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www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Polymorphisms

in

methylenetetrahydrofolate

reductase

and

cystathionine

beta-synthase

in

oral

cancer

---

a

case---control

study

in

southeastern

Brazilians

Andressa

Barbosa

a

,

Marcelo

dos

Santos

a

,

José

Roberto

Vasconcelos

de

Podestá

b

,

Sônia

Alves

Gouvêa

c

,

Sandra

Ventorin

Von

Zeidler

d

,

Iúri

Drumond

Louro

a

,

Melissa

de

Freitas

Cordeiro-Silva

e,

aUniversidadeFederaldoEspíritoSanto(UFES),DepartamentodeCiênciasBiológicas,Vitória,ES,Brazil

bHospitalSantaRitadeCássia,DivisãodeCirurgiadeCabec¸aePescoc¸o,ProgramadePrevenc¸ãoeDetecc¸ãoPrecocedeCâncer

Oral,Vitória,ES,Brazil

cUniversidadeFederaldoEspíritoSanto(UFES),DepartamentodeCiênciasFisiológicas,Vitória,ES,Brazil dUniversidadeFederaldoEspíritoSanto(UFES),DepartamentodePatologia,Vitória,ES,Brazil

eFaculdadeCatólicaSalesianadoEspíritoSanto,Vitória,ES,Brazil

Received1April2015;accepted2October2015 Availableonline17December2015

KEYWORDS

Oralsquamouscell carcinoma; Methylenetetra-hydrofolate reductase; Cystathionine beta-synthase; Genetic polymorphism

Abstract

Introduction:Oralsquamous cellcarcinoma (OSCC)is aseriouspublichealth problem,due toitshighmortalityrateandworldwiderisingincidence.OSCCsusceptibilityismediatedby interactions between geneticand environmental factors. Studiessuggest thatgenetic vari-antsencodingenzymesinvolvedinfolatemetabolismmaymodulateOSCCriskbyalteringDNA synthesis/repairandmethylationprocess.

Objective:Thegoalsofthisstudyweretoevaluatetheassociationofthreegenotypic polymor-phism(MTHFRC677T,MTHFRA1298CandCBS844ins68)andoralcancerriskinsoutheastern Braziliansandevaluatetheinteractionsbetweenpolymorphismsandclinicalhistopathological parameters.

Methods:Thiscase---controlstudyincluded101casesand102controlsinthestateofEspírito Santo,Brazil.MTHFRgenotypingwasdonebyPCR-RFLP(polymerasechainreaction--- restric-tionfragmentlengthpolymorphism)andCBSgenotypingbyPCR(polymerasechainreaction) analysis.

Pleasecitethisarticleas:BarbosaA, dosSantosM,de PodestáJR, GouvêaSA,Von ZeidlerSV, LouroID,et al.Polymorphisms in

methylenetetrahydrofolatereductaseandcystathioninebeta-synthaseinoralcancer---acase---controlstudyinsoutheasternBrazilians.Braz JOtorhinolaryngol.2016;82:558---66.

Correspondingauthor.

E-mail:melissafcs@yahoo.com.br(M.F.Cordeiro-Silva).

http://dx.doi.org/10.1016/j.bjorl.2015.10.012

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Results:MTHFRC677Tpolymorphismwasassociatedwithlymphnodeinvolvement.Genotype CT+TT acted asa protective factor. MTHFR A1298C AC+CC genotype was associated with tumor differentiation, and possiblywith a betterprognosis.In risk analysis,no correlation wasobservedbetweengenotypesandOSCC.

Conclusion: WeconcludedthatMTHFRC677T,MTHFRA1298CandCBS844ins68polymorphisms werenotassociatedwithOSCCriskinsoutheasternBrazilians;however,wesuggestaprognosis effectassociatedwithMTHFRC677TandA1298CpolymorphismsinOSCC.

