GenomicsimplicatesadaptiveandinnateimmunityinAlzheimer’sandParkinson’sdiseases

Runninghead:ImmunesystemandneurodegenerativediseasesAuthors:SarahAGagliano,PhD1,2,3JennieGPouget,PhD2,3,4JohnHardy,PhD5JoKnight,PhD6,2,3,4,*MichaelRBarnes,PhD7,*MinaRyten,MBPhD5,*MichaelEWeale,PhD1,*Affiliations:1:King'sCollegeLondon,Dept.ofMedical&MolecularGenetics8thFloor,TowerWing,Guy'sHospital,LondonSE19RT,UnitedKingdom2:CampbellFamilyMentalHealthResearchInstitute,CentreforAddictionandMentalHealth,250CollegeStreet,Toronto,ON,M5T1R8,Canada3:InstituteofMedicalScience,UniversityofToronto,1King’sCollegeCircle,Room2374,Toronto,ON,M5S1A8,Canada4:DepartmentofPsychiatry,UniversityofToronto,250CollegeStreet,8thFloor,Toronto,ON,M5T1R8,Canada5:InstituteofNeurology,UniversityCollegeLondon,QueenSquare,LondonWC1N3BG,UnitedKingdom6:DataScienceInstituteandFacultyofHealthandMedicine,FurnessCollege,LancasterUniversity,Lancaster,LA14YG,UnitedKingdom7:WilliamHarveyResearchInstitute,BartsandTheLondonSchoolofMedicineandDentistry,QueenMaryUniversityofLondon,CharterhouseSquare,LondonEC1M6BQ,UnitedKingdom*Equalauthorship Correspondingauthor:SarahAGaglianogsarah6221@gmail.comCharactersinthetitle:79Charactersintherunninghead:43WordsintheAbstract:234WordsinIntroduction:538WordsinDiscussion:695WordsinTotal(excludingabstract,references&figlegends):3,795Numberoffigures:2Numberofcolourfigures:2Numberoftables:1

AbstractObjectives:Weassessedthecurrentgeneticevidencefortheinvolvementofvariouscelltypesandtissuetypesintheaetiologyofneurodegenerativediseases,especiallyinrelationtotheneuroinflammatoryhypothesisofneurodegenerativediseases.Methods:Weobtainedlarge-scalegenome-wideassociationstudy(GWAS)summarystatisticsfromParkinson’sdisease(PD),Alzheimer’sdisease(AD),andamyotrophiclateralsclerosis(ALS).Weusedmultiplesclerosis(MS),anautoimmunediseaseofthecentralnervoussystem,asapositivecontrol.WeappliedstratifiedLDscoreregressiontodetermineiffunctionalmarksforcelltypeandtissueactivity,andgenesetlistswereenrichedforgeneticheritability.Wecomparedourresultstothosefromtwogene-setenrichmentmethods(IngenuityPathwayAnalysisandenrichr).Results:Therewerenosignificantheritabilityenrichmentsforannotationsmarkinggenesactivewithinbrainregions,buttherewereforannotationsmarkinggenesactivewithincell-typesthatformpartofboththeinnateandadaptiveimmunesystems.WefoundthisforMS(asexpected)andalsoforADandPD.Thestrongestsignalswerefromtheadaptiveimmunesystem(e.g.Tcells)forPD,andfromboththeadaptive(e.g.Tcells)andinnate(e.g.CD14:amarkerformonocytes,andCD15:amarkerforneutrophils)immunesystemsforAD.AnnotationsfromtheliverwerealsosignificantforAD.Pathwayanalysisprovidedcomplementaryresults.Interpretation:ForAlzheimer’sandParkinson’sdisease,wefoundsignificantenrichmentofheritabilityinannotationsmarkinggeneactivityinimmunecells.IntroductionNeurodegenerativediseases–includingAlzheimer’s(AD),amyotrophiclateralsclerosis(ALS),andParkinson’sdisease(PD)–arepersonallydevastatingandanincreasingburdenonhealth-caresystemsworldwide.Recentlytherehasbeenmuchprogressinidentifyinggeneticvariantsassociatedwithneurodegenerativediseases.InthelatestADmeta-analysis19lociinadditiontothewell-establishedAPOElocuswerepinpointed.1ThelatestALSmeta-analysisidentifiedthreeALS-associatedloci2andthelatestPDmeta-analysisbroughtthetotalnumberofestablishedPDlocito26.3Despiteprogressinidentifyinggenetichitsintheseneurodegenerativediseases,theunderlyingprocessesorcell-typesmediatingthepathologyremainuncertain.Asgenome-wideassociationstudies(GWASs)havegrowninsizeandpower,sohasthequalityandscopeoffunctionalinformationthatcanbeusedtoannotatethegenomewithrelevantgenomicandepigenomicmarkslinkedtotheregulationofgeneexpression.Previousstudieshavedemonstratedenrichmentofdisease-associatedvariants(fornumerousdiseases)withfunctionalgenomicannotations,includingDNaseIhypersensitivesites,transcriptionfactorbindingsites,histonemodifications,andexpressionquantitativetraitloci(eQTLs).4–7Theseannotationsvarydependingoncell/tissue-type.Giventhemanywaysinwhichcomplexdiseases

