Meta-Analysis of 74,046 Individuals Identifies 11 New Susceptibility Loci for Alzheimer's Disease

© 2013 Nature America, Inc. All rights reserved. in each study ( study each in SNPs per data set. We excluded SNPs that did not pass quality control from types 2010) to December impute for genotypes up to 11,863,202 on based data from sequence freeze 4 August 2010 and haplo phased release interim data (2010 from haplotype Project the 1000 Genomes Supplementary Note A A full list of authors and affiliations appears at the end of the paper. ( Consortium the Genetic and Environmental Risk in Alzheimer’s Disease (GERAD) (CHARGE) Consortium, the European Alzheimer’s InitiativeDisease Epidemiology (EADI) and Genomic in Research Aging and Heart for Cohorts the (ADGC), Consortium Genetic Disease Alzheimer’s 2). (stage controls 11,312 and cases ( by up followed genotyping of 1) 11,632 SNPs showing moderate of evidence (stage association controls 37,154 and cases 17,008 totaling of 4 GWAS a meta-analysis conducted of ancestry samples European banner of I-GAP (International Genomics of Alzheimer’s Project), we power. of GWASUnder the statistical meta-analysis to increase scale with tified associated regions genomic LOAD other nine identified ancestry disease Alzheimer’s for factor risk genetic major a is E) apolipoprotein (encoding gene autonomy.The of loss to leading functions, cognitive in tion affecting the elderly. The disease manifests with progressive deteriora primarily disorder neurological devastating a is disease Alzheimer’s associated combined 1 In set were disease published (7,055,88 ancestry. wide We portion (LOAD) Eleven susceptibility loci for Alzheimer’s disease Meta-analysis of 74,046 individuals identifies 11 new Nature Ge Nature Received 13 March; accepted 27 September; published online 27 October 2013; P 9

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doi:10.1038/ng.280 ing ing SNPs within the meta-analysis was 1.087 for the full set of SNPs and 1.082 after exclud to study. each for correction factor the The control genomic inflation as performed meta-analysis of analyzed the results after applying genomic control were dosages genotype in the described set, data each In Methods). Online SNPs; (7,055,881 sets data all 40% across and samples control cases the of disease Alzheimer’s the of 40% least at in imputed successfully or genotyped either SNPs included meta-analysis Our and stage 2 data sets, which represent a secondary discovery effort, effort, discovery secondary a represent which sets, data 2 stage and 1. stage in as association of direction and ( of significance level a nominal at Alzheimer’s with SNPs in disease ses 2, stage 2,562 were associated signi genome-wide ficance with Alzheimer’sat disease risk in stage 1. associated Additionally, in been analy had 80 SNPs, 116 these Of ( testing multiple for correction Bonferroni strict a were after 2 stage in that risk 2 disease Alzheimer’s with and associated 1 significantly stages in association of direction and allele risk ( States Note United Supplementary the and UK the Sweden, Spain, Italy, Hungary, Greece, Germany, Finland, Belgium, Austria, from nating of controls and European 11,312 cases 8,572 included we considered 11,632 SNPs for association analysis. The stage 2 sample iSelect technology. After quality control procedures (Online Methods), From the initial set of SNPs, 14,445 could be genotyped using Illumina ( 45,409,039–45,412,650) P in given are region for results not each are Detailed the genes. causative these potentially SNP. associated However, that are aware we significantly most the to samples Asian and through candidate gene approaches and in a GWAS combining ADGC SORL1 SLC24A4 susceptibility genetic factors as GWAS by identified previously been had ( level significance genome-wide the reached that tions (see ( significance genome-wide of level specified pre a at disease Alzheimer’s with associated SNPs of kb 500 within value less than 1 × 10 × 1 than less value The results from stages 1 and 2 and from the combined stage 1 stage combined the from and 2 and 1 stages from results The a with SNPs 1 stage all genotyping for selected we 2, stage In the to addition In Supplementary Fig. 1 Fig. Supplementary had previously been identified as an gene Alzheimer’s identified disease been had previously 3– 7 - ad ie ( five and , RIN3 2 and Supplementary Note Supplementary 1 Supplementary Figures 2 Figures Supplementary 0 APOE DSG2 . Genes attributed to a signal were those closest closest those were signal a to attributed Genes . APOE ). We observed 116 SNPs showing the same same the showing SNPs 116 observed We ). HLA-DRB5 −3 n locus (chr. 19: and 45,409,039–45,412,650) ) represent newly associated loci ( loci ) associated represent newly , excluding SNPs flanking flanking SNPs excluding , = 19,532; see URLs for database access). access). database for URLs see 19,532; = for quantile-quantile plots). quantile-quantile for locus, 14 genomic regions had associa had regions genomic 14 locus, P < 0.05), having the same risk allele allele risk same the having < 0.05), – HLA-DRB1 ( Supplementary Table Supplementary 2 P – < 5 × 10 × 5 < 7 . , , PTK2B s r e t t e l P APOE

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© 2013 Nature America, Inc. All rights reserved. in red. Red diamonds represent SNPs with the the with SNPs represent diamonds Red red. in shown are genes associated newly and black, in shown GWAS are by identified previously Genes ( significance genome-wide for threshold The controls). 37,154 and cases (17,008 disease Alzheimer’s with association wide 1 Figure of of 3 the from for away kb 5.5 approximately signal a detected ( rs9381040 we these, Among association of ( evidence suggestive with loci 13 identified also sets data 2 stage and and Methods (Online identify P causative potential genes, we all SNPsalso examined with association except at There was no significant heterogeneity across studies at any of the loci, in are provided LOAD loci identified the in level combined analysis significance ( genome-wide the reached loci new seven (LD, age disequilibrium link SNPs in surrounding from by data supported not was and SNP single a for data on based was locus this for association of evidence to replicate 1. Inability in stage level cance signifi the genome-wide that surpassed loci all replicated nominally in shown are AAO, ageatonset;AAE,examination. T s r e t t e l smallest P P able able 1 < 5× 10 5× < < 5 × 10 1 10 × 1 < h rsls rm h cmie sae 1 stage combined the from results The TREM2 TREML2 Stage 2 Stage 1

N United States UK Sweden Spain Italy Hungary Greece Germany Finland Belgium Austria Country N GERAD EADI CHARGE ADGC cis P Consortium Description Description of the consortium data sets used for stage 1 and stage 2 Manhattan plot of stage 1 for genome- 1 for stage of plot Manhattan DSG2 values in the overall analysis. overall the in values −8 . . expression quantitative trait locus ( −8 TREM2 that were within 500 kb of the top SNP at each locus to to locus each at SNP top the of kb 500 within were that and 24 kb away from the 5 the from away kb 24 and P ) is indicated by the red line. line. red the by indicated ) is −6 = 6.3 × 10 × 6.3 = Table ( ( ) Table upeetr Tbe 4 Table Supplementary Table was recently reported to to reported recently was 2 . With the exception of of exception the With . 2 Supplementary Table 3 Supplementary 17,008 10,273 and 8,572 2,121 1,729 3,177 2,243 1,315 −7 r 2 2 N N 572 490 797 125 256 972 422 878 210 > 0.8; ), which is located located is which ), ). ). More detailed results for the seven newly Alzheimer’s diseasecases Supplementary Supplementary Figs. 12 Supplementary Fig. 7b Supplementary Percent Percent women women 61.9 57.6 61.7 66.3 66.5 68.0 63.3 63.9 68.0 66.1 61.0 64.0 64.9 63.6 59.4

