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
Original
article
Uncommon
non-oncogenic
HPV
genotypes,
TP53
and
MDM2
genes
polymorphisms
in
HIV-infected
women
in
Southern
Brazil
Ludmila
Gonc¸alves
Entiauspe
a,b,
Fabiana
Kömmling
Seixas
a,c,
Emily
Montosa
Nunes
a,
Fernanda
Martins
Rodrigues
a,
Odir
A.
Dellagostin
a,c,
Tiago
Collares
a,c,
Mariângela
Freitas
da
Silveira
b,d,∗aMolecularandCellularOncologyResearchGroup,BiotechnologyUnit,TechnologyDevelopmentCenter,UniversidadeFederaldePelotas (UFPel),Pelotas,RS,Brazil
bPost-GraduateProgrammeinEpidemiology,UniversidadeFederaldePelotas(UFPel),Pelotas,RS,Brazil cPost-GraduateProgrammeinBiotechnology,CDTec,UniversidadeFederaldePelotas(UFPel),Pelotas,RS,Brazil dMaternalandChildDepartment,FacultyofMedicine,UniversidadeFederaldePelotas(UFPel),Pelotas,RS,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received11November2013 Accepted11July2014
Availableonline1September2014
Keywords: HPVinfection HIVinfection Cervicalcancer Epidemiology
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b
s
t
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c
t
Background:ItisbelievedthatHumanPapillomavirus(HPV)andHumanImmunodeficiency Viruscoinfectioncontributestoincreasetheriskforcervicalintraepithelialinjuries.Several factorsmaycontributetocervicalcancer(CC)development,includinggeneticvariantssuch asTP53andMDM2genepolymorphisms.
Materialsandmethods:AhundredHIV-infectedwomenwereexaminedforHPVdetection anditsgenotypes,aswellasthefrequenciesoftheSNPsArg72ProandSNP309andtheir associationswithCCriskfactors.NestedPolymeraseChainReaction(nPCR)wasusedfor HPVdetectionandPCR-RFLPforTP53andMDM2SNP309genotyping.
Results:HPVDNAwasdetectedin68%ofsamples.Ahigherfrequencyoflow-riskHPV geno-types(66.7%)wasobservedwhencomparedtohigh-riskgenotypes(33.3%).Ninedifferent HPVgenotypeswereidentified,withthehighestprevalenceofHPV-6,followedbyHPV-16 and31.p53Arg72ArgandSNP309TGgenotypewerethemostprevalent.HPVgenotyping wasperformedbysequencing.
Conclusion: ThedataobtainedsuggestthatHIV-infectedwomenaremoresusceptibleto beinfectedbylow-riskHPV(LR-HPV)genotypesthanbyhigh-risk(HR-HPV),andPro72Pro ofTP53geneandTGofMDM2SNP309genotypes apparentlyseemtobeprotective fac-torsamongHIV-infectedwomenforHPVacquisitionandHR-HPVinfection,respectively, inasampleofSouthernBrazilianwoman.Futureinvestigationsinlargerpopulationsare necessarytobetterunderstandthepotentialrolesoftheseSNPsandthebehaviorof non-oncogenicHPVgenotypesinHIV-mediatedimmunosuppressioncases.
©2014ElsevierEditoraLtda.Allrightsreserved.
∗ Correspondingauthorat:CentrodePesquisasEpidemiológicas,RuaMarechalDeodoro,1160–3◦Piso,BairroCentro,Cep:96020-220,
CaixaPostal464,PelotasCity,RS,Brazil.
E-mailaddress:maris.sul@terra.com.br(M.F.daSilveira).
