TITLE: Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome AUTHOR(S): Oliveira, Marisa; Lert-itthiporn, Worachart; Cavadas, Bruno; Fernandes, Verónica; Chuansumrit, Ampaiwan; Anunciação, Orlando; Casademont, Isabelle; ... Suriyaphol, Prapat; Pereira, Luisa; Sakunta ... CITATION: Oliveira, Marisa ...[et al]. Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome. PLOS Neglected Tropical Diseases 2018, 12(2): e0006202. ISSUE DATE: 2018-02-15 URL: http://hdl.handle.net/2433/230493 RIGHT: © 2018 Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp RESEARCHARTICLE Joint ancestry and association test indicate two distinct pathogenic pathways involved in classical dengue fever and dengue shock syndrome MarisaOliveira1,2,3,4,WorachartLert-itthiporn5,BrunoCavadas1,2,3,VerońicaFernandes1,2, AmpaiwanChuansumrit6,OrlandoAnunciação2,IsabelleCasademont4,7,FannyKoeth4,7, MarinaPenova4,7,8,KanchanaTangnararatchakit6,ChieaChuenKhor9,10, a1111111111 RichardPaul4,7,11,PridaMalasit12,13,FumihikoMatsuda7,8,EtienneSimon-Lorière4,7,11, a1111111111 PrapatSuriyaphol5,LuisaPereira1,2,14‡*,AnavajSakuntabhai4,7,11‡* a1111111111 1 i3S—InstitutodeInvestigaçãoeInovaçãoemSau´de,UniversidadedoPorto,Porto,Portugal,2 Institutode a1111111111 PatologiaeImunologiaMoleculardaUniversidadedoPorto(IPATIMUP),Porto,Portugal,3 Institutode a1111111111 CiênciasBiome´dicasAbelSalazar(ICBAS),UniversidadedoPorto,Porto,Portugal,4 FunctionalGenetics ofInfectiousDiseasesUnit,InstitutPasteur,Paris,France,5 BioinformaticsandDataManagementfor Research,OfficeforResearchandDevelopment,FacultyofMedicineSirirajHospital,MahidolUniversity, Bangkok,Thailand,6 DepartmentofPediatrics,FacultyofMedicine,RamathibodiHospital,Mahidol University,Bangkok,Thailand,7 PasteurKyotoInternationalJointResearchUnitforIntegrativeVaccinomics, Kyoto,Japan,8 CenterforGenomicMedicine,KyotoUniversityGraduateSchoolofMedicine,Kyoto,Japan, OPENACCESS 9 GenomeInstituteofSingapore,A-STAR,Singapore,Singapore,10 DepartmentofBiochemistry,National Citation:OliveiraM,Lert-itthipornW,CavadasB, UniversityofSingapore,Singapore,Singapore,11 CNRS,Unite´deRechercheAssocieé3012,Paris, FernandesV,ChuansumritA,AnunciaçãoO,etal. France,12 DengueHemorrhagicFeverResearchUnit,OfficeforResearchandDevelopment,Siriraj Hospital,FacultyofMedicine,MahidolUniversity,Bangkok,Thailand,13 MedicalBiotechnologyUnit, (2018)Jointancestryandassociationtestindicate NationalCenterforGeneticEngineeringandBiotechnology,NationalScienceandTechnologyDevelopment twodistinctpathogenicpathwaysinvolvedin Agency,Pathumthani,Thailand,14 DepartmentofPathology,FacultyofMedicine,UniversityofPorto,Porto, classicaldenguefeveranddengueshock Portugal syndrome.PLoSNeglTropDis12(2):e0006202. https://doi.org/10.1371/journal.pntd.0006202 ‡LPandASjointlydirectedthiswork. *luisap@ipatimup.pt(LP);anavaj.sakuntabhai@pasteur.fr(AS) Editor:BenjaminAlthouse,InstituteforDisease Modeling,UNITEDSTATES Received:August9,2017 Abstract Accepted:January2,2018 Ethnicdiversityhasbeenlongconsideredasoneofthefactorsexplainingwhythesevere Published:February15,2018 formsofdenguearemoreprevalentinSoutheastAsiathananywhereelse.Herewetake Copyright:©2018Oliveiraetal.Thisisanopen advantageoftheadmixedprofileofSoutheastAsianstoperformcoupledassociation- accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,which admixtureanalysesinThaicohorts.Fordengueshocksyndrome(DSS),thesignificanthap- permitsunrestricteduse,distribution,and