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2 . Percent Percent INPP5D women women 54.0 29.2 62.8 55.3 55.7 69.0 34.1 53.1 59.3 59.5 43.3 51.8 60.7 57.8 58.6 Controls neurotransmitters that increase calcium flux and the downstream downstream the and flux or calcium receptors increase that neurotransmitters neuropeptide-activated between intermediate an be 5 Table Supplementary inde the confirmed the data of 2 pendence stage the in analysis conditional (iii) ( loci two the between exists peak recombination a (ii) and ( LD in not are genes two these of associ each strongly within SNPs most ated two the (i) because independent are signals from away kb 130 approximately only metabolism of the amyloid precursor andprotein (APP) trafficking to LOADaberrant connects directly that gene LOAD first the dominant and sporadic forms of Alzheimer’sautosomal both disease of risk increased with associated is it noteworthy,as

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DVANCE ONLINE PUBLICATION ONLINE DVANCE 6 PTK2B ZCWPW1 NME8 PTK2B EPHA1 Chromosom 7 CLU PICALM CD2AP ( genes disease Alzheimer’s fluid spinal tau, a for biomarker of levels phosphorylated cerebral of study a in ficance disease signi This region genome-wide reached also Alzheimer’s developing of risk increased fourfold to three- with associated p.Arg47His) (encoding variant rare a carry for this signal ( signal for this were unable to we define which gene(s) 6, are responsible chromosome on region (HLA) of antigen organization the human leukocyte disease Parkinson and sclerosis multiple of both risk and histocompatibility and, interestingly, with region is associated with immunocompetence DR II, class (encoding major histocompatibility complex, the in was ciation significance in European samples. European in significance associatedgenewasatgenome-widethat this L(DLR class) 1). Our data clearly demonstrated SORL1 association ( association PTK2

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© 2013 Nature America, Inc. All rights reserved. (sodium/potassium/calcium exchanger), member 4). The The 4). member exchanger), (sodium/potassium/calcium disease Alzheimer’s in implicated the in significance genome-wide with SNPs two associated For instance, PTK2B the in signals are multiple there that possibility the however, exclude memory of formation the in process central a region, 1) ammonis (cornu CA1 hippocampal the in potentiation long-term signaling (MAPK) kinase signals regulating neuronal activity such as mitogen-activated protein SNP T Nature Ge Nature and hair and development iris in involved protein a encodes gene rs10498633 rs11218343 rs28834970 rs9271192 New locireachinggenome-widesignificanceinthediscoveryanalysis rs3865444 rs4147929 rs10792832 rs983392 rs9331896 rs11771145 rs10948363 rs6733839 rs6656401 Known GWAS-defined associatedgenes e a Chr., chromosome. rs7274581 rs17125944 rs10838725 rs1476679 rs2718058 rs190982 rs35349669 New locireachinggenome-widesignificanceinthecombineddiscoveryandreplicationanalysis rs8093731 Calculated withrespecttotheminorallele. SNPs showingthebestlevelofassociationaftermeta-analysisstages1and2. able able 2 The fourth locus was was locus fourth The PTK2B a -

CLU S g g ummary ummary of stage 1, stage 2 and overall for meta-analyses - n CLU Chr. 20 14 11 18 14 11 19 19 11 11 region that are functionally connected to a single gene. gene. single a to connected functionally are that region etics 2 8 6 8 7 6 2 1 7 7 5 region are eQTLs for the gene 234068476 121435587 143110762 127892810 207692049 100004446 47487762 55018260 53400629 47557871 37841534 88223420 29088958 92926952 27195121 32578530 51727962 85867875 59923508 27467686 Position

1063443 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE SLC24A4 b 1 6 . . CASS4 FERMT2 CELF1 ZCWPW1 NME8 MEF2C INPP5D DSG2 RIN3 SLC24A4 SORL1 PTK2B HLA-DRB1 HLA-DRB5 CD33 ABCA7 PICALM MS4A6A CLU EPHA1 CD2AP BIN1 CR1 PTK2B Closest gene f Cochran’s 1 (encoding solute carrier family 24 24 family carrier solute (encoding 8 c ( is involved in the induction of of induction the in involved is

Supplementary Table 3 Supplementary - – Major/minor Q test. alleles A/G A/G G/A G/A A/G G/A A/G G/A G/T A/C C/A T/C T/C T/C T/C C/T C/T T/C T/C T/C C/T g Not replicatedinstage2. DPYSL2 0.083 0.092 0.316 0.287 0.373 0.408 0.488 0.017 0.217 0.039 0.366 0.276 0.307 0.190 0.358 0.403 0.379 0.338 0.266 0.409 0.197 MAF

d 1 7 that has been . We cannot, We . cannot, (0.82–0.92) (1.07–1.19) (1.04–1.11) (0.89–0.96) (0.90–0.96) (0.89–0.95) (1.03–1.10) ( (0.87–0.94) (0.70–0.83) (1.07–1.14) (1.07–1.16) (0.88–0.94) (1.10–1.20) (0.85–0.91) (0.87–0.93) (0.84–0.89) (0.87–0.93) (1.07–1.14) (1.17–1.25) (1.12–1.22) 0.43–0.67) (95% CI) SLC24A4 1.11 0.91 1.14 0.88 0.90 0.86 0.90 1.10 1.21 1.17 0.87 1.13 1.08 0.92 0.93 0.92 1.07 0.54 0.90 0.76 1.10 b OR Build 37,assemblyhg19. ).

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S 1.6 ×10 5.1 ×10 1.7 ×10 6.5 ×10 2.8 ×10 9.6 ×10 8.8 ×10 3.1 ×10 1.7 ×10 7.7 ×10 1.6 ×10 1.0 ×10 6.7 ×10 7.4 ×10 1.3 ×10 2.5 ×10 9.6 ×10 4.6 1.5 ×10 5.0 ×10 3.3 ×10 NPs NPs reaching genome-wide significance after stages 1 and 2 P Meta in the the in analysis. combined the in significance genome-wide reached loci new seven 1, stage in pathology tau-mediated to connected with interacts the directly called gene another is SNP associated strongly most the of vicinity the in in the brain and may be involved in neural development neural in involved be may and brain the in hypertension developing of risk the with associated being to addition in humans in variation color skin value × The strongest association at one of these new loci was intronic intronic was loci new these of one at association strongest The significance genome-wide reaching loci four these to addition In 10

−8 −8 −9 −16 −11 −17 −10 −8 −26 −15 −6 −5 −6 −6 −5 −6 −5 −8 −7 −11 −9 RIN3 ZCWPW1 c Genes located (0.94–1.04) (1.10–1.24) (0.81–0.89) (0.86–0.94) (0.82–0.90) (0.86–0.95) (1.04–1.15) (1.18–1.29) (1.14–1.28) (0.82–0.96) (1.08–1.26) (1.04–1.14) (0.85–0.94) (0.87–0.95) (0.89–0.98) (1.05–1.15) ( (0.88–0.98) (0.70–0.88) (1.06–1.17) (1.06–1.18) 0.80–1.28) (95% CI) (encoding Ras and Rab interactor 3), and its gene product 1.12 0.99 1.17 0.85 0.90 0.86 0.90 1.09 1.24 1.21 0.89 1.17 1.09 0.89 0.91 0.93 1.10 1.01 0.93 0.78 1.11 OR

Stage 2 gene (encoding zinc finger, CW type with PWWP PWWP with type CW finger, zinc (encoding gene e ± 100kbofthetopSNP. 4.2 ×10 6.9 ×10 9.9 ×10 1.1 ×10 4.5 ×10 4.5 ×10 2.8 ×10 4.1 ×10 3.4 ×10 7.9 ×10 4.1 ×10 1.6 ×10 4.1 ×10 9.7 ×10 6.3 ×10 3.4 ×10 5.7 ×10 9.0 7.8 ×10 4.0 ×10 4.3 ×10 P Meta × value BIN1 10

−5 −1 −8 −11 −6 −10 −5 −4 −19 −11 −3 −4 −4 −6 −5 −3 −5 −1 −3 −5 −6 gene product (0.91–0.96) (1.11–1.19) (0.85–0.89) (0.87–0.92) (0.84–0.89) (0.88–0.93) (1.07–1.13) (1.18–1.25) (1.14–1.22) (0.84–0.92) (1.09–1.19) (1.05–1.11) (0.89–0.94) (0.90–0.95) (0.90–0.95) (1.05–1.11) ( (0.88–0.94) (0.72–0.82) (1.08–1.13) (1.08–1.15) 0.62–0.86) (95% CI) 1.11 0.94 1.15 0.87 0.90 0.86 0.90 1.10 1.22 1.18 0.88 1.14 1.08 0.91 0.93 0.93 1.08 0.73 0.91 0.77 1.10 OR 19 d 2 , Average inthediscoverysample. 2 3

0 .

