Rare Coding Variants in PLCG2, ABI3, and TREM2 Implicate Microglial

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. A A full list of authors and affiliations appears at the end of the paper. ( ponent Late-onset Alzheimer’s disease (LOAD) has a substantial genetic com contributes evidence Alzheimer’s network and altering susceptibility OR variant MAF p.Ser209Phe, 0.0059, variant variants observed 2 additional genotyping ( exome In disease We Alzheimer’s disease implicate innatemicroglial-mediated immunity in Rare coding variants in Nature Ge Nature Received 1 July 2016; accepted 16 June 2017; published online 17 July 2017; odds genotyped 16,097 LOAD cases and 18,077 cognitively normal elderly elderly LOADnormal 16,097 and cognitively cases 18,077 genotyped Project Alzheimer’s of ( (IGAP) Genomics International the from subjects to sequencing alternative cost-effective a is This sample. independent large a in genotypes imputed and genotyping using follow-up with type a large sample with microarrays targeting known exome variants disease Alzheimer’s with associated variants rare have that genes of number con also risk variants disease Rare to tribute heritability. disease of proportion a only polygenic highly is risk and known, P

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mon variants using a logistic regression model in model mon each sample regression cohort using a variants logistic com We 5%). < analyzed (MAF rare or frequency low were 176,955 (MAF common were 26,947 polymorphic, were 203,902 ( control quality passed variants 241,551 total, In model. additive an assuming Methods) (Online meta-analy sis single-variant a in combined were consortia from multiple Data microarray. HumanExome Illumina the using controls nominal evidence of association ( association of evidence nominal multiple ( species and ( controls in 0.008 and in cases 0.011 of MAF a with stages, all across risk LOAD increasing for factor ABI3). The scription showed Phe209 allele consistent evidence ( this gene ( Tables10 associationatfor evidence nominal ( species several and 0.0093 in controls. The reference allele (Pro522) is conserved across ciated with decreased risk of LOAD, showing a MAF of 0.0059 in cases Supplementary Fig. 3 10 × Tables8 and7 association signals with LOAD ( the using Table 6 Supplementary imputed were controls Haplotype resource Reference 8,345 Consortium and cases independent 6,652 for and genotypes where 3 associations stage to effect of significant directions consistent genome-wide single-nucleotide with forward (SNVs) carried variants We Methods). (Online tation from derived genotypes using trols, con 21,921 and cases LOAD 14,041 in association for these tested ( loci risk known excluding 10 SeqMeta with were combined and data test score the using ants were analyzed METAL using data combined and P = 4.56 × 10 × 4.56 = −4 The second new association is a missense change p.Ser209Phe p.Ser209Phe change missense a is association new second The We identified four rare coding variants with genome-wide significant We cluster plots for reviewed ( variants showing association , , Supplementary Table 12 −10 ) and identified 43 candidate variants ( variants 43 candidate ) and identified ABI3 , OR = 0.68) in0.68) = OR , 3 and 1 Supplementary Fig. 5 ( Supplementary Fig. 2 Fig. Supplementary 6 −10 11 ). The first is a missensevarianta firstis The p.Pro522Arg). ( Supplementary Fig. 4 Fig. Supplementary ), and we found no otherfoundindependentwenoand ),association at , OR = 1.43) in in 1.43) = OR , Supplementary Fig. Supplementary 7 , , and , and PLCG2 ). Supplementary Table 9 Supplementary Table 5 Table Supplementary Fig. 1 Fig. ). The reference allele is conserved across P (phospholipase C < 5 × 10 TREM2 ). Supplementary Table 3 Table Supplementary ABI3 b P ). 3 , , = 5.22 × 10 × 5.22 = P 0 Table . Rare and low-frequency vari low-frequency and Rare . de novo de = 1.52 × 10 × 1.52 = (B3-domain-containing tran (B3-domain-containing −8 ). Gene-wide analysis showed analysis Gene-wide ). ). Gene-wide ). analysis Gene-wide showed ) ( Table 32 2 Supplementary Table 4 Supplementary , , , 3 genotyping and impu and genotyping Supplementary Fig. 6 Fig. Supplementary 3 (Online Methods (Online and −5 2 γ ). This variant is asso 2) ( 2) and −4 ) ( )

( s r e t t e l Supplementary Supplementary ). In stage 2, we we 2, stage In ). Fig.1 Supplementary Supplementary ). Of these, these, Of ). ≥ a 5%), and and 5%), , P Table P = 5.38 = < 1 × 2 ), ), ------, ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Supplementary Table 13 Table Supplementary ( association gene-wide remaining the ( interesting 10 × remained 6.3 signal the but association, wide gene- the diminished analysis the from variants p.Arg62His the and × 10 TREM2 ( variants risk ent independ are p.Arg47His and p.Arg62His that confirmed analyses ( disequilibrium linkage TREM2 rs75932628 and rs143332484 include data from stage 1, stage 2, and stage 3 ( stage and 2, stage 1, stage from data include rs143332484 and rs75932628 ( the at association 3 ( stage and 2, stage 1, stage from data include rs72824905 for presented Data purple. in indicated 1 Figure reported previously the for association of evidence observed also 9 Figs. Supplementary ( controls in 0.0089 and cases in 0.0143 of MAF a TREM2 at association significant genome-wide for evidence first the report ( ( stages subsequent in replicate to failed this ( association suggestive independent an Tables 10 disease Initiative(EADI)( Consortium, theAlzheimer’s DiseaseGeneticConsortium(ADGC),theCohortsforHeartand AgingResearchinGenomicEpidemiology(CHARGE),andtheEuropeanAlzheimer’s Data arefromtheGeneticandEnvironmentalRiskforAlzheimer’s Disease(GERAD)/Defining Genetic,PolygenicandEnvironmentalRiskforAlzheimer’s Disease(PERADES) Stage 1–3total Stage 3 Total Stage 2 Total Stage 1 T s r e t t e l gray.in shown are information n a Plotted Supplementary Table 7 Supplementary able able 1 SNPs

= 84,493). ( = 84,493). log (P value)

− LOAD with association suggestive of reports Following 10 10 0 2 4 6 8 −15 r 2 ) ) ( 0.2 0.4 0.6 0.8 s

coding variant p.Arg62His ( showed a genome-wide significant association ( association significant showed a genome-wide rare variant p.Arg47His ( p.Arg47His variant rare MIR785 −3 Association plots of of plots Association 81.6 Supplementary Tables 10 Supplementary and ummary ummary of the consortium data sets used for stages 1–3 , , CMI 4 MIR6504 P P LOC100129617 Burden c 11 ) Regional plot of identified association at the the at association identified of plot ) Regional Position onchr.16(Mb) ABI3 ). ). The 81.8 = 4.1 × 10 × 4.1 = Supplementary Fig. 11 Fig. Supplementary rs72824905 S PLCG PLCG upplementary Note) locus. Top hit rs616338 is indicated in purple. Data presented for rs616338 include data from stage 1, stage 2, and stage 3 stage and 2, stage 1, stage from data include rs616338 for presented Data purple. in indicated is Top rs616338 locus. hit 2 B4GALNT2 and and 82.0 Supplementary Table14 Supplementary ). 2 ), suggesting that there are additional risk risk additional are there that suggesting ), SDR42E PLCG2 HSD17B2 1 10 −3 GERAD/PERADES genotype MPHOSPH ). No single SNV was responsible for for responsible was SNV single No ). , and and , 82.2 , , Table CHARGE, genotype CHARGE, GERAD/PERADES 6 gene, adjacent to ABI3 EADI, genotype ADGC, P and . = 1.55 × 10 Supplementary Table 13 Table Supplementary Consortium CHARGE 82.4 ADGC Supplementary Fig. 11 Fig. Supplementary , and , and 2 Supplementary Fig. 8 Fig. Supplementary 11 ). These variants are not in in not are variants These ). in silico 0 20 40 60 80 100 in silico ). Removal of ). Removal p.Arg47His ). Gene-wide analysis of of analysis Gene-wide ).

P TREM2 Recombination rate (cM/Mb) rate Recombination combined Plotted b SNPs −log (P value) −14 10 10 ), and conditional conditional and ), 0 2 4 6 8 46.8 , , OR = 1.67), with Fig. 1 Fig. . . ( ABI3 PRAC HOXB13 MIR3185 PRAC = 1.68 × 10 × 1.68 = a TTLL6 CALCOCO 2 ) Regional plot of identified association at the the at association identified of plot ) Regional 1 P ATP5G1 SKAT , , contained 2 c UBE2Z TREM2 47.0 P , , SNF8 SKAT-O Table 34 GI IGF2BP P Position onchr.17(Mb) = 1.42 , 3 ), but but ), ). We ). 5 1 and and , we we , 47.2 locus. Top hit rs75932628 is indicated in purple. Data presented for for presented Data purple. in indicated is Top rs75932628 locus. hit B4GALNT2 9 −4 n = , rs616338 2 1 48,402 21,921 11,787 18,077 controls 8,345 3,246 1,839 5,049 8,101 7,002 2,974 1 - ) ABI3 , ABI3 PHOSPHO1 GNGT

FLJ40194 Z 2 MIR6129 47.4 We also observed a different suggestive common variant signal in in ( study LOAD case–control signal another variant common suggestive different a observed also We (GWAS) tau (P-tau) tau and fluid of phosphorylated cerebrospinal ciation with LOAD near in variants analyzed are variants common when enriched also biology of areas response, protein cholesterolparticles, efflux, B cell differentiation and immune all using pathways previously.9,816 used The top here pathways are related to lipo presented data variant rare the on analyses pathway ( × 10 for immune response ( cluster. After for correction multiple testing, enrichment we observed gene-wide combine to Fisher’smethod and Methods (Online clusters pathway Alzheimer’s eight disease–associated these 1%) to reanalyze are also present in rare variants, we used the rare variant data (MAF < To in common test observed enrichments biological variants whether disease Alzheimer’s with associated variants common in enriched LOC102724596 P NF652 = 9.20 × 10 × 9.20 = We previously identified eight gene pathway clusters significantly significantly clusters pathway gene eight identified Wepreviously n PH LOC101927207 −5 = 80,733 and 53,042, respectively). SNVs with missing LD missing with SNVs respectively). 53,042, and = 80,733 B MIR6165 NGFR ), hemostasis ( ), hemostasis 47.6 LOC100288866 NXPH 3 7 3 ( TREM2 SPOP FAM117A SLC35B Supplementary Table 16 Supplementary r 2 0.2 0.4 0.6 0.8 −4 1 0 20 40 60 80 10 a ), and protein folding ( folding protein and ),

DVANCE ONLINE PUBLICATION ONLINE DVANCE 0 Recombination rate (cM/Mb) rate Recombination asso variant a common . We reported previously P c Plotted SNPs n = × 2.10 10 −log (P value) P 37,022 14,041 16,097 = 84,905). ( = 84,905). n

1010 15 20 25 = 8.64 × 10 6,652 7,836 1,434 4,049 1,391 8,706 6,000 0 5 TREM2 cases 722 PLCG2 40.8 Supplementary Table 15 Table Supplementary , , in study a association genome-wide locus. Top hit rs72824905 is Top rs72824905 locus. hit −3 LOC101929555 −3 b ), clathrin–AP2 adaptor complex ), clathrin–AP2 Position onchr.6(Mb) P ) Regional plot of identified identified of plot ) Regional ), ), cholesterol transport ( = 6.3 × 10 × 6.3 = ). 41.0 TSPO2 APOBEC UNC5CL P P OARD1 NFYA rs7593262 = 0.02). We also performed We performed 0.02). = also ADCY10P1 values for all genes in each each in genes all for values 2 TREM2 TREML TREM2 TREML TREML3P TREML4 41.2 1 TREML5P 2 8 TREM1

−7 NCR2 Nature Ge Nature ) 2 85,133 14,997 35,962 19,623 34,174 15,708 n 3,968 3,273 9,098 9,492 8,974 . 41.4 total LINC01276 ). We used used We ). FOXP4 MIR464 41.6 MDFI r 2 0.2 0.4 0.6 0.8 P 1 n = 3.84 etics 0 20 40 60 80 100

