Genome-wide association studies in women of African ancestry identified 3q26.21 as a novel ...

12 Pages·2017·0.75 MB·English

HumanMolecularGenetics,2016,Vol.25,No.21 4835–4846
doi:10.1093/hmg/ddw305
AdvanceAccessPublicationDate:4September2016
AssociationStudiesArticle
ASSOCIATION STUDIES ARTICLE
Genome-wide association studies in women of African
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ancestry identified 3q26.21 as a novel susceptibility n
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locus for oestrogen receptor negative breast cancer d
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Dezheng Huo1, Ye Feng2, Stephen Haddad3, Yonglan Zheng4, Song Yao5, ttp
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Yoo-Jeong Han4, Temidayo O. Ogundiran6, Clement Adebamowo7, ://a
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Oladosu Ojengbede8, Adeyinka G. Falusi9, Wei Zheng10, William Blot10, de
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Qiuyin Cai10, Lisa Signorello11, Esther M. John12,13, Leslie Bernstein14, .o
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Jennifer J. Hu15, Regina G. Ziegler16, Sarah Nyante17, Elisa V. Bandera18, .co
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Sue A. Ingles2, Michael F. Press19, Sandra L. Deming10, /hm
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Jorge L. Rodriguez-Gil15, Katherine L. Nathanson20, Susan M. Domchek20, /artic
Timothy R. Rebbeck21, Edward A. Ruiz-Narva(cid:2)ez3, le-a
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Lara E. Sucheston-Campbell22, Jeannette T. Bensen17, Michael S. Simon23, tra
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Anselm Hennis24,25, Barbara Nemesure25, M. Cristina Leske25, Stefan Ambs26, t/25
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Lin S. Chen1, Frank Qian4, Eric R. Gamazon27,28, Kathryn L. Lunetta29, 1/4
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Nancy J. Cox27, Stephen J. Chanock16, Laurence N. Kolonel30, 5/2
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Andrew F. Olshan17, Christine B. Ambrosone5, Olufunmilayo I. Olopade4,*, 25
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Julie R. Palmer3,* and Christopher A. Haiman2,* 0 b
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1DepartmentofPublicHealthSciences,UniversityofChicago,Chicago,IL,USA, 2DepartmentofPreventive es
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Medicine,KeckSchoolofMedicineandNorrisComprehensiveCancerCenter,UniversityofSouthern n
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California,LosAngeles,CA,USA, 3SloneEpidemiologyCenter,BostonUniversity,Boston,MA,USA, 4Centerfor 4
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C5RlionsiwcaelllCPaanrckeCraGnecneertiIcnsstaintudteG,loBbuafflaHloe,aNltYh,,UDSeApa,r6tDmeepnatrtomfMenetdoicfiSnuer,gUenryiv,eCroslilteygoefoCfhMiceadgioc,inCeh,iUcangiove,IrLs,itUySoAf, pril 20
1
Ibadan,Ibadan,Nigeria, 7DepartmentofEpidemiology&PreventiveMedicine,UniversityofMaryland, 9
Baltimore,MD,USA, 8CenterforPopulationandReproductiveHealth,CollegeofMedicine,Universityof
Ibadan,Ibadan,Nigeria, 9InstituteforMedicalResearchandTraining,CollegeofMedicine,Universityof
Ibadan,Ibadan,Nigeria, 10DivisionofEpidemiology,DepartmentofMedicine,VanderbiltEpidemiologyCenter,
Vanderbilt-IngramCancerCenter,VanderbiltUniversity,Nashville,TN,USA, 11CancerPreventionFellowship
Program,NationalCancerInstitute,Bethesda,MD,USA, 12CancerPreventionInstituteofCalifornia,Fremont,
CA,USA, 13DepartmentofHealthResearch&PolicyandStanfordCancerInstitute,StanfordUniversitySchool
ofMedicineStanford,CA,USA, 14DivisionofCancerEtiology,DepartmentofPopulationSciences,Beckman
Received:April1,2016.Revised:July21,2016.Accepted:August26,2016
VCTheAuthor2016.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:[email protected]
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ResearchInstituteofCityofHope,Duarte,CA,USA, 15SylvesterComprehensiveCancerCenterandDepartment
ofPublicHealthSciences,UniversityofMiamiMillerSchoolofMedicine,Miami,FL,USA, 16DivisionofCancer
EpidemiologyandGenetics,NationalCancerInstitute,Bethesda,MA,USA, 17DepartmentofEpidemiology,Gillings
SchoolofGlobalPublicHealth,andLinebergerComprehensiveCancerCenter,UniversityofNorthCarolina,Chapel
Hill,NC,USA, 18TheCancerInstituteofNewJersey,NewBrunswick,NJ,USA, 19DepartmentofPathology,Keck
SchoolofMedicineandNorrisComprehensiveCancerCenter,UniversityofSouthernCalifornia,LosAngeles,
CA,USA, 20DepartmentofMedicine,UniversityofPennsylvania,Philadelphia,PA,USA, 21DanaFarberCancer
Institute&HarvardT.H.ChanSchoolofPublicHealth,Boston,MA,USA, 22CollegesofPharmacyand
VeterinaryMedicine,OhioStateUniversity,Columbus,OH,USA, 23KarmanosCancerInstitute,Departmentof
Oncology,WayneStateUniversity,Detroit,MI,USA, 24ChronicDiseaseResearchCentre,TropicalMedicine
ResearchInstitute,UniversityoftheWestIndies,Bridgetown,Barbados, 25DepartmentofPreventiveMedicine, D
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StateUniversityofNewYorkatStonyBrook,StonyBrook,NY,USA, 26LaboratoryofHumanCarcinogenesis, w
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NationalCancerInstitute,Bethesda,MD,USA, 27DivisionofGeneticMedicine,DepartmentofMedicine, loa
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VanderbiltUniversity,Nashville,TN,USA, 28AcademicMedicalCenter,UniversityofAmsterdam,Amsterdam, ed
TheNetherlands, 29DepartmentofBiostatistics,BostonUniversitySchoolofPublicHealth,Boston,MA,USA fro
