Genome-wide association and HLA region fine-mapping ... · Genome-wide association and HLA region...

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Genome-wideassociationandHLAregionfine-mappingstudiesidentify

susceptibilitylociformultiplecommoninfections

ChaoTian,1*BethannSHromatka,1AmyKKiefer,1NicholasEriksson,1JoyceYTung,1DavidA.

Hinds,1**123andMe,MountainView,California,UnitedStatesofAmerica.

*CorrespondingauthorsEmailaddress:*CT:[email protected]**DAH:[email protected]

.CC-BY-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted September 9, 2016. . https://doi.org/10.1101/073056doi: bioRxiv preprint

https://doi.org/10.1101/073056

http://creativecommons.org/licenses/by-nd/4.0/

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ABSTRACT

Weperformed23genome-wideassociationstudiesforcommoninfections,including

chickenpox,shingles,coldsores,mononucleosis,mumps,hepatitisB,plantarwarts,

positivetuberculosistestresults,strepthroat,scarletfever,pneumonia,bacterial

meningitis,yeastinfections,urinarytractinfections,tonsillectomy,childhoodear

infections,myringotomy,measles,hepatitisA,rheumaticfever,commoncolds,rubellaand

chronicsinusinfection,inmorethan200,000individualsofEuropeanancestry.Forthe

firsttime,genome-widesignificantassociations(P<5x10-8)wereidentifiedformany

commoninfections.Theassociationsweremappedtogeneswithkeyrolesinacquiredand

innateimmunity(HLA,IFNA21,FUT2,ST3GAL4,ABO,IFNL4,LCE3E,DSG1,LTBR,MTMR3,

TNFRSF13B,TNFSF13B,NFKB1,CD40)andinregulationofembryonicdevelopmental

process(TBX1,FGF,FOXA1andFOXN1).Severalmissensemutationswerealsoidentified

(inLCE5A,DSG1,FUT2,TBX1,CDHR3,PLG,TNFRSF13B,FOXA1,SH2B3,ST5andFOXN1).

MissensemutationsinFUT2andTBX1wereimplicatedinmultipleinfections.Weapplied

fine-mappinganalysistodissectassociationsinthehumanleukocyteantigenregion,which

suggestedimportantrolesofspecificaminoacidpolymorphismsintheantigen-binding

clefts.Ourfindingsprovideanimportantsteptowarddissectingthehostgenetic

architectureofresponsetocommoninfections.


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Introduction

Infectiousdiseases,thesecondleadingcauseofdeathworldwide,representpersistent

challengestohumanhealthduetoincreasingresistancetoestablishedtreatments,lackof

life-savingvaccinesandmedicationsindevelopingcountries,andincreasingdistribution1.

Studieshavelinkedsusceptibilitytoinfectiousagentstocancers,autoimmunediseases,

anddrughypersensitivity.Humanpapillomavirusesareassociatedwithmultiplecancers2;

rubellaandmumpsinfectionhavebeenlinkedtodevelopmentoftype1diabetes(T1D)in

children3;andriskofdevelopingmultiplesclerosis(MS)wasgreateramongpatientswith

herpeszoster(shingles)thaninmatchedcontrols4.Reactivationofchronicpersistent

humanherpesviruseshasbeenlinkedtodrug-inducedhypersensitivity5.Thus,infectious

diseaseshaveaprofoundimpactonourhealth,bothdirectlyaswellasthrough

connectionswithotherdiseases.Nevertheless,geneticstudiesofcommoninfectious

diseaseslagsomewhatbehindthoseofothermajorcomplexdiseases.Fewgenome-wide

associationstudies(GWAS)havebeenundertakenforcommoninfectiousdiseasesoflower

mortalityorforinfectiousdiseasesforwhichvaccinesareavailable6.

Inthisstudy,weconducted23imputedGWASswithmorethan200,000European

researchparticipantswhoweregenotypedonthe23andMeplatformandwereaskedto

reportontheirhistoryofinfections(Table1andSupplementarynote).Classicalhuman

leukocyteantigen(HLA)locihavebeenprototypicalcandidategeneticsusceptibilityto

multipleinfectiousdiseases7.WefurtherimputedandtestedtheHLAclassicalallelesand

aminoacidpolymorphismstodissectindependentHLAsignalsformultiplecommon

infectionsthathavesignificantHLAassociationsinourGWASs.


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Results

Intotal,59genome-widesignificant(GWS)regions(P<5x10-8)werediscovered(Table

2,seealsoSupplementaryFigure1andSupplementaryFigure2forGWASManhattanplots

andregionalplots).TheHLAregionissignificantlyassociatedwith13oftheinfectionsthat

westudiedandwedissectedindependentsignalsineachassociation(Figure1andTable3,

seealsoSupplementaryFigure3forHLAregionalplots).Inthelatersections,wedescribe

theresultforeachphenotypeindetail.

Geneticcorrelations

ThepairwiseLDscorecorrelationonGWASstatistics(Figure2,SupplementaryTable2)

showedsignificantpositivegeneticcorrelationsbetweenstrepthroatandtonsillectomy(rg

=0.85,se=0.09,P=1.89x10-21),betweenchildhoodearinfectionandmyringotomy(rg=

0.88,se=0.14,P=1.27x10-9),andbetweenchickenpoxandshingles(rg=0.56,se=0.16,P

=4x10-4),whicharepairsofrelatedphenotypes.Ahighlysignificantgeneticcorrelation

wasfoundbetweentonsillectomyandchildhoodearinfections(rg=0.42,se=0.05,P=8.19

x10-15).Tonsillitisandearinfectionsaresometimescomorbidsymptoms.Aprevioustwin

studysuggestedasubstantialoverlapingeneticfactorsinfluencingvariationsinliabilityto

earinfectionandtonsillitis8.InourGWASs,weidentifiedthemissensemutation(N397H)

inTBX1asasignificantsusceptibilitylocusforboth.Significantpositivecorrelationswere

alsoobservedforpneumoniawithchronicsinusinfections(rg=0.72,se=0.19,P=1.0x

10-4),childhoodearinfections(rg=0.39,se=0.07,P=1.47x10-8),coldslastyear(rg=

0.57,se=0.12,P=2.66x10-6)andstrepthroats(rg=0.69,se=0.13,P=1.0x10-7).These

areallupperrespiratoryinfections.Thesamevirusesthatcausecoldsandsorethroat(if

theseinfectthethroat,sinusesandupperrespiratorytract)canalsocausepneumonia(if

thesereachthelungs).Wealsosawahighgeneticcorrelationbetweenurinarytract


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infection(UTI)andyeastinfections(rg=0.68,se=0.08,P=4.30x10-16).Therewasa

phenotypiccorrelation(Pearsonr=0.41,P<2.2x10-16)amongwomenwhoreportedboth

UTIandyeastinfectionsinourcohort.Thesetwoinfectionscanhavesimilarsymptoms

andbothcausediscomfortinvaginalarea,butdifferentinfectiousagentscausethem.The

geneticcorrelationfromLDscoreregressionremovesthecorrelationinGWASsummary

statisticsduetothecorrelationinphenotypesandthussuggestsasharedhostgenetic

susceptibilitytoUTIandyeastinfectionsinfemales.

Chickenpox(Varicella-zostervirus,Herpesvirusesfamily)

Chickenpox,characterizedbyred,itchybumpsontheskin,isahighlycontagiousdisease

causedbyprimaryinfectionwithvaricella-zostervirus(VZV).Aftertheinitialchickenpox

infection,thevirusremainsdormantinthenervoussystem,thoughinapproximately20%

ofpeopleitreactivatesandmanifestsasshingles(seebelow).Weidentifiedindependent

associationsinHLA-AandHLA–BintheclassIregion.OnconditionalanalysiswithinHLA-

A,theaminoacidpolymorphismHLA-A-Gly107(P=3.77x10-10)accountedfortheHLA

alleleassociation(HLA-A*02:01,P=1.08x10-9,conditionalp-valueis0.90)inthisinterval.

AlthoughHLA-A-Gly107isnotlocatedinthepeptide-bindingcleft,itisinhighLDwith

HLA-A-Asp74,HLA-A-Gly62andHLA-A-V95(r2>0.95),whichareallinthepeptide-binding

cleft.InHLA-B,onlyrs9266089(P=1.00x10-10)metthethresholdforGWS.

Shingles(Varicella-zostervirus,Herpesvirusesfamily)

Shingles,alsoknownasherpeszoster,ischaracterizedbyapainfulband-shapedrash9

andiscausedbyreactivationoftheVZV(seeabove).Weidentifiedmultipleindependent

HLAsignalsforshingles.WithinHLA-A,theaminoacidpolymorphismHLA-A-Arg97(P=

2.75x10-22),locatedinthepeptide-bindinggroove,accountedformostoftheSNPeffect

(rs2523815,P=1.83x10-22,conditionalp-valueis0.04)andtheHLAalleleeffect(HLA-


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A*02:01,P=8.91x10-20,conditionalp-valueis0.06).HLA-A*02:01isimplicatedinboth

ourchickenpoxandshinglesGWASs.Theaminoacidassociationsaredifferent,butHLA-A-

Arg97isinhighLDwithHLA-A-Gly107(r2=0.73).WithinHLA-B,rs2523591(P=1.74x

10-27)hadastrongerassociationthanHLAclassicalvariants.Therewasasignificant

residualeffectforrs2523591afterconditioningonHLAallele(conditionalp-valueis1.71x

10-9)oraminoacid(conditionalp-valueis3.13x10-5)associationsinHLA-Bregion.The

SNPrs2523591isinLDwithrs9266089(r2=0.24,D’=0.98),whichisaGWSsignalin

chickenpoxGWAS,suggestingpotentialsharedgeneticfactorsforchickenpoxandshingles.

AfteradjustingforthesignalsinHLA-AandHLA-Bregions,weobservedindependent

secondarysignalsintheclassIIIregion(rs41316748,P=1.44x10-12)andintheclassII

regionindexbyDRB1-PheSerHis13(P=5.9x10-10),whichisinthepeptide-bindingcleftof

HLA-DRB1.

WeidentifiedavariantupstreamofIFNA21(rs7047299,P=1.67x10-8)asaGWS

associationwithshingles.Noneofthevariantswithin500kbandinmoderateLD(r2>0.6)

withtheindexSNPrs7047299werecoding,norweretheyreportedasexpression

quantitativetraitloci(eQTL).IFNA21,amemberofthealphainterferongeneclusterat

band9p21,encodedtypeIinterferonandismainlyinvolvedininnateimmuneresponse

againstviralinfection.Ithavebeenshowntobeinvolvedinthepathogenesisofrubella10,

andmayalsoinfluencesusceptibilitytoasthmaandatopy11.

