Mitochondrial DNA ancestry, HPV infection and the risk of cervical cancer in a multiethnic

16 Pages·2017·1.95 MB·English

RESEARCHARTICLE
Mitochondrial DNA ancestry, HPV infection
and the risk of cervical cancer in a multiethnic
population of northeastern Argentina
InesBadano1,2☯*,DaianaJ.Sanabria1,2☯,MariaE.Totaro1,SamaraRubinstein3,Juan
A.Gili4,DomingoJ.Liotta1,MariaA.Picconi5,RodolfoH.Campos6,TheodoreG.Schurr3
1 LaboratoriodeBiolog´ıaMolecularAplicada,FacultaddeCienciasExactas,Qu´ımicasyNaturales,
UniversidadNacionaldeMisiones,Misiones,Argentina,2 ConsejoNacionaldeInvestigacionesCient´ıficasy
Te´cnicas(CONICET),BuenosAires,Argentina,3 LaboratoryofMolecularAnthropology,Departmentof
a1111111111
Anthropology,UniversityofPennsylvania,Philadelphia,UnitedStatesofAmerica,4 Laboratoriode
a1111111111
Epidemiolog´ıaGene´tica,Direccio´ndeInvestigacio´nCEMIC-CONICET,BuenosAires,Argentina,5 Servicio
a1111111111 deVirusOncoge´nicos,DepartamentodeVirolog´ıa,INEI-ANLIS“Dr.CarlosG.Malbra´n”,BuenosAires,
a1111111111 Argentina,6 Ca´tedradeVirolog´ıa,FacultaddeFarmaciayBioqu´ımica,UniversidaddeBuenosAires,
a1111111111 BuenosAires,Argentina
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS
Abstract
Citation:BadanoI,SanabriaDJ,TotaroME,
RubinsteinS,GiliJA,LiottaDJ,etal.(2018)
Background
MitochondrialDNAancestry,HPVinfectionandthe
riskofcervicalcancerinamultiethnicpopulationof MisionesProvinceinnortheasternArgentinaisconsideredtobearegionwithahighprevalence
northeasternArgentina.PLoSONE13(1):
ofHPVinfectionandahighmortalityrateduetocervicalcancer.Thereasonsforthisepidemio-
e0190966.https://doi.org/10.1371/journal.
logicaltrendarenotcompletelyunderstood.Togaininsightintothisproblem,weexploredthe
pone.0190966
relationshipbetweenmitochondrialDNA(mtDNA)ancestry,HPVinfection,anddevelopmentof
Editor:MariaLinaTornesello,FondazioneIRCCS
cervicallesions/cancerinwomenfromthecityofPosadasinMisionesProvince.
IstitutoNazionaledeiTumori,ITALY
Received:October28,2017
Methods
Accepted:December22,2017
Twohundredandsixty-onewomen,including92casesofpatientsdiagnosedwithcervical
Published:January12,2018
lesionsand169controls,wereanalyzed.mtDNAancestrywasassessedthroughHVS1
Copyright:©2018Badanoetal.Thisisanopen
sequencing,whilethedetectionandtypingofHPVinfectionwasconductedthroughnested
accessarticledistributedunderthetermsofthe
multiplexPCRanalysis.Multivariatelogisticregressionwasconductedwiththeresulting
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and datatoestimatetheoddsratios(ORs)adjustedbysocio-demographicvariables.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Results
DataAvailabilityStatement:Allrelevantdataare
Thestudyparticipantsshowed68.6%Amerindian,26.1%Europeanand5.3%African
withinthepaperanditsSupportingInformation
files.AllHVS-1sequencefilesareavailablefrom mtDNAancestry,respectively.MultipleregressionanalysisshowedthatwomenwithAfrican
theGenBankdatabase(accessionnumbers mtDNAswerethreetimesmorelikelytodevelopacervicallesionthanthosewithNative
KY344532-KY344740).
AmericanorEuropeanmtDNAs[ORof3.8(1.2–11.5)forancestryandORof3.5(1.0–12.0)
Funding:ThisworkwassupportedbytheAgencia forLhaplogroups],althoughtheassociatedpvalueswerenotsignificantwhentestedunder
NacionaldePromocio´nCient´ıficayTecnolo´gica,
morecomplexmultivariatemodels.HPVinfectionandthedevelopmentofcervicallesions/
Argentina(GrantnumberPICT-2012-0761);the
cancerweresignificantforalltestedmodels,withthehighestORvaluesforHPV16[ORof
Comite´EjecutivodeDesarrolloeInnovacio´n
Tecnolo´gica,GobiernodeMisiones,Argentina 24.2(9.3–62.7)]andHPV-58[ORof19.0(2.4–147.7)].