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and 30EpidemiologyProgram,UniversityofHawaiiCancerCenter,Honolulu,HI,USA h
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*oTfoCwhihcaogmo,cCohrriecsapgoon,IdLe,nUcSeAs.hEomualdil:bfeoalodpdardeess@emdeadt:icOinluef.ubnsdm.uilcahyiocaI.gOo.leodpua,dJeu,lMieBRB.SP,aClmenetre,rSfcoDr,CSllionniceaElpGiedneemtiicoslo&gyInCneonvtaetri,oBnoisntoGnloUbnailvHeresailttyh,,BUonstivoenrMsitAy, s://a
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USA.Email:[email protected],ChristopherA.Haiman,PhD,DepartmentofPreventiveMedicine,KeckSchoolofMedicineandNorrisComprehensive ad
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CancerCenter,UniversityofSouthernCalifornia,LosAngeles,CA,USA.Email:[email protected] m
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Abstract m
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Multiplebreastcancerlocihavebeenidentifiedinpreviousgenome-wideassociationstudies,buttheyweremainlycon- m
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ductedinpopulationsofEuropeanancestry.WomenofAfricanancestryaremorelikelytohaveyoung-onsetandoestrogen /a
receptor(ER)negativebreastcancerforreasonsthatareunknownandunderstudied.Toidentifygeneticriskfactorsforbreast rtic
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cancerinwomenofAfricandescent,weconductedameta-analysisoftwogenome-wideassociationstudiesofbreastcancer; -a
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onestudyconsistsof1,657casesand2,029controlsgenotypedwithIllumina’sHumanOmni2.5BeadChipandtheotherstudy s
included3,016casesand2,745controlsgenotypedusingIlluminaHuman1M-DuoBeadChip.Thetop18,376singlenucleotide trac
polymorphisms(SNP)fromthemeta-analysiswerereplicatedinthethirdstudythatconsistsof1,984AfricanAmericans t/2
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casesand2,939controls.WefoundthatSNPrs13074711,26.5KbupstreamofTNFSF10at3q26.21,wassignificantlyassociated /2
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withriskofoestrogenreceptor(ER)-negativebreastcancer(oddsratio[OR]¼1.29,95%CI:1.18-1.40;P¼1.8(cid:2)10(cid:3)8).Functional /4
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annotationssuggestthattheTNFSF10genemaybeinvolvedinbreastcanceraetiology,butfurtherfunctionalexperiments 35
areneeded.Inaddition,weconfirmedSNPrs10069690wasthebestindicatorforER-negativebreastcancerat5p15.33 /25
(OR¼1.30;P¼2.4(cid:2)10(cid:3)10)andidentifiedrs12998806asthebestindicatorforER-positivebreastcancerat2q35(OR¼1.34; 25
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P¼2.2(cid:2)10(cid:3)8)forwomenofAfricanancestry.Thesefindingsdemonstratedadditionalsusceptibilityallelesforbreastcancer 10
canberevealedindiversepopulationsandhaveimportantpublichealthimplicationsinbuildingrace/ethnicity-specificrisk by
predictionmodelforbreastcancer. gu
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Introduction appeartobespecificforER-negativedisease(19,21).Becauseof A
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AsthemostcommoncanceramongwomenbothintheUnited itsaggressivenature,thereisanunmetclinicalneedforpredic- ril 2
tivegeneticmarkersofsusceptibilitytoER-negativebreastcan- 0
Statesandintheworld(1),breastcancerisaheterogeneousdis- 1
cer,especiallyamongwomenofAfricanancestry. 9
ease with several molecular subtypes defined by biomarkers
such as oestrogen receptor (ER). Oestrogen receptor-negative
breastcancerismorelikelytobeearly-onsetandisassociated
Results
with worse clinical outcomes. African Americans are more
likelytohaveER-negativebreastcancer,forreasonsthatremain Thestudyconsistsofadiscoverystageandavalidationstage.
unknown and understudied (2). Indigenous Africans have an In the discovery stage, we conducted a meta-analysis of two
even higher proportion of ER-negative breast cancer than GWAS (Table 1). One study (GWAS of Breast Cancer in the
African Americans (3,4), suggesting that previously unrecog- AfricanDiaspora,ROOTconsortium)consistsof1,657casesand
nizedgeneticfactorsplayanimportantroleinthisracialdiffer- 2,029controls,whiletheotherstudy(AfricanAmericanBreast
ence. Multiple genome-wide association studies (GWAS) of Cancer Consortium, AABC) consists of 3,016 cases and 2,745
breastcancerhavebeenconductedbutmainlyinpopulationsof controls. Genotyping was conducted using the Illumina
Europeanancestry,whichhaverevealedmorethan90common HumanOmni2.5-8v1 and Illumina Human1M-Duo BeadChip in
susceptibility loci (5–21). However, only a few of these loci ROOT and AABC studies, respectively. To combine genotyping
HumanMolecularGenetics,2016,Vol.25,No.21 | 4837
Table1.CharacteristicsofstudyparticipantsintheGWASofbreastcancerforwomenofAfricanancestry
No.ofsubjects Mean(SD)ofageinyears Oestrogenreceptor(n,%)
Study StudyName Case Control Case Control Positive Negative
ROOTconsortium
NBCS NigerianBreastCancerStudy 711 624 48(12) 45(12) 99(70) 42(30)
BNCS BarbadosNationalCancerStudy 92 229 57(15) 55(13)
RVGBC RacialVariabilityinGenotypicDeterminants 145 257 46(11) 41(12) 25(48) 27(52)
ofBreastCancerRiskStudy
CCPS ChicagoCancerProneStudy 394 387 47(12) 45(11) 140(45) 171(55)
BBCS BaltimoreBreastCancerStudy 95 102 54(14) 52(13) 44(49) 45(51)
SCCS SouthernCommunityCohortStudy 220 430 57(9) 57(9) 66(36) 118(64)
subtotal 1657 2029 49(12) 48(13) 374(48) 403(52) D
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AABCconsortium w
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MEC MultiethnicCohortStudy 694 990 66(9) 67(9) 408(70) 176(30) lo