Coldsores(type1herpessimplexvirus,Herpesvirusesfamily)

OverhalfoftheUSpopulationsuffersfromcoldsores(herpessimplexlabialis),which

aremostcommonlycausedbytheherpessimplexvirustypeI(HSV-1)12.Oncesomeone

hasbeeninfected,thevirususuallycannotbeeliminatedandliesdormantinnervecells

whereitmayreactivateyearslater.AlthoughmostpeopleareinfectedwithHSVatsome


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pointintheirlives,itisnotclearwhyonlysomepeoplesufferfromcoldsoreoutbreaksand

reactivation13.WeidentifiedtwoindependentGWSSNPassociationsintheHLAclassI

region.Oneis2kbupstreamofPOU5F1indexedbyrs885950(P=7.47x10-13)andthe

otherisupstreamofHCP5indexedbyrs4360170(P=3.41x10-9).WealsofoundaGWS

associationwithHLA-B-ThrGly45inthepeptide-bindingcleftoftheHLA-Bprotein(P=

4.91x10-12).UponconditioningonHLA-B-ThrGly45,thetwoSNPshadsignificantresidual

effects(conditionalp-valuesare6.28x10-9and1.32x10-5).However,theeffectofHLA-B-

ThrGly45waslargelyremoved(conditionalp-valueis0.001)afterconditioningonthetwo

SNPs.

Mononucleosis(Epstein-Barrvirus,Herpesvirusfamily)

Over90%oftheworld’sadultpopulationischronicallyinfectedwithEpstein-Barrvirus

(EBV).Primaryinfectionatchildhoodisusuallyasymptomaticorwithonlymildsymptoms.

Primaryinfectionlaterinlifeisoftenaccompaniedbyinfectiousmononucleosis(IM)14,

commonlyreferredtoas“mono”andcharacterizedbyfever,tonsillitis,andfatigue.A

longitudinalstudyofIMinacohortof2,823,583Danishchildrenfoundevidenceoffamilial

aggregationofIMthatwarrantsGWASonIMdiseaseetiology14.WeidentifiedtheHLA

classIregionupstreamofHCP5asaGWSassociation(indexedbyrs2596465,P=2.48x10-

9).NoGWSHLAalleleoraminoacidvariantwasidentified.

Mumps(mumpsvirus)

Mumpsisanillnesscausedbythemumpsvirus.Thereisnodiseasemodifying

treatmentformumpsinfection.Itisararediseaseindevelopedcountriessincethe

introductionofroutinevaccination;however,outbreakscanstilloccurandmumpsremains

asignificanthealththreatindevelopingcountries15.WeidentifiedfourindependentGWS

associationswithmumps.


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VariationinHLA-A,intheclassIregion,wassignificantlyassociatedwithmumps.The

aminoacidpolymorphismHLA-A-Gln43(P=2.51x10-17)accountedformostoftheSNP

association(rs114193679,P=2.23x10-17,conditionalp-valueis0.19)andtheHLAallele

association(HLA-A*02:05,P=1.73x10-17,conditionalp-valueis0.26)intheHLA-Aregion.

WeidentifiedFUT2(indexedbyrs516316,P=9.63x10-72)andST3GAL4(indexedby

rs3862630,P=1.21x10-8)ashighlysignificantassociationswithmumps.Bothbelongto

theglycosphingolipid(GSL)biosynthesispathway,whichwasthemostsignificant

MAGENTA-analyzedpathway(Table4:P=1x10-4andFDR=0.003)identifiedusing

mumpsGWASdata.ThispathwayalsoincludestheABOgene(indexedbyrs8176643,P=

5.81x10-5).Glycosphingolipids(GSLs),foundinthecellmembranesofbacteriaalltheway

tohumans,playimportantbiologicalrolesinmembranestructure,host-pathogen

interactions,cell-cellrecognition,andmodulationofmembraneproteinfunction.Genetic

variationinthehumanFUT2genedetermineswhetherABObloodgroupantigensare

secretedintobodyfluids16.Detailedfunctionalannotationsofthegenesidentifiedinour

mumpsGWASareprovidedinSupplementarytable3.Thehigh-riskallelers516316(C)is

incompleteLDwithrs601338(A),anonsensevariantinFUT2thatencodesthe“non-

secretor”(se)allele,whichhasbeenreportedtoprovideresistancetoNorovirus17and

susceptibilitytoCrohn’sdisease18,19andT1D20.Thesurfaceglycoproteinofmumpsvirus,

hemagglutinin-neuraminidase,attachestosialicacidreceptorsandpromotesfusionand

viralentryintohostcells21.ABOantigenscanmodulatetheinteractionbetweenpathogens

andcellsurfacesialicacidreceptors22;thispresentsaplausiblemechanismbywhich

secretorstatuscouldmodifysusceptibilitytomumpsinfections.ST3GAL4encodesa

memberoftheglycosyltransferase29family,agroupofenzymesinvolvedinprotein

glycosylation.


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Wealsoidentifiedasignificantassociationbetweenmumpsandavarianton

chromosome14(rs11160318,P=4.56x10-12)thatislocatedintheintergenicregion

upstreamofBDKRB2.TheroleofBDKRB2inmumpssusceptibilityisnotclear,butthe

BDKRB2producthashighaffinityforintactkinins,whichmediateawidespectrumof

biologicaleffectsincludinginflammation,pain,vasodilation,andsmoothmuscle

contractionandrelaxation23.

HepatitisB(hepatitisBvirus)

ChronichepatitisBvirus(HBV)infectionisachallengingglobalhealthproblem.Several

GWASshavebeencarriedouttoidentifygeneticlociinvolvedinHBVsusceptibilityand

immuneresponsetohepatitisBvaccine,andtheHLA-DRB1-HLA-DQB1regionhasbeen

consistentlyshowntobeGWS24–26.InourGWASofHBVinfectionweidentifiedaGWS

associationwithrs9268652(P=3.14x10-9,inHLA-DRA),consistentwiththeassociation

oftheHLAclassIIregionwithHBVsusceptibility.Finemappinganalysisfailedtoidentify

GWSassociationswithHLAclassicalvariants,butDQB1*06:02wasmoderatelyassociated

withhepatitisB(P=5.47x10-6).HLA-DQB1*06:02isprotectiveagainstHBVinourdata,

whichisconsistentwithpreviousstudiesthattheDRB1*15:01-DQB1*06:02haplotypeis

associatedwithprotectionfromtype1autoimmunehepatitisanddevelopmentof

hepatocellularcarcinoma27.AlthoughchronicinfectionwiththeHBVhasbeenlinkedtothe

developmentofhepatocellularcarcinomaformorethan30years28,themechanismby

whichHBVinfectionleadstohepatocellularcarcinomaisunclear.

HepatitisA(hepatitisAvirus)

InfectionwithhepatitisAvirus(HAV)canbeasymptomaticorresultinanacuteillness

characterizedbyflu-likesymptomsandjaundice.Seriouscomplicationsarerareandthe

hepatitisAvaccineiseffectiveforprevention29.Nolargecohortstudyhasbeendonefor


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HAVinfection.OurGWASshowedasuggestiveassociationwithIFNL4,indexedby

rs66531907(P=5.7x10-8,OR=1.23,1kbupstreamofIFNL4).IFNL4islocatedupstream

ofIFNL3(alsoknownasIL28B).Thehigh-riskallelers66531907(C)inourhepatitisA

GWASisinalmostcompleteLDwithrs8099917(T)(r2=0.992),whichhasbeenassociated

withprogressiontochronichepatitisCinfection(HCV)andpoorresponsetoHCVtherapy

inmultiplestudies30.

Plantarwarts(humanpapillomavirus)

Thehumanpapillomavirus(HPV)causesplantarwarts,whichoccuronthesolesofthe

feet.SomestrainsofHPVarealsoassociatedwithcertainformsofcancer2.Weidentified

twoGWSassociationswithplantarwart.OurfirstassociationwaswiththeHLA-DRB1in

classIIregion.TheaminoacidpolymorphismDRB1_PheSerTyr13(P=1.84x10-39),inthe

peptidebindingcleftsofHLA-DRB1,washighlysignificant.Conditioningonit,theeffectsof

HLA-DRB1*15:01(P=4.48e-14,conditionalp-valueis0.36),HLA-DRB1*04:01(P=4.88x

10-18,conditionalp-valueis0.001),andrs9272050(P=9.66x10-31,conditionalp-valueis

0.06)werelargelyremoved,butasmallresidualeffectremainedforrs28752534(P=1.50

x10-30,conditionalp-valueis2.15x10-8).

WealsoidentifiedaGWSassociationwithrs6692209(P=5.25x10-9)nearLCE3Einthe

EpidermalDifferentiationComplex(EDC)on1q21.TheEDCcomprisesaremarkable

densityofgenefamiliesthatdeterminedifferentiationofthehumanepidermis31.Thehigh-

riskallelers6692209(T)isinhighLD(r2=0.86)withrs2105117(A)(P=2.98x10-8inour

GWAS),amissensevariantinLCE5A.Variantsinlatecornifiedenvelope(LCE)geneshave

beenimplicatedinGWASsofatopicdermatitis(rs3126085-A,P=6x10-12,r2=0.047with

rs6692209)32andpsoriasis(rs4085613-A,P=7x10-30,r2=0.002withrs6692209)33,

althoughtheindexSNPsidentifiedinthoseGWASswereinlowLDwithouridentified


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region.Theskinistheprimaryinterfacewiththeexternalenvironmentandprovidesa

criticalfirstbarrieroftheinnateimmunedefensetoinfections34.Whilestudieshavelinked

theepidermaldifferentiationgenestovariousskindiseases,theroleoftheLCEproteinsin

epidermalbiologyhasnotbeenextensivelystudied.OnestudysuggestedthatLCEproteins

ofgroups1,2,5and6areinvolvedinnormalskinbarrierfunction,whileLCE3genes

encodeproteinsthatareinvolvedinbarrierrepairafterinjuryorinflammation35.

PositiveTBtest(Mycobacteriumtuberculosis)

Tuberculosisisacommonand,inmanycases,lethalinfectiousdiseasecausedbyvarious

strainsofmycobacteria,usuallyMycobacteriumtuberculosis.Achestx-rayandasampleof

sputumareneededforadiagnosisofactiveTBdisease36.ThephenotypeusedinourGWAS

wasdefinedbyaskingcustomerswhethertheyhadhadapositiveresponse(redbumps)to

atuberculosis(TB)skintest.ApositiveTBtestindicatesthatapersonhasatsometime

beenexposedtoandinfectedwithTBbacteria.AnegativeTBtestindicatesthatlatentTB

infectionorTBdiseaseisunlikely.InourGWASofpositiveTBtests,thecaseshaveeither

activeTBdisease,latenttuberculosisinfections,orwerevaccinatedagainstTB,while

controlsmayormaynothavebeenexposedtoTBbacteriaandhaveneverbeeninfected.

