OGG1 Mutations and Risk of Female Breast Cancer: Meta-Analysis and Experimental Data

16 Pages·2015·2.18 MB·English

Hindawi Publishing Corporation
Disease Markers
Volume 2015, Article ID 690878, 16 pages
http://dx.doi.org/10.1155/2015/690878
Research Article
OGG1 Mutations and Risk of Female Breast Cancer:
Meta-Analysis and Experimental Data
KashifAli,1IshratMahjabeen,1MaimoonaSabir,1
HumeraMehmood,2 andMahmoodAkhtarKayani1
1CancerGeneticsLaboratory,DepartmentofBiosciences,COMSATSInstituteofInformationTechnology,ParkRoad,
ChakShahzad,Islamabad44000,Pakistan
2NuclearMedicineOncology&RadiotherapyInstitute(NORI),Islamabad44000,Pakistan
CorrespondenceshouldbeaddressedtoMahmoodAkhtarKayani;[email protected]
Received5January2015;Accepted9April2015
AcademicEditor:HollySoares
Copyright©2015KashifAlietal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
InfirstpartofthisstudyassociationbetweenOGG1polymorphismsandbreastcancersusceptibilitywasexploredbymeta-analysis.
Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing.
󸀠
Fifteenmutationswereobserved,whichincludedfiveintronicmutations,foursplicesitemutations,two3UTRmutations,three
missensemutations,andanonsensemutation.Significantly(𝑝<0.001)increased(∼29fold)breastcancerriskwasassociatedwith
asplicesitevariantg.9800972T>Gand3󸀠UTRvariantg.9798848G>A.Amongintronicmutations,highest(∼15fold)increasein
breastcancerriskwasassociatedwithg.9793680G>A(𝑝<0.009).Similarly∼14-foldincreasedriskwasassociatedwithVal159Gly
(𝑝 < 0.01),∼17-foldwithGly221Arg(𝑝 < 0.005),and∼18-foldwithSer326Cys(𝑝 < 0.004)inbreastcancerpatientscompared
withcontrols,whereasanalysisofnonsensemutationshowedthat∼13-fold(𝑝<0.01)increasedbreastcancerriskwasassociated
withTrp375STOPinpatientscomparedtocontrols.Inconclusion,asignificantassociationwasobservedbetweenOGG1germline
mutationsandbreastcancerrisk.ThesefindingsprovideevidencethatOGG1mayprovetobeagoodcandidateofbetterdiagnosis,
treatment,andpreventionofbreastcancer.
1.Introduction acids)andisoform𝛽-OGG1(424aminoacids)proteins.The
first 316 amino acids are common for both isoforms, while
8-Oxoguanine DNA glycosylase 1 (OGG1) is an important the C-termini vary considerably. OGG1 has two important
protein in base excision repair (BER) pathway which plays domains; the OGG-N domain containing mitochondrial
a key role in maintaining genome integrity and preventing localizationsignal(MLS,position9–26)partiallycontributes
cancerdevelopment[1].OGG1isencodedbytheOGG1gene tothe8-oxoG-bindingpocketandtheHhH-GPDdomain(a
andisanimportantproteinactingasakeyenzymeinBER helix-hairpin-helixstructuralelementfollowedbyaGly/Pro-
pathway. It initiatesthe process by recognizingand directly richloopandaconservedasparticacid)containingnuclear
removing 8-hydroxy-2-deoxyguanine (8-OHdG) adducts localizationsignal(NLS,335–342)providesboththecatalytic
from damaged DNA by releasing the modified base and and DNA-binding functions of the DNA glycosylase [4].
generatinganAPsite[2].TheOGG1geneislocatedinchro- The human OGG1 protein structure reveals another highly
mosome 3p26.2 and this region of genome has frequently conserved motif which corresponds to the helix-hairpin-
been detected missing or deleted in various tumors, partic- helix (HhH) motif and is characteristic of the active site of
ularly lung, colon, stomach, kidney, oesophageal, prostate, endonuclease III family of DNA glycosylases/AP lyases [5].
and breast tumors, suggesting the loss of OGG1 function Inactivation of the OGG1 gene may lead to a higher risk of
as a possible contributor to tumorigenesis and loss of het- cancerbecausecellswithaccumulated8-OH-Gadductsstill
erozygosity of markers [3]. There are two major isoforms retain the ability to proliferate and a substantial increase of
of human OGG1, that is, isoform ∞-OGG1 (345 amino spontaneousmutationfrequencieshasbeenclearlyidentified
2 DiseaseMarkers
in the DNA of mutant mice, bearing transgenic gene when ethnicity matched cancer-free healthy female individuals as
exposedtoexogenouscarcinogensorendogenousoxidative controls. Patients belonging to different areas of Pakistan
species [6]. Theseobservationssuggest thatOGG1 actsasa wererecruitedfromNuclearMedicine,OncologyandRadio-
majorproteininpathwayresponsibleforremovalof8-oxoG therapy Institute (NORI) and Pakistan Institute of Medical
or8-OH-Gadducts[7]. Sciences (PIMS), Islamabad Pakistan, while controls were
OGG1 gene is highly polymorphic among humans and selectedrandomlyandvoluntarilyfromgeneralpopulation.