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CARE Women’sContraceptiveandReproductive 357 215 49(8) 48(8) 183(58) 130(42) d
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WCHS WoEmxpeenr’isenCcirecsleStoufdHye,aLlotghAStnugdeylescomponent 261 239 50(10) 50(9) 131(62) 80(38) d fro
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SFBCS SanFranciscoBayAreaBreastCancerStudy 165 220 56(12) 55(12) 84(63) 50(37) h
NC-BCFR NorthernCaliforniasiteoftheBreastCancer 424 50 50(9) 49(9) 219(64) 121(36) ttp
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FamilyRegistry ://a
CBCS CarolinaBreastCancerStudy 635 589 51(12) 52(11) 272(46) 317(54) c
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PLCO Prostate,Lungs,ColorectalandOvarian 56 116 68(7) 68(6) 14(70) 6(30) d
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CancerScreeningTrialCohort m
NBHS NashvilleBreastHealthStudy 304 182 54(11) 52(10) 143(69) 65(31) ic.o
WFBC WakeForestUniversityBreastCancerStudy 120 144 55(12) 55(11) 66(61) 43(39) up
subtotal 3016 2745 55(12) 58(13) 1520(61) 988(39) .co
AMBERconsortium m
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BWHS BlackWomen’sHealthStudy 752 2249 54(10) 53(11) 412(65) 218(35) m
CBCS CarolinaBreastCancerStudy 727 61 51(11) 53(9) 444(64) 252(36) g/a
WCHS Women’sCircleofHealthStudy 505 629 54(11) 51(10) 237(70) 103(30) rtic
subtotal 1984 2939 53(11) 53(10) 1093(66) 573(34) le
Total 6657 7713 53(12) 53(13) 2987(60) 1964(40) -ab
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t/2
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resultsfromthetwostudies,weimputedgenotypesforsingle associated with ER-negative breast cancer (T-allele, OR¼1.30; 1
nucleotide polymorphisms in the 1000 Genomes Project using P¼2.4(cid:2)10(cid:3)10),butonlyweaklyassociatedwithER-positivedis- /48
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IMPUTE2(22).Quantile-quantileplotsofP-valuesshowedlittle ease(OR¼1.08,P¼0.03).SNPrs12998806wasimputedinROOT 5/2
inflation of test statistics in ROOT, AABC, or in the meta- and AABC (imputation information score r2>0.99) and 5
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analysis (all genomic control inflation factor k’s<1.04; rs13074711 was imputed in ROOT (r2¼0.987). We genotyped 59
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Supplementary Material, Fig. S1), indicating that there was no these two SNPs in 96 samples from ROOT using sequencing 0
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residualcrypticpopulationsubstructureintheanalysis.Results technique, and found that the genotyping results were 100% y
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of the meta-analysis are summarized in the Manhattan plots and 99% in concordance with imputation for rs12998806 and u
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(Fig.1).Thetop20,000SNPsfromthemeta-analysisofoverall rs13074711, respectively, suggesting that the results based on s
breastcancerorER-specificdiseasewereconsideredforstage2 imputedSNPswerereliable. t on
replicationgenotypinginanindependentsampleof1,984inva- The rs13074711 SNP is located 26.5Kb upstream of the 04
sive cases and 2,939 controls assembled by the African Tumour Necrosis Factor Superfamily, Member 10 (TNFSF10) A
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American Breast Cancer Epidemiology and Risk (AMBER) con- gene and the association signal for ER-negative breast cancer ril 2
sortium(Table1).Ofthe(cid:4)20,000SNPs,18,376SNPspassedthe mapstoalinkagedisequilibrium(LD)blockof(cid:4)100Kbbetween 0
1
designstagefortheIlluminacustomchipandweresuccessfully 172.2Mb and 172.3Mb on chromosome 3, which covers the 9
genotyped. TNFSF10 gene (Fig. 2A, Supplementary Material, Fig S2). The
InthecombinedanalysisofGWASdiscoveryandvalidation TNFSF10gene,alsoknownasTRAILorAPO2L,encodesacyto-
stages,associationsofSNPsinthreeregionsattainedgenome- kine that belongs to the tumour necrosis factor ligand family.
widesignificance(Table2),withoneoftheseregions(3q26.21) TNFSF10 cytokine preferentially induces apoptosis in tumour
not identified in previous GWAS. All three SNPs were signifi- cellsortransformedcells,butisnottoxictonormalcells(23).
cantly associated with overall risk of breast cancer, but the The TNFSF10 gene is expressed in a wide range of tissues, in-
strengthoftheassociationvariedbybreastcancersubtype.SNP cludingbloodlymphocytes,breast,prostate,lungs,minorsali-
rs12998806at2q35wasassociatedwithER-positivebreastcan- varygrandandspleen(24).Interestingly,triple-negativebreast
cer(G-allele,OR¼1.34;P¼2.2(cid:2)10(cid:3)8),butnotwithER-negative cancer cell lines have been shown to be very sensitive to
disease(OR¼0.99).SNPrs13074711at3q26.21wassignificantly TNFSF10-induced apoptosis, while ER-positive cell lines were
associated with ER-negative breast cancer (T-allele, OR¼1.29; resistanttoTNFSF10-inducedapoptosis(25).Becauseofitsim-
P¼1.8(cid:2)10(cid:3)8), but only weakly with ER-positive breast cancer portantroleintumourapoptosis,severalTNFSF10receptorago-
(OR¼1.10, P¼0.0094). Similarly, rs10069690 at 5p15.33 was nists or recombinant forms of TNFSF10 are in clinical
4838 | HumanMolecularGenetics,2016,Vol.25,No.21
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Figure1.Manhattanplotsof–log10ofP-valuesinthemeta-analysisofROOTandAABCforoverall,ERþandER-breastcancerrisk.
development as molecularly targeted therapy for cancers (26). andER-negativebreastcancerremainedstatisticallysignificant
Thus,TNFSF10geneisaplausiblecausalgeneintheregion. (p¼0.002)thoughtheadjustedoddsratiowassmaller(Table3).