PLOSONE|https://doi.org/10.1371/journal.pone.0190966 January12,2018 1/16
mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
(BecasCEDIT-Res.31/12toDJS);theMinisterio Conclusion
deSaluddelaNacio´n(MSAL)Argentina-
HPVinfectionremainsacentralriskfactorforcervicalcancerinthePosadaspopulation.
Comisio´nNacionalSaludInvestiga-Becas¨Carrillo
-Oñativia¨(Res.699/13toDJS);andCONICET ThepotentialroleofAfricanmtDNAancestryopensanewavenueforfuturemedicalassoci-
Argentina(Res.4838/13toDJSandRes. ationstudiesinmultiethnicpopulations,andwillrequirefurtherconfirmationinlarge-scale
Resolucio´nDNo3993/12toIB)andCodon-Code
studies.
AlignerLicenseGrant-USA(2016).Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe
manuscript.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Thehumanpapillomavirus(HPV)isasmall,nonenvelopedviruswithacirculardouble-
strandedDNAgenome(ofapproximately8kb)thatbelongstothefamilyPapillomaviridae
[1].Itinfectsstratifiedsquamousepithelia(mucosalandcutaneous),whereitcancauseneo-
plasiasorpersistasymptomatically.Inparticular,HPVgenitalinfectionsbycertaintypes
(identifiedashigh-risk)canleadtothedevelopmentofcervicallesionsandcervicalcancer
[2,3].Currently,morethansixteenHPVstrainsareidentifiedashigh-risktypes(HPV-HR)
forthedevelopmentofcervicalcancer,andareclassifiedasGenusAlpha-Papillomavirus,spe-
ciesA6,A7andA9[1–3].
Cervicalcanceristhesecondmostcommonfemalecancerinwomenaged15to44from
acrosstheworld.Incidenceratesvarywithindifferentgeographicregions,beinghigherin
AfricathaninEurope(27.6casesper100,000vs.11.4/100,000),andintermediateintheAmer-
icasandAsia(14.9/100,000and12.7/100,000respectively)[4].Thesedifferenceshavebeen
attributedtothesocio-culturalcharacteristicsofthepopulationsintheseregionsandthelack
ofeffectivescreeningprograms[4].However,thegeneticbackgroundofthehostpopulation
mayfurtherinfluencetherateofcervicalcancer[5–9].
Sincetheearly1990s,epidemiologicalstudiesaddressingtheroleof“ethnicity”(i.e.,genetic
ancestry)inthedevelopmentofcervicalcancerhaveindicatedthatAfrican-Americansand
Amerindians(“LatinosorHispanics”)fromtheUnitedStatesareatahigherriskofdeveloping
cervicalcancerthan“whites”[10,11].However,thesestudiesclassifiedpopulationsbasedon
self-reportedorigin,surnameorskincolor[10],characteristicsthathavebeenshowntobe
poormarkersofgeneticancestryindiversepopulationsofLatinAmericasuchasthosefrom
BrazilandArgentina[12,13].Moreover,afteradjustmentbysocio-economicstatus,suchasso-
ciationstendtodisappear,indicatingthatsocialfactors(ratherthanethnicityorancestry)
haveamoreimportantinfluenceoncervicalcancerincidence[10,14].Forthesereasons,the
potentialinfluenceofgeneticancestryontheprevalenceofcervicalcancerwithindifferent
populationshasnotyetbeencomprehensivelyaddressed.
TheanalysisofmitochondrialDNA(mtDNA)sequencevariationcanbeusedtoassessthe
maternalgeneticancestryofanindividual[15].ThemtDNAisa16,569-base-paircircular
double-strandedmoleculecontaining37genesthatplayanimportantroleincellularenergy
productionandapoptosis[16].Anindividual’smtDNAcanbeplacedinahaplogroup(mater-
nallineage)basedonthesetofpolymorphismsorsinglenucleotidepolymorphisms(SNPs)
presentinitssequence.Basedonnumerousstudies,itisnowclearthatmanyhaplogroups
originatedinspecificareasoftheworldand,thus,predominateinlocalpopulations.Forexam-
ple,fourlineages(L0,L1,L2,andL3)arecommoninAfricanandAfrican-derivedpopulations
[17],nine(H,I,J,K,T,U,V,WandX)inEuropeanpopulations[18],andeight(A,B,C,D,E,
F,GandM)inAsianpopulations[19,20],withfourAsian-derivedhaplogroups(A2,B2,D1
andC1)beingspecifictoNativeAmericans[21,22].