http://dx.doi.org/10.1016/j.bjid.2014.07.005
Introduction
Itisestimatedthatcervicalcancer(CC)isthesecond most commontype ofcancer inthe Brazilianfemalepopulation andthethirdcause ofdeathinwomen.1Today,it is
possi-bletoconfirmthatCCdevelopmentiscloselyassociatedwith theHumanPapillomavirus(HPV)presenceandpersistence,2
withHPVDNAdetectedinupto99.7%ofinvasiveCCcases acrosstheworld.3TheWorldHealthOrganizationattributes
tocervicalinfection withHPV-16or -18about70% of inva-siveCCcasesinBrazil.4Cervicalintraepithelialneoplasia(CIN)
developmentandcervicalcancerinHPV-infectedwomenare associatedwithriskfactors,suchasyoungage,highnumberof sexualpartnersthroughoutlife,earlysexualdebut,smoking, geneticsvariants,amongothers.5,6 Moreover,co-infections,
suchasthosewithHumanImmunodeficiencyVirus(HIV)and
Chlamydiatrachomatisbacterium,maybeinvolvedasco-factors to CC development, acting as adjuvants of the neoplastic process.7,8
The polymorphism on codon 72 (Arg72Pro) of TP53
tumor suppressor gene has been extensively investigated regardingassociation with a wide range ofcancers world-wide. In HPV-infected cells, the E6 oncoprotein binds to p53 protein and promotes its degradation through an ubiquitin proteolytic system altering the p53 activity in someprocesses,suchastumorigenesis, transcription regu-lation, telomerase activation,and apoptosis,thus resulting in deregulation of the cell cycle.9 Previous studies have
shown that the Arg72Arg genotype is related to a higher risk for CC development when compared to the Pro72Pro genotype.10
StudieshaveshownthataSingleNucleotidePolymorphism (SNP)on promoterregionofMDM2 gene,the SNP309 (Tto Gchange onnucleotide309ofthefirst intron),resultsina higherlevelofMDM2mRNAandMDM2protein,and conse-quentreductionofthep53pathway.11TheSNP309occursat
arelativelyhighfrequencyinthegeneralpopulation,andit wasshown thatit presentsa strongassociation with HPV-mediatedcervicalcarcinogenesis.12
Basedonthe presenteddata,this studyaimedto inves-tigate the HPV infection spectrum, to identify the most prevalent genotypes,todeterminethe frequenciesofSNPs Arg72Pro and MDM2 SNP309, and their association with possibleriskfactors forviralpersistenceand forthe devel-opment of pre-neoplastic and neoplastic lesions of the uterinecervixinHIV-infectedwomen. Thestudy was con-ducted in patients of the Service of Specialized Care for HIV/AIDS,CollegeofMedicine,FederalUniversityofPelotas (Servic¸o de Assistência Especializada em HIV/SIDA – SAE-UFPel).
Materials
and
methods
Studytype,characterizationandsamplesize
Thiswasacross-sectionalstudyconsistingof100HIV-infected women,whichweresequentiallyrandomlyinvitedto partic-ipate.Theeligibilitycriteriawere:notbeinginthemenstrual
period,agedbetween18and45years,andnotbeingpregnant. Womenwhohadundergonecervicalconizationand/or hys-terectomywereexcluded.TheEpi-Info6.0softwarewasused tocalculatetherequiredsamplesizefortheprevalencestudy, usinganestimatedHPVprevalenceof80%,witha95% confi-denceleveland80%testingpower.
Logistics
Allparticipantscompletedastandardizedquestionnairewith the purpose of obtaining information about the patient and other relevant epidemiological and socio-demographic variables. An adapted questionnaire from an intervention study with HIV-infected (HIV+) women conducted at SAE-UFPel was used.13 The Research Ethics Committee of the
College of Medicine of the Federal University of Pelotas approved the present study by June 2009, and informed consentwas obtainedfrom all participants. All procedures wereconductedaccordingtotheHelsinkiDeclaration guide-lines.
SamplecollectionandDNAextraction
Cytobrusheswerecollectedcontainingcervicalsecretion sam-ples from the endocervical region. The collected samples werestoredineppendorftubescontaining300LofCellular Lysis Solution(PUREGENETMGentraSystems Kit)androuted
inanappropriatedcontainertotheLaboratoryofFunctional Genomics (Center for Technology Development – CDTec-UFPel) for viral molecular detection by Polymerase Chain Reaction(PCR).BeforeDNAextraction,thesampleswere sub-jected to enzymatic digestion with 1.5L of Proteinase K (10mg/mL)andincubatedfor16hatroomtemperature.The extracted DNA wasperformed accordingto manufacturer’s specifications(PUREGENETMGentraSystemsKit).