lotypesarelocatedingenescodingforphospholipaseCmembers(PLCB4addedtoprevi- reproductioninanymedium,providedtheoriginal ouslyreportedPLCE1),relatedtoinflammationofbloodvessels.Fordenguefever(DF),we authorandsourcearecredited. foundevidenceofsignificantassociationwithCHST10,AHRR,PPP2R5EandGRIP1 DataAvailabilityStatement:Datahasbeen genes,whichparticipateinthexenobioticmetabolismsignalingpathway.Weconducted depositedattheEuropeanGenome-Phenome functionalanalysesforPPP2R5E,revealingbyimmunofluorescenceimagingthatthecoded ArchiveundertheaccessionEGAS00001002756. proteinco-localizeswithbothDENV1andDENV2NS5proteins.Interestingly,onlyDENV2- Funding:Theresearchleadingtotheseresultshas NS5migratedtothenucleus,andadeletionofthepredictedtop-linkingmotifinNS5abol- receivedfundingfromtheEuropeanCommission SeventhFrameworkProgramme[FP7/2007-2013] ishedthenucleartransfer.Theseobservationssupporttheexistenceofdifferencesbetween fortheDENFREEprojectunderGrantAgreement serotypesintheircellulardynamics,whichmaycontributetodifferentialinfectionoutcome no.282378.MOhasaPhDgrantfromFCT(The risk.ThecontributionoftheidentifiedgenestothegeneticriskrenderSoutheastandNorth- PortugueseFoundationforScienceand eastAsianpopulationsmoresusceptibletobothphenotypes,whileAfricanpopulationsare Technology—SFRH/BD/95626/2013).I3Sis PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 1/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome financedbyFEDER—FundoEuropeude bestprotectedagainstDSSandintermediatelyprotectedagainstDF,andEuropeansthe DesenvolvimentoRegionalfundsthroughthe bestprotectedagainstDFbutthemostsusceptibleagainstDSS. COMPETE2020—Competitivenessand InternationalizationOperationalProgramme (POCI),Portugal2020,andbyPortuguesefunds throughFCT/Ministe´riodaCiência,Tecnologiae Inovaçãointheframeworkoftheproject"Institute Authorsummary forResearchandInnovationinHealthSciences" (POCI-01-0145-FEDER-007274).Thefundershad DenguefeverisendemicintropicalandsubtropicalareasofEastAsiaandAmerica,but noroleinstudydesign,datacollectionand globalizationandclimatechangesareintroducingvectorandvirustothenaïveregionsof analysis,decisiontopublish,orpreparationofthe EuropeandNorthAmerica.Inthisworkweconductedastatisticallyrobust,coupledasso- manuscript. ciation-admixturetestintwodenguecohortsfromThailand(classicaldenguefever,DF, Competinginterests:Theauthorshavedeclared anddengueshocksyndrome,DSS)andapublishedVietnamese(DSSonly)cohort.We thatnocompetinginterestsexist. identifiednewcandidategenesassociatedwithDFriskandconfirmedknowngenefamily associationwithDSSrisk.InDF,phosphatasecontroliscrucial,includingthroughbind- ingtoviralproteins,asweshowedforPPP2R5Eproteinco-localizationwithDENV1and DENV2-NS5proteinswithinlivercellsanddifferentialcellularlocalizationsalongtime. InDSS,cytokinedynamics,inflammationandactivationofvascularendotheliumcellsare dominantfeatures.Theparticulargeneticriskconferredbythesegenesindicatesthat SoutheastandNortheastAsiansarehighlysusceptibletobothphenotypes,whileAfricans arebestprotectedagainstDSS,andEuropeansbestprotectedagainstDFbutthemostsus- ceptibleagainstDSS. Introduction Denguevirus(DENV)isthemostcommonmosquito-borneviralinfection,infectingapproxi- mately390millionpeopleperyearworldwidewithonequarterdevelopingdenguedisease (MIM:614371)[1].Symptomsrangefromundifferentiatedfever,classicaldenguefever(DF) toshocksyndrome(DSS;hemorrhage,plasmaleakageandvitalorganimpairment)[2]. Recent-omicapproachesprovideunbiasedgenomicinsightsintomechanismsassociated withdenguedisease.Therehasbeenonlyonepublicationonclassicalgenomewideassociation study(GWAS)ofdengue[3]comparedtoaconsiderablenumberoftranscriptomicstudies [4–7].Thereasonforthisdiscrepancyisthatcohortsofthousandsofindividualsarerequired forGWAStoreachgenomewidesignificance.TheGWASworkconductedonacohortof Vietnamesechildren[3]included2,008DSSsamplesversus2,018controls,replicatedin1,737 versus2,934,andfoundSNPsingenesMICBandPLCE1associatedwithDSSphenotype. Lately,analyticalimprovementsbasedonadmixturemappinghavereducedthesamplesize requirementfromthousandstohundredsofindividualsorevenfewer[8].Mosthumanpopu- lationshavesomedegreeofancestryadmixture,whichbringstogetherhaplotypesthatoccur atdifferentfrequenciesinparentalpopulations.Admixturemappinganalysestheseblocks acrossthemosaicdescendantchromosomesandallowstocomparetheirdistributionbetween caseandcontrolcohorts.ThelowernumberofblockscomparedwithindividualSNPsreduces considerablythestatisticalburden.Wehavesuccessfullyconductedsuchanadmixturestudy indenguecohortsfromCuba[9],andidentifiedtwogenesinvolvedinlipidmetabolismwhich showedtobeprotectiveagainsttheriskofdenguehemorrhagicfever,aprotectionconferred bytheAfricaninheritedancestry.WhereasforRXRAgenetherewasalreadyfunctionalevi- denceofitsinvolvementininfection[10],wealsodemonstratedfunctionallybyshRNAthat theknockdownofOSBPL10genehadasignificantnegativeimpactinDENVreplicationrate [9]. PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 2/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Epidemiologicreportshaveshowntheexistenceofethnicdifferencesinsusceptibilityto denguefevernotonlyinCuba[11]butalsoinMalaysia[12]wheretheincidenceratebyethnic groupwas3.7:1:1.3forChinese,MalaysandIndians,respectively,intheyears1970’sand 1980’s,althoughnocross-evaluationwasperformedwithothersocio-demographicfactors.In thepresentstudy,wetakeadvantageoftheadmixedprofileofSoutheastAsians(inthenexus betweenSouth,NortheastandSoutheastAsia)toperformcoupledassociation-admixtureanal- yses(BMIX;[13])ofcase/controlcohortsofdenguepatients:Thaidenguepatientswhodevel- opedDF(n=252)orDSS(n=159),andacontrolblooddonorgroup(n=290);andthe publishedVietnamesedataset(2018controlsand2008DSSpatients;[3]).Althoughtheadmix- tureintheregionhasbeentakingplacealongtime,sincethefirstarrivalofmodernhuman aftertheout-of-Africamigration,aconsiderablemigrationfromsouthChinabeganinthe15th centuryandincreasedinthe19thand20thcenturies,mainlytowardsThailandwhereabout 40%ofthepopulationhassomeChineseadmixtureand14%areidentifiableThaiChinese [14].ThisisasimilarscenariototheadmixturethattookplaceintheAmericas,wherethese localadmixtureinferencetoolshavebeensuccessfullyapplied[9,15,16].Wewereableto identifydistinctcandidategenesconferringsusceptibility/resistancetotheriskofDFandDSS, arguinginfavorofindependentpathogenicmechanismsfortheestablishmentofthetwophe- notypes.WefurtherconfirmedthatoneDFcandidategenecodesforahumanproteinthatco- localizeswiththeDENV1andDENV2-NS5proteins,and,inthelattercase,transientlyrelo- catedfromthecytoplasmtothenucleus.Wealsoinferredtherelativeworldwidegeneticrisks contributedbythedetectedcandidategenesbasedontheirfrequenciesforthesusceptible/ resistanthaplotypes. Results