentrate,nightmares,and depression.Theseeffectscan beobserved inapproximately 40% ofpatients inthe early daysorweeksoftreatment.4 _{Inadditiontotheseeffects,it} isreportedthatdrugrashesoccurin27%oftheadultsand 45% ofthe children, occurringaround the second weekof treatment.5 _{Theincreasedfrequencyofdrugallergy inHIV} patientscanbeattributedtoimmunesystemdysregulation orvulnerabilitytooxidativestress.Drugexposureisa neces-sarycomponent,butnottheonlycausativeagentthatinduces drugallergy.Allergyalsodependsonindividualsusceptibility. Inthis way,individualsusceptibilityisthoughttobe multi-factorial,includinggeneticfactors.3,5_{Somestudiesofgenetic} predispositiontoantiretroviralallergyhaveshownastrong associationbetweenthepresenceofHLA-B*5701and hyper-sensitivitytoabacavir.6,7_{Thus,screeningforthisallelebefore} the initiation of antiretroviral therapy could be important intheidentificationofpatientsatincreasedriskforallergic reactiontoabacavir.8_{Othergeneticstudieshaveshownthat} polymorphismofgenesthatregulatecytokineproductioncan affecttheindividualimmuneresponse.9

Interleukin-10 (IL-10) isan importantanti-inflammatory cytokine secreted byvarious cells ofthe immune system, including Tlymphocytes, macrophages, dendriticcells and monocytes.Thiscytokinehasimmunoregulatoryeffectssuch asinhibitionofpro-inflammatorycytokinesIL-1,IL-6,IL-12, IL-18andtumornecrosisfactorTNF,aswellasco-stimulatory molecules on antigen-presenting cells.10 _{Single nucleotide} polymorphisms (SNP) atpositions −1082(A/G), −819 (T/C), −592(C/A)oftheproximalpromoterregionoftheIL-10gene may affect the cytokine in vitro production.11,12 _The pres-enceoftheAalleleatposition−1082seemstobeassociated with low levels of IL-10 production, and occurs indepen-dentlyofpolymorphismsatotherpositions.11_Theaimofthe presentstudyistoinvestigatetheassociationbetween aller-gicreactiontoefavirenzandpolymorphisminthepromoter regionoftheIL-10geneatposition−1082G>AinHIV-infected patients.

Thisisacasecontrolstudyinvolving63HIV-infectedadult patients on Highly Active Antiretroviral Therapy (HAART) receiving care at Hospital São José Infectious Diseases in Fortaleza, Ceará, Brazil. Case definition of drug allergy to efavirenzincludedrash,skineruption,urticaria,and/or ery-themafollowingexposuretoefavirenz.Additionally,efavirenz withdrawalledtoacompleteremissionoftheallergicreaction. The assistant physicians of patients suspect of having an allergic reaction to efavirenz were to complete a drug change/modificationrequestform(ADRreportinginform)in ordertodiscontinueefavirenzandswitchtoanotherregimen. From2006to2012,therewere84patientswithdrugreaction reportingformswhowerecontactedovertelephonecallsand invitedtoparticipateinthestudy.Ofthose,only21eligible patientswerelocatedandagreedwithbloodcollection.There wereninemalesand12females,aged24–71years.Thecontrol groupconsistedof32malesand10females,aged20–67years, HIV-infectedpatientsonHAART-containingefavirenz forat leastsixmonthswithoutanyevidenceofadversecutaneous reactions tothe drug.All the participantssignedawritten informed consent. Theproject wasapprovedby the Ethics CommitteeinResearch,HospitalSãoJosédeDoenc¸as Infec-ciosas,number025/2011.