WeidentifiedmultipleindependentHLAassociations.InthemostsignificantHLA-DRA-

DQA1region,conditioningonHLA-DRB1-Leu67(P=1.14x10-29)andHLA-DQA1-His129

(P=1.56x10-13)removedtheeffectofDQA1*01:02(P=7.04x10-22,conditionalp-valueis

0.54),DQA1*03:01(P=3.99x10-10,conditionalp-valueis0.36),andrs3135359(P=8.82x

10-21,conditionalp-valueis0.06);however,theeffectofrs2894257(P=8.16x10-36,

conditionalp-valueis1.2x10-7)remainedsignificant.Uponconditioningonthesignalsin

theHLA-DRA-DQA1region,secondarysignalsweredetectedintheclassI(HLA-B*08:01,

P=8.43x10-14)andclassIIIregions(rs148844907inC6orf47,P=2.21x10-12).


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Strepthroat(groupAstreptococcusbacteria)

Strepthroat(alsocalledstreptococcaltonsillitisorstreptococcalpharyngitis)is

characterizedbyfever,headache,severesorethroat,swollentonsilsandlymphnodes,and

iscausedbygroupAstreptococcus(GAS)bacteria37.Itisroutinelytreatedwithantibiotics.

Thesymptomsofuntreatedstrepthroattypicallyresolvewithinafewdays,butcan

sometimescausecomplicationssuchasrheumaticfeverandkidneyinflammation.We

foundaHLA-BclassIregion(indexSNPrs1055821,P=7.69x10-11)asaGWSassociation

withstrepthroat.HLA-B*57:01wasalmostGWS(P=5.06x10-8).HLA-DRB1_CysTyr30in

classIIregionalsoshowedasignificanteffect(P=2.14x10-9).Conditioningonrs1055821,

theeffectofHLA-B*57:01wasremoved(conditionalp-valueis0.84)andtheeffectofHLA-

DRB1_CysTyr30wassubstantiallydecreased(conditionalp-valueis0.005).

WealsofoundanGWSassociationwithasmallinsertion,rs35395352,in1p36.23(P=

3.90x10-8,effect=0.03).Noneofthevariantswithin500kbandinmoderateLD(r2>0.6)

withthisSNPwerecoding.However,twovariantshavebeenreportedascis-eQTLs:

rs7548511(P=7.84x10-8,effect=0.03,riskalleleA,r2=0.96withrs35395352)isan

eQTLforENO1(eQTLp-valueis8x10-5)inlymphoblastoid38andrs11121129(P=9.62x

10-8,effect=0.03,riskalleleG,r2=0.96withrs35395352)isaneQTLforTNFRSF9(eQTL

p-valueis4.4x10-4)inlymphoblastoid39.TheproductofENO1,Alphaenolase,hasbeen

identifiedasanautoantigeninHashimoto’sencephalopathy40andaputativeautoantigenin

severeasthma41andBehcet’sdisease42.TheTNFRSF9-encodedproteinisamemberofthe

TNF-receptorsuperfamily,whichcontributestotheclonalexpansion,survival,and

developmentofTcells.Thesame1p36.23regionhasbeenimplicatedinGWASsof

psoriasis(rs11121129,riskalleleA,P=1.7x10-8,r2=0.96withrs35395352)43.

Interestingly,thestrepthroatriskallelers11121129-Aisprotectiveagainstpsoriasis.


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Studiesfoundthatexacerbationofchronicpsoriasiscanbeassociatedwithstreptococcal

throatinfections,andtheT-cellsgeneratedbypalatinetonsilscanrecognizeskinkeratin

determinantsinpatients’blood44.

Scarletfever(groupAstreptococcusbacteria)

Scarletfever(alsoknownasscarlatina)iscausedbygroupAstreptococcus(GAS)andis

characterizedbyabrightredrashthatcoversmostofthebody,sorethroat,andfever.It

wasamajorcauseofdeathbeforethediscoveryofeffectiveantibiotics.WefoundaGWS

associationbetweenscarletfeverandtheHLAclassIIregion.Theaminoacid

polymorphismHLA-DQB1-Gly45(P=6.35x10-14),locatedinthepeptide-bindingcleftof

HLA-DQB1,accountedfortheSNPassociation(rs36205178,P=9.49x10-14,conditionalp-

valuesis0.83)andtheHLAalleleassociation(HLA-DQB1*03:01,P=2.98x10-14,

conditionalp-valuesis0.14)inthisregion.Althoughscarletfeverdevelopsinsomepeople

withstrepthroat,wedidnotobserveanycrossdiseaseeffectbetween‘scarletfever’and

‘strepthroat’inouranalysis.

Pneumonia(multipleorigins)

Pneumoniaischaracterizedbyinflammationofthealveoliinthelungsandcanbe

causedbyavarietyoforganisms,includingbacteria,viruses,andfungi.Themostcommon

causeofbacterialpneumoniainadults–isolatedinnearly50%ofcommunity-acquired

cases--isStreptococcuspneumonia45.WefoundaGWASassociationbetweenpneumonia

andaHLAclassIregionindexedbyrs3131623(P=1.99x10-15).Conditioningon

rs3131623,theresidualeffectsofHLA-B-SerTrpAsn97(P=2.37x10-12,conditionalp-value

is9.33x10-4)andHLA-DRB1-LS11(P=4.10x10-11,conditionalp-valueis3.89x10-6),both

inthepeptide-bindingcleft,remainedsignificant.Therewasalsoaresidualeffectleftfor


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rs3131623(conditionalp-valueis6.94x10-5)afterconditioningonthetwoaminoacid

associations.

Bacterialmeningitis(Streptococcuspneumoniae,GroupBStreptococcusbacteria

andmanyotherbacteria)

Bacterialmeningitisisasevereinfectioncausingsubstantialneurologicalmorbidityand

mortalityworldwide.Streptococcuspneumoniaeistheleadingcauseofbacterial

meningitisandisassociatedwitha30%mortalityrate46.WeidentifiedaGWSassociation

withCA10(carbonicanhydraseX)indexedbyrs1392935(P=1.19x10-8,intronicofCA10).

Noneofthevariantswithin500kbandinmoderateLD(r2>0.6)withrs1392935were

coding,norweretheyreportedasaneQTL.However,rs1392935fallswithinanenhancer

thatisdefinedinthefetalbrain.TheproteinencodedbyCA10isanacatalyticmemberof

thealpha-carbonicanhydrasesubgroupanditisthoughttoplayaroleinthecentral

nervoussystem,especiallyinbraindevelopment47.

Yeastinfection(Candidaspp.)

Ayeastinfection,alsocalledcandidiasis,isusuallyalocalizedfungalinfectionoftheskin

ormucosalmembranes,typicallyaffectingtheoralcavity,esophagus,gastrointestinaltract,

urinarybladder,toenails,orgenitalia48.Mostyeastinfectionsarecausedbyspeciesof

Candida,oftenCandidaalbicans.WefoundGWSassociationsbetweenyeastinfectionsand

variantsinDSG1(rs200520431,P=1.87x10-16).TheindexSNPrs200520431isintronic

andinhighLD(r2>0.8)withmultiplemissensemutations(rs8091003,rs8091117,

rs16961689,rs61730306,rs34302455)inDSG1.TheDSG1geneproductisacalcium-

bindingtransmembraneglycoproteincomponentofdesmosomesinvertebrateepithelial

cells.Itconnectsthecellsurfacetothekeratincytoskeletonandplaysakeyrolein

maintainingepidermalintegrityandbarrierfunction49.Thisglycoproteinhasbeen


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identifiedastheautoantigenoftheautoimmuneskinblisteringdiseasepemphigus

foliaceus50andhomozygousmutationsinDSG1havebeenshowedtoresultinsevere

dermatitis,multipleallergies,andmetabolicwastingsyndrome49.Avariantdownstreamof

PRKCH(indexedbyrs2251260,P=3.46x10-10)wasalsosignificantlyassociatedwith

yeastinfections.Noneofthevariantswithin500kbandinmoderateLD(r2>0.6)withthe

indexSNPrs2251260werecoding,norweretheyreportedasaneQTL.However,

rs2251260fallswithinstrongenhancersdefinedinhepatocellularcarcinoma.PRKCHisa

memberofafamilyofserine-andthreonine-specificproteinkinaseandispredominantly

expressedinepithelialtissues.ThePRKCHproteinkinasecanregulatekeratinocyte

differentiation51.Wealsofoundasignificantassociationwithavariantinthe14q32.2gene

desert(rs7161578-T,p=4.04x10-8).TheindexSNPisinhighLDwithmanyenhancer

sequencesdefinedinepidermalkeratinocytes.Avariantinthesameregion(rs7152623-A,

p=3x10-15,r2=(+)0.98withrs7161578-T)wasimplicatedinaorticstiffness52.

UrinaryTractInfection(Escherichiacoli,Staphylococcussaprophyticus,rarely

viralorfungal)

About80%-85%ofurinarytractinfections(UTIs)arecausedbyEscherichiacoli(E.

coli),and5-10%arecausedbyStaphylococcussaprophyticus53.Veryrarely,virusesor

fungimaycauseUTIs54.UTIsoccurmorecommonlyinwomenthanmen55andinour

dataset,significantlymorefemalethanmalecustomersreportedhavingtwoormoreUTIs.

WefoundanassociationbetweenUTIsandvariantsinPSCA(indexedbyrs2976388,P=

3.27x10-10).TheindexSNPrs2976388fallswithinstrongenhancersdefinedinepidermal

keratinocytes.ItisalsoinhighLD(r2=0.95)withrs2294008(P=1.01x10-9),whichisin

the5’-UTRpromotersequenceofPSCAandcancausechangesintranscriptionalrepressor

CTCFmotif.ThePSCAgenevariantsconferredriskofUTIonlyinfemalewhenwefurther


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testedtheeffectinfemaleandmalecohortseparately(P<1e-11infemale,andP>0.1in

male).PriorGWASshavefoundassociationsbetweenthePSCAgeneandduodenalulcer

(rs2294008-C,p=2e-33)andbladdercancer(rs2294008-T,p=4x10-11)56,57.PSCA

encodesaglycosylphosphatidylinositol-anchoredcellmembraneglycoproteinofunknown

function.Thisglycoproteinwasinitiallyidentifiedasaprostate-specificcell-surfacemarker

58andisoverexpressedinalargeproportionofprostatecancers,butalsodetectedin

bladderandpancreaticcancers57.

WealsoidentifiedavariantintronicofFRMD5(rs146906133,P=2.02x10-8)thatisin

LDwithrs138763871(r2=0.8,p=1.75x10-6inourGWAS),amissensevariantinSTRC

(R1521W).TheroleofFRMD5orSTRCinUTIsisunclear.

Tonsillectomy(multipleorigins)

Tonsilsareconsideredthefirstlineofdefenseagainstrespiratoryinfections.Previous

twinstudieshavesuggestedasubstantialgeneticpredispositionforrecurrenttonsillitis59.

