Prevalence of IFNL3 gene polymorphism among blood donors and its relation to genomic profile of ancestry in Brazil

brazjinfectdis2016;20(6):619–622

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Prevalence

of

IFNL3

gene

polymorphism

among

blood

donors

and

its

relation

to

genomic

profile

of

ancestry

in

Brazil

Silvia

Renata

Cornelio

Parolin

Rizzo

_,

_Diana

_Gazito

_,

_Henrique

_Pott-Junior

_,

Flavia

Roche

Moreira

Latini

_,

_Adauto

_Castelo

a_{Associac¸ãoBeneficentedeColetadeSangue(Colsan),SãoPaulo,SP,Brazil}

b_{UniversidadeFederaldeSãoPaulo(Unifesp),DepartamentodeMedicina,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3January2016 Accepted13October2016 Availableonline25October2016

Keywords:

Ancestryinformativemarkers ChronichepatitisCvirusinfection Direct-actingantivirals

Singlenucleotidepolymorphisms

a

b

s

t

r

a

c

t

Therecentdevelopmentofinterferon-freeregimensbasedondirect-actingantiviralsfor thetreatmentofchronichepatitisCvirusinfectionhasbenefitedmanybutnotallpatients. Somepatientsstillexperiencetreatmentfailure,possiblyattributedtounknownhostand viral factors,suchasIFNL3gene polymorphism.Thepresentstudy assessedthe preva-lenceofrs12979860-CC,rs12979860-CT,andrs12979860-TTgenotypesoftheIFNL3gene, anditsrelationshipwithancestryinformativemarkersin949adultBrazilianhealthyblood donors.Racewasanalyzedusingancestryinformativemarkersasasurrogatefor ances-try.IFNL3genewasgenotypedusingtheABITaqMan singlenucleotidepolymorphisms genotypingassays.Theoverallfrequencyofrs12979860-CCgenotypewas36.9%.The contri-butionofAfricanancestrywassignificantlyhigheramongdonorsfromthenortheastregion inrelationtosoutheastdonors,whereastheinfluenceofEuropeanancestrywas signifi-cantlyhigherinsoutheastdonors.Donorswithrs12979860-CCandrs12979860-CTgenotypes hadsimilarancestrybackground.ThecontributionofAfricanancestrywashigheramong rs12979860-TT genotypedonors in comparisonto both rs12979860-CC and rs12979860-CTgenotypes.Theprevalenceofrs12979860-CCgenotypeissimilartothatfoundinthe US,despitetheBrazilianancestryinformativemarkersadmixture.However,intermsof ancestry,rs12979860-CTgenotypewasmuchclosertors12979860-CCindividualsthanto rs12979860-TT.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:acastelof42@gmail.com(A.Castelo). http://dx.doi.org/10.1016/j.bjid.2016.10.002

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

620

Introduction

ChronichepatitisC(CHC)virusinfectionisaglobaldisease that imposes a major global health burden. In a recently publishedstudy,theworldprevalenceofanti-HCVwas esti-matedat1.6%(1.3–2.1%),correspondingto115(92–149)million infections.1

Evidence suggests that the ability to controlviral repli-cation is related to viral and host factors. Several studies havedemonstratedthatastronginitialinnateandadaptive immuneresponseagainsthepatitisCvirus(HCV)favorsviral clearance.2–4_{Thus,variationingenesinvolvedintheimmune}

responsemaycontributetotheabilitytoclearthevirus.2

In 2009, single nucleotide polymorphisms (SNP) of the IFNL3gene(rs12979860)wererelatedtonaturalclearanceof HCV.3,5_{Accordingtothisgenome-widestudy,rs12979860-CC}

genotype was associated with spontaneous HCV infection resolutionamongindividualsofbothEuropeanandAfrican ancestry.FurtherinvestigationshaveimplicatedIFNL3gene polymorphismsinthedevelopmentofspontaneous resolu-tionofacuteinfectionandtherapeuticcureofHCV-infected patientstreatedwithinterferon-basedregimens.6

Before2011,interferon-based therapywasthe standard-of-carefor treatingHCV. Recently,interferon-freeregimens basedondirect-actingantivirals(DAAs)haverevolutionized thetreatmentofCHC.Comparedtoconventional interferon-basedregimens,DAAshavedemonstratedgreatersustained virologicalresponse(SVR)rates,shortertreatmentduration, increasedtolerability,andlowerincidenceofadverseevents.7

Nonetheless,somepatientsstillexperiencetreatmentfailure duetoon-treatmentviralbreakthroughorrelapseafter cessa-tionoftherapy,possiblyattributedtounknownhostandviral factors,suchasIFNL3genepolymorphism.