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
Inadditiontotheirphylogeographichistories,thereisgrowinginterestintherelationship
betweenmtDNAhaplogroupsandthedevelopmentofvarioustypesoffemalecancers.The
presenceoftheAmerindianhaplogroupD5hasbeenassociatedwithbreastcancer[23],the
EuropeanhaplogroupUKwithvulvarcancer[24],andtheAmerindianhaplogroupB2with
cervicalcancer[25].Thesefindingssuggestthatmaternalgeneticancestrycouldplayarolein
theincidenceofthesediseases.
Similarly,anunderstandingofmatrilinealancestryisimportantforassessingdiseasepreva-
lenceandsusceptibilityinArgentina.InArgentina,alargeproportionofthepopulationhas
NativeAmericanancestry(45%and70%forthecentralandnorthernareas,respectively)fol-
lowedbyEuropean(50to29%)andAfrican(<3%)ancestry[26,27].Nevertheless,theeffect
ofmitochondrialgeneticancestryoncervicalcancerinthiscountryisnotcurrentlyknown.
ThisisalsothecaseforMisionesProvince,whichislocatedinthenortheasterntipof
ArgentinaandsharesinternationalborderswithParaguayandBrazil.Fromanepidemiologi-
calpointofview,thisprovinceisconsideredtobearegionwithahighprevalenceofHPV
infectionandmortalityrateofcervicalcarcinoma(33–43%and12/100,000individuals,respec-
tively)comparedtootherareasofthecountry,suchasBuenosAires(30%and3/100,000)[28,
29].Itscurrentpopulationisalsotheproductofgenerationsofintermixingbetweenvarious
groups,includingNativeAmericans,whooriginallyinhabitedthispartofSouthAmerica,
Spanishconquerors,andalargeEuropeanimmigrantpopulationthatarrivedinthe1930s
[30].FurtheradmixturehasoccurredthroughrecentimmigrationfromborderingSouth
AmericancountriessuchasBrazilandParaguay[30].
Despitethiscomplexhistory,thegeneticbackgroundofthispopulationhasbeenpoorly
studied[31–33].Moreover,thegeneticinfluencesonHPVinfectionandcervicalcarcinoma
incidenceintheregionhavenotbeenexamined.Therefore,theobjectiveofthisstudywasto
exploretherelationshipbetweenmtDNAancestry,HPVinfectionandthedevelopmentofcer-
vicallesionsandcancerinwomenfromthismultiethnicregionofnortheasternArgentina.
Materialsandmethods
Ethicstatement
ThisstudywasconductedwiththeapprovalofTheEthicsCommitteeoftheDr.Ramo´nMada-
riagaHospital,Posadas,Argentina(DepartamentodeDocenciaeInvestigacio´n,Comite´de
Bioe´tica,HospitalDr.Ramo´nMadariaga,Posadas,Misiones).Allexperimentswereperformed
incompliancewithinstitutionalguidelinesandinaccordancewithethicalstandardsofthe
DeclarationofHelsinski.
Studydesign
Weconductedaretrospectivestudyofgeneticriskfactorsinvolvedinthedevelopmentofcer-
vicallesionsandcancerinwomenfromPosadasinMisionesProvince.Thestudysamples
wereobtainedfromwomenattendingtodifferenthealthcentersandclinicsinthecitybetween
2005–14.ThestudysampleswerearchivedattheLaboratoriodeBiolog´ıaMolecularAplicada
(LaBiMAp)oftheFacultaddeCienciasExactas,Qu´ımicasyNaturalesoftheUniversityofMis-
iones.Foreachsample,adatabasecontaininginformationaboutparticipantage,Papcytology,
dateandthelocationofthesamplingcenter,healthcaresystem,nationalityandplaceofresi-
dencewasrecorded.InformationabouttheHPVstatusoftheparticipantswasretrievedfrom
thedatabase(n=128)orassessedinthisstudy(n=136)(seethe“HPVdetectionandtyping”
sectionbelow).
AllsamplesfromtheLaBiMApwereobtainedwithinformedconsentinthecontextofpre-
viousepidemiologicalstudies,withnoneofthemaddressingmtDNAcharacterization[8,29].
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
TheEthicsCommitteeoftheDr.Ramo´nMadariagaHospital,Posadas,Argentina,approved
theuseofthesebiologicalsamplesforthisstudy.