3 3 Recombination rate (cM/Mb) rate Recombination 6 6 - - . . © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. and and ( variants or rare common for either ment Table 15 ( analysis set gene variant rare this ( common analysis previous the variant both from signals association for enriched and including genes, 56 containing network interaction interactions teinprotein 15 Table Supplementary MAF MAF P S upregulated in LOAD human cortex and in two amyloid precursor precursor amyloid two in and cortex human LOAD in upregulated MS4A with loci the same expression pattern associated included and astrocytes, ( cells endothelial oligodendrocytes, neurons, in expression limited and cells microglia in expression high with cortex, brain human in enrichment. the driving is work ( 1%) < (MAF here described variants rare for ( variants common LOAD-associated enrich for ment strong a showed these and two modules, in four these present of more or genes 151 identified We genes. immune-response risk LOAD with ciated asso variants common for enriched are that modules gene four fied rs143332484, 95.7%forrs72824905,and81.9%rs616338(OnlineMethods intervals, see Data include MAF MAF OR P S N MAF MAF OR P S N MAF OR P S N MAF OR Effect allele Gene Protein variation Position (bp) Chr. SNV T Nature Ge Nature of levels for corrected However, when models. mouse (APP) protein N able able 2 tage 1 tage 1–3meta-analysis tage 3 tage 2 TREM2 identi networks coexpression brain normal of analysis Previous controls cases controls cases controls cases controls cases 17 TREM2 gene cluster, and and cluster, gene ), suggesting that the 56-gene ( 56-gene the that ), suggesting s ). The remaining 95 genes only have nominally significant enrich ummary ummary of stages 1, 2 and 3 and combined results meta-analysis for , , P S n value,oddsratios(OR),minorallelefrequency(MAF)incasesandcontrols,numberofsubjectsincludedeachanalyticalstage.ForOR95%confidence upplementary ABI3 ( etics Fig. Fig. , and and , Supplementary Fig. 12 Fig. Supplementary P

2 50 10 × 5.0 = and Online Methods). This subset is strongly strongly is subset This Methods). Online and ADVANCE ONLINE PUBLICATION ONLINE ADVANCE T PLCG2 2 able 7 HLA-DRB1 3 , 3711 7 ). We then used a set of high-quality pro high-quality of set a used Wethen ). to construct, from these 151 genes, an an genes, 151 these from construct, to . These four modules are enriched for for enriched are modules four These . . ConcordanceforalternateallelecarriergenotypesbetweenimputedversuscalledSNPsinstage3was75.2%rs75932628,91.1% have a common expression pattern pattern expression common a have −6 ; ; P upeetr Tbe 17 Table Supplementary . . 3.02 ×10 5.38 ×10 rs75932628 41,129,252 1.23 ×10 4.38 ×10 = 1.08 × 10 × 1.08 = PLCG2 Supplementary Table 18 Supplementary Arg47His 35,831 30,018 80,733 14,884 TREM2 0.004 0.003 0.006 0.002 0.004 0.001 0.003 0.002 2.37 2.46 2.46 2.58 6 T Supplementary Tables 15 Supplementary ) 3 8 , , . Other known LOAD- known Other . −12 −24 −6 −8 ABI3 P = 4.0 × 10 × 4.0 = −7 P ; ; , and and , 11 × 10 × 1.17 = Supplementary Supplementary

PLCG2 TREM2 SORL1 −6 , , ) 3 ABI3 ) and and ) ) ) net 6 rs143332484 and and , , the 1.55 ×10 41,129,207 2.45 ×10 3.66 ×10 3.48 ×10 are are −6 Arg62His 33,786 53,042 15,288 TREM2 3,968 0.006 0.014 0.010 0.015 0.012 0.009 0.014 0.008 ------, ; 1.55 3.97 1.58 1.67

6 S T upplementary lates multiple pathways leading to T cell activation cell T to leading pathways multiple lates in the through participation WAVE2 complex risk with signaling interferon-mediated via response ABI3 signaling cellular in function of gain and loss of mix complex a is result The region. regulatory autoinhibitory cSH2 the (APLAID) Genomic deletions PLAID (PLAID) and missense and mutations (APLAID) affect autoinflammation and (PLAID) ulation PLCG2 and in tissues, brain IP the While NF- the initiate which (RasGRPs), proteins guanyl-nucleotide-releasing Ras C (PKC) protein kinase and molecules, two major signaling activates Ca plasmic reticulum where it binds to ligand-gated ion channels to increase cyto IP glycerol). IP messengers PIP (PLC enzyme aling ( microgliosis to related be to appeared expression in changes these genes, microglial other −14 −3 −7 −9 Functional annotation ( annotation Functional PLCG2 2 2 (1-phosphatidyl-1 . ABI3 has an important role in actin cytoskeleton organization organization cytoskeleton actin in role important an has ABI3 . INPP5D ( upeetr Fg 14 Fig. Supplementary variants also cause antibody deficiency and immune dysreg immune and deficiency antibody cause also variants κ B and mitogen-activated protein kinase (MAPK) pathways. pathways. (MAPK) kinase protein B and mitogen-activated S ( T NVs NVs at able 6 2+ Supplementary Fig. 13 Fig. Supplementary 3 3 . . DAG remains bound to the membrane plasma where it is released into the cytosol and acts at the endoplasmic endoplasmic at the acts and cytosol the into released is ( –DAG–Ca 3 P (myoinositol 1,4,5-trisphosphate) and DAG and (diacyl 1,4,5-trisphosphate) (myoinositol = 2.2 × 10 ). Chr., chromosome. P < 5 × 10 γ PLCG2 2) that hydrolyzes the membrane phospholipid phospholipid membrane the hydrolyzes that 2) 5.38 ×10 rs72824905 81,942,028 2.48 ×10 1.35 ×10 1.19 ×10 Pro522Arg d 35,831 33,786 84,905 15,288 PLCG2 0.008 0.006 0.011 0.006 0.006 0.009 0.006 0.007 Supplementary Tables 19 Supplementary 0.69 0.70 0.65 0.68 -myoinositol 4,5-bisphosphate) to secondary 2+ 16 G −10 signaling pathway is active in many cells cells many in active is pathway signaling Supplementary Table 21 Table Supplementary is primarily expressed in microglial cells. cells. in is expressed microglial primarily ), ), a gene previously implicated in LOAD −8 −10 −4 −5 −2 ) has a role in the innate immune immune innate the in role a has ) ) encodes a transmembrane sign transmembrane a encodes ) 4 4 0 1 . . , , a complex that regu ABI3 4.56 ×10 47,297,297 8.37 ×10 2.16 ×10 1.75 ×10 Ser209Phe rs616338 and and 4 35,831 33,786 84,493 14,876 s r e t t e l 2 0.010 0.008 0.010 0.010 0.013 0.008 0.011 0.008 ABI3 3 1.41 1.42 1.58 1.43 . is coexpressed coexpressed is 17 9 ) suggests that that suggests ) T . 20 −10 ). −5 −5 −2 3 9 - - - - . © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Trem2 its, resulting in microglial activation and proliferation and activation microglial in resulting its, depos amyloid and debris membrane of recognition in participates PLC ity influences and activity PI3K activates turn in which of signaling, Syk and in ZAP70 activation results activation Receptor DAP12. with complex immune-receptor-signaling an forms protein ( microglia brain of membrane loci risk variant common PLCG2 homology. mauve, mining; text green, light databases; cyan, experiments; magenta, coexpression; black, co-occurrence; blue, dark fusion; gene red, proteins: corresponding the linking evidence of type the to refer edges of Colors risk. disease Alzheimer’s with associated variants rare and common 2 Figure s r e t t e l TREM2 4 3 . . In activation induces TREM2–DAP12 M2-like microglia, , and , and -homozygous-knockout or or -homozygous-knockout

ARHGAP24 Protein–protein interaction network (using high-confidence human interactions from the STRING database) of 56 genes enriched for both both for enriched genes 56 of database) STRING the from interactions human high-confidence (using network interaction Protein–protein encodes a transmembrane receptor present in the plasma plasma the in present receptor transmembrane a encodes ABI3 AB13 are highlighted by red circles, circles, red by highlighted are ARHGAP30 ARHGDIB CYBA 2 , GMIP 5– FGD3 7 NCKAP1L are highlighted by black circles. black by highlighted are HMHA1 NCF4 upeetr Fg 15 Fig. Supplementary Trem2 RAC2 -heterozygous-knockout -heterozygous-knockout SYK , , CSF1R DOCK2 45– , and , and LY96 ). TREM2 TREM2 ). 4 LY86 γ 7 2 activ 2 . When When . TYROBP 4 HCK 4 and HCLS1 - - LPAR6 are highlighted by blue circles, and and circles, blue by highlighted are FCGR2A RGS19 immune-response genes and includes includes and genes immune-response function TREM2 in decrease may or loss in region resulting this signaling, TREM2 in abolish or attenuate disruption Any protein. the of extra region ligand-binding cellular conserved the encode to predicted is which mark are plaques with reduced edly associated microglia of number and size with crossed are mice CD14 INPP5D RGS18 The 56-gene interaction network identified here is enriched in in enriched is here identified network interaction 56-gene The FCER1G PLCG2 SYK CARD9 PLCB2 LPAR5 P2RY13 46 HCST ITGAM TREM2 , 4 a 7 DVANCE ONLINE PUBLICATION ONLINE DVANCE . APBB1IP TREM2 CXCL16 4 7 CX3CR1 . TYROBP ADORA3 APP CSF1R C3 BLNK risk variants are located within exon 2, 2, exon within located are variants risk C3AR1 transgenics that develop plaques, the the plaques, develop that transgenics PTPN6 INPP5D CD4 SPI1 CD33 CMTM , , TREM2 RGS10 SPI1 7 , and , and B2M , , SCIN

PLCG2 CD86 Nature Ge Nature CD33 CFD CEBPA IKZF1 LYZ identified as identified , , ABI3 SERPINA1 IRF8 TREM2 n , , etics SPI1 - - , ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. University. Ulster Garden Villages, AS, ARUK, the American Federation for Aging and the MRC. ARUK and the Big FundLottery provided support to Nottingham London and Maudsley NHS Foundation Trust and by King’s College London Mental Health and the Biomedical Research Unit for Dementia at the South College London was supported by the NIHR Biomedical Research Centre for Unit. The University of Southampton acknowledges support from the AS. King’s Biomedical Centre and NIHR SquareQueen Dementia Biomedical Research of Neurodegenerative was collection Disease supported by the UCLH/UCL from the MRC. Patient recruitment for the MRC Prion Unit/UCL Department Williams is an associate director. Cambridge University acknowledges support acknowledges the support of the UK Dementia Research Institute, of which J. a donation from the Moondance Charitable Foundation. Cardiff University the PG2014-1), Welsh Assembly Government (grant SGR544:CADR), and grant (JPND; Alzheimer’sMR/L501517/1), Research UK (ARUK; grant ARUK- supported by the European Joint Programme for Neurodegenerative Disease Sims(R. MR/L023784/1) is an AS Research Fellow). Cardiff University was also and the Medical Research Council (MRC; grants MR/K013041/1, G0801418/1, Cardiff University was supported by the Alzheimer’s (AS; grantSociety RF014/164) GERAD/PERADES. online version of the pape Note: Any Supplementary Information and Source Data files are available in the t the in available are references, and codes accession statements of including Methods, and data availability any associated Me for data. sequence comprehensive heritability the of resolution Alzheimer’s bybe will facilitated disease larger sample sizes and more Complete sites. intergenic and exonic sites with lower MAF and/or additional size, or intronic effect be may there variants, rare For effect. number small of large variants a common of to due be may heritability remaining The disease. account for a of portion small the ‘missing of heritability’ Alzheimer’s here described variants The studies. genetic LOAD knowledge, from our emerge to to target, druggable classically first the represents PLC In addition, conclusion. this supports analysis neurodegeneration of consequence stream and is a not simply down to disease leading pathway of a part causal in LOAD is directly response microglial the that evidence additional provides work This response. immune innate common a of part are TREM2 work net expression brain disease–related Alzheimer’s an in gene hub a targets trial, clinical disease Alzheimer’s 3 phase a in currently nilvadipine, drug antihypertensive Notably,the genes associated is affected by the INPP5D–CD2AP complex SYK regulates amyloid- models mouse in phenotype anti-inflammatory an to profile tory inflamma microglial the shifts and proliferation microglial inhibits signaling microglial activated mouse models of Alzheimer’s disease, synaptic pruning associates disease with Alzheimer’s of models mouse transgenic ing in consistently Alzheimer’sobserved brain disease in humans and dysfunction microglial to related disease matter white a spheroids, with lopathy in mutations tion of genome-wide significant signals identified by IGAP significant gene-wide a association has with that Alzheimer’s state disease activation microglial in factor scription INPP5D Nature Ge Nature Acknowledgments he pape he We identified three rare coding variants in in variants coding rare three identified We tho 3 8 that encodes the TREM2 complex protein DAP12. protein complex TREM2 the encodes that with genome-wide significant associations with LOAD that that LOAD with associations significant genome-wide with , , r d . CSF1R s n 4 8 etics . Activated microglial cells surround plaques , , 2 We thank all individuals who participated in this study. , and mediates phosphorylation of PLC of phosphorylation mediates and , SYK