Tonsillectomyisasurgerytoremovethetonsilstypicallyduetorepeatedoccurrenceof

tonsillitisortonsilhypertrophy.OurtonsillectomyGWASrevealed35independent

chromosomeregionsreachingGWS.WeobservedstrongHLAassociationsatHLA-Binthe

classIregion.TheaminoacidpolymorphismHLA-B-Val97(P=1.44x10-21),inthepeptide-

bindingcleftoftheHLA-Bprotein,accountedformostoftheSNPeffect(rs41543314,P=

5.36x10-21,conditionalpvalueis0.06)andHLAalleleeffect(HLA-B*57:01,P=6.29x10-

22,conditionalpvalueis0.05)inthisinterval.Wealsonotedadditionalindependentsignals

intheclassIIandclassIIIregions.WithinHLA-DRB1intheclassIIregion,theeffectof

rs140177540(P=5.02x10-15,conditionalp-valueis0.001)waslargelyremovedafter

conditioningonDRB1_LeuValGly11(P=1.02x10-12)inthepeptide-bindingcleftofthe

HLA-DRB1protein.


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Wedetected34additionalGWSassociations(Table2andSupplementaryTable4),

includingsignalsfromgenesencodingtumornecrosisfactors/receptorssuperfamily

members(LTBR,TNFRSF13B,TNFSF13B,CD40,TNFAIP3),myotubularin(MTMR3),

chemokines(CXCL13),mitogen-activatedproteinkinases/regulators(IGFBP3,SPRED2,

DUSP10,ST5,MAPK3),SRChomologydomainbindingproteins(SBK1,ST5,SH2B3),

disintegrins(ADAM23,ADAMTS10),varioustranscriptionfactors(NFKB1,HOXA2,FOXA1,

IKZF1,MDFIC,TFEC,RERE,TBX1,FOXN1),andsignalingmolecules(WNT2,GNA12,ITSN1).

Manyofthesegenesmaptopathwaysinvolvedinimmuneandinflammatoryprocesses,

whileothersareimportantregulatorsofembryonicdevelopment(e.g.mostofthe

transcriptionfactors),andafewarepotentiallyinvolvedinplateletproductionor

hemostasis(GNA12,SH2B3,RAP1GAP2,ADAM23,ADAMTS10).Whenwetestedthe

significantlociforenrichmentincanonicalpathways,thetopover-representedpathway

was‘IntestinalimmunenetworkforIgAproduction’(Table5,P=2x10-6andFDR=3.5e-3).

ThegenesthatcontributedsignificantlyinthispathwayincludeLTBR(rs10849448,P=

2.35x10-35),TNFRSF13B(rs34557412,missensevariant,P=2.66x10-21),TNFSF13B

(rs200748895,P=1.20x10-17)andCD40(rs6032664,P=7.31x10-12).IgAisanantibody

thatplaysacriticalroleinmucosalimmunity.Tonsilsbelongtonasopharyngeal-associated

lymphoidtissuesandthegenerationofBcellsisconsideredoneofthemajortonsillar

functions;secretorydimericIgAproducedbytheBcellsiscapableofpreventing

absorptionandpenetrationofbacteriaand/orvirusesintotheupperrespiratorytract

mucosa60.TonsillectomyhasbeenshowntosignificantlydecreaselevelsofserumIgAand

salivarysecretoryIgAlevels61.Otheridentifiedsignificantsignalingpathwaysinclude

‘TACIandBCMAstimulationofBcellimmunesystem’(P=9x10-4andFDR=0.078),

‘ceramidesignalingpathway’(P=3x10-4andFDR=0.08)and‘Glucocorticoidreceptor


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regulatorynetwork’(P=5x10-4andFDR=0.099).ThecontributinggenesareTNFSF13B

(rs200748895,P=1.2x10-17),TNFRSF13B(rs34557412,P=2.66x10-21),NFKB1

(rs23052,P=4.54x10-14)andMAPK3(rs12931792,P=4.06x10-8).

Althoughtonsillectomyhasbeenperformedforover100years,possibleimmunological

effectsofthisprocedureremaincontroversial.Manyreportshavedemonstratedthatin

certainpatientstonsillectomyisaneffectivetherapyforpsoriasis44andrheumatoid

arthritis(RA)62;therationaleforthiseffectisunknown.The‘Intestinalimmunenetwork

forIgAproduction’pathwaywasalsoidentifiedasthemostsignificantpathwayinarecent

GWASofIgAN63.Inthatstudy,theyalsolinkedtheintestinalmucosalinflammatory

disordersandinflammatoryboweldisease(IBD)withriskofIgAN.Ourtonsillectomy

GWASshowedsignificantoverlapoftheidentifiedlociwiththoseautoimmuneand

inflammatorydisorders64.WefoundfiverisklocithatarealsoassociatedwithRA(HLA,

TNFIP3,CD40,SPRED2andSH2B3);fourrisklocithatarealsoassociatedwithIBD(HLA,

MTMR3,TNFAIP3andCD40)andulcerativecolitis(HLA,GNA12,MAPK3andNFKB1);three

risklocithatarealsoassociatedwithpsoriasis(HLA,SLC12ABand1p36.23);andtworisk

loci(HLAandMTMR3)thataresharedwithIgAN.Weobservedbothconcordantand

opposingeffectscomparedtotheseimmune-mediateddiseases.Ourresultsmayhelpto

elucidatetheconnectionbetweentonsillectomyandthesediseasesandmayprovide

insightintoclinicalmarkersthatcouldbeusedasindicatorsoftonsillectomyasatherapy

forthesediseases.

Childhoodearinfection(multipleorigins)

Althoughearinfectionscanhaveabacterialorigin,theyareoftensecondarytoanupper

respiratoryinfectionsuchasacoldorsorethroat,whichcanbecausedbyvirusesor

bacteria65.Childrenaremorelikelythanadultstohaveearinfectionsbecausetheir


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Eustachiantubesarelesseffectiveatdrainingfluidandtheirimmunesystemsarenotfully

developed.InourGWAS,weidentified14regionsthatweresignificantlyassociatedwith

childhoodearinfection.SignalsintheHLAregionmappedtoHLA-DRB1inclassIIregion.

OurstrongestassociationwithinHLA-DRB1waswithHLA-DRB1-Gln96inthepeptide-

bindinggroove.AfterconditioningonHLA-DRB1-Gln96(P=2.11x10-12),theeffectsof

rs4329147(P=9.55x10-12,conditionalpvalueis0.27)andDQB1*06:02(P=5.79x10-10,

conditionalpvalueis0.85)wereeliminated.

Wefound13additionalGWSsignals(Table2andSupplementaryTable5).Although

MAGENTApathwayanalysisdidnotidentifysignificantpathways(Table6),variantsin

FUT2andABO,whichwereinvolvedintheglycosphingolipidbiosynthesispathwayand

wereimplicatedinourmumpsGWAS,werealsosignificantlyassociatedwithchildhoodear

infection.Theriskallelers681343(C)(P=3.51x10-30)isasynonymousmutationinFUT2

andisinalmostcompleteLDwithrs601338(G)(r2=0.9993),the‘secretor’(se)alleleis

associatedwithhigherriskofchildhoodearinfectioninourdata.Thisisconsistentwitha

previousreportinwhichsecretorswereover-representedamongpatientswithupper

respiratoryinfections66.Oursecondmostsignificantassociationwaswithavariantin

TBX1(rs1978060,P=1.17x10-19).Thelow-riskallelers1978060(A)isinLD(r2=0.45)

withrs72646967(C),amissense(N397H)mutationinTBX1thatwasalsoimplicatedin

ourtonsillectomyGWAS.T-boxgenesencodetranscriptionfactorsthatplayimportant

rolesintissueandorganformationduringembryonicdevelopment.Inmice,TBX1haplo-

insufficiencyintheDiGeorgesyndrome(22q11.2deletionsyndrome)regionhasbeen

showedtodisruptthedevelopmentofthepharyngealarcharteries67andthemiddleand

outerear68,69.Wealsoidentifiedothergenesinvolvedindevelopmentalprocesses,

includingMKX(rs2808290,P=5.09x10-16),FGF3(rs72931768,P=2.63x10-9)andAUTS2


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(rs35213789,P=3.75x10-9).TheMKXproductisanIRXfamily-relatedhomeobox

transcriptionfactorandhasbeenshowntoplayacriticalroleintendondifferentiationby

regulatingtypeIcollagenproductionintendoncells70.FGF3isamemberofthefibroblast

growthfactorfamily,whichisinvolvedinavarietyofbiologicalprocessesincluding

embryonicdevelopment,cellgrowth,morphogenesis,tissuerepairandinvasion.Studyof

similargenesinmouseandchickensuggestedtheroleininnerearformation71..AUTS2has

beenimplicatedinneurodevelopmentandisacandidategeneforautismspectrum

disordersanddevelopmentaldelay72,73.Finally,weidentifiedmissensemutationsinCDHR3

(rs114947103,P=5.40x10-9)andPLG(rs73015965,P=3.78x10-8).TheindexSNP

rs114947103isinalmostcompleteLDwithrs6967330(r2=0.97),amissensevariantin

CDHR3,thatwasrecentlyidentifiedinaGWASasasusceptibilitylocusforasthma74.The

biologicalroleofCDHR3isnotclear,butitishighlyexpressedinairwayepitheliumand

belongstothecadherinfamilythatisinvolvedincelladhesionandepithelialpolarity74.

MutationsinthePLGgenecouldcausecongenitalplasminogendeficiency,whichresultsin

inflamedgrowthsonthemucousmembranes.StudiesinmicehaveshowedthatPLGplays

anessentialroleinprotectingagainstthespontaneousdevelopmentofchronicotitismedia

(middleearinfection)andhavealsosuggestedthepossibilityofusingPLGforclinical

therapyofcertaintypesofotitismedia75.

Myringotomy(multiplecauses)

Myringotomyisasurgicalprocedureinwhichatinyincisioniscreatedintheeardrumto

allowthereleaseofmiddle-earfluid.Beforetheinventionofantibiotics,myringotomy

withouttubeplacementwasacommontreatmentforsevereorfrequentacuteotitis

media76.ThesamevariantinTBX1thatwasassociatedwithtonsillectomyandchildhood


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earinfection(seeearlier)wasalsofoundtobesignificantlyassociatedwithmyringotomy

(rs1978060,P=3.34x10-10).


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Discussion

Weidentifiedthegreatestnumberofindependentassociationsfortonsillectomy(n=35)

andchildhoodearinfection(n=14),tworelativelynonspecificphenotypes.Formanyofthe

infections,weonlyidentifiedHLAassociations.Thesefindingsmaybeduetoourrelatively

largersamplesizesforthetwotraitsandmayalsobeduetothefactthattonsillectomyand

childhoodearinfectionsareheterogeneousphenotypes,influencedbyavarietyof

pathogensandalsobyanatomicalabnormalities.Peopleundergotonsillectomyfor

differentreasons,includingrecurrenttonsillitis,obstructivesleepapnea,andnasalairway

obstruction77.Thecausesofchildhoodearinfectionsarealsodiverse,involvingdifferent

typesofbacteriaandviruses,andeardevelopmentaldefects78.VariantsintheTBX1gene,

whichisessentialforinnereardevelopment68,69,wereassociatedwithbothtonsillectomy

andinnerearinfections.Manygenesthatareinvolvedinembryoticdevelopmentwerealso

identifiedinourtonsillectomyandchildhoodearinfectionGWASs.Thevulnerabilityto

tonsillectomyandchildhoodearinfectionsmayalsobelargelyinherited.Therewere

earlierstudiescalculatedthatheredityaccountforover70%ofsusceptibilitytorecurrent

earinfectionsinchildren79,80.Previoustwinstudieshadsuggestedasubstantialgenetic

predispositionforrecurrenttonsillitis59andsleep-disorderedbreathing81.Accordingto

theLDscoreregressiononourGWASdata,tonsillectomyandchildhoodearinfectionshave

relativelyhigherheritabilityontheobservedscalethanmostoftheotherinfectionswe

studies.Wedidnotcalculatetheliabilityscaleheritabilityduetothelackofpopulation

prevalenceinformation.