Venipunctureof4mLofwholebloodwascollectedinatube containingethylenediaminetetraaceticacidEDTAasan anti-coagulant.DNAwasextractedusingacommercialextraction kit,BiopurExtractionKitMiniPlusSpin–250(Biopur,Brazil), accordingtothemanufacturer’sinstructions.Polymorphism ofIL-10(−1082G>A)wasdetectedbyPCR-RFLP,aspreviously described byKochetal.13 _{Thereactionwasperformedina} totalvolumeof20␮Lreactionsolution,usingTopTaqMaster Mix(Qiagen,Germany)withadditionof0.01%bovineserum albumin (BSA), primer at concentration of 20pmol/␮L and approximately25ngofDNAtemplate.Thepolymerasechain reaction(PCR)wasperformedinathermocycler(Eppendorf, Germany)underthefollowingconditions:initialdenaturation at94◦Cfor4min,followedby35cyclesof94◦Cfor40s,56◦C for35sand72◦Cfor40s.Thefinal extensionwasdoneat 72◦Cfor5min.ThePCRproductsof377bpwerethendigested withrestrictionendonucleaseXagI(Fermentas,Lithuania)at 37◦Cfor16h.The280and97bpfragmentscorrespondedtothe −1082Aalleleand253,97and27bptothe−1082Gallele.The analysisofthedigestionproductswasdonebyelectrophoresis on6%polyacrylamidegelaftersilverstaining(Fig.1).

Thestatistical analyseswere performed usingthe com-mercialsoftwareGraphPadPrismversion5.00forWindows, (GraphPadPrism,Inc.,LaJolla,CA,USA).TheChi-squaretest wasusedtotestfortheassociationbetweenefavirenzallergy andgenotypicandallelicfrequencies.Oddsratio(OR)witha 95%confidenceintervalwascalculated.Ap-value<0.05was consideredofstatisticalsignificance.

Intheanalysisoftheallelefrequency,astatistically sig-nificant difference was found comparing case and control groups(p=0.009,Table1).AhigherfrequencyoftheAallele wasfoundintheefavirenz-allergicgroup.ThegenotypeAA at the position −1082 for IL-10 was much more frequent among efavirenz-allergic patients than in the non-allergic group (p=0.019, Table1). TheORindicatedthat the proba-bility thatan individual would presentan allergicreaction tothedrugwas3.625timesgreaterwhentheincaseofAA

brazj infect dis.2014;18(4):445–448

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Lane M: 100bp marker; Lane A: PCR product 377bp; Lanes 1-4: Samples -1082AA homozygote; Lanes 5-6: Samples -1082GA heterozygote.

Fig.1–PCR-RFLPforanalysisofthepolymorphismIL-10genepromoter(−1082G/A).

genotype. AAgenotype wasidentified in62% of efavirenz-allergicpatients.Ontheotherhand,thegenotypeGGorGA attheposition−1082forIL-10wasidentifiedin69%of non-allergicpatients.AccordingtoTurneretal.,11_{thecarriageof} theAAgenotypeatposition−1082isoneofthefactorswhich determineslower levels ofIL-10.Our resultsdemonstrated thatindividualswiththepresenceofthe−1082Aalleleand the −1082AAgenotype are moreprone to suffer allergy to efavirenz.

Itisobservedthatsomepatientstreatedwithvariousdrugs mayexhibitvariabilityintherapeuticresponsetothedrugand mayhavesusceptibilitytosomeadversereactions. Pharmaco-geneticsisgainingwideapplicabilityinclinicalpharmacology oncethestudyofgeneticfactorsmaycontributetothe effec-tivenessandsafetyofdrugtherapy.14,15

Saag et al.7 _{evaluated the detection ofHLA-B*5701 as a} markerforhypersensitivitytoabacavirafterconfirmationof drugallergybypatchtestinginBlackandCaucasianpatients livingintheUnitedStates.Theresultsofthesurveyshowed 100%sensitivityforHLA-B*5701asamarkerofdrugallergy toabacavirinbothgroups.Someotherstudiessuggestthat screeningforHLA-B*5701shouldbeperformedbefore start-ingtheabacavirtherapy,therebyreducingtheriskofallergic reactionstothisdrug.8

Several studies associating polymorphism in the pro-moterofIL-10andincreasedpathogenesisinsomeinfectious diseases have been carried out. Patients with toxoplasmic retinochoroiditisweremorefrequentlycarriersofthe−1082A allele (AA+GA genotypes) than controls. It was also sug-gestedthatthegenotypeGAwasassociatedwithtoxoplasma retinochoroiditis.16 _{Inanother study, itwas foundthat the}

polymorphisminthepromoterofIL-10(−1082G>A)was asso-ciatedwiththedevelopmentofcardiomyopathyinindividuals withChagasdisease.Furthermore,thisstudycould demon-stratethatlowexpressionofthecytokinewasassociatedwith worsecardiacfunction.17