There appeared to be a second association signal in the Interestingly,theriskalleleofrs9833271existsonlyinAfrican
3q26.21/TNFSF10 region: rs9833271 was associated with ER- populations. Furthermore, a missense variant in exon 1 of
negativebreastcancer(A-allele,OR¼1.39,P¼3.7(cid:2)10(cid:3)5)andit TNFSF10(rs6763816,c.97G>A,Val33Ile)hasafrequencyof0.04
wasinweakLDwiththetopsignalSNP,rs13074711(r2¼0.06). in African populations, but is monomorphic in other popula-
After adjusting rs13074711, the association between rs9833271 tions;theminoralleleofthismissensevariantwasassociated
HumanMolecularGenetics,2016,Vol.25,No.21 | 4839
with reduced risk of ER-negative breast cancer (OR¼0.75;
cancer Pvalue 0.30 0.990.56 0.84 0.24 4.1E-070.0038 1.8E-08 0.0048 2.3E-060.00162.4E-10 riPns¼1g30f0o.07r04r17s211)13.0w7O4et7rhe1e1nr(oTSalNobnlPegse3r).thstaattiswtiecarellyisnignmifiocdaenrtataefteLrDadjwuistth-
ER-negativebreast OR(95%CI) 0.88(0.70-1.12) 1.00(0.86-1.16)1.07(0.85-1.34) 0.99(0.88-1.10)0.51 1.11(0.93-1.33) 1.37(1.21-1.55)1.30(1.09-1.56) 1.29(1.18-1.40)0.16 1.27(1.08-1.50) 1.31(1.17-1.47)1.31(1.11-1.54)1.30(1.20-1.41)0.94 sgeSithiruyoaopnltTwphbhlwaeeledotmeotrtheedehnxeaa(pt2tbsar7srrse)eoyesacovsMisieraotratnbacetraeldarnsqiiwincaguelina,tr(2h-iFnfia8itgcst)i,th.asaoSnebt3cutievi)ta.SxetWNnperoPdteetsrsaSaisiiNnlntisoPobntslhreoeioexcnafaufl3smTsqtuN2eitn6F(une.Semc2QFde11To/tT0TuLhNN)reinFF(pSaTSpFonFae1s1abr0s0lliiyepbershixe4ils----,
pression by serving as enhancer variants. The top associated
ncer Pvalue 0.12 3.0E-070.022 2.2E-08 0.09 0.00170.049 0.0094 0.21 0.110.370.03 SotoNfnhPei,gHrhs31h3liy0ss7tio4nn7ee121H7,3aanlcydestiyintlseatL4iDomn-loi(nHnko3emKd2e7StAhNcyP)lsafetwiaoetnurer(Helso3cKtah4taMetdea1irn)eacrnehdgairhoaincss-- Dow
ER-positivebreastca OR(95%CI) 1.21(0.95-1.54) 1.45(1.26-1.67)1.24(1.03-1.49) 1.34(1.21-1.48)0.27 0.86(0.73-1.02) 1.18(1.06-1.31)1.15(1.00-1.32) 1.10(1.02-1.19)0.007 1.11(0.94-1.31) 1.08(0.98-1.19)1.06(0.93-1.20)1.08(1.01-1.16)0.91 tSNhltrsieisiNauoot1relnmiPt7ssac,a6tabeili0cnnllr1ylss8a,som97t8(fsdaM9at3ur)erm3Ytocna2nOtmhl7asgs)1aloai,n(tberTerycnrweaeehsebaraioaplsdsenefitetlch4cdeaea)Tmel.rsnilNnTioecfaFheneltSarertlcFosatce1utcnel0h(ralslFserltciesegl(r(eiHdi.rnopsMSef2t6iiNB7nEto(Th)6CPn.4e3s)aT7s8fanaceD1hnlc6oe)Sdt.,esoNTesnbrhPerhrt(scseeGoa1oanmAw6nstc8htTdeie4neArmr5eoa37tyrs)ref9osaoalb8oeenul,ilcpmnsneiicdaldtaeerhtiinnnepieontddg--f. nloaded from https://a
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ncer Pvalue 0.22 0.00110.076 0.00017 0.53 1.9E-060.015 2.1E-06 0.0049 0.00220.0494.8E-06 rliinsn1et3hs0e7o43f7qe12v16id.2ce1onurceledgsioasnultgeagrnedmstbutrhletaiaptsltethceamnaocstesirfoscr.iisaTktaiomkneanbyetbtoewgemeethendevira,atretihadenbstyes ademic.o
ncer Overallbreastca OR(95%CI) 1.09(0.95-1.26) 1.20(1.08-1.34)1.14(0.99-1.31) 1.15(1.07-1.24) 1.03(0.93-1.14) 1.23(1.13-1.34)1.15(1.03-1.28) 1.14(1.08-1.21) 1.15(1.04-1.26) 1.13(1.04-1.22)1.11(1.00-1.23)1.13(1.07-1.19) mrbrreser1ggeo3uiasTo3oslhan8tmet7icon0eaaf4gsn2s(cid:4)Tc2oe8Niqcnr73Fia5KStitnhFbi1ois(p0bnSrerugesetepviwgngpiioneeolueeanemnslxhapeG2arrn1Woest7usabA.sn8reiSy5ode5nncrMosMtah1nbas2rdsto9aueou9nrcc8gidtia8heal0dt2,ee61ndm7wFh.iw9aaag4insin2tchlcSeMyo4rEnb)iaR.nfico-ntppnVievooadiscptrihiiutteaoirlsvnoa.ae--t up.com/hmg/artic
betweenthreelociandbreastcancerriskinAfricanancestry,bytypeofbreastca ImputationAlleles score(reference/SNPPosition*StudyRAFtest) rs12998806217893763A/GROOT0.9950.87 AABC0.9920.86AMBERTyped0.86 Combined0.86Pforheterogeneity0.57 rs13074711172267803C/TROOT0.9870.67 AABCTyped0.70AMBERTyped0.69 Combined0.69Pforheterogeneity0.04 rs100696901279790C/TROOTTyped0.63 AABCTyped0.59AMBERTyped0.60Combined0.60Pforheterogeneity0.88 genreceptor;OR,allelicoddsratio;CI,confidenceinterval;RAF,risk(test)allelefrequency. ws(tpAwAtAwMac(pEr(o2r(Pkr3O3hissahqnurnfoao2fmf3aih6a14oeRmrrtr3trsdo¼n)5s04tIiiihjtoToi5eGcccwe¼eAse04taeK0mphrtaahircwrF62n-hn.irv1biennent6ocLBab99eaodmee..ea)3afD1lPr67gl.gsralnsa,pe)nN2o5b95iOeoseaakEuoFnao,bs01narlnymosneuirewnsecpgp0lngslerrPoerth(yae’ee0uoaoo.gaod2ssocss¼en6sStctepuls9fwpkttaihlc5t9eie5Cn,unrnpr0e,aun3tarep6facy)seEli.dac2tf.0dnsao9a0ato1ne1eiUl>nio)eoky0(t0c5ntunx3nw,c9ipeln9p30dtwcpsoapg(,ybSaaar)(3re8.resnirS.uN5oi2nsrn1seiarr7sotsusa¼ntsi,TcshP0sopFrpg3,(ftp1(esisNO4ocooSn0ewn2osE0iipsoi2rarui.dusokP)Rosaa0Rt3nsecln1ennrpetltw6o11i-¼alttbyeddaomppd9h2)rkiw(iatydtnc11in6ol9hetepes(seeAaa6.i9hf99sr11wedonmnet9s0osis,8a262tes1tpwtctiiit8hwKti;qeoadag2thcKtwuhav0hehrnbmn3nPo)riaueble6.satyia)S5entis¼nnrhs4,.bsfiuaetNrbafia4rIisMrcL(nT0slrbGnoiweinPl34y1oanDten.dhrnsadaF3s0v2ue3cneaetteesB0t)s9ad3uMrdausa.tEberctPo27rw8lstiklsudirloyiItae5)5ntyc7no,aintw,eiorrctsaaii0ecna(ore(crnbnat4Glno4t3ksetesp,rhataaunhgh23lc-sisbnoemeaatcaFeS4aeoesoca(factmelilwNrnStcmK,oeogltnme1rLhtceiuhddr.Pnraebrh0aDuFelairrposcse)gp0tseSiierilAitepnnhg)slk0aoran6lhpadawT.fkealrgaspn)rreoeireaGs.nEtsnebinuodSewmues(gtcoRetgoi3Tnmt7lAoduarscniTaAieI3eti)htseocmGnilfnh.tsroe)nhsaatriaenig,nwtFaunimiotttTaruecaYBEbtiitadnefiLnoooanhhdenPetRuorRiosysDnnnnhnddyyeee2ssr-ttfI,, le-abstract/25/21/4835/2525910 by guest on 04 April 2019