. . e SLC24A4 2.9 ×10 3.0 ×10 1.1 ×10 9.3 ×10 6.1 ×10 2.8 ×10 1.1 ×10 5.2 ×10 6.9 ×10 5.7 ×10 2.5 ×10 7.9 ×10 1.1 ×10 5.6 ×10 4.8 ×10 3.2 ×10 3.2 ×10 1.0 5.5 ×10 9.7 ×10 7.4 ×10 2 2 P , , a protein that may be × Meta value Overall 10 is also expressed expressed also is s r e t t e l −12 −6 −15 −26 −16 −25 −13 −11 −44 −24 −8 −9 −8 −10 −9 −8 −8 −4 −9 −15 −14

14, 2.4×10 28, 6.1×10 10, 3.0×10 38, 3.9×10 10, 3.0×10 0, 5.4×10 0, 6.9×10 0, 9.4×10 0, 9.8×10 1, 4.5×10 0, 4.9×10 0, 7.8×10 0, 9.9×10 0, 7.6×10 0, 7.0×10 0, 9.2×10 0, 6.4×10 0, 8.0×10 0, 6.3×10 0, 8.3×10 2 1 0, 9×10 P . Of note, note, Of . I 2 value (%),

−1 −1 −1 −1 −1 −1 −1 −2 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −2 −1 © 2013 Nature America, Inc. All rights reserved. during activity-dependent refinement of synaptic connectivity and thus formation mental retardation, stereotypic movements, epilepsy and cerebral mal severe with associated are locus Mutations atthis 2). factor enhancer APP of metabolism GRB2, with along modulate, to of the Alzheimer’s disease genes previously identified by GWAS has been shown protein to interact with CD2AP,encoded the whose but corresponding gene brain, is one the in levels low at expressed is on 145 2. kDa) chromosome polyphosphate-5-phosphatase, ( of ( ortholog family However, the protein. encoded the of function the about known is Little 4). member family protein scaffolding Cas ing described recently was toxicity tau-mediated the angiogenesis of mediator is and important an modulation, shape cell and the assembly in actin involved of is orchestration integrins, activates cell to structures, adhesion localizes matrix protein corresponding Its brain. the in expressed ciliary (ref. 6 type dyskinesia primary for responsible is which 8), member family NM23 which may affect long-term neuronal viability in Alzheimer’s disease kinase–activatingMAPreduced domain),ingexpressiondeaththe of genes ( ZCWPW1 tein family that regulates pre-mRNA alternative splicingpro the of member a is product gene whose 1), member family like causal. be may region this in gene which morphogenesis neuronal generally, more corresponding gene in mice brain affects size, neurite elongation and, the in gene didate ( genes ten about contains and large is data our in risk disease Alzheimer’s lation regu epigenetic protein modulates corresponding domain 1), whose controls). 37,154 2 Figure s r e t t e l and learning maymemory facilitate hippocampal-dependent Table We identified a seventh signal adjacent to at identified was locus Another at 20 chromosome on signal fifth a Weidentified The adjacent A to was signal discrete observed A second locus was within the CD2AP –log10 (P) 10 15 rspia melanogaster Drosophila 0 5 2 Supplementary Fig. 10a 4 27.0 2 Supplementary Fig. 9b Fig. Supplementary rs28834970

. However, the region defined by all the SNPs associated with with by SNPs the all associated . However, defined region the FERMT2 ) that is involved in actin dynamics actin in involved is that ) Regional plot for the the for plot Regional 3 locus, the regioninterestofthelargecontains andlocus,is about ten (CMS), a known Alzheimer’s disease susceptibility gene gene susceptibility disease Alzheimer’s known a (CMS), 4 . The MEF2C protein limits excessive synapse formation synapse excessive limits protein MEF2C The . STMN4 TRIM35 gene (encoding fermitin family member 2) is is 2) member family fermitin (encoding gene p130CAS 27.2 PTK2 ZCWPW1 B 2 8 ). CHRNA2 EPHX2 Position onchr.8(Mb) ) binds to CMS, the the CMS, to binds ) PTK2B 27.4 region is is region ortholog of of ortholog ). Among these genes is ). Another interesting possible can possible interesting Another ). CELF1 SCARA CL - U CLU 3 rs9331896 gene (encoding CUGBP, Elav- INPP5D locus (17,008 cases and and cases (17,008 locus NYAP1 27. 2 CCDC2 ESCO 2 9 MEF2C 6 5 . An association between between association An . . Our data do not resolve resolve not do data Our . 3 FERMT2 NME8 1 2 30 5 . PB . , as disruption of the the of disruption as , K (encoding inositol inositol (encoding Drosophila MIR4287 (encoding myocyte SCARA5 (encoding NME/ (encoding 27. Drosophila 3 3 CASS4 ( MADD . 8 2 fit1/fit2 6 . As with the C8orf80 ELP r ortholog ortholog 0. 0. 0. 0. 2 (encod 3 INPP5D (encod 3 2 4 6 8 5 3 CASS CASS .