is also mutated in cancer cells. Epidemiologic studies have Theinclusioncriterionforthecontrolswasageandethnicity
linked single nucleotide polymorphisms (SNP) in DNA matched healthy female individuals with absence of prior
glycosylase and BER core protein genes to human cancer history of cancerous or precancerous lesions. Patients and
risk including breast cancer [8, 9]. The OGG1 gene has at controls suffering from any other familial disease (dia-
leasttwenty-fivevalidatedsequencevariantsthatreportedly betes, blood pressure, and cardiovascular, renal, or hepatic
changeaminoacidoftheproteinbutSer326Cys(rs1052133) impairment)wereexcludedfromthisstudy.Afterobtaining
hasbeenextensivelyinvestigatedforitsassociationwithdif- informedandwrittenconsent,eachindividualwaspersonally
ferenttypesofcancerriskincludingesophageal[10],lung[11], interviewed using the specifically designed questionnaire.
stomach [12], thyroid [2], laryngeal [13], colorectal [14, 15], Information regarding age, age at menarche, menopausal
andpancreaticcancer[9].TheresultsaboutOGG1polymor- status, menopausal age, family history, ethnic group, and
phisms are contradictory and further studies involving dif- tobacco use was collected from both patients and control
ferentpopulationsarerequired.Presentstudyisdesignedto individuals.Detailsregardinghormonalreceptorstatusand
observethemutationalspectrumofOGG1anditsassociation histopathological findings were also recorded for clinical
withdifferentenvironmental,clinical,andhistopathological characterization of patients in first or follow-up meetings.
parameterinbreastcancerpatientsinPakistanipopulation. Standardvenipuncturewasusedtocollect5mLofperipheral
Initially a meta-analysis was performed involving previous blood in EDTA containing tubes from patients and control
studiesandthentheresultswerecomparedtoobtainaclear individualsandwasstoredat−20∘Cuntilfurtheruse.
pictureabouttheroleofOGG1variationsinbreastcancer.
2.3.DNAExtractionandPolymeraseChainReaction(PCR).
2.MaterialsandMethods Genomic DNA was extracted from leucocytes, using stan-
dardphenol-chloroformextractionmethodasdescribedby
2.1. Search Strategy and Selection Criteria for Meta-Analysis. Baig et al. [16] with minor alterations. Freshly extracted
A comprehensive literature search was conducted using DNA was quantified by spectrophotometry and yield gel
Pub Med database for all eligible studies (updated from electrophoresis and stored at −20∘C till further processing.
January 2007 to November 2014) reporting OGG1 poly- Human OGG1 exon sequence was taken from Ensemble.
morphism/mutations, using the following search strategy: Primersweredesignedusingprimer3softwareandchecked
cancer,OGG1,polymorphisms,mutations,andgeneticvari- for their specificity using BLAST. Whole coding region
ations.Therewasnorestrictiononsamplesize,ethnicityof including exon intron boundaries of approximately 60bp
population, language, or type of report. All eligible studies sequenceofOGG1wasinvestigatedtoidentifynovel,already
were retrieved and checked for other relevant studies. The reported, and any splice site variation. Each PCR reaction
literature retrieval was performed in duplication by two wasperformedina10𝜇Lreactionmixturecontaining1𝜇Lof
independent reviewers. Studies were included only if they genomicDNA(approximately50ng)templates,1𝜇L(10mM)
met the following criteria: (1) case-control studies which of each primer, 1𝜇L nuclease-free water, and 5𝜇L PCR
evaluatedtheassociationbetweenOGG1polymorphismsand master mix (Thermo Scientific) containing 0.05U/𝜇LTaq
cancer risk; (2) studies using DNA extracted from blood DNA polymerase, reaction buffer, 4mM MgCl2, 0.4mM of
∘
samplesofcancerpatientsandalsofromhealthyindividuals eachdNTP.PCRconditionswereinitialmeltingstepat94 C
∘
usedascontrolsforcomparison;(3)studiesusinganyofthe for5min,35cycleseachcomprisedof94 Cfor45sec,exon
∘
mutation detection techniques (e.g., PCR-RFLP, PCR-SSCP, specificannealingtemperaturefor1minand72 Cfor1min.
∘
ARMS-PCR,andqRT-PCRarrays);(4)studiespublishedas It was followed by a final extension step at 72 C for 10min
fullarticlesinEnglish. and finally held at 4∘C. 2𝜇L of PCR products along with
A number of studies were excluded on the basis of the loading dye were electrophoresed on a 2% agarose gel and
following points. (1) Studies using cancer cell lines, tumor stainedwithethidiumbromide.100bpladderwasalsoloaded
samples, serum, or saliva samples were not included. (2) asstandardforquantificationofamountandconfirmationof
Review articles and previous meta-analysis were also not PCRproductsize.