IntheHLAregion,wefoundthatviraldiseases—e.g.,chickenpox,shingles,coldsores,

mononucleosis(allcausedbyherpesvirus)aswellasmumps(causedbymumpsvirus)—

weremainlyassociatedwithvariationinclassImolecules(Figure1).Thebacterial


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diseases—specificallyhavingapositiveTBtest(causedbyMycobacteriumtuberculosis),

scarletfever(causedbyGAS),andchildhoodearinfection(mostlycausedbybacteria)—

weremainlyassociatedwithvariationinclassIImolecules.Tonsillectomyandpneumonia,

causedbyeitherbacteriaorviruses,wereassociatedwithbothclassIandclassII

molecules.Theseobservationsareconsistentwithpreviousknowledgeaboutantigen

presentation;virusesmostlyreplicatewithinnucleatedcellsinthecytosolandproduce

endogenousantigensthatarepresentedbyclassIMHCmolecules,whilebacteriagrow

extracellularlyandaretakenupbyendosomalcompartmentswheretheyareprocessedfor

presentationbyclassIIMHCmolecules82.Thesetwointracellularpathwaysofprotein

processingmaynotbecompletelyseparate.ActivationofCD8+,HLAclassI-restrictedT-

cellsbyexogenousantigenshasbeenreported83andHLAclassII-restrictedCTLsthat

recognizeendogenouslysynthesizedantigen,suchasHBVenvelopeantigens,havealso

beendescribed84,85.

Somediseases,however,donotfollowthecommonprinciplesofantigenpresentation.

Plantarwarts(causedbyHPV)aredifferentfromothervirus-causingdiseases.HPV

infectionsareexclusivelyintraepithelialandthereisnoviraemiaorcytolysisinthe

infectioncycle86.TheprimaryresponsetoHPVantigensismorelikelytobeinitiatedbythe

antigen-presentingcells(APCs)ofsquamousepithelia,theLangerhanscells(LCs).LCs

captureantigensbymacropinocytosisandreceptor-mediatedendocytosis,andthen

initiateclassIIprocessingofexogenousantigens87,whichexplainswhyourplantarwarts

GWASmainlyidentifiedassociationswithMHCclassIImolecules.Inothercases,our

resultssuggestevenmorecomplexinteractions.Strepthroat(causedbyGASbacteria)has

bothMHCclassIandclassIIassociations.Someofourreportedstrepthroatcasesmay

representsecondarybacterialinfectionsinresponsetoaviralupperrespiratoryinfection,


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meaningthatthediscoveredMHCclassIassociationsareactuallywiththevirusinfection

thatinitiatedtheprocess88.Sincemostsorethroatsarecausedbyviruses89,another

possibilityisthatsomeofthereportedstrepthroatcasesactuallyhadamisdiagnosedor

misreportedviralinfection.AfinalpossibilityisthatHLAclassImoleculescanbind

bacterialpeptidesderivedfromexogenousproteinsthatareinternalizedbyendocytosisor

phagocytosis,aprocesscalledcross-presentation85,90.

WefailedtodetectanyGWSassociationforcertaininfectiousdiseases,includingrubella,

measles,chronicsinusinfection,thecommoncold,orrheumaticfever.ItisknownthatHLA

affectsresponsestomeaslesandrubellavaccinations91,92andmaythusalsobeassociated

withsusceptibility.Onelimitationofourstudythatmightaffectfindingswithrespectto

measlesandrubellaisourlackofdataonvaccinationstatus.Sincethereisnowacommon

vaccinationformeaslesandrubella,misclassifyingvaccinatedpeopleascontrolswould

theoreticallyreduceourpowertodetectassociations.Thislimitationmightalsoaffectthe

powerofourshinglesGWAS.ThecontrolsusedfortheshinglesGWASwerenotfilteredfor

apositivehistoryofchickenpox,thoughhavingchickenpoxisaprerequisiteforshingles.

Anotherreasonmightbetheheterogeneityofthephenotype.Coldsarecausedbyseveral

differentviruses(mostlyrhinovirusesandcoronaviruses),andlargersamplesizesmaybe

requiredtoidentifyallelesonlyassociatedwithspecificpathogens.

Althoughadditionalstudieswillberequiredtovalidateourassociations,ourfindings

areanimportantsteptowardsdissectingthehostgeneticcontributiontovariationin

responsetoinfections.Researchinsightsintoinfectiousdiseaseswillhelptoderivenew

diagnosticapproachesandperhapsnewtherapiesandpreventions.Thisstudymayalso

helpustounderstandsomeoftheautoimmunedisordersthatareassociatedwithvarious

infectiontriggers.Manyoftheidentifiedassociationswerealsofoundinautoimmune


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diseases.Onepostulatedconnectionbetweeninfectiousdiseasesandimmunological

disordersisthattheimmunecellsthatareactivatedinresponsetoapathogenepitopeare

alsocross-reactivetoselfandleadtodirectdamageandfurtheractivationofotherarmsof

theimmunesystem93.


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Methods

Subjects

WeconductedGWASsof23infectiousdiseases.Allparticipantsweredrawnfromthe

customerbaseof23andMe,Inc.,apersonalgeneticscompany.Allindividualsincludedin

theanalysesprovidedinformedconsentandansweredsurveysonlineaccordingtoour

humansubjectsprotocol,whichwasreviewedandapprovedbyEthical&Independent

ReviewServices,aprivateinstitutionalreviewboard(http://www.eandireview.com).

AllparticipantswereofprimarilyEuropeanancestry(>97%Europeanancestry)as

determinedthroughananalysisoflocalancestry94.Amaximumsetofunrelatedindividuals

waschosenusingasegmentalidentity-by-descent(IBD)estimationalgorithm95.

Individualsweredefinedasrelatediftheysharedmorethan700cMofIBD,including

regionswherethetwoindividualsshareeitheroneorbothgenomicsegmentsIBD.This

levelofrelatedness(involving~20%ofthehumangenome)correspondsapproximatelyto

theminimalexpectedsharingbetweenfirstcousinsinanoutbredpopulation.

Table1showsourdiscoverysamplesizes,drawnfrommorethan650,000genotyped

customerswhoreportedviaweb-basedquestionnaireswhethertheyhadbeendiagnosed

withoneormoreinfectiousdiseases.(Formoredetailonsurveyquestionsusedtocollect

thephenotypedata,seetheSupplementaryNotes).

GenotypingandSNPimputation

DNAextractionandgenotypingwereperformedonsalivasamplesbyNationalGenetics

Institute(NGI),aCLIA-certifiedlaboratorythatisasubsidiaryofLaboratoryCorporationof

America.Samplesweregenotypedononeoffourgenotypingplatforms.TheV1andV2

platformswerevariantsoftheIlluminaHumanHap550+BeadChipandcontainedatotalof


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about560,000SNPs,includingabout25,000customSNPsselectedby23andMe.TheV3

platformwasbasedontheIlluminaOmniExpress+BeadChipandcontainedatotalofabout

950,000SNPsandcustomcontenttoimprovetheoverlapwithourV2array.TheV4

platformincurrentuseisafullycustomarrayofabout950,000SNPsandincludesalower

redundancysubsetofV2andV3SNPswithadditionalcoverageoflower-frequencycoding

variation.Samplesthatfailedtoreach98.5%callratewerere-analyzed.Individualswhose

analysesfailedrepeatedlywerere-contactedby23andMecustomerservicetoprovide

additionalsamples,asisdoneforall23andMecustomers.

ParticipantgenotypedatawereimputedusingtheMarch2012“v3”releaseofthe1000

GenomesProjectreferencehaplotypes96.Wephasedandimputeddataforeachgenotyping

platformseparately.First,weusedBEAGLE97(version3.3.1)tophasebatchesof8,000to

9,000individualsacrosschromosomalsegmentsofnomorethan10,000genotypedSNPs,

withoverlapsof200SNPs.WeexcludedSNPswithminorallelefrequency(MAF)<0.001,

Hardy-WeinbergequilibriumP<1×10−20,callrate<95%,orlargeallelefrequency

discrepanciescomparedtotheEuropean1000GenomesProjectreferencedata.Frequency

discrepancieswereidentifiedbycomputinga2x2tableofallelecountsforEuropean1000

Genomessamplesand2000randomlysampled23andMecustomerswithEuropean

ancestry,andidentifyingSNPswithachisquaredP<10−15.Weimputedeachphased

segmentagainstall-ethnicity1000Genomeshaplotypes(excludingmonomorphicand

singletonsites)usingMinimac298withfiveroundsand200statesforparameter

estimation.

Forthenon-pseudoautosomalregionoftheXchromosome,malesandfemaleswere

phasedtogetherinsegments,treatingthemalesasalreadyphased;thepseudoautosomal

regionswerephasedseparately.Wethenimputedmalesandfemalestogetherusing


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Minimacaswiththeautosomes,treatingmalesashomozygouspseudo-diploidsforthe

non-pseudoautosomalregion.

GWASanalysis

Forcasecontrolcomparisons,wetestedforassociationusinglogisticregression,

assumingadditivealleliceffects.Forquantitativetraits,associationtestswereperformed

usinglinearregression.Fortestsusingimputeddata,weusetheimputeddosagesrather

thanbest-guessgenotypes.Weincludedcovariatesforage,gender,andthetopfive

principalcomponentstoaccountforresidualpopulationstructure.Theassociationtestp-

valuewascomputedusingalikelihoodratiotest,whichinourexperienceisbetterbehaved

thanaWaldtestontheregressioncoefficient.ResultsfortheXchromosomewere

computedsimilarly,withmencodedasiftheywerehomozygousdiploidfortheobserved

allele.

GeneticcorrelationsWecomputedLDscoresaspreviouslydescribed99usingthesamplesinthe1000

GenomesProjectreferencepanel(phase3verion5a)withaMAFcutoffof5%.We

calculatedthepairwisegeneticcorrelation(rg)ontheGWASsummarystatistics(including

MHCregion)usingLDscoreregression99.Theclustermapofgeneticcorrelation,baseon

thelowerboundofthe95%confidentintervalofcalculatedrg,wasplottedusing’Nearest

PointAlgorithm’implementedinpython“Searborn”package

(https://stanford.edu/~mwaskom/software/seaborn/index.html).Wefoundno

significantnegativecorrelationsinourdata,thuswesetallnegativevaluestozero.