Towardthatend,datafromarecentlypublishedsubgroup analysis of only eight weeks of ledipasvir plus sofosbuvir forgenotype1HCVshowed SVRrates varyingsignificantly accordingtors12979860genotype.Inrs12979860-CCgenotype patientsSVRratewereinexcessof98%,whereasSVRrates were 95.1% and 90.9%,inrs12979860-CT and TT genotype, respectively(p=0.02).Thoseresultsprompteddiscussionon therelationshipofpolymorphismsneartheIFNL3geneand SVRtoDAA-basedregimens.8

PatientsofEuropeanancestryhaveasignificantlyhigher probability of being cured with interferon-based regi-mens than patients of African ancestry. The frequency of rs12979860-CCgenotypeissubstantiallygreaterinEuropean thaninAfricanpopulations.Thisgeneticpolymorphismalso explains approximately half of the difference in response ratesbetweenAfrican-Americansand patientsofEuropean ancestry.5

Brazilians are one of the most heterogeneous popula-tionsintheworld,resulting offivecenturiesofinterethnic crosses between European, Africans, and autochthonous Amerindians.9 _{A growing number of publications have}

reported the use of ancestry-informative markers (AIMs) whose allele frequency varies significantly between popu-lations of distinct geographic origins in order to estimate individualadmixtureandtoidentifypopulationsubstructure. In moststudies, theseAIMs consisted ofsingle-nucleotide

polymorphisms(SNPs),butinsertion-deletionpolymorphisms (INDELs)ofsmallDNAfragmentsandshorttandemrepeats havealsobeenused.10–13

Thus,theobjectivesofthepresentstudyweretoassessthe prevalenceofrs12979860-CC,rs12979860-CT,and rs12979860-TT genotypes of the IFNL3 gene and its relationship with ancestrymarkersinaBrazilianpopulation.

BetweenJanuary2ndandJuly31st2011,949healthyadult blooddonorswereselectedfromtheColsanbloodbank net-work,inSãoPaulo,Brazil.Ofthose,420wereoriginallyfrom thenortheastregionand529fromthesoutheastregion.This studywasapprovedbyResearchEthicsCommitteeofthe Fed-eralUniversityofSãoPaulo(UNIFESP).

Samplesofperipheralbloodfromeachsubjectwere col-lectedin0.5MEDTAtubes,andgenomicDNAwasextracted frommononuclearcellsusingDNAbloodMinikit(QIAamp, Qiagen, Inc.,Valencia,CA, USA)according tothe manufac-turer’sinstructions.

The rs12979860 was genotyped using the ABI Taq-Man SNP genotyping assays (Applied Biosystems, Foster City, CA, USA). Primers that contemplated polymorphism were IL28BFoward: 5 GCCTGTCGTGTACTGAACCA 3 and IL28BReverse:5GCGCGGAGTGCAATTCAAC3(EurofinsMWG Operon,Hunstsville,AL,USA),TaqMan®_{MGBProbes(Applied}

Biosystems,FosterCity,CA,USA)foreachallelewhichwere labeledwithadifferentfluorophore:5TGGTTCGCGCCTTC3 –VICand5CTGGTTCACGCCTTC3–FAM.5_Thetotal

reac-tionvolumewas8.5␮Lcontaining:1.1␮LH2O;genotyping5␮L

TaqManMasterMix(AppliedBiosystems,FosterCity,CA);5pM primers;25pMprobe(AppliedBiosystems,FosterCity,CA)and cycledinPCRmachineReal®_{time7500(AppliedBiosystems,}

FosterCity,CA,USA)accordingtostandardcyclingasfollows: denaturationat95◦Cfor10min,followedby40cyclesof dena-turationat92◦Cfor15s,annealingandextensionat60◦Cfor 1min.TheresultswereanalyzedbyusingSoftwareSystem 7500(AppliedBiosystems,FosterCity,CA).

GeneticancestrywasdeterminedbyanalyzingAIMs.DNA samplesweretypedfor48biallelicINDELsusingthree16-plex PCRreactionsaspreviouslydescribed.10_{DNAfragmentswere}

separatedusinganABIPRISM3130GeneticAnalyzer(Applied Biosystems)andanalyzedwithGeneMapperIDv3.2software (AppliedBiosystems).10

Also, at the time of blood donation participants were askedtoinformtheirperceivedrace,asCaucasianor African-descendent.