TP53genepolymorphismanalysis
TheArg72PropolymorphismofTP53genewas analyzedby PCRusingthe72Aand72SprimersdescribedbyLinetal.14The
amplificationconditionsconsistedofaninitialdenaturation at94◦Cfor3minfollowedby40cyclesat94◦Cfor30s,57◦C for30s,72◦Cfor30s,andafinalextensionat72◦Cfor3min. The PCR product obtained was subjected to enzymatic digestion by RFLP (Restriction Fragment Length Polymor-phism) using the Bst UI restriction enzyme for restriction fragments analysis.Thefragmentsobtainedwere analyzed byagarosegel electrophoresis(2.5%agaroseinTBE buffer), stainedwithGelRedTM(BiotiumInc.,CA)andobservedunder
ultravioletlight(UV).Thep53genotypeswerecharacterized accordingtoLinetal.14
MDM2SNP309analysis
TheMDM2T/GSNP309polymorphismwasanalyzedbyPCR usingtheprimersdescribedbySotomaaetal.15Thereaction
wasperformedwithinitialdenaturingat94◦Cfor10min fol-lowedby30subsequentcyclesat94◦Cfor30s,60◦Cfor1min, 72◦Cfor1min,andafinalextensionat72◦Cfor10min.The PCRproductswereanalyzedbyagarosegelelectrophoresis(2%
agarosegelinTBEbuffer),stainedwithGelRedTM(BiotiumInc.,
CA)andobservedunderUV.Thegenotypingwasperformedby RFLPusingtheMspA1lrestrictionenzyme.15
HPVDNAdetectionbyPCR
TheHPVDNA detectionwas performedbythe nested PCR (nPCR) technique, which is performed in two stages. The first stage used a pair of external primers (MY09/11) pre-viously described by Manos et al.,16 and the second stage
usedapairofinternalprimers(GP5/6)describedbySnijders etal.17Both reactionswere performedwithinitial
denatur-ing at 95◦C during 9min and final extension at 72◦C for 5min.18,19Thereactionconditionsforthefirststageconsisted
of40subsequentcyclesof94◦Cduring30sfordenaturation, 45◦Cduring30sforannealing,and72◦Cfor30sfor exten-sion.Forthesecondstagewereused40subsequentcyclesof 95◦Cfor1min,55◦Cfor1min,and72◦Cfor1minfor dena-turation, annealing and extension, respectively.19 The PCR
productsobtainedweresubjectedtoagarosegel electrophore-sis(1.0%and2.0%agaroseforthefirst andsecond rounds, respectively)andobservedunderUV.Areactionpositive con-trol (RPC) consisting of 450pb corresponding to HeLa cells (HPV-16)andanegativecontrol(NC)withoutanyDNAwere used.
SequencingofthePCRproducts
TheHPVDNA-positivesamplesweresequencedfor identifi-cationofthepresentgenotypes.Theproductsobtainedfrom nPCRwerepurifiedusingtheIllustraTMGFXTMPCRDNAand
GelBandPurificationKit(GEHealthCare)andsequencedona MegaBACE500(GEHealthcare).
Sequenceanalyses
Forwardandreversesequenceswerealignedanda consen-sussequencewasobtainedforeachcaseusingVECTORNTI®
10.0software.Thegenotypesweredeterminedcomparingthe consensussequence withreferencesequencesdeposited in DNAdatabasesthroughthenucleotideBasicLocalSearchTool (BLASTn).Forthesampleseditionandalignment,theVECTOR NTI®10.0softwarewasused.
Dataanalyze
Data was entered by two different data clerks with the softwareEpi-Info® Windowsversioninordertomakea
fur-ther comparison and, thus,ensure better data quality. An automaticdatacheckingwasperformedatthetimeof typ-ing using theEpi-Info® Check functionforconsistency and
amplitude checking. To identify and correct coding, proof-reading and typing inconsistencies, a data cleaning was performed by obtaining frequencies of the collected vari-ables. The analysis was performed using the SPSS® v16.0
software.Chi-squaretests(2)were usedfortheunivariate
analyses. Multivariate logistic regression analysis was also performed.Variablesremainedatthemodeliftheirlevelof significancewasbelow0.20,aspotentialconfoundersforthe
nextlevel.Ap-value<0.05wasconsideredstatistically signif-icant.
Results
Mostparticipantswereagedbetween40and45years(53%), werewhite(51%),45.3%hadbetweenfiveandeightyearsof education,were marriedorinastablerelationship(64.4%), andtheoverridingmajorityhadpercapitaincomeofone mini-mumwage(96.6%).Regardingbehavioralvariables,59.6%were non-smokers,60%reportednothavingconsumedalcoholin thelastfourweeks,63.3%reportedhavingusedacondomat lastsexualintercourse,56%wereagedbetween12and15years atsexualdebut,40.8% hadmorethanninesexualpartners throughoutlife,andover90%reportedneverhadexchanged sexformoneyand/ordrugs.
