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Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

Abstract

We identified rare coding variants associated with Alzheimer's disease in a three-stage case–control study of 85,133 subjects. In stage 1, we genotyped 34,174 samples using a whole-exome microarray. In stage 2, we tested associated variants (P < 1 × 10−4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, we used an additional 14,997 samples to test the most significant stage 2 associations (P < 5 × 10−8) using imputed genotypes. We observed three new genome-wide significant nonsynonymous variants associated with Alzheimer's disease: a protective variant in PLCG2 (rs72824905: p.Pro522Arg, P = 5.38 × 10−10, odds ratio (OR) = 0.68, minor allele frequency (MAF)cases = 0.0059, MAFcontrols = 0.0093), a risk variant in ABI3 (rs616338: p.Ser209Phe, P = 4.56 × 10−10, OR = 1.43, MAFcases = 0.011, MAFcontrols = 0.008), and a new genome-wide significant variant in TREM2 (rs143332484: p.Arg62His, P = 1.55 × 10−14, OR = 1.67, MAFcases = 0.0143, MAFcontrols = 0.0089), a known susceptibility gene for Alzheimer's disease. These protein-altering changes are in genes highly expressed in microglia and highlight an immune-related protein–protein interaction network enriched for previously identified risk genes in Alzheimer's disease. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to the development of Alzheimer's disease.

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Figure 1: Association plots of PLCG2, ABI3, and TREM2.
Figure 2: Protein–protein interaction network (using high-confidence human interactions from the STRING database) of 56 genes enriched for both common and rare variants associated with Alzheimer's disease risk.