Results

and

discussion

Ourstudyevaluatedthelinkbetweenraceandpolymorphisms neartheIFNL3(formerlyknownasIL28B)geneamonghealthy voluntaryblooddonorsusingAIMasasurrogatefor ances-try.Atotalof949blooddonorswereincluded, 420(44.25%) fromthenortheastregionand529(55.75%)fromthe south-eastregion;328(34.6%)femalesand621(65.4%)males.The overallfrequencyofrs12979860-CCgenotypewas36.9%,with a tendency for greater prevalence among donors born in the southeast(39.3%)than inthe northeast(33.8%;p=0.08) regions.TheaverageAIMofAfricanancestrywassignificantly higher amongdonors from the northeast region (0.279) in

brazj infect dis.2016;20(6):619–622

621

relationtothesoutheast(0.225;p<0.001),whereastheaverage AIMofEuropeanancestrywassignificantlyhigherin south-eastdonors.Theancestrybackgroundwassimilar between rs12979860-CCgenotypeandrs12979860-CTgenotypedonors. Africanancestrywashigheramongrs12979860-TTgenotype donorsincomparisontobothrs12979860-CC genotypeand rs12979860-CTdonors.

In the present study, the prevalence of rs12979860-CC, rs12979860-CT, and rs12979860-TT genotypes were 36.9%, 49.1%and14%,respectively.Thers12979860-CCgenotypein southeasterndonors(39.3%)tendedtobemorefrequentthan amongnortheasterndonors(33.8%;p=0.08).Onthecontrary, rs12979860-TTgenotypewasmoreprevalentinnortheastern donors(17.4%)thaninsoutheasterndonors(11.3%;p=0.01). Aprevious Brazilianstudy conductedin thenortheast city ofSalvador,Bahia,evaluated222HCVinfectedpatientsand foundthattheprevalenceofrs12979860-CC(24.4%)waslower, ofrs12979860-TT(20.8%)higher,andofrs12979860-CT(54.7%) wassimilartoourstudyfindings.Theencountered discrep-ancyofrs12979860-CCandrs12979860-TTprevalenceinthe twostudiescouldbeduetohigherrepresentationofblacks inthe Salvador study.Indeed,the self-reported prevalence ofAfrican-descendantsinourstudy(11.4%)wasmuchlower thanthatoftheSalvadorstudy(36.8%).9

A study in 88 Japanese HCV infected patients showed a prevalence of 64.3% of rs12979860-CC and 38.7% of rs12979860-CT genotypes. No rs12979860-TT genotype was encountered.14_{InChina,threelargestudieswithhepatitisB}

infectedpatientsfoundratesofrs12979860-CCgenotypeover 85%andrs12979860-TTgenotypearound5%.14–17

IntheUnitedStates, wheremiscegenationremainsvery low,theprevalenceofrs12979860-TTgenotype amongHCV infected self-reported African descendants is significantly greater than that found in self-reported Caucasians, 37% and 12%, respectively.18 _{Interestingly the prevalence of}

rs12979860-CTgenotypeinCaucasians(49%)wassimilarto thatofAfricandescendants(51%).

TwostudiesconductedinSpainevaluated283and119 HCV-infectedpatients.Theratesofrs12979860-CCgenotypewere 44.7%and41.1%,respectively.19,20 Theseratesaresimilarto thosefoundinGermany(40.2%),21_{butlowertotheprevalence}

foundinEgypt(54%), anintermediaryvaluebetweenrates foundamongAsiansandCaucasians.22

OurresultsshowthatBrazilianadulthealthyblooddonors haveaprevalence(36.7%)ofrs12979860-CCgenotypesimilar to that found in the US, despite its heterogeneous popu-lationmix. Theprevalenceofrs12979860-CT genotypewas alsosimilartothatofother westerncountries.However,in termsofancestry,rs12979860-CTgenotypewasmuchcloser tors12979860-CCindividualsthantors12979860-TT. Accord-ingly, one could question whether the likelihood of viral clearanceamongrs12979860-CTBrazilianindividualsiscloser tothatobservedinrs12979860-CCthaninrs12979860-TT indi-viduals.Thesame wouldalsoapplyfortheHCVtreatment response.

Theoverallfrequencyofrs12979860-TTgenotypeobserved in our study was 14.0%, nearly the same as other west-ern countries. In these cases, treatment failure due to on-treatment viral breakthrough or relapse after cessation oftherapymay be attributedtoan ineffectiveendogenous

interferon responseunabletopromoteviral clearanceand, hence,raisetheemergenceofRAVs.

Despitethelargesamplesize,ourstudyresultsmaynot necessarilyberepresentativefortheentireBrazilian popula-tion.

Finally,theroleofCTgenotypeinviralclearanceand treat-ment response should befurther evaluatedin populations withsignificantandprolongedinterethniccrossessuchasthe Brazilianpopulation.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WeareindebttoColsanforthelogisticsupportandlaboratory tests.

r

e

f

e

r

e

n

c

e

s

1.GowerE,EstesCC,HindmanS,Razavi-ShearerK,RazaviH. Globalepidemiologyandgenotypedistributionofthe hepatitisCvirus.JHepatol.2014;611Suppl:S45–57.