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© 2013 Nature America, Inc. All rights reserved. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial M.A.P.-V., L.J.L., L.A.F., C.M.v.D., C.V.B., S. J.W.,Seshadri, G.D.S. and P.A. D. Harold, A.L.D.,A.C.N., C. Bellenguez, J.C.B., A.V.S., B.M.P.,R.M., J.L.H., S. J.W.,Seshadri, G.D.S. and P.A. L.-S.W.,E.R.M., B.M.P., L.J., J.L.H., P.A.H., M.A.P.-V., L.J.L., L.A.F., C.M.v.D., V.M.,B.N.V., Y.K., C.-F.L., B.K., C. Reitz, A.L.F., N.A., J.R.G., R.F.A.G.d.B., W.A.K., K.L.L., G.J.,A.C.N., C. Bellenguez, A.L.D., J.C.B., G.W.B., T.A.T.-W., A.V.S., V.C., M.A.I., V.M. and J.W. B.K., C. Reitz, J.R.G., O.V., W.A.K., L.-S.W.,K.L.L., E.R.M., K.L.H.-N., B.M.P., M.L., B.G.-B., T.A.T.-W.,G.R., N.J., A.V.S., V.C., M.A.I., D.Z., Y.K., B.N.V., C.-F.L., A.G., C.A.I.-V., D. Harold, A.C.N., R. Sims, G.J., C. Bellenguez, A.L.D., J.C.B., G.W.B., A. Brice, D. Hannequin, M.H., M.R., D.R.,K.R., C.G. and C.V.B. O.C., H. Soininen, S. Mead, D.A.B., L.F., C.H., P. Passmore, T.J.M., K. Bettens, H. Hakonarson, S.P., M.M.C., M.I., V.A., S.G.Y., E.C., C. P.Razquin, Pastor, I.M., H. Hampel, A.P., M. Bullido, F. Panza, P.C., B.N., M. Mayhaus, L. Lannfelt, C. R.C., Brayne, D.G., M. Mancuso, F.M., S. Moebus, P.M., M.D.Z., W.M., P. Bossù, G.S., P. Proitsi, J.C., S. Sorbi, F.S.-G., N.C.F., J.H., M.C.D.N., P. Bosco, P.B.-G., B.M., A.J.M.,R.G., D.W., J.G.,E.R., P.S.G.-H., J.C., A.L., A. Bayer, M.T., S. Lovestone, F.J.M., D.C.R., K.S., A.M.G., N.F., M.G., K. Brown, M.I.K., L.K., O.H., J.D.B., D. Campion, P.K.C., C. T.B.,Baldwin, C.C., D. Craig, V.D., J.A.J., J.W., G.D.S. and P.A. M.M.N., B.M.P., L.J., J.L.H., M.L., L.J.L., L.A.F., A.H., C.M.v.D., S. Seshadri, J.F.P., M.A.N., J.S.K.K.,K.R., E.B., L.-S.W., M. M.R., Boada, J.-F.D., C. Tzourio, M.C.O., L.B.C., D.A.B., T.B.H., R.F.A.G.d.B., T.J.M., J.I.R., K.M., T.M.F., W.A.K., P.S.G.-H., L.Y., M.M.C., D. Beekly, F.Z., O.V., S.G.Y., W.G., M.J.O., K.M.F., P.V.J., G.E., K.S., A.M.G., N.F., M.J.H., M.I.K., E.B.L., A.J.M., C.D., S.T., S. Love, E.R., S.-H.C., P.H., V.G., C. C.C., Baldwin, C. Berr, O.L.L., P.L.D.J., D.E., L. Letenneur, B.G.-B., N.J.,G.R., V.C., C. Thomas, D.Z., Y.K., A.G., H. Schmidt, M.L.D., M.-T.B., Acquisition of data: M.L., M.A.P.-V., L.J.L., L.A.F., C.M.v.D., V.M., S. J.W.,Seshadri, G.D.S. and P.A. T.B.H., J.I.R., W.A.K., R. Schmidt, A.H., M. R.M., B.M.P.,Boada, J.L.H., P.A.H., A.V.S., M.A.I., H. Schmidt, A.L.F., V.G., O.L.L., D.W.T., D. Blacker, T.H.M., Study concept and design: CHARGE was also supported by Erasmus Medical Center and Erasmus University. Institutes of Health grant numbers are listed in the including grants U01 AG032984 and R01 AG033193 (additional US National by the US National Institutes of Health, National Institute on Aging (NIH-NIA), UK (ARUK) and the Welsh government. andADGC CHARGE were supported (Chicago, Illinois) grant provided some funds to each consortium for analyses. meetings and communication for IGAP, and the Alzheimer’s Association and the Alzheimer’s Association (Chicago, Illinois) grant supported in-person The French National Foundation on Alzheimer’s and Disease Related Disorders of Innovative Strategies for a Transdisciplinary Approach to Alzheimer’s Disease). of Excellence Program Investment for the Future) DISTALZ grant (Development Rotary. This work has developed andbeen supported by the LABEX (Laboratory Pasteur de INSERM, Lille, FRC (Fondation pour la Recherche sur and le Cerveau) involved the Centre National de Génotypage and was supported by the Institut Foundation on Alzheimer’s and Disease Related Disorders. Data management the patients and chipstheir families. iSelect were funded by the French National This work was made possible by the generous participation of the control subjects, Nature Ge Nature COM AU Acknowledgments

Complete acknowledgments are in detailed the GERAD was supported by the Wellcome Trust, the MRC, Alzheimer’s Research T olnwrh P. Hollingworth, S. Seshadri, D. Harold, J.-C. Lambert, E. Genin, E.H. Corder, 429–435 (2011). 429–435 CD33 disease. (2009). PICALM CR1 inheritance. families. onset late in disease Alzheimer’s H P O ET associated with Alzheimer’s disease. Alzheimer’s with associated R R and I CONT NG soitd ih lhie’ disease. Alzheimer’s with associated J. Am. Med. Assoc. Med. Am. J. n t al. et Statistical Statistical analysis and interpretation: FI t al. et etics CD2AP Mol. Psychiatry Mol. et al. et t al. et N l et al. et RIBU .

A APOE t al. et Genome-wide association study identifies variants at variants identifies study association Genome-wide J.-C.L., C.A.I.-V., D. Harold, R. Sims, C. Bellenguez, G.J., Sample Sample contribution: Genome-wide analysis of genetic loci associated with Alzheimer with associated loci genetic of analysis Genome-wide NC ee oe f plppoen tp 4 lee n te ik of risk the and allele 4 type E apolipoprotein of dose Gene

r ascae wt Azemrs disease. Alzheimer’s with associated are Genome-wide association study identifies variants at variants identifies study association Genome-wide T ADVANCE ONLINE PUBLICATION ONLINE ADVANCE IA n Azemr ies: mjr ee ih semi-dominant with gene major a disease: Alzheimer and omn ains at variants Common I ONS J.-C.L., C.A.I.-V., D. Harold, A.C.N., A.L.D., J.C.B., L I NTE

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© 2013 Nature America, Inc. All rights reserved. S Palmi V Jonsson O E Robert Alzheimer Alzheimer Centre Educacional (ACE). Institut Català de Aplicades, Neurociències Barcelona, Spain. USA. Pennsylvania, Philadelphia, du Humain Polymorphisme (CEPH), Paris, France. The Netherlands. 19 University, Nashville, Tennessee, USA. Zurich, TechnischeEidgenössische of Hochschule (ETH)/University Zurich, Zurich, Switzerland. Florida, USA. Department of Medicine, University of Washington, Seattle, Washington, USA. USA. Massachusetts, Genetics), Boston University School of Public Health, Boston, USA. Massachusetts, Epidemiology and Perelman Biostatistics, School of Medicine, University of USA. Pennsylvania, Philadelphia, Pennsylvania, 7 Medicine and Clinical Medical Neurosciences, Research Council (MRC) Centre for Genetics Neuropsychiatric & Genomics, Cardiff University, Cardiff, UK. Center, Rotterdam, The Netherlands. 1 L Peter A Holmans M L M K T T T Hilkka C M John R PilottoAlberto S C ( D John Hardy Petroula Proitsi Anthony Bayer M Alison L Philip Vilmundur Joseph Florence Pasquier M Badri V Albert G Rebecca Jean- s r e t t e l Department Department of and Biostatistics Epidemiology, Perelman School of Medicine, University of USA. Pennsylvania, Philadelphia, Pennsylvania, INSERM, U744, Lille, France. E udha usanne usanne uc uc atiana amara B Harris indsay A Farrer i- Department Department of Radiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands. katerina Rogaeva homas J aren Ritchie ristina ristina Razquin onsortium onsortium (A ary ary aniela aniela tto Valladares ercè Boada elanie arkus inerva inerva Ilyas A S D an L C W etenneur I) N L S harles harles W M D G S G 87 K eshadri Vardarajan Beecham

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D mith G N oebus Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami, Miami, Florida, USA.