Populationdemographics
PosadasisthecapitalofMisionesProvinceandcurrentlyhasapopulationof324,758people
[34].Ofthese,106,141arewomenofreproductiveage(morethan19yearsold)[34],withthe
mortalityrateforcervicalcancerbeing12/100,000[28].Todate,nosystematicsurveyof
geneticdiversityintheinhabitantsofthiscityhasbeenundertaken,andthepossibleexistence
ofassociationbetweensocio-demographicvariablesandmtDNAancestryisunknown.Such
associationshavebeenreportedforotherLatin-Americanpopulations,suchasthatofUru-
guay[13,35],andtheirexistenceshouldbeaddressed,sincetheycouldaffecttheoutcomeof
geneticassociationstudies[35].
Inthisstudy,sampleswererecruitedfromhealthcenterslocatedatthreelocationswithin
thecity,includingtheDowntownareaandthe1stand2ndUrbanBelts.Socio-demographic
variablesfromeachlocationwerenotrecordedalthough,basedonourownrecords,wecan
brieflydescribethemasfollows.TheDowntownareaischaracterizedbyprivatepractices,
withtheresidentpopulationbeingofupper-middleincomestatusandhavinghealthinsur-
ance.Bycontrast,the1stand2ndUrbanBeltscontainthemaincityhospitalandacommunity
healthcenter(respectively).Botharepublichealthcenterswithanattendingpopulationof
middle-lowincomestatuswhosememberslargelylackhealthinsurance.Toaccountforthe
potentialeffectofsamplecenterlocation,thosesociodemographicvariablesshowingassocia-
tionswithmtDNAancestrywereincludedinthemultivariateanalysisofthisstudy(seethe
“AssociationAnalysis”section).
Nomenclature
Thecytologicalclassificationsusedinthisstudywereasfollows:NILM:negativeforintrae-
pitheliallesionandmalignancy;L-SIL:low-gradesquamousintraepitheliallesion;H-SIL:
high-gradesquamousintraepitheliallesion;CIS:carcinomainsitu;andISCC:invasivesqua-
mouscellcarcinoma[36].ThenotationH-SIL+wasusedtogroupH-SIL+CIS+ISCC
patients.
Studygroups
Inthisstudy,134patientsdiagnosedwithcervicallesions(60L-SIL,59H-SILand15withcer-
vicalcancerinsitu/invasive)wereselectedwithoutpersonalidentifiersfromthelaboratoryreg-
istry.Theyrepresentedabout80%ofthepatientsrecordedasHSIL+fortheperiodbetween
2005–14.Atotalof186controlsubjectswereselectedfromthesamedatabase,withalloftheir
samplesbeingacquiredduringthesameperiodasthoseofthecasepatientsunderconditions
ofanonymity.DNAsfortheControlsandL-SILsampleswereobtainedfromcervicalscrapes,
whereasthosefortheH-SIL+groupwereextractedfrombiopsiesofformalin-fixed,paraffin-
embeddedtissues(fixedbiopsies).Althoughtheinitialsamplesizewas320individuals,com-
pletegenotypingtoestimatemtDNAancestrywassuccessfulin261ofthem(81.6%),which
comprisesthefinalnumberanalyzedinthisstudy(seethe“MitochondrialDNAAnalysis”
section).
MitochondrialDNAanalysis
mtDNAhaplotypesweredefinedthroughdirectsequencingoftheHVS1regionofthecontrol
region(np16024–16383).Briefly,theHVS1wasPCRamplifiedusingprimers15977-F
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
(5´-CCA CCA TTA GCA CCC AAA GC-3´)and16552-R(5´-TAA GGG GAA CGT
GTG GGC-3´).Positiveampliconswerepurifiedusingcommercialkits(ADNPuriPrep-GP
kit,InbioHighway)anddirectlysequencedusingtheoriginalprimersthroughsequencingser-
vicesusingaBeckmanCoulterCEQ2000XLDNAAnalysisSystem(Cromatida,Argentina).A
totalof320sampleswereprocessedand280werepositiveformtDNA(87.5%).Ofthese,qual-
itysequenceswereobtainedfrom261samples(93.2%).ThemtDNAsequenceswereedited
andalignedusingCodonCodealignersoftwarev3.0.1(CodonCodeCorporation).HVS1hap-
lotypeswereclassifiedintohaplogroupsbyusingHaploGrepandPhylotreebuild17.0[37,38].
mtDNAhaplotypesandtheirhaplogroupstatusareshowninS1Table.HVS-1sequenceswere
depositedinGenBankunderaccessionnumbers:KY344532—KY344740.