2.ElliotLN,LloydAR,ZieglerJB,FrenchRA.Protectiveimmunity againsthepatitisCvirusinfection.ImmunolCellBiol. 2006;84:239–49.

3.PostJ,RatnarajahS,LloydAR.Immunologicaldeterminants oftheoutcomesfromprimaryhepatitisCinfection.CellMol LifeSci.2009:733–56.

4.DustinLB,CashmanSB,LaidlawSM.Immunecontroland failureinHCVinfection-tippingthebalance.JLeukocBiol. 2014;96:1–14.

5.GeD,FellayJ,ThompsonAJ,etal.GeneticvariationinIL28B predictshepatitisCtreatment-inducedviralclearance. Nature.2009;461:399–401.

6.RembeckK,LaggingM.ImpactofIL28B,ITPAandPNPLA3 geneticvariantsontherapeuticoutcomeandprogressionof hepatitisCvirusinfection.Pharmacogenomics.

2015;16:1179–88.

7.StahmeyerJT,RossolS,KrauthC.Outcomes,costsand cost-effectivenessoftreatinghepatitisCwithdirectacting antivirals.JCompEffRes.2015;11:1–11.

8.O’BrienTR,LangKuhsKA,PfeifferRM.Subgroupdifferences inresponseto8weeksofledipasvir/sofosbuvirforchronic hepatitisC.OpenforumInfectDis.2014;1,ofu110. 9.PenaSDJ,diPietroG,Fuchshuber-MoraesM,etal.The

genomicancestryofindividualsfromdifferentgeographical regionsofBrazilismoreuniformthanexpected.PLoSONE. 2011;6.

10.SantosNPC,Ribeiro-RodriguesEM,Ribeiro-dos-SantosÂKC, etal.Assessingindividualinterethnicadmixtureand populationsubstructureusinga48-insertion-deletion(INSEL) ancestry-informativemarker(AIM)panel.HumMutat. 2010;31:184–90.

11.PritchardJK,StephensM,RosenbergNA,DonnellyP. Associationmappinginstructuredpopulations.AmJHum Genet.2000;67:170–81.

12.FalushD,StephensM,PritchardJK.Inferenceofpopulation structureusingmultilocusgenotypedata:linkedlociand correlatedallelefrequencies.Genetics.2003;164:1567–87.

622

13.PimentaJR,ZuccheratoLW,DebesAA,etal.Colorand genomicancestryinBrazilians:astudywithforensic microsatellites.HumHered.2006;62:190–5.

14.AsahinaY,TsuchiyaK,MuraokaM,etal.Associationofgene expressioninvolvinginnateimmunityandgeneticvariation ininterleukin28Bwithantiviralresponse.Hepatology. 2012;55:20–9.

15.PengLJ,GuoJS,ZhangZ,ShiH,WangJ,WangJY.IL28B rs12979860polymorphismdoesnotinfluenceoutcomesof hepatitisBvirusinfection.TissueAntigens.2012;79:302–5. 16.LeeDH,ChoY,SeoJY,etal.Polymorphismsnearinterleukin

28BgenearenotassociatedwithhepatitisBvirusclearance, hepatitisBeantigenclearanceandhepatocellularcarcinoma occurrence.Intervirology.2013;56:84–90.

17.LiW,JiangY,JinQ,etal.Expressionandgenepolymorphisms ofinterleukin28BandhepatitisBvirusinfectioninaChinese Hanpopulation.LiverInt.2011;31:1118–26.

18.ThompsonAJ,MuirAJ,SulkowskiMS,etal.Interleukin-28B polymorphismimprovesviralkineticsandisthestrongest

pretreatmentpredictorofsustainedvirologicresponsein genotype1hepatitisCvirus.Gastroenterology.2010;139. 19.Montes-CanoMA,García-LozanoJR,Abad-MolinaC,etal.

Interleukin-28Bgeneticvariantsandhepatitisvirusinfection bydifferentviralgenotypes.Hepatology.2010;52:33–7. 20.Rivero-JuárezA,EspejoÁC,Perez-CamachoI,etal.

AssociationbetweentheIL28BgenotypeandhepatitisCviral kineticsintheearlydaysoftreatmentwithpegylated interferonplusribavirininHIV/HCVco-infectedpatientswith genotype1or4.JAntimicrobChemother.2012;67:202–5. 21.SarrazinC,SusserS,DoehringA,etal.ImportanceofIL28B

genepolymorphismsinhepatitisCvirusgenotype2and3 infectedpatients.JHepatol.2011;54:415–21.

22.ShakerOG,SadikNAH.Polymorphismsininterleukin-10and interleukin-28BgenesinEgyptianpatientswithchronic hepatitisCvirusgenotype4andtheireffectontheresponse topegylatedinterferon/ribavirin-therapy.JGastroenterol Hepatol.2012;27:1842–9.