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Acknowledgements

GERAD/PERADES. We thank all individuals who participated in this study. Cardiff University was supported by the Alzheimer's Society (AS; grant RF014/164) and the Medical Research Council (MRC; grants G0801418/1, MR/K013041/1, MR/L023784/1) (R. Sims is an AS Research Fellow). Cardiff University was also supported by the European Joint Programme for Neurodegenerative Disease (JPND; grant MR/L501517/1), Alzheimer's Research UK (ARUK; grant ARUK-PG2014-1), the Welsh Assembly Government (grant SGR544:CADR), and a donation from the Moondance Charitable Foundation. Cardiff University acknowledges the support of the UK Dementia Research Institute, of which J. Williams is an associate director. Cambridge University acknowledges support from the MRC. Patient recruitment for the MRC Prion Unit/UCL Department of Neurodegenerative Disease collection was supported by the UCLH/UCL Biomedical Centre and NIHR Queen Square Dementia Biomedical Research Unit. The University of Southampton acknowledges support from the AS. King's College London was supported by the NIHR Biomedical Research Centre for Mental Health and the Biomedical Research Unit for Dementia at the South London and Maudsley NHS Foundation Trust and by King's College London and the MRC. ARUK and the Big Lottery Fund provided support to Nottingham University. Ulster Garden Villages, AS, ARUK, the American Federation for Aging Research, and the Northern Ireland R&D Office provided support for Queen's University, Belfast. The Centro de Biología Molecular Severo Ochoa (CSIS-UAM), CIBERNED, Instituto de Investigación Sanitaria la Paz, University Hospital La Paz, and the Universidad Autónoma de Madrid were supported by grants from the Ministerio de Educación y Ciencia and the Ministerio de Sanidad y Consumo (Instituto de Salud Carlos III) and by an institutional grant of the Fundación Ramón Areces to the CMBSO. We thank I. Sastre and A. Martinez-Garcia for DNA preparation, and P. Gil and P. Coria for their recruitment efforts. The Department of Neurology, University Hospital Mútua de Terrassa, was supported by CIBERNED, Instituto de Salud Carlos III, Madrid, Spain, and acknowledges M.A. Pastor (University of Navarra Medical School and Center for Applied Medical Research), M. Seijo-Martinez (Hospital do Salnes), and R. Rene, J. Gascon, and J. Campdelacreu (Hospital de Bellvitage) for providing DNA samples. The Hospital de la Sant Pau, Universitat Autònoma de Barcelona, acknowledges support from the Spanish Ministry of Economy and Competitiveness (grant PI12/01311) and from the Generalitat de Catalunya (2014SGR-235). The Santa Lucia Foundation and the Fondazione Ca' Granda IRCCS Ospedale Policlinico, Italy, acknowledge the Italian Ministry of Health (grant RC 10.11.12.13/A). The Bonn samples are part of the German Dementia Competence Network (DCN) and the German Research Network on Degenerative Dementia (KNDD), which are funded by the German Federal Ministry of Education and Research (grants KND: 01G10102, 01GI0420, 01GI0422, 01GI0423, 01GI0429, 01GI0431, 01GI0433, 04GI0434; grants KNDD: 01GI1007A, 01GI0710, 01GI0711, 01GI0712, 01GI0713, 01GI0714, 01GI0715, 01GI0716, 01ET1006B). M.M.N. is a member of the German Research Foundation (DFG) cluster of excellence ImmunoSensation. Funding for Saarland University was provided by the German Federal Ministry of Education and Research (BMBF), grant 01GS08125, to M. Riemenschneider. The University of Washington was supported by grants from the US National Institutes of Health (R01-NS085419 and R01-AG044546), the Alzheimer's Association (NIRG-11-200110), and the American Federation for Aging Research (C. Cruchaga was recipient of a New Investigator Award in Alzheimer's disease). Brigham Young University was supported by the Alzheimer's Association (MNIRG-11-205368), the BYU Gerontology Program, and the US National Institutes of Health (R01-AG11380, R01-AG021136, P30-S069329-01, R01-AG042611). We also acknowledge funding from the Institute of Neurology, UCL, London, who was supported in part by ARUK via an anonymous donor, and by a fellowship to R.G. The participation of D.S., M.U., and C. Masullo in the study was completely supported by Ministero della Salute, IRCCS Research Program, Ricerca Corrente 2015–2017, Linea 2 'Malattiecomplesse e Terapie Innovative' and by the '5 × 1000' voluntary contribution. AddNeuromed is supported by InnoMed, an Integrated Project funded by the European Union's Sixth Framework Programme priority FP6-2004-LIFESCIHEALTH-5, Life Sciences, Genomics, and Biotechnology for Health. We are grateful to the Wellcome Trust for awarding a Principal Research Fellowship to D.C.R. (095317/Z/11/Z). M. Riemenschneider was funded by BMBF NGFN grant 01GS08125. B.N. was supported by the Fondazione Cassa di Risparmio di Pistoia e Pescia (grants 2014.0365, 2011.0264, 2013.0347). H. Hampel is supported by the AXA Research Fund, the Fondation Universite Pierre et Marie Curie, and the 'Fondation pour la Recherche sur Alzheimer', Paris, France. The research leading to these results has received funding from the program 'Investissements d'Avenir', ANR-10-IAIHU-06 (Agence Nationale de la Recherche-10-IA Agence Institut Hospitalo-Universitaire-6.

CHARGE. Infrastructure for the CHARGE Consortium is supported in part by National Heart, Lung, and Blood Institute grant HL105756 and for the neurology working group by AG033193 and AG049505.

Rotterdam (RS). The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, the Netherlands Organization for Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the municipality of Rotterdam. The authors are grateful to the study participants, the staff from the Rotterdam Study, and the participating general practitioners and pharmacists. Generation and management of the Illumina exome chip v1.0 array data for the Rotterdam Study (RS-I) was executed by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine (http://www.glimdna.org/), Erasmus MC, Rotterdam, the Netherlands. The Exome chip array data set was funded by the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO)-sponsored Netherlands Consortium for Healthy Aging (NCHA; project 050-060-810); the Netherlands Organization for Scientific Research (NWO; project 184021007); and by the Rainbow Project (RP10; Netherlands Exome Chip Project) of Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL; http://www.bbmri.nl). Generation and management of GWAS genotype data for the Rotterdam Study (RS-I, RS-II, RS-III) was executed by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands. The GWAS data sets are supported by the Netherlands Organization of Scientific Research NWO Investments (175.010.2005.011, 911-03-012), the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, the Research Institute for Diseases in the Elderly (014-93-015; RIDE2), and the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) Netherlands Consortium for Healthy Aging (NCHA), project 050-060-810. This study makes use of an extended data set of RS-II and RS-III samples based on Illumina Omni 2.5 and 5.0 GWAS genotype data. This data set was funded by the Genetic Laboratory of the Department of Internal Medicine, the Department of Forensic Molecular Biology, and the Department of Dermatology, Erasmus MC, Rotterdam, the Netherlands. We thank M. Jhamai, S. Higgins, and M. Verkerk for their help in creating the exome chip database; C. Medina-Gomez, L. Karsten, and L. Broer for quality control and variant calling; M. Jhamai, M. Verkerk, L. Herrera, M. Peters, and C. Medina-Gomez for their help in creating the GWAS database; and L. Broer for the creation of the HRC-imputed data. Variants were called using the best practice protocol developed by M.L. Grove and colleagues as part of the CHARGE Consortium Exome Chip central calling effort. The work for this manuscript was further supported by ADAPTED: Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development (115975); the CoSTREAM project (http://www.costream.eu/); and funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 667375.