included.(3)Studiesondiseasesotherthancancerwerealso
excludedfrompresentstudy. 2.4. Mutational Screening and Sequence Analysis. Single
stranded conformational polymorphism (SSCP) assay was
2.2. Collection of Blood Samples. Present study was con- used for mutational analysis of PCR products. Samples
ductedwithapriorapprovalfromethicalcommitteesofboth with altered electrophoretic mobility were reamplified in
COMSATS Institute of Information Technology Islamabad a separate reaction and were analyzed by direct sequenc-
(CIIT) and collaborating hospitals. A total of 925 subjects ing to confirm and characterize the nature of muta-
wereenrolledinpresentstudyincluding530femalepatients tions/polymorphisms. Control (normal) samples were also
with histological confirmed breast cancer and 395 age and sequenced along with cancerous samples to compare the
DiseaseMarkers 3
sequencingresults.DNAsequencingwascarriedoutbyMC individuals (including 14844 patients and 17782 controls),
lab(USA).ResultsofDNAsequencingwereanalyzedusing thirty-fivestudiesinvolving19594individuals(including9071
BioEditsoftware(version7.0.5)andAlamutvisualinteractive patients and 10523 controls) concluded a contributory role
biosoftware(version2.4-5). of OGG1 polymorphism to different type of cancers while
in twenty-six studies involving 12812 individuals (including
2.5. Data Analysis. 𝜒2-test, Fisher’s exact test, and Pearson 5663 patients and 7149 controls) no association of OGG1
correlation coefficient were used to analyze the differences polymorphismtocancersusceptibilitywasobservedandonly
in selected demographic variables, family history, smoking one study involving 220 individuals (including 110 patients
status, tumor types, tumor grades, ER/PR, and HER-2/nue and 110 controls) suggested negative or protective role of
status by using the Graph Pad Prism 5. Pearson’s correla- OGG1polymorphismagainstcancer(Table1).Insummary,
tion coefficient was used to assess the correlations among when all the eligible studies were pooled into the meta-
the observed mutations and clinical and histopathological analysis of OGG1 mutations, 60.1% individuals showed an
parameters. Missense mutations were analyzed in silico associationofOGG1mutationswithdifferenttypesofcancers
via Alamut biosoftware (version 2.4-5) for prediction of while 39.3% individuals showed no association and 0.7%
the pathogenicity caused by point mutations, PhyloP for individuals showed a negative or protective role of OGG1
conservation level of mutated nucleotides, and amino acids mutationsagainstcancer.
alongwithGranthamdistanceforphysicochemicalchanges Secondpartofpresentstudyinvolved925subjectsinclud-
inaminoacidstructure. ing 530 breast cancer patients and 395 cancer-free healthy
individualsascontrolusedformutationalanalysisofOGG1
3.Results gene.Meanageofpatientsandcontrolswascalculatedas46.4
(±11.59) and 42.80 (±12.96) years, respectively (see Supple-
In first part of study a meta-analysis was performed to mentaryTables1and2inSupplementaryMaterialavailable
evaluatetheassociationbetweenOGG1polymorphismsand online at http://dx.doi.org/10.1155/2015/690878). In present
cancersusceptibilityespeciallyasriskfactorofbreastcancer. study,all(eight)exonsofOGG1werescreenedcomprehen-
Based on our search criteria, 152 studies relevant to the sivelyforanynovelorreportedgermlinemutationsinvolving
role of OGG1 mutations/polymorphisms on cancer/disease SSCP followed by direct sequence analysis of suspected
susceptibility were identified. 90 studies of total 152 were samples.Fifteendifferenttypesofmutationswereobserved,
󸀠
excludedonthebasisofthefollowingreasons. which included five intronic, four splice site, two 3 UTR,
(i) Five studies were review/meta-analysis, (ii) 8 studies and four missense mutations. Among identified mutations,
were involving only general healthy population, (iii) 18 one intronic mutation (g.9793680G>A, rs55846930) and
studieswereinvolvingOGG1mutationsinpatientsotherthan two missense mutations (Gly221Arg, TMP ESP 3 9796483
cancerous, for example, diabetes, cataract, endometriosis, andSer326Cys,rs1052133)havealreadybeenreportedwhile
andsoforth,(iv)14studiesusedDNAsamplesfromtissues remaining twelve mutations were novel. Four novel muta-
other than blood samples of cancer patients, and (v) 45 tions (g.9792260 insert T; g.9793748G>A; g.9798336T>G;
studieswereolderthanJanuary2007. g.9798349T>A) were observed in intronic regions, four
As a result, a total of 62 relevant studies (involving mutations(g.9792109delT,g.9798307T>G,g.9798502T>G&
32626individualsincluding14844patientsand17782healthy g.9800972T>G) were observed in splice site regions, two
controlindividuals)mettheinclusioncriteriaforthecurrent mutations(g.9798848G>A,g.9798896T>C)wereobservedin
meta-analysis. Among them, most of studies used PCR- 3󸀠UTR, one missense mutation (g.9793544T>G, Val159Gly)
RFLP(48)andothertechniques(12)fordetectionofalready was observed in exon 3, and one nonsense mutation
reported one polymorphism Ser326Cys in cancer. Only (g.9807669G>A, Trp375STOP) was observed in exon 8
two studies used techniques for the detection of reported (Figure1).