SNPFunctionAnnotation

ToexplorewhetheranyofthesignificantSNPsidentifiedmightlinktofunctional

mutationsorhavepotentialregulatoryfunctions,weusedtheonlinetoolsHaploReg


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(http://www.broadinstitute.org/mammals/haploreg/haploreg.php)toconfirmthe

locationofeachSNPinrelationtoannotatedprotein-codinggenesand/ornon-coding

regulatoryelements.Wequeriedonlythevariantsthatwerewithin500kbofandin

moderateLD(r2>0.6)withtheindexSNP.

ImputationofHLAclassicalallelesandrespectiveaminoacidvariations

HLAimputationwasperformedwithHIBAG100,anattributebaggingbasedstatistical

methodthatcomesasafreelyavailableRpackageandincludesapre-fitclassifier.This

classifierwastrainedfromalargedatabaseofindividualswithknownHLAallelesandSNP

variationwithintheHLAregion.Over98%ofthetaggingSNPsusedinHIBAGwere

genotypedandpassedqualitycontrolon23andMe’splatform.Weimputedallelicdosage

forHLA-A,B,C,DPB1,DQA1,DQB1,andDRB1lociatfour-digitresolution.Weusedthe

defaultsettingsofHIBAGandtheruntimefor100,000sampleswasabout10hoursonour

cluster.

UsinganapproachsuggestedbyP.deBakker101,wedownloadedthefilesthatmapHLA

allelestoaminoacidsequencesfromhttps://www.broadinstitute.org/mpg/snp2hla/and

mappedourimputedHLAallelesatfour-digitresolutiontothecorrespondingaminoacid

sequences;inthiswaywetranslatedtheimputedHLAallelicdosagesdirectlytoaminoacid

dosages.Weencodedallaminoacidvariantsinthe23andMeEuropeansamplesasbiallelic

markers,whichfacilitateddownstreamanalysis.Forexample,position45ofHLA-Bprotein

hadfivedifferentalleles(E:Glu,M:Met,T:Thr,K:Lys,G:Gly),wefirstencodedtheposition

usingfivebinarymarkers,eachcorrespondingtothepresenceorabsenceofeachallele

(e.g.HLA-B-Gly45tagsGlyatposition45ofHLA-Bprotein).Forpositionshavingthreeor

morealleles,wealsocreatedmarkersthattagmultiplealleles,eachcorrespondingtothe

presenceorabsenceofthemultiplealleles(e.g.HLA-B-ThrGly45tagsThrandGlyat


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position45ofHLA-Bprotein).Thus,wecreatedbinaryindicatorsforallpossible

combinationsofaminoacidvariants.Weusethisnamingconventionforaminoacid

polymorphismsthroughoutthispaper.

Weimputed292classicalHLAallelesatfour-digitresolutionand2395bi-allelicamino

acidpolymorphisms.SimilartotheSNPimputation,wemeasuredimputationqualityusing

r2,whichistheratiooftheempiricallyobservedvarianceofthealleledosagetothe

expectedvarianceassumingHardy-Weinbergequilibrium.Theimputationquality(r2)of

thetopassociatedHLAallelesandaminoacidsareinSupplementaryTable6.

HLAregionfinemapping

TotestassociationsbetweenimputedHLAallele/aminoaciddosagesandphenotypes,

weperformedlogisticorlinearregressionusingthesamesetofcovariatesusedintheSNP-

basedGWASforthatphenotype.Weappliedaforwardstepwisestrategy,withineachtype

ofvariant,toestablishstatisticallyindependentsignalsintheHLAregion.Withineach

varianttype(e.g.SNP,HLAallele,andHLAaminoacid),wefirstidentifiedthemoststrongly

associatedsignals(lowestp-value)foreachdiseaseandperformedforwarditerative

conditionalregressiontoidentifyotherindependentsignalsiftheconditionalp-valuewas

<5x10-8.Allanalysescontrolledforsexandfiveprincipalcomponentsofgeneticancestry.

Thep-valueswerecalculatedusingalikelihoodratiotest.Theiterativeconditional

regressiondissectedHLAsignalintoindependentHLAassociations.Withineachidentified

HLAlocus(HLA-A,B,C,DPB1,DQA1,DQB1,andDRB1),Wefurthercarriedoutreciprocal

analyses,whicharetheconditionalanalysesacrossvariantstypes,toseeiftheassociation

canbeattributedtotheaminoacidpolymorphismwithineachHLAlocus.

Pathwayanalysis


https://doi.org/10.1101/073056


31

Inordertobetterunderstandhowmultiplegenesinthesamepathwaymaycontribute

tocertaininfections,weperformedpathwayanalysisusingMeta-AnalysisGene-set

EnrichmentofvariaNTAssociations(MAGENTA)102,whichtestsforenrichmentofgenetic

associationsinpredefinedbiologicalprocessesorsetsoffunctionallyrelatedgenes,using

GWASresultsasinput.Weusedgenesetsof1320canonicalpathwaysfromtheMolecular

SignaturesDatabase(MsigDB)compiledbydomainexperts103anddefaultsettingsof

MAGENTAtool.


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32

Acknowledgements:

Wethankthecustomersof23andMeforparticipatinginthisresearchandthe

employeesof23andMefortheircontributionstothiswork.

Conflictofinterest:

Allauthorslistedwere/areemployedby23andMe,andownstockorstockoptionsin

23andMe.


https://doi.org/10.1101/073056


33

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FigureTitlesandLegends

Figure1.SummaryofIndependentHLASignals

Thestrongestassociatedsignals(red)andputativeindependentsecondarysignals

(blue)areshownforeachdiseasealongwiththeirlocationwithintheregion.Independent

secondarysignalsweredefinedasthosewithresidualconditionalassociationusingthe

samesignificantthresholdastheprimaryassociationsignalmentionedinthemaintext.

Figure2.Heatmapshowingthegeneticcorrelations


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44

Eachsquare[i,j]showsthelowerboundofthe95%confidenceintervalof

estimatedgeneticcorrelation(rg)betweentraitiandtraitj,whereiindexesrowandj

indexescolumns.Darkercolorsrepresentlargergeneticcorrelations.Allnegativevaluesto

zero.Phenotypeswereclusteredby’NearestPointAlgorithm’implementedinpython

“Seaborn”package.


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45

TablesTable1D

iscoverycohortcharacteristics

PhenotypeswithsignificantG

WASfindings

Chickenpox

ShinglesColdsores

Mononucleosis

Mum

psHepatitisB

Cases

107,769

16,711

25,108

17,457

31,227

1,425

Control

15,982

118,152

63,332

68,446

54,153

218,180

Plantarw

artsPositiveTB

Strepthroat aScarletfever

Pneumonia

Bacterialmeningitis

Cases

24,994

4,426

[qt]5

2,487

6,812

40,600

842

Control

37,451

84,290

22,017

113,837

90,039

82,778

Yeastinfections a

UTIfrequency

aTonsillectom

yChildhoodearinfection

Myringotom

y

Cases

[qt]5

2,218

[qt]3

5,000

60,098

46,936

4,138

Control

10,235

33,478

113,323

74,874

85,089

Phenotypesw

ithnosignificantGWASfindings

Measles

HepatitisA

Rheumaticfever

#ofColdslastyear aRubella

ChronicsinusinfectionCases

38,219

2,442

1,115

[qt]4

3,826

12,000

5,291

Control

47,279

217,137

88,076

15,720

71,597

79,622

a.Strepthroat,yeastin

fections,urinarytractin

fection(UTI)frequencyandnumberofcoldslastyearwerequantita

tivetraits([qt]).


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46

Table2:Genome-w

ideSignificantAssociationsforEachDisease

PhenotypeCytoband

GenecontextVariants

PositionAlleles

aFreq

OR/effect b

(95%CI)

P-valueChickenpox

6p21.33

[HLA]

rs9266089

31320509

G/A

0.85

1.12

(1.1-1.14)

1.00E-10

Shingles6p21.33

[HLA]

rs2523591

31326960

G/A

0.58

1.14

(1.13-1.16)

1.74E-27

9p21.3

[IFNA21]

rs7047299

21167465

A/G

0.56

1.07

(1.06-1.09)

1.67E-08

Coldsores6p21.33

[HLA]

rs885950

31140152

C/A

0.42

1.08

(1.07-1.09)

7.47E-13

Mononucleosis

6p21.33

[HLA]

rs2596465

31412948

T/C

0.47

1.08

(1.06-1.09)

2.48E-09

Mum

ps

19q13.33

[FUT2]

rs516316

49206145

C/G

0.48

1.25

(1.24-1.27)

9.63E-72

6p22.1

[HLA]

rs114193679

29925103

G/C

0.99

1.72

(1.62-1.84)

2.23E-17

14q32.2

C14orf132--[]--B

DKRB2

rs11160318

96659919

G/A

0.68

1.1

(1.08-1.11)

4.56E-12

11q24.2

[ST3GAL4]

rs3862630

126243649

T/C

0.12

1.13

(1.1-1.15)

1.21E-08

HepatitisA

19q13.2

[IFNL4]

rs66531907

39740675

A/C

0.21

1.23

(1.14-1.33)

5.70E-08

HepatitisB

6p21.32

[HLA]

rs9268652

32409056

G/A

0.74

1.32

(1.25-1.38)

3.14E-09

Plantarwarts

6p21.32

[HLA]

rs9272050

32599071

G/A

0.38

1.19

(1.17-1.21)

9.66E-31

1q21.3

CRCT1--[]--L

CE3E

rs6692209

152506444

T/C

0.62

1.08

(1.06-1.09)

5.25E-09

PositiveTBTest

6p21.32

[HLA]

rs2894257

32433276

C/G

0.53

1.36

(1.33-1.39)

8.16E-36

Strepthroat6p21.33

[HLA]

rs1055821

31321919

T/G

0.06

0.08

(0.06-0.09)

7.69E-11

1p36.23

ERRFI1---[]---S

LC45A1

rs35395352

8266829

D/I

0.72

0.03

(0.03-0.04)

3.90E-08

Scarletfever6p21.32

[HLA]

rs36205178

32627938

C/T

0.81

1.24

(1.21-1.28)

9.49E-14

Pneumonia

6p21.33

[HLA]

rs3131623

31418810

T/A

0.85

1.1

(1.09-1.12)

1.99E-15

Bacterialmeningitis

17q21.33

[CA10]

rs1392935

49939129

G/A

0.91

1.78

(1.6-1.99)

1.19E-08

Yeastinfections

18q12.1

[DSG1]

rs200520431

28918197

D/I

0.07

0.11

(0.09-0.12)

1.87E-16

14q23.1

PRKCH

rs2251260

62023767

T/C

0.17

0.05

(0.04-0.06)

3.46E-10

14q32.2

[]---C14orf177

rs7161578

98586162

T/C

0.39

0.04

(0.03-0.04)

4.04E-08

UTIfrequency

8q24.3

JRK-[]-P

SCA

rs2976388

143760256

G/A

0.56

0.04

(0.04-0.05)

