stage 1 exome-chip genotyping in gerad/perades, adgc …...w ashu2 whicap combined or or 1.85 1.56...

Supplementary Figure 1

IGAP study design.

Stage 1 exome-chip genotyping in GERAD/PERADES, ADGC and CHARGE cohorts. Stage 2 genotyping and imputed genotyping of independent GERAD/PERADES, CHARGE and EADI samples. Stage 3 in silico analysis using ADGC HRC imputed GWAS data.

Nature Genetics: doi:10.1038/ng.3916


Quantile–quantile plots for the minimally adjusted (‘unadjusted’) model single-variant association analyses in SeqMeta.

(a) Plot showing all variants with minor allele count (MAC) ≥4, genomic inflation factor ( ) = 1.188. (b) Plot showing

variants with MAC ≥4 and MAF <0.05, genomic inflation factor ( ) = 1.066. (c) Plot showing variants with MAC ≥4 and

excluding all associations in the APOE region, genomic inflation factor ( ) = 1.102. (d) Plot showing variants with MAC ≥4,

with MAF <0.05, and excluding all associations in the APOE region, genomic inflation factor ( ) = 1.054. Although there is

some evidence of inflated association statistics, this inflation is consistent with inflation observed in previous exome array studies (Jeroen et al. Nat. Genet. 45, 197–201, 2013) and has been shown in multiple studies to be dramatically reduced with the exclusion of common variants, which on the exome array are mostly previously reported variants with strong associations in GWAS empaneled on the exome array, and regions of strong association, such as the APOE region in Alzheimer’s disease.


PLCG2−rs72824905−C

Study

ADC7

CHOP

Miami

NorthShore

WashU

AGES

CHS

FHS

RS

GERAD v1.0

GERAD v1.1

GERAD/PERADES

ACE

IGAP_Swe

IGAP_Finland

IGAP_Bel

IGAP_Aus_Ger

IGAP_Gre

IGAP_Italy

IGAP_Spa

IGAP_UK

IGAP_USA

IGAP_Can

EADI_France

RS1

RS2

ACT

ADC1

ADC2

ADC3

ADC6

ADNI

CHAP

GSK

LOAD

MAYO

MIRAGE

MTV

NBB

OHSU

ROSMAP

ROSMAP2

TARC1

TGEN2

UMVUMSSM_B

UPITT

WASHU

WASHU2

WHICAP

Combined OR

OR

0.74

0.57

1.11

0.62

0.58

0.47

0.8

0.58

0.73

0.52

0.93

0.8

0.9

0.5

11.98

0.66

0.54

2.42

0.25

0.84

1.04

0.56

0.4

0.84

0.8

0.37

0.76

4.61

0

0.34

0.3

0.4

0.25

0.44

0.79

0.75

0.24

1.57

0.18

2.92

0.6

0.7

0.53

0.53

0.92

0.85

0.08

0.3

0.28

0.68

0.50 0.80 1.0 1.25 2.0 3.0


Forest plot of rs72824905 (PLCG2) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

Cohort-specific odds ratios (ORs) are provided and denoted by blue squares, with 95% confidence intervals (CIs) denoted by blue lines. Individual study OR and combined OR are calculated using the SeqMeta package. The combined OR estimate for each cohort is represented by the blue diamond, where diamond width corresponds to 95% confidence interval bounds. Square and diamond heights are inversely proportional to the precision of the OR estimate.



PLCG2 conservation at human position 522 (p.P522R) across human, chimpanzee, rhesus monkey, mouse, rat, rabbit, horse, dog, and elephant.

The proline residue conserved across these species is indicated in red.



Regional plot of the conditional analysis performed at the PLCG2 locus in the stage 1 data set.

When conditioning on top hit rs72824905, indicated in purple, the most significant association is seen with rs200506549 (P = 6.52 × 10–4, MAF = 0.0019).


ABI3−rs616338−C

Study

ADC7

CHOP

Miami

NorthShore

WashU

AGES

CHS

FHS

RS

GERAD v1.0

GERAD v1.1

GERAD/PERADES

ACE

IGAP_Swe

IGAP_Finland

IGAP_Bel

IGAP_Aus_Ger

IGAP_Gre

IGAP_Italy

IGAP_Spa

IGAP_UK

IGAP_USA

IGAP_Can

EADI_France

RS1

RS2

ACT

ADC1

ADC2

ADC3

ADC6

ADNI

CHAP

GSK

LOAD

MAYO

MIRAGE

MTV

NBB

OHSU

ROSMAP

ROSMAP2

TARC1

TGEN2

UMVUMSSM_B

UPITT

WASHU

WASHU2

WHICAP

Combined OR

OR

2.29

1.82

2.12

1.06

1.17

1.78

0.79

0.3

1.75

1.28

1.99

1.21

1.82

1.54

2.3

0.69

1.42

6.68

1.75

1.35

1.52

1.32

3.18

1.47

1.17

0.56

0.29

0.37

33.87

3.74

0.24

2.86

0.1

1.69

2.6

1.71

0.22

1.46

1.85

6.35

83.91

8.79

1.01

1.12

1.71

1.51

0.26

2.64

1.43

0.50 0.80 1.0 1.25 2.0 3.0


Forest plot of rs616338 (ABI3) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

Cohort-specific odds ratios (ORs) are provided and denoted by blue squares, with 95% confidence intervals (CIs) denoted by blue lines. Individual study OR and combined OR are calculated using the SeqMeta package. The combined OR estimate for each cohort is represented by the blue diamond, where diamond width corresponds to 95% confidence interval bounds. Square and diamond heights are inversely proportional to the precision of the OR estimate.



ABI3 conservation at human position 209 (p.S209F) across multiple species.

The serine amino acid conserved across species is indicated in red. Note that the reference allele at this site encodes a phenylalanine and is the rare allele. The common allele encodes a serine and is shown for the human sequence above. The serine residue conserved across these species is indicated in red.



Regional plot of the conditional analysis performed at the ABI3 locus in the stage 1 data set.

When conditioning on the top hit rs616338, indicated in purple, the most significant association is seen with rs141826857 (P = 1.89 × 10–5, MAF = 0.00018).


TREM2−rs143332484−C

Study

ADC7

CHOP

Miami

NorthShore

WashU

AGES

CHS

FHS

RS

GERAD v1.0

GERAD v1.1

GERAD/PERADES

ACE

IGAP_Swe

IGAP_Finland

IGAP_Bel

IGAP_Aus_Ger

IGAP_Gre

IGAP_Italy

IGAP_Spa

IGAP_UK

IGAP_USA

IGAP_Can

EADI_France

RS1

RS2

ACT

ADC1

ADC2

ADC3

ADC6

ADNI

CHAP

GSK

LOAD

MAYO

MIRAGE

MTV

NBB

OHSU

ROSMAP

ROSMAP2

TARC1

TGEN2

UMVUMSSM_B

UPITT

WASHU

WASHU2

WHICAP

Combined OR

OR

1.85

1.56

3.2

1.4

1.32

0.34

0.94

2.33

1.11

1.51

2.06

3.51

7.92

1.73

4.27

0.99

2.49

2

3.42

17.61

1.75

1.04

1.72

0.31

8.42

1.7

3.81

0.34

0.67

1.34

3.01

0.64

1.7

7.82

>100

0.92

1.67

0.50 0.80 1.0 1.25 2.0 3.0


Forest plot of rs143332484 (TREM2) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

Cohort-specific odds ratios (ORs) are provided and denoted by blue squares, with 95% confidence intervals (CIs) denoted


by blue lines. Individual study OR and combined OR are calculated using the SeqMeta package. The combined OR estimate for each cohort is represented by the blue diamond, where diamond width corresponds to 95% confidence interval bounds. Square and diamond heights are inversely proportional to the precision of the OR estimate.