Humanpapillomavirusdetectionandtyping
HPVdetectionwasperformedusingPCRamplificationwithL1consensusprimersMY09–
MY11[39].HPVDNA-positivesamplesweretypedbyE6-NestedMultiplexPCR(E6-NMPX)
withcocktailsofprimersC-1(high-riskHPVs16,18,31,45,and59)andC-2(high-riskHPVs
33,56,52,58andlow-riskHPVs6and11)[40]todeterminethetypeofstrain(s)presentin
thesamples.PositivesamplesthatwerenotrevealedaspositivebyE6-NMPXamplification
wereleftas“HPVundetermined”.
Associationanalysis
ThedistributionofmtDNAhaplogroupsorHPVtypesbetweenstudygroupswascompared
incontingencytablesbyusingχ2ortwo-tailedFisherexacttestforcellcountsbelowfive.Simi-
larly,thedistributionofmtDNAhaplogroupsaccordingtosocio-demographicvariables(age,
samplecenterlocation,healthcaresystem,nationalityandplaceofresidence)wastestedin
contingencytables(χ2ortwo-tailedFisherexacttest),withtheexceptionofage,whichwas
testedusingtheMediantest.Thosevariablesfoundtobesignificantwereincludedinthemul-
tivariateanalysis.
MultivariateordinallogisticregressionwasusedtoestimatetheORand95%confidence
intervals(CIs).Forthesetests,eachvariablewasclassifiedasfollows:(1)Cytologicaldiagnosis,
includingthreecategoriesofprogressivenature[NLIM,LSILandHSIL+];(2)mtDNAances-
try,includingthreecategories[Amerindian,EuropeanandAfrican];(3)mtDNAhaplogroups,
whichincludedeightcategories(A,B,C,D,HV,JT,UK,L),withhaplogroupsoccurringata
frequencylessthan5%being“clustered”intolargermonophyleticcladessuchasUK,JT,HV
andL(L0,L1,L2andL3)called“phylogroups”;and(4)HPVtypes,including10categories
[HPVnegative,HPVundetermined,HPV6/11,HPV56,HPV58,MultipleInfections,HPV52,
HPV33,otherHPV-HRandHPV16],inwhichthoseHPVsoccurringatfrequencieslessthan
5%wereclusteredintolargergroups(otherHPV-HR,includingspeciesA7:18,45and59).
Fortheanalysis,HPVtypesweretestedbyorderingthemaccordingtotheirincreaseinthe
ORaccordingtoMuñozetal.,2003[3].Thefinalmodelsofanalysisincluded(I)Papcytology
andHPVinfection,(II)PapcytologyandmtDNAancestry,adjustedbysocio-demographic
variables,(III)PapcytologyandmtDNAhaplogroups,adjustedbysocio-demographicvari-
ables,(IV)Papcytology,mtDNAancestryandHPVinfection,adjustedbysocio-demographic
variables,and(V)Papcytology,mtDNAhaplogroupsandHPVinfection,adjustedbysocio-
demographicvariables.OrderedlogisticregressionproducedaunifiedORvalue,takingunder
considerationthethreeprogressivestagesofPapcytology.Allcalculationsweremadeusing
Stata14.0(StataCorpLLC,Texas,USA,2015).ForestplotsweremadeusingGraphPadPrism
version7.0dforMacOSX(GraphPadSoftwareInc,LaJollaCaliforniaUSA,www.graphpad.
com).
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
Results
Studygroups
Twohundredandsixtyonesamples,including92casesofpatientsdiagnosedwithcervical
lesions(50L-SIL,35H-SILand7cervicalcancerinsitu/invasive)and169controlsubjects
wereanalyzed.TheaverageageoftheH-SIL+caseswas32.1years(agerange20–54years),
thatoftheL-SILcaseswas31.5years(20–50years),andthatofcontrolswas36.0years(10–83
years).
Humanpapillomavirustypingdistributionandcervicallesions
Atotalof130sampleswereidentifiedaspositiveforHPV(49.8%)and11differentviraltypes
(16,18,31,33,45,52,56,58,59,6,11)wereidentifiedinthem.HPVinfectionwashigherin
womenwithcervicallesions(70%)comparedtothosewithoutlesions(37.9%),apatterncon-
sistentwithitsroleincervicalcancerdevelopment.Briefly,HPV16infectionhadthehighest
frequencyamongstudygroups(9.5%ofNILM;22%ofL-SILsand57.1%ofH-SIL+),followed
byHPV6/11(3.0%ofNILM;20%ofL-SILsand16.7%ofH-SIL+).Multipleinfections(double
andtriple)werepresentin11.1%ofwomenwhile8%oftheviralinfectionsremaineduntyped.
DetailsaboutHPVtypesandtheirfrequenciesareshowninTable1.