© 2015 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE

Carcinoma epidermoideoral; Metilenotetrahidrofolato redutase;

Cistationina beta-sintase; Polimorfismo genético

Polimorfismosemmetilenotetrahidrofolatoredutase,cistationinabeta-sintase nocâncerdeboca---umestudodecaso-controlenoSudestebrasileiro

Resumo

Introduc¸ão: Ocarcinomaespinocelularoral(CECO) trata-sedeum importanteproblemade saúdepública,devidoàelevadataxademortalidadeeincidênciacrescenteemtodoomundo. AsusceptibilidadeaoCECOémediadaporinterac¸õesentrefatoresgenéticoseambientais. Estu-dossugeremqueasvariantesgenéticasquecodificamasenzimasenvolvidasnometabolismo dofolatopodemmodularoriscodeCECO,alterandoasíntese/reparac¸ãodoDNAeoprocesso demetilac¸ão.

Objetivo: Os objetivos deste estudo foram avaliar a associac¸ão de três polimorfismos genotípicos (MTHFR C677T, MTHFR A1298C e CBS 844ins68) e o risco de câncer bucal em brasileirosdaregiãoSudeste,eavaliarasinterac¸õesentrepolimorfismoseparâmetros clínico-histopatológicos.

Método: Esteestudodecaso-controleincluiu101casose102controlesnoestadodoEspírito Santo,Brasil.A genotipagemdopolimorfismoMTHFRfoirealizada porPCR-RFLP (Reac¸ãode PolimeraseemCadeia---PolimorfismonoComprimentodeFragmentodeRestric¸ão)eadoCBS poranálisedaPCR(Reac¸ãodePolimeraseemCadeia).

Resultados: OpolimorfismoMTHFRC677Tfoiassociadoaoenvolvimentodegânglioslinfáticos. OgenótipoCT+TTatuoucomoumfatorprotetor.OgenótipoMTHFRA1298CAC+CCfoi associ-adoàdiferenciac¸ãodotumore,possivelmente,aumprognósticomelhor.Naanálisederisco, acorrelac¸ãoentreosgenótiposeoCECOnãofoiobservada.

Conclusão:ConcluímosqueospolimorfismosMTHFRC677T,MTHFRA1298CeCBS844ins68não estãoassociadosaoriscodeCECOnosbrasileirosdaregiãoSudeste;noentanto,sugerimosum efeitoprognósticoassociadoaospolimorfismosMTHFRC677TeA1298CemCECO.

© 2015 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Oral squamous cell carcinoma (OSCC) is the eighth most commonhumancancerworldwide.1InBrazil,nearly15,290

newcasesoforal cancerareexpectedin2014,andinthe Southeast it is the fourth among men and tenth among women.2OSCCisamultifactorialdisease,affectedby

noto-riousenvironmentalfactorssuchasalcoholandtobacco,as wellasgeneticfactors,ofwhichlittleis known. Polymor-phismsincertaingenes mayconfersusceptibilitytoOSCC development.Studies have shown a relationship between polymorphismsofgenesinvolvedinfolatemetabolismand OSCCriskduetotheirinfluenceonmethylation,synthesis andDNArepair.3---7

MTHFR gene encodes the methylenetetrahydrofolate reductase enzyme, that is important for intracellu-lar folate homeostasis and the irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-MTHF) into 5-methyltetrahydrofolate (5-MTHF). Polymorphisms C677T andA1298CintheMTHFRgenemaybeassociatedwithoral cancersusceptibilityduetochangesincatalyticactivity.The C677Tpolymorphismresultsinanenzymewith65percentof thewild-typehomozygoteactivityforheterozygotesand30 percentforhomozygotesofthevariantallele.8,9TheMTHFR

A1298Cpolymorphismislocalizedintheregulatorydomain region.10Homozygous1298Cindividualshaveapproximately

thesameenzymeactivityofthosewhoareheterozygous.11

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offolatefortheproduction ofthymidylateandpurinefor DNAsynthesisandrepair.12

TheCBSgeneencodescystathioninebetasynthase(CBS), also involved in the folate pathway, which mediates the conversionofhomocysteinetocystathionine.CBS844ins68 polymorphismhasbeenassociatedwithshortersurvivaltime inheadandnecksquamouscellcarcinomapatients.13

There-fore, this study aimed to investigate the frequency and association ofMTHFRand CBSpolymorphisms inoral can-cersusceptibilityinthepopulationofthestate ofEspírito Santo, Brazil and its potential impact on the prognostic outcome.