TREM2−rs75932628−C

Study

ADC7

CHOP

Miami

NorthShore

WashU

AGES

CHS

FHS

RS

GERAD v1.0

GERAD v1.1

GERAD/PERADES

ACE

IGAP_Swe

IGAP_Finland

IGAP_Bel

IGAP_Aus_Ger

IGAP_Gre

IGAP_Italy

IGAP_Spa

IGAP_UK

IGAP_USA

IGAP_Can

EADI_France

RS1

RS2

ACT

ADC1

ADC2

ADC3

ADC6

ADNI

CHAP

GSK

LOAD

MAYO

MIRAGE

MTV

NBB

OHSU

ROSMAP

ROSMAP2

TARC1

TGEN2

UMVUMSSM_B

UPITT

WASHU

WASHU2

WHICAP

Combined OR

OR

1.67

3.03

2.31

1.38

1.45

4.07

2.01

4.13

1.89

1.35

1.94

2.69

4.47

0.62

2.33

2.77

4.19

3.34

0.47

2.15

2.63

4.06

12.45

0.09

0.85

7.82

>100

8.13

1.68

2.59

0.79

3.03

0.78

0.14

2.01

3.35

3.53

7.86

0.74

85.11

2.94

2.46

0.50 0.80 1.0 1.25 2.0 3.0


Forest plot of rs75932628 (TREM2) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

Cohort-specific odds ratios (ORs) are provided and denoted by blue squares, with 95% confidence intervals (CIs) denoted by blue lines. Individual study OR and combined OR are calculated using the SeqMeta package. The combined OR estimate for each cohort is represented by the blue diamond, where diamond width


corresponds to 95% confidence interval bounds. Square and diamond heights are inversely proportional to the precision of the OR estimate.


b

a

c


Regional plots of the conditional analyses performed at the TREM2 locus in the stage 1 data set.

(a) When conditioning on rs75932628 (p.R47H), indicated in purple, the most significant association is seen with


rs143332484 (p.R62H) (P = 3.38 × 10–9). (b) When conditioning on rs143332484 (p.R62H), indicated in purple, the most significant association is seen with (rs75932628 (p.R47H) P = 5.12 × 10–12). (c) When conditioning on both rs143332484 (p.R62H) and rs75932628 (p.R47H), both indicated in purple, the most significant association is seen with rs143539514 (P = 1.51 × 10–3).



Gene expression profiles (RNA-seq) of PLCG2, ABI3, and TREM2 from transcriptome data from six cell types from human temporal lobe cortex (pink) and transcriptome data from seven cell types from mouse whole cortex (blue).

(a–c) Across species, PLCG2 (a), ABI3 (b), and TREM2 (c) show high expression in microglia/macrophage cells (Zhang et al. Neuron 89, 27–53, 2016). Figure downloaded from http://web.stanford.edu/group/barres_lab/brainseq2/brainseq2.html.


http://web.stanford.edu/group/barres_lab/brainseq2/brainseq2.html


Schematic of the PLCG2 protein from the Protein Data Bank.

The location of p.P522R upstream of a SH2 domain is indicated by the dashed blue line.



Schematic of the ABI3 protein from the Protein Data Bank.

The location of p.S209F is indicated by the dashed blue line.



Schematic of the TREM2 protein from the Protein Data Bank.

The location of p.R62H is indicated by the dashed blue line and that of p.R47H is indicated by the solid blue line.


1

RarecodingvariantsinPLCG2,ABI3andTREM2implicatemicroglial-mediatedinnateimmunityinAlzheimer’sdisease1. SampleCohorts…………………………………………………………………………….22. QualityControlandAnalyses………………………..….…………………………103. SingleVariantFindings…………………………………………………………………114. Gene-wideFindings………………………..….………………………..………….….125. GeneExpression………………………………………………………………………….126. FunctionalAnnotation………………………..….…………………………………..137. References………………………..….………………………………………………..…..158. SupplementaryTableLegends………………………..….……………………….23


2

1.SampleCohortsGERAD/PERADES:

Stage1:CasesandcontrolswererecruitedbytheMedicalResearchCouncil(MRC)GeneticResourceforLOAD(CardiffUniversity;InstituteofPsychiatry,London;UniversityofCambridge);theAlzheimer’sResearchUK(ARUK)Collaboration(UniversityofNottingham;UniversityofManchester;UniversityofSouthampton;UniversityofBristol;Queen’sUniversityBelfast);MRCPRIONUnit,UniversityCollegeLondon,UK;UniversityofOxford,UK;WashingtonUniversity,StLouis,UnitedStates;CompetenceNetworkofDementia(CND)andDepartmentofPsychiatry,UniversityofBonn,Germany;UniversityofHalle,Germany;UniversityHospital,Saarland,Germany;UniversityMedicalCentre,Hamburg,Germany;UniversityDulsburg-Essen,Germany;UniversidadAutônomadeMadrid,Spain;UniversidadAutônomadeBarcelona,Spain;UniversityofCantabriaandIDIVAL,Santander,Spain;UniversityofNavarra,Pamplona,Spain;SantaLuciaFoundation,Rome,Italy;AristotleUniversity,Thessaloniki,Greece;CIBERNED,Madrid,Spain;CSIC-UAM,Madrid,Spain;HospitalUniversitarioCentralAsturias,Oviedo,Spain.

Stage2:CasesandcontrolswererecruitedbytheMRCGeneticResourceforLOAD;

MRCPRIONUnit,UniversityCollegeLondon,UK;SantaLuciaFoundation,Rome,Italy;CIBERNED,Madrid,Spain;CSIC-UAM,Madrid,Spain;HospitalUniversitarioCentralAsturias,Oviedo,Spain;ARUKcollaboration;KingsCollegeLondon,London,UK;UniversityofPerugia,Perugia,Italy;CatholicUniversityofRome,Rome,Italy;RegionalNeurogeneticCentre(CRN),ASPCatanzaro,LameziaTerme,Italy;MemoryclinicandResearchCenter,InstitutCatalàdeNeurociènciesAplicades,Barcelona,Spain;UniversityofMilan,Milan,Italy;UniversityofBonn,Bonn,Germany;QueensUniversity,Belfast,NorthernIreland;UniversityofDuisburg-Essen,Germany;KlinikumderUniversitätMünchen,Munich,GermanyandGermanCenterforNeurodegenerativeDiseases(DZNE,Munich),Munich,Germany;UniversityofBristol,Bristol,UK;CardiffUniversity,Cardiff,UK;UniversityofSouthampton,SouthamptonUK;UniversityofNottingham,Nottingham,UK;MayoClinic,Jacksonville,Florida,USA.

Alllate-onsetAlzheimer’sdisease(LOAD)caseswereagedover60andmetcriteriaforeitherprobable(NINCDS-ADRDA,DSM-IV)ordefinite(CERAD)AD.AllelderlycontrolswerescreenedfordementiausingtheMiniMentalStateExamination(MMSE)orADAS-cog,weredeterminedtobefreefromdementiaatneuropathologicalexaminationorhadaBraakscoreof2.5orlower.Controlsampleswerechosentomatchcasesamplesforage,gender,ethnicityandCountryoforigin.Informedconsentwasobtainedforallresearchparticipants,andtherelevantindependentethicalcommitteesapprovedstudyprotocols.CHARGE:

Stage1:AgeGene/EnvironmentSusceptibility–Reykjavikstudy(AGES):TheAGESstudyhas

beendescribedpreviously1.Thestudywasinitiatedin2002toexaminegeneticsusceptibilityandgene/environmentinteractionsrelatedtodiseaseanddisabilityinoldage.TheAGESstudyiscomprisedof5764individualsdrawnfromtheReykjavikStudy,apopulation-basedcohortcomprisedofindividualsbornbetween1907and1935and


3

followedsince1967bytheIcelandicHeartAssociation.3219individualschosenrandomlyamong5307AGESindividualswith‘mid-life’dataavailablefromtheReykjavikStudyweregenotypedonagenome-wideassociation(GWA)array.2983werefurthergenotypedfortheEC.AgewascodedinyearswheretheageofcaseswastheageatthevisitwhereLOADwasfirstdiagnosedandtheageofcontrolswastheageatthelastvisitindividualwasstillfreeofLOADpathology.