TheresultsoftheassociationanalysisareshowninTable2.ThehighestORvalueswere
foundforHPV16[OR=18.3(7.6–44.3)],HPV58[OR=13.8(2.0–95.4)]andHPV6/11
[OR=9.3(2.9–30.0)].OthersignificantassociationsincludedHPV6/11[OR=9.3(2.9–30.0)]
andotherHPV-HR[OR=5.9(1.5–22.1)].
MitochondrialDNAancestryandsocio-demographicvariables
ThemtDNAancestryofthestudypopulationwas68.6%Amerindian,26.1%Europeanand
5.3%Africaninorigin,respectively.TherewerestatisticallysignificantdifferencesinmtDNA
Table1. FrequencyofHPVtypesinthestudygroups.
NLIM LSIL HSIL+ Total pvaluea
(169) (50) (42) (261)
HPVpositive 64(37.9) 35(70.0) 31(73.8) 130(49.8) <0.001
HPVNegative 105(62.1) 15(30.0) 11(26.2) 131(50.2)
HPVtypesb
16 16(9.5) 11(22.0) 24(57.1) 51(19.5) <0.001
18 1(0.6) 5(10.0) 0(0.0) 6(2.3) <0.001
31 4(2.4) 3(6.0) 2(4.8) 9(3.4) 0.409
33 10(5.9) 3(6.0) 1(2.4) 14(5.4) 0.645
45 3(1.8) 0(0.0) 0(0.0) 3(1.1) 0.438
52 8(4.7) 2(4.0) 1(2.4) 11(4.2) 0.791
56 13(7.7) 4(8.0) 1(2.4) 18(6.9) 0.450
58 2(1.2) 2(4.0) 2(4.8) 6(2.3) 0.257
59 1(0.6) 3(6.0) 0(0.0) 4(1.5) 0.016
6/11 5(3.0) 10(20.0) 11(16.7) 26(8.4) <0.001
Multiple-Infections 13(7.7) 10(20.0) 6(14.3) 29(11.1) >0.040
HPV-Undetermined 16(9.5) 5(10.0) 0(0.0) 21(8.0) >0.111
apvaluesforthedistributioninacontingencytables(χ2orfisherexacttest).Significantpvaluesareshowninboldface.
bHPVinfectioncountsandfrequenciesincludetypesinsingleandmultipleinfections.
https://doi.org/10.1371/journal.pone.0190966.t001
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
Table2. AssociationanalysisbetweenHPVinfectionanddevelopmentofcervicallesions.
HPV O.R. CI95% pvalue
HPVnegative 1 Ref
HPVpositive 4.0 2.3–6.9 <0.001
HPVtypesa
16 18.3 7.6–44.3 <0.001
33 1.3 0.3–5.1 0.696
52 0.6 0.1–5.3 0.667
56 1.5 0.4–5.9 0.582
58 13.8 2.0–95.4 0.008
6/11 9.3 2.9–30.0 <0.001
OthersHPV-HRb 5.9 1.5–22.1 0.009
Multiple-Infections 4.5 2.0–10.0 <0.001
HPV-Undetermined 1.1 0.4–3.3 0.840
aOrderedlogisticregressionforPapcytologyandHPVinfection(ModelI).Significantassociationsareshownin
boldface.
bIncluding:HPV18,HPV59,HPV45(specieA7).
https://doi.org/10.1371/journal.pone.0190966.t002
ancestryamongthestudygroups,withwomenhavingEuropeanmtDNAsbeingmorefre-
quentlyfoundinthecervicallesiongroups(L-SILandH-SIL+)thanthosewomenwithAmer-
indianmtDNAs(p<0.05).Inaddition,weexploredtheeffectofsocio-demographicvariables
onthemtDNAdistributionwithinthispopulation(Table3).Notably,mtDNAancestrywas
associatedwithsamplecenterlocation,healthcaresystemandnationality,butnotwithplaceof
residenceorage.Basedontheseresults,thesesignificantvariableswereincludedinamultivar-
iateanalysis.
Table3. mtDNAancestry,cervicallesionsandsocio-demographicvariablesforthePosadaspopulation.