as well as novel mutations in cancer, one involved high Significantlyincreasedbreastcancerriskwasfoundasso-
resolutionmelting(HRM)analysisandotheroneusedPCR- ciated with different mutations when compared with con-
SSCP. Of all eligible studies, the majority of studies were trols(Table2).Threeintronicmutations(g.9792260insert T;
on head and neck, lung, and colorectal cancers whereas g.9798336T>G; and g.9798349T>A) and one 3󸀠UTR muta-
only6studiesevaluatedtheOGG1polymorphisminbreast tion (g.9798896T>C) were also detected in control sam-
cancer. The majority of studies were from Caucasian (17), ples but their frequency was significantly high in patients
Chinese (16), and Indian (14) populations while only one (𝑝 < 0.05). Significantly (𝑝 < 0.001) increased (∼29
studywasfromPakistanipopulationinvolvingheadandneck fold) breast cancer risk was found associated with a splice
cancer patients. Moreover, only 4 of the available studies site variant g.9800972T>G (OR = 28.85, 95% CI = 3.87 to
used patient sample size more than or equal to 500 and 207.7)and3󸀠UTRvariantg.9798848G>A(OR=29.20,95%
remaining 94% of studies used fewer number of patient CI = 33.98 to 213.74). Among intronic mutations, highest
samples. Only 3 studies recruited purely population based (∼15fold)increaseinbreastcancerriskwasassociatedwith
(PB)controlswhileallotherstudiesinvolvedhospitalbased g.9793680G>Avariation(OR=14.65,95%CI=1.95to109.9;
(HB) controls. Findings of all previous studies investigated 𝑝 < 0.009).Similartrendwasobservedinalldetectedmis-
for this meta-analysis were contradictory regarding associ- sense mutations in breast cancer patients when compared
ation of OGG1 polymorphisms to increased risk of cancer withcontrolsand∼14-foldincreasedriskwasassociatedwith
susceptibility. Out of selected 62 studies involving 32626 Val159Gly (OR = 13.68, 95% CI = 1.82 to 102.9; 𝑝 < 0.01),
4 DiseaseMarkers
ancerriskassociation AssociatedAssociatedAssociatedAssociatedNotassociatedNotassociated Notassociated NotassociatedAssociatedAssociatedNotassociatedNotassociatedNotassociatedAssociatedNotassociatedAssociatedNotassociatedAssociatedAssociatedNotassociatedNotassociatedAssociatedAssociatedNotassociatedAssociatedNotassociated Associated NotassociatedNotassociatedAssociatedAssociatedAssociatedAssociatedNotassociatedNotassociatedNotassociatedAssociatedAssociatedAssociatedNotassociatedNotassociatedNotassociatedNegativelyassociatedNotassociatedAssociated
C
C
>
G
m. Mutations Ser326CysSer326CysSer326CysSer326CysSer326CysSer326Cys>>c.-18GT,c.-23AG,>>GC,c.-45GA,c.-63Ser326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysAsp267Asn,Ser279GlyIle253Phe,Ala399GluSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326CysSer326Cys
3
s 5
phi c.-
r
mo ysis
rtingOGG1poly Techniques PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPTaqManassays HRManalysis PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPiPLEXPCR-RFLPPCR-RFLPSNParraysPCR-RFLPTaqManassayPCR-RFLPPCR-RFLPTaqManassayPCR-RFLPPCR-RFLPPCR-CTPP PCR-SSCP PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPARMS-PCRARMS-PCRPCR-RFLPPCR-RFLPSequencingPCR-RFLPPCR-RFLPPCR-RFLPRT-PCRPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPMeltingcurveanal
o
p ol
re ntr
dies ofco HBHBB/PBHBHBHB HB HBB/PBHBHBB/PBHBHBB/PBHBHBHBHBHBHBHBHBHBHBHB B/PB HBB/PBHBHBPBPBHBHBHBHBB/PBHBHBHBHBHBHBHB
blestu nalysis Source H H H H H H H
databaseforalleligi G1germlinemutationaSamplesizePatientsControls251251214210402477924736857983206 518777 124126104158118132110116106116——212250402479204209165250346361278278598545382379455443415511231300358716194245 300300 108195192224401533350225195250212250834582201439676253253182245336370209215100106150150110110114200507425
uresearchinPubMed PreviousstudieswithOG Cancertype BladdercancerLivercancerGastriccancerColorectalcancerLungcancerPterygium Breastcancer LungcancerEndometrialcancerProstatecancercolorectalcarcinomaGastriccancerCervicalcancerUrothelialbladdercancerThyroidcancerGallbladdercancerLungcancerBreastcancerHeadandneckcancerNasopharyngealcancerLungcancerLungcancerLymphoblasticleukemiaNasopharyngealcancerLungcancerBreastcancer HeadandneckCancer GastricCancerProstatecancerBreastcancerHeadandneckcancerProstatecancerBladdercancerBladdercancerlungadenocarcinomaColorectalcancerLaryngealcancerColorectalcancerColorectalColorectalcancerBreastcancerEndometrialcancerLungcancerColorectalcancerBreastcancer