Methods

Samples

In this case---control study, blood samples were collected from101 patients with conclusive histopathological diag-nosticoforal squamouscellcarcinoma obtainedfromthe HeadandNeck Divisionof theHospitalSanta Ritade Cás-sia, Brazil.Of this total,69 individuals wereclassified by skincolorandotherphysicaltraits:22aswhite(Caucasians, mainly Portuguese descendents); 32 as ‘‘pardos’’ (ethnic mixture of Europeans, Africans and Amerindians), and 15 asblack(Africandescendants),basedontheofficial Brazil-ian census categorization. All patients were residents of thestateEspíritoSanto(ES,Brazil)andrandomlyrecruited from2011to2013.Theinclusioncriteriaduringthisstudy werepatients of both genders, over 35 years of age who acceptedtoparticipateinthestudy.Theexclusioncriteria werepatientswithSCCinothersitesandthosewhoreceived radiotherapy,chemotherapy,surgeryoranyothertreatment priortorecruitment.

Thecontrolgroupwascomposedby102individuals resid-inginES,Brazil,whowerereferredforclinicalassessment andhadanegativecancerfamilialhistoryanddidnotshow pre-malignantormalignantorallesionsatthetimeof sam-plecollection. Thecontrolgroupwasmatchedbyageand gender.As onlypartialdatawasavailablefor thecontrols regardingthehabitssuchsmokingandalcoholusage,itwas notincludedformatchingwiththecases.Allsubjects pro-vided signed informed consents approved by institutional review boards.This work waspreviously approvedby the localresearchethicscommittee(CEPProtocoln◦318/2011). Clinicopathological features of patients analyzed were tumorstage(earlystageI---IIandlatestageIII---IV),size(T1, T2,T3andT4),nodalstatus(positiveN+andnegativeN0) accordingtoTNMClassification,14histolopathologicalgrade

(well,moderatelyandpoorlydifferentiatedtumors)15 and

smokinghabit. Allrequiredinformationabout clinicaland histopathological parameters was obtained from medical records.Participantswerenotclassifiedintoethnicgroups orskincolor.

Genotypingassays

Genomic DNA was isolated by phenol-chloroform extrac-tion.TheMTHFRC677TandMTHFRA1298Cpolymorphisms weregenotypedbythe PCR-RFLP(polymerase chain reac-tion---restrictionfragmentlengthpolymorphism)method,as

previouslydescribed.8,11Allreactionsincludedpositiveand

negativecontrols.Approximately20percentofthesamples were randomly selected to repeat the genotyping proce-dure.Thereproducibilitywas100percent.

The C→T transition creates a restriction site for

the enzyme Hinf I. PCR product (198bp) was digested usingHinf1 andvisualized byelectrophoresis in 8percent polyacrylamidegelsandsilvernitratestaining.PCRproducts includedasingle198bpfragmentforwild-typehomozygotes (CC);198bp,175bpand23bpfragmentsforheterozygote (CT),and175and23bpformutanthomozygote(TT).

The MTHFR1298AC polymorphismeliminates theMboII restrictionsite.

Wildgenotype(AA) producedfive fragmentsof56, 31, 30, 28 and 18bp, whereas heterozygous (AC) yielded six

Table1 Clinicalcharacteristicsofcancerpatientsand con-trolsubjects.

Characteristics Patients Controls p-value

n(%) n(%)

Gender

Female 20(19.8) 20(19.6) 0.972 Male 81(80.2) 82(80.4)

Age,years

≤55 47(46.5) 56(54.9) 0.233

>55 54(53.5) 46(45.1)

Ethnicgroup

Whites 22(31.9) ---

---‘‘Pardos’’ (mixedrace)

32(46.4)

---Blacks 15(21.7)

---Tobaccoexposure

Consumer 73(72.3) 0(0.0) ---Non-consumer 28(27.7) 0(0.0) Unknowna 0(0.0) 102(100.0)

Tumorstage

Earlystage(I,II) 25(24.7) --- ---Advanced(III,

IV)

76(75.3)

---Tumorsizec

T1 12(11.9) --- ---T2 22(21.8)

---T3 17(16.8) ---T4 50(49.5)

---Nodalstatusc

N0 50(49.5) --- ---N+ 51(50.5)

---Histopathologicalgrade

Well 25(24.7) --- ---Moderately 29(28.7)

---Poorly 5(5.0) ---Notavailableb 42(41.6)

---Total 101(49.8) 102(50.2)

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Table2 DistributionofMTHFRandCBSgenotypesamongoralcancerpatientsandcontrolgroups.