79 oate C G 21 ontine enetic enetic and unstan öthen 23 25 , L arrasquillo Centre Centre National de Génotypage, Institut Génomique, Commissariat à l’Energie Atomique, Evry, France. 44 6 udnason 62 80 , , 50 13 , , Jean-François ambert 11 , 66 93 142 17 , 130 DGC , , Amanda J C 23 , , 49 125 41 Department Department of Boston Biostatistics, University School of Public Health, Boston, USA. Massachusetts, , , Francisco J , 112 17 58 12 M 88 14 9 , , , , 46 , 6 106 éline éline Bellenguez , , , , John 18 , 13 – , 2 , , D M 110 42 , , 145 , , 135 , , , S , , Patrizia , , 120 65 , 11 111 59 , aria aria M 6 , M 47 34 28 , , Vincent , K 84 L 9 113 teven teven M ebby , , , , Pau Pastor 14 , , Agustin Ruiz aria aria Bullido , , Yoichiro , , John , , , ) , , aura Fratiglioni N , , , Vincent 141 athryn – , , , , Paul Hollingworth ark , , ikko Hiltunenikko , , 146 , , 87 , , Benjamin L 2 W anuel 36 1 C 17 , , M Université Université Lille 2, Lille, France. K O athalie athalie Fiévet – E 24 ina ina C , , D , , , C aroline 3 alter A , Julie , arolien Bettens 142 nofre C 103 nvironmental Risk in Alzheimer’s Institute Institute for Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria. 18 ichelangelo ichelangelo , D andida andida L W eborah eborah Blacker 145 5 ollinge G Department Department of Epidemiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands. ohorts ohorts for Heart and Aging Research in , , athrop 17 K ominique , , G M , , Younkin C

M Faculty Faculty of Medicine, University of Iceland, Reykjavik, Iceland. T , , C M L M eller allacher C M C arlos arlos yers suang

ornelia ornelia L ayhaus W D ecocci C artin artin Ingelsson D houraki K arla arla A Ibrahim-Verbaas orón K unetta G 106 artigues ombarros 94 amatani illiams eramecourt D 74 60 ukull G 21 raff 63 1 – 25 eniz eniz , , , – 23 C , , Pascale Barberger- 96 107 renier-Boley , , , 70 S 22 112 3 51 Department Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, , , 1 C ruchaga 89 , , M 103 andro , , Francesco Panza 66 60 117 100 , , , C M , M 3 G , , Ignacio 11 139 , , arole L 32 , , , , , , , Peter , 1 ancuso 113 ampion D 118 N 6 , , yungah yungah Jun 53 M arie- , John – ei ei Yu M van , , , , , , 136 50 E 114 145 3 , , 22 aranjo avid avid L 6 D , , , , , aria aria M 54 atthew J Huentelman , , liecer liecer 108 , Alfredo , Ramirez Alfredo , ars , , 133 C E C , , Bruce idier idier Hannequin , , S , , Jerome I Rotter D C 146 115 argaret A Pericak-Vance den den R T harlene harlene 2 D 41 orbi 71 3 live live Holmes , , 109 , , Richard hiao-Feng Institut Institut Pasteur de Lille, Lille, France. ufouil S 28 101 C hérèse hérèse Bihoreau S L 85 D uijn , , , , , , 37 t t 42 M

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52 104 Hamilton- , , D , , Bernadette C , , onovan , , Paola Bossù , , odd , Robert , Robert udny , , , Arfan Ikram homas H S , , , N 86 , 27 S O 137 affarra 16 43 65 e C M eung-Hoan , , Victoria Alvarez G anchez- 20 S ajaf Amin , , Department Department of Molecular Physiology and Biophysics, Vanderbilt , , livier Hanonlivier , , , linton Baldwin G tefano 67 E GE Netherlands Netherlands Consortium for Healthy Aging, Leiden, enomic enomic , , C 15 26 ichael ichael 13 43 D L uropean uropean Alzheimer’s errish Taub Institute on Alzheimer’s Disease and the Aging Brain, , , Jordi hristophe , , 23 , , , O E enis enis 14 esley Jonesesley M 18 a T G RA D 6 iriksdottir 4 , DVANCE ONLINE PUBLICATION ONLINE DVANCE Icelandic Icelandic Heart Association, Kopavogur, Iceland. wen 145 98 , , ricia A ricia Department Department of Neurology, Erasmus MC University Medical , , S aier de Bruijn C avid avid L L chmidt , 11 G M D G 99 N larke enore J E aura B , 146 6 6 122 E , , Joshua arcia 127 vans 6 C ) ill , , Helena , Benedetta , Benedetta elson M osley Jrosley 49 5 29 , 87 , , 73 145 pidemiology ( pidemiology , , W larimon 4 C 102 & Philippe Amouyel K , , 56 , c C , – , 39 , , Alzheimer’s , , 123 5 M G T hoi 82 129 31 T elley elley , , Adam allon G , , , laudine laudine Berr 19 , , , , Harald Hampel 48 77 131 hornton- K zourio uinness 6 , , ichael ichael A , , Annette 13 S , , ianfranco ianfranco C 18 , , Jonathan L , , , , 9 C 4 , , K 11 ristelle ristelle Brown 20 abrina Pichler , , , , M 22 antwell S , auner 105 , , 116 N , , 5 arol Brayne C T evin evin W 9 , , , , imon D K , M Fondation Fondation Jean Dausset, Centre d’Etude S 37 Department Department of Medicine (Biomedical 53 20 atthias Riemenschneider ick ick 12 C D Bis 15 68 im im Becker chmidt , , Fanggeng Zou , , ristel 25 an Rujescu ei ei , , Cardiovascular Health Cardiovascular Research Unit, Faber , , , Peter Passmore Functional Functional Genomics Center hristiane hristiane Reitz D C iana iana Zelenika 54 , 134 S N Memory Memory Clinic of Fundació 69 G 43 C eth eth