The TP53 genotypingshowed that, out ofthe 100 sam-plesanalyzed,43% presentedArg72Progenotype,39%were Arg72Arg and 18% were Pro72Pro. The alleles frequency showed82%fortheA(AA+AP)alleleand61%fortheP(AP+PP) allele.TheMDM2 SNP309genotyping showeda higher fre-quencyfortheTGgenotype(45%).Moreover,theT(TT+TG) allelewasthemostfrequent(84%)comparedtotheG(GG+TG) allele(61%).
HPVinfectionwasobservedin68%ofthesamples.Nine dif-ferentHPVgenotypeswerefound,withahigherprevalence ofHPV-6,followedbyHPV-16,HPV-31,HPV-11,HPV-18, HPV-35,HPV-45,HPV-56andHPV-81.Elevensamplescouldnotbe genotypedbysequencing,andthesewerecategorizedas HPV-X.AccordingtotheepidemiologicclassificationofHPVtypes associatedwithcervical cancer,33.3%ofthe sampleswere high-riskoncogenicHPV(HR-HPV)and 66.7%werelow-risk oncogenicHPV(LR-HPV).20
Table1showstheunivariateanalysis,havingHPV infec-tionasoutcomerelatedtosocioeconomic,demographicand behavioral variables. There was no statistically significant associationwithanyofthevariables.Table2showsthe fre-quencydistributionofgenotypesandallelesofArg72Proand MDM2SNP309.TheArg72ArggenotypeoftheTP53genewas themostprevalentamongtheHPV+group(45.6%), predomi-natingA(AA+AP)allele(82.4%)comparedtoP(AP+PP)allele (54.4%). The MDM2 SNP309 presented the TG genotype as the mostprevalent (50%), as well as the T (TT+TG) allele (83.8%)whencomparedtotheG(GG+TG)allele(66.2%).The LR-HPVinfectedgroupshowedfrequenciessimilartothose shownbytheHR-HPVinfectedgroup,exceptforsome vari-ables.RegardingtheTP53genepolymorphism,itwasobserved that52.6%oftheHR-HPVinfectedsamplesand47.4%ofthe LR-HPVinfectedsamplespresentedtheArg72Arggenotype. Inaddition,theA(AA+AP)allelewasmorefrequentinboth infections(LRandHR)whencomparedtotheP(AP+PP)allele (p<0.05).Furthermore,theMDM2SNP309genotypingshowed a higher frequency of TT genotype (42.1%) in the HR-HPV group,aswellashigherfrequencyofTGgenotype(65.8%)in theLR-HPVgroup(p<0.05).TheT(TT+TG)allelewasmore frequent when compared tothe G(GG+TG) allele in both groups.
Themultivariateanalysesshowednostatistically signifi-cantdifferencesandtheirresultsarenotreportedhere.
Table1–InteractionofHPVinfectionandtheoncogenicpotentialriskonthevariablesofHIV-positivewomen.
Variables HPVinfection
Positive(%) Negative(%) RR(IC95%)
Age(years) 18–24 2.9 3.1 0.71(0.06–8.54) 25–30 5.9 18.8 0.23(0.05–0.97) 31–35 17.6 15.6 0.86(0.25–2.88) 36–39 16.2 18.8 0.65(0.20–2.11) 40–45 57.4 43.8 1 p=0.342 Skincolor White 55.9 40.6 2.02(0.82–4.99) Non-white 44.1 59.4 1 p=0.155 Schooling(years) >5 23.1 13.3 2.43(0.66–8.99) 5–8 46.2 43.3 1.50(0.57–3.89) >8 30.8 43.3 1 p=0.378 Maritalstatus Married/consensualunion 63.2 68.8 1 Single 22.1 18.8 0.99(0.26–3.78) Separated/divorced/widowed 14.7 12.5 1.35(0.27–6.67) p=0.865
Percapitaincome(minimumwages)
0–1 96.5 96.8 0.91(0.80–10.53)