DiagnosisofLOADinAGES–TheFolsteinMMSEandtheDigitSymbolSubstitutionTest(DSST)wereadministeredtoallparticipantsandpersonswhoscoredbelowapre-determinedthresholdonthesetests(≤23ontheMMSEor≤17ontheDSST)wereadministeredasecond,diagnostictestbattery.BasedonperformanceontheTrailsBandtheReyAuditoryVerbalLearningtest(RAVLT),asubsetoftheseindividualswithaRAVLTscore≤18orTrailsBscore≥8(ratiooftimetakenforTrailsB/TrailsAcorrectedforthenumbercorrect)wentontoathirdstep,whichincludedaneurologicalexaminationandastructuredinformantinterviewaboutmedicalhistoryandsocial,cognitive,anddailyfunctioning.MRIwasacquiredasapartofthecorestudyprotocol.Apanelthatincludedageriatrician,neurologist,neuropsychologist,andneuroradiologistreachedaconsensusdiagnosisofdementiabasedontheDiagnosticandStatisticalManualofMentalDisorders,FourthEdition(DSM-IV)guidelines2.Therewere319casesofdementiadiagnosedinthefirst5764AGESparticipantsandofthese123alsohadgenotypingandbrainMRI.Internationaldiagnosticguidelines,includingtheNationalInstituteofNeurologicalandCommunicativeDisordersandStroke–AlzheimerDiseaseandRelatedDisordersAssociation(NINCDS-ADRDA)criteriaforprobableandpossibleAlzheimerDiseaseandtheAlzheimer’sDiseaseDiagnosisandTreatmentCenter’s(ADDTC)StateofCaliforniacriteriaforprobableandpossiblevasculardementia(VaD)withorwithoutAD,werefollowed.TheAGESstudyidentified3subtypes:possible/probableADwithoutVaD(includedinanalysis),mixedAD(casesthatmetcriteriaforbothADandVaD,includedinanalysis),and,possible/probableVaDorotherdementiawithoutAD(excludedfromanalysis).3316individualsparticipatedinthefollow-upvisit(AGES-2)andwereexaminedusingthesameprotocolasusedduringtheAGES-1visitfordiagnosisofdementiaandAD.Controlswerethosestillfreeofdementiaandmildcognitiveimpairmentatlastassessment.Studyapproval–TheAGESstudywasapprovedbytheIcelandicNationalBioethicsCommittee(VSN00-063),andbytheNationalInstituteonAgingIntramuralInstitutionalReviewBoard.Informedconsentwasobtainedfromallparticipants.

CardiovascularHealthStudy(CHS):TheCHSisapopulation-basedcohortstudyofriskfactorsforcoronaryheartdiseaseandstrokeinadults≥65yearsconductedacrossfourfieldcenters3.TheoriginalpredominantlyCaucasiancohortof5201personswasrecruitedin1989-1990fromrandomsamplesoftheMedicareeligibilitylists;subsequently,anadditionalpredominantlyAfrican-Americancohortof687personswasenrolledforatotalsampleof5888.Bloodsamplesweredrawnonallparticipantsattheirbaselineexamination;DNAwasextractedfrombloodfromparticipantswhodonatedDNAsamplesforstorageandprovidedinformedconsentforparticipationinDNAstudies(~95%ofallCHSparticipants).AlthoughCHSisapopulation-basedsampleweempiricallyestimatedcrypticrelatednessbasedongenotypesofaLD-prunedsetofcommonECvariants.ForthisweusedPLINKv1.074(http://pngu.mgh.harvard.edu/purcell/plink/).Weidentifiedclustersof


4

individualswith‘PI_HAT’>0.15or‘Z0’<0.4(‘PI_HAT’istheempiricalestimateoftwicethekinshipcoefficientandZ0istheempiricalestimateoftheprobabilityofsharingzeroallelesidenticalbydecent).Amongtheseclusters,wekeptonlyoneindividualforanalysis,givingpreferencetocasesovercontrols.Covariatesinthemodelswereageinyears,sex,andfieldcenter.AgewastheageatLOADdiagnosisforcasesortheageatlastfollow-upevaluationforcontrols.

DiagnosisofLOADinCHS–TheADsampleforCHSincludedallprevalentcasesidentifiedin1992andincidenteventsidentifiedbetween1992andDecember2006.Briefly,personswereexaminedannuallyfromenrolmentto1999,andtheexaminationincludeda30minutesscreeningcognitivebattery5.In1992-94andagain,in1997-99,participantswereinvitedtoundergobrainMRIanddetailedcognitiveandneurologicalassessmentaspartoftheCHSCognitionStudy5.Personswithprevalentdementiawereidentified,andallotherswerefolloweduntil1999forthedevelopmentofincidentdementiaandAD.Sincethen,CHSparticipantsattheMarylandandPennsylvaniacentershaveremainedunderongoingdementiasurveillance6.Beginningin1988/89,allparticipantscompletedtheModifiedMini-MentalStateExamination(3MSE)andtheDSSTattheirannualvisits,andtheBentonVisualRetentionTest(BVRT)from1994to1998.TheTelephoneInterviewforCognitiveStatus(TICS)wasusedwhenparticipantsdidnotcometotheclinic.FurtherinformationoncognitionwasobtainedfromproxiesusingtheInformantQuestionnaireforCognitiveDeclineintheElderly(IQCODE),andthedementiaquestionnaire(DQ).SymptomsofdepressionweremeasuredwiththemodifiedversionoftheCenterforEpidemiologyStudiesDepressionScale(CES-D).In1991-94,3608participantshadanMRIofthebrainandthiswasrepeatedin1997-98.TheCHSstaffalsoobtainedinformationfromparticipantsandnext-of-kinregardingvisionandhearing,thecircumstancesoftheillness,historyofdementia,functionalstatus,pharmaceuticaldruguse,andalcoholconsumption.Dataoninstrumentalactivitiesofdailyliving(IADL),andactivitiesofdailyliving(ADL)werealsocollected.Personssuspectedtohavecognitiveimpairmentbasedonthescreeningtestslistedaboveunderwentaneuropsychologicalandaneurologicalevaluation.Theneuropsychologicalbatteryincludedthefollowingtests:theAmericanversionoftheNationalReadingtest(AMNART),Raven’sColouredProgressiveMatrices,CaliforniaVerbalLearningTest(CVLT),amodifiedRey-Osterreithfigure,theBostonNamingtest,theVerbalfluencytest,theBlockdesigntest,theTrailsAandBtests,theBaddeley&PapagnoDividedAttentionTask,theStroop,DigitSpanandGroovedPegboardTests.Theresultsoftheneuropsychologicalbatterywereclassifiedasnormalorabnormal(>1.5standarddeviationsbelowindividualsofcomparableageandeducation)basedonnormativedatacollectedfromasampleof250unimpairedsubjects.Theneurologicalexamincludedabriefmentalstatusexamination,aswellasacompleteexaminationofothersystems.TheexamineralsocompletedtheUnifiedParkinson’sDiseaseRatingScale(UPDRS)andtheHachinskiIschemicScale.Aftercompletingtheneurologicalexam,theneurologistclassifiedtheparticipantasnormal,havingmildcognitiveimpairment(MCI),ordementia.Internationaldiagnosticguidelines,includingtheNINCDS-ADRDAcriteriaforprobableandpossibleADandtheADDTC’sStateofCaliforniacriteriaforprobableandpossiblevasculardementia(VaD)withorwithoutAD,werefollowed.CHSidentified3subtypes:possible/probableADwithoutVaD(categorizedaspureAD,includedinanalysis)andmixedAD(forcasesthatmetcriteriafor


5

bothADandVaD,includedinanalysis),and,possible/probableVaDwithoutAD(excludedfromcurrentstudy).

FraminghamHeartStudy(FHS):TheFHSisathreegenerationalprospectivecohortthathasbeendescribedindetailpreviously7–9.Individualswereinitiallyrecruitedin1948inFramingham,MA,USAtoevaluatecardiovasculardiseaseriskfactors.Thesecond-generationcohort(5,124offspringoftheoriginalcohort)wasrecruitedbetween1971and1975.Thethird-generationcohort(4095grandchildrenoftheoriginalcohort)wascollectedbetween2002and2005.6946European-AmericanindividualsweregenotypedusingtheEC.Participants≤60yearsatthetimeofblooddrawforDNAextractionwereexcludedpriortoanalysis.Becausethestatisticaltestsuseddidnotaccountforfamilystructure,weexcludedrelatedparticipants.Usinggenome-wideidentity-by-descent,wefirstidentified7pairsofrelatedcases,andexcludedtheyoungerofthetwoineachpair,ortheonewiththemostmissingdata.Wethenexcluded151controlswhowererelatedtocases,andfinally,weexcluded439controlsrelatedtoothercontrols,applyingthesameage/missingdataruleasforrelatedcases.Covariatesusedwereageinyearsandsex,whereagewastheageatLOADdiagnosisforcasesortheageatlastfollow-upevaluationforcontrols.DiagnosisofLOADinFHS–FHSparticipantswerescreenedateachbiennialexaminationforpossiblecognitivedeclinethroughanumberofmechanisms,includingmeasuresoftheFolsteinMini-MentalStatusExamination(MMSE)10,referralbyFHSstaffandphysiciansatregularclinicexams,byself,familyorprimarycarephysician,referralfollowinghealthupdatesorancillarystudiesbyotherFHSworkinggroups,andreferralfromneuropsychologicaltestingincludedindedicatedproject.Participants“flagged”asbeingatriskfordevelopingdementiaunderwentcompleteneuropsychologicalevaluation.Iftheneuropsychologicaltestingorneurologicalevaluationsuggestedadeclineincognitivefunction,andothersourcesofdatacouldnotclarifyifthepersonhadMCIorAD,weadministeredastructuredfamilyinterview.Wethendeterminedwhethereachpersonfulfilledcriteriaforadiagnosisofdementia,theprobabledateofonset,andtypeofdementiaataconsensusreviewconductedbyapanelcomprisingatleastonebehaviouralneurologistandoneneuropsychologist.ParticipantswithdementiametcriteriaoutlinedintheFourtheditionoftheDiagnosticandStatisticalManualofMentalDisorders(DSM-IV)criteria2,andwererequiredtohavesymptomsforatleast6months.ParticipantswithADmetNINCDS-ADRDAcriteriafordefinite,probable,orpossibleAD11.