CSF1R r ADVANCE ONLINE PUBLICATION ONLINE ADVANCE . , and and , β production and tau hyperphosphorylation as hrdtr dfue leukoencepha diffuse hereditary cause TYROBP 5 2 . Pharmacological targeting of targeting CSF1R . Pharmacological ( Fig. Fig. 5 2 SYK 5 ). ). 46 encoded by two LOAD- 5 and is in the proximity , SPI1 47 as well as as well as , PLCG2 57 5 1 , is a central tran central a is γ . In the brains of of brains the In . 5 online version of version online 2, as an enzyme, an as enzyme, 2, 8

2 . Our network network Our . . . Loss-of-func , , 49 γ ABI3 2 (ref. (ref. 2 TYROBP , 5 0 , a find , and and , 5 6 5 5 3 4 ). ). ------, . ,

to M. Riemenschneider. The University of Washington was supported by grants German ofFederal Ministry Education and Research (BMBF), grant 01GS08125, excellence ImmunoSensation. Funding for Saarland University was provided by the M.M.N. is a member of the German Research Foundation (DFG) cluster of 01GI0711, 01GI0712, 01GI0713, 01GI0714, 01GI0715, 01GI0716, 01ET1006B). 01GI0429, 01GI0431, 01GI0433, 04GI0434; grants KNDD: 01GI0710, 01GI1007A, Education and Research (grants KND: 01G10102, 01GI0420, 01GI0422, 01GI0423, Dementia (KNDD), which are funded by the German ofFederal Ministry Competence Network and (DCN) the German Research Network on Degenerative (grant RC The 10.11.12.13/A). Bonn samples are part of the German Dementia IRCCS Policlinico,Ospedale Italy, acknowledge the Italian of Ministry Health (2014SGR-235). The Santa Lucia Foundation and the Fondazione Ca’ Granda Competitiveness (grant PI12/01311) and from the Generalitat de Catalunya Barcelona, acknowledges support from the Spanish of Ministry Economy and providing DNA samples. The Hospital de la Sant Pau, Universitat Autònoma de and R. Rene, J. Gascon, and J. Campdelacreu (Hospital de forBellvitage) for Applied Medical Research), M. (HospitalSeijo-Martinez do Salnes), acknowledges M.A. Pastor (University of Navarra Medical School and Center supported by CIBERNED, Instituto de Salud Carlos III, Madrid, Spain, and The Department of Neurology, University Hospital Mútua de Terrassa, was for DNA preparation, and P. Gil and P. forCoria their recruitment efforts. Ramón Areces to the CMBSO. We thank I. Sastre and A. Martinez-Garcia (Instituto de Salud Carlos III) and by an institutional grant of the Fundación the Ministerio de Educación y Ciencia and the Ministerio de Sanidad y Consumo Paz, and the Universidad Autónoma de Madrid were supported by grants from CIBERNED, Instituto de Investigación Sanitaria la Paz, University Hospital La University, The Belfast. Centro de Biología Molecular Severo Ochoa (CSIS-UAM), Research, and the Northern Ireland R&D providedOffice support for Queen’s RS-III) RS-III) was executed by the Human Genotyping Facility of the Genetic management of GWAS genotype data for the Rotterdam Study (RS-I, RS-II, Infrastructure Netherlands (BBMRI-NL; Netherlands Exome Chip Project) of Biobanking and Biomolecular Research Research Scientific (NWO; project and184021007); by the Rainbow Project (RP10; for Healthy Aging (NCHA; project the 050-060-810); Netherlands Organization for Organization for Research Scientific (NWO)-sponsored Netherlands Consortium Erasmus MC, from the Netherlands Genomics Initiative (NGI)/Netherlands was funded by the Laboratory ofGenetic the Department of Internal Medicine, org Laboratory ofGenetic the Department of Internal Medicine ( Rotterdam Study (RS-I) was executed by the Human Genotyping Facility of the Generation and management of the Illumina exome chip v1.0 array data for the Rotterdam Study, and the participating general practitioners and pharmacists. Rotterdam. The authors are grateful to the study participants, the staff from the Welfare and Sports, the European Commission XII), (DG and the municipality of (RIDE), ofthe Ministry Education, Culture and forthe Science, Ministry Health, and Development (ZonMw), the Research Institute for in Diseases the Elderly Erasmus University, Rotterdam, the Netherlands Organization for Health Research Rotterdam (RS). working group by AG033193 and AG049505. National Heart, Lung, and InstituteBlood grant HL105756 and for the neurology CHARGE. Nationale de la Recherche-10-IA Agence Institut Hospitalo-Universitaire-6. funding from the program ‘Investissements d’Avenir’, ANR-10-IAIHU-06 (Agence Alzheimer’, Paris, France. The research leading to resultsthese has received Universite Pierre et Marie Curie, and the ‘Fondation pour la Recherche sur H. 2013.0347). Hampel is supported by Research the AXA Fund, the Fondation Fondazione Cassa di Risparmio di Pistoia e Pescia (grants 2011.0264, 2014.0365, was funded by BMBF NGFN grant B.N.01GS08125. was supported by the Principal Research Fellowship to D.C.R. M. (095317/Z/11/Z). Riemenschneider Biotechnology for Health. We are grateful to the Wellcome Trust for awarding a Programme priority FP6-2004-LIFESCIHEALTH-5, Life Genomics, Sciences, and InnoMed, an Integrated Project funded by the European Union’s Sixth Framework and by the ‘5 × 1000’ voluntary contribution. AddNeuromed is supported by Ricerca Corrente Linea 2015–2017, 2 ‘Malattiecomplesse e Terapie Innovative’ was completely supported by Ministero della Salute, IRCCS Research Program, by a fellowship to The R.G. participation of D.S., M.U., and C. Masullo in the study UCL, London, who was supported in part by ARUK via an anonymous donor, and R01-AG042611). We also acknowledge funding from the Institute of Neurology, National Institutes of Health (R01-AG11380, R01-AG021136, P30-S069329-01, Association the (MNIRG-11-205368), BYU Gerontology Program, and the US Alzheimer’s Brigham Youngdisease). University was supported by the Alzheimer’s for Aging Research (C. Cruchaga was recipient of a New Investigator Award in the Alzheimer’s Association and(NIRG-11-200110), the American Federation from the US National Institutes of Health and(R01-NS085419 R01-AG044546), / ), ), Erasmus MC, Rotterdam, the Netherlands. The Exome chip array data set Infrastructure for the CHARGE Consortium is supported in part by The Rotterdam Study is funded by Erasmus Medical Center and http://ww w.bbmri.n http://www.gl l ). ). Generation and s r e t t e l

imdna.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. AG017173, R01 AG025259, R01 AG048927, R01AG33193), Columbia University University (P30 AG013846, U01 AG10483, R01 CA129769, R01 MH080295, R01 R01AG041797), Banner Sun Health Research Institute (P30 AG019610), Boston NACC (U01 AG016976), NIA LOAD (Columbia University) (U24 AG026395, Storage Site (NIAGADS) at the University of Pennsylvania (U24-AG041689-01), and distributed by the National Institute on Aging Alzheimer’s Data Disease participation made this work possible. Data for this study were prepared, archived, used in this study, as well as the patients and their families, helpwhose and (NIA), were used in this study. We thank the contributors who samplescollected agreement grant (U24 AG21886) awarded by the National Institute on Aging (NCRAD), which Disease receives government support under a cooperative and RC2 AG036528. Samples from the National forRepository Cell Alzheimer’s NIA) supported this work through the following grants: U01 ADGC, AG032984, ADGC. y Competitividad, Spain) and ‘La Obra Caixa’Social (Barcelona, Spain). (Acción Estratégica en Salud, Instituto de Salud Carlos III, Ministerio de Economía Instituto de Salud Carlos III Project. A. Ruiz is supported by grant PI13/02434 Dementia SpanishGenetics Consortium 430 (DEGESCO). CIBERNED is an ACE collaborates with CIBERNED and is one of the participating centers of and her family for their support of the Fundació ACE research programs. Fundació all patients for their participation in this project. We are obliged to T. Port-Carbo A. Espinosa, G. Ortega, M. Tarragona, C. Abdelnour, and D. Sanchez. We thank A. M. Mauleon,Rosende-Roca, L. Vargas, O. M. Alegret, Rodriguez-Gomez, Fundació ACE. Disorders and Stroke (R01-NS017950). Institute on Aging (R01AG049607) and the National Institute of Neurological and Seshadri A.L.D. were also supported by additional grants from the National Institute on Aging: AG033193, U01-AG049505, and AG008122 S. (S. Seshadri). This HHSN268201500001I). study was also supported by grants from the National and Institute’sBlood Framingham Heart Study (contracts N01-HC-25195 and Framingham Heart Study. represent ofviews the official the US National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily investigators and institutions can be found at Research Center.Diabetes California Endocrinology A list full of CHS principal Diabetes Kidney Research Disease Center (DRC) grant DK063491 to the Southern CTSI grant andUL1TR000124, National Institute of Diabetes and Digestive and supported in part by the National Center for Advancing Translational Sciences, the National Institute on Aging (NIA). The provision of genotyping data was R01AG023629, R01AG15928, and R01AG20098 and by U01AG049505 from and Stroke (NINDS). Additional support was provided by R01AG033193, with additional contribution from the National Institute of Neurological Disorders grant fromU01HL080295 the National Heart, Lung, and InstituteBlood (NHLBI), N01HC85080, N01HC85081, N01HC85082, N01HC85083, and N01HC85086 and N01HC55222, HHSN268200800007C, N01HC85079, HHSN268201200036C, Health Study Cardiovascular (CHS). and the Althingi (the Icelandic Parliament). NIA Intramural Research Program, Hjartavernd (the Icelandic Heart Association), with HHSN271201200022C contributions from NEI, NIDCD, and NHLBI, the AGES. grant agreement 667375. the European Union’s Horizon 2020 research and innovation programme under (115975); the projectCoSTREAM ( ApolipoproteinDisease Pathology for Treatment Elucidation and Development The work for this manuscript was supported further by ADAPTED: Alzheimer’s colleagues as part of the CHARGE Consortium Exome Chip central calling effort. Variants were using called the best practice protocol developed by M.L. Grove and the GWAS database; and L. Broer for the creation of the HRC-imputed data. M. Verkerk, L. Herrera, M. Peters, and C. Medina-Gomez for their help in creating L. Karsten, and L. Broer for quality control and variant M. calling; Jhamai, M. Verkerk for their help in creating the exome chip database; C. Medina-Gomez, Erasmus MC, Rotterdam, the Netherlands. We thank M. Jhamai, S. Higgins, and Department of Forensic Molecular Biology, and the Department of Dermatology, was funded by the Laboratory ofGenetic the Department of Internal Medicine, the samples onbased Illumina Omni 2.5 and 5.0 GWAS genotype data. This data set This 050-060-810. study makes use of an extended data set of RS-II and RS-III Research (NWO) Netherlands Consortium for Healthy Aging (NCHA), project Netherlands Genomics Initiative (NGI)/Neth (P50 AG008702, R37 AG015473), Duke University (P30 AG028377, AG05128), The AGES study has funded bybeen NIA contracts N01-AG-12100 and The US National Institutes of Health, National Institute on Aging (NIH- We sincerely acknowledge the collaboration of S. Ruiz, This work was supported by the National Heart, Lung, http://www.costream.eu This research was supported by contracts https://chs-nhlbi.o / ); ); and funding from rg / .