arise,andforhumanbraindiseases,thewell-recognizedcellularheterogeneityofthebrain,pinpointingcell-typesofinterestisimportanttofurtherunderstandpathogenicity.Effortstoobtainbrainsamples(themostobviouslyrelevanttissueforneurodegenerativediseases)foreQTLanalysesareongoing.8–13Therehasbeenarecentproliferationintheavailabilityofcell-typeandtissue-specificannotations,includingbraintissue,forexamplethroughtheRoadmapEpigenomicsProject14andthePsychEncodeProject.11Nevertheless,obtaininglargenumbersofpost-mortemhumanbrainsremainschallenging,andcurrenteQTLanalysesarelikelytobeunderpowered.CharacterizationofeQTLsandDNAregulatoryelementsinbloodisacomplementaryapproach.Theneuroinflammatoryhypothesisofneurodegenerativediseasespositsthatdysregulationoftheimmunesystemisanimportantfactorintheaetiologyofthesediseases.15,16Thereislittledoubtthatmultiplesclerosis(MS)isanimmune-mediateddisease1718,19Wethereforeusethisdiseaseasapositivecontrolwithregardtoexpectedenrichmentinheritabilityforannotationsfromimmunecells.Thereisextensivefunctionalandclinicalevidencethatimmunedysfunctionplaysakeyroleinthepathogenesisoftherelapse-remittingphaseofMS.20,21ForAD,Yokoyamaetal.22showedthateightvariantswereassociatedwithbothADandimmune-mediateddiseases,andthereisfurtherevidencefrompathwayanalysis1,23,24andfromanimalmodels.25ForPD,theroleoftheimmunesystemhasbeensuggestedthroughpathwayanalysis26,27,animalmodels28,andvariantsintheHLAregionreachingstatisticalsignificanceingenome-wideassociationstudies.3,29ForALS,thereisevidenceofimmuneabnormalities.30Nevertheless,theextenttowhichtheimmunesystemisinvolvedinneurodegenerativediseasessuchasAD,ALSandPD,andthepotentialrolesplayedbytheinnateandadaptiveimmunecomponents,remainunestablished.Finucaneetal.31introducedstratifiedLDscoreregressionasamethodforpartitioningtheinferredheritabilityfromGWASsummarystatistics.Theydeterminedwhethergeneticheritabilityin17GWASswasenrichedwithinvariousfunctionalannotationswhichreflectedpartsofthegenomethatwereactiveinanumberoftissuesandcell-types.Weappliedthismethodologytofourdiseases(MS,AD,ALS,PD)totestforenrichmentofheritability,bothusingFinucaneetal.’s31cell-typegroupannotationsandusingadditionalannotationsfrombrainandimmunecellsandfrompublishedsetsofbrainandimmune-relatedgenes.32