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© 2013 Nature America, Inc. All rights reserved. Correspondence should Correspondence be addressed to P.A. ( 144 142 National Institute on Aging, Bethesda, Maryland, USA. and Génome Québec Innovation Centre, Montreal, Quebec, Canada. Washington, Seattle, Washington, USA. Sciences and Society, Karolinska Institute–Alzheimer’s Disease Research Center (KIADRC), Stockholm, Sweden. Human Genetics, Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany. Bordeaux, Centre Hospitalier (CHU) Universitaire de Bordeaux, Bordeaux, France. Medical University of Graz, Graz, Austria. USA. of Texas Health Science Center at Houston, Houston, Texas, USA. Young University, Provo, Utah, USA. National Institute on Aging, Bethesda, Maryland, USA. 123 UMR 7225, Université Pierre et Marie Curie, Centre de Recherche de l’Institut du Cerveau et de la Moëlle Epinière (CRICM), Hôpital de la Salpêtrière, Paris, France. of Washington, Seattle, Washington, USA. Stockholm, Sweden. Alzheimer’s Disease Center, Rush University Medical Center, Chicago, Illinois, USA. School, Boston, USA. Massachusetts, of Epidemiology, Harvard School of Public Health, Boston, USA. Massachusetts, Molecular Genetics, Indiana University, Indiana, Indianapolis, USA. Santander, Spain. 108 Center for Applied Medical Research, University of Navarra School of Medicine, Pamplona, Spain. Seattle, Washington, USA. USA. Pennsylvania, 101 99 Madrid, Spain. Biomédica Investigación en Red sobre (CIBERNED), Madrid, Enfermedades Neurodegenerativas Spain. Italy. Ludwig Maximilians University, Munich, Germany. Sciences, University of Cagliari, Cagliari, Italy. of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy. Informatics, Biometry and Epidemiology, University Hospital Essen, University Essen, Duisburg-Essen, Germany. Unit, Biostatistics Cambridge, UK. Cambridge, UK. Italy. Weston Institute Laboratories, of Neurology, London, UK. Disease, University College London Institute of Neurology, London, UK. 77 and Child Health), University of Florence, Florence, Italy. Disease, Neurodegenerative University College London Institute of Neurology, London, UK. Aristotle University of Greece. Thessaloniki, Thessaloniki, (VAPSHCS), Seattle, Washington, USA. Diseases (CIBERNED), Barcelona, Spain. Sant Pau (IIB Sant Pau). Sant Pau Hospital. Universitat Autònoma de Barcelona, Barcelona, Spain. Heath Park, Cardiff, UK. University of Toronto, Toronto, Ontario, Canada. of Bristol Institute of Clinical School Neurosciences, of Clinical Sciences, Frenchay Hospital, Bristol, UK. Boston, USA. Massachusetts, 62 and Society, Karolinska Institutet and Stockholm University, Stockholm, Sweden. 59 Queen’s Medical Centre, University of Nottingham, Nottingham, UK. Research Institute, Phoenix, Arizona, USA. Antwerp, Belgium. Institute for Medical Research, University of Cambridge, Cambridge, UK. London, UK. Aging, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA. School, Boston, USA. Massachusetts, Translational Genomics, NeuroPsychiatric Institute for the Department Neurosciences, of Neurology & Psychiatry, Brigham and Women’s Hospital and Harvard Medical Hôpital La Colombière, Montpellier, France. Group, Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University, Belfast, UK. Missouri, USA. Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany. Medicine, University of Washington, Seattle, Washington, USA. Sciences, Mount Sinai School of Medicine, New York, New York, USA. Medicine, New York, New York, USA. Washington, USA. Paris Descartes, Sorbonne Paris V, Broca Hospital, Geriatrics Department, Paris, France. 29 New York, USA. Department of Neurology, Columbia University, New York, New York, USA. Nature Ge Nature Clinical Clinical Medicine–Neurology, University of Eastern Finland, Kuopio, Finland. Department Department of Immunology, Hospital Dr.Universitario Negrin, Las Palmas de Gran Canaria, Spain. Department of Medicine and Partners Center for Personalized Genetic Medicine, Division of Genetics, Brigham and Women’s Hospital and Harvard Medical School, Alzheimer’s Disease Research Center, University of Pittsburgh, Pittsburgh, USA. Pennsylvania, Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany. Center Center for Cognitive Disorders, Azienda Unita Sanitaria Local (AUSL), Parma, Italy. Centre Centre Hospitalier Régional de Universitaire Lille, Lille, France. Department of Neurology, Boston University School of Medicine, Boston, USA. Massachusetts, Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital de la Paris, Pitié–Salpêtrière, France. Neurology Neurology Service, Marqués de Valdecilla University Hospital, University of Cantabria and Instituto de Formación e Marqués Investigación de Valdecilla (IFIMAV), Department of Public Uppsala Health/Geriatrics, University, Uppsala, Sweden. 94 82 130 Centro Centro de Biología Molecular Severo Ochoa, Consejo Superior Autónoma de de Científicas–Universidad Investigaciones Madrid, Madrid, Spain. Oxford Healthy Aging Project (OHAP), Clinical Trial Service Unit, University of Oxford, Oxford, UK. Hospital Hospital Universitari Vall de d’Hebron–Institut Recerca, Universitat Autònoma de Barcelona. (VHIR-UAB), Barcelona, Spain. 50 n etics INSERM, INSERM, U897, Victor Segalen University, Bordeaux, France. 97 42 28 84 Departement Departement of Geriatrics, Center for Aging Brain, University of Bari, Bari, Italy. Hope Hope Center Program on Protein Aggregation and WashingtonNeurodegeneration, University School of Medicine, St. Louis, Missouri, USA. Centre Centre National de Reference pour les Malades Alzheimer Jeunes (CNR-MAJ), Centre Hospitalier Régional de Universitaire Lille, Lille, France. 109 33 University University of Milan, Fondazione Cà Granda, IRCCS Ospedale Policlinico, Milan, Italy. 54 103 Department Department of Global Health, University of Washington, Seattle, Washington, USA. 119 Laboratory Laboratory of Institute Neurogenetics, Born-Bunge, University of Antwerp, Antwerp, Belgium. CIBERNED, CIBERNED, Marqués de Valdecilla University Hospital, University of Cantabria and IFIMAV, Santander, Spain.

Department Department of Mayo Neuroscience, Clinic, Florida, Jacksonville, USA. Division Division of Clinical School Neurosciences, of Medicine, University of Southampton, Southampton, UK. ADVANCE ONLINE PUBLICATION ONLINE ADVANCE 67 105 Department Department of Clinical University Neurosciences, of Cambridge, Cambridge, UK. 63 Genetica Genetica Molecular, Hospital Central Universitario Asturias, Oviedo, Spain. Department Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami, Miami, Florida, USA. 87 127 Full Full lists of members and affiliations appear in the 35 116 47 Department Department of Psychiatry, Mount Sinai School of Medicine, New York, New York, USA. Human Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA. 71 138 Program Program in Medical and Population Genetics, Broad Institute, Boston, USA. Massachusetts, Department Department of Medicine University (Geriatrics), of Mississippi Medical Center, Jackson, Mississippi, USA. 132 70 Department Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA. 121 Vanderbilt Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, USA. 56 Geriatric Geriatric Research, Education and Clinical Center (GRECC), Veteran Puget Administration Sound Health Care System 45 Department Department of Psychiatry, University of Halle, Halle, Germany. Mercer’s Institute for Research on Aging, St. James Hospital and Trinity College, Dublin, Ireland. [email protected] Medical Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. Departments Departments of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, USA. Pennsylvania, 91 66 Department Department of Psychiatry, University of Frankfurt, Frankfurt am Main, Germany. Institute Institute of Primary Care and Public Health, Cardiff University, Neuadd Meirionnydd, University Hospital of Wales, 93 Gerontology Gerontology and Geriatrics Research Laboratory, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 141 125 76 81 73 Department Department of Epidemiology, Boston University School of Public Health, Boston, USA. Massachusetts, Faculty of Medicine, Imperial College, St. Mary’s Hospital, London, UK. Centro Centro di Ricerca, Trasferimento e Alta Formazione DENOTHE, University of Florence, Florence, Italy. Istituto Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Associazione Oasi Maria Santissima Srl, Troina, Clinical Clinical and Behavioral Neurology, Fondazione Santa Lucia, Rome, Italy.