AncestryofThaiandVietnamesecohorts Allanalyzedindividualshavesomedegreeofadmixture(Fig1;S1Fig).TheNortheast AsianbackgroundisdominantinVietnam(77.3%)anddecreasesinThailand(56.4%),incon- trasttotheSoutheastAsiancomponent,whichincreasesfrom20.7%inVietnamto35.1%in Thailand.TheSouthAsianinfluenceis8.5%inThailandand2.0%inVietnam.Withinthe denguecohorts,weobservedastatisticallysignificantincreaseintheSoutheastAsianback- groundinThailandforbothDF(4.1%increase;p-value=1.25x10−7)andDSS(4.8%increase; p-value=5.90x10−8)comparedtoThaicontrol. DSScohortsanalyses WebeganbycheckingiftheBMIXresultsonthepublishedVietnamesecohort[3]arein accordancewiththeresultsfromtheclassicalassociationmapping,atestoftherobustness ofthealgorithm.BMIXindicatesalsotheassociationofDSSwithMICBandPLCE1genes (Table1,Fig2A,S1andS4Tables).TheidentifiedregionsurroundingMICBencompasses sevensignificantSNPs,placedalong165,080bp,fromthedownstreamMICAtotheupstream LTBgene,aregionhighlyrichingenes.Threelinked(S2Fig)SNPsinMICBhavethemostsig- nificantp-values,formingtheprotectivehaplotypeGTT(OR=0.77;p-value<0.0001),which isthemostfrequenthaplotypeinworldwidepopulations(Fig3C).ThesusceptibleMICBhap- lotypeACC(OR=1.39;p-value<0.0001)ismorefrequentinEuropeansandSouthAsians (0.18to0.34).ThetwoSNPsfoundforPLCE1reachedsignificantp-valuesandarealmostin completelinkage(S5Fig).TheDSSprotectivePLCE1haplotype(CG;OR=0.75;p-value< 0.0001)ismorefrequent(Fig3B)inNortheastAsia(0.12–0.28)andSoutheastAsia(0.19),fol- lowedbyEurope(0.04–0.14)andabsentinAfrica. PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 3/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Fig1.GlobalancestryinferredthroughRFMixwhenusingthreeparentalancestries(South,Northeastand SoutheastAsian)fortheglobaldataset.Eachverticallinerepresentsanindividual,andthethreecoloursrepresent theproportionofthethreeparentalpopulationsineachgenome(lightorangeforSouthAsian,darkorangefor SoutheastAsianandblueforNortheastAsian). https://doi.org/10.1371/journal.pntd.0006202.g001 WefurtheranalyzedtheThaiDSSvs.controlcohort(Table1,Fig2B,S2andS5Tables), andobtainedareliablesignalofsixlinked(S3Fig)significantSNPsforPLCB4(phospholipase C,beta4;S4AFig),ageneinthesamefamilyasPLCE1,andparticipatinginmanycommon pathways,suchasdendriticcellmaturation,PI3KsignalinginBlymphocytesandPPARA/ RXRAactivation.TheDSSprotectivePLCB4haplotype(GAGAGG;OR=0.58;p-value= Table1. Oddsratios(ORs),95%confidenceintervalsandYatesp-values(correctedforcontinuity)oftheχ2testforthesignificanthaplotypes/SNPsinthepheno- typeandpopulationsforwhichassociationwasdetected. Protectivehaplotype Susceptiblehaplotype Gene Frequency Frequency OR(95CI) Pvalue Frequency Frequency OR(95CI) Pvalue Control Case Control Case VietnameseDSSstudy MICB rs2534666-rs2855807-rs3132468 G-T-T A-C-C 0.78 0.73 0.77(0.70–0.85) <1.0x10−4 0.13 0.18 1.39(1.23–1.57) <1.0x10−4 PLCE1 rs3740360-rs2274223 C-G A-A 0.27 0.22 0.75(0.68–0.84) <1.0x10−4 0.69 0.75 1.30(1.18–1.43) <1.0x10−4 ThaiDSSstudy PLCB4 rs16995800-rs2299676-rs7269910-rs1997696-rs6133707-rs6056595 G-A-G-A-G-G A-G-A-C-A-A 0.17 0.10 0.58(039–0.88) 1.3x10−2 0.61 0.70 1.48(1.10–1.98) 1.1x10−2 ThaiDFstudy CHST10 rs4850931-rs1030902-rs2241811-rs2241810-rs4149518-rs2241809-rs4149510-rs4851313-rs3828193 C-T-C-T-A-C-G-G-G T-G-T-C-G-T-A-A-T 0.26 0.17 0.59(0.44–0.79) 5.2x10−4 0.62 0.74 1.78(1.37–2.31) <1.0x10−4 AHRR rs6555205-rs2721020 