MethodsWeobtainedGWASsummarystatisticsforthreeneurodegenerativediseases:AD,1ALS2andPD.3WeusedMS33asapositivecontrol,asitisadiseaseaffectingthebrainwithknownimmuneaetiology.AllstudieswereconductedinEuropeanpopulations,andaresummarizedinTable1.ForAD,whichisatwo-stagestudy,weonlyuseddatafromthefirststage.(SeeBox1fordetailsonthisstudy.)WedidnotstudyHuntington’sdisease(whichhasothergeneticmodifiersinadditiontotheprimaryHTTlocus)andfrontotemporaldementia,becausethecurrentGWASsamplesizes

forthesediseasesaremodest,andthusthedatasetswereconsideredtobeinsufficientlypoweredforouranalyses.31Box1.InternationalGenomicsofAlzheimer'sProject(IGAP)isalargetwo-stagestudybasedupongenome-wideassociationstudies(GWAS)onindividualsofEuropeanancestry.Instage1,IGAPusedgenotypedandimputeddataon7,055,881singlenucleotidepolymorphisms(SNPs)tometa-analysefourpreviously-publishedGWASdatasetsconsistingof17,008Alzheimer'sdiseasecasesand37,154controls(TheEuropeanAlzheimer'sdiseaseInitiative–EADItheAlzheimerDiseaseGeneticsConsortium–ADGCTheCohortsforHeartandAgingResearchinGenomicEpidemiologyconsortium–CHARGETheGeneticandEnvironmentalRiskinADconsortium–GERAD).Instage2,11,632SNPsweregenotypedandtestedforassociationinanindependentsetof8,572Alzheimer'sdiseasecasesand11,312controls.Finally,ameta-analysiswasperformedcombiningresultsfromstages1&2.Weestimatedpairwisegeneticcorrelationsamongthefourdiseasesusingcross-traitLDscoreregression.34WethenappliedstratifiedLDscoreregressiontodetermineifvariousfunctionalcategories(cell-typegroups,annotationsatthetissue/celllevelforbrainorimmunecells,andsetsofbrainandimmunegenelists)wereenrichedforheritability.LDscoreregressionexploitstheexpectedrelationshipsbetweentrueassociationsignalsandlocalLDaroundthemtocorrectoutsystematicbiasesandarriveatunbiasedestimatesofgeneticheritabilitywithinagivensetofSNPs(herestratifiedaccordingtotheirfunctionalcategory).31FollowingFinucaneetal.31,weaddedannotationsindividuallytothebaselinemodel;weusedHapMapProjectPhase3SNPsfortheregressionand1000GenomesProjectEuropeanpopulationSNPsforthereferencepanel;weonlypartitionedtheheritabilityofSNPswithminorallelefrequency>5%;andweexcludedtheMHCregionfromanalysis.ThehighLDandstrongassociationsignalswithintheMHCregionresultinadominatingeffectonLDscoreregression,andforthepurposesofouranalysesexcludingthisregionresultsinaconservativeapproach.Thegroupedcell-typeannotationsprovidedbyFinucaneetal.31aretheunionofhistonemarksfor10broadcategoriesincludingcentralnervoussystem(CNS),cardiovascular,immune/hematopoietic,andliver.FortheseanalyseswecorrectedformultipletestingoffourGWASsacross10cell-typegroups(4x10=40hypothesestested),resultinginaBonferronisignificancethresholdofp=1.2x10-3.WethenextendedtheanalyticalapproachofFinucaneetal.31inthefollowingways.Firstly,weobtainedadditionalannotationinformation.WeobtainedhistonemarksandDNaseIhypersensitivesitesdatafromtheRoadmapEpigenomicsConsortium;14weobtainedeQTLsderivedfrombrainregionsfromtheUKBrainExpressionConsortium10andtheGTExConsortium;9andweobtainedpromotercaptureHiCarrayexpressdatainCD34(amarkerofimmaturehematopoieticcells)cellsfromGM12878(reference:E-MTAB-2323).35Wealsoconsideredtwogene-