Genotypes Total Patients Controls p-value

n(%) n(%) n(%)

MTHFRC677T

CC 100(49.2) 50(49.5) 50(49.0) 0.438

CT 86(42.4) 45(44.6) 41(40.2)

TT 17(8.4) 6(5.9) 11(10.8)

MTHFRA1298C

AA 113(55.7) 60(59.4) 53(52.0) 0.541

AC 80(39.4) 36(35.6) 44(43.1)

CC 10(4.9) 5(5.0) 5(4.9)

CBS844ins68

NN 163(80.3) 76(75.2) 87(85.3) 0.112

NI 38(18.7) 24(23.8) 14(13.7)

II 2(1.0) 1 1

Total 203(100.0) 101(49.8) 102(50.2)

N,non-insertion;I,insertion.

fragmentsof84,56,31,30,28and18bp,andhomozygous mutants (CC) produced four fragments of 84, 31, 30 and 18bp.

CBS 844ins68 polymorphisms were characterized by differential size separation after PCR, as previously described.16 Thepolymorphicalleleresultsfromthe

inser-tion of 68bp at exon 8. The major allele (I)presented a 239bp fragment, and the normal allele (N) presented a 171bpfragment.

Statisticalanalysis

Genotypic frequencies were tested for Hardy---Weinberg equilibrium (HWE). The chi-squareand Fisher exact tests were used for association analysis, and confirmation was obtainedbytheLillieforstest(significanceconsideredwhen

p<0.05).Multivariatelogisticregressionwasusedtoobtain oddsratio(OR)andconfidenceintervals(95%CI).Statistical calculations were performed using the Epi InfoH v 3.4.3,

2007 software. Linkage disequilibrium (LD) and halotype analysiswereconductedbyHaploviewsoftware.

Results

Characteristicsof101oralcancerpatientsand102controls areshowninTable1.Significantdifferencesbetweengroups werenotobserved(p>0.05).

GenotypefrequenciesforMTHFRC677T,A1298CandCBS

844ins68incontrols andoralcancerpatientsareshownin

Table2.

Genotype distribution of the three genetic polymor-phismswasnotsignificantlydifferentbetweenoralcancer andcontrolgroup(p>0.05)(Table2).MTHFRC677T,A1298C andCBS844ins68allelefrequenciesincontrolsandoral can-cerpatientsareshowninTable3.Alltestedpolymorphisms areintheHWE.

Haplo View software was used for conducted LD and haplotypeanalysisonallelesof MTHFRC677TandA1298C

Table3 DistributionofMTHFRandCBSallelesamongtheoralcancerpatientsandcontrolgroups.

Allele Patients Controls p-value

n(%) HWE n(%) HWE

2 p-value 2 p-value

MTHFRC677T

C 145(71.8) 1.006 0.316 141(69.1) 0.348 0.555 0.556

T 57(28.2) 63(30.9)

MTHFRA1298C

A 156(77.2) 0.018 0.893 150(73.5) 1.193 0.275 0.387

C 46(22.8) 54(26.5)

CBS844ins68

N 176(87.1) 0.357 0.550 188(92.2) 0.260 0.610 0.096

I 26(12.9) 16(7.8)

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Table4 LinkagedisequilibriumandhaplotypeanalysisforallelesofC677TandA1298Cpolymorphisms.

Haplotype Cases 2n=202

Controls 2n=204

2 p-value OR(95%CI)

C-A 301 291 --- --- 1

C-C 191 195 0.173 0.676 0.946(0.817---1.364)

T-A 231 213 0.141 0.706 1.048(0.745---1.220)

T-C 103 117 1.040 0.307 0.851(0.861---1.602)

Table5 ClinicopathologicalcharacteristicsofpatientswithOSCCandrelationwiththeMTHFRpolymorphismsstudied.