T-C C-T 0.25 0.16 0.54(0.40–0.74) 1.2x10−4 0.67 0.79 1.89(1.43–2.48) <1.0x10−4 PPP2R5E rs3829766-rs6573513-rs743221-rs7144210 A-C-G-G GTAA 0.43 0.32 0.62(0.48–0.79) 2.0x10−4 0.39 0.50 1.59(1.25–2.03) 2.0x10−4 GRIP1 rs1480010 T C 0.17 0.11 0.56(0.39–0.80) 1.6x10−3 0.83 0.89 1.79(1.26–2.56) 1.6x10−3 https://doi.org/10.1371/journal.pntd.0006202.t001 PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 4/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Fig2. ManhattanplotsofBMIXanalysisinVietnameseDSSvsControl(A),ThaiDSSvsControl(B)andThaiDFvsControl(C)forNortheastandSoutheast Asianancestries.Theredlinerepresentsthesignificancethreshold.TheproteincodinggeneswithsignificantlyassociatedSNPsareidentified. https://doi.org/10.1371/journal.pntd.0006202.g002 0.013)israreinmostworldwidepopulations(Fig3A),reachingthehighestfrequenciesin Africa(0.21–0.28).OnlyonePLCE1SNP(rs2274223)waspresentinthechipusedintheThai denguecohortanditdidnotreachsignificance. Individually,theconventionalassociationstudywithPCAcorrectionforpopulationstrati- ficationinThaiDSSvs.controlcouldnotidentifyanycandidategenewhencorrectingfor multipletest(S6AFig–thetwosingledoutsignificantSNPsarespurioussignalsaslinkedSNPs donotdisplaysignificantp-values).Wealsotested10runsofpseudodatasets,permutating caseandcontrollabels(S1Text).NoSNPissignificantintheassociationtests,andtheBMIX PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 5/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Fig3.Worldwide(fromthe1000Genomesdatabase)andThaidenguecohorts(control,DFandDSS)frequencies forsignificantlyassociatedhaplotypesinthevariousgenes.A-PLCB4;B-PLCE1;C-MICB;D-CHST10;E-AHRR; F-GRIP1;G-PPP2R5E.Theprotectiveandcausativehaplotypesarehighlighted. https://doi.org/10.1371/journal.pntd.0006202.g003 algorithmidentifiedspurioussignificantSNPs(mostlyisolatedindifferentchromosomes)that donotreplicatebetweenrunsandthataredifferentfromthecase-controlcomparison.Overall posteriorp-valueswerealsolowerinthepseudodatasets.Thehigherspuriousdetectionsin BMIXthanintheassociationtestagreewiththefactthatthestatisticalburdenofthelocal ancestrytestisconsiderablylowerthantheonefortheassociationtest,whichraisesthepossi- bilityofdetectingapositivesignal.Therandomnessbetweenrunsreflectsthehighvariability betweenindividualsinadmixturepercentagesandindistributionofancestryblocksalongthe genomes.Thisarguesforadouble-carefulinterpretationofBMIXresultsinthecontextofthe disease.FortheThaiDSSvs.control,thefactthatthePLCB4genebelongstothesamefamily ofthepreviouslyindependentlyidentifiedPLCE1geneisanimportantadditionalevidencefor consideringthatgeneastrongcandidateinDSSphenotype. CalculatingthegeneticriskofDSSaccordingtotheworldwidepopulationfrequencyofthe phospholipaseCandMICBprotectiveandsusceptiblehaplotypes(Fig4A),itcanbeobserved thatAfricananddescendentCaribbeanpopulationsarebestprotected,whileEuropean,Asian andLatinAmericanpopulationsaremoresusceptibletoDSS. PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 6/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Fig4. Geneticriskforthevariousworldwideregionsbyconsideringanadditivemodelofprotectiveandcausativehaplotypes/SNPsforDSS(A)andDF(B). Median(middleline),mean(littlesquare),95%confidenceinterval(whiskers)andextremevalues(crosses)areindicated. https://doi.org/10.1371/journal.pntd.0006202.g004 WegenotypedtwooftheBMIX-identifiedsignificantlyassociatedPLCB4SNPsinfurther