sets.AlltheseannotationsarelistedinSupplementaryTable1.Secondly,inordertoreducethemultipletestingburden,wecombinedinformationacrossthefourdifferenthistonemarksandtheDNaseIhypersensitivemarksinordertocreateanaggregatesetofregulatorymarksforeachcelltype.Thisaggregationannotationwasobtainedbasedonasimpleunionoperation:foreachtissueorcell-type,aSNPwaslabeledas‘annotated’basedonwhetheritpossessedanyrelevanthistonemarkorDNaseIhypersensitivemark.BothDNaseIandhistonemarksareknowntoreflectactiveregionsofthegenome,motivatingtheiraggregationinordertocreateageneralmarkofgenomicactivity.DNaseIsitesareassociatedwithanopenchromatinstructure,anddifferenthistonemarksaremarkersofactivepromoters(H3K4Me3+H3K27Ac)oractiveenhancers(H3K4Me1+H3K27Ac)regions.Forbraintissue,wedefinedaunionsetofhistonemarksplusDNaseIhypersensitivesitesfromtheRoadmapEpigenomicsConsortium14usingthesameaggregationprocedureasabove.Thisprocessingresultedinoneannotationperbrainregion(10annotations).WegroupedeQTLsacrossallbrainregions,buttreatedtheeQTLsfromtheUKBrainExpressionConsortium10andtheGTExConsortium9separately(resultingintwoannotations).BoththeGTExandUKBECanalysesincludedbrainregionshighlyrelevanttoMS,ADandPD,namelywhitematter,hippocampus,temporalcortexandsubstantianigra.Amongstspecificimmunecellsweassessedthehistonemarkspreviouslydescribed,31andalsothehistonemarksandDNaseIhypersensitivesitedatafromtheRoadmapEpigenomicsConsortiumforimmuneandbloodcells,14andwetooktheunionforeachcell-typeasdescribedabove(threehistonemarksandDNaseIhypersensitivesite).Thisresultedin20annotationsfromFinucaneetal.31and14annotationsfromRoadmap.Additionally,wedefinedfourimmunecell-typeannotationsbasedonpromotercaptureHiCarrayexpressdatainCD34fromGM12878(reference:E-MTAB-2323).35Thedataforthepreyandbaitwereanalysedseparatelyforinteractionsbetweencapturedpromoterandcapturedpromoterinteractionsandforcapturedpromoterandallotherregions,whichresultedinfourannotations.Theabovecell/tissue-typespecificannotationsresultedinamultipletestingcorrectionforfourGWASsacross50(12brain+38immune)annotations(4x50=204hypothesestested).ThuswesetaBonferronisignificancethresholdofp=2.5x10-4fortheseanalyses.Notethattherearecorrelationswithintheimmuneandbrainannotations,makingourBonferronicorrectionsomewhatconservativeWealsoappliedheritabilityenrichmentanalysistotwosetsofgenes;onewithknownbrainandonewithknownimmunefunction.Weusedabraingenelistof2,635genespreviouslydescribedbyRaychaudhurietal.36,andanimmunegenelistof973genespreviouslydescribedbyPougetetal.32Braingenesweredefinedas

thosefulfillinganyofthefollowingcriteria:preferentialexpressioninthebraincomparedtoothertissues,“neural-activity”annotationinPanther,“learning”annotationinIngenuity,and“synapse”annotationinGeneOntology.Immunegenesweredefinedasthosewithan“immuneresponse”annotationinatleastthreeofthefollowingdatabases:KyotoEncyclopediaofGenesandGenomes,GeneOntology,Ingenuity,andImmunologyDatabaseandAnalysisPortal.SNPswereannotatedtogenesusinga50kbwindow,andabaselinelistofallgenesusingthis50kbwindowwasincludedinthemodelaspreviouslydescribed.32Finally,wecontrastedtheaboveheritabilityenrichmentanalyseswithacomplementaryapproachbasedongene-setenrichmentanalysis.WeusedIngenuityPathwayAnalysis(IPA)(www.ingenuity.com)toidentifypathwayenrichmentamonggenesassociatedwithdifferentneurologicaltraitsforcanonicalpathways.Canonicalpathwaysarestructuredpathways.DatafromthedifferentphenotypeswereintegratedandsubjectedtonetworkanalysisviaIPAtoidentifypathwayenrichment.Enrichednetworksareorderedby–logp-value,basedonaFisherExacttestp-value.37ForeachdiseaseweincludedSNPswithap-value<5x10-4,andexcludedSNPsintheMHCregionduetothelongstretchesofLDinthisregion.WealsoperformedapathwayanalysislookingatKEGGpathwaysusingenrichr38,39inordertocompareresults.ResultsThereislimitedevidenceofpairwisegeneticcorrelationamongthefourdiseasesusingcross-traitLDscoreregression.ThelackofanAD-PDpairwisecorrelationhasalreadybeenreported,aswellasbetweenAD-MSandPD-MS.40WealsofoundnostatisticallysignificantevidenceforgeneticcorrelationbetweenALS-AD(0.2,p=0.08),ALS-PD(-0.08,p=0.01),andALS-MS(-0.04,p=0.7).Forthegroupedcell-typeanalysisfromFinucaneetal.,31themostsignificantenrichmentwasseenfortheimmune/hematopoieticcategoryforMS(10.1,p=3.8x10-13),confirmingtherecognizedroleoftheimmunesysteminthisdisease.ThiscategorywasalsosignificantlyenrichedforheritabilityofAD(5.5,p=2.4x10-7),inadditiontoliver(10.5,p=1.1x10-5),andtheseADsignalsremainedsignificantevenaftertheremovalofAPOE(chr19:44905754-44909393)(5.5,p=2.5x10-7and10.5,p=1.1x10-5respectively).ForALSandPD,therewerenosignificantlyenrichedfunctionalcategories(Fig1).Atthetissuelevel,noneoftheenrichmentsweresignificantforthebrainannotations.ThemostsuggestivesignalwasfortheinferiortemporalregioninAD(4.9,p=6.6x10-4).Forthecell-specificimmuneannotationsassessedrelatingtoboththeinnateandadaptiveimmunesystems,therewassignificantenrichmentforMSheritabilityandtoalesserextentforADandPD.TherewasnoenrichmentofheritabilityforALS,thesmallestdatasetinourstudy(SupplementaryTable1,Fig2).StrongMSsignalsforheritabilityenrichmentwerefoundinallimmunecellcategories,