the the BrightFocus Foundation (M.P.-V., P.S.G.-H.A2111048). is supported by the Research and Development, the Biomedical Laboratory Research Program, and the 05-14147), US Department of Veterans Affairs Administration, ofthe Office provided by the Alzheimer’s Association (L.A.F., M.P.-V.,IIRG-08-89720; IIRG- grant NS39764, NIMH MH60451, and by GlaxoSmithKline. Support was also Price BrainBryan Bank at Duke University Medical Center is funded by NINDS and Washington University (P50 AG005681, P01 AG03991). The Kathleen University of Wisconsin (P50 AG033514), Vanderbilt University (R01 AG019085), AG027944, AG021547, AG019757), University of Washington (P50 AG005136), Southwestern (P30 AG012300), University of Miami (R01 AG027944, AG010491, AG02365), University of Southern (P50 California AG005142), University of Texas University of Pittsburgh (P50 AG005133, AG030653, AG041718, AG07562, of Michigan (P50 AG008671), University of Pennsylvania (P30 AG010124), AG019724), University of Kentucky (P30 AG028383, AG05144), University (P50 AG005131), University of San California, Francisco (P50 AG023501, P01 of Los AngelesCalifornia, (P50 AG016570), University of San California, Diego (P30 AG010129), University of (P50 Irvine California, AG016573), University AG016582), University of Arizona (R01 AG031581), University of California, Davis AG30146), TGen (R01 UniversityNS059873), of Alabama at Birmingham (P50 University (P30 AG010161, R01 AG019085, R01 AG15819, R01 AG17917, R01 Oregon Health & UniversityScience (P30 AG008017, R01 AG026916), Rush 1RC2AG036502, 1R01AG035137), Northwestern University (P30 AG013854), AG08051, UL1 RR029893, 5R01AG012101, 5R01AG022374, 5R01AG013616, of Medicine (P50 AG005138, P01 AG002219), New York University (P30 Hospital (P50 AG005134), Mayo Clinic (P50 AG016574), Mount Sinai School Hopkins University (P50 AG005146, R01 AG020688), Massachusetts General UO1 HG004610, UO1 HG006375), Indiana University (P30 AG10133), Johns UniversityEmory (AG025688), Group Health Research Institute (UO1 AG006781, Conseil Régional Conseil du Nord-Pas-de-Calais. The Three-City Study was performed Européenne de ERDF Lille), (European Regional Development Fund), and the Approach to Alzheimer’s including Disease) funding from MEL (Métropole DISTALZ grant (Development of Innovative Strategies for a Transdisciplinary by the LABEX (Laboratory of Excellence Program Investment for the Future) Centre National de Génotypage. This work has developed andbeen supported Alzheimer’s and Disease Related Disorders, the Institut Pasteur de andLille, the EADI. thank D.S. Snyder and M. Miller from the NIA who are members. ADGC ex-officio the Laboratory for Neuro Imaging at the University of WeSouthern California. Study at the University of San California, Diego. ADNI data are disseminated by Education, and the study is coordinated by the Alzheimer’s Cooperative Disease The grantee organization is the Northern InstituteCalifornia for Research and by the Foundation for the National Institutes of Health ( support ADNI clinical sites in Canada. Private sector contributions are facilitated Therapeutics. The Canadian Institutes of Health Research provide funds to TakedaPiramal Imaging; Servier; Pharmaceutical Company; and Transition Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer, Inc.; Lundbeck; Merck & Co., Inc.; Meso LLC.; Diagnostics, Scale NeuroRx Research; Johnson & Johnson Pharmaceutical Research & Development, LLC.; Lumosity; IXICO, Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Roche, Ltd., and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Eisai, Inc.; Elan the Alzheimer’s Foundation;Drug Discovery Araclon Biotech; BioClinica, generous contributions from the following: AbbVie, Alzheimer’s Association; National Institute of Biomedical Imaging and and Bioengineering, through ADNIW81XWH-12-2-0012. is funded by the National Institute on Aging, the Institutes of Health grant U01 AG024904 and Department of Defense award de Barcelona. ADNI data andcollection sharing were funded by US National and the Institut de Neuropatologia, Anatomia Servei Patologica, Universitat International Parkinson Fonds, Internationale Stiching Alzheimer Onderzoek), Research, Brain Net Europe, Hersenstichting Nederland Breinbrekend Werk, Netherlands Brain Bank (funding via numerous sources including Stichting MS Bristol NHS Trust Research and Innovation Department and DeNDRoN), the (HEFCE), Alzheimer’s Research UK (ARUK), and BRACE as well as the North numerous sources including the Higher Education Funding Council for England Medical Research the Council), South West Dementia Brain Bank (funding via the MRC London Brain Bank for Neurodegenerative (funding via Diseases the via the Medical Research NHS Council, local andtrusts, Newcastle University), samples from the following sites: the Newcastle Brain Tissue Resource (funding Institute, the Medical Research Council, and the state of Arizona and also includes M.J.H., the Banner Alzheimer’s Foundation, the Johnnie B. Byrd Sr. Alzheimer’s The Science. TGen was series also funded by NIA grant AG041232 to A.J.M. and of Health Research. Genotyping of the TGEN2 cohort was supported by Kronos Wellcome Trust, the Howard Hughes Medical Institute, and the Canadian Institutes This work was supported by INSERM, the National Foundation for a DVANCE ONLINE PUBLICATION ONLINE DVANCE http://www.fnih.org

Nature Ge Nature n / etics ). ). © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. G.D.S. ADGC. P.A.H., J. Williams. D. Harold, E. Majounie, P.A.H. J. B., J.R.G., Hardy, A.G., J. Chapman, M. Hill. generation: D. Craig, J. Johnston, M. Gill, B.L., A. Lynch, N.C.F., D.S., ARUK Consortium. D. Carrell, S. Sorbi, S. Moebus, M.U., A. Pilotto, J. Kornhuber, P. Bosco, S. Todd, Mayhaus, W.G., A. Lleó, J.F., R. Blesa, I.S.B., K. Brookes, C. Cupidi, R.G.M., C. Fenoglio, M. M. Serpente, Arcaro, C. Caltagirone, M.D.O., A.C., S.P., M. M. Daniilidou, J.U., Y.P., J.T.H., J.L., J. Turton, A.M.H., M. Aguilar, R.C., C. Thomas, A.M., R. Marshall, M.D.F., A. Hodges, B.V., H. Soininen, I.K., C.H., J.P., K.M., P. Passmore, D.R., S.O.-C., J. Kauwe, M. Dunstan, A. Braddel, A.A., C.E.S., J. Collinge, D.M., M.T., J. Clarimón, R. Sussams, S. Lovestone, S. Mead, M. Mancuso, U.B., A.D., O.P., B.N., M. Riemenschneider, C. R.H., Brayne, D.C.R., M.N., R. Munger, M.J.B., E.C., V.A., M. Gallo, A.C.B., M. Dichgans, D.G., E. Scarpini, G. Spalletta, P. Bossù, E. Sacchinelli, P.S.-J., F.J., J. Morris, C. Corcoran, J. Tschanz, A.F.-G., I.G., H. Hampel, P.M., V.B., M. Scherer, A. M.L., S.R.-H., Braae, C. Masullo, J. Williams, A. Ramirez, M.K.L., C. Medway, K. Brown, B.M., P. Proitsi, P. Pastor, M.J.O., P.A.H., J. Williams. GERAD/ Michael J. Fox Foundation for Parkinson’s Research. 0011, 05-901, and 1001 to the Arizona Parkinson’s Consortium), and Disease the Research Center), the Arizona Biomedical Research Commission (contracts 4001, Arizona Department of (contract Health211002, Arizona Alzheimer’sServices Institute on Aging (P30 AG19610 Arizona Alzheimer’s Core Disease Center), the and Tissue Resource for Parkinson’s and Disease Related Disorders), the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain Arizona. Mayo Foundation. NINDS grant R01 NS080820, the CurePSP Foundation, and support from the AG006576, U01 AG006786, R01 AG025711, R01 AG017216, and R01 AG003949, NIA grants P50 AG016574, R01 AG032990, U01 AG046139, R01 AG018023, U01 Health Institute) for sharing human tissue. AG046139-01 (T.E.G., N.E.-T., N.P., S.G.Y.). We thank T.G. (Banner SunBeach this work would not have possible. been This work was supported by NIH/NIA human brain donations, we thank all patients and their families, without whom AMP AD University of Florida/Mayo Clinic/Institutes of Systems Biology. Alzheimer Foundation. and the Karolinska Institutet, the Swedish Brain Foundation, and the Swedish Training and Research Clinical (ALF) the between Stockholm County Council Victoria’sQueen Foundation of Freemasons, the Regional Agreement on Medical Foundation, the Swedish Research King Council (521-2010-3134), Gustaf V and by the Swedish Brain Power network, the Marianne and Marcus Wallenberg University Hospital, and the Nordic Center of Excellence in Neurodegeneration. Health Research Council of the Academy of Finland, EVO grant 5772708 of Kuopio Clinics at the Hospital Network Antwerp and University Hospitals Leuven. of the Institute Born-Bunge, and the Departments of Neurology and Memory Antwerp site thank the personnel ofFacility, the VIB Service Genetic the Biobank Research Special Fund of the University of Antwerp, AuthorsBelgium. from the of the Flemish Government, the Research Foundation Flanders (FWO), and the Foundation for Alzheimer Research (SAO-FRA), a Methusalem Excellence Grant Interuniversity Attraction Poles program of Policy the Science Belgian the Office, for Genomic Analysis LAG-BRC, Institut Pasteur de Lille. The Three-City Biological Bank was developed and maintained by the Laboratory Joint Programme for Neurodegenerative grant(JPND: Disease MR/L501517/1). received an unconditional grant from Eisai and was supported by the European ‘Cohortes et de Collections Données Biologiques’ program. The Genopole Lille projects), Fondation de France, and the joint French of Ministry Research/INSERM Nationale de la Recherche (ANR supported and the COGINUT COVADIS des Produits de Santé, the Aquitaine and Bourgogne regional councils, Agence Santé, MGEN, Institut de la Longévité, Agence Française de SanitaireSécurité Caisse Nationale Maladie des Travailleurs Direction de Salaries, Générale la preparation and initiation of the study. The 3C Study was also funded by the and Sanofi-Synthelabo. The Fondation pour la Recherche Médicale funded the as part of collaboration INSERM, between Victor Bordeaux Segalen II University, Nature Ge Nature D. Blacker, R.S.D., T.J.F., M.P.F., B.G., M.I.K., M.J.K.,R.M.H., C.D.K., E.B.L., R.B.L., AU Sun Health Research Institute Brain and Body Donation Program of Sun City, The Mayo Clinic Brain Bank. Swedish sample collection Finnish sample collection. Belgium sample collection. T H Sample contribution: O Study design or conception: The Brain and DonationBody Program is supported by the National R R PERADES. PERADES. R. Sims, S.H.-H., P.H.,R.R., R.T., T.M., N.D., A. Ramirez, J. Williams, CONT n etics RIBU Study supervision/management: Study design or conception:

T ADVANCE ONLINE PUBLICATION ONLINE ADVANCE I ONS Sample contribution: A.R.W., S. Mukherjee, P.K.C., R.C.B., P.M.A., M.S.A., . . Sample was collection supportedfinancially in part Financial support for this project was provided by the Research at the Antwerp site is funded in part by the Manuscript preparation: Data was collection supported through funding by L.A.F., J. Haines, R. Mayeux, M.A.P.-V., L.-S.W., Analysis: M.M.N., W.M., S. Herms, A.J.F., R. Sims, V.E.-P., M.C.O’D., R. Sims, A. Ramirez, J. Williams. R. Sims, S. Taylor, L.J., R. Sims, N.B., M.V.,

