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

20 Pages·2017·6.43 MB·English

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,Vero´nicaFernandes1,2,
AmpaiwanChuansumrit6,OrlandoAnunciac¸ão2,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—InstitutodeInvestigac¸ãoeInovac¸ãoemSau´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´deRechercheAssocie´e3012,Paris,
FernandesV,ChuansumritA,Anunciac¸ãoO,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.
*[email protected](LP);[email protected](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
Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
financedbyFEDER—FundoEuropeude bestprotectedagainstDSSandintermediatelyprotectedagainstDF,andEuropeansthe
DesenvolvimentoRegionalfundsthroughthe bestprotectedagainstDFbutthemostsusceptibleagainstDSS.
COMPETE2020—Competitivenessand
InternationalizationOperationalProgramme
(POCI),Portugal2020,andbyPortuguesefunds
throughFCT/Ministe´riodaCiência,Tecnologiae
Inovac¸ãointheframeworkoftheproject"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].
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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.
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Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
Fig1.GlobalancestryinferredthroughRFMixwhenusingthreeparentalancestries(South,Northeastand
SoutheastAsian)fortheglobaldataset.Eachverticallinerepresentsanindividual,andthethreecoloursrepresent
theproportionofthethreeparentalpopulationsineachgenome(lightorangeforSouthAsian,darkorangefor
SoutheastAsianandblueforNortheastAsian).
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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
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Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
Fig2. ManhattanplotsofBMIXanalysisinVietnameseDSSvsControl(A),ThaiDSSvsControl(B)andThaiDFvsControl(C)forNortheastandSoutheast
Asianancestries.Theredlinerepresentsthesignificancethreshold.TheproteincodinggeneswithsignificantlyassociatedSNPsareidentified.
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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
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Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
Fig3.Worldwide(fromthe1000Genomesdatabase)andThaidenguecohorts(control,DFandDSS)frequencies
forsignificantlyassociatedhaplotypesinthevariousgenes.A-PLCB4;B-PLCE1;C-MICB;D-CHST10;E-AHRR;
F-GRIP1;G-PPP2R5E.Theprotectiveandcausativehaplotypesarehighlighted.
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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.
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Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
Fig4. Geneticriskforthevariousworldwideregionsbyconsideringanadditivemodelofprotectiveandcausativehaplotypes/SNPsforDSS(A)andDF(B).
Median(middleline),mean(littlesquare),95%confidenceinterval(whiskers)andextremevalues(crosses)areindicated.
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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).
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Fig5. SignificantlyalteredgeneexpressionforPPP2R5E(A),GRIP1(B)andAHR(C)inThaidenguecohortalongthecourseofdiseasefroma
transcriptomedatasetforwholeblood[17].Significantp-valuesareindicated.D)Schemeofthexenobioticmetabolismsignallingpathway(basedonIngenuity
databaseinformation),highlightingthethreenucleartranscriptionfactors:theconstitutiveactivereceptor(CAR);thepregnaneXreceptor(PXR);andthearyl
hydrocarbonreceptor(AHR).
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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.
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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
Independentpathogenesesandcandidategenesindenguefeveranddengueshocksyndrome
Fig6.ConfocalimagingofPPP2R5EandNS5fromDENV1andDENV2.A.ThemainlinkmotifsinNS5proteinsfromDENV1andDENV2.B.Subcellular
localizationofPPP2R5EinHuh7controlcells.C.SubcellularlocalizationofPPP2R5EinHuh7controlandafter24h,48hand72hoftransfectionwithDENV2-NS5
protein.D.SubcellularlocalizationofmutatedPPP2R5EinHuh7controlandafter24h,48hand72hoftransfectionwithDENV2-NS5protein.E.Subcellular
localizationofPPP2R5EinHuh7controlandafter24h,48hand72hoftransfectionwithDENV1-NS5protein.GreenimmunofluorescenceindicatesPPP2R5E,red
indicatesNS5,blueflagsnucleus.Yellowsignalsindicateco-localizationofNS5andPPP2R5E,andwasobtainedbyoverlappingthetwopanels.Original
magnification,×630.
https://doi.org/10.1371/journal.pntd.0006202.g006
WereinforcedtheassociationofphospholipaseCgenefamilywiththeDSSphenotypein
Thaipatients,ashadbeenfoundinVietnameseDSSpatients[3],butthistimethegene
detectedwasPLCB4insteadofPLCE1.Theseenzymeshavebeenimplicatedinahighnumber
ofsignaltransductionpathways[19]andonimmuneregulation[20].Morerecently,Linetal.
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006202 February15,2018 10/20