includingbothadaptiveandinnatecelltypes.SignificantADsignalswerefoundinallimmunecellcategoriesexceptforthenon-T-cell/non-B-cellcomponentoftheadaptiveimmunesystem.ForPD,onlytwoannotationspassedthemultipletestingthreshold:primaryThelpercellsPMA-IstimulatedandprimaryTregulatorycellsfromperipheralblood(5.2and5.4,respectively,p=0.0002forboth),butseveralotherimmuneannotationsweresuggestive.ConsistentwithpreviousapplicationsoftheLDscoreregressionmethod,weincludedtheannotationsseparatelyintheregressionmodel.Thismeansthatthatenrichmentsininnateimmunecellscouldinprinciplebeduetooverlapinannotationwithadaptiveimmunecell-types,andviceversa.Toassessthisissue,wedeterminedthedegreeofannotationoverlapbetweenallpairsofimmunecelltypesinourstudy(SupplementaryTable2).Wefoundthedegreeofoverlapbetweeninnateversusadaptivecellsrangedfrom0.06%(forCD14:amarkerformonocytesversusCD20:amarkerofBlymphocytes)to12%(forperipheralbloodmononuclearprimarycellversusprimaryTcellsfromcordblood),suggestingalargedegreeofindependencebetweenadaptiveandinnatecellmarks.Tofurtherinvestigatethisissue,wecarriedoutdeeperanalysesonarepresentativeadaptivecelltype(primaryTcellsfromcordblood)andarepresentativeinnatecelltype(CD15:amarkerforneutrophils),bothofwhichdisplayedstrongheritabilityenrichmentsignalsinAD.Theannotationoverlapbetweenthesetwocelltypeswas6.7%(SupplementaryTable2).WhenweincludedbothannotationssimultaneouslyintheLDscoreregressionmodel,wefoundthatbothcelllinesremainedsignificantlyenrichedforMS(22.0,p=7.4x10-20and17.2,2.3x10-5,respectively).Similarly,forAD,bothcelllinesremainedsignificantlyenriched(8.7,p=1.7x10-7and14.3,p=7.3x10-6,respectively).NeitherofthesecelllineshadreachedsignificanceforPDorALSinthemodelswheretheywereinputtedseparately,norweretheysignificantwhenincludedsimultaneouslyintothemodel.TheenrichmentresultsforprimaryTcellsfromcordbloodandCD15whenincludedsimultaneouslyinthemodelforPDare4.9,p=1.1x10-3and7.0,p=5.2x10-3,respectively;andforALS3.5,p=0.03and3.1,p=0.37respectively.Overall,theseanalysesprovideduswithre-assurancethatweweredetectingindependentsignalsinadaptiveversusinnateimmunecelltypes.Ourheritabilityenrichmentanalysiswithinbrain-relatedandimmune-relatedgenesetsalsoprovidedstrongevidenceforasignalintheimmunegeneset,andnotinthebraingeneset(SupplementaryTable1).Asexpected,thestrongestimmunegenesignalwasforMS(1.6,p=4.6x10-14).WehavepreviouslyreportedtheenrichmentofthisimmunegenelistinthesameMSdataset,usinganearlierversionofLDscoreregression.32TheimmunegenelistwasalsoenrichedforheritabilityinAD(5.2,p=4.8x10-4),andtheeffectsinPDandALSweresuggestivebutwouldnotsurvivemultipletestingcorrection(4.5,p=0.02and2.5,p=0.03,respectively).Thebraingenelistwasnotsignificantlyenrichedinanyoftheneurodegenerativediseasesassessed(amongtheotherthreediseasesenrichmentrangesfrom0.9to1.9,p>0.04forallthree).