2 3.5 3.2 1
p=0.944
Familyincome(minimumwages)
Upto1 29.8 32.3 0.18(0.02–1.72) Upto2 33.3 45.2 0.15(0.01–1.33) Upto3 21.1 19.4 0.22(0.02–2.18) Over4 15.8 3.2 1 p=0.307 Cigarettesmoking Currentsmoker 41.2 38.7 0.90(0.37–2.15) No-smoker 58.8 61.3 1 p=0.817
Alcoholconsume(last4weeks)
Atleastonceaweek 17.6 25.0 0.75(0.26–2.12) Lessthanonceaweek 23.5 12.5 2.00(0.59–6.77)
Never 58.8 62.5 1
p=0.375
Useofcondom(lastsexualactivity)
Yes 62.7 65.6 1
No 37.3 34.4 0.88(0.36–2.12)
p=0.776
Sexualactivitybegan(years)
<15 51.5 65.6 1.71(0.47–6.19) 16–18 38.2 15.6 5.19(1.16–23.17)
≥19 10.3 18.8 1
p=0.062
Sexualpartners(inlife)
Upto1 3.0 3.1 1
2–4 39.4 25.0 1.07(0.90–12.94) 5–8 18.2 28.1 1.75(0.62–4.87) ≥9 39.4 43.8 0.71(0.24–2.11)
Table1(Continued)
Variables HPVinfection
Positive(%) Negative(%) RR(IC95%)
Give/receiveddrugsand/ormoneyforsex
Yes 80.0 20.0 1.93(0.20–18.07)
No 67.4 32.6 1
p=0.555
LastPaptest
Never 8.6 13.3 0.25(0.03–1.86)
<3years 70.7 76.7 1
>3years 20.7 10.0 0.35(0.07–1.76) Donotremember 14.7 6.2 0.80(0.11–5.77)
p=0.336
Visualinspectionwithaceticacid(VIA)
Positive 10.4 6.7 1.63(0.31–8.37)
Negative 89.6 93.3 1
p=0.553
VisualinspectionwithLugol’siodine(VILI)
Positive 10.4 6.7 1.63(0.31–8.37)
Negative 89.6 93.3 1
p=0.553
Paptestresults
Normal 60.7 65.5 1
Inflammatory 31.1 27.6 0.77(0.13–4.39) Altered 8.2 6.9 0.95(0.15–5.95)
p=0.905
CD4countingcells(cells/mm3)
<350 25.4 20.0 0.73(0.25–2.10)
>350 76.3 80.0 1
p=0.565
Table2–InteractionofArg72ProandMDM2SNP309ontheHPVinfectionandoncogenicriskstatus.
HPVinfection p HPVgenotypes p
HPV-positiven(%) HPV-negativen(%) High-riskan(%) Low-riskan(%)
P53genotypes Arg72Arg 31(45.6) 8(25) 10(52.6) 18(47.4) Arg72Pro 25(36.8) 18(56.2) 0.11 6(31.6) 14(36.8) 0.91 Pro72Pro 12(17.6) 6(18.8) 3(15.8) 6(15.8) P53alleles Argininecarriers 56(84.2) 26(81.2) 0.89 16(84.2) 32(84.2) 1.00 Prolinecarriers 37(554.4) 24(75) 0.04 9(47.4) 20(52.6) 0.70 MDM2genotypes TT 23(33.8) 16(50) 8(42.1) 9(23.7) TG 34(50) 11(34.4) 0.26 6(31.6) 25(65.8) 0.04 GG 11(16.2) 5(16.2) 5(26.3) 4(10.5) MDM2alleles Thyminecarriers 57(83.8) 27(84.4) 0.94 14(73.7) 34(89.5) 0.12 Guaninecarriers 45(66.2) 16(50) 0.12 11(57.9) 29(76.3) 0.15 a Totalof57samples.
Discussion
StudieshaveshownthatHPVinfectionismostcommonin HIV-infectedwomenanditisbelievedthatHIVcontributesto increaseHPVviralloadandhenceitspersistence,increasing theriskforcervicalintraepithelial lesions.8 TheHPV
infec-tion prevalenceinthis study was68%. Theseobservations
areconsistentwithameta-analysisstudyreportedbyClifford et al.,whoobservedhigherHPVprevalenceinHIV-infected womeninBrazilandMexico(57.3%)comparedtotheglobal prevalence(36.5%).21
Inthisstudywomenolderthan40years,withlowincome, early sexual debut and high number of sexual partners throughoutlifeshowedgreaterrisktoacquireHPVinfection.