TheassociationofthepolymorphismofIL-10with hyper-sensitivity reactions to drugs is promising. A study of polymorphismsinthepromoterofIL-10atthepositions−819 and −592 wasconducted inthe French population. It was foundthatatopicwomenpresentingthegenotypesCTorTT attheposition−819andthegenotypeCAorAAattheposition −592hadhigherriskfordevelopingimmediate hypersensitiv-ityreactionto␤-lactams.Thisfindingisrelevantbecausethe alleles−819Tand−592Aareassociatedwithlower expres-sionofIL-10.InregardtotypeIhypersensitivity,asIL-10has animmunomodulatoryactivity,itsreductionenablestheTh2 profile,withconsequentstimulationofIL-4, IL-9,andIL-13. ThesecytokinesfavortheproductionofIgEwhichculminates withimmediatehypersensitivity.18

Qiao et al.19 observed an association between levels of specificIgE,cytokinesandpolymorphismsofIL-4C/Tand IL-4R␣Q576Rinpatientswithpenicillinallergy.Accordingtothe authors,thealleleIL-4R␣*Q576waspresentmorefrequently inallergicpatientsthanincontrolsubjects.Furthermore,it wasdemonstratedthatthealleleIL-4R␣*Q576wasassociated withtheexpressionofsometypesofpenicillin-specificIgE, whichincludedbenzylpenicilloyl,phenoxomethylpenicillanyl andampicillanyl.

Thedifferentformsofexanthems(rashes)arethemost fre-quentdrugreactions.Someappearrapidlyafterdrugintake, for example, immediate-type reactions like urticaria and

Table1–GenotypicandallelicfrequenciesofIL-10−1082G>Aamongpatientswhopresentedallergytoefavirenzand amongcontrols.

Polymorphism Genotypicfrequency(%) Allelicfrequency

Genotype Allergic Controls pvalue Allele Allergic Controls pvalue

AA 13(61.90%) 13(30.95%) 0.019 A 0.79 0.55 0.009

IL-10(−1082) GA 07(33.34%) 20(47.62%) G 0.21 0.45

GG 01(4.76%) 09(21.43%)

Genotypicfrequency:2_{=5.534;oddsratio(OR)=3.625;95%confidenceinterval(CI)=1.210–10.860.}

Thep-valuefor genotype frequency iscalculatedbyIL-10 −1082AA vs IL-10−1082GA/GG. Allelic frequency: 2_{=6.787;OR=3.029; 95%}

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angieodema.Othersappearhoursordaysafterdrugintake; theyarethe so-calleddelayed-typereactions andcomprise abroad spectrumofclinical and distinct histopathological features.20_{Inourstudy,thehighfrequencyof−1082AA} geno-typeintheIL-10gene promoterisprobablyenabling TCD4 Th1and/orTh2profiles.ReductionofIL-10levelspromotes increasedactivityofCD4+TlymphocytesoftheTh1andTh2 profiles,whichwouldproduceagreaterquantityofIFN-␥and IL-4respectively.12

Somelimitationsofourstudyare relatedtothehigh of refusalrateofpatientstojointhestudy.Variousfactorsappear toberelevant, suchas lowsocioeconomic status, distance fromthepatient’sresidencetothehospital,inabilityto under-stand the purpose of the study, fear of exposinghis (her) diseasestatustoothers.Wecouldnotperformskinteststo confirmdrugallergytoefavirenzbecauseoflogisticalreasons [severalreadingsinthecaseofepicutaneoustests(24h,48h and72h)].

Ourdatacouldsuggestthatpolymorphismsinregulatory regionsofcytokineproductionareamongthefactorsthatmay contributetoanindividualsusceptibilitytodrugallergy.Other studiesarenecessarytoproveourfindings.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ThisstudywasfinanciallysupportedbytheConselhoNacional deDesenvolvimentoCientíficoeTecnológico(CNPq,process 554970/2010-4).

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