Finally,wecomparedtheaboveresultstoanIngenuityIPApathwayenrichmentanalysis,bothwithincanonicalpathways(SupplementaryTable3a)andwithindiseases/biologicalfunctionsincludingcancer-relatedfunctions(SupplementaryTable3b).Wealsocomparedourresultstoanenrichrpathwayenrichmentanalysis(SupplementaryTable3c).Remarkably,fortheIPAcanonicalpathwayanalysis,allthesignificantpathwayssaveone("AldosteroneSignalinginEpithelialCells")werefoundtobeconnectedtoeitheradaptiveorinnateimmuneresponse.Specificexamplesincluded:inMS(e.g.Thelpercelldifferentiation,roleofmacrophages,fibroblastsandendothelialcellsinRA,Bcellreceptorsignaling,dendriticcellmaturation,PI3KsignalinginBlymphocytes,CD40signaling;PKCθsignalinginTlymphocytes,NF-κBactivationbyviruses);inPD(e.g.dendriticcellmaturation–sharedwithMS,graft-versus-hostdiseasesignaling,alteredTcellandBcellsignalinginrheumatoidarthritis);inAD(IL-8signaling,IL-12signalingandproductioninmacrophages,FcepsilonRIsignaling,Fcγreceptor-mediatedphagocytosisinmacrophagesandmonocytes,roleofpatternrecognitionreceptorsinrecognitionofbacteriaandviruses,naturalkillercellsignaling);andinALS(e.g.NF-κBsignaling).ThevalueoftheIPAmethodwasalsodemonstratedinprovidingsignificantsignalsforotherpathwayspreviouslyimplicatedinthepathogenesisofAD,includingCREBsignalinginneurons,41neuregulinsignaling,andErbBsignaling.42FortheIPAdiseases/biologicalfunctionsanalysis,variouscancerscameupasmoststronglysignificantforallthedisorders.Cancerhasbeenshowntobecorrelatedwithmultipleimmunedisorders43,andthereisevidenceofcancerandneurodegenerativedisorders,suchasPD,sharingcommonpathways.44Theenrichranalysisrevealedmanysignificantimmune-relatedpathways,inlinewiththeIPAcanonicalpathwaysanalysis.DiscussionMultiplelinesofevidencesuggestasignificantcontributionofvariantsexhibitingfunctionalmarksforchromatinaccessibility(i.e.histonemarks,DNaseIhypersensitivesites)inimmunecelltypestotheheritabilityoftwoneurodegenerativediseases,namelyADandPD.AnnotationsfromimmunecellsaremostsignificantlyenrichedfortheheritabilityofMS,aknownautoimmunediseasewhichactedasapositivecontrolinourinvestigations.17ImmuneannotationsarealsoconsistentlyenrichedbuttoalesserdegreeforAD(withinvolvementfromboththeinnateandadaptiveimmunesystems),andsomecell-specificimmuneannotations(T-cells)weresignificantlyenrichedforPD.AlackofresultsfromtheALSdatasetcouldbeattributedtothisdatasetbeingsmallerthantheotherdatasetsinvestigated(Table1).Theseresultsprovidefurthersupportfortheneuroinflammatoryhypothesisofneurodegenerativedisease,15,16andhighlightthepotentialutilityofimmunemodulatingagents,suchasthosecurrentlyusedinMSforthetreatmentofADandPD.However,oneneedstobecautiouswithinterpretingthesecell/tissue-typespecificresultsintheabsenceoffunctionalandotherstudies.Wenotethatifwecorrectforthe17GWASsassessedinFinucaneetal.31aswellasthefourGWASsweassessedhereforthe10cell-typegroups((17+4)x10=210