Rotterdamstudy(RS):TheRSisanongoingprospectivepopulation-basedcohortstudy,focusedonchronicdisablingconditionsoftheelderly12.ThestudycomprisesanoutbredethnicallyhomogenouspopulationofDutchCaucasianorigin.Therationaleofthestudyhasbeendescribedindetailelsewhere12.Insummary,7983menandwomenaged55yearsorolder,livinginOmmoord,asuburbofRotterdam,theNetherlands,wereinvitedtoparticipate.3163individualsweregenotypedfortheEC.Thiscohortwasextendedwith3,011participantswhohadbecome55yearsofageorhadmovedintothedistrictsincethestartofthestudy(RSII).

IntheRStherearesomesmallfamiliesduetoinclusionofparentsaswellaschildrenlivingbothinOmmoord.FrompairsofsubjectswithempiricalIBD>0.4onewasexcluded,withapreferenceofkeepingcases.Inthestage2in-silicoreplication,related


6

subjectswerealsoexcluded,withapreferencetokeepcasesovercontrols.Agewascodedinyearsforageofonsetforcasesandageatcensoringorageatlastscreeningforcontrols.DiagnosisofLOADinRS–IntheRSparticipantswerescreenedforprevalentdementiaatbaselineusingathree-stageprocessdescribedindetailelsewhere13.Thosefreeofdementiaremainedundersurveillanceforincidentdementia,adeterminationmadeusingrecordslinkageandassessmentatthreesubsequentre-examinations.WeincludedallprevalentcasesandallincidenteventsuptoJanuary1st2014.ScreeningwasdonewiththeFolsteinMini-MentalStatusExamination(MMSE)10andtheGeriatricMentalSchedule(GMS)14organiclevelforallpersons.Screen-positives(MMSE<26orGMSorganiclevel>0)underwenttheCAMDEX15.Personswhoweresuspectedofhavingdementiaunderwentmoreextensiveneuropsychologicaltesting.Whenavailable,imagingdatawereused.Inaddition,allparticipantshavebeencontinuouslymonitoredformajorevents(includingdementia)throughautomatedlinkageofthestudydatabasewithdigitizedmedicalrecordsfromgeneralpractitioners,theRegionalInstituteforOutpatientMentalHealthCareandthemunicipality.InadditionphysicianfilesfromnursinghomesandgeneralpractitionerrecordsofparticipantswhomovedoutoftheOmmoorddistrictwerereviewedtwiceayear.Forsuspecteddementiaevents,additionalinformation(includingneuroimaging)wasobtainedfromhospitalrecordsandresearchphysiciansdiscussedavailableinformationwithaneurologistexperiencedindementiadiagnosisandresearchtoverifyalldiagnoses.Dementiawasdiagnosedinaccordancewithinternationallyacceptedcriteriafordementia(DiagnosticandStatisticalManualofMentalDisorders,RevisedThirdEdition,DSM-III-R16),andADusingtheNINCDS-ADRDAcriteriaforpossible,probableanddefiniteAD11.TheNationalInstituteofNeurologicalDisordersandStroke–AssociationInternationalepourlaRechercheetl’EnseignementenNeurosciences(NINDSAIREN)criteriawereusedtodiagnosevasculardementia.Thefinaldiagnosiswasdeterminedbyapanelofaneurologist,neurophysiologist,andresearchphysicianandthediagnosesofADandVaDwerenotmutuallyexclusive.

StudyApproval–TheRotterdamStudyhasbeenapprovedbytheMedicalEthicsCommitteeoftheErasmusMCandbytheMinistryofHealth,WelfareandSportoftheNetherlandsimplementingtheWetBevolkingsonderzoek:ERGO(PopulationStudiesAct:RotterdamStudy).Allparticipantsprovidedwritteninformedconsenttoparticipateinthestudyandtoobtaininformationfromtheirtreatingphysicians.Datacanbeobtaineduponrequest.RequestsshouldbedirectedtowardsthemanagementteamoftheRotterdamStudy([email protected]),whichhasaprotocolforapprovingdatarequests.Becauseofrestrictionsbasedonprivacyregulationsandinformedconsentoftheparticipants,datacannotbemadefreelyavailableinapublicrepository.

Stage2:HRCimputeddataintheRotterdamStudy:TheRotterdamStudyIandRotterdam

StudyIIwereimputedtotheHaplotypeReferenceConsortiumreference(HRC)panel17,18.Imputationwasperformedontheweb-serviceprovidedbytheMichiganImputationserver(dateofpipeline17-12-2015).PreviouslydescribedgenotypeQCwasperformedpriortoimputations19.Inshortgenotypeswerepre-phasedwithSHAPEIT220andimputedusingMinimac3.Imputedgenotypeswithlowimputationquality(Rsq<0.5)wereexcluded.Subjectsincludedinthestage1analysiswereexcludedfromthestage2analysis.Inthe


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RotterdamStudyIIonlycontrolswithanage>75wereincludedtodecreasethecasetocontrolratio.

GenotypedData:Anadditional3,273case-controlsampleswereobtainedforreplicationfromcentersinAustria(1center)andSpain(1center).ClinicaldiagnosesofprobableADwereallestablishedaccordingtotheDSM-III-RandNINCDS-ADRDAcriteria.ControlsweredefinedassubjectswithoutDMS-III-Rdementiacriteriaandwithintegrityoftheircognitivefunctions(MMS>25).ADGC:

Stage1:CasesandcontrolsweretakenfrommultipleADGCdatasets21,22andpartitionedintofivesubsetsforgenotypingandsubsequentanalyses.Thefivesubsetsincluded:(1)7,091individualsselectedfrommultipleADGCdatasetsweregenotypedattheRobertS.BoasCenterforGenomicsandHumanGenetics,FeinsteinInstituteforMedicalResearch,Manhasset,NewYork(NorthShore);(2)2,024individualsfromtheADGC“UMVUMSSM”datasetweregenotypedattheJohnP.HussmanInstituteforHumanGenomics,UniversityofMiami,Miami,Florida(Miami);(3)1,374individualsfromtheADGC“WashU”datasetweregenotypedatWashingtonUniversity,St.Louis,Missouri(WashU);(4)6,082individualsfrommultipleAlzheimer’sDiseaseCenter(ADC)genotypingwavesweregenotypedattheCenterforAppliedGenomics,TheChildren’sHospitalofPhiladelphia,Philadelphia,Pennsylvania(CHOP);and(5)all1,528subjectsintheseventhwaveofADCsamplesweregenotypedatCHOP(ADC7).Perindividualsourcestudies,allsubjectswererecruitedunderprotocolsapprovedbytheappropriateInstitutionalReviewBoards.CaseslivingattimeofrecruitmentwereadjudicatedaspossibleorprobableADpriortoanalysesaccordingtoNINCDS/ADRDAcriteria11whereasaffectionstatusofalldeceasedcaseswasconfirmedthroughautopsy.Sampleswithage-at-onsetorage-at-examlessthan60years,missingcovariates,orcontrolswithMMSE<26werecensored.