AGES. The AGES study has been funded by NIA contracts N01-AG-12100 and HHSN271201200022C with contributions from NEI, NIDCD, and NHLBI, the NIA Intramural Research Program, Hjartavernd (the Icelandic Heart Association), and the Althingi (the Icelandic Parliament).

Cardiovascular Health Study (CHS). This research was supported by contracts HHSN268201200036C, HHSN268200800007C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, and N01HC85086 and grant U01HL080295 from the National Heart, Lung, and Blood Institute (NHLBI), with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided by R01AG033193, R01AG023629, R01AG15928, and R01AG20098 and by U01AG049505 from the National Institute on Aging (NIA). The provision of genotyping data was supported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR000124, and National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. A full list of CHS principal investigators and institutions can be found at https://chs-nhlbi.org/. The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

Framingham Heart Study. This work was supported by the National Heart, Lung, and Blood Institute's Framingham Heart Study (contracts N01-HC-25195 and HHSN268201500001I). This study was also supported by grants from the National Institute on Aging: AG033193, U01-AG049505, and AG008122 (S. Seshadri). S. Seshadri and A.L.D. were also supported by additional grants from the National Institute on Aging (R01AG049607) and the National Institute of Neurological Disorders and Stroke (R01-NS017950).

Fundació ACE. We sincerely acknowledge the collaboration of S. Ruiz, M. Rosende-Roca, A. Mauleon, L. Vargas, O. Rodriguez-Gomez, M. Alegret, A. Espinosa, G. Ortega, M. Tarragona, C. Abdelnour, and D. Sanchez. We thank all patients for their participation in this project. We are obliged to T. Port-Carbo and her family for their support of the Fundació ACE research programs. Fundació ACE collaborates with CIBERNED and is one of the participating centers of Dementia Genetics Spanish Consortium 430 (DEGESCO). CIBERNED is an Instituto de Salud Carlos III Project. A. Ruiz is supported by grant PI13/02434 (Acción Estratégica en Salud, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain) and Obra Social 'La Caixa' (Barcelona, Spain).