For

Data

11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional reprints/index.htm at online available is information permissions and Reprints version of The authors declare competing interests:financial details are available in the Study supervision/management: B.G.-B., P.A., J.-C.L. J.-F. Deleuze, F. Garzia, F. Golamaully, G. Septier., R.O A. Haapasalo, V.S. A.K.-S., V. Giedraitis, L. Kilander, R. Brundin, L.C., S. Helisalmi, A.M.K., C.V.B., S.E., R.V., P.P.D.D., A.S., P.S.-J., C.M.F., Y.A.B., H.T.,F.S.G., C. Forsell, L. Lilius, M.C.D.N., L.F., L. Keller, F. Panza, P. L. Caffarra, Lannfelt, M.I., C.G., O.C., J.-C.L., V.D., N.F., O.H., C. Dufouil, A. Brice, B.K.R., Dubois, K.S., M. Hiltunen, D. Hannequin, F. Pasquier, C. Berr, J.-F. D.Dartigues, Campion, C. Tzourio, P.A., EADI. supervision/management: Analysis: E.B., C.J.O’D., M.B., S. Ahmad, S.M.-G., H.H.A., I.H., L.T., O.S.-G., N.A., V.E., H. Schmidt, J. Jakobsdottir, A.V.S., J.A.B., M.F., X.J., H. Lin, L.A.C., D.L., Q.Y., T.A., M.A.I., L.J.L. O.L.L., J.J.H., A.L.F., A. Hofman, D.A.B., P.L.D.J., B.M.P., V. Gudnason, M.B., M.L.G., C.L.S., F.J.W., T.H.M., A.S.B., M.E.G., G.E., R. Schmidt, H. Schmidt, W.T.L., A.L.D., L.J.L., N.A., C.M.v.D., S. Seshadri. CHARGE. management: S.B., G.J., K.L.L., C.R. Analysis: P.W., C.T.B., C.C.D.,R.M.C., E.A.C., J.R.G., D.C.Marsh, W.P., T.A.T.-W., C.B.W.T.E.G., H. Hakonarson, T.W.B., B. Dombroski, W.A.K., N.E.-T., L.B.C., D. Beekly, J. Malamon, C.C.F., H. Li, J.D.Burgess, M. N.D.P.,Allen, P. Chakrabarty, X.W., K.C.W., J. Williamson, T.S.W., R.L.W., C.-E.Y., L.Y. R.E.T.,R.H.S., J.Q.T., J.C.T., V.M.V.D., L.J.V.E., H.V.V., M.A.S., A.J.S.,J.P.V., J.A. Schneider, L.S.S., W.W.S., S.W., A.G.S., J.A. K.A.W.-B., Sonnen, S. Spina, R.A.S., W.W.P., H.P., J.F.Q., A. Raj, M. B.R., Raskind, J.M.R., E.D.R., H.J.R.,E.R., R.N.R., J.W.M., J.C.M., J.R.M., A.J.M., S.O’B., J.M.O., V.S.P., J.E.P., H.L.P., E.P.,F.M., A. Pierce, E. Masliah, W.C.M., S.M.M., A.N.M., A.C.M., M. Mesulam, B.L.M., C.A.M., N.W.K.,R.K., J.H.K., F.M.L., J.J.L., J.B.L., A.I.L., G.L., A.P.L., C.G.L., D.C.Marson, L.E.H., L.S.H., R.L.H., M.J.H., C.M.H., B.T.H., G.P.J., E.A., L.-W.J., A. K., J.A.K., D.W.F., M.R.F., S.F., T.M.F., D.R.G., M. D.H.G.,Gearing, J.H.G.,R.C.G., N.R.G.-R., S.L.C., H.C.C., D.G.C., D.H.C., C. DeCarli, M. Dick, R.D., D.A.E., K.M.F., K.B.F., A. Boxer, J.R.B., J.M.B., J.D.Buxbaum, N.J.C., C. Cao, C.S.C., C.M.C., M.M.C., S. Asthana, C.S.A., L.L.B., T.G.B., J.T.B., E.H.B., T.D.B., B.F.B., J.D.Bowen, A.M.G., C. Cruchaga, S.G.Y., D.W.D., W.A.K., N.E.-T., L.G.A., S.E.A., R.L.A., T.J.M., R.C.P., J.S.R.,E.M.R., D.R.R., M. Sano, P.S.G.-H., D.W.T., C.-K.W., S.J.v.d.L., A. Ruiz, J.C.B., J. Jakobsdottir, V.C., C.C.W., C.M.v.D., S. Seshadri. G., N.A., V.C., C.C.W., S.-H.C., J.D., Y.C., S. Li, A.L.D. COM

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Paris Paris Descartes, Paris, France. Neurology and Psychiatry, Brigham and Women’s Hospital, Boston, USA. Massachusetts, of Neurology, Kuopio University Hospital, Kuopio, Finland. 25 Department of Neurology, Columbia University, New York, New York, USA. Genetics Center, University of Texas Health Science Center at Houston, Houston, Texas, USA. 21 Fundació ACE, Institut Català de Aplicades, Neurociències Barcelona, Spain. Department of Molecular Genetics, VIB, Antwerp, Belgium. and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, USA. Pennsylvania, Philadelphia, Boston, USA. Massachusetts, University of Bonn, Bonn, Germany. Washington, USA. de Génomique, Centre National de Génotypage, Evry, France. France. Determinants of Diseases, Aging-Related Lille, France. and University Biostatistics, of Pennsylvania Perelman School of Medicine, USA. Pennsylvania, Philadelphia, 2 1 and and Rare Disease Centre, University of Bari Aldo Moro, Bari, Italy. and Biomedical Sciences, Queen’s University, Belfast, UK. Department of Basic Medicine, and Neuroscience, Sense Organs, University of Bari Aldo Moro, Bari, Italy. Biomedicine, Computational Department of Medicine, Boston University School of Medicine, Boston, USA. Massachusetts, Medical Centre, University of Nottingham, Nottingham, UK. Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet and Stockholm University, Stockholm, Sweden. Institute, Herston, Queensland, Australia. Institute Neuroscience, of Psychiatry, Psychology and King’sNeuroscience, College London, London, UK. Rouvray, Sotteville les Rouen, France. 40 de Gran Canaria, Spain. Valdecilla’ University Hospital (University of Cantabria and IFIMAV), Santander, Spain. Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy. Iceland, Reykjavik, Iceland. of Bonn, Bonn, Germany. D Peter Passmore Philip M S Rebecca D Benedetta Antonio D M M Ronald M T C C HaapasaloAnnakaisa Rose s r e t t e l 1 Jean-François Jonathan L Hakon Hakonarson Jean- Philippe Amouyel C Department Department of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands. Institute of Medicine Psychological and Clinical MRC Neurosciences, Centre for Genetics Neuropsychiatric and Genomics, Cardiff University, Cardiff, UK. 0 imon indsay A Farrer Departments Departments of Medicine, Geriatrics, Gerontology and Neurology, University of Mississippi Medical Center, Jackson, Mississippi, USA. Department of Neurology, Columbia University, New York, New York, USA. Division of Medical Genetics, University Hospital and Department of Biomedicine, University of Basel, Basel, Switzerland. hor Aspelund

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erce Boada N , 20 O 282 Institute Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland. 110 RA M , , Julie Immunology Immunology Biomarkers Group, Genentech, South San Francisco, California, USA. athalie athalie Fievet ’ 264 T 234 101 D 1 ontine , Alfredo , Ramirez Alfredo 43 235 , , Reinhard Heun aner , , DES onovan L , , M 251 , , , , Bruno 120 2 , , Jerome I Rotter 89 3 109 M L auner Department Department of and Biostatistics for Epidemiology/Center Clinical Epidemiology , T , 38 108 271 35 27 argaret A Pericak-Vance ,

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 169 Alzheimer’s Disease Center, Emory University, Atlanta, Georgia, USA. Neuropathology, Banner Sun Health Research Institute, Phoenix, Arizona, USA. University Medical Center, Chicago, Illinois, USA. USA. Massachusetts, USA. Education and Clinical Center (GRECC), University of Wisconsin, Madison, Wisconsin, USA. Indiana University School of Medicine, Indiana, Indianapolis, USA. Healthcare System (VAAAHS), Ann Arbor, Michigan, USA. 121 Research on Aging, St. James Hospital and Trinity College, Dublin, Ireland. Erlangen, Germany. University Hospital of Essen, University Essen, Duisburg-Essen, Germany. Health), University of Florence, Florence, Italy. University of Washington School of Medicine, Seattle, Washington, USA. Barcelona, Barcelona, Spain. Germany. University of Milan, Fondazione Ca’ Granda, IRCCS Ospedale Policlinico, Milan, Italy. 108 Disorders Department, Medical University of Lodz, Lodz, Poland. Psychiatry, Psychology and King’sNeuroscience, College London, London, UK. 103 Mathematics and Statistics, Utah State University, Logan, Utah, USA. University School of Medicine, St. Louis, Missouri, USA. Psychiatry, Washington University School of Medicine, St. Louis, Missouri, USA. University of Pittsburgh, Pittsburgh, USA. Pennsylvania, Indiana, Indianapolis, USA. for Neuropathology, General Massachusetts Hospital, USA. Charlestown, Massachusetts, University of Pittsburgh, Pittsburgh, USA. Pennsylvania, Washington, USA. Rome, Italy. Medicine, Houston, Texas, USA. 85 General Massachusetts Medical Hospital/Harvard School, Boston, USA. Massachusetts, University Graz, Graz, Austria. 80 Sciences, Pharmaceutical University of Iceland, Reykjavik, Iceland. University, Baltimore, Maryland, USA. Center, Dallas, Texas, USA. Primary Medical Care, University Medical Centre Hamburg, Germany.Hamburg-Eppendorf, CNR-Maj, Lille, France. on Aging, Bethesda, Maryland, USA. Épinière (ICM), Département de Neurologie, Hôpital de la Paris, Pitié-Salpêtrière, France. Universités, Université Pierre et Marie Curie, Paris, France. Germany. University of Parma, Parma, Italy. Translational Research in Disease, Neurodegenerative Department of University Neuroscience, of Florida, Gainesville, Florida, USA. (CIBERNED), Instituto de Neurodegenerativas Salud Carlos III, Madrid, Spain. Madrid, Madrid, Spain. Italy. IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy. Nature Ge Nature Laboratory Laboratory Medicine, University of California, Davis, Sacramento, California, USA. Center on Aging, College of Public Health, Department of Epidemiology, University of Kentucky, Lexington, Kentucky, USA. Washington, Seattle, Washington, USA. Institute, Phoenix, Arizona, USA. 173 Harvard Medical School, Boston, USA. Massachusetts, Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, USA. Massachusetts, University of California, San Diego, La Jolla, California, USA. University of Kentucky, Lexington, Kentucky, USA. 162 Center, Miami Beach, Florida, USA. Neurological Disorders, University of California, Irvine, Irvine, California, USA. Irvine, California, USA. 156 University of Southern California, Los Angeles, California, USA. USA. Research Center, Seattle, Washington, USA. University, St. Louis, Missouri, USA. York, USA. and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, USA. North Carolina, USA. USA. Seattle, System/GRECC, Washington, USA. 139 School of Medicine, Pittsburgh, USA. Pennsylvania, Indiana, USA. 126 Institute Institute of Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria. Schools of Life Sciences and Medicine, University of Nottingham, Nottingham, UK. Department Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA. Department of Disease, Neurodegenerative MRC Prion Unit, UCL Institute of Neurology, London, UK. Memory Unit, Department of Neurology, Hospital Mútua Universitario Terrassa, Terrassa, Barcelona, Spain. Department Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA. Department of Neurology, Mayo Clinic, Florida, Jacksonville, USA. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA. Department of Neurology, Ralph H. Johnson VA Medical Center, Charleston, South Carolina, USA. Cognitive Neurology and Alzheimer’s Disease Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. Department of Neurology, Indiana University, Indiana, Indianapolis, USA. 56 153 143 131 Department Department of Neurology, Rouen University Hospital, Rouen, France. Department Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina, USA. Department of Neurology, University of California, San Francisco, San Francisco, California, USA. Wisconsin Alzheimer’s Disease Research Center, Madison, Wisconsin, USA. 111 65 148 Department Department of Public Uppsala Health/Geriatrics, University, Uppsala, Sweden. 88 Memory Memory Unit, Neurology Department and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Autonomous University of n Department Department of Psychiatry, Mount Sinai School of Medicine, New York, New York, USA. 128 Department Department of Neurology, University of Washington, Seattle, Washington, USA. etics Department Department of Psychiatry, University of Pennsylvania Perelman School of Medicine, USA. Pennsylvania, Philadelphia, 90 118 Office Office of Strategy and Measurement, University of North Texas Health Science Center, Fort Worth, Texas, USA. 145 133

Istituto Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Associazione Oasi Maria Santissima Srl, Troina, Italy. 158 58 72 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE University University of Kansas Alzheimer’s Disease Center, University of Kansas Medical Center, Kansas City, Kansas, USA. Department Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA. Instituto Instituto de Sanitaria Investigación Hospital la Paz (IdiPAZ), Madrid, Spain. Department Department of Pharmacology and University Neuroscience, of North Texas Health Science Center, Fort Worth, Texas, USA. Department Department of Neurology, University of California, Davis, Sacramento, California, USA. 75 95 112 Department Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany. Department Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 82 Department Department of Epidemiology, Harvard School of Public Health, Boston, USA. Massachusetts, 87 Regional Regional Neurogenetic Centre (CRN), ASP Catanzaro, Lamezia Terme, Italy. 175 63 Experimental Neuropsychiatry Laboratory,Experimental Neuropsychiatry IRCCS Santa Lucia Foundation, Department of Clinical and Behavioural Neurology, Center Center for Cognitive Disorders AUSL, Parma, Italy. 161 Department Department of Pathology, Duke University, Durham, North Carolina, USA. 150 70 77 Centre Centre Hospitalier de Universitaire Lille, Epidemiology and Public Health Department, Lille, France. Rush Rush Institute for Healthy Aging, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA. 177 USF USF Health Byrd Alzheimer’s Institute, University of South Florida, Tampa, Florida, USA. Institute Institute of Social Medicine, Occupational Health and Public Health, University of Leipzig, Leipzig, Germany. Department Department of Medicine (Medical Genetics), University of Washington, Seattle, Washington, USA. 141 152 Department Department of Neurology, Mayo Clinic, Rochester, Minnesota, USA. 115 Department Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami, Miami, Florida, 135 IRCCS IRCCS ‘Don Carlo Gnocchi’, Florence, Italy. 164 138 Rush Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, Illinois, USA. Department Department of Psychiatry, New York University, New York, New York, USA. 172 Department Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. 97 92 100 123 Department Department of University Pathology of (Neuropathology), Pittsburgh, Pittsburgh, USA. Pennsylvania, Department Department of Human Genetics, University of Pittsburgh, Pittsburgh, USA. Pennsylvania, Department of Neurology, University of Pittsburgh, Pittsburgh, USA. Pennsylvania, 68 Department Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, USA. 166 Michigan Michigan Alzheimer Disease Center, Ann Arbor, Michigan, USA. Institut Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A) and Institut du Cerveau et de la Moelle