Table1:Literat Population(Locationandethnicity)Asian,JapaneseAsian,IndianCaucasian,TurkishCaucasian,TurkishLatino-AfricanAmericansAsian,Chinese Asian,Chinese Asian,ChineseCaucasian,TurkishSouthAustralianCaucasian,TurkishCaucasian,TurkishEuropeanAsian,NorthIndianEuropean,SpanishAsian,ChineseCaucasian,TurkishAsian,KoreanAsian,IndianNorthAfricanEuropeanAsian,ChineseAsian,ChineseAsian,ChineseAsian,TaiwanAsian,Chinese Asian,Pakistani Asian,IndianAsian,IndianAsian,ChineseAsian,IndianAsian,IndianAsian,IndianCaucasian,TurkishAsian,ChineseAsian,KoreanEuropean,PolishCaucasianPolishRussian,BelarusAustralian/PolishCaucasian,PolishEuropean,PolishEuropean,BelgianAsian,IndianAsian,Thai
008[63]011[64]
22
Authorandyear Arizonoetal.,2008[29]Boseetal.,2013[30]Canbayetal.,2010[31]Canbayetal.,2011[14]Changetal.,2009[32]Chenetal.,2010[33] Chenetal.,2011[34] Chengetal.,2012[35]Cincinetal.,2012[36]Dhillonetal.,2011[37]Enginetal.,2010[38]Enginetal.,2011[39]Farkasovaetal.,2008[40]Gangwaretal.,2009[41]´Garcıa-Quispesetal.,2011[2]Jiaoetal.,2007[42]Karahaliletal.,2008[43]Kimetal.,2013[44]Kumaretal.,2011[22]Laantrietal.,2011[45]Letkovaetal.,2013[46]Lietal.,2011[47]Lietal.,2011[48]Lietal.,2013[49]Liuetal.,2010[50]Luoetal.,2014[17] Mahjabeenetal.,2011[19] Maliketal.,2010[51]Mandaletal.,2012[52]Hsuetal.,2010[53]Mitraetal.,2011[54]Mittaletal.,2012[55]Mittaletal.,2012[55]Narteretal.,2009[56]Ouyangetal.,2013[57]Parketal.,2007[58]Pawlowskaetal.,2009[59]Przybylowskaetal.,2013[60]Ramaniuketal.,2014[61]Reevesetal.,2012[62]Romanowicz-Makowskaetal.,Romanowicz-Makowskaetal.,deRuycketal.,2007[65]Sameeretal.,2012[66]Sangrajrangetal.,2008[67]
DiseaseMarkers 5
n
o
cerriskassociati NotassociatedNotassociatedAssociatedAssociatedNotassociatedAssociatedAssociatedAssociatedAssociatedNotassociatedAssociatedAssociatedAssociatedAssociatedAssociatedAssociatedAssociated
n
a
C
ns ysysysysysysysysysysysysysysysysys
o CCCCCCCCCCCCCCCCC
ati 2626262626262626262626262626262626
Mut er3er3er3er3er3er3er3er3er3er3er3er3er3er3er3er3er3
SSSSSSSSSSSSSSSSS
y
Techniques PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPassspectrometrTaqManAssay
M
ol
ntr
o B
ofc HBHBHBHBHBPBHBHBHBHBHBHBHBHBHBB/PHB
e H
c
ur
o
S
Table1:Continued. mlinemutationanalysisSamplesizePatientsControls167186109217265280100100941423023017320497131732553354024605404104107272350400266266193197572574
er
g
GG1 mia
O
Previousstudieswith Cancertype MelanomaThyroidcancerHeadandneckcancerColorectalcancerEndometrialCancerGallbladdercancerGallbladdercancerutelymphoblasticleukeGastriccancerEsophagealcancerUrothelialcarcinomaLungadenocarcinomaLaryngealcancerHepatocellularcancerProstatecancerProstatecancerRenalcellcarcinoma
Ac
y)
PopulationcationandethnicitEuropean,ItalianCaucasianCaucasianPolishCaucasian,PolishCaucasian,PolishAsian,IndianAsian,IndianCaucasian,PolishAsian,ChineseAsian,IndianAsian,TaiwaneseAsian,ChineseAsian,ChineseAsian,ChineseAsian,KoreanAmericanAsian,Chinese
o
L
(
Authorandyear Santonocitoetal.,2012[68]Santosetal.,2012[69]Sliwinskietal.,2011[70]Sliwinskietal.,2009[71]Sobczuketal.,2012[72]Srivastavaetal.,2010[73]Srivastavaetal.,2009[74]Stanczyketal.,2011[75]Sunetal.,2010[76]Upadhyayetal.,2010[77]Wangetal.,2011[78]Xueetal.,2013[79]Yangetal.,2008[80]Yuanetal.,2012[81]Yunetal.,2012[82]Zhangetal.,2010[83]Zhaoetal.,2011[84]
6 DiseaseMarkers
T C A A A A T C T T C C A A A A C C
C T T T T A A T A
g.9792260ins_T CTTTTAATAM g(r.9s5759834668903G0>)A Intron 3 TTCCAAAAAGAATTCCTTMW g.9793748G>A TTCCCCAAAGAAAACCCCM
Intron 1 CTTT AATAW Intron3 W
(a) (b) (c)
G C A G G G G G C A T G G A A G A A C C G G G A G T
g.9792109del T CCGG GAGTM
g.9798336T>G GCAGGGGGCM g.9798349T>A ATGGAAGAAM splice site Intron1 CCGGTGAGTW
Intron 5 GCAGTGGGCW Intron 5 ATGGTAGAAW
(d) (e) (f)
T G G G G G A G G G C G G G G A G T G A G G G G A G G
sgp.9li7c9e8 s3it0e7 ITn>trGon5 TTGGGGGG GTGGAAGGGGMW sgp.9li7c9e8 s5it0e2 ITn>trGon6 GGCCGGGGGTGGAAGGTTMW sgp.9li8c0e0 s9it7e2 ITn>trGon7a GGAAGGGGGTGGAAGGGGMW
(g) (h) (i)
T C T C C C C C C A T G G G G G A G
C C T G A A C A A
g.9798848G>A CCTGAACAAM g.9798896T>C TCTCCCCCCM g.9793544T>G ATGGGGGAGM
3’UTR CCTGGACAAW 3’UTR TCTCTCCCCW Exon 3 ATGGTGGAGW
(j) (k) (l)
Figure1:Continued.