39 German German Center for Diseases Neurodegenerative (DZNE), Bonn, Germany. 129 145 140 111 58 Division Division of Epidemiology, University of Texas Health Sciences Center at Houston, Houston, Texas, These These authors contributed equally to this work. 37 Department Department of Human Genetics, University of Pittsburgh, Pittsburgh, USA. Pennsylvania, Laboratory Laboratory of Epidemiology and Population Sciences, Intramural Research Program, Geriatric Geriatric Department, Landspitali University Hospital, Reykjavik, Iceland. 79 CNR-MAJ, CNR-MAJ, INSERM, U1079, Rouen University Hospital, Rouen, France. 53 Department Department of Molecular Institute Neuroscience, of Neurology, London, UK. 27 Neurodegenerative Brain Neurodegenerative Diseases Group, Department of Molecular Genetics, VIB, 90 Gertrude Gertrude H. Sergievsky Center, Department of Neurology, Columbia University, New York, 113 41 51 Section Section of Neuroscience and Clinical Pharmacology, Department of Biomedical ) ) or J.W. ( Department Department of Psychiatry, Washington University School of Medicine, St. Louis, Departamento Departamento de Genómica Estructural, Neocodex, Seville, Spain. Department Department of Neurology, Kuopio University Hospital, Kuopio, Finland. 115 61 102 134 Group Group Health Research Institute, Group Health, Seattle, Washington, USA. Department Department of Psychiatry, General Massachusetts Medical Hospital/Harvard 118 30 Center Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, INSERM, INSERM, U708, Victor Segalen University, Bordeaux, France. 100 Institute Institute of Human Genetics, University of Bonn, Bonn, Germany. Department Department of Geriatric Medicine, Karolinska University Hospital Huddinge, [email protected] S Department Department of Psychiatry, Saarland University Hospital, Homburg, Germany. 32 upplementary upplementary Note Department Department of Epidemiology, University of Washington, Seattle, 75 NEUROFARBA (Department of Psychology,Neuroscience, Drug Research 104 National National Alzheimer’s Coordinating Center, University of Washington, 49 60 98 143 107 Institute Institute of Psychiatry, King’s College London, Denmark Hill, Aging Aging Research Center, Department of Neurobiology, Care Sciences Department Department of University Neuroscience, of Parma, Parma, Italy. 69 78 Center Center for Medical Systems Biology, Leiden, The Netherlands. CIBERNED, CIBERNED, Instituto de Salud Carlos III, Madrid, Spain. 124 Center Center for Networker Biomedical Research in Neurodegenerative Dementia Dementia Research Centre, Department of Neurodegenerative 136 96 85 34 Laboratory Laboratory of Intramural Neurogenetics, Research Program, 106 65 Instituto Instituto de Sanitaria Investigación Hospital la Paz (IdIPaz), Neurological Neurological Clinic, University of Pisa, Pisa, Italy. Department Department of Mount Neuroscience, Sinai School of 83 Department Department of Neurobiology, Karolinska Institutet, Care 133 Tanz Centre for Research in Disease, Neurodegenerative 68 Institute Institute of Public Health, University of Cambridge, Neurogenetics Laboratory,Neurogenetics Division of Neurosciences, . . 88 Neurology Neurology Department. Biomedical Research Institute Centre Centre de Mémoire de Ressources et de Recherche de ). Urban Urban Epidemiology, Institute for Medical 137 89 55 Section Section of Gerontology and Geriatrics, Department Department Department of Health Service, University of Neurogenomics Division, Neurogenomics Translational Genomics 146 These These authors jointly directed this work. 44 INSERM, INSERM, U1061, Faculty of Medicine, 36 128 120 Department Department of Genetics and Genomic 92 Human Human Genetics Center, University Department Department of Pathology, University 126 Department Department of Psychiatry, 110 48 74 122 131 Department Department of Biology, Brigham Rush Rush Institute for Healthy MRC MRC Prion Unit, Department of Department Department of Medical and INSERM INSERM UMRS 975, CNRS 72 Department Department of Neurology, Department Department of Neurology, 40 Institute Institute for Medical 139 57 Institute Institute of Genetics, 112 McGill McGill University 38 s r e t t e l 52 135 117 Department Department of Institute Institute of Cambridge Cambridge 46 80 95 Institute Institute of Rush Rush 114 Program Program in 31 Reta Reta Lilla Centro Centro de 64 University University Department Department University University

86 43

MRC MRC Ageing Ageing © 2013 Nature America, Inc. All rights reserved. Weinberg equilibrium. In total, 798 SNPs showed a highly significant significant highly a showed SNPs 798 total, In Hardy- of equilibrium. assumption an Weinberg avoid to permutations 4,500,000 and performing by PLINK, with performed was test allelic The individuals. (see Finnish of controls exclusion after and 10,750 control”) on quality sample 2 “Stage estimated were data 2 stage for data. 2 frequencies stage and Allele panel reference EUR Project Genomes frequency 1000 allelic the in between discrepancies for tested first We genotyping. in biases potential detect to SNPs 12,446 remaining the to applied were measures trol compared to data Project 1000 ( Genomes being found in a heterozygous state ( ( no signal having (i) intensity SNPs the of result a as SNPs 1,999 additional an of further exclusion the to led failure Genotyping attempted. SNPs was 14,445 genotyping of which number for final a to leading synthesis, oligonucleotide failed SNPs 2,287 process, production Illumina the During production. microarray for selected were 0.4 to equal or superior score Illumina an exhibiting SNPs microarray. A of total 16,732 an to iSelect develop website Illumina a devoted to submitted was SNPs 19,532 of list A replication. for selected were analysis and exhibiting risk Alzheimer’s disease control. with ated quality SNP 2 stage and design microarray iSelect analysis. 1 stage SNPs in 7,055,881 of number final a to led step selection quality data present only in a limited number of data sets of small size. This last for studies some SNPs between us homogeneity to by increase poor excluding allowed approach This negatives). (false sets data of number limited more a in power statistical adequate reached have could SNPs those if even interest, potential of SNPs of removal the to led have could studies all in present only SNPs hand, On studying other the positives. of risk the false increased greatly have could study one least at in available SNPs all was analyzing SNP Indeed, present. given the which in samples of number the maximizing and SNPs of number total the maximizing between compromise best the threshold represented This meta-analysis. the in included were samples control of 40% consortium. of each by members for access website I-GAP SNP to an internal data set was 8,131,643. Each consortium uploaded summarized results for each | ficient coef regression logistic showing SNPs of exclusion the After observed. were results similar very and control, quality for set data EADI the to applied were SNPTEST (PLINK analyses these for software analogous but different used consortia Alzheimer’s disease data, Cox proportional hazards models were used. The four ( stratification population possible for adjust to components principal and sex age, for covariates including model regression logistic a by analyzed was age were that retained were set. data 1 at least in present SNPs 8,133,148 of maximum a procedures, these After excluded. also were <1% of MAF a with SNPs Similarly, analyses. from excluded were equivalent), be to described estimates quality two these (with respectively IMPUTE2, or MaCH by indicated as 0.3, than less of estimates with SNPs set, data each In 2010). December from from 4 data freeze August on haplotypes and 2010 the based sequence phased release interim (2010 Project Genome 1000 the in ancestry European of ples to impute from derived sam of with haplotypes the genotypes all participants (ref. excluded; were <95% of rates call with (SNPs collection sample each and individual each for finalized were criteria analysis. 1 stage for selection SNP and Imputation boards. review institutional appropriate by the approved and were reviewed proxy,populations or for other all protocols study and the guardian a from legal caregiver, impairment, cognitive substantial with those and of description (see full the I-GAP data and sets) in in are described studies case-control All ONLINE Nature Ge Nature Project Genomes 1000 the between frequency allele of terms in difference upeetr Tbe 2 Table Supplementary SNPs genotyped or imputed in at least 40% of Alzheimer’s cases and disease dos genotype LOAD of with association the set, data case-control each In 8 4 . Written informed consent was obtained from study participants or, . from study participants for Writtenwas obtained consent informed 2) or MaCH/Minimac or 2) β | | > 5 or 4

5 ME , , ProbABEL n etics TH P n value equal to of 0 number or SNPsequal maximum 1, value the in any = 559), (ii) not being polymorphic ( polymorphic being not (ii) 559), = ODS 4 6 or R; ). For the three CHARGE cohorts with incident incident with cohorts CHARGE three the For ). 4 3 software ( software Supplementary Table Supplementary 2 n = 248) or (iv) having mismatched alleles Table 1 Supplementary Table 2 Table Supplementary n Supplementary Note Supplementary = 16). Finally,con quality several , , in the P Supplementary TablesSupplementary 1 value < 1 × value 10 P R values were computed computed were values 2 After quality control control quality After Supplementary Note Supplementary or info score quality quality score info or ). Three of these tools tools of ). Three these n = 1,176), (iii) only only (iii) 1,176), = SNPs associ SNPs ), IMPUTE2 IMPUTE2 ), −3 ) was used used was ) in stage 1 in stage 4 , 7 4 - - - - - ,