155 Department Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, USA. 107 Department Department of Neurology, Medical University of South Carolina, Charleston, South Carolina, USA. 55 125 79 Department Department of Health Sciences, Psychiatry for the Elderly, University of Leicester, Leicester, UK. 168 Section Section of Gerontology and Geriatrics, Department of Medicine, University of Perugia, Perugia, 170 National National Alzheimer’s Coordinating Center, University of Washington, Seattle, Washington, USA. 102 Department Department of Medical and Molecular Genetics, Indiana University, Indiana, Indianapolis, USA. Neurogenetics Program, Neurogenetics University of California, Los Angeles, Los Angeles, California, USA. Division Division of Genetics, Department of Medicine and Partners Center for Personalized Genetic 114 Fundació Fundació per la Recerca Biomèdica i Social Mútua Terrassa, Terrassa, Barcelona, Spain. 117 120 NEUROFARBA (Department of Psychology,Neuroscience, Drug Research and Child 57 127 Department Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Department Department of Molecular UCL, Neuroscience, Institute of Neurology, London, UK. 160 60 Department Department of Neurology, University Hospital La Paz, Universidad Autónoma de 105 137 Department Department of Radiology and Imaging Sciences, Indiana University, Indianapolis, 122 99 National National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA. Wien Center for Alzheimer’s Disease and Memory Disorders, Mount Sinai Medical INSERM INSERM U558, University of Toulouse, Toulouse, France. 180 Hope Hope Center Program on Protein Aggregation and WashingtonNeurodegeneration, Departments Departments of Psychiatry, Neurology, and Psychology, University of Pittsburgh Geriatric Geriatric Research, Education and Clinical Center (GRECC), VA Ann Arbor 147 132 Department Department of Boston Biostatistics, University, Boston, USA. Massachusetts, 110 81 64 Department Department of Mount Neuroscience, Sinai School of Medicine, New York, New 84 Department Department of Medicine (Genetics Program), Boston University, Boston, Department Department of Neurology, Clinical Division of Medical Neurogeriatrics, 94 Department Department of Psychiatry and Psychotherapy, University Hospital, Saarland, Department Department of Psychiatry, Martin Luther University Halle-Wittenberg, Halle, 86 Department Department of Neurology, Catholic University of Rome, Rome, Italy. 69 Department Department of Pathology and Laboratory Medicine, Indiana University, 66 74 Alzheimer’s Disease and Memory Disorders Center, Baylor College of 130 Laboratory Laboratory of Epidemiology and Population Sciences, National Institute AXA AXA Research Fund and UPMC Chair, Paris, France. Department Department of Psychiatry, University of Texas Southwestern Medical 89 116 Department Department of Medicine, University of Wisconsin, Madison, Wisconsin, Department Department of Epidemiology, University of Washington, Seattle, 163 Institute Institute for Medical Informatics, Biometry and Epidemiology, 174 Sanders-Brown Center Sanders-Brown on Aging, Department of Biostatistics, Neurogenomics Division, Neurogenomics Translational Genomics Research 157 149 171 59 Department Department of Neurology, University of California, Irvine, Centro Centro de Biomédica Investigación en Red de Enfermedades Department Department of Pathology and Immunology, Washington 109 Department Department of Neurology, General Massachusetts Hospital/ 144 Department Department of and Pathophysiology Transplantation, 113 Department Department of Medicine, Duke University, Durham, 104 176 Department Department of Psychiatry and Behavioral Sciences, Department Department of Old Age Psychiatry, Institute of Department Department of Genome Sciences, University of 159 142 Institute Institute for Memory Impairments and 76 Swedish Swedish Medical Center, Seattle, Washington, 134 Department Department of Neurology, Johns Hopkins Department Department of Behavioral Sciences, Rush 179 96 124 83 Alzheimer’s Disease Research Center, Department Department of Pathology and Department Department of Psychiatry, 165 Indiana Indiana Alzheimer’s Disease Center, 151 91 62 Department Department of Neurosciences, 154 Fred Fred Hutchinson Cancer Department Department of Psychiatry, Department Department of Neuroscience, 140 119 Department Department of Neurology, 129 VA Puget Sound Health Care Mercers Mercers Institute for 146 Geriatric Geriatric Research, 71 106 178 Department Department of Genetics INSERM INSERM UMRS 1171, 93 136 Elderly Elderly and Psychiatric C.S. C.S. Kubik Laboratory Sanders-Brown Sanders-Brown 67 101 Civin Civin Laboratory for s r e t t e l Sorbonne Sorbonne 73 98 Department Department of 78 Department Department of Department Department of 61 Faculty Faculty of Center Center for 167 Emory Emory 11 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. should should be addressed to S.J.v.d.L. ( Neuroepidemiology, UMR 897, Bordeaux, France. Netherlands. Biology and Brigham Neuroscience, Young University, Provo, Utah, USA. de Universitario Palencia, Palencia, Spain. Centre Neurosciences, for Applied Medical Research, University of Navarra School of Medicine, Pamplona, Spain. University, Montpellier, France. and Resources Center, CMRR of Montpellier, Department of Neurology, Hospital Gui de Chauliac, Montpellier, France. University Medical Center, New York, New York, USA. Alzheimer’s Disease Research Center, Gainesville, Florida, USA. School Neurosciences, of Medicine, University of Southampton, Southampton, UK. Manchester, Manchester, UK. King’s College London, London, UK. 262 Texas, USA. University of Sacred Hearth, Rome, Italy. Italy. Medical Research, University of Cambridge, Cambridge, UK. Klinikum der Universität München, Munich, Germany. Clinical Center (GRECC), University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA. 252 University of Arizona, Phoenix, Arizona, USA. Netherlands. Erasmus University Medical Center, Rotterdam, the Netherlands. of Central Asturias, Oviedo, Spain. Center, Torrance, California, USA. Seattle, Washington, USA. of Washington, Seattle, Washington, USA. France. A-ICM), Institut du Cerveau et de la Moelle Épinière (ICM), Paris, France. d’Alzheimer (IM2A), Département de Neurologie, Hôpital de la AP–HP,Pitié-Salpêtrière, Paris, France. Hospital, Genetics, Paris, Pitié-Salpêtrière France. UMR 7225, Sorbonne Universités, UPMC Université Paris 06, UMRS 1127, Institut du Cerveau et de la Moelle Épinière, Paris, France. France. Lille Lille (Centre Mémoire de Ressources et de Recherche), Lille, France. Karolinska Institutet, Stockholm, Sweden. Medicine, Genetics Unit, Karolinska University Hospital Huddinge, Stockholm, Sweden. Antwerp, Antwerp, Belgium. Department of Neurology, Miller School of Medicine, University of Miami, Miami, Florida, USA. Hill, North Carolina, USA. Psychiatry and Behavioral Sciences, Duke University, Durham, North Carolina, USA. University, New York, New York, USA. University of California, Los Angeles, Los Angeles, California, USA. Center on Aging, Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky, USA. Perelman School of Medicine, USA. Pennsylvania, Philadelphia, Novartis Institutes for Biomedical Research, Cambridge, USA. Massachusetts, Southern California, Los Angeles, California, USA. Texas, USA. Disease, Neurodegenerative University of Toronto, Toronto, Ontario, Canada. University, New York, New York, USA. Medicine, Aurora, Colorado, USA. 202 Science Center, Fort Worth, Texas, USA. USA. Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. Jolla, California, USA. York, New York, USA. Michigan, USA. USA. 188 Boston University, Boston, USA. Massachusetts, University of California, Irvine, Irvine, California, USA. Oregon, USA. 181 s r e t t e l 1 2

Department Department of Medical Sciences, Institute of Biomedicine (iBiMED), University of Aveiro, Aveiro, Portugal. Departments of Psychiatry, Medicine, and Family and Community Medicine and South Texas Veterans Health Geriatric Administration Research Education and Department Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA. Department of Neurobiology and Behavior, University of California, Irvine, Irvine, California, USA. Department of Ophthalmology, Boston University, Boston, USA. Massachusetts, 257 199 190 238 230 PharmaTherapeutics Clinical Research, PharmaTherapeutics Pfizer Worldwide Research and Development, Cambridge, USA. Massachusetts, Department Department of Neurology, Washington University, St. Louis, Missouri, USA. Cleveland Clinic Lou Ruvo Center for Brain Health, Cleveland Clinic, Cleveland, Ohio, USA. Sorbonne Sorbonne Universités, Université Pierre et Marie Curie, Hôpital de la AP–HP,Pitié-Salpêtrière, Paris, France. University University of Bordeaux, Neuroepidemiology, Bordeaux, France. 260 209 279 248 183 Department Department of Psychiatry, Charité University Medicine, Berlin, Germany. Department Department of Rush Pathology (Neuropathology), University Medical Center, Chicago, Illinois, USA. 192 Department Department of Epidemiology and Case Biostatistics, Western Reserve University, Cleveland, Ohio, USA. Arizona Alzheimer’s Consortium, Phoenix, Arizona, USA. Department Department of Neurology, Portland Veterans Affairs Medical Center, Portland, Oregon, USA. Department Department of Psychiatry, Johns Hopkins University, Baltimore, Maryland, USA. 194 196 Department Department of Neurology, University of Miami, Miami, Florida, USA. School School of Nursing Northwest Research Group on Aging, University of Washington, Seattle, Washington, USA. 221 242 224 Department Department of Pathology, Oregon Health & Science University, Portland, Oregon, USA. 265 Institute Institute for Translational Genomics and Population Sciences, Los Angeles BioMedical Research Institute at Harbor-UCLA Medical Laboratory Laboratory for Cognitive Neurology, Department of Neurology, University of Leuven, Leuven, Belgium. 273 3rd 3rd Department of Neurology, Medical School, Aristotle University of Greece. Thessaloniki, Thessaloniki, 204 243 [email protected] 245 Memory Memory Research and Resources Center, CMRR de Bordeaux, Bordeaux, France. 264 Alzheimer’s Disease Center, New York University, New York, New York, USA. Centro Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain. 218 206 Human Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA. Institute Institute of Brain, Behaviour and Mental Health, Clinical and Cognitive Neuroscience Research Group, University of 201 Department Department of Psychiatry, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. Department of Neurology, University of California, Los Angeles, Los Angeles, California, USA. 259 240 227 276 Department Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA. Departments Departments of Neurology, Pharmacology, and TexasNeuroscience, Tech University Health Science Center, Lubbock, Centre Centre for Public Health, University of Iceland, Reykjavik, Iceland. 251 Institute Institute of Clinical Medicine/Neurology, University of Eastern Finland, Kuopio, Finland. Center Center for Applied Genomics, Children’s Hospital of USA. Pennsylvania, Philadelphia, Philadelphia, 187 Department Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 281 211 234 Department Department of Neuropsychology, University of California, San Francisco, San Francisco, California, USA. These These authors contributed equally to this work. Department Department of Pathology, University of Washington, Seattle, Washington, USA. 270 INSERM INSERM U1061, La Colombière Hospital, Montpellier, France. 254 185 Department Department of Health Services, University of Washington, Seattle, Washington, USA. German German Center for Diseases Neurodegenerative (DZNE), Munich, Germany. Department Department of Neurology, Boston University, Boston, USA. Massachusetts, 256 ), ), J. Williams ( Department Department of Experimental and Clinical Medicine, Neurological Institute, University of Pisa, Pisa, 269 214 247 217 Center Center for Translational and Systems Neuroimmunology, Department of Neurology, Columbia Department Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA. Netherlands Netherlands Consortium on Health Aging and National Genomics Initiative, Leiden, the 229 Taub Institute on Alzheimer’s Disease and the Aging Brain, Department of Pathology, Columbia University University Paris Descartes, EA 4468, AP–HP, Hôpital Broca, Geriatrics Department, Paris, 278 249 237 231 [email protected] Department Department of Neurology, Erasmus MC University Medical Center, Rotterdam, the 208 Banner Banner Alzheimer’s Institute, Phoenix, Arizona, USA. INSERM, INSERM, CNRS, UMRS 975, Institut du Cerveau et de la Moelle Épinière (ICM), Paris, INSERM, INSERM, Neuroepidemiology, UMR 897, Bordeaux, France. 198 213 Department Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Department Department of Pathology, University of Southern California, Los Angeles, California, Department Department of Pathology and Laboratory Medicine, University of Pennsylvania 182 267 200 220 Department Department of Neurology, Oregon Health & Science University, Portland, Department Department of Psychiatry, University of Oxford, Oxford, UK. Internal Internal Medicine, Division of Geriatrics, University of North Texas Health Department Department of Genetics, University of North Carolina at Chapel Hill, Chapel 261 226 Institute Institute of Public Health, University of Cambridge, Cambridge, UK. Department Department of Neurobiology, Care Sciences and Society, KIADRC, 195 223 203 Department Department of Pathology, University of California, San Diego, La Department Department of Neurology and Memory Clinic, Hospital Network Department Department of Neurology, University of Colorado School of ) ) or G.D.S. ( 193 191 189 282 Department Department of Medicine–Pulmonary, New York University, New 236 Department Department of Pathology, University of Michigan, Ann Arbor, a Department Department of Neurology, Emory University, Atlanta, Georgia, These These authors jointly directed this work. Correspondence DVANCE ONLINE PUBLICATION ONLINE DVANCE Institut Institut des TranslationnellesNeurosciences de Paris (IHU- 184 253 263 244 216 [email protected] Department Department of Pathology and Laboratory Medicine, 241 Institute Institute for Stroke and Dementia Research, Institute Institute of Psychiatry, Psychology and Neuroscience, Molecular Genetics Laboratory-Hospital, Molecular University Genetics Laboratory-Hospital, 205 275 Department Department of Pathology and Laboratory Medicine, 210 Group Group Health Research Institute, Group Health, 274 222 Department Department of Epidemiology, Columbia Department Department of Neurology, Complejo Asistencial 235 272 Department Department of Psychiatry, University of Neurogenetics Laboratory,Neurogenetics Division of Evelyn Evelyn F. McKnight Brain Institute, 280 239 Institut Institut de la Mémoire et de la Maladie Department Department of Neurology, Montpellier University University of Bordeaux, Department Department of Family Medicine, University 246 Department Department of Internal Medicine, 258 250 Institute Institute of Neurology, Catholic 212 186 255 225 Department Department of Psychiatry, 228 207 197 Translational Medicine, Department Department of Pathology, 233 Cambridge Cambridge Institute for Department Department of Geriatric CHU CHU Lille, Memory Center of Tanz Centre for Research in Department Department of Neurology, 232 266 AP–HP, Department of INSERM INSERM U1127, CNRS Division Division of Clinical