Sauvagetetal.inastudyconductedwithwomenwithaverage ageof39yearsfromruralareasofIndia,foundan associa-tionwithlowincome,earlyageatfirst pregnancy,andlow educational level.22 They suggested that constant increase
inHPVprevalenceinmiddle-agedwomen mayresultfrom reducedabilityofthehosttoeliminateHPVinfection, nutri-tionaldeficiencies,orfromthevirusabilitytobecomelatent andbereactivated duetoafailure ofimmunesurveillance or hormonalfactors associatedtoolder age. Moreover,the HR-HPVprevalence usuallyshows a constant decline with the increasing age seen in high-income countries, in con-trasttotheU-shapedcurvesreportedinsomeLatin-American countries.Here,anincreasedprevalenceofHPVinfectionin theagegroupof35–44yearsoldhasbeenreportedina cross-sectionalstudyperformedinelevencountrieswithdifferent cervicalcancerincidences.23
Thegenotyping bysequencing showed the presence of ninedifferentHPVgenotypes,withahigherprevalencefor HPV-6,followed byHPV-16and -31 inthis study. Although moststudiesshowthatHPV-16genotypeisthemostprevalent worldwide,21thisdataisinlinethereportbyCorrêaetal.,who
observedaprevalenceof63.9%ofHPV-6genotypefollowedby HPV-16genotypeasthesecondmostprevalent(48.5%).24Levi etal.observedsimilardata,withhigherprevalenceof HPV-6genotypeinHIV-infectedwomen(87%).25McKenzieetal.,
inameta-analysisstudy,observedthatdifferentgeographic areas,suchasZambia,BrazilandRochester(NewYork,United States),show differentHR-HPVinfections byless prevalent genotypeswhencomparedtothepopulationingeneral.26
Fur-thermore,theyshowthatcervicalmalignancyhasadifferent behaviorinHIV-infectedpatients,aswellasbenign manifes-tationsofHPV.
Apparently,theHIVinfectionexertsamultifactorial onco-genic function in cervical cancer development, interfering in the immune function and also in the direct promotion ofthelesion development.TheHIV-1 Tatproteinseems to promotethecellularproliferationandpositivelyregulatethe E6and E7HPVgenes, responsible for themalignant cellu-lartransformation.27,28 Recentdataindicatethatsignificant changesoccurintheuterinecervixinHPV/HIVco-infected womensuggestingthattheimmuneresponseappearstobe negativelyregulated,exceptforthe RANTESprotein (Regu-latedonActivation,NormalTcellExpressedandSecreted), which has been found to be increased.29 RANTES levels
werefoundsignificantlyincreasedinplasmaandtissuefrom patientswithbreastcancer andcervical cancer,suggesting thatpositiveregulationofRANTESandotherchangesin cer-vical tissue associated withHPV/HIV co-infection(such as thosemediatedbyHIV-1Tatprotein)wouldpermitless aggres-sive HPV genotypes to cause malignant transformation.30
Someauthorssuggestthatthisvariationofgenotypesisdue tosocioeconomicandbehavioralcharacteristics,whichmay bedifferentinHIV-infectedwomen,besidesthegeographic region where the research is conducted.31 With regard to
HPV infection stratified by genotype, further studies com-paring innate immunity between HIV+ and HIV− women arenecessary.Thesestudieswillhelptoindividualize treat-ment and prevention inthe group of womenat increased risk of morbidity and mortality due to HPV-related dis-eases.
TheabilityofthepersistentHPVinfectiontocause malig-nanttransformationisassociatedtotheviralDNAintegration into thegenomeofthehost.32However,somestudieshave
challenged this statement. These studies indicate that the necessityofintegrationforthecarcinogenesisvarieswiththe HPVgenotype(forexample,only50%oftheHPV-16genome integrates with the host, while HPV-18 needs to integrate 94%).33Thisdistinctionisparticularlyimportantforthe
com-prehensionofthe diseaseetiologyinHIV-infectedpatients, sincethese are commonlyinfected byHPVgenotypes that requireintegrationwiththehostgenomeforthe carcinogene-sisdevelopment.Furthermore,recentstudieshavedescribed theassociationbetweentheintegrationrateandthecervical dysplasiaseverity.34
Concerning the Arg72Pro TP53 gene polymorphism, the present study found no statistically significant relation betweenobservedgenotypes,frequencyoftheArg andPro allelesandHPVpresence.Moreover,itisworthnotingthatthe controversialresultsregardingtheassociationbetweenTP53
genepolymorphismandcervicalcancercanoccurduetosome factors,suchasdifferencesintheTP53polymorphismamong ethnicalgroups,samplesize,choiceofthecontroltobeused andDNAobtainingsource.35Foranexample,astudyusing normalcervicalsamplesandhigh-andlow-risklesion sam-plesfailedtoshowanassociationbetweenthepresenceof thepolymorphismandfrequencyoftheArgandProalleles withincreasedriskforCC,12suggestingthattheassociation
betweentheTP53genepolymorphismandthedevelopmentof HPV-associatedcervicalneoplasiaisimprobable.However,ina meta-analysisstudyperformedoneyearlater,itwasobserved significantlyhigheroddsofprogressionfromSILtoCCwith thep53Argallele[OR1.37;95%CI,1.15–1.62;p<0.001]in HPV-positiveindividuals.36
Thereare afewstudiescorrelatingMDM2SNP309toCC susceptibility,althoughSNP309presentsarelativelyhigh fre-quencyinthegeneralpopulationanditspresencehasbeen associatedwiththeaccelerationoftumorigenesisandtime oftumoronset.10 Inthis study,TTandTGgenotypes were
significantly morefrequentin HR-HPVandLR-HPV groups, respectively,butpresenceorfrequencyoftheTandGalleles wasnotsignificantlyassociatedwithHPVinfection.Thereis aninconsistencyregardingtheroleofMDM2SNP309in sus-ceptibility tothe CC development. Nunobiki et al. founda prevalenceof78.8%fortheTGgenotypeamonghigh-grade squamous intraepithelial lesion(HSIL)sampleswhen com-paredwithlow-gradesquamousintraepitheliallesion(LSIL) samplesandcontrols,amongHR-HPVpositivesamplesand, specially,theHPV-16and-18genotypes.11However,therewas
noassociationbetweenthesamplesandthefrequenciesofT andGalleles.Meissneretal.inastudyconductedin North-easternBrazilforidentificationoftheMDM2SNP309in72CC samplesand100normalsamplesbyPCRfoundno statisti-callysignificantassociationbetweentheSNP309andtherisk andearlydiagnosisforCC.37Also,nodifferencewasobserved
forthefrequencyofallelesorgenotypesamongthegroupof patientsdiagnosedwithCCatanearlyage(below40years) and thegroupofolderpatients.Ontheotherhand, Arvan-tisandSpandidosthroughtheanalysisofmRNAexpression profilesof24G1-andS-phasecheckpointgenesin35cervical carcinomassamples,26HSILsamples,33LSILsamples,and
28normalcervicalsamples(usedascontrols),showedthat theMDM2wasfoundpositivelyregulatedinSIL,suggesting thatMDM2geneisapotentialcandidateforcervical neopla-siadevelopment.38 AlthoughthisdatasuggestthatHR-HPV
infectionandtheMDM2SNP309maybeassociatedwith cer-vicalcarcinogenesis,inourstudytherewerenosamplesfrom patientswithCC,whichcanbeconsideredalimitingfactor forthesetwoassociationstypes,sincethepolymorphismsof bothMDM2andTP53genesareassociatedwithincreasedrisk forCCandnotwithsusceptibilitytoHPVinfection.
AbetterunderstandingoftheHPVinfectionevolutionin thesewomen,theviralpersistenceoccurrence,andpossible factors associated to the malignant cervical lesions devel-opmentare necessaryforestablishingmoreappropriateCC screeningroutines in this population. This is essential for thepropositionofmoreappropriatemonitoringstrategiesand treatmentaccordingtotheBrazilianhealthservicereality,as wellaspatients.
Conclusion
Current recommendations are partly based on the knowl-edgeofprofessionalsaboutCCmanagementandHIVinfection in women. The data obtained in this study suggest that HIV-infectedwomenaremoresusceptibletobeinfectedby theLR-HPVgenotypesthan bythehigh-riskHR-HPV. Addi-tionally, Pro72Pro of TP53 gene and TG of MDM2 SNP309 genotypesapparently seem tobe protectivefactors among HI-infectedwomen forHPVacquisition and HR-HPV infec-tion,respectively,inasampleofSouthernBrazilianwoman. Moreover,furtherstudiesarenecessaryfortheinvestigation ofthebehavioroflow-riskgenotypesinfectingHIV-mediated immunosuppressedcases.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TheauthorsaregratefultoBrazilianNationalResearch Coun-cil(CNPq).
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