Features MTHFRC677Tgenotype

CC CT TT p-value CT+TT p-value

n(%) n(%) n(%) n(%)

Tumorstage

Earlystage(I,II) 12(24.0) 11(24.4) 2(33.3) 0.880 13(25.5) 0.862 Advanced(III,IV) 38(76.0) 34(75.6) 4(66.7) 38(74.5)

Tumorsizea

T1 5(10.0) 5(11.1) 2(33.3) 0.569 7(13.7) 0.716 T2 12(24.0) 10(22.2) 0(0.0) 10(19.6)

T3 10(20.0) 6(13.3) 1(16.7) 7(13.7) T4 23(46.0) 24(53.3) 3(50.0) 27(52.9)

Nodalstatusa

N0 19(38.0) 28(62.2) 3(50.0) 0.062 31(60.8) 0.022 N+ 31(62.0) 17(37.8) 3(50.0) 20(39.2)

Histopathologicalgrade

Well 10(20.0) 15(33.3) 0(0.0) 0.192 15(29.4) 0.238 Moderately 12(24.0) 14(31.1) 3(50.0) 17(33.3)

Poorly 4(8.0) 1(2.2) 0(0.0) 1(2.0) Notavailableb 24(48.0) 15(33.3) 3(50.0) 18(35.3)

Features MTHFRA1298Cgenotype

AA AC CC p-value AC+CC p-value

n(%) n(%) n(%) n(%)

Tumorstage

Earlystage(I,II) 14(23.3) 10(27.8) 1(20.0) 0.860 11(26.8) 0.689 Advanced(III,IV) 46(76.7) 26(72.2) 4(80.0) 30(73.2)

Tumorsizea

T1 6(10.0) 6(16.7) 0(0.0) 0.747 6(14.6) 0.512 T2 16(26.7) 5(13.9) 1(20.0) 6(14.6)

T3 10(16.7) 6(16.7) 1(20.0) 7(17.1) T4 28(46.7) 19(52.8) 3(60.0) 22(53.7)

Nodalstatusa

N0 30(50.0) 19(52.8) 1(20.0) 0.386 20(48.8) 0.904 N+ 30(50.0) 17(47.2) 4(80.0) 21(51.2)

Histopathologicalgrade

Well 8(13.3) 14(38.9) 3(60.0) 0.037 17(41.5) 0.007 Moderately 18(30.0) 10(27.8) 1(20.0) 11(26.8)

Poorly 5(8.3) 0(0.0) 0(0.0) 0(0.0) Notavailableb 29(48.3) 12(33.3) 1(20.0) 13(31.7)

aTNMclassification.

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polymorphisms(Table4).Wefoundthattherewasno differ-enceforthehaplotypesinthetwogroups(p>0.05),which suggested that thehaplotypes donot increase therisk of cancer.

Patientswere evaluatedby ethnicity,but aprevalence of polymorphic allelein ethnicgroups was not observed. TheprevalenceoftheCallele(MTHFRC677Tpolymorphism) washigher in the three ethnicgroups (white --- 63.6 per-cent;black---73.3percent,and‘‘pardos’’---82.8percent). FortheMTHFRA1298Cpolymorphism,theAallele predom-inated inallethnicgroups (white---72.7percent;black ---83.3percent,and‘‘pardos’’---76.6percent),andfinally,the presenceofthewild-typealleleforCBS844ins68 polymor-phismwasalsomoreprevalentinallethnicgroups(white ---86.4percent;black--- 86.7percent,and‘‘pardos’’--- 89.1 percent). These data demonstrate the ample mixture of racesinthecasegroup.

Interaction between genotypes and clinicopathological features was further analyzed (Tables 5 and 6). MTHFR

C677T polymorphism is associated with positive lymph nodes, and the combination of CT+TTgenotypes acts as a protective factor. Multivariate analysis (95% CI) consid-ering tumorsizerevealed thatthecombination of CT+TT genotypes generates a risk of lymph node metastasis three times smaller than the CC genotype (p=0.012) (Table7).