Thaicontrol(n=244)andcase(n=20)samples(S7Table),andthers1997696SNPpresentsa p-valueoverthesignificancethresholdofatraditionalGWAS(p=4.7x10-8). Denguefevercohortanalysis WhencomparingThaiDFvs.control,adistinctivegeneticsignaturewasobtained.Threegenes locatedondifferentchromosomeshadatleasttwoSNPsabovetheBMIXsignificantposterior probabilitythresholdof0.5(Fig2C),forminghaplotypes(S7,S8andS9Figs).CHST10codes forcarbohydratesulfotransferase10(S4CFig),hasninesignificantSNPs(Table1,S3andS6 Tables),formingtheprotectivehaplotypeCTCTACGGG(OR=0.59;p-value=0.0005), whereasthehaplotypeTGTCGTAATincreasedriskofDF(OR=1.78;p-value<0.0001).The protectivehaplotypeisfrequentinSouthAsianpopulations(0.38–0.57),whereasthesusceptible haplotypeisfrequentinNortheastAsia(0.61–0.74)andveryrareintheAfricanpopulations (Fig3D).AHRR(S4DFig)codesforaryl-hydrocarbonreceptor(AHR)repressor,hastwosignif- icantSNPs,andsimilarlytoCHST10,theprotectiveAHRRhaplotype(TC—OR=0.54;p- value=0.0001)ismorefrequentinSouthAsianandAfricanpopulations(between0.40–0.60) andtheoppositehaplotype(CT—OR=1.89;p-value<0.0001)ismorefrequentinNortheast Asianpopulations(0.57–0.78)(Fig3E).PPP2R5E(S4FFig)codesforproteinphosphatase2 (PP2A),regulatorysubunitB’,epsilonisoform(alsoknownasPP2A-B56),hasfoursignificant SNPs,whoseprotectivehaplotype(ACGG—OR=0.62;p-value=0.0002)showedhighfre- quencyinSouthAsianpopulations(0.76–0.86),whileAfricanpopulationshavethelowestfre- quencyofthishaplotype(0.09–0.20)(Fig3G).Interestingly,theproteinscodedbythesethree genes,andbyanothergene,GRIP1(S4EFig)thatcodesforglutamatereceptorinteractingpro- tein1,withonesignificantSNP(T;OR=0.56;p-value=0.0016),areinvolvedinthexenobiotic metabolismsignalingpathway(Fig5D).TheGRIP1protectivealleleismorefrequentinSouth Asianpopulations(0.19–0.32)andabsentinAfrica(Fig3F). Again,individually,theconventionalassociationstudywithPCAcorrectionforpopulation stratificationcouldnotidentifyanycandidategenewhencorrectingformultipletest(S6B Fig). PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 7/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Fig5. SignificantlyalteredgeneexpressionforPPP2R5E(A),GRIP1(B)andAHR(C)inThaidenguecohortalongthecourseofdiseasefroma transcriptomedatasetforwholeblood[17].Significantp-valuesareindicated.D)Schemeofthexenobioticmetabolismsignallingpathway(basedonIngenuity databaseinformation),highlightingthethreenucleartranscriptionfactors:theconstitutiveactivereceptor(CAR);thepregnaneXreceptor(PXR);andthearyl hydrocarbonreceptor(AHR). https://doi.org/10.1371/journal.pntd.0006202.g005 ThecontributiontothegeneticrisktoDF,inferredfromanadditivemodelcombiningthe protectiveandsusceptiblehaplotypesofthefourxenobiotic-relatedgenes(Fig4B),indicates highestprotectioninEuropeanandSouthAsianpopulationsandhighestriskinNortheast andSoutheastAsians.AfricanandLatinAmerican/Caribbeanpopulationshaveanintermedi- ateriskconferredbythesegenestoDF. ThegenotypingofsixSNPsinthesefourgenesinadditionalThaicontrols(n=245)and cases(n=55)improvesp-valuesofatraditionalassociationtestinthetotalcohorttolevelsof 10−5infourSNPsand10−4intwoSNPs(S7Table).ThesevaluesaresignificantafterBonfer- ronicorrectionfortheset-testofsixSNPs. PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 8/20 A Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome Weanalyzedtheexpressionofthesegenesinthexenobioticpathwayinatranscriptome