Amerindian European African pvaluea
(n=180) (n=67) (n=14)
Papcytology NILM 130 32 7
LSIL 30 18 2 0.002
HSIL+ 20 17 5
Age Mean 35.1 34.3 32.1
Median 32.5 33.0 32.0 0.619b
SD 11.3 9.3 5.6
Range 18–83 20–66 22–38
SampleLocation Downtown 54 51 4
1stUrbanBelt 32 8 3 <0.001
2ndUrbanBelt 94 8 7
Nationality Argentinian 169 66 11
Others 11 1 3 0.013
PlaceofLiving Posadas 166 62 11
Countryside 3 4 0 0.175
HealthCareSystem Public 126 16 10
Private 54 51 4 <0.001
Note:Somecolumnsdonotadduptothetotalbecauseofmissingdata.
apvaluesforthedistributioninacontingencytables(χ2orfisherexacttest).Significantpvaluesareshowninboldface.
bpvaluefortheMedianTest.
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
MitochondrialDNAhaplogroupsandcervicallesions
Weidentified17differentmtDNAhaplogroupsinthestudypopulation,allofwhichcorre-
spondedtothemajormaternallineagesthathavecontributedtoArgentineanhistory.These
includedA2,B2,C1,D1(Amerindian),H,HV,I,J,K,T,U,V,X2(European)andL0,L1,L2,
L3(African)(S1Table).mtDNAhaplogroups(orphylogroups)withafrequency>5%are
showninTable4.TherewerestatisticallysignificantdifferencesinthedistributionofEuro-
peanphylogroupJT,whichoccurredmorefrequentlyinthecervicallesionsgroups(p<0.05).
However,thisphylogroupwasnotfoundtobeassociatedwiththedevelopmentofcervical
lesionsintheadjustedmultivariateanalysis.
Multivariatelogisticregressionmodels
TheassociationbetweenmtDNAancestryandcervicallesiondevelopmentwasestimated
usinglogisticregressionmodelsadjustedbysignificantsocio-demographicvariables(sample
centerlocation,healthcaresystemandnationality).TheresultsareshowninFig1.Inthispop-
ulation,womenwithAfricanmaternalancestryweremorelikelytodevelopacervicallesion
thanthosehavingAmerindianorEuropeanmaternalancestry,withanORof3.8(1.2–11.5)
andapvalueof0.018.Thisassociationremainedpositiveatthelevelofphylogroupwithan
ORof3.5(1.0–12.0)forhaplogroupL,althoughthesignificanceofthisfindingwasweak,with
apvalueof0.043.DetailsabouttheORestimatesareprovidedinS2andS3Tables.
WerananadditionaltestthatincludedHPVinfectionintheregressionmodels(ModelIV:
Papcytology,mtDNAethnicancestryandHPVinfection,adjustedbysocio-demographicvar-
iables;andModelV:Papcytology,mtDNAhaplogroupsandHPVinfection,adjustedby
socio-demographicvariables).TheresultsareshowninFig2.Bothanalysesshowedthat
womencarryingAfricanmtDNAswerenearlythreetimesmorelikelytodevelopacervical
lesionthanthosehavingNativeAmericanorEuropeanmtDNAs,withaORof3.2(0.8–12.2)
forModelIVandaORof2.5(0.6–10.7)forModelV,althoughtheassociatedpvalueswere
notsignificant(0.085and0.229respectively).DifferentfrommtDNAinfluence,thedevelop-
mentofcervicallesionwasconsistentlyassociatedwithHPVinfectionbyHPV16atanORof
Table4. FrequencyofmtDNAhaplogroupsandphylogroupsinthestudygroups.
NLIM LSIL HSIL+ TOTAL pvaluea
(169) (50) (42) (261)
A 37(21.9) 9(18.0) 7(16.7) 53(20.3) 0.680
B 26(15.4) 5(10.0) 6(14.3) 37(14.2) 0.631
C 45(26.6) 11(22.0) 4(9.5) 60(23.0) 0.061
D 21(12.4) 4(8.0) 3(7.1) 28(10.7) 0.482
SubtotalAmerindian 130(76.9) 30(60.0) 20(47.6) 180(69.0) <0.001
HV 20(11.8) 10(20.0) 7(16.7) 37(14.2) 0.306
JT 3(1.8) 4(8.0) 5(11.9) 12(4.6) 0.009
UK 8(4.7) 3(6.0) 4(9.5) 15(5.7) 0.489
SubtotalEuropean 32(18.9) 18(36.0) 17(40.5) 67(25.7) 0.003
SubtotalAfricanb 7(4.1) 2(4.0) 5(11.9) 14(5.4) 0.121
Othersc 2(1.2) 2(4.0) 1(2.4) 5(1.9) 0.307
Total 169(100.0) 50(100.0) 42(100.0) 261(100.0)
apvaluesforthedistributioninacontingencytables(χ2orfisherexacttest).Significantpvaluesareshowninboldface.
bHaplogroupsL0,L1,L2andL3.