DiseaseMarkers 7
G G C A G G C T A C A A T G C C G C G C T G A A C C A
(gT.9M79P6_4E8S3PG_>3A_9 E7x9o6n4 843) GGGGCCGAGGGGCCTTAAMW Eg.x9o7n9867d7 (3rCs1>0G52133) CCAAAATTGCCCCCGGCCMW Eg.x9o8n0 78669G>A GGCCTTGGAGAACCCCAAMW
(m) (n) (o)
Figure1:SequencingelectropherogramofpolymorphismsofOGG1.(a),(b),(c),(d),and(e)areintronicmutations((a)g.9792260insertion
ofTinIntron1,(b)g.9793680G>A(rs55846930)substitutioninIntron3,(c)g.9793748G>AsubstitutioninIntron3,(d)g.9798336T>G
substitutioninIntron5,and(e)g.9798349T>AsubstitutioninIntron5).(f),(g),(h),and(i)aresplicesitemutations((f)g.9792109deletion
ofTatSplicesiteregionofIntron1,(g)g.9798307T>GsubstitutioninsplicesiteregionofIntron5,(h)g.9798502T>Gsubstitutioninsplice
site region of Intron 6, and (i) g.9800972T>G substitution in splice site region of Intron 7a). (j) and (k) are substitutions in 3󸀠UTR ((j)
g.9798848G>Asubstitutionin3󸀠UTR(k)g.9798896T>Csubstitutionin3󸀠UTR).(l),(m),(n),and(o)aremissensemutations((l)missense
mutationVal159Glyshowingg.9793544T>GsubstitutioninExon3resultinginchangeofcodonfromGTGtoGGGencodingaminoacid
ValineinsteadofGlycine,(m)missensemutationGly221Arg(TMP ESP 3 9796483)showingg.9796483G>AsubstitutioninExon4resulting
inchangecodonfromGGGtoAGGencodingtheaminoacidGlycineinsteadofArginine,(n)missensemutationSer326Cys(rs1052133)
(CM993185)showingg.9798773C>GsubstitutioninExon6dresultinginchangeofcodonfromTCCtoTGCencodingtheaminoacid
SerineinsteadofCysteine,and(o)nonsensemutationTrp375STOP∗showingg.9807669G>AsubstitutionsinExon8resultinginchangeof
codonfromTGGtoTGAterminatingtheproteininsteadofencodingtheTryptophanaminoacid).
∼17-foldwithGly221Arg(OR=16.85,95%CI=2.26to125.53;
𝑝 < 0.005), and ∼18-fold with Ser326Cys (OR = 18.45,
95%CI=2.49to136.99;𝑝<0.004)inbreastcancerpatients
comparedwithcontrols,whereasanalysisofnonsensemuta-
tionshowedthat∼13-fold(OR=12.90,95%CI=1.71to97.28;
𝑝 < 0.01) increased breast cancer risk was associated with
Trp375STOPinpatientscomparedtocontrols.
Missense mutations Val159Gly, Gly221Arg, and
Ser326Cys were observed in protein domains HhH-GPD
and 8-oxoguanine DNA-glycosylase (Supplementary
Table 3). Missense and nonsense mutations were also
analyzed via Alamut biosoftware (version 2.4.5) to check #1GLY159.A
the conservation levels of mutated nucleotides and amino #1ARG221.A
acids along with in silico predictions about Align GVGD
score, Grantham distance, SIFT score and Mutation Taster
(Table3).MutationTasterpredictedtwomissensemutations
(Val159Gly and Gly221Arg) and one nonsense mutation Figure2:SuperimposedproteinstructureofmutatedOGG1with
(Trp375STOP) as potentially disease causing (𝑝 = 1.0). itswildtype.Wild-typeOGG1protein(grey)andmutatedOGG1
Greater physiochemical difference in protein structure was protein(blue).SuperimposedstructureofmutatedOGG1protein
predicted in case of nonsense mutation Trp375STOP that showing the location of two observed mutations, Val159Gly and
resultedintruncatedproteinchaindue toreplacement ofa Gly221Arg. Wild-type protein model obtained from protein data
bank.StructurewaspredictedusingautomatedSwissmodel.Two
moderately conserved amino acid Tryptophan with a stop
structures, wild and mutated, were aligned using UCSF chimera
codon(Granthamdistance=170).Proteinmodelingoftwo
software.
detected mutations (Val159Gly, Gly221Arg) of OGG1 and
comparison with wild-type OGG1 protein has concluded
that no major conformational change occurs due to these
mutations while one nonsense mutation (Trp375STOP) significantly higher in patients with invasive ductal carci-
resultedintruncationofprotein(Figure2). noma(𝑝 < 0.0001),negativeER(𝑝 < 0.001),andnegative
Association of observed mutations was also correlated PR status (𝑝 < 0.01). All observed OGG1 mutations were
withdifferentclinicopathologicalparametersincludingfam- found significantly correlated with tumor types (𝑟 =
ily history, menopausal age, and HER-2/nue and ER/PR −0.333∗∗∗; 𝑝 < 0.0001), ER status (𝑟 = 0.739∗∗; 𝑝 <
status. Frequency of OGG1 mutations was observed to be 0.001), and PR status (𝑟 = −0.155∗; 𝑝 < 0.01) of breast
8 DiseaseMarkers
Table2:MutationsandtheirallelefrequenciesobservedintheOGG1geneinbreastcancerpatients.