271 ) Nature Ge Nature Memory Memory Research 219 277 268 215 Department Department of Departments Departments of Florida Florida Sanders-Brown Sanders-Brown n etics © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. on the basis of call rate, excluding variants with >10% missingness, and and missingness, >10% filtered with were variants excluding Variants rate, call inspection. of basis visual the on via heterozygosity excess and missingness autosomal >10% with samples excluding rate, call sample for controls). 9,095 cases, (7,755 samples, replication the of subset a in France, Paris, CNG, the at the Agena MassARRAYBioscience platform or Thermo Fisher TaqMan assays plot An inspection. additional nine variants were genotyped successfully using in (rs147163004 variant successfully one with were genotyped, variants Twenty-one France. Paris, de (CNG), National Centre Génotypage the and Germany, Bonn, Brain, and Life at performed was Genotyping MassARRAY platform. Bioscience Agena on the genotyping status. disease quality participants’ to and blinding with performed were control genotyping sample All procedures. control quality and calling See sets. data all over alleles reference and alternate the harmonized we analysis, to Prior ing. components three principal were adjusted for as covariates test in association the first (>6 s.d. analyses; outliers from the were mean) from excluded further in EIGENSTRAT analysis principal-component using ined of the five subsets (NorthShore, Miami, WashU, CHOP, and ADC7) was exam variants with >0.01 were evaluated for departure from Hardy–Weinberg equilibrium and any rates <95% and filtering out of samples with call rate <95%. Variants with MAF control applied procedures to GWAS, exclusion including of variants with call quality standard a TaqMan using underwent samples assay. typed on all Data in (p.Arg47His) rs75932628 TREM2 variant, One v1.0. Illumina BeadChip the on HumanExome sets) data ADC7 and (CHOP CHOP and WashU, Miami, proportions ( equilibrium Hardy–Weinberg expected the form deviated that significantly those and individuals the of 5% over removal in (vii) called not and variants controls; of over retained cases with 0.45), > (IBS) state by relatives first-degree on the basis of relatedness calculated genetically (identity mismatches; (v) removal of one individual from duplicate pairs; (vi) removal sex of with individuals of removal (iv) (<95%); rate call genotype low with ants of individuals with low genotype completion rate (<90%); (iii) removal of vari removal (ii) samples; problematic of removal GWAS and with data checking previously described been have procedures control quality study. central at The each and Houston control procedures for the genotype data were both atcentrally performed UT at from 11 samples of studies University the Texas HSC at Houston 62,266 with called jointly were studies four all for genotypes the calls, otype Toof gen rare v1.0. the variant quality the increase BeadChip HumanExome array. 1.1 version the for remained 239,814 and analysis for 1,375 controls) remained. The version 1.0 array had variants available 244,412 and LOAD 1,907 cases 1.1, version controls; and LOAD 1,599 4,093 cases 1.0, (version controls elderly 2,974 and cases LOAD 6,000 control, quality After factor, inflation control on genomic the impact maximum the had covariates as components principal three first the including EIGENSTRAT, variants with ants with a separation score <0.4, and Hardy–Weinberg equilibrium excluding ing variants with >1% missingness, genotype cluster separation excluding vari (non-European ancestry). Variants were filtered on the basis of call rate exclud (IBD) descent by identity with related pair each of one excluding relatedness ance, on gosity based outliers excluding <1% and >1% MAF separately, sex discord rate call excluding samples with excess >1% autosomal heterozy missingness, used for control Quality genotype calling. filters were implemented for sample zCall or GenomeStudio in algorithm clustering 2.0 version GenTrain v1.0 ( BeadChip HumanExome Illumina with Germany, Bonn, Brain, and Life at performed Genotyping and quality control. ONLIN doi: P < 1 × 10 × 1 < EA/EAE ad C qaiy control. quality ACE and GERAD/PERADES 2. Stage ADGC. CHARGE. 10.1038/ng.3916 ≥ , clustered poorly across all ADGC cohorts and was therefore regeno and was therefore cohorts ADGC all across poorly , clustered 0.125 (the level expected for first cousins), and population outliers outliers population and cousins), first for expected level (the 0.125 E E Genotyping was performed in subsets at four centers: NorthShore, at NorthShore, four centers: in subsets was performed Genotyping Twenty-two variants were successfully designed for replication replication for designed successfully were variants Twenty-two −6 M ). All four CHARGE cohorts were genotyped for the Illumina Illumina the for genotyped were cohorts CHARGE four All P P E HWE HWE THO n Supplementary Table 3 Supplementary = variants) 247,870 or v1.1 ( < 10 < 1 × 10 6 D 1 . Minimum quality control included (i) concordance concordance (i) included control quality Minimum . S −6 were excluded. Population substructure within each −4 . . Ten principal components were using extracted

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with 39,235,157 SNPs that allows imputation down to an unprecedented unprecedented an to down 0.00008. of MAF imputation allows that SNPs 39,235,157 with panel reference haplotype HRC the to of Miami and Vanderbilt University. in All samples used stage 3 were imputed cases and 115 controls from second-wave 84 samples ascertained atwith the Universitytogether genotyped were (TARCC2) subjects TARCC Second-wave BeadChip. HumanOmniExpress-24 the Illumina using were genotyped trols) and 74 wave con cases (172 in second the subjects chip, while 6.0 microarray 1M platform. TARCC first-wave subjects were genotyped Illumina using the the Affymetrix on genotyped was NBB while array, OmniExpress-24 the on Illumina genotyped were sets data WHICAP and CHAP sets. data WHICAP and CHAP,TARCC, NBB, the described for except been sets, data have most for used before detail in arrays genotyping The above. described as sets previously using methods. determined described were components Principal control. quality 10 × 1 < controls and cases cases and controls separately) < 1 × 10 (in equilibrium Hardy–Weinberg >5%, missingness of basis the on excluded were Variants panels. genotyped the within present variants chromosome X for heterozygous males were as excluded, were genotypes missing three than more with Samples separately. panel each in performed was control quality controls. or cases either with variants excluding equilibrium, Hardy–Weinberg independent analysts confirmed the meta-analysis results. meta-analysis the confirmed analysts independent Three information. MAF the to contribute they as excluded not were studies individual in variants Monomorphic estimates. maximum-likelihood the to ‘one-step’ a as approximation interpreted be can errors standard and ficients coef seqMeta The meta-analyzed. were studies individual for objects Rdata *. the SeqMeta, of functions ‘skatOmeta()’ and ‘singlesnpMeta()’ the Using SNP vidual scores, MAF, and a matrix for covariance each unit of aggregation. indi the SKAT analyses: meta-analyze to information necessary the contain objects *.Rdata These objects. *.Rdata in captured were ‘prepScores()’, which the function through score statistics generated analyses cohort-level pipeline, SeqMeta the In package. R meta-analysis. SeqMeta the in meta-analyzed were variants Rare inverse-variance-weighted fixed-effects a using METAL in cognition. normal with controls for interview at last age the and components. Age was defined as the age at onset of principal clinical symptoms three for cases first the and origin of country as well as sex, and age for ates covari included which ‘adjusted’ an model, (ii) and analysis, pal-component from princi components principal three and of first the stratification, origin country using population possible for adjustment minimal included which (i) an two ‘unadjusted’ models: under model, CHARGE consortia and ADGC the in center contributing each for and consortium GERAD/PERADES the (MAF variation rare for package seqMeta and test score the using estimation maximum-likelihood menting imple and 1%) > (MAF variants low-frequency and common for modeling Analysis. in reported SNPs is replicated for genotypes chip exome and genotypes imputed 3 stage between Concordance and alternate allele homozygotes) was >88.5% on average ( was >99%, while concordance among alternate allele genotypes (heterozygotes genotypes to those directly genotyped by the exome array; overall concordance imputed comparing by variants critical for quality imputation we the 1, examined stage of part as genotyped been also available. had samples genotypes these of array subset a of As density regional the on based varying vidual indi each for imputed SNPs of number actual the with imputed, were SNPs 39,235,157 samples, all Across imputed. were cognition normal with viduals indi 17,176 and cases disease Alzheimer’s 16,175 for arrays genotyping SNP ( edu Server Imputation Michigan the on elsewhere extensively that described have been Prior to imputation, all genotype data underwent quality control procedures 3. Stage IGAP and EADI QC. Meta-analysis for common and low-frequency variants was undertaken undertaken was variants low-frequency and common for Meta-analysis / ) running Minimac3 running )