esses. On the basis of data for all of these SNPs, we excluded individuals who who individuals excluded we SNPs, these of all for data of basis the On esses. our data control us refine that genetic quality proc allowed various to further SNPs included to were supplementary 2. of stage devoted 11,632 These which Stage 2 sample quality control. low. be to considered was association 1 stage P exhibited SNPs untagged the of majority vast the SNP.Because ing a with associated and only 471 variant were ( genotyped not by tagged another successfully <0.3. was score info their if quality imputation low of be to considered were SNPs sets, data For 2. imputed stage in analyzed were and 1 country at in least quality typing countries. all from data in quality genotyping low with SNPs 16 of removal the to led steps control quality These populations). European across hits significant and suggestive for assessed missingness in 10 × 1 than lower was controls the if or if individuals, the the of 10% than more for data genotype missing had it if set data country a in quality genotyping low of considered was SNP A country. analysis. the from excluded were and ( data 2 stage and panel reference EUR as implemented in PLINK. Analysis was adjusted for age, sex and principal principal and sex age, for adjusted was Analysis PLINK. in implemented regression as logistic using model, additive an under SNPs genotyped quality consortia. 4 the of each by undertaken analyses the between observed was matching perfect expected, as and used software the of independently generated were results METAL meta-analysis: pack for software used Two were website. ages I-GAP the on available files data the loading Each consortium performed ansets. independent stage data 1 meta-analysis after across down statistics summary the combining before estimated factors the of errors standard the β with meta-analysis variance-weighted inverse analysis. Statistical quality sample After 2. stage in analysis for available were controls) analysis. the from excluded control ( were age of years 25 set. data country each within and removed were so as iteratively to a obtain sample of individuals unrelated related be to considered were 0.2 than greater IBD with pairs in Individuals LD. minimize to selected and >1% of MAF SNPs with autosomal 6,764 using was computed for all pairs of in individuals data from separately,each country Similarly, IBD was excluded. genotype of missing proportion greater the with individual the Otherwise, excluded. were individuals both discordant, individuals were duplicated for data clinical If duplicates. be to considered were of pairs all for in a computed and using PLINK, pair individuals with IBD greater than 0.98 individuals was (IBD) descent by Identity axes. two first the approach clustering Bayesian a applying by tified ancestry. Individuals with of evidence non-European were ancestry then iden genetic their to axes define PCA two onto first the were projected individuals software 4.2 EIGENSOFT from SMARTPCA function on (PCA) analysis a HapMapprincipal-component performed 2 data with the Using a of panel ancestry-informative 261 had occurred. that sample mixing suggesting in were sex observed, of number discrepancies abnormal an whom for plate single a from individuals 93 removed Wealso on X. by 40 SNPs PLINK on chromosome as estimated sex genetic using ined ( identified showed were or individuals related sex) and Duplicated ancestry. (genetic non-European of evidence data genetic of basis the on estimated sex and sex had more than 3% missing genotypes, showed a between discrepancy reported -coefficient scaled by the square roots of study-specific genomic inflation inflation genomic study-specific of roots square the by scaled -coefficient values between 1 × between 10 values Of note, of the 7,086 SNPs that we were unable to successfully genotype, genotype, successfully to unable were we that SNPs 7,086 the of note, Of After SNP quality control, 11,632 SNPs were considered to be of high geno SNP Other control quality in separately steps were data for performed each For stage 2, association tests were performed for each country for all high- for all country for each were performed tests 2, For association stage than less controls and data clinical missing with individuals Finally, P value for the Hardy-Weinberg test in controls was lower than 1 × 10 × 1 than lower was controls in test Hardy-Weinberg the for value Supplementary TableSupplementary 10 P value for the test for differences in missingness between cases and and cases between missingness in differences for test the for value Supplementary TableSupplementary 10 For the stage 1 meta-analysis, we undertook fixed-effects fixed-effects we Forundertook the 1 stage meta-analysis, P −3 value at least 10 times higher than that of the miss the of that than higher times 10 least at value and 1 × 10 −6 The iSelect microarray contained 33,368 SNPs, (see (see ), ), 19,884 individuals (8,572 cases and 11,312 ). ). Briefly, in discrepancies sex were exam −4 Supplementary Table 9 Supplementary (92%), the likelihood of a missing true the likelihood (92%), P < 1 × 10 × 1 < 4 9 and GWAMA and −5 4 ; ; 8 Supplementary Fig. 18 Fig. Supplementary to their coordinates on on coordinates their to 4

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© 2013 Nature America, Inc. All rights reserved. and were located in a 500-kb window upstream or downstream of the top top the of downstream or upstream window 500-kb a in located were and ( ficance signi genome-wide reached that SNPs all selected We first expression. gene and loci I-GAP top the in SNPs between associations reported explored we Annotation of I-GAP top SNPs for eQTLs. analyses. 2 stage and 1 stage combined after level significant genome-wide software LocusZoom with generated results. the on heterogeneity study of effect and heterogeneity generated Cochran’s performed we point, At country. this by data 2 stage and GERAD EADI, CHARGE, ADGC, across statistics summary combining by shown). not (data by country performed was analysis when obtained those to similar were Results meta-analysis. by fixed-effects results the bined 2 in for stage center and each com separately Weanalysis the performed also countries. different the from results 2 stage combine to performed then was as implemented in METAL. Using meta-analysis this approach, a fixed-effects approach variance inverse then the using were meta-analysis by fixed-effects results combined and separately, analyzed were and samples Genotyped German SNPTEST. imputed in implemented as data missing for tests score data. For using tests likelihood were imputed data performed association sets, GWAS on based was samples 2 stage Bonn for PCA 0.05. than lower was test and analysis was further adjusted on principal components if the coordinates PCA in between cases and Difference controls were tested for the separately. first four principal components, country each from on individuals performed was PCA SMARTPCA, Using necessary. when components, doi: PAF was calculated using the Levin equation Levin the using PAF calculated was was data 1 stage the in signal association the of representation graphic A We finally generated inverse fixed-effects variance–weighted meta-analyses 10.1038/ng.2802 P value value ≤ 5 × 10 × 5 −8 ) in the combined stage 1 and stage 2 analysis analysis 2 stage and 1 stage combined the in ) I 2 estimates with METAL to evaluate the possible 5 1 for all the loci of interest reaching a reaching interest of loci the all for To gain further biological insights, 5 2 . P value of this Q test for test - 50. 49. 48. 47. 46. 45. 44. 43. 42. lowest the with one the selected SNPs and eQTL reported of number the counted we then database, in this as defined association of eQTL type and each gene eQTL For reported each URLs). (see laboratory Pritchard the from database SNPs eQTL in the of these each with associated expression ( locus each at SNP 52. 51.

äi R & ors AP GAA sfwr fr eoewd ascain meta- association genome-wide for software GWAMA: A.P. Morris, & R. Mägi, of meta-analysis efficient and fast METAL: G.R. Abecasis, & Y. Li, C.J., Willer, C. eigenanalysis. Bellenguez, and structure Population D. Reich, & A.L. Price, N., Patterson, Aulchenko, Y.S., Struchalin, M.V. & van Duijn, C.M. ProbABEL package for genome- multipoint new A P. Donnelly, & G. McVean, S., Myers, B., Howie, J., Marchini, S. Purcell, sequence using MaCH: G.R. Abecasis, & P. Scheet, J., Ding, C.J., Willer, Y., Li, Howie, B.N., Donnelly, P. & Marchini, J. A flexible and accurate genotype imputation Levin, M.L. The occurrence of lung cancer in man. in cancer lung of occurrence The M.L. Levin, R.J. Pruim, analysis. scans. association genomewide studies. association (2012). genome-wide in samples Genet. PLoS genotypes. data. imputed of of analysis association wide imputation via studies association Genet. Nat. genome-wide for method analysis. linkage based genotypes. unobserved and haplotypes estimate Epidemiol. to data genotype and 5 studies. association genome-wide of generation next the for method 9 results. scan , e1000529 (2009). e1000529 , , 531–541 (1953). 531–541 , BMC Bioinformatics BMC

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