Patients Controls
Mutation/exon Allelefrequency Allelefrequency aOddsratio(95%CI) b𝑝value
Chr3(GRCh37) Number Number
Minor/major Minor/major
g.9792260insert TIntron1 34 T0.09/0.91 07 T0.35/0.65 3.80(1.67to8.66) 0.001
g.9793680G>AIntron3(rs55846930) 19 A0.05/G0.95 00 A00/G1.0 14.65(1.95to109.90) 0.009
g.9793748G>AIntron3 14 A0.04/G0.96 00 A00/G1.0 10.70(1.40to81.64) 0.02
g.9798336T>GIntron5 10 G0.03/T0.97 06 G0.40/T0.60 1.25(0.45to3.46) 0.67
g.9798349T>AIntron5 34 A0.09/T0.91 02 A0.10/T0.90 13.30(3.18to55.70) 0.0004
g.9792109delTsplicesiteIntron1 26 0.07/T0.93 00 00/T1.0 20.07(2.71to148.53) 0.003
g.9798307T>GsplicesiteIntron5 16 G0.04/T0.96 00 G00/T1.0 12.11(1.60to91.70) 0.01
g.9798502T>GsplicesiteIntron6 18 G0.05/T0.95 00 G00/T1.0 13.68(1.82to102.90) 0.01
g.9800972T>Gsplicesiteintron7a 36 G0.10/T0.90 00 G00/T1.0 28.85(3.87to207.70) 0.001
g.9798848G>A3󸀠UTR 37 A0.10/G0.90 00 A00/G1.0 29.20(3.98to213.74) 0.001
g.9798896T>C3󸀠UTR 48 C0.13/T0.87 03 C0.15/T0.85 12.85(3.97to41.56) <0.0001
g.9793544T>GExon3,Val159Gly 18 G0.05/T0.95 00 G00/T1.0 13.68(1.82to102.90) 0.01
g.9796483G>AExon4,Gly221Arg
22 A0.06/G0.94 00 A00/G1.0 16.85(2.26to125.53) 0.005
(TMP ESP 3 9796483)
g.9798773C>GExon6dSer326Cys
24 G0.06/C0.94 00 G00/C1.0 18.45(2.49to136.99) 0.004
(rs1052133)(CM993185)
g.9807669G>AExon8,Trp375STOP 17 A0.05/G0.95 00 A00/G1.0 12.90(1.71to97.28) 0.01
OR:oddsratio;CI:confidenceinterval. aORsforlogisticregressionanalysis. b𝑝<0.05,by𝜒2-testfortrend.
cancerpatientsbutanonsignificantcorrelationwasobserved carcinogenesis.Resultsofcurrentmeta-analysisrevealedthat
between all mutations and HER-2/neu status (𝑟 = 0.318, 60outof62selectedstudiesfocusedonlyonOGG1mutation
𝑝 = 0.12) of breast cancer patients (Table4). As shown (Ser326Cys) for its role in carcinogenesis and the majority
in Table5, significantly increased breast cancer risk was of studies (60%) concluded association of this mutation
associated with an intronic (g.9793680G>A, 𝑝 < 0.03), with different cancers (Table1). But mutations other than
a splice site (g.9798502T>G, 𝑝 < 0.03), and a missense Ser326Cys, in the same domain orother domainsof OGG1
(Ser326Cys, 𝑝 < 0.009) mutation in patients with family singlyorincombinationmayalsobeimportantininitiation
history as compared to controls. Correlations between fre- and developmentofcancer as reported by Mahjabeen et al.
quency of OGG1 mutations and menopausal age of breast [13].Moreoverstudiesinvolvingrelativelylargerpopulation
cancer patients (Table5) revealed that frequencies of three for exploration of different OGG1 mutations (novel as well
intronic mutations (g.9792260 ins T; g.9793680G>A; and as reported) in relation to other clinicohistopathological
g.9798349T>A), two splice site mutations (g.9792109delT parameters may also be needed for their role in cancer
and g.9800972T>G), two 3󸀠UTR mutations (9798848G>A development.
andg.9798896T>C),andonemissensemutation(Ser326Cys) Second part of present study is designed to screen all
weresignificantlyhigher(𝑝 < 0.05)inpatientswithearlier intronic and exonic regions of OGG1 gene in 925 individ-
menopause (≤50 years) compared to controls and patients uals including 530 breast cancer patients and 395 controls
withlatemenopause(>50years).Table6showedassociation using PCR-SSCP followed by sequencing. A total of fifteen
of OGG1 mutations with smoking status of patients and mutations were identified in patients and in some control
controls. Statistically significant (𝑝 < 0.05) association of individuals. Eleven mutations were observed in different
OGG1mutations(Ser326Cys,g.9792109delT,g.9800972T>G, noncoding regions of OGG1 gene including five mutations
g.9792260 ins T, and g.9798848G>A) was observed with inintronicregions,fourmutationsindonorsplicesite,and
patients having smoking history compared to patients and twomutationsin3󸀠UTRregions.Amongthese,12mutations
controlswithnosmokinghistory. were novel and three were already reported (rs55846930,
TMP ESP 3 9796483, and rs1052133). Frequencies of these
4.Discussion observedspicesitemutationswerefoundsignificantlyhigher
in patients as compared to control individuals suggesting
OGG1isanimportantgeneofBERpathwaywhichencodes theirassociationwithbreastcarcinogenesis.Observedmuta-
the enzyme responsible for the excision of 8-oxoguanine tionswerealsoanalyzedbyAlamutbiosoftware(version2.4-