datasetincludingpatientssampledduringacutephaseofDF,DHFandconvalescencecom- paredwithcontrols[17].CHST10andAHRRexpressionsdidnotsignificantlychangeduring dengueinfection(S10Fig),however,therewasasignificantincreaseinPPP2R5Eexpression andasignificantdecreaseinAHR(negativelyregulatedbyAHRR)andGRIP1expressionsdur- ingacutedengueinfection(Fig5A,5Band5C).Thesefindingsarefurtherevidencethat PPP2R5E,GRIP1andAHRcanbeinvolvedindengueinfectionanddevelopmentofdengue disease.WefurthercheckedintheGTExdatabaseiftheDFcandidateSNPsactaseQTLs. AllcandidateprotectiveallelesinPPP2R5EandAHRRgenessignificantlyreducetheexpres- sionoftherespectiveproteins(S11Fig).ThecandidateSNPinGRIP1geneisnotaneQTL intheGTExcohort,andthetwoeQTLs(rs11176317andrs12322014)closetothecandidate rs1480010arenotinLDwithit.AstheGTExcohortismainlyofEuropeanancestry,wecan- notascertainifthisGRIP1SNPorotherlinkedSNPscanbeeQTLsinAsianpopulations. Immunofluorescenceco-localizationimagingofPPP2R5EandNS5protein fromDENV1and2 TherecentidentificationofconservedmotifsthatprovidebindingspecificitytothePP2A-B56 phosphatase[18]ledustofurthertestthehypothesisofthepotentialbindingofthisregulatory regionofPP2AproteintoDENVproteins.Webeganbyperforminganinsilicosearch[19]for thehigh-affinityLxxIxEmotifaswellastheintermediate-andlow-affinitymotifsinthepro- teinreferencesequencesofthefourDENVserotypes(S8TableandFig6A).NS5presents betweenthreeandsixmotifsinallfourDENVserotypes,andatleasttwoofthesemotifs (LxxIxEandLxxVxE)arehighlyconserved.Otherviralproteinsalsobearmotifs,butaremore heterogeneousbetweenDENVserotypes. WethentestedthehypothesisthatPP2A-B56caninteractwithNS5byconductingconfocal immunofluorescenceco-localizationtests.WetransfectedHuh7cellswithamammalian expressionplasmidcontainingDENV2-NS5taggedwithanorangefluorescenceprotein.We fixedandstainedwithantibodyagainstPPP2R5Eat24h,48hand72haftertransfection.In non-transfectedcells,PPP2R5Eislocalizedinthecytoplasm(Fig6B).At24hofpost-transfec- tion,bothPPP2R5EandNS5arelocalizedinthecytoplasm,butby48htheybothco-localize inthenucleus,andat72hPPP2R5EreturnstothecytoplasmwhileNS5remainsinthenucleus (Fig6C).WethendeletedthexLxxIxEmotifinourDENV2-NS5vector(Fig6D)andtrans- fectedcellsinthesameway.Thedeletionofthismotifpreventedthetranslocationoftheviral NS5proteintothenucleus(Fig6D). Testifyingtotheexistenceofdifferencesbetweenserotypes,theimmunofluorescenceco- localizationtestbetweenPPP2R5EandDENV1-NS5(Fig6E)showedthatthetwoproteins co-localizeinthecytoplasm,buttheentranceinthenucleusisalmostnegligible,andlittle accumulationofNS5canbedetectedinthenucleusat72h. Discussion Oursuccessfulassociation-admixtureanalysesinThaipopulationhaveprovidedevidencethat differentgenes/pathwayscontributetothegeneticsusceptibilityorresistancetodifferentout- comeofdengueinfection.Wesuggestthatxenobioticsandlipidmetabolism,aswellasinterac- tionofviralproteinstothesemoleculesandtoitsphosphatases,arecriticalinthedevelopment ofclassicalDF,whereasmoresevereformsofdenguearecausedbyoverreactiveimmunity leadingtocytokinestormand/ordefectinendothelialcelldysfunctionandcoagulation system. PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 9/20

Title Joint ancestry and association test indicate two distinct pathogenic pathways involved in

Similar Title Joint ancestry and association test indicate two distinct pathogenic pathways involved in