ADGC. The US National Institutes of Health, National Institute on Aging (NIH-NIA) supported this work through the following grants: ADGC, U01 AG032984, and RC2 AG036528. Samples from the National Cell Repository for Alzheimer's Disease (NCRAD), which receives government support under a cooperative agreement grant (U24 AG21886) awarded by the National Institute on Aging (NIA), were used in this study. We thank the contributors who collected samples used in this study, as well as the patients and their families, whose help and participation made this work possible. Data for this study were prepared, archived, and distributed by the National Institute on Aging Alzheimer's Disease Data Storage Site (NIAGADS) at the University of Pennsylvania (U24-AG041689-01), NACC (U01 AG016976), NIA LOAD (Columbia University) (U24 AG026395, R01AG041797), Banner Sun Health Research Institute (P30 AG019610), Boston University (P30 AG013846, U01 AG10483, R01 CA129769, R01 MH080295, R01 AG017173, R01 AG025259, R01 AG048927, R01AG33193), Columbia University (P50 AG008702, R37 AG015473), Duke University (P30 AG028377, AG05128), Emory University (AG025688), Group Health Research Institute (UO1 AG006781, UO1 HG004610, UO1 HG006375), Indiana University (P30 AG10133), Johns Hopkins University (P50 AG005146, R01 AG020688), Massachusetts General Hospital (P50 AG005134), Mayo Clinic (P50 AG016574), Mount Sinai School of Medicine (P50 AG005138, P01 AG002219), New York University (P30 AG08051, UL1 RR029893, 5R01AG012101, 5R01AG022374, 5R01AG013616, 1RC2AG036502, 1R01AG035137), Northwestern University (P30 AG013854), Oregon Health & Science University (P30 AG008017, R01 AG026916), Rush University (P30 AG010161, R01 AG019085, R01 AG15819, R01 AG17917, R01 AG30146), TGen (R01 NS059873), University of Alabama at Birmingham (P50 AG016582), University of Arizona (R01 AG031581), University of California, Davis (P30 AG010129), University of California, Irvine (P50 AG016573), University of California, Los Angeles (P50 AG016570), University of California, San Diego (P50 AG005131), University of California, San Francisco (P50 AG023501, P01 AG019724), University of Kentucky (P30 AG028383, AG05144), University of Michigan (P50 AG008671), University of Pennsylvania (P30 AG010124), University of Pittsburgh (P50 AG005133, AG030653, AG041718, AG07562, AG02365), University of Southern California (P50 AG005142), University of Texas Southwestern (P30 AG012300), University of Miami (R01 AG027944, AG010491, AG027944, AG021547, AG019757), University of Washington (P50 AG005136), University of Wisconsin (P50 AG033514), Vanderbilt University (R01 AG019085), and Washington University (P50 AG005681, P01 AG03991). The Kathleen Price Bryan Brain Bank at Duke University Medical Center is funded by NINDS grant NS39764, NIMH MH60451, and by GlaxoSmithKline. Support was also provided by the Alzheimer's Association (L.A.F., IIRG-08-89720; M.P.-V., IIRG-05-14147), the US Department of Veterans Affairs Administration, the Office of Research and Development, the Biomedical Laboratory Research Program, and the BrightFocus Foundation (M.P.-V., A2111048). P.S.G.-H. is supported by the Wellcome Trust, the Howard Hughes Medical Institute, and the Canadian Institutes of Health Research. Genotyping of the TGEN2 cohort was supported by Kronos Science. The TGen series was also funded by NIA grant AG041232 to A.J.M. and M.J.H., the Banner Alzheimer's Foundation, the Johnnie B. Byrd Sr. Alzheimer's Institute, the Medical Research Council, and the state of Arizona and also includes samples from the following sites: the Newcastle Brain Tissue Resource (funding via the Medical Research Council, local NHS trusts, and Newcastle University), the MRC London Brain Bank for Neurodegenerative Diseases (funding via the Medical Research Council), the South West Dementia Brain Bank (funding via numerous sources including the Higher Education Funding Council for England (HEFCE), Alzheimer's Research UK (ARUK), and BRACE as well as the North Bristol NHS Trust Research and Innovation Department and DeNDRoN), the Netherlands Brain Bank (funding via numerous sources including Stichting MS Research, Brain Net Europe, Hersenstichting Nederland Breinbrekend Werk, International Parkinson Fonds, Internationale Stiching Alzheimer Onderzoek), and the Institut de Neuropatologia, Servei Anatomia Patologica, Universitat de Barcelona. ADNI data collection and sharing were funded by US National Institutes of Health grant U01 AG024904 and Department of Defense award W81XWH-12-2-0012. ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer's Association; the Alzheimer's Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Eisai, Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche, Ltd., and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO, Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development, LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer, Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research provide funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (http://www.fnih.org/). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer's Disease Cooperative Study at the University of California, San Diego. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California. We thank D.S. Snyder and M. Miller from the NIA who are ex-officio ADGC members.

EADI. This work was supported by INSERM, the National Foundation for Alzheimer's Disease and Related Disorders, the Institut Pasteur de Lille, and the Centre National de Génotypage. This work has been developed and supported by the LABEX (Laboratory of Excellence Program Investment for the Future) DISTALZ grant (Development of Innovative Strategies for a Transdisciplinary Approach to Alzheimer's Disease) including funding from MEL (Métropole Européenne de Lille), ERDF (European Regional Development Fund), and the Conseil Régional du Nord-Pas-de-Calais. The Three-City Study was performed