Table2.Association Cytoband,gene 2q35 TNFSF103q26.21 TERT5p15.33 *NCBIBuild37position. Abbreviation:ER,oestro ratriacshssanll11envea00elclA00eyre66Aysro99iBassf66Cti99omr500sfcp1ioEla01SanRn05Nr-sd6/nPotT9oerEE6tgwRRt9iauh-T0antmasifievtosgerop(2atbrwbh1tesri)eovvee.mieraTbovsheubeetsnersdctelasyoaiintnnfsritcAedAdencifeAcrragnaiBnwtctthCaciohfine,roierscfaoduhinafngsciiEgnnsReReocsOs-lctanutOriiaydenTGt.gegiWadoattnnhidAvdaabSetteAatbtmMwhrfreeeBeoateETmasnR--t
4840 | HumanMolecularGenetics,2016,Vol.25,No.21
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Figure2.The3q26.21/TNFSF10locustaggedbyrs13074711.(A)TheregionalManhattanplotshowsthe–log10PvaluesfortestingSNPsin250kbregionwithER-breast 0
1
cancerriskinthemeta-analysisoftheROOT,AABCandAMBERstudies.Thecoloursdepictthestrengthofthecorrelation(r2)betweenSNPrs13074711andtheSNPs 9
testedintheregion.ThecorrelationisestimatedusingtheAfricanpanelofthe1000GenomesProjectdata.Thebluelineindicatestherecombinationratesincenti-
morganspermegabase.(B)AnalysisofregulationenhancerwithdatafromENCODEthroughUCSCGenomeBrowser,includinghistonemodificationmarksfor
H3K4Me1andH3K27Acofsevencelltypes,transcriptionfactorbindingsitesandDNasehypersensitivitysitesofhumanmammaryepithelialcells(HMEC),breastcan-
cercells(MCF7,T-47D).ChromosomalcoordinatesareinNCBIbuild37.
Discussion
inpreviousGWASofotherpopulations.Functionalannotation
In the largest breast cancer GWAS conducted in women of of the associated variants suggests that the nearby TNFSF10
Africanancestrywithmorethan14,000casesandcontrols,we gene may be the causal gene underlying this association. We
have identified 3q26.31 locus as a novel risk region for ER- also found and confirmed breast cancer risk variants that are
negativebreastcancer.ThesecondsignalSNPandamissense specific for African Americans at two known susceptibility re-
variantin3q26.31locusonlyexistsinAfricanancestrypopula- gions.Thesefindingsshowedthatgenome-widescansofcom-
tions,whichmayexplainwhythislocushadnotbeendetected mon and rare genetic variants carried out in diverse
HumanMolecularGenetics,2016,Vol.25,No.21 | 4841
Table3.Selectedvariantsin3q26/TNFSF10region:theirassociationwithoestrogen-receptornegativebreastcancer,andtheirlinkagedisequi-
libriumwithrs13074711(thetopSNPintheregion)
Variant Position* Allele† r2inYRI r2inCEU Study RAF OR(95%CI) Pvalue AOR(95%CI)‡ Pvalue‡
rs6763816 Val33Ile C/T 0.08 NA ROOT 0.06 0.92(0.66-1.28) 0.62 0.98(0.68-1.39) 0.90
Exon1 AABC 0.05 0.73(0.56-0.94) 0.014 0.85(0.65-1.11) 0.22
AMBER 0.06 0.60(0.41-0.87) 0.0079 0.65(0.44-0.98) 0.038
Combined 0.06 0.75(0.62-0.89) 0.0012 0.83(0.69-1.01) 0.058
rs16845798 13767 G/A 0.44 0.75 ROOT 0.73 1.03(0.85-1.24) 0.79 0.88(0.67-1.16) 0.37
AABC 0.74 1.25(1.10-1.42) 6.7E-04 0.95(0.78-1.15) 0.57
Combined 0.74 1.17(1.06-1.30) 0.0031 0.92(0.79-1.08) 0.33
rs17601879 16833 C/T 0.25 0.32 ROOT 0.81 1.00(0.81-1.22) 0.98 0.87(0.67-1.14) 0.32
AABC 0.81 1.24(1.08-1.43) 0.0025 0.97(0.80-1.16) 0.71
D
Combined 0.81 1.16(1.03-1.30) 0.014 0.93(0.80-1.09) 0.38 o
w
rs7650827 18258 C/T 0.36 0.32 ROOT 0.78 1.04(0.85-1.27) 0.69 0.92(0.70-1.22) 0.58 n
AABC 0.80 1.27(1.10-1.46) 7.9E-04 0.98(0.81-1.19) 0.86 loa
Combined 0.79 1.19(1.06-1.34) 0.0029 0.96(0.82-1.13) 0.65 de
d
rs9809402 22074 C/T 0.25 0.09 RAOAOBCT 00..3302 11..1149((01..9066--11..3354)) 00..105031 11..1005((00..9922--11..3230)) 00..3403 from
Combined 0.31 1.17(1.07-1.29) 0.0011 1.07(0.96-1.19) 0.22 h
rs7627427 23005 G/C 0.25 0.09 ROOT 0.29 1.04(0.87-1.25) 0.66 1.00(0.83-1.21) 0.99 ttp
s
AABC 0.29 1.19(1.05-1.35) 0.0053 1.07(0.94-1.22) 0.29 ://a
Combined 0.29 1.14(1.03-1.26) 0.011 1.05(0.94-1.17) 0.38 c
a
rs9833915 24723 G/A 0.75 1.0 ROOT 0.60 1.06(0.90-1.26) 0.47 0.93(0.69-1.26) 0.64 de
m
AABC 0.61 1.26(1.12-1.42) 8.1E-05 0.95(0.77-1.15) 0.58
ic
Combined 0.61 1.20(1.09-1.32) 2.5E-04 0.94(0.80-1.11) 0.47 .o
u
rs13074711 26505 C/T 1.0 1.0 ROOT 0.67 1.11(0.93-1.33) 0.24 NA NA p
.c
AABC 0.69 1.37(1.21-1.55) 4.1E-07 o
m
AMBER 0.69 1.30(1.09-1.56) 0.0038 /h
Combined 0.69 1.29(1.18-1.40) 1.8E-08 m
g
rs9833271 37244 G/A 0.06 NA ROOT 0.07 1.63(1.19-2.24) 0.0024 1.59(1.15-2.18) 0.0046 /a
AABC 0.06 1.32(1.06-1.64) 0.012 1.18(0.94-1.47) 0.15 rtic
AMBER 0.06 1.32(0.95-1.82) 0.095 1.23(0.89-1.71) 0.21 le
-a
Combined 0.06 1.39(1.19-1.63) 3.7E-05 1.28(1.09-1.50) 0.002 b
s
tra
*†breafseerepnacireuapllsetlree/atemstoafltlehlee.transcriptionstartingsiteofTNFSF10. ct/25
‡conditionalanalysisadjustedforSNPrs13074711. /21
Abbreviation:NA,notapplicable;OR,allelicoddsratio;CI,confidenceinterval;RAF,risk(test)allelefrequency;YRI,YorubainIbadan,Nigeria;CEU,Utahresidentswith /4
8
NorthernandWesternEuropeanancestry. 35
/2
5
2
5
9
1