We observed thatthe A1298C polymorphism is related to tumor differentiation. AC+CC genotypes were more frequent in well-differentiated tumors, whereas the AA genotype wasmore frequent in moderately or poorly dif-ferentiatedtumors(p=0.007)(Table5).

Table 6 Clinicopathological characteristics of patients withOSCCandrelationwiththeCBSpolymorphismstudied.

Features CBS844ins68genotype

Non-insertion Insertion p-value

n(%) n(%)

Tumorstage Earlystage

(I,II)

18(23.7) 7(28.0) 0.664

Advanced (III,IV)

58(76.3) 18(72.0)

Tumorsizea

T1 8(10.5) 4(16.0) 0.792 T2 18(23.7) 4(16.0)

T3 13(17.1) 4(16.0) T4 37(48.7) 13(52.0)

Nodalstatusa

N0 40(52.6) 10(40.0) 0.273 N+ 36(47.4) 15(60.0)

Histopathologicalgrade

Well 19(25.0) 6(24.0) 0.462 Moderately 22(28.9) 7(28.0)

Poorly 5(6.6) 0(0.0) Notavailableb 30(39.5) 12(48.0)

a TNMclassification.

b Notavailable(notconsideredinthestatisticalcalculations).

Table7 Multivariateanalysisofthenodalstatus,according totumorsizeandMTHFRC677Tpolymorphism.

Nodalstatus(N)a

Variable Multivariateanalysis

OR(95%CI) p-value

Tumorsize(T)a

T1,T2 1

T3 3.07(0.87---10.87) 0.083 T4 6.46(2.32---17.95) <0.001

MTHFRC677T

CC 1

CT+TT 0.32(0.13---0.77) 0.012

OR,oddsratio;CI,confidenceinterval.

a TNMclassification.

There was no statistically significant association betweenCBS844ins68polymorphismandanalyzedvariables (Table6).

Discussion

Folate deficiency has been associated with diseases such ascancer.Therefore,theroleofgeneticpolymorphismsof folatemetabolismenzymeshasbeeninvestigatedinseveral cancer types.17,18 Among these enzymes, we investigated MTHFRandCBSinOSCC.

Ourstudyhasreportedforthefirsttimetheassociation betweenlymphnodemetastasisandMTHFRC677T polymor-phism combination CT+TT, acting as a protective factor (p=0.022). Furthermore, other studies have suggested a betterprognosisfororalcancerpatientswithCTorTT geno-types.Tsaietal.19 showedthat 677CT+TTpatients hada

lowerriskofmetastasiscomparedwiththosewithCC,and Sailasreeetal.20showedimprovedsurvival.

Theprotectiveeffectcouldbeduetoitsdecreased effi-ciency forDNA methylation.21 MTHFRconverts 5,10-MTHF

to5-MTHF.The 5,10-MTHF isusedfor conversionof dUMP todTMP,whereas5-MTHF isthe methyldonorfor synthe-sisofmethionineandS-adenosylmethionineinmethylation reactions.22IndividualswithMTHFR677TTandMTHFR677CT

genotypes show enzymes with decreased activity,8 hence

they tend to accumulate 5,10-MTHF causing a change in the pathway, leading to lowering of DNA methylation.21

A low level of genomic DNA methylation could decrease thechanceofpromoterhypermethylationincancer-related genesandleadtofewermutationsbyspontaneous deami-nationof5-methylcytosine(5mC).Hypermethylationfavors cancerinitiationandprogressionbysilencingtumor suppres-sorgenes or DNArepair genes.23 Mostmutations found in

cancersareC→Ttransitionsat CG:CGsequences,due to

highfrequenciesofspontaneousdeaminationof5mC.24,25

Lack of cytosine methylation could prevent C→T

muta-tions.