Stage3:HRC-ImputedADGCGWASdatasets:Stage3replicationincludedgenotype

probabilitiesfromimputationtotheHaplotypeReferenceConsortium(HRC)referencepanels17,18onallADGCsamplesnotgenotypedontheexomechipandfromdatasetswithmorethan50samplesremainingafterexcludingexomechip-genotypedsamples.TheseincludedsamplesfromtheAdultChangesinThought(ACT)/ElectronicMedicalRecordsandGenetics(eMERGE)study;theNationalInstituteonAging(NIA)AlzheimerDiseaseCenters(ADCs)(waves1-3and6);theAlzheimerDiseaseNeuroimagingInitiative(ADNI)Study;theMulti-SiteCollaborativeStudyforGenotype-PhenotypeAssociationsinAlzheimer’sDisease(GenADA)Study;theUniversityofMiami/VanderbiltUniversity/Mt.SinaiSchoolofMedicine(UM/VU/MSSM);theMulti-InstitutionalResearchinAlzheimer'sGeneticEpidemiology(MIRAGE)Study;OregonHealthandScienceUniversity(OHSU);theNCRAD/NIA-LOADStudy;theTranslationalGenomicsResearchInstituteseries2(TGEN2)dataset;theMayoClinicJacksonville;theRushUniversityReligiousOrdersStudy/MemoryandAgingProject(ROSMAP)andChicagoHealthandAgingProject(CHAP);theUniversityofPittsburgh(UP);WashingtonUniversity(WU)inSt.Louis;theTexasAlzheimer’sResearchandCareConsortium(TARCC);theNetherlandsBrainBank(NBB);andtheWashingtonHeights-InwoodColumbiaAgingProject(WHICAP).Detaileddescriptionsofthe


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ascertainmentandevaluationofsubjectsintheACT/eMERGE,ADCwaves,ADNI,GenADA,UM/VU/MSSM,MIRAGE,OHSU,NCRAD/NIA-LOAD,TGEN2,Mayo,ROSMAP,UP,andWUcohortshavebeenprovidedelsewhere21,22;briefdescriptionsincludedherenoteanydifferencesbetweendatausedinthisstudyanddatausedinpreviousstudiesbytheADGCandIGAPstudy,includingshortsummariesoftheCHAP,TARCC,NBB,andWHICAPdatasets.AnalyseswererestrictedtoindividualsofEuropeanancestryduetotheinsufficientnumberofnon-EuropeansamplesavailableforimputationinHRC.AllsubjectswererecruitedunderprotocolsapprovedbytheappropriateInstitutionalReviewBoards.

ChicagoHealthandAgingProject(CHAP):CHAPisanon-goingcommunitybasedstudyofindividualsfromageographicallydefinedcommunityof3neighbourhoodsinChicago,Illinois(MorganPark,WashingtonHeights,andBeverly),with6,158participantsinthefirstphaseofthestudy(78.7%overall;80.5%oftheblacks,74.6%ofthewhites)23.Datawerecollectedincyclesofapproximately3years;eachconsistingofanin-homeinterviewofallparticipantsandclinicalevaluationofarandom,stratifiedsample.Thebaselinecyclemeasureddiseaseprevalenceandprovidedriskfactordatapriortoincidentdiseaseonset.Acohortof3,838personsfreeofADwasidentified;729personsweresampledforbaselineclinicalevaluation.Personsinthedisease-freecohorthadeithergoodcognitivefunctionatbaseline,orifcognitivefunctionwasintermediateorpoor,werefreefromADatthebaselineclinicalevaluation.Thisdisease-freecohortwasevaluatedforincidentdiseaseafteranaverageof4.1years.Samplingforincidentclinicalevaluationwasbasedonage,sex,race,andchangeincognitivefunction(i.e.,stableorimproved,smalldecline,orlargedecline).ThesamplesetavailableintheADGCforgeneticanalysesincluded32ADcasesand197personsfreeofADattimeoflastassessment(allsubjectswereage65yearsorolderatlastassessment).

NetherlandsBrainBank(NBB):TheNBBisadepartmentoftheNetherlandsInstituteforNeuroscience,aninstituteoftheRoyalNetherlandsAcademyofArtsandSciences.TheNBBisanon-profitorganizationthatcollectshumanbraintissuefromdonorswithavarietyofneurologicalandpsychiatricdisordersandbraintissuefromnon-diseaseddonors,aswellasanonymizedsummariesofdonors'medicalrecordstobemadeavailableforneuroscienceresearch24.ThesamplesetavailableintheADGCforgeneticanalysesincluded215pathologically-confirmedADcasesand85subjectsfreeofAlzheimer’spathologyatautopsy.Allcaseswereage65yearsorolderattimeofdiagnosis,andallcontrolswereage65yearsorolderattimeofdeath.

TexasAlzheimer’sResearchandCareConsortium(TARCC):TheTARCCisacollaborativeAlzheimer’sresearcheffortdirectedandfundedbytheTexasCouncilonAlzheimer’sDiseaseandRelatedDisorders(theCouncil),aspartoftheDarrellKRoyalTexasAlzheimer’sInitiative.ComposedofBaylorCollegeofMedicine(BCM),TexasTechUniversityHealthSciencesCenter(TTUHSC),UniversityofNorthTexasHealthScienceCenter(UNTHSC),theUTSouthwesternMedicalCenteratDallas(UTSW),UniversityofTexasHealthScienceCenteratSanAntonio(UTHSCSA),TexasA&MHealthScienceCenter(TAMHSC),andtheUniversityofTexasatAustin(UTA),thisconsortiumwascreatedtoestablishacomprehensiveresearchcohortofwellcharacterizedsubjectstoaddressbetter


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diagnosis,treatment,andultimatelypreventionofAD25.Theresultingprospectivecohort,theTexasHarrisAlzheimer'sResearchStudy,containsclinical,neuropsychiatric,genetic,andbloodbiomarkerdataonmorethan3,000participantsdiagnosedwithAlzheimer'sdisease(AD),mildcognitiveimpairment(MCI),andcognitivelynormalindividuals.Longitudinaldata/samplecollectionandfollow-uponparticipantsoccursonanannualbasis.Twowavesofcase-controldatafromTARCCwereexaminedaspartofgeneticanalysesintheADGC.DatafromtheTARCCincluded323casesand181controlsinthefirstwave,with84casesand115controlsinthesecondwave.AllTARCCsubjectsweregreaterthan65yearsofageatdiseaseonset(cases)oratlastdisease-freeexam(non-cases).

TheWashingtonHeights-HamiltonHeights-InwoodColumbiaAgingProject(WHICAP):WHICAPisacommunity-basedlongitudinalstudyofaginganddementiaamongelderly,urban-dwellingresidents26,27.Beginningenrolmentin1989,WHICAPhasfollowedmorethan5,900residentsover65yearsofage,includingwhite,AfricanAmerican,andHispanicparticipants.Detailedclinicalassessmentswereperformedatapproximately24-monthintervalsoverthe7yearsoftheinitialstudy.AllinterviewswereconductedineitherEnglishorSpanish.Thechoiceoflanguagewasdecidedbythesubjectinordertoensurethebestperformance,andthemajorityofassessmentswereperformedinthesubject’shome,whichincludedmedical,neurological,andneuropsychologicalevaluations.Resultsoftheneurological,psychiatricandneuropsychologicalassessmentswerereviewedinaconsensusconferencecomprisedofneurologists,psychiatrists,andneuropsychologists.Basedonthisreviewallparticipantswereassignedtooneofthreecategories:dementia,cognitiveimpairmentornormalcognitivefunction.ThesamplesetavailableintheADGCforgeneticanalysesincluded73ADcasesand570subjectswithnormalcognitivefunction.EADI:

Stage2:The2,012ADcaseswereascertainedbyneurologistsfromBordeaux,Dijon,Lille,Montpellier,Paris,Rouen,andwereidentifiedasofEuropeanancestry.ClinicaldiagnosisofprobableADwasestablishedaccordingtotheDSM-III-RandNINCDS-ADRDAcriteria21,28.The6,502Controlswereselectedfromthe3CStudy29.Thiscohortisapopulation-based,prospective(10-yearsfollow-up)studyoftherelationshipbetweenvascularfactorsanddementia.IthasbeencarriedoutinthreeFrenchcities:Bordeaux(southwestFrance),Montpellier(southeastFrance)andDijon(centraleasternFrance).

Anadditional11,109case-controlsampleswereobtainedforreplicationfromcentersinBelgium(1center),Finland(1center),Italy(8centers),Spain(5centers),Sweden(2centers)andCanada(1center).ClinicaldiagnosesofprobableADwereallestablishedaccordingtotheDSM-III-RandNINCDS-ADRDAcriteria.ControlsweredefinedassubjectswithoutDMS-III-Rdementiacriteriaandwithintegrityoftheircognitivefunctions(MMS>25).

Forfullsamplecharacteristicsinstage1andstages2+3seeSupplementaryTables1

and2respectively.FordetailsofthestudydesignseeSupplementaryFigure1.