hypothesestested),boththeimmune/hematopoieticandlivercategoriesremainsignificantforAD.TheroleoftheimmunesysteminADpathogenicityhasbeenpreviouslyshown22,25andpreviouspathwayanalysisoftheADGWASweassessedhereshowedenrichmentinimmune-relatedpathways.24Findingsarestrongestfortheinnateimmuneresponse,forinstanceassociationwiththeTREM2gene,whichinbraincellsareprimarilyexpressedonmicroglia.45,46OurfindingsfurthersupporttheroleofimmunevariationinADsusceptibility.Interestingly,usingLDscoreregression,ADwasfoundtobenotsignificantlycorrelatedwithavarietyofimmunediseases.34Thislackofcorrelationcouldbebecausewhenconsideringtheentiregenomethesignalcomingfromthecorrelatedlocibetweenthediseasesisdiluted,ortheimmunevariantsinvolvedinADaredifferentfromthoseinvolvedinotherimmunediseases.Microglia,themainimmunecelltypeinthebrain,haveadifferentdevelopmentaltrajectoryseparatefromtheperipheralimmunesystem.47Theuniquemechanismsofimmunesurveillanceinthebrain48,49alsomakesimmunediseasesofthebrainbiologicallydistincttoperipheralimmunediseases,butthereismuchevidencethatdisruptionofthebrain’simmunesurveillanceiscriticaltothe“viciouscycle”ofworseningpathologyseeninneurodegeneration.50Ouranalysissuggestsapredominantlyepigenomicmechanismforimmunedysregulationinneurodegenerativedisease,andifconfirmedthismaybeoftherapeuticrelevance,asmanydrugsareknowntoactthroughthismechanism.Some,suchashistonedeacetylaseinhibitors,couldpotentiallybeefficaciousinneurodegenerativediseases.51FunctionalmarksfromliverwerealsoenrichedfortheheritabilityofAD.ThisresultagreeswithfindingsintheliteratureofthecontributionoflipidmetabolismthroughliverXreceptors(LXR)totheinitiationandprogressionofthisdisease.52,53CanonicalpathwayanalysisshowedenrichmentofADassociationsinCREBsignalinginneurons,andalsoIL8andIL12signaling(whichareCREBregulated),supportingtheimmunehypothesisinAD,andpointingtointerleukinsignalingasapotentialCREB-responsivemechanism.54Ourresultsdonotprovidestatisticallysignificantevidencethatvariantsoverlappingwithfunctionalannotationsfromthebraincontributeexcessivelytotheheritabilityofneurodegenerativediseases.Thebraindoubtlessplaysanimportantroleinthegeneticaetiologyofthesediseases.Thelackofbrainannotationenrichmentcouldbeduetodatabeingbasedonfewsamplesforthebrain.Furthermore,thebrainisaveryheterogeneoustissue.Datafrombrainregionscontainamixtureofdifferentcelltypessuchasmicrogliaandneurons.Singlecellsamplingmayreducethisheterogeneityinthefuture.55Thisanalysisshouldberevisitedasbrainannotationinformationimproves.