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theAAgenotype hadahigher frequencyin moderatelyor poorlydifferentiatedtumors.Althoughtheprognosticvalue of the histological grade is controversial in OSCC, some studieshave suggestedthat poorlydifferentiated carcino-mastendtometastasizeandareassociatedwithdecreased survivalrates.26 However,thereisnoconsensusinthe

lit-eratureaboutwhetherhistologicaltumorgradingisagood isolatedparameter of prognosis value. Tumor histopatho-logical grade, together with additional prognosticfactors andTNMstagingmightprovidebettersupportfortreatment decision.

However,inthisstudywithasoutheasternBrazilian pop-ulation, we did not observe association between MTHFR

C677T,MTHFRA1298CandCBS844ins68polymorphismsand oral cancersusceptibility. Some studieshave alsofound a non-significantreducedrisk for MTHFR677TTgenotype in oral cancer.27,28 In contrast, Sailasree et al.20 had found

thatC677Twasassociatedwithpredispositiontooralcancer witha significant reduced risk for CT+TT genotype indi-viduals. Meta-analysis studies29,30 have shown a marginal

association or no associations of MTHFR C677T polymor-phismwithoralcancerrisk.Theconflictingresultsregarding the associations between MTHFR C677T polymorphisms and risks for OSCC may be due to different ethnici-ties,subtypesand regionaldietaryand localcarcinogens’ exposure.

ForA1298Cpolymorphism,somestudies agreewithour results,includingameta-analysisstudy19,20,31showingalack

ofassociationwithoralcancerrisk.However,meta-analysis demonstratedthatCallelehasapossiblepreventiverolefor oralcancer.29

Inourstudy,theCBS68bpinsertionallele(I)wasneither associatedwithOSCCrisknorwithheterozygousgenotype (I/N)or polymorphic homozygousgenotype (I/I), corrobo-ratingtheresultsofGalbiattietal.,32butunlikeresultsfor

othertumortypessuchasprostateandupper gastrointesti-naltractcancers.33,34

MTHFRC677Tpolymorphismgenotypeprevalencevaries toa great extent amongdifferent human populations. In Indians,thefrequencyofTTgenotypeisbelow1percent,20

whereas among Mexicans, it is above 30 percent; the TT genotype in our controls showed a 10.8 percent fre-quency, which is comparable to previous reports from Chinese and Puerto Rican populations,27,35---37 and also in

regions of Brazil.38---40 The frequencies of TT genotype

demonstratedin studies withpopulations in southeastern Brazil range from4.4 percentto 14 percent in the state of São Paulo.39,41,42 Control prevalence of variant MTHFR

A1298C genotypes (CC) in our study was 4.9 percent, in agreementwithstudies in northeasternBrazil and alsoin otherpopulationsoftheworld,suchasChinese,Japanese, Polish, Italian and Americans19,43---48; however, there was

little difference from frequencies observed in the state of São Paulo, with frequencies of 6.1 percent and 8.8 percent.41,42

Inourstudy,theCBS68bpinsertionallele(I)wasfoundin 7.8percentofthecontrolpopulation. Asimilarfrequency (7%)wasobserved in thePakistanipopulation49;however,

theallelicfrequencywashigherinanotherstudyconducted in São Paulo, Brazil.32 As found in another study,32 CBS

844ins68polymorphism wasnotassociatedwithclinical or histopathologicalfeaturesinourstudy.

Conclusion

Inconclusion,MTHFRC677TCTandTTgenotypeswere asso-ciatedwithlymphnodeinvolvement,actingasaprotective factorinOSCC,andtheMTHFRA1298CAC+CCgenotypewas associatedwithtumor differentiation,whichmaybe asso-ciatedwitha betterprognosis.However,the resultsneed tobe confirmed inlarger studiesof patients andcontrols matchedbysmokinghabit.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ABS wassponsored by a CAPES scholarship.We thank the HeadandNeckDivisionoftheSantaRitadeCássiaHospital, ES,Brazilforhelpingwithsamplecollection.

References

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Imagem

Table 1 Clinical characteristics of cancer patients and con- con-trol subjects.
Table 2 Distribution of MTHFR and CBS genotypes among oral cancer patients and control groups.
Table 5 Clinicopathological characteristics of patients with OSCC and relation with the MTHFR polymorphisms studied.
Table 6 Clinicopathological characteristics of patients with OSCC and relation with the CBS polymorphism studied.

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