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2.QualityControlandAnalysesAPOEConditionalAnalyses

AsexpectedsignificantevidenceforassociationwithLOADwasidentifiedattheAPOElocuswithtwenty-twovariants.Anintronicproxyforthers429358variantdeterminingtheAPOEε4genotype(rs769449,OR=2.88,P<1x10-500,r2withrs429358=0.82),andtheexonicvariantAPOEε2genotypes(rs7412,OR=0.43,P=2.7x10-105)showedthestrongestassociations.Performingtwoconditionalmeta-analyses,adjustingforindependentlydeterminedAPOEgenotypesinallcohorts,oneadjustingforAPOEε4(coded0,1,2)asecondadjustingforAPOEε2(coded0,1,2),diminishedallassociationsignalsidentifiedwithallthegeneticvariantswithintheAPOEregion,thereforethese22variantswerenotconsideredfurther.Theleadvariantrs769449reducedfromP<1x10-500toP=1.1x10-5,whenadjustingforAPOEε4,andrs7412fromP=2.7x10-105toP=0.07,whenadjustingforAPOEε2.AdditionalQualityControl Twohundredsevenvariantsshowedsuggestiveevidenceforassociation(P≤0.0001)inanyofthefourmeta-analysesofthediscoverydataset.Onehundredandeighty-fivevariants,independentofAPOEε4andε2,werecarriedforwardforadditionalqualitycontrolthatinvolvedareviewofallstudyspecificgenotypeclusterplots.Wherevariantgenotypeclusterscouldbeimproved,theseweremanuallyre-clustered.Variantswhosegenotypeclustersweredeemedtoopoorforaccurategenotypecallingwereexcludedfromre-analysis.Re-calledvariantswerere-analysedaspreviouslydetailed.Afterre-analysistwentyvariantsthatnolongershowednominallysignificantassociation(P>0.05)wereexcluded.Wealsoexcludedseventy-onevariantsthathadaminorallelecount(MAC)oflessthan4,orthosevariantsthatwereobservedtobepolymorphicinonlyoneanalysiscohort,afterrecalling.Oftheremainingvariants50werecommon(MAF≥0.05),andtheobservedassociationswerenearknowngenome-widesignificantloci(SupplementaryTable5).Forty-threerarevariantslocatedoutsideoftheAPOEregionwereeligibleforreplicationandconsideredforadditionalgenotypingandinsilicoreplication(SupplementaryTable4).PreviouslyDescribedRiskLoci

WeobservedassociationatcommoncodingvariantsforanumberofADrisklocipreviouslyidentified(SupplementaryTable5).VariantsinAPOE,CLUandCR1showedgenome-widesignificantassociation(P<5x10-8)intheunadjustedanalysis,whilecommonvariantsnearBIN1,MS4A6A,CD33,HLA-region,ABCA7andINPP5Dshowedsuggestiveassociation(P<5x10-4).PreviouslydescribedgeneswithevidenceforassociationwithAD(TREML2,UNC5C,TTC3,PLXNA4,PLD3,MTHFR,CYP2D6,ADAM10,ZNF628,AKAP9,CD33,TRIP4,MAPT,SQSTM1,ATP5H/KCTD2)orfamilialADgenes(APP,PSEN1,PSEN2)areshowninSupplementaryTable5.Gene-wideAnalysis

VariantswereallocatedtogenesaccordingtheRefSeqdatabase.Variantswereassignedtogenesiftheywerelocatedwithinthegenomicsequencelyingbetweenthestartofthefirstandtheendofthelastexonofanytranscriptcorrespondingtothatgene,as


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definedbyNCBI.Gene-wideanalyseswereexaminedusingtheunifiedmethodimplementedinSKAT-O,wheretheoptimallinearcombinationoftheburdenandSKATtestsisimplemented36.Asinthesinglevariantanalyses,associationwithdiseasewastestedforineachcohortsetincludingthestudyspecificcovariatesunderboththeadjustedandunadjustedmodels.Analyseswereperformedincluding‘ALL’variants,variantswithaMAF<5%andvariantswithaMAF<1%.Testswererestrictedtoindividualgeneswithtwoormorepolymorphicvariants.Studyspecificresultswerecombinedinameta-analysisusingtheseqMetapackage.Variantswithingenesshowingstatisticallysignificantevidenceforassociation(P<2.5x10-6)underwentadditionalclusterplotinspectionandpoorlyperformingvariantswereremovedfromtheanalysis.PowerCalculations

Iftheallelefrequenciesincasesis0.003andincontrolsis0.001,thenthepowertodetectthisrarevariantwith5000casesand2500controlsat5%significancelevelis70%.Ifthenumberofcontrolsis18000,thenthepowerisincreasedupto98%atα=0.05and28%atα=1e-6(toaccountfor30,000genes).Thispowercalculationsareperformedusingfunctionpower.fisher.test()inRstatisticalsoftware.LinkageDisequilibriumCalculations

Linkagedisequilibrium(LD)calculationswereperformedusingPLINKv1.94andtheGERADv1.0dataset.HighD’valuesandlowr2valueswereidentifiedforalltheLDpairstested(SupplementaryTable14).ThisdiscrepancyinLDmeasuresistobeexpectedwhenanalysingrarevariants.TheD’calculationestimatesco-presenceoftheminoralleleatoneSNVcomparedtoareferencealleleatanotherSNV,whiler2isameasureofthecorrelationbetweenthepresenceorabsenceofaparticularalleleatthefirstSNVandthepresenceorabsenceofaparticularalleleatthesecondSNVandisthereforeaffectedbyallelefrequency.Forbi-allelicmarkers,themostcommonlyusedmeasuresforLDisr237,whichindicatesindependenceofthetestedSNVassociations.3.SingleVariantFindings

OutsideoftheAPOEregion,andexcludingtheknowncommonriskloci,fourSNVsreachedgenome-widesignificantevidenceforassociation(P<5x10-8),underboththeunadjustedandadjustedanalysismodels.SeeSupplementaryTables7and8respectively.

Aforestplotoftheassociationidentifiedatrs72824905inPLCG2isgiveninSupplementaryFigure3.Weidentifiedasecondindependent(r2=1.5x10-5)suggestivesignalwithstrongeffectwithinPLCG2atsynonymousSNVrs200506549(Pdiscovery=5.8x10-4,OR=2.0,MAF=0.0017).However,explorationintheStage3sample(N=12,616)didnotreplicatethisassociation(P=0.76,OR=0.89,MAF=0.0016).Allstage1associationstestedatthePLCG2geneareshowninSupplementaryTable9.

Aforestplotoftheassociationidentifiedatrs616338inABI3isgiveninSupplementaryFigure6.Allstage1associationstestedattheABI3geneareshowninSupplementaryTable12.


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Aforestplotoftheassociationidentifiedatrs143332484andrs75932628inTREM2aregiveninSupplementaryFigures9and10respectively.Allstage1associationstestedattheTREM2geneareshowninSupplementaryTable13.Itshouldbenotedthatthe61%(9.6%GERAD/PERADES,100%ADGC,81.8%CHARGEand33.7%EADI)ofthesamplesutilizedinthisstudyoverlapswiththatoftheGuerreiroetal.38,andthatRSstage1plusRS1stage3samplesoverlapwithJonssonetal.39,inwhichR47HrobustlyassociatedwithADstatus.

Anadditional3SNVsshowsuggestiveevidenceforassociation(Pcombined<1x10-6)withconsistentdirectionofeffectacrossstages(SupplementaryTables7and8).ConditionalAnalyses

ConditionalanalyseswereundertakenatthePLCG2,ABI3andTREM2lociusingtheGCTAtool40(usingthedefaultparameters)andthestage1unadjustedsummarystatisticsasinput.DatafromtheGERADv1.0datasetwasusedtocalculatethebackgroundLD.TheGERADv1.0datasetwasutilisedtoestablishLD(NGERADv1.0=5692).Wedidnotidentifysignificantorsuggestiveassociation(P<1x10-5)independentofthegenome-widesignificant(GWS)hits.Whenconditioningonrs72824905inPLCG2,thetophitisrs200506549,P=6.52x10-4(SupplementaryFigure5).Whenconditioningonrs616338inABI3,thetophitisrs141826857inB4GALNT2,P=1.89x10-5(SupplementaryFigure8),thisassociationdidnotreplicateinthestage2analysis(Pstage2=9.89x10-1,Pcombined=1.68x10-4).Whenconditioningonrs75932628inTREM2,rs143332484remainssignificantlyassociatedwithdiseaseat(P=3.38x10-9)(SupplementaryFigure11a),theoppositeisobserved,withrs75932628showingsignificantassociationwithdiseasewhenconditioningonrs143332484(P=5.12x10-12)(SupplementaryFigure11b).Whenconditioningonbothrs143332484andrs75932628inTREM2,thetophitisrs143539514,P=1.51x10-3,OR=1.84,MAF=0.0039(SupplementaryFigure11c).4.Gene-wideFindings

OutsideoftheAPOEregion(definedas1MBaroundtheAPOElocus),inboththeMAF<5%andMAF<1%unadjustedanalyses,onlytheTREM2geneshowedstatisticallysignificantevidenceforassociation,withMAF<5%Pgene-wide=1.42x10-15andMAF<1%Pgene-wide=1.01x10-13(SupplementaryTable10).Removalofthep.R47Handp.R62Hvariantsfromtheanalysesdiminishesthegene-wideassociation(P>2.5x10-6).However,theSKAT-OtestremainssuggestivewithP=6.3x10-3,andifaburdentestwasappliedP=4.1x10-3,suggestingthatmoreraredamagingvariantsincreasingriskonADmaybepresentinTREM2.IntheadjustedanalysisanovelassociationwiththeCBLN3geneisidentifiedwith2SNVsatthislocus(SupplementaryTable11).Bothvariantsinthisgeneareextremelyrareandthisfindingrequiresfurtherreplication.