Insummary,ourresultssuggestasignificantcontributionofvariantsthatexhibitchromatinaccessibilitymarksinimmunecellstotheheritabilityoftwoneurodegenerativediseases,namelyADandPD.AcknowledgementsWewouldliketothankAndySingletonandMikeNallsforaccesstotheParkinson’sdiseasesummarystatistics.WewouldalsoliketothankIsabellaFoghandJohnPowellforprovidingaccesstotheALSsummarystatistics.TheSLAGEN(ItalianConsortiumfortheGeneticsofALS)andALSGEN(InternationalConsortiumofAmyotrophicLateralSclerosisGenetics)ConsortiacontributedALS/controlssamples.ThemultiplesclerosissummarystatisticswereaccessedthroughImmunobase(https://www.immunobase.org/).WethankBorbalaMifsudforadviceonHiCdatasets.WealsothankJayneDanskaforinputontheimmuneinterpretation.Fundingforthiswork:SAGwasfundedthroughtheWestonBrainInstituteInternationalFellowshipinNeuroscience.JGPwasfundedbyFulbrightCanada,WestonCanada,andBrainCanadathroughtheCanadaBrainResearchFund,apublic-privatepartnershipestablishedbytheGovernmentofCanada.MRBwasfundedthroughtheNationalInstitutesforHealthResearch(NIHR)aspartoftheportfoliooftranslationalresearchoftheNIHRBiomedicalResearchUnitatBarts.JH,MRandMEWwerefundedthroughMedicalResearchCouncil(MRC)grantnumberG0901254/1.WethanktheInternationalGenomicsofAlzheimer'sProject(IGAP)forprovidingsummaryresultsdatafortheseanalyses.TheinvestigatorswithinIGAPcontributedtothedesignandimplementationofIGAPand/orprovideddatabutdidnotparticipateinanalysisorwritingofthisreport.IGAPwasmadepossiblebythegenerousparticipationofthecontrolsubjects,thepatients,andtheirfamilies.Thei–SelectchipswasfundedbytheFrenchNationalFoundationonAlzheimer'sdiseaseandrelateddiseases.EADIwassupportedbytheLABEX(laboratoryofexcellenceprograminvestmentforthefuture)DISTALZgrant,Inserm,InstitutPasteurdeLille,UniversitédeLille2andtheLilleUniversityHospital.GERADwassupportedbytheMedicalResearchCouncil(Grantn°503480),Alzheimer'sResearchUK(Grantn°503176),theWellcomeTrust(Grantn°082604/2/07/Z)andGermanFederalMinistryofEducationandResearch(BMBF):CompetenceNetworkDementia(CND)grantn°01GI0102,01GI0711,01GI0420.CHARGEwaspartlysupportedbytheNIH/NIAgrantR01AG033193andtheNIAAG081220andAGEScontractN01–AG–12100,theNHLBIgrantR01HL105756,theIcelandicHeartAssociation,andtheErasmusMedicalCenterandErasmusUniversity.ADGCwassupportedbytheNIH/NIAgrants:U01AG032984,U24AG021886,U01AG016976,andtheAlzheimer'sAssociationgrantADGC–10–196728.AuthorContributionsS.A.G.,M.R.,M.R.B.,J.K.,andM.E.W.areresponsibleforconceptandstudydesign.S.A.G.analyseddata,M.R.B.conductedthepathwayanalysis,andJ.G.P.analyseddata

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Figures

Fig1.Enrichmentofcell-typegroupsasusedinFinucaneetal.2015.Theblackdashedlinesat-log10(P)=2.9isthecutoffforBonferronisignificance.

Fig2.Enrichmentofimmunecellannotations.Theblackdashedlinesat-log10(P)=3.6isthecutoffforBonferronisignificance.Whitebars=tissue;purplebars=CD34(markerofimmaturehematopoieticcells-notstrictlyadaptiveorinnate);lightbluebars=markerofTcells;darkbluebar=markerofBcells;royalbluebars=cellsoftheadaptiveimmunesystem;pinkbars=cellsoftheinnateimmunesystemTablesTable1.DescriptionoftheGWASssummarystatisticsNeurodegenerativedisease

PMID Cases Controls Cohorts

Parkinson’sdisease(PD)

25064009

13,708 95,282 15

Alzheimer’sdisease(AD)

24162737 17,008 37,154 19

Amyotrophiclateralsclerosis(ALS)

24256812 7,177 8,393 8

Multiplesclerosis(MS)

21833088

9,772 17,376 23

Supplementaryfiles:SupplementaryTable1.Annotationenrichmentresults.RedcellsmarkenrichmentthatsurvivedBonferronicorrection.SupplementaryTable2.Overlapamongchromatinaccessibilityannotationsforimmunecells.Themaindiagonalshowsgenomecoverage(basepairs)forthatcelltype.Theupperoff-diagonalshowstheoverlapcoverage(basepairs)forthatcell-type-pair.Theloweroff-diagonalshowstheproportionofoverlapcoverageforthatcell-typepair.SupplementaryTable3.[a]IngenuityPathwayAnalysis(IPA)resultsforcanonicalpathways.RedcellsmarkenrichmentthatsurvivedBonferronicorrection.[b]IngenuityPathwayAnalysis(IPA)resultsforcancer-relatedfunctions.RedcellsmarkenrichmentthatsurvivedBonferronicorrection.[c]enrichrKEGGpathwayresults.Multiplicationofthep-valuecomputedusingtheFisherexacttestwiththez-scoreofthedeviationfromtheexpectedrankasdescribedintheenrichrpaper.38