cHaplogroupsI,WandX.
https://doi.org/10.1371/journal.pone.0190966.t004
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
Fig1.MultivariateregressionanalysisformtDNAandcervicalcancer.Legend:AForestPlotshowingoddsratio
valuesand95%confidenceintervalsfor:(A)ModelII—PapcytologyandmtDNAancestry,adjustedbysocio-
demographicvariablesand,(B)ModelIII—PapcytologyandmtDNAhaplogroups,adjustedbysocio-demographic
variables.Thex-axisrepresentstheoddsratio(circlesandsquare)and95%confidenceintervals(whiskers).The
dashedverticallineindicatesanORvalueof1.Non-significantvaluesareshownasasmallcircle.Significantvaluesare
shownassquare,withtheOR(CI95%)asalegendintothefigure.DetailsonotherOR(CI=95%)andpvaluesare
providedintheSupplementaryInformation(S2andS3Tables).AmerindianancestryandhaplogroupAwerenot
plottedsincetheywereusedasreferencevalues(OR=1).
https://doi.org/10.1371/journal.pone.0190966.g001
24.2(9.3–62.7),andHPV-58atanORof19.0(2.4–147.7),amongothertypes,acrossalltested
models(Fig2).DetailsabouttheORestimatesareprovidedinS4andS5Tables.
Discussion
Multi-ethnicpopulationsofferanopportunitytotesttheeffectsofancestryondiseasewithin
thesamepopulation[41].Basedonhistoricalrecords,thepopulationofPosadaswasasuitable
candidatewithwhichtousethisapproach,althoughitsgeneticcompositionwasnotwell
known.ThisstudyrevealedconsiderablematernalgeneticdiversityinthePosadaspopulation,
with68.6%oftheparticipantshavingAmerindian,26.1%Europeanand5.3%African
mtDNAs,respectively.Thisgeneralgeneticprofileissimilartothatpreviouslyreportedfor
northeasternArgentina[26,27],butdifferedfromthereportednationalaverageandfromthat
oflargercitiesinArgentinasuchasLaPlataandCo´rdoba(withanEuropeancomponentof
nearly50%)[42,43].Overall,thegeneticstructurewithinthecountryisanimportantissuefor
futurenationwidemedicalstudies.
Moreover,thereisgrowingevidencethatmtDNAvariationisdeeplystructuredinworld-
widepopulationsandalsomoresusceptibletofalse-positivefindingsinassociationstudies
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mtDNAancestry,HPVinfectionandcervicalcancerinArgentina
Fig2.MultivariateregressionanalysisformtDNA,HPVinfectionandcervicalcancer.Legend:AForestPlot
showingtheoddsratiovaluesand95%confidenceintervalsfor:(A)ModelIV—Papcytology,mtDNAancestryand
HPVinfection,adjustedbysocio-demographicvariablesand(B)ModelV—Papcytology,mtDNAhaplogroupsand
HPVinfection,adjustedbysocio-demographicvariables.Thex-axisrepresentstheoddsratio(circles,square,others)
and95%confidenceintervals(whiskers).ThedashedverticallineindicatestheORvalueof1.Non-significantvalues
areshownasasmallfilledcircle.Significantvaluesareshownassquares(HPV33),diamonds(otherHPV-HR),
asterisks(HPV58),triangles(HPV16),invertedtriangles(HPV6/11),andunfilledcircles(multipleinfections),withthe
OR(CI=95%)asalegendintothefigure.DetailsaboutotherOR(CI=95%)andpvaluesareprovidedinthe
SupplementaryInformation(S4andS5Tables).AmerindianancestryandhaplogroupAwerenotplottedsincethey
wereusedasareferencevalues.
https://doi.org/10.1371/journal.pone.0190966.g002
thanautosomalSNPs[44,45].Inthisregard,weobservedsignificantdifferencesbetweenthe
maternallineagedistributionandseveralsocio-demographicvariables(samplecenterlocation,
healthcaresystemandnationality),afeaturesharedwithotherLatinAmericanpopulations
[12,35].Hence,controllingthesevariablesthroughtheuseofadjustedORsandmultivariate
analysiswasnecessarytoreducepotentialfalsepositives.
InArgentina,therearenopreviousreportsontherelationshipbetweenmtDNAancestry
andcervicalcancer,andonlythreepublicationshaveaddressedthisissueinthepublishedlit-
erature[25,46,47].Amongthem,theriskofdevelopingcervicalcancerhasbeenlinkedto
AmerindianhaplogroupB2inMexico[OR1.6(1.05–2.58)][25],toAsianhaplogroupMin
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