3.27E-10

15q15.3

[FRMD5]

rs146906133

44262951

T/C

1.00

0.38

(0.32-0.45)

2.02E-08

Tonsillectomy

12p13.31

[LTBR]

rs10849448

6493351

A/G

0.25

1.13

(1.12-1.14)

2.35E-35

22q12.2

[MTMR3]

rs201112509

30324654

I/D

0.69

1.12

(1.11-1.13)

3.39E-34


https://doi.org/10.1101/073056


47

17p11.2

[TNFRSF13B]

rs34557412

16852187

G/A

0.01

1.59

(1.52-1.67)

2.66E-21

6p21.33

[HLA]

rs41543314

31322690

G/A

0.03

1.23

(1.2-1.26)

5.36E-21

13q33.3

[TNFSF13B]

rs200748895

108960380

D/I

0.03

1.26

(1.23-1.29)

1.20E-17

7p12.3

IGFBP3---[]

rs80077929

46094089

T/C

0.11

1.11

(1.1-1.13)

1.74E-15

4q24

[NFKB1]

rs230523

103462038

T/C

0.67

1.07

(1.06-1.08)

4.54E-14

7p15.2

HOXA1-[]-H

OXA2

rs6668

27138183

T/C

0.37

1.06

(1.06-1.07)

1.71E-13

14q21.1

MIPOL1-[]--F

OXA1

rs148131694

38025098

T/C

0.44

1.07

(1.06-1.08)

7.86E-13

4q24

[TET2]

rs1391439

106151642

G/A

0.40

1.06

(1.05-1.07)

2.76E-12

2p14

[SPRED2]

rs201473667

65562862

D/I

0.47

1.06

(1.05-1.07)

5.39E-12

20q13.12

NCOA5--[]-C

D40

rs6032664

44739419

A/T

0.26

1.07

(1.06-1.08)

7.31E-12

7p12.2

C7orf72---[]--IK

ZF1

rs876037

50308692

T/A

0.68

1.06

(1.05-1.07)

1.12E-11

13q21.33

[KLHL1]

rs9542155

70579527

T/C

0.32

1.06

(1.05-1.07)

1.92E-11

7p22.2

[GNA12]

rs2644312

2841164

G/A

0.71

1.06

(1.05-1.07)

2.73E-11

3q21.2

[SLC12A8]

rs1980080

124911841

C/T

0.33

1.06

(1.05-1.07)

3.16E-11

16p11.2

[SBK1]

rs141876325

28330968

I/D

0.28

1.06

(1.05-1.07)

4.72E-11

1q41

DUSP10---[]---H

HIPL2

rs12126292

222165909

G/T

0.12

1.09

(1.07-1.1)

7.21E-11

4p15.2

LGI2--[]-S

EPSECS

rs10939037

25114601

A/G

0.56

1.05

(1.05-1.06)

1.42E-10

12q24.12

[SH2B3]

rs3184504

111884608

T/C

0.50

1.05

(1.04-1.06)

3.07E-10

4q21.1

[CXCL13]

rs7685785

78440425

C/T

0.89

1.09

(1.07-1.1)

4.52E-10

7q31.2

MDFIC---[]---T

FEC

rs2023703

114944919

C/A

0.33

1.05

(1.05-1.06)

7.79E-10

19p13.2

ADAMTS10---[]--A

CTL9

rs2918308

8789912

A/C

0.84

1.07

(1.06-1.08)

1.10E-09

17p13.3

[RAP1GAP2]

rs67968065

2729247

D/I

0.52

1.05

(1.04-1.06)

2.08E-09

21q22.11

[ITSN1]

rs200746495

35052995

D/I

0.87

1.07

(1.06-1.09)

4.45E-09

2q33.3

[ADAM23]

rs1448903

207308961

G/A

0.09

1.08

(1.07-1.1)

5.13E-09

1p36.23

RERE--[]--E

NO1

rs12068123

8894346

G/A

0.45

1.05

(1.04-1.06)

5.60E-09

11p15.4

[ST5]

rs11042055

8756856

A/G

0.43

1.05

(1.04-1.06)

6.96E-09

20q13.33

[SAMD10]

rs41278232

62606617

G/A

0.89

1.08

(1.06-1.09)

7.54E-09

9q34.2

ABO-[]--S

URF6

rs635634

136155000

T/C

0.20

1.06

(1.05-1.07)

8.47E-09

17q11.2

[FOXN1]

rs62066768

26858395

A/G

0.08

1.09

(1.07-1.11)

1.09E-08

6q23.3

OLIG3---[]---T

NFAIP3

rs11757201

138003822

C/G

0.20

1.06

(1.05-1.07)

2.97E-08


https://doi.org/10.1101/073056


48

16p11.2

MAPK3--[]--C

ORO1A

rs12931792

30156398

G/A

0.54

1.06

(1.05-1.07)

4.06E-08

7q31.2

[WNT2]

rs200608253

116917244

I/D

0.52

1.05

(1.04-1.05)

4.07E-08

22q11.21

[TBX1]

rs41298830

19752943

A/C

0.77

1.06

(1.05-1.07)

4.95E-08

ChildhoodEarInfections

19q13.33

[FUT2]

rs681343

49206462

C/T

0.52

1.11

(1.1-1.12)

3.51E-30

22q11.21

[TBX1]

rs1978060

19749525

G/A

0.60

1.09

(1.08-1.1)

1.17E-19

10p12.1

RAB18--[]--M

KX

rs2808290

27900882

C/T

0.51

1.07

(1.07-1.08)

5.09E-16

15q26.2

SPATA8---[]---L

INC00923

rs7174062

97452829

G/A

0.73

1.08

(1.07-1.09)

3.49E-14

6p21.32

[HLA]

rs4329147

32566577

T/C

0.83

1.11

(1.09-1.13)

9.55E-12

9q34.2

[ABO]

rs8176643

136149095

D/I

0.36

1.06

(1.05-1.07)

3.67E-11

2p16.1

[EFEMP1]

rs1802575

56093204

G/C

0.87

1.09

(1.07-1.1)

1.47E-10

2p24.1

NT5C1B-RDH14---[]---

OSR1

rs5829676

19274784

D/I

0.40

1.06

(1.05-1.07)

1.78E-10

11q13.3

FGF3---[]--A

NO1

rs72931768

69829717

G/C

0.88

1.09

(1.07-1.1)

2.63E-09

7q11.22

[AUTS2]

rs35213789

69268012

C/T

0.74

1.06

(1.05-1.07)

3.75E-09

7q22.3

[CDHR3]

rs114947103

105658927

C/T

0.18

1.07

(1.06-1.08)

5.40E-09

8q22.2

NIPAL2--[]--K

CNS2

rs13281988

99372329

C/G

0.31

1.06

(1.05-1.07)

9.84E-09

3p21.31

[BSN]

rs67035515

49656530

I/D

0.18

1.07

(1.05-1.08)

1.56E-08

6q26

[PLG]

rs73015965

161127501

G/A

0.01

1.43

(1.34-1.53)

3.78E-08

Myringotom

y22q11.21

[TBX1]

rs1978060

19749525

G/A

0.60

1.17

(1.14-1.2)

3.34E-10

a. TheallelesforSNParerepresentedashigh-riskallele/low-riskallele;

b. Strepthroat,p

lantarwartsandUTIfrequencyarequantita

tivetraits,forwhichwereportedtheeffectsizeinsteadofth

eoddsratio.


https://doi.org/10.1101/073056


49

Table3:HLAFine-m

appingResultsforEachDisease

PhenotypeGene

CytobandVariants

bPosition

Allelesc

FreqOR/

Effect d(95%

CI)Con.

P-valueP-value

Chickenpox[HLA-A]

6p22.1

A_Gly107

29911093

GW

0.71

1.09

(1.07-1.1)

3.77E-10

3.77E-10

6p22.1

A*02:01

29911953

NA

0.27

0.92

(0.91-0.93)

1.08E-09

1.08E-09

[HLA-B]

6p21.33

rs9266089

31320509

G/A

0.85

1.12

(1.1-1.14)

1.00E-10

1.00E-10

Shingles

[HLA-A]

6p22.1

rs2523815

29840243

A/G

0.64

1.12

(1.11-1.14)

6.17E-20

1.83E-22

6p22.1

A_Arg97

29911063

IRM

0.36

0.88

(0.87-0.9)

2.75E-22

2.75E-22

6p22.1

A*02:01

29911953

NA

0.27

0.88

(0.87-0.89)

1.72E-12

8.91E-20

[HLA-B]

6p21.33

B*44:02

31323318

NA

0.09

0.81

(0.79-0.82)

3.54E-23

3.54E-23

6p21.33

B*57:01

31323318

NA

0.04

1.22

(1.18-1.25)

1.03E-08

1.81E-10

6p21.33

B_Trp147

31324051

WL

0.95

0.92

(0.89-0.94)

6.67E-10

1.24E-03

6p21.33

B_GluMet45

31324602

EMTKG

0.52

1.11

(1.1-1.13)

9.51E-16

1.65E-18

6p21.33

rs2523591

31326960

G/A

0.58

1.14

(1.13-1.16)

1.74E-27

1.74E-27

[TNXB]

6p21.33

rs41316748

32019512

C/T

0.04

1.19

(1.15-1.23)

1.02E-08

1.44E-12

6p21.32

DRB1*11:01

32552079

NA

0.06

0.79

(0.77-0.82)

9.84E-10

2.96E-11

6p21.32

DRB1_PheSerHis13

32552131

FSHYGR

0.66

0.92

(0.91-0.94)

1.22E-09

5.90E-10

Coldsores

POU5F1-[]--

HCG27

6p21.33

rs885950

31140152

C/A

0.42

1.08

(1.07-1.09)

7.47E-13

7.47E-13

[HLA-B]

6p21.33

B_ThrGly45

31324602

EMTKG

0.23

0.91

(0.9-0.92)

4.91E-12

4.91E-12

MICA--[]--

MICB

6p21.33

rs4360170

31430359

A/G

0.91

1.13

(1.11-1.16)

3.38E-09

3.41E-12

Mononucleosis

MICA--[]--

MICB

6p21.33

rs2596465

31412948

T/C

0.47

1.08

(1.06-1.09)

2.48E-09

2.48E-09

Mum

ps[HLA-A]

6p22.1

A_Gln43

29910660

QR

0.99

1.71

(1.6-1.82)

2.51E-17

2.51E-17

6p22.1

A*02:05

29911953

NA

0.01

0.56

(0.52-0.6)

1.73E-17

1.73E-17

6p22.1

rs114193679

29925103

G/C

0.99

1.72

(1.62-1.84)

2.23E-17

2.23E-17

HepatitisB

[HLA-DRA]

6p21.32

rs9268652

32409056

G/A

0.74

1.32

(1.25-1.38)

3.14E-09

3.14E-09

Plantarwarts

[HLA-DRB1]

6p21.32

DRB1*04:01

32552079

NA

0.08

1.21

(1.19-1.24)