5.GeneExpression

RNAsequencingwasalsousedtomeasuregeneexpressionlevelsinbrainsfromCRND8transgenicmousemodelat3,6and12monthsofage(n=12,12and14,


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respectively);PS1APPmodelatage12months(n=11)andwildtype(WT)miceat3,6and12monthsofage(n=12,12and10,respectively).Basedonourpreliminarydatawhichshowedexpressionchanges>2-foldininnateimmunitygenesbetweenTgvs.Non-Tgmice,basedonconservativeestimateofvarianceandgroupsizesof10,wehavean80%powerintheRNAseqstudiestodetecteffectsizesof1.8,2.2and2.8atanα<0.05,0.01and0.001.AllmicewerehousedinSPFconditionsinthesamefacility,fedstandardmousechow,andeuthanizedbyC02asphyxiation.Brainsweredissectedtoremovethecerebellumandmidbrain,andthe"forebrains"wereprocessedforRNAextractionandsequencinginamanneranalogoustothatdescribedforthehumanbrainsamples.Transgenicanimalsandtheirnon-transgeniclittermatesunderwentRNAseqinthesamebatch,whichincludedanimalsfrombothsexesandandallagegroupsassessed.Samplesweresequencedastriplicatesperlaneandrandomizedacrosstheflowcellsbyageandtransgene(+vs.-).RNAseqprocessingincludingalignmentandqualitycontrolwasdoneonallmousesamplesinanautomatedfashion.ThemouseRNAsequencingdataoverviewandanalyticmethodsareavailableatSynapsepagessyn3157182andsyn3435792,respectively.Multivariablelinearregressionwasusedtotestforassociationofgeneexpressionlevelswithtransgenicstate(Dx).Inallanalyses,adjustmentsweremadeonlyforsexandRNAintegritynumber(RIN),givenlimitedsamplesize.Meannormalizedgenereadcountsandstandarddeviations(sd)forthetransgenic(Tg)andWTgroupsareshown(SupplementaryTable20).TheRNAseqdatausedintheanalyseshavebeennormalizedusingConditionalQuantileNormalization(CQN)viatheBioconductorpackagecqn;accountingforsequencingdepth,genelength,andGCcontent.CQNapproximateslog2(RPKM)exceptattheextremesoftheexpressiondistribution.ThegeneexpressiondatashownhereinhavemeanCQN>-1.Levelsofall3genesincreasewithagebuttoagreaterextentforTgmiceforTrem2andAbi3.All3genesaresignificantlyhigherinCRND8brainsat12months.Trem2andAbi3arealsosignificantlyhigherinCRND8miceat6monthsandPS1APPmiceat12months.6.FunctionalAnnotation

ToinvestigatethefunctionaleffectofindexSNVsrs72824905andrs616338,thesurroundingsequencewasanalysedtoidentifypotentialcis-effects.VariantsinLD(r2>0.7)withtheindexSNVswereidentifiedusingHaploRegv4.148usingtheEuropeanpopulationfrom1000GenomesPhase149forLDcalculation.Additionally,theCommonGeneHaplotypeAllelesfeatureintheUniversityofCalifornia,SantaCruz(UCSC)genomebrowser50(https://genome.ucsc.edu),generatedfromimputationofthe1000GenomesPhase1data,wasusedtoidentifyvariantsonthesamehaplotypebackgroundastheindexSNVs.Thisapproachidentified8additionalvariantsthatmaybetaggedbytheindexSNVs(SupplementaryTable21).In-silicofunctionalanalysisofthevariantswasconductedusingAnnovar51andthefollowingdatabases:RefSeq52release69wasusedtoannotatevariantstogenes.TranscriptionfactorbindingsitescomputedwiththeTransfacMatrixDatabasev0.7(http://www.gene-regulation.com/pub/databases.html)weresourcedfromtheUCSCgenomeannotationtracks53fortheFeb2009assemblyofthehumangenome(http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/).ThesnoRNAandmiRNAtrack,basedonthemiRBaseandsnoRNABaserelease54–58,aswellastheTargetScanS59–61microRNAsbindingsitetrack,weresourcedfromtheaboveUCSCassemblyandusedtoidentifyvariantsoverlappingmicroRNAsortheirregulatorysites.VariantspreviouslyidentifiedbypublishedGWASandcollectedintheCatalogofPublishedGenome-Wide


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AssociationStudiesattheNationalHumanGenomeResearchInstitute(NHGRI,accessedMarch2015)62wereflaggedusingdatafromthecorrespondingUCSCtrack.VariantswerealsoannotatedusingthedbNSFPv30adatabase63,64thatcompilespredictionsandconservationscoresfrom20sources,theCLINVARdatabaseofvariantswithclinicalsignificance65,andfunctionalpredictiontoolsGWAVA66release70,CADD67v1.0andDANN68.Finally,variantswereinvestigatedfortheireffectongeneexpressionusingeQTLdatafromBRAINEAC69,HaploRegv4.1andthosereportedbyKnightandco-workers70.PLCG2

PLCG2encodesphospholipaseCγ2(PLCγ2),anenzymeresponsibleforligand-mediatedsignallingincellsofthehematopoieticsystem,andplaysakeyroleintheregulationofimmuneresponses.Thep.P522RvariantidentifiedwithinPLCG2residesinaregionoftheproteinhighlyconservedacrosshuman,chimp,rhesusmonkey,mouse,rat,rabbit,horse,dogandelephant(SupplementaryFigures4and13).Functionalannotationsuggeststhattheprotectivevariant,whichencodesforanarginineresidue,affectschromatinstructureandpotentiallyproteinfolding.AswellasassociatingwithautoimmunediseasesPLAIDandAPLAID71,PLCG2hasbeenshowntoassociatewithinflammatoryconditionssuchasInflammatoryboweldisease72.ABI3

ThefunctionofABI3(previouslyknownasNESH)isfarfromunderstood.EarlystudiesindicatedthatoverexpressionofABI3ledtoareductionincellmotilityandreducedmetastasisinaninvivocancermodelattributedtoanapparentinteractionwithp21activatedkinase73.Whilstthisstudydidnotdemonstrateanimpactoncellproliferation,subsequentstudyofbothABI3andABI3BP(ABI3bindingprotein),reportedanimpactofitsexpressiononproliferationaswellasinvivocancercellgrowth74.ThesetumoursuppressingrolesforABI3areinterestinginthecontextofobservedlowexpressionofABI3incancercells75.GiventheassociationwehavemadebetweenABI3polymorphismswiththedevelopmentofAlzheimer’sdisease,thekeycontributionofABI3totheaetiologyofthediseaseandwhetheritisattributabletoalterationsincellgrowthandadhesion/migrationorotherwiseunknownfunctionsremainscompletelyunknown.Theriskvariantp.S209F,whichencodesaphenylalanineresidueispredictedtobedeleterious67,thevariantliesinaregionoftheproteinhighlyconservedacrosshuman,chimp,rhesusmonkey,mouse,rat,rabbit,dogandelephant(SupplementaryFigure7),whichisthoughttohavearoleinalteringchromatinstructure(SupplementaryFigure14).TREM2

TREM2isaTypeItransmembranereceptorproteinexpressedonmyeloidcells76,77,inthebrain,primaryTREM2expressionisonmicroglia.TREM2actstocontrolregulationofphagocytosisandsuppressionofinflammatoryreactivitysignallingpathways78–80.TREM2hasshowngeneticassociationwithmultipledementias81–85,includingAD38,39,andhasalsoshowndifferentialexpressioninAβplaque-associatedversusAβplaque-freetissuefromtransgenicmice86.Bothp.R47Handp.R62HarelocatedinaIg-likeV-typedomain(SupplementaryFigure15),suggestingthatthesevariantsaffectligandbinding/signaltransductionofTREM2.