(8-oxoG), a mutagenic base byproduct which occurs as a 5) which predicted that skip of Exons 1, 5, 6, and 7 is very
result of exposure to reactive oxygen species (ROS) [11]. In likely as mutations were observed in donor splice site areas
first part of study a meta-analysis was designed to explore of respective exons. Since splice site regions in a gene are
the association between OGG1 polymorphisms and breast involved in the processing of precursormRNA into mature
DiseaseMarkers 9
hamdistance,andMutationTasterprediction. nthamdist.(forphysicochemicalMutationTasterdifferenceb/waminoacids)(pvalue)(0–215) Diseasecausing109(moderate)𝑝=1.0() Diseasecausing125(moderate)𝑝=0.999() Polymorphism112(moderate)𝑝=1.0() 170(large)Diseasecausing𝑝=1.0Proteintruncation() deleterious(C65)toleastlikelydeleterious(C0)GV(Granthammicalchanges;215=largechanges).InsilicopredictionswereMutationTaster:diseasecausingvariants(pvalue=1.0),might
Table3:MutationsinOGG1geneinbreastcancerpatientswithconservationscore,SIFTscore,AlignGVGDscore,Grant ChangeinnucleotideAminoacidchangeGraChangeinSIFTscoreAlignGVGDscorewithitsconservationlevelwithitsconservationcodon(median)(C0–C65)−PhyloPscore[14.1;6.4]level(upto13species)>g.9793544TGVal159GlyC0(GV:197.52-GD:DeleteriousGTGtoGGGHighlyconservednucleotideModeratelyconserved0.01(2.95)72.75)4.97phyloP:aminoacid>g.9796483GAGly221ArgToleratedGGGtoAGGModeratelyconservednucleotideModeratelyconservedC0(GV:161.50-GD:19.250.28(2.95)2.38phyloP:aminoacid>g.9798773CGSer326CysToleratedC0(GV:353.86-GD:TCCtoTGCNotconservednucleotideWeaklyconserved0.19(2.95)0.00)0.28phyloP:aminoacid>g.9807669GATrp375STOPDeleteriousC0(GV:197.52-GD:TGGtoTGAWeaklyconservednucleotideModeratelyconserved0.01(2.95)72.75)0.12phyloP:aminoacid−PhyloPwasusedasaconservationscoreratingthenucleotidesfrom“notconserved”(14.1)to“highlyconserved”(6.4).AlignGVGDscore:mostlikelyvariation)andGD(Granthamdeviation).TheGranthamdistancewasusedtoevaluatephysicochemicalchangesinaminoacids(0=nophysicoche<>performedusingPolyPhen-2(PolymorphismPhenotyping-2),SIFT(SortingIntolerantfromTolerant)score:0.05deleterious,0.05tolerated,and<notbediseasecausing(pvalue0.99).
10 DiseaseMarkers
HER-2/neustatus−ve+veNumber(%)Number(%)24(14.46)9(7.44) 15(9.14)4(3.3) 7(4.27)7(5.78)5(3.04)4(3.3)16(9.76)13(10.74)17(10.36)12(9.91)8(4.87)6(4.96)12(7.32)6(4.96)13(7.93)16(13.22)16(9.76)8(6.61)15(9.14)11(9.09)3(1.83)12(9.91) 7(4.27)6(4.96) 6(3.66)7(5.78) 7(4.27)6(4.96)0.3180.12 lobularcarcinoma;ER:estrogen
nts. asive
eustatusinbreastcancerpatie PRstatus−ve+veNumber(%)Number(%)28(13.66)6(4.96) 16(7.8)2(1.65) 5(2.43)9(7.44)8(3.9)2(1.65)21(10.24)13(10.74)18(8.78)8(6.61)09(4.39)6(4.96)9(4.39)9(7.44)14(6.82)16(13.22)16(7.8)13(10.74)11(5.36)12(9.91)16(7.8)2(1.65) 10(4.87)8(6.61) 19(9.27)3(2.48) 5(2.28)14(11.57)∗−0.155 0.01 ductalcarcinomainsitu;ILC:inv
ypes,ER/PRstatus,andHER-2/n ERstatus−ve+veNumber(%)Number(%)20(9.57)12(10.62) 10(4.78)9(7.96) 10(4.78)4(3.35)6(2.87)3(2.65)18(8.61)13(11.50)16(76.55)10(10.62)9(4.30)7(6.19)16(76.55)2(2.21)19(9.09)13(11.50)24(11.48)13(11.50)29(13.87)14(12.38)12(5.65)6(5.30) 15(7.18)5(4.42) 12(5.65)8(7.08) 9(4.30)7(6.19)∗∗0.739 0.001 DC:invasiveductalcarcinoma;DCI:∗∗∗∗∗𝑝<𝑝<5,0.01,0.001.
t I0
nsandtumor ILCumber(%)4(10.25) 2(5.13) 1(2.56)4(10.25)5(12.82)6(15.38)003(7.69)1(2.56)3(7.69)5(12.82)00 2(5.13) 1(2.56) 2(5.13) allysignificant;∗𝑝<eptor2.0.
betweenOGG1mutatio TypeoftumorIDC)Number(%)N24(11.11) 8(3.70) 5(2.31)6(2.77)21(9.72)16(7.4)10(4.63)11(5.09)28(12.96)30(13.89)17(4.63)14(6.48) 14(6.48) 15(6.94) 4(1.85)∗∗∗−0.333 0.00010.05isconsideredstatisticpidermalgrowthfactorrec
Table4:Correlation Mutation/exonDCISChr3(GRCh37)Number(%g.9792260insertTIntron16(7.69)>g.9793680GAIntron39(11.54)(rs55846930)>g.9793748GAIntron38(10.25)>g.9798336TGIntron500>g.9798349TAIntron58(10.25)g.9792109delTsplicesiteIntron14(5.13)>g.9798307TGsplicesiteIntron56(7.69)>g.9798502TGsplicesiteIntron64(5.13)>g.9800972TGsplicesiteintron7a3(3.85)󸀠>UTRg.9798848GA300󸀠>UTRg.9798896TC38(10.25)>g.9793544TGExon3,Val159Gly4(5.13)>g.9796483GAExon4,Gly221Arg5(6.41)ESP39796483)(TMP>g.9798773CGExon6dSer326Cys6(7.69)(rs1052133)(CM993185)>g.9807669GAExon8Trp375STOP7(8.97)aCorrelationbpvalue2ab𝑝𝜒𝑝<Pearsoncorrelationcoefficient;valuefortest;receptor;PR:progesteronereceptor;HER-2/nue:humane