Stage 1. Stage Replication was performed using genotypes from 23 ADGC data data ADGC 23 from genotypes using performed was Replication We tested association with LOAD using logistic regression regression logistic LOAD using with We association tested Variants were in genotyped three different panels, and 63 , 6 −5 4 . Genotypes from genome-wide, high-density high-density genome-wide, from Genotypes . , for each country cohort. All variants passed passed variants All cohort. country each for , ≤ Supplementary Table 6 Supplementary 1%). Analyses were conducted globally in in globally conducted were Analyses 1%). −5 32 , , and missingness differential between , https://imputation 3 3 , which includes 64,976 haplotypes haplotypes 64,976 includes , which 2 , 7 . Imputation was performed . Imputation was performed Nature Ge Nature P n HWE . = 13,000 samples). server.sph.umich. < 1 × 10 × 1 < n etics −5 in in ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. total total number of protein–protein the interactions for given each gene, random chance, sets of by 151 expected than larger was network this whether Totest interactions. protein high-confidence by connected genes 56 of network a in The largest protein network interaction resulting from this procedure resulted follows: as generated were networks interaction protein Then, ( database STRING the of (v10) version latest the from high-confidence score (confidence > of 0.7) human protein–protein list interactions was downloaded a First, performed. was analysis interaction protein them for between relationships future study, genes biological and these clarify To identify etiology. disease Alzheimer’s genes to relevance of genes co-expressed contains 151 thus of set This association. GWAS IGAP for enriched strongly present genes in two or more were of four particularly modules these four modules was also found to be enriched for immune-related genes. The 151 ants associated with Alzheimer’s disease risk in the IGAP GWAS. Each of these vari common for enriched were that modules gene four identified networks Protein interaction analysis. ( results variant common the parallel closely they and response, and immune differentiation, B cell efflux, cholesterol particles, all 9,816 pathways used previously. The top pathways are related to lipoprotein analysis. the test both from the LD genes and between genes dropping from less significant enrichment the in bias potential any removes procedure conservative highly SKAT-O significant more the with gene other, the each of Mb 1 within genes pathway of pair each for and, removed was region the analysis, secondary a However,as genes. pathway between LD for low degree of LD the Given test. Fisher’s using of were probability combined interest combined the common variation data set in pathways enriched of excess significant most the yielded this as sequence, genomic surrounding without were apply defined Genes also to rare variants. 15 were for in tested rare this study enrichment ( variation clusters previously identified as enriched for association in the IGAP data set analysis. enrichment set gene and Pathway in variants in and variants only and below, described procedures analytical package. R SeqMeta the in IGAP undertaken was Swedish Meta-analysis clusters. and Italian the in included was one and set data EADI1 the meta-analysis mega IGAP the of format the Following participants. Spanish only including ACE cohort the and with the IGAP only French EADI1 cohort cohorts, including participants the in country by and cohort GERAD/PERADES the in globally undertaken in indicated groups analysis above, the in described models two the under conducted were Analyses package. 2 See provided. this size sample larger the given model unadjusted the used analysis primary The analyses. adjusted the in included included in the unadjusted analyses, and 15,272 cases and 17,115 controls were were controls 17,967 and cases 16,160 Therefore, analyses. adjusted the from excluded were and available information age have not did controls) 852 and Nature Ge Nature number total same the have to chosen gene each with generated, were genes for quantile–quantile plots of unadjusted and adjusted analyses. adjusted and unadjusted of plots quantile–quantile for ) to test whether the biological enrichments observed in common variants variants common in observed enrichments biological the whether test ) to 5. 4. 3. 2. 1. We also performed pathway analyses on the rare variant data presented using 3. Stage Stage 2. In the consortium, GERAD/PERADES 1,740 participants (888 LOAD cases TREM2

with a protein corresponding to any gene already in the network. the in already any gene to corresponding protein a with interaction protein high-confidence a shows protein corresponding its Repeat, choosing each of the 151 genes in turn as the seed gene. seed the as turn in genes 151 the of each choosing Repeat, network. the in genes of number Note the added. be can genes more no until 2 step Repeat if it add network to the of genes, 151 set in the gene remaining For each ‘seed’ gene). (the network the start to gene a Choose We LOAD with the score using test and association seqMeta tested The association analyses performed followed stage 1 and stage 2 stage and 1 stage followed performed analyses association The PLCG2 were examined. For gene-based testing, 10 variants in in variants 10 testing, gene-based For examined. were n etics 6 5 , and 13 variants in in variants 13 and , between rare variants, our primary analyses did not control Previous analysis of normal brain co-expression 2 3 , four principal components were included for included were components , principal four 7 . Gene-wide . SKAT-OGene-wide Supplementary Table 2 Table Supplementary TREM2 were examined. were The eight biological pathway pathway biological eight The Supplementary Table 16 Supplementary P value was removed. This This removed. was value Supplementary Figure Figure Supplementary P Supplementary Table Supplementary values for the variants htt p://string-db.org . Analyses were were Analyses . ABI3 , , ABI3 PLCG2 APOE , 35 35 , / ). 3 ). ). 7 - ,

expression levels in the temporal cortex of 80 subjects with pathologically pathologically with subjects 80 of cortex temporal the in levels expression 2.78 × 10 were the of it. set omitting run Both 151 ( genes analyses enrichment <1%, gene-wide significant that Given genes. interacting 56 of set a to signal refined this has analysis network protein the furthermore, and, GWAS, variant in common the observed signal of biological the replication show convincing ( genes the between LD the excluding analysis conservative ( genes 56 of subset in the set of 151 module overlap genes ( overlap in genes of set the 151 module that the 56 network genes account for most of observed signal the enrichment It seen <1%. be can MAF with variants rare the in for association enrichment 10 × ( genes 151 of set the Both above. described method gene enrichment set the using <1% MAF with for variants in tested signal were association network of the enrichment not but overlap module the in genes 95 of set genes. overlap module 151 of set the in observed signal enrichment 17 Table in ( genes network 95 interaction but protein not the overlap module the the on performed also was analysis same The SNPs). those ing P ALIGATOR GWAS using IGAP the in enrichment association for tested was genes work network. protein the of analysis enrichment set Gene bias. without genes 151 of set original the in that to relative tested in of the set can genes be 56 network association of genetic Thus, the strength gene. each for associations variant GWASrare of or strength the to agnostic is and data interaction protein on only relies network interaction protein the interaction network as large as 56 genes. Noteprotein a that the yielded procedure them for of generatingnone and generated, were sets gene random 1,000 network. 56-gene observed the to compared was network such largest the of Protein networks were generated for each gene as above,described and the size data. actual the in gene corresponding the as interactions protein–protein of has been shown to correlate with increasing neuritic plaque burden plaque neuritic increasing with correlate to shown been has rate (FDR) < Similarly, discovery 0.05). burden high (false to adjacent sites H3K9ac two at acetylation lower and sites CpG four at ylation ( Zhang by reported as microglia in and project ROADmap by the reported blood whole both of expression of levels high the with are consistent findings These C.C.W.,data). unpublished and 2,600, respectively, out of the 11,500 expressed genes) (P.L.D.J., D.A.B. and 1,740 (ranked cohorts from these of from the cortex 11 subjects macrophages genes) However,expressed 13,484 of out 12,965 (ranked autopsy prospective with levels in the dorsolateral prefrontal cortex of subjects from two studies of aging genes are these at (508 persons): expressed low individuals characterized cally genes expression. Gene study. biological future for ment for genetic association signal. These genes are therefore strong candidates enrich show but also interaction, only protein and not co-expression are through related that genes of network a defines it that is set gene the reducing for rationale the Thus, susceptibility. disease Alzheimer’s to important most are that genes the on work such concentrate to important therefore It is sive. TREM2 to restricted not is networks these in susceptibility disease to variants rare of that the contribution of suggesting signal, enrichment showed significant still ( enrichment significant nominally only have genes 95 remaining the as genes, 151 of set in the observed signal of for 56 of most accounts subset genes enrichment the n -value thresholds for defining significant SNPs contain genes thus (and the significant for defining thresholds -value = 714) PLCG2 AMP-AD gene expression data. expression gene AMP-AD The set of 56 network genes, the set of 151 module overlap genes, and the the and genes, overlap module 151 of set the genes, network 56 of set The −6 PLCG2 ) and the subset of 56 genes ( genes 56 of subset the and ) . Biological follow-up of genetic results is labor intensive and expen and intensive labor is results genetic of follow-up Biological . 6 ). It can be seen that the 56 network genes account for most of the the of most for account genes network 56 the that seen be can It ). −3 and and 8 ABI3 and H3K9ac acetylation ( t al. et ) ) and of the subset 56 genes ( 6 6 P and and , as was done in the original pathway analysis, using a range of range a using analysis, pathway original the in done was as , ABI3 = 0.043). Both the set of 151 genes ( genes 151 of set the Both 0.043). = and 3 8 . From the same brain tissue, we examined methylation methylation examined we tissue, brain same the From . We examined mRNA expression of the newly associated associated newly the of expression mRNA Weexamined ABI3 PLCG2 in relation to increased global neuritic plaque and tangle tangle and plaque neuritic global to increased relation in P PLCG2 = 2.98 × 10 × 2.98 = P value ( value in post-mortem brain tissue from neuropathologi from tissue brain post-mortem in Supplementary Table 17 Table Supplementary were more microglia/ in expressed highly purified and and P = 1.01 × 10 × 1.01 = RNA sequencing was used to measure gene to was measure used RNA sequencing ABI3 −7 n APOE P ) show significant enrichment under a under enrichment significant show ) = 676) data and found differential meth = 1.08 × 10 × 1.08 = in peripheral monocytes, spleen, and and spleen, monocytes, peripheral in Supplementary Table 17 Supplementary P = ( 0.010) region and correcting for possible possible for correcting and region −13 ) among variants with MAF MAF with variants among ) −7 Supplementary Table Supplementary 18 ). Thus, the rare variants variants rare the Thus, ). ) show highly significant significant highly show ) P = 5.15 × 10 × 5.15 = TREM2 doi: The set of 56 net 56 of set The TREM2 Supplementary Supplementary 10.1038/ng.3916 has a highly highly a has ). Again, ). the Again, −5 expression expression 6 ) and the the and ) 9 P . = 1.17 1.17 = P 6 = 7 ------) .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. accessed by applying directly to Cardiff University. Stage 1 ADGC data are are data ADGC 1 Stage University. Cardiff to directly applying by accessed in provided are Data availability. genes that given microgliosis, quence of changes in cell types that occur with Alzheimer’s most disease, likely are a elevations likely that these conse This suggests model). (comprehensive gene counts but for adjusting cell-type-specific after is abolished model), significance this (simple type cell for correcting before cortex temporal disease TREM2 neuropathology. Alzheimer’s disease with occur that changes number for cell lial), ( genes cell-type-specific five for markers type cell brain and covariates, these all for adjust we model, comprehensive sex, RNA number integrity tissue (RIN), source, and RNA-seq flow cell. In the death, at age for adjust we model, simple the In models. different two using diagnosis disease Alzheimer’s with levels expression gene of association for to was used test regression linear syn3163039, Multivariable and syn6090802. syn6126114, respectively: pages, Synapse following the at available are ods meth analytic and control, quality overview, data sequencing RNA human by two s.d. The in groups divided means the between within-group difference at and 0.59 0.49, sample any Assuming two without samples of pathologies. 100 per group,neurodegenerative two- controls 76 and disease Alzheimer’s confirmed pathologically with subjects 80 had cohort human post-mortem our control, quality After ( Synapse in Accelerating Medicines Partnership-AD (AMP-AD) knowledge portal housed the in deposited are data sequencing RNA human The Institute. Health Sun Banner the and Bank Brain Clinic Mayo the from obtained pathologies tive neurodegenera any without controls 76 and disease Alzheimer’s confirmed doi: Stage 1 data (individual level) for the GERAD exome chip cohort can be be can cohort chip exome GERAD the for level) (individual data 1 Stage 10.1038/ng.3916 OLIG2 1 5 ( t , , test, same s.d., we would have 80% power to sizes effect of detect 0.40, Supplementary Fig. 12 and Supplementary Table 19 Supplementary and 12 Fig. Supplementary PLCG2 (oligodendroglia), (oligodendroglia), https://www.sy Supplementary Table 6 Supplementary , and and , P Summary statistics for statistics Summary the 43 identified genetic associations < 0.05, 0.01, and 0.001, respectively, where effect size is the size effect where respectively, and 0.001, 0.01, < 0.05, ABI3 TREM2 expression is significantly higher in Alzheimer’s Alzheimer’s in higher significantly is expression napse.org/#!Synapse: GFAP , , PLCG2 (astrocyte), (astrocyte), . , and and , CD68 (microglia), (microglia), ABI3 ENO2 syn2580853/wiki/6672 are microglia-enriched microglia-enriched are (neuron)) to (neuron)) account CD34 ). (endothe 2 ). ). - - - - 66. 65. 64. 63. 62. 61. 60. 59. data). (human syn3163039 and data) (mouse syn3435792 platform software from Sage Bionetworks under Synapse and IDs syn3157182 Alzheimer’s Disease (AMP-AD) knowledge portal that is hosted in the Synapse in Partnership Medicines Accelerating the through investigators qualified all request. upon available are data 2 3 Stage and primary stage laws. to Icelandic owing are not available US cohorts and to Erasmus University for Rotterdam data. AGES primary data all for dbGaP to applying by accessible are data data CHARGE 1 Stage repository. qualified-access NIA/NIH-sanctioned and NIAGADS- a in deposited 69. 68. 67. A detailed description of the Mayo Clinic RNA-seq data is available to to available is data RNA-seq Clinic Mayo the of description detailed A

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