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8.SupplementaryTableLegendsSupplementaryTable1.Fulldescriptionofthedifferentstage1samplesfromtheGERAD/PERADES,ADGCandCHARGEconsortia.SupplementaryTable2.Fulldescriptionofthedifferentstage2andstage3samples/datasetsfromtheGERAD/PERADES,ADGC,CHARGEandEADIconsortia.SupplementaryTable3.Detailsofstage1callingsoftware(s)andqualitycontrolmetricsappliedacrosstheADGC,CHARGEandGERAD/PERADEScohorts.SupplementaryTable4.Tableof43variantseligibletobetakenforwardfromstage1,meetingP<1x10-4beforere-clusteringandP<0.05afterre-clustering.TheORforanumberofSNVsareextremelyhighduetoacombinationofthe‘one-step’approximationoftheeffectestimatefromthescore-testandtherarityoftheminorallele.SupplementaryTable5.ObservedassociationsatpreviouslyidentifiedGWSADriskloci.VariantsinAPOE,CLUandCR1showedgenome-widesignificantassociation(P<5x10-8)intheunadjustedanalysis,whilecommonvariantsnearBIN1,MS4A6A,CD33,HLA-region,ABCA7andINPP5Dshowedsuggestiveassociation(P<5x10-4).Also,rareandcommonvariationinpreviouslydescribedriskloci(TREML2,UNC5C,TTC3,PLXNA4,PLD3,MTHFR,CYP2D6,ADAM10,ZNF628,AKAP9,CD33,TRIP4,MAPT,SQSTM1,ATP5H/KCTD2,APP,PSEN1,PSEN2).ExcludingCD33commonvariantrs3865444,nosignificantevidenceforassociationwithLOADwasidentified.TheORforanumberofSNVsareextremelyhighduetoacombinationofthe‘one-step’approximationoftheeffectestimatefromseqMetaandtherarityoftheminorallele.SupplementaryTable6.ConcordanceofalternateallelecarriergenotypesforallreplicatedSNPsamongsampleswithbothexomechipgenotypingandwithGWASimputedtoHRC.Forcomparison,imputedgenotypeswereassignedifprobabilityofagivengenotypeexceeded0.9.Wherepercentconcordanceisabsent,SNPswereimputedwithhighprobabilityasmonomorphicacrossallsamplesexamined.SupplementaryTable7.ResultsofunadjustedanalysisoftheSNVsidentifiedaseligibleforreplicationinstage1.Resultsshowp-value,oddsratio,minorallelefrequencyandnumberofindividualsforeachstageofthestudy,aswellasthefinalcombinedanalysis.TheORforanumberofSNVsareextremelyhighduetoacombinationofthe‘one-step’approximationoftheeffectestimatefromtheseqMetaandtherarityoftheminorallele.SupplementaryTable8.ResultsofadjustedanalysisoftheSNVsidentifiedaseligibleforreplicationinstage1.Resultsshowp-value,oddsratio,minorallelefrequencyandnumberofindividualsforeachstageofthestudy,aswellasthefinalcombinedanalysis.TheORforanumberofSNVsareextremelyhighduetoacombinationofthe‘one-step’approximationoftheeffectestimatefromtheseqMetaandtherarityoftheminorallele.


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SupplementaryTable9.UnadjustedassociationwithsinglenucleotidevariationwithinthePLCG2geneonchromosome16.SupplementaryTable10.ResultsofunadjustedSKAT-Ogene-wideanalysisoftheSNVsinstage1.ResultsshownumberofSNVsincludedinanalysisatMAF≤0.01andMAF≤0.05andtheirrespectivep-valuesforallSNVswithP<1x10-5ateitherMAFthreshold.Tablealsoshowsgene-wideanalysisofPLCG2(P>1x10-5).SupplementaryTable11.ResultsofadjustedSKAT-Ogene-wideanalysisoftheSNVsinstage1.ResultsshownumberofSNVsincludedinanalysisatMAF≤0.01andMAF≤0.05andtheirrespectivep-valuesforallSNVswithP<1x10-5ateitherMAFthreshold.Tablealsoshowsgene-wideanalysisofPLCG2andABI3(P>1x10-5).SupplementaryTable12.UnadjustedassociationwithsinglenucleotidevariationwithintheABI3geneonchromosome17.SupplementaryTable13.UnadjustedassociationwithsinglenucleotidevariationwithintheTREM2geneonchromosome6.SupplementaryTable14.LinkagedisequilibriumcalculationsgeneratedfortheobservedSNVassociationsatthePLCG2andTREM2loci.SupplementaryTable15.EnrichmentfortheIGAPpathwayclustersbasedoncombininggene-widep-valuesfromvariantswithMAF<0.01withFisher’smethod.Theclustersrepresentingtheimmuneresponse,cholesteroltransport,hemostasis,Clathrin/AP2adaptorcomplexandproteinfolding,surviveBonferronifor8tests(p<0.00625).AconservativeanalysisremovingtheAPOEregionandthemoresignificantofanypairofgeneslessthan1Mbapart(toremovepotentialbiasresultingfromLDbetweengenes)isalsoshown.SupplementaryTable16.Significantpathways(FDR<0.01)fromananalysisoftherarevariantdata(MAF<1%)onall9,816pathwaysoriginallyanalysedintheIGAPGWAS.SupplementaryTable17.ALIGATORenrichmentanalysisofthe151genesintheoverlapofimmune-relatedgeneexpressionmodulesintheIGAPGWAS,stratifyingbymembershipoftheproteininteractionnetwork.Arangeofp-valuecutoffswereusedtodefinesignificantSNPs(andthegenescontainingthem).“Top5%”referstothetop5%ofgenesbeingcountedassignificant(correspondingtoSNPP<8.32x10-4)andwastheprimaryanalysisintheoriginalpathwayanalysisoftheIGAPdata.

SupplementaryTable18.Listofthe56genesintheprotein-proteininteractionnetwork,withgenebasedp-valuesintheIGAPcommonvariantGWASandinthepresentrarevariantstudy(unadjustedmodel).


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SupplementaryTable19Differentialexpressionofgenes(DEG)inhumantemporalcortex.Meannormalizedgenereadcountsandstandarddeviations(sd)fortheADandcontrol(con)groupsareshown.EffectofADdiagnosis(Dx.Beta,Dx.SE=standarderror),significanceofADdiagnosiseitheruncorrected,orcorrectedusingFDR-basedqvaluesareshown.All3genesaresignificantlyhigherinADtemporalcortexpriortocorrectingforcelltypes(Simplemodel),butthissignificanceisabolishedafteradjustingforcell-specificgenecounts(Comprehensivemodel).ThissuggeststhattheseelevationsarelikelyaconsequenceofchangesincelltypesthatoccurwithAD,mostlikelymicrogliosisgiventhatTREM2,PLCG2andABI3aremicroglia-enrichedgenes.SupplementaryTable20.Differentialexpressionofgenes(DEG)inbrainsfromCRND8transgenicmousemodelat3,6and12monthsofage(n=12,12and14,respectively);PS1APPmodelatage12months(n=11)andwildtype(WT)miceat3,6and12monthsofage(n=12,12and10,respectively).Meannormalizedgenereadcountsandstandarddeviations(sd)forthetransgenic(Tg)andWTgroupsareshown.Effectoftransgenicstate(Dx.Beta,Dx.SE=standarderror),significanceofTgstateeitheruncorrected,orcorrectedusingFDR-basedqvaluesareshown.Levelsofall3genesincreasewithagebuttoagreaterextentforTgmiceforTrem2andAbi3.All3genesaresignificantlyhigherinCRND8brainsat12months.Trem2andAbi3arealsosignificantlyhigherinCRND8miceat6monthsandPS1APPmiceat12months.SupplementaryTable21.FunctionalannotationofthePLCG2andABI3GWSSNVsandvariantsinLD(r2>0.7).AssociatedSNVsarehighlightedinblue.Interestingfindingsarehighlightedinred.Interpretationofdataisviathehandbookoftherelevantdatabase.


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