populations can reveal additional susceptibility alleles for prevention. In particular, theses data can be used to improve 0
b
breastcancer,andpopulation-specificriskvariantsareimpor- riskpredictionmodelforaggressiveformsofbreastcancerindi- y
g
tantforbuildingappropriateriskpredictionmodels. versepopulations.Weprovideseverallinesofevidencethatthe u
e
Interestingly, the associations with the three breast cancer associationat3q26.31regionmaybemediatedbyregulationof s
risk susceptibility loci depend on ER status, which reinforces TNFSF10geneexpression,andTNFSF10isimportantfortumour t on
the need for even larger discovery efforts for etiologically dis- apoptosis. Further functional experiments will be needed to 04
tinct breast cancer subtypes. The risk allele frequency for confirm whether the TNFSF10 gene is a susceptibility gene for A
p
rs10069690in5p15/TERTlocusisgreaterinAfricanpopulations ER-negativebreastcancerandtofullyunderstandhowgenetic ril 2
(0.62)thaninEuropeanancestrypopulations(0.27),andtherisk variantsaffectriskofbreastcancerindiversepopulationsand 0
1
allele of rs9833271 exists only in African populations. These whytheassociationsvarybybreastcancersubtype. 9
may contribute to the higher proportion of ER-negative breast
cancerinwomenofAfricanancestry.Bycontrast,theriskallele
frequencyforrs13074711issmallerinAfricanpopulations(0.61) MaterialsandMethods
thaninEuropeanancestrypopulations(0.89),andtheriskallele
Studypopulations
frequencyforrs12998806isgreaterinAfricanpopulations(0.78)
than in women of European ancestry (0.66), suggesting that All the studies included in the GWAS have been approved by
thesetwolocimaynotaccountforracialdifferencesinsubtype theircorrespondingInstitutionalReviewBoards.Informedcon-
distributionbutmoredefinitivestudiesarewarranted. sent has been obtained from the participants. The discovery
The aetiology of ER-negative breast cancer is largely un- stage(stage1)ofthestudyisameta-analysisofgenome-wide
known,andourstudyfindingsthattheassociationforallthree association studies in two breast cancer consortia among
toplocivariesbyERstatusprovideimportantinsightintodis- womenofAfricanancestry.TheROOT(GWASinBreastCancer
tinctbiologicalpathwaysforER-positiveandER-negativebreast in the African Diaspora) consortium consists of six epidemio-
cancers, which will benefit both breast cancer treatment and logic studies and the AABC (African American Breast Cancer)
4842 | HumanMolecularGenetics,2016,Vol.25,No.21
singHaploReg(37) Motifschanged bythevariant Irf,Pax-5,XBP-1 Pou2f2,Pou5f1 SEF-1 HNF4,Mef2,Pax-2 6alteredmotifs 8alteredmotifs 29alteredmotifs DMRT2
u
TNFSF10n3q26/region Proteinsbound 7boundproteins(ERALPHA_Aand GATA3inT-47D)GATA1,GATA3(T-47D) FOXA1(T-47D)GATA3(T-47D) cells. Dow
i a n
onofselectedvariants DNAse hypersensitivity 26tissues(HMEC.35,HEMC) 9tissues(HEME.35,HMEC) Cervix(HELAS3) Skin,Cervix 6tissues(HMEC.35,HMEC) 4tissues(HMEC) mammaryductalcarcinom loaded from https://ac
s(27,28))andregulatoryelementannotati PromoterEnhancerhistone histonemarks†marks 6tissues(HMEC.35,24tissuesMYO)(HMEC.35, MYO,HMEC)3tissues7tissues(HMEC.35,MYO,HMEC) Blood,GIandbreast(HEMC.35,MYO)10tissues(HMEC.35, MYO,HMEC)(HMEC,MYO) 4tissues12tissues(HMEC.35,MYO,HMEC)3tissues(HMEC.35, MYO)7tissues(HMEC.35, MYO,HMEC)8tissues(HMEC.35,MYO,HMEC) MEC:mammaryepithelialprimarycells;T-47D: ademic.oup.com/hmg/article-abstract/25
analysi brain brain brain brain ycells;H /21/483
TNFSF10Table4.Correlationofselectedvariantswithexpressionof(fromeQTL VariantPosition*AllelesMinorallelefrequencyeQTL Ref/AltAFRAMRASNEUR rs6763816Exon1,C/T0.04000BrainVal33Ile rs1684579813767A/G0.340.140.120.12Brain rs1760187916833T/C0.280.10.120.08Blood, rs765082718258T/C0.270.10.120.08Blood, rs980940222074C/T0.240.340.120.44Blood, rs762742723005G/C0.230.330.120.44Blood, rs983391524723A/G0.450.130.120.11Brain rs1307471126505T/C0.390.120.120.11Brain rs983327137244G/A0.05000 TNFSF10*basepairupstreamofthetranscriptionstartingsiteof.†Normalmammaryorbreastcancercelllinesareindicatedinparenthesis. HMEC.35:breastvarianthumanmammaryepithelialcells;MYO:breastmyoepithelialprimar 5/2525910 by guest on 04 April 2019
HumanMolecularGenetics,2016,Vol.25,No.21 | 4843
consortium consists of nine epidemiological studies (34). The samples;theaverageconcordanceratewas99.95%(>99.93%for
replicationstage(stage 2)ofthestudyisbasedonthe AMBER all pairs). We also eliminated SNPs with genotyping concor-
(African American Breast Cancer Epidemiology and Risk) con- dancerates<98%basedonthereplicates(n¼11,701).Thefinal
sortium, which consists of four epidemiologic studies (35). analysis dataset included 1,043,036 SNPs genotyped on 3,016
Selectedcharacteristicsofstudyparticipantsforeachstudyare casesand2,745controls,withanaverageSNPcallrateof99.7%
summarizedinTable1.Thestudydesignforeachstudyisde- andaveragesamplecallrateof99.8%.Hardy-Weinbergequilib-
scribedintheSupplementaryMaterials. rium (HWE) was not used as a criterion for removing SNPs at
thisstage.