4.88E-18

4.88E-18

6p21.32

DRB1*15:01

32552079

NA

0.13

1.14

(1.12-1.15)

3.50E-19

4.48E-14

6p21.32

DRB1_PheSerTyr13

32552131

FSHYGR

0.63

0.85

(0.84-0.86)

1.84E-39

1.84E-39


https://doi.org/10.1101/073056


50

6p21.32

rs28752534

32585068

T/C

0.64

1.11

(1.1-1.13)

9.28E-09

1.50E-30

6p21.32

rs9272050

32599071

G/A

0.38

1.19

(1.17-1.21)

9.65E-31

9.66E-31

PositiveTBTest

[HLA-B]

6p21.33

B*08:01

31323318

NA

0.11

1.29

(1.24-1.33)

1.73E-11

8.43E-14

[C6orf47]

6p21.33

rs148844907

31628397

A/T

0.01

1.92

(1.74-2.12)

4.95E-10

2.21E-12

[HLA-DRA-

DQA1]

6p21.32

rs3135359

32390578

C/T

0.71

1.21

(1.18-1.24)

2.63E-13

8.82E-21

6p21.32

rs2894257

32433276

C/G

0.53

1.36

(1.33-1.39)

8.16E-36

8.16E-36

6p21.32

DRB1_Leu67

32551969

LIF

0.41

1.3

(1.27-1.33)

1.14E-29

1.14E-29

6p21.32

DQA1*01:02

32608660

NA

0.20

0.75

(0.73-0.78)

7.04E-22

7.04E-22

6p21.32

DQA1*03:01

32608660

NA

0.10

1.27

(1.22-1.32)

1.59E-09

3.99E-10

6p21.32

DQA1_His129

32609872

QHx

0.66

1.19

(1.16-1.22)

2.40E-13

1.56E-13

Strepthroat[HLA-B]

6p21.33

rs1055821

31321919

T/G

0.06

0.08

(0.06-0.09)

7.69E-11

7.69E-11

[HLA-DRB1]

6p21.32

DRB1_CysTyr30

32552080

CYLGRH

0.83

-0.04

(-0.05--

0.04)

2.14E-09

2.14E-09

Scarletfever[HLA-DQB1]

6p21.32

rs36205178

32627938

C/T

0.81

1.24

(1.21-1.28)

9.49E-14

9.49E-14

6p21.32

DQB1*03:01

32630853

NA

0.20

0.83

(0.81-0.86)

2.98E-14

2.98E-14

6p21.32

DQB1_Gly45

32632724

GE

0.80

1.19

(1.16-1.22)

6.35E-14

6.35E-14

Pneumonia

[HLA-B]

6p21.33

B*08:01

31323318

NA

0.11

0.91

(0.9-0.92)

2.87E-11

2.87E-11

6p21.33

B_SerTrpAsn97

31324201

STWRNV

0.34

0.94

(0.93-0.95)

2.37E-12

2.37E-12

MICA--[]--

MICB

6p21.33

rs3131623

31418810

T/A

0.85

1.1

(1.09-1.12)

1.99E-15

1.99E-15

[HLA-DRB1]

6p21.32

DRB1_LeuSer11

32552137

LSVGDP

0.52

0.94

(0.94-0.95)

1.42E-09

4.10E-11

Tonsillectomy

[HLA-B]

6p21.33

rs41543314

31322690

G/A

0.03

1.23

(1.2-1.26)

5.36E-21

5.36E-21

6p21.33

B*57:01

31323318

NA

0.04

1.22

(1.19-1.24)

6.29E-22

6.29E-22

6p21.33

B_Val97

31324201

STWRNV

0.04

1.21

(1.19-1.24)

1.44E-21

1.44E-21

[LST1,LST1]

6p21.33

rs1052248

31556581

A/T

0.28

1.05

(1.04-1.06)

3.69E-08

3.15E-17

[HLA-DRB1]

6p21.32

DRB1_LeuValGly11

32552137

LSVGDP

0.42

1.06

(1.05-1.07)

1.67E-09

1.02E-12

6p21.32

rs140177540

32569453

D/I

0.47

1.06

(1.05-1.07)

2.92E-12

5.02E-15

GRM4--[]-

HMGA1

6p21.31

rs201220830

34195176

D/I

0.05

1.11

(1.09-1.14)

1.32E-08

3.65E-08

ChildhoodEarInfections

[HLA-DRB1]

6p21.32

DRB1_Gln96

32549612

EHYxQ

0.17

0.92

(0.91-0.93)

2.11E-12

2.11E-12

6p21.32

rs4329147

32566577

T/C

0.83

1.11

(1.09-1.13)

9.55E-12

9.55E-12


https://doi.org/10.1101/073056


51

6p21.32

DQB1*06:02

32630853

NA

0.13

0.92

(0.91-0.94)

5.79E-10

5.79E-10

a. Weidentify

independentassociationsforeachcategoryofvariants:SNP,HLA(classicalallele)andAA(HLAaminoacid),separatelyusing

iterativeconditio

nalregression.Foraminoacidpolymorphism,th

elabelspecifie

stheaminoacidandthepositio

n.Forexample,‘A-Val95’

meansaminoacidValatpositio

n95ofth

eHLA-Aprotein.W

eusethisnamingconventionthroughoutth

epaper,whichiscomposedfrom

thenameofth

eHLAprotein(A),followedbythethree-lettercodefortheaminoacid(Val)anditslocationintheprotein(95).Theamino

acidpolymorphismishighlightedas‘red’ifth

esignalin

thatgeneregioncanbeattributetoitb

yreciprocalanalysis.

b. TheallelesforSNParerepresentedasriskallele/non-riskallele;th

ealleleforHLAclassicalalleleisrecordedas‘NA’,w

hichmeansit

eitherhasordoesnothasthetestedallele;th

eallelesforHLAaminoacidpolymorphismsareallth

epossibleaminoacids(one-letter

code)atth

atpositio

n.

c. Strepthroat,p

lantarwartsandUTIfrequencyarequantita

tivetraits,forwhichwereportedtheeffectsizeinsteadofth

eoddsratio.

d. The‘Cond.P-value’columncontainsconditio

nalp-valuesthatareafterite

rativeconditio

nalregressionwithineachcategoryofvariants

(SNP,im

putedHLAallelesandim

putedHLAaminoacidpolymorphisms).The‘P-value’columncontainstheunconditio

nalassociation

testp-values.


https://doi.org/10.1101/073056


52

Table4:MEGAN

TAPathwayAnalysisonM

umpsGW

AS

Pathway

Pathway.

PvaluePathw

ay.FD

RGene

Gene.Pvalue

SNP

SNP.Pvalue

Glycosphingolipidbiosynthesis

1.00E-04

0.0029

FUT1

0.00E+00

rs584768

2.38E-62

FUT2

0.00E+00

rs516316

9.63E-72

ST3GAL4

1.12E-08

rs3862630

1.21E-08

B3GNT1

4.20E-02

rs7947391

2.42E-04

GCNT2

4.34E-02

rs147839826

7.16E-05

B3GNT2

4.46E-02

rs116508040

2.60E-04

ABO

4.97E-02

9:136149709:AC_A

5.81E-05

Themostsignific

antpathway(genesets)withagenesetenrichmentFDR<0.01arepresented.Thegenesthatcontaingenome-wide

signific

antSNPsintheGWASanalysisarehighlightedred.

Table5:MEGAN

TAPathwayAnalysisonTonsillectom

yGWAS

Pathway

Pathway.

PvaluePathw

ay.FD

RGene

Gene.Pvalue

SNP

SNP.Pvalue

Intestinalimmune

networkforIgA

production2.00E-06

3.50E-03

LTBR

0.00E+00

rs10849448

2.35E-35

TNFSF13B

0.00E+00

rs200748895

1.20E-17

TNFRSF13B

0.00E+00

rs34557412

2.66E-21

CD40

3.19E-10

rs6032664

7.31E-12

ICOS

1.40E-03

rs231779

1.52E-06

CXCR4

4.75E-03

rs2680880

6.91E-06

CCL27

7.23E-03

rs12350154

6.71E-06

TNFRSF17

8.69E-03

rs456178

1.13E-05

IL6

9.25E-03

rs57561814

7.45E-06

IL15RA

1.63E-02

rs11256464

6.99E-06

TACIandBCMA

stimulationofB

9.00E-04

7.80E-03

TNFSF13B

0.00E+00

rs200748895

1.20E-17

TNFRSF13B

0.00E+00

rs34557412

2.66E-21


https://doi.org/10.1101/073056


53

cellimmune

responses.NFKB1

9.08E-14

rs230523

4.54E-14

TNFRSF17

8.69E-03

rs456178

1.13E-05

TRAF2

1.21E-02

rs17250694

1.56E-05

Ceramide

signalingpathway

3.00E-04

7.95E-03

TNFRSF1A

0.00E+00

rs10849448

2.35E-35

NFKB1

9.08E-14

rs230523

4.54E-14

MAPK3

3.78E-06

rs12931792

4.06E-08

MAP2K2

4.75E-04

rs350906

5.34E-07

MYC

1.13E-03

rs13279820

1.44E-06

TRAF2

1.21E-02

rs17250694

1.56E-05

MADD

3.01E-02

rs2856661

3.76E-05

CTSD

3.13E-02

rs7929314

3.17E-05

CASP8

3.15E-02

rs190209406

3.72E-05

Glucocorticoidreceptorregulatorynetw

ork

5.00E-04

9.93E-03

NFKB1

9.08E-14

rs230523

4.54E-14

MAPK3

3.78E-06

rs12931792

4.06E-08

NCOA2

9.50E-04

rs1481046

5.65E-07

SELE

1.66E-03

rs6691453

5.38E-07

BGLAP

1.94E-03

rs2075165

2.52E-06

IL6

9.25E-03

rs57561814

7.45E-06

MAPK11

1.20E-02

rs4076566

1.23E-05

SMARCA4

1.41E-02

rs9797720

1.58E-05

SPI1

2.48E-02

rs11604680

3.38E-05

FKBP5

3.06E-02

rs74853132

1.68E-05

ICAM1

3.12E-02

rs74956615

4.07E-05

PCK2

3.34E-02

14:24604399:AAC_A

5.33E-05

Themostsignific


signific



https://doi.org/10.1101/073056


54

Table6:MEGAN

TAPathwayAnalysisonChildhoodEarInfectionsGW

AS

Pathway

Pathway.

PvaluePathw

ay.FD

RGene

Gene.Pvalue

SNP

SNP.Pvalue

Catalysisofthetransferof

organicanionsfrom

onesideofamem

branetotheother

9.00E-04

0.0062

SLCO1A2

1.44E-03

rs10841784

2.76E-06

SLCO1B1

3.45E-03

rs2417971

5.67E-06

ABCC3

4.74E-03

rs186049592

1.82E-05

SLC22A6

3.43E-02

rs67754977

1.20E-04

SLC22A8

3.80E-02

rs67754977

1.20E-04

Themostsignific


signific



https://doi.org/10.1101/073056


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