Association of the XPD and XRCC3 gene polymorphisms with oral squamous cell carcinoma in a Northeastern Brazilian population: A pilot study

Short

communication

Association

of

the

XPD

and

XRCC3

gene

polymorphisms

with

oral

squamous

cell

carcinoma

in

a

Northeastern

Brazilian

population:

A

pilot

study

Joabe

dos

Santos

Pereira

a

_,

_Fabrícia

_Lima

_Fontes

b

_,

_Silvia

_Regina

_Batistuzzo

_de

_Medeiros

b

_,

Roseana

de

Almeida

Freitas

c

_,

_Lélia

_Batista

_de

_Souza

c

_,

_Márcia

_Cristina

_da

_Costa

_Miguel

c,

_*

a_{DepartamentofPathology,FederalUniversityofRioGrandedoNorte,Natal,RioGrandedoNorte,Brazil}

b_{DepartmentofCellularBiologyandGenetics,FederalUniversityofRioGrandedoNorte,Natal,RioGrandedoNorte,Brazil}

c_{Post-GraduationProgramofOralPathology,DepartamentofDentistry,FederalUniversityofRioGrandedoNorte,Natal,RioGrandedoNorte,Brazil}

ARTICLE INFO

Articlehistory:

Received13February2015

Receivedinrevisedform6October2015 Accepted19December2015

Keywords:

DNArepair

Geneticpolymorphisms Oralcancer

Restrictionfragmentlengthpolymorphisms Squamouscellcarcinoma

ABSTRACT

Objective:toevaluatetheassociationbetweenXPDandXRCC3polymorphismsandoralsquamouscell carcinoma(OSCC).

Design:thesampleconsistedof54casesofOSCCand40casesofinflammatoryfibroushyperplasia(IFH). Genotypesweredeterminedbythepolymerasechainreaction-restrictionfragmentlength polymor-phism(PCR-RFLP)method.

Results:XPD-Lys/GlnwasmorecommoninIFH(n=28;70%)thaninOSCC (n=24;44.4%)(OR:0.3;

p<0.05).XPD-Glnwasmorefrequentinhigh-gradelesions(0.48)thaninlow-gradelesions(0.21)(OR: 3.4;p<0.05).TheGln/GlngenotypewasassociatedwithIIIandIVclinicalstages(OR:0.07;p<0.05).

XRCC3-Metwas morefrequentin OSCC(0.49) thaninIFH(0.35) (OR:2.6;p<0.05).TheMet/Met genotypewasassociatedwiththepresenceofmetastases(OR:8.1;p<0.05)andwithIIIandIVclinical stages(OR:0.07;p<0.05).

Conclusions:inthissample,thefrequencyofXPD-GlninIFHsuggeststhatthisvariantmayprotectagainst OSCC.ThepresenceoftheXRCC3-MetalleleseemstocontributetothedevelopmentofOSCC,metastases andmoreadvancedstagesintheselesions.

ã2015ElsevierLtd.Allrightsreserved.

1.Introduction

Oralcancerhasbeenthesubjectofmanystudiesbecauseitisan importantcauseofmorbidityandmortalityworldwide.Themost common type of oral cancer is oral squamous cell carcinoma (OSCC),whichaccountsforapproximately90%ofallmalignantoral neoplasms(Syrjanen,2005).Animportantaspectoforal carcino-genesis is individual genetic susceptibility, which is based on differencesintheindividual’sabilitytometabolizecarcinogensas aresultofthepresenceofdifferenttypesofpolymorphismsthat

mayormaynotfavorthedevelopmentofcancer(daSilvaetal., 2011).Inthisrespect,DNArepairgenesareessentialtomaintain theintegrityofthegenome(Huang,Chang,Liu,Lin,&Hsia,2010). AcommonpolymorphismintheXPDgeneischaracterizedbya

nucleotidechangeincodon751,whichresultsinthesubstitution of the amino acid Lys for Gln. This polymorphism has been associated withtheoccurrenceof differentneoplasms(Dufloth, Arruda,Heinrich,Schmitt,&Zeferino,2008;Zhuetal.,2014).On theotherhand,oneofthepolymorphismsfoundintheXRCC3gene ischaracterizedbyanon-conservativesubstitutionoftheamino acidThrforMetincodon241(exon7).Thispolymorphismhas beenstudiedregardingitsinfluenceonthesensitivitytoradiation andinductionofDNAdamage(Gokkusuetal.,2013).Theobjective of this study was to evaluate the association between the frequencyoftheXPD-Lys751Gln(rs13181)andXRCC3-Thr241Met (rs861539)polymorphismsandtheclinical–pathologicalprofileof aseriesofOSCCcases.

* Corresponding author at: Universidade Federal do Rio Grande do Norte, DepartamentodeOdontologia,ProgramadePós-GraduaçãoemPatologiaOral,Av. SenadorSalgadoFilho,1787,LagoaNova,Natal/RN,CEP59056-000,Brazil. Fax:+558432154138.

E-mailaddress:[email protected](M.C.daCostaMiguel).

http://dx.doi.org/10.1016/j.archoralbio.2015.12.004 0003-9969/ã_{2015ElsevierLtd.Allrightsreserved.}

–

ContentslistsavailableatScienceDirect

Archives

of

Oral

Biology

2.Materialsandmethods

2.1.Sample

The project was approved by the Ethics Committee of the FederalUniversityofRioGrandedoNorte(ProtocolNo.76510)and was conductedin accordancewith theDeclaration of Helsinki. Writteninformedconsentwasobtainedfromeach subject.The sampleconsistedof54casesofOSCCand40casesofinflammatory fibroushyperplasia(IFH)ascontrol.Inflammatoryfibrous hyper-plasia was chosen as thecontrol since it is a reactional (non-neoplastic)lesioncausedbyinjuriesthattriggerachronicprocess characterized by the formation of excess repair tissue (Zarei, Chamani,&Amanpoor,2007;Santos,Nonaka,Pinto,&deSouza, 2011).Histologicalmalignancygradingwasevaluatedaccordingto parametersproposedbyBryne(1998)andadaptedbydaSilveira etal. (2010). Briefly,four morphologic features wereanalyzed: degree of keratinization, cellular pleomorphism, pattern of invasion,andinflammatorycellinfiltration.Eachonereceiveda score ranging from 1 to 4. Cases with a total score 8 were classified aslow-grade, while those>8 wereclassifiedas high-grade.Clinicalparameters(metastasisandtumor-node-metastasis stage)werealsoevaluated.

2.2.DNAextraction

DNAwasextractedfromparaffinblocksusingChelex1001_resin

(BioRad, Hercules, CA, USA) and the QIAamp DNA Minikit1

(Qiagen,Hamburg,Germany),accordingtomanufacturer instruc-tions. Theblocks were deparaffinized withxylene, followedby enzymaticdigestionwithproteinaseK(Invitrogen,GrandIsland, NY,USA).Next,200

m

lAEbuffer(10mMTris–HCl,0.5mMEDTA, pH9.0)ordistilledwaterwasadded.

2.3.PCRamplification

Amplifications were performed in a DNA thermal cycler (Mastercycler1 _{pro, Eppendorf, Hamburg, Germany). Protocols}

forPCRwereadaptedfromdeSouzaetal.(2011).PCRamplification oftheLys751GlnpolymorphismoftheXPDgenewascarriedoutin

afinalvolumeof28

m

lcontaining2

m

lgenomicDNA(124.5ng/ml), 0.5

m

l(0.1nM)ofeachprimer,and25

m

lPlatinumPCRSuperMix1

(Invitrogen Co., Carlsbad, CA, USA). PCR amplification of the Thr241MetpolymorphismoftheXRCC3genewascarriedoutina

finalvolumeof34

m

lcontaining3

m

lgenomicDNA(124.5ng/ml), 0.5

m

l(0.1nM)ofeachprimer,and30

m

lPlatinumPCRSuperMix1_.

PrimersPCO3+ and PCO4+, flanking a sequence of the human

b

-globingene,wereusedaspositivecontrol(Soares,Oliveira,de Souza, Costa Ade, & Pinto, 2008). Amplification of the human

b

-globingenefragmentwascarriedoutinafinalvolumeof28

m

l containing2

m

lgenomicDNA(124.5ng/ml),0.5

m

l(0.1nM)ofeach primer,and25

m

lPlatinumPCRSuperMix1_{.Thecharacteristicsof}

theprimersusedarespecifiedinTable1.

ThedigestionofPCRproductswasperformedaccordingtothe manufacturer’sinstructions.FortheLys751Glnpolymorphismof theXPDgene,theamplifiedproductsweredigestedinamixture containing5

m

loftheamplifiedsample,2

m

l10XNEBuffer3(New EnglandBiolabs,Ipswich,MA,USA),0.3

m

l(20,000U/ml)PstI,and 12.7

m

l Milli-Qwater. For the Thr241Met polymorphism of the

XRCC3gene,theamplifiedproductsweredigested ina mixture containing4

m

loftheamplifiedsample,2

m

l10XNEBuffer4,2

m

l BSA(100

m

g/ml),0.5

m

lNlaIII(10,000U/ml),and13.5

m

lMilli-Q water.

2.4.Statisticalanalysis

ThedatawereanalyzedusingtheStatistical Packagefor the Social Sciences for Windows(SPSS 20.0, Chicago, IL, USA). The resultswereanalyzedstatisticallyusingPearson’schi-squaretest, adoptingalevelofsignificanceofp< 0.05.Theoddsratiowasalso calculated todeterminethe degreeanddirectionof correlation between the variables studied, considering a 95% confidence intervalandasignificantdifferencewhenp< 0.05.

3.Results

Theclinical–pathologicalcharacteristicsoftheselectedsample are summarized in Table 2. The frequency distribution of all genotypes was in Hardy–Weinberg equilibrium (p>0.05) for

patientswithOSCCandcontrols.

3.1.AnalysisofalleleandgenotypefrequenciesforXPD

ComparisonofOSCCsandIFHsshowedahigherfrequencyof the Lys/Gln genotype in the latter, with this difference being statisticallysignificant(p=0.033).TheriskofdevelopingOSCCwas lowerinpatientscarryingtheLys/Glngenotypecomparedtothose withtheLys/Lysgenotype(OR:0.373;95%CI0.148–0.936)(Table3). PatientscarryingtheGlnalleleweremorethanthreetimesmore likely to develop high-grade lesions (OR: 3.409; 95%CI 1.101– 10.56). Analysis of the frequency of the Gln allele revealed a significantassociationbetweenthisalleleandhigh-gradelesions (p=0.031).Gln/GlngenotypewasassociatedwithIIIandIVclinical stages (p=0.01).Patients carrying Lys/Lys genotypedisclosed a

Table1

Geneticpolymorphisms,fragmentsizesandprimerssequences.a

Gene Polimorphism Size Primers(50_–30₎

Forward Reverse

XPD Lys751Gln

(rs13181) 161pb CTGCTCAGCCTGGAGCAGC AAGACCTTCTAGCACCACC

XRCC3 Thr241Met

(rs861539)

194pb AAGAAGGTCCCCGTACTGCT CTCACCTGGTTGATGCACAG

b-globin – 110pb CTTCTGACACAACTGTGTTCACTAGC TCACCGCAACTTCATCCACGTTCACC

a_{PrimerssequencesforXPDgenepolymorphismwereobtainedfromSliwinskietal.(2011)thoseforXRCC3genepolymorphismwerebuiltusingPrimer3}1_software

(0.4.0version,availableathttp://primer3.sourceforge.net/),andthoseforhumanb-globinwereobtainedfromSoaresetal.(2008).

Table2

Gender,metastasisandhistologicalgradeofmalignancyofthesample.

Gendern(%) Metastasisn(%) Gradationn(%) Presence Absence Highgrade Lowgrade Female 12(22.2) 3(13.6) 9(28.1) 3(10.7) 9(34.6) Male 42(77.8) 19(86.4) 23(71.9) 25(89.3) 17(65.4) Total 54(100) 22(100) 32(100) 28(100) 26(100)

lowerrisk topresentmore advancedstages compared tothose withGln/Glngenotype(OR:0.073;95%CI0.007–0.725)(Table4).

3.2.AnalysisofalleleandgenotypefrequenciesforXRCC3

For XRCC3, Thr/Met and Met/Met genotypes disclosed an increased risk for developing OSCC (OR: 2.550; 95%CI 0.964– 6.744 and OR:3.375; 95%CI 0.812–14.02, respectively), but the differencewasnotsignificant(p>0.05).TheMetallelicvariantwas more frequent in OSCCs (0.49) than in IFHs (0.35) and this differencewassignificant(p=0.037).(Table3).

Themostfrequentgenotypesinpatients withoutmetastases wereThr/MetandThr/Thr(p=0.037).PatientscarryingtheMet/ Metvariantshowedamorethan8-foldhigherriskofdeveloping metastaticlesions(OR:8.167;95%CI1.027–64.97).StagesIIIandIV were associated with Met/Met genotype (p=0.012). Thr/Thr genotypedisclosedalowerrisktopresentmoreadvancedstages comparedtoMet/Metgenotype(OR:0.071;95%CI0.008–0.649) (Table4).

Table3

XPDandXRCC3genotypesaccordingtolesion.

Lesion OR CI(95%) pvalue*

OSCCn(%) IFHn(%)

XPDGenotypes

Lys/Lys 23(42.6) 10(25) Ref. Lys/Gln 24(44.4) 28(70) 0.373 0.148–0.936 0.033 Gln/Gln 7(13) 2(5) 1.522 0.267–8.656 0.634 Glnfrequence 0.35 0.4 0.449 0.183–1.101 0.077

XRCC3Genotypes

Thr/Thr 10(18.9) 15(38.5) Ref. Thr/Met 34(64.2) 20(51.3) 2.550 0.964–6.744 0.056 Met/Met 9(17) 4(10.3) 3.375 0.812–14.02 0.087 Metfrequence 0.49 0.35 2.688 1.046–6.904 0.037 Abbreviations:OSCC,oralsquamouscellcarcinoma;IFH,inflammatoryfibrous hyperplasia;OR,oddsratio;CI,confidenceinterval.

* _Pearson

’schi-squaretest.

Table4

XPDandXRCC3genotypesaccordingtometastasis,histologicgradingofmalignancy,andclinicalstaging.

XPDGenotypes Metastasis OR CI(95%) pvalue*

Presencen(%) Absencen(%)

Lys/Lys 11(50) 12(37.5) Ref.

Lys/Gln 6(27.3) 18(56.2) 0.3636 0.106–1.250 0.104

Gln/Gln 5(22.7) 2(6.2) 2.727 0.436–17.05 0.273

Glnfrequence 0.36 0.34 0.6000 0.199–1.804 0.361

XPDGenotypes Gradation OR CI(95%) pvalue*

Highgraden(%) Lowgraden(%)

Lys/Lys 8(28.6) 15(57.7) Ref.

Lys/Gln 13(46.4) 11(42.3) 2.216 0.684–7.179 0.181

Gln/Gln 7(25) 0(0) – – –

Glnfrequence 0.48 0.21 3.409 1.101–10.56 0.031

XPDGenotypes Clinicalstaging OR CI(95%) pvalue*

IandIIn(%) IIIandIVn(%)

Lys/Lys 16(50) 7(31.8) Ref.

Lys/Gln 15(46.9) 9(40.9) 0.729 0.217–2.454 0.609

Gln/Gln 1(3.1) 6(27.3) 0.073 0.007–0.725 0.010

Glnfrequence 0.27 0.48 0.467 0.150–1.450 0.184

XRCC3Genotypes Metastasis OR CI(95%) pvalue*

Presencen(%) Absencen(%)

Thr/Thr 3(13.6) 7(22.6) Ref.

Thr/Met 12(54.5) 22(71) 1.273 0.277–5.847 0.756

Met/Met 7(31.8) 2(6.5) 8.167 1.027–64.97 0.037

Metfrequence 0.59 0.41 1.847 0.420–8.121 0.412

XRCC3Genotypes Gradation OR CI(95%) pvalue*

Highgraden(%) Lowgraden(%)

Thr/Thr 3(10.7) 7(28) Ref.

Thr/Met 19(67.9) 15(60) 2.956 0.651–13.42 0.150

Met/Met 6(21.4) 3(12) 4.667 0.672–32.38 0.110

Metfrequence 0.55 0.42 3.241 0.736–14.27 0.1087

XRCC3Genotypes Clinicalstaging OR CI(95%) pvalue*

IandIIn(%) IIIandIVn(%)

Thr/Thr 8(25.8) 2(9.1) Ref.

Thr/Met 21(67.7) 13(59.1) 0.404 0.074–2.205 0.285

Met/Met 2(6.5) 7(31.8) 0.071 0.008–0.649 0.012

Metfrequence 0.40 0.61 0.287 0.055–1.515 0.125

Abbreviations:OR,oddsratio;CI,confidenceinterval.

* _{Pearson’schi-squaretest.}

4.Discussion

The present results regarding the frequency of XPD gene polymorphismshowedapossibleprotectiveroleoftheGlnallelic variantagainstthedevelopmentofOSCC.Similarresultshavebeen reportedinstudiesinvestigatingtumorsatdifferentsites.(Casson, 2005;Mechanicetal.,2005;Sliwinskietal.,2011)Althoughthe specificfunctionofthepolymorphicvariantsofXPDisnotfully

understood,thesubstitutionofnucleotidesincodon751produces adramatic changein theelectricconfigurationof theresulting aminoacid(Casson,2005).Takentogether,thepresentresultsand the findings of other studies suggest a protective role of this polymorphismagainstthedevelopmentofOSCC.Incontrast,some studiesdemonstrated anassociation of theXPDgene polymor-phismwithanincreasedriskofdevelopingheadandneckcancer (Yeetal.,2006;Liuetal.,2007).Itshouldbenotedthatthepresent study is a preliminary study and the first to investigate the distributionoftheXPDLys751Glnpolymorphisminpatientswith

OSCCandacontrolgroupfromNortheasternBrazil.

With respect to the histological grade of malignancy, the presenceoftheGlnvariantwasassociatedwithamorethan three-fold increase in the risk of developing high-grade lesions. Regardingclinicalstage, Gln/Gln genotype was associated with tumorspresentingmoreadvanced stages.Similarly, Wangetal. (2011)observedanassociationbetweentheLys/GlnandGln/Gln genotypesanda higherriskofdevelopinghigh-gradeurothelial carcinoma.Theyalsofoundahigherriskfortumorstage,although nosignificant.ThesameassociationwasreportedbySobtietal. (2012)forbladdercarcinomas,butthisassociationwassignificant. OtherstudiesfoundnocorrelationbetweentheXPDgenotypeand the histological grade (Mandal, Gangwar, Mandhani, & Mittal, 2010;Szkanderaetal.,2013)orstage(Sliwinskietal.,2009;Agalliu etal.,2010)ofsometumors.Thesedivergentresultsmayberelated tothebiologicaldiversityofmalignanttumorsofdifferentorigins andassociated withetiological factorsother thansmoking and alcohol,asobservedinOSCC.Inthisrespect,thepresentresults suggestthat thevariantgenotype couldbeassociatedwiththe inhibitionofthedevelopmentofOSCC.Itsassociationwith high-gradeand advanced stagelesionsis a findingthat needstobe confirmedinfuturestudiesinvolvinglargersamples.

Analysis of thefrequencyoftheXRCC3 Thr241Met

polymor-phismindicatesthatthispolymorphismmaybeafacilitatorofthe developmentofOSCC.Resultssimilartothosehavebeenreported intheliterature(Werbroucketal.,2008).Evidenceindicatesthat theThr/Metvariantresidesintheadenosinetriphosphate-binding domainofXRCC3,theonlydomainwithknownfunctionalactivity (Manuguerra et al., 2006). This fact suggests that the change causedbytheXRCC3Thr241Metpolymorphismisassociatedwith other proteins, especially Rad51 (Werbrouck et al., 2008). The productofXRCC3isaRad51paralognecessaryforthebindingand

stabilizationof the latter during DNA repair. Additionally, this polymorphismmaybeinlinkagedisequilibriumwithotherfactors sincevariousgenesareinvolvedin DNArepair(Ladeira,Viegas, Carolino,Gomes,&Brito,2013).

SomestudiesextractingDNAfromperipheralbloodfoundno associationbetweentheXRCC3Thr241Metpolymorphismandthe developmentofOSCC(Yenetal.,2008;Gresneretal.,2012).The differences between the present results and studies in the literaturemay be related to parameters such as differences in DNAextraction(paraffin-embeddedmaterialorperipheralblood), specificityoftumorsites,ethnicdifferencesingenotype distribu-tion, and interactions with other cancer susceptibility genes (Mechanicetal.,2005).

Withrespecttothepresenceorabsenceofmetastases,theMet/ Metgenotypewasassociatedwithamorethan8-foldincreasein theriskof developingmetastases in OSCC.Similarresultshave

beenreportedbyKrupaetal.(2009),suggestingthattheMetallelic variantsomehowdoesnotonlyinfluencethedevelopmentofOSCC as shown previously, but also the development of tumor metastases.Toourknowledge,thisisthefirststudytoinvestigate and identify a correlation between the XRCC3 Thr241Met

polymorphismandtheriskofmetastasisinOSCC.

Concerning about clinical stage, the Met/Met genotype was associatedwithahigherriskofdevelopingIIIandIVstages.Others studiesfoundnocorrelationbetweentheXRCC3genotypesand clinicalstage(Krupaetal.,2011;Zhuetal.,2012).Accordingto Krupaetal.(2011),differentfindingsforXRCC3Thr241Metcould

berelated tocomplex interactionsbetweenthis polymorphism andothers,underlinedbymechanismsnotyetdescribed.

Thepresentstudyhassomelimitations,likethedifficultyin obtainingmaterialfromparaffinblocksofsufficientquantityand quality for DNA extraction and amplification of the regions of interest.However,thepresentresultscouldserveasabasisfornew studiesinvolvinglargersamples.

Finally,consideringthebroadpresenceandvariabilityofSNPs betweenindividuals,aswellastheincreasingevidenceoftheir influenceonthedevelopmentofdifferentdiseases,theadequate understandingofthepathologicalmechanismsofthesevariants andoftheinteractionwithotherSNPswillcontributetotheearly detection of groups that are at risk of developing malignant neoplasms,includingOSCC,andthustothepreventionofthese diseases.

Conflictofinterest

Theauthorshavenoconflictofinterest.

Ethicalapproval

Thepresentstudywasindependentlyreviewedandapproved bytheEthicsCommitteeoftheFederalUniversityofRioGrandedo Norte(CEP/UFRN)(PermitNo.76510).

Funding

TheuniquesourceoffundingwasthePostGraduationProgram inOralPathologyfromFederalUniversityofRioGrandedoNorte.

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