Genome-widegenotypingandqualitycontrol
Imputationandtechnicalvalidationbysequencing
Genotyping inthe ROOT consortium wasconducted using the
GenotypeimputationfortheROOTconsortiumwasconducted
IlluminaHumanOmni2.5-8v1array.Genotypingwasattempted
bytheUniversityofWashingtonGeneticsCoordinatingCenter D
foratotalof3,909studysamples,ofwhich3,859weresuccess- (GCC)usingtheIMPUTE2software(23).Followingfiltersforge- ow
ful.The3,859studysampleswerederivedfrom3,774subjects, n
notypeimputation,includingnoambiguityinstrandalignment lo
with85subjectswithduplicatesamples.Aftergenotyping,qual- a
andabilitytobemappedtoNCDBbuild37,2,018,833SNPswere d
itycontrolanalysis wascarried outby checking chromosomal e
selected as the basis for imputation. Using the 1000 Genomes d
anomalies,participantrelativeness,populationstructure,miss- Project phase I integrated variant set as the reference panel fro
ing call rates, batch effects, duplicate sample discordance, m
(October2011release),20,109,249SNPswereimputed.Ofthese, h
Mendelian errors, Hardy-Weinberg equilibrium, and duplicate 16,147,413havepassedtheimputationqualityfilter(imputation ttp
SNP probes. First, duplicate samples from 85 pairs were ex- s
cluded.Chromosomalanomalies>5Mbandanomaliesonchro- score>0.3)andwereincludedalongwiththegenotypedSNPsin ://a
the final association analysis. Genotype imputation in AABC c
mosomeswherethesumoftheanomalylengths>10Mbwere wasconductedusingIMPUTE2software(23)toacosmopolitan ad
e
identified.Nosubjectswereexcludedforthisreasonbutallge- panelofall1000GenomeProjectsubjects(March2012release). m
notypes were set to missing for regions with chromosome To provide technical validation, we sequenced 96 samples ic.o
anomalies. The relatedness between each pair of participants u
that have been included in the final analysis of the ROOT p
was evaluated by the identical by descent (IBD) analysis. .c
GWAS. We conducted whole genome sequencing on the o
According to the IBD analysis, samples from 59 subjects were m
Illuminaplatform,withaveragedepthof30x.Theconcordance /h
excludedbecausetheywerepossiblyrelativesofothersubjects. m
ratebetweenwholegenomesequencingandarray-basedgeno-
g
Additionally, 20 subjects (18 African Americans and 2 African typingwas99.5%. /a
Barbadian)wereexcludedbecauseprincipalcomponentsanaly- rtic
sisshowedthattheywereclusteredwiththeCEU/TSIHapMaps le
-a
(i.e.highpercentageofEuropeanancestry).Wefurtherfiltered Genotypinginthereplicationstage bs
out7sampleswithamissingcallrate>2%and2sampleswith tra
GenotypingoftheAMBERcasesandcontrolswasperformedon c
pofos3s,6ib8l6estuubmjeocutsrc(1o,n65ta7mcainsaetsioann.dA2f,t0e2r9thcoensetroexlsc)luwseiorensin,calutodteadl theIlluminaHumanExomeBeadchipv1.1withadditionalcus- t/25
forthefinalanalysis. tomcontentattheCenterforInheritedDiseaseResearch(CIDR). /21
Thestandardcontentofthisarrayincludesmorethan240,000 /4
A total of 2,379,855 SNP probes were attempted in Illumina 8
Hsius,m3a2n,1O9m2SnNi2P.5s-8wvi1thartreacyh.nInicathleerSrNorPsl,e1v1e9l,q92u4alSitNyPcsonwtirtohlamnianloyr- cTowdeinngtyvathrioaunstasn,adsvwaerilalnatssaindceensttirfiyeidnfionrmthaetiAvAeBmCa/RrkOeOrsT(AmIMetsa)-. 35/25
2
allelefrequency¼0and5,631duplicateSNPswereexcluded.We analysis,including2ormorevariantsperassociatedregionfor 59
thetop(cid:4)5000loci,wereincludedaspartofthecustomcontent. 1
furtherfilteredout99,511SNPswithmissingcallrate>¼2%,563 0
Of 405,555 SNPs attempted for genotyping, 381,212 were re- b
SNPswith>1discordantcalls,1,078SNPswith>1Mendelianer- y
leasedbyCIDRand299,873oftheseremainedafterremovingSNPs g
rors and 4,591 SNPs with Hardy-Weinberg equilibrium test u
P<10(cid:3)4.Aftertheseexclusions,2,116,365SNPremained. that were monomorphic (n¼70,761), were positional duplicates, es
Genotyping in the AABC consortium was conducted using were on the Y chromosome, had Hardy-Weinberg Equilibrium t on
the IlluminaHuman1M-Duo BeadChip. Of the 5,984 samples (HWE) P<10(cid:3)4, had call rate<0.98, had>1 Mendelian errors in 04
available to the AABC consortium (3,153 cases and 2,831 con- trios from HapMap (http://hapmap.ncbi.nlm.nih.gov/), or had>2 A
trols), we attempted genotyping of 5,932, removing samples dadisdciotirodnaanltcvaalrlsiainntsduupsliincagtethsaem1p0l0e0s.IGMePnUomTEe2swPahsausseedItroefiemrepnuctee pril 2
(n¼52)withDNAconcentrations<20ng/ul.Followinggenotyp- 0
ing,weremovedsamplesbasedonthefollowingexclusioncri- panel(5/21/20111000Gdata,December2013haplotypereleasein 19
IMPUTE2site).Forthemeta-analysis,18,376SNPspassedquality
teria:1)unknownreplicates((cid:5)98.9%geneticallyidentical)that
controlandincludedinthestudy.
we were able to confirm (only one of each duplicate was re-
moved,n¼15);2)unknownreplicatesthatwewerenotableto
confirm through discussions with study investigators (pair or
Statisticalanalysis
triplicateremoved,n¼14);3)sampleswithcallrates<95%after
asecondattempt(n¼100);4)sampleswith(cid:6)5%Africanances- IntheROOTGWAS,weanalysedgenotypedSNPsandimputed
try(n¼36);and,5)sampleswith<15%meanheterozygosityof SNPswithimputationscore>0.3usingSNPTESTsoftwaretoac-
SNPsintheXchromosomeand/orsimilarmeanalleleintensi- countforuncertaintyinimputation(36).Weexaminedtheasso-
tiesofSNPsontheXandYchromosomes(n¼6)(thesearelikely ciationofeachSNPandbreastcancerriskusingunconditional
tobemales). logisticregression,adjustingforage,studysiteandthefirstfour
In the analysis, we removed SNPs with<95% call rate eigenvectorsfromprincipalcomponentanalysis.Thefirstfour
(n¼21,732)orminorallelefrequencies(MAFs)<1%(n¼80,193). eigenvectors were used to control for population stratification
Toassessgenotypingreproducibility,weincluded138replicate asonlythefirst4eigenvectorswereassociatedwithcasestatus.
4844 | HumanMolecularGenetics,2016,Vol.25,No.21
Oddsratios(OR)and95%confidenceintervals(CI)werecalcu- U01-CA161032. DH and OIO were also supported by American
latedfromthemultivariablelogisticregressions.Alltestsofsta- Cancer Society (MRSG-13-063-01-TBG and CRP-10-119-01-CCE).
tisticalsignificanceweretwo-sided.Usingsimilarmethods,we AABCwassupportedbyaDepartmentofDefenseBreastCancer
conducted an analysis to compare ER-positive breast cancers Research Program Era of Hope Scholar Award to CAH
with controls and compare ER-negative breast cancers with [W81XWH-08-1-0383] and the Norris Foundation. Each of the
controls,inordertoidentifysubtype-specificSNPs. participating AABC studies was supported by the following
In the AABC GWAS, we analysed genotyped SNPs and im- grants: MEC (National Institutes of Health grants R01-CA63464
puted SNPs with imputation score>0.3 and minor allele fre- and R37-CA54281); CARE (National Institute for Child Health
quency>0.01. Unconditional logistic regression was used to andDevelopmentgrantNO1-HD-3-3175,K05CA136967);WCHS
examine the association between each SNP andoverall breast (U.S. Army Medical Research and Material Command
cancerrisk,ER-positiveandER-negativebreastcancerrisksepa- (USAMRMC) grant DAMD-17-01-0-0334, the National Institutes
rately.Age,thefirst10eigenvectorsfromprincipalcomponent ofHealthgrantR01-CA100598,andtheBreastCancerResearch
analysis,andstudysitewereadjustedforinthelogisticregres- Foundation; SFBCS (National Institutes of Health grant R01-
D
sion.Tobeconservative,allthefirst10eigenvectorswereused CA077305 and United States Army Medical Research Program o
w
to control population stratification. The SFBCS and NC-BCFR grantDAMD17-96-6071);NC-BCFR(NationalInstitutesofHealth n
lo
studies were conducted in the same San Francisco Bay Area grant U01-CA069417); CBCS (National Institutes of Health a
d
population and were combined as one study site in the Specialized Program of Research Excellence in Breast Cancer, e
d
analyses. grant number P50-CA58223 and Center for Environmental fro
In the replication study, principal components were com- Health and Susceptibility National Institute of Environmental m
h
putedusingthesmartpcaprogramintheEIGENSOFTpackage. Health Sciences, National Institutes of Health, grant number ttp
No eigenvectors from the principal component analysis were P30-ES10126); PLCO (Intramural Research Program, National s
strongly associated with case status after controlling for the CancerInstitute,NationalInstitutesofHealth);NBHS(National ://a
c
matching factors, DNAsource andstudy. ORsand95% CIsfor Institutes of Health grant R01-CA100374). The Breast Cancer ad
e
each SNP were estimated in the analyses adjusted for study, Family Registry (BCFR) was supported by the National Cancer m
age,geographicregion,DNAsourceandeigenvectors5,6and8, Institute, National Institutes of Health under RFA-CA-06-503 ic.o
whichwereassociatedwithcasesstatusatP<0.10.Regression and through cooperative agreements with members of the u
p
coefficients(logoddsratios)andtheirstandarderrors,fromthe Breast Cancer Family Registry andPrincipal Investigators. The .c
o
two GWAS and the replication study were combined using a content of this manuscript does not necessarily reflect m
/h
fixedeffectmeta-analysismethod. theviewsorpoliciesoftheNationalCancerInstituteoranyof m
g
thecollaboratingcentresintheBCFR,nordoesmentionoftrade /a
names, commercial products, or organizations imply endorse- rtic
Functionalannotation ment by the U.S. Government or the BCFR. AMBER was sup- le
-a
ported by the National Cancer Institute grants P01-CA151135, b
Foreachtopvariantandregionidentifiedinthisstudy,weused s
HaploReg(37)andUSCSGenomeBrowsertoexplorefunctional R01-CA098663, R01-CA058420, UM1-CA164974, R01-CA100598, tra
c
annotations of noncoding variants. Chromatin states (pro- UM1-CA164973,R01-CA54281,P50-CA58223,andtheUniversity t/2
Cancer Research Fund of North Carolina. Pathology data were 5
moters and enhancers), conserved regions, variant effect on /2
obtained from numerous state cancer registries (Arizona, 1
regulatorymotifs,andproteinbindingsiteswereassessedfrom /4
California, Colorado, Connecticut, Delaware, District of 8
available data from the ENCODE (38) and the Roadmap 3
Epigenomics Consortium (39). Data from breast tumour cell Columbia, Florida,Georgia,Hawaii, Illinois, Indiana,Kentucky, 5/2
Louisiana, Maryland, Massachusetts, Michigan, New Jersey, 5
lines (MCF-7, T-47D) and normal mammary epithelial cells 2
(HMEC,MYO,vMHEC)wereemphasized. New York, North Carolina, Oklahoma, Pennsylvania, South 591
Carolina, Tennessee, Texas, Virginia). The results reported do 0
Dataonexpressionquantitativetraitlocus(eQTL)analysisin b
bloodwereobtainedfromWestraetal.(27),inwhicheQTLswere not necessarily represent their views or the views of the y g
identifiedinameta-analysisin5,311peripheraluntransformed NationalInstitutesofHealth. ue
s
bloodsamplesandreplicationanalysisinanother2,775samples t o
(27). Data on eQTL analysis in brain were obtained from (cid:4)400 References n 0
autopsiedsubjects(28).AnalysisoneQTLinbreasttumourswas 4
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Funding
Receptor-defined subtypes of breast cancer in indigenous
The ROOT Consortium was supported by National Cancer populationsinAfrica:asystematicreviewandmeta-analy-
InstitutegrantsR01-CA142996,P50-CA125183,R01-CA89085and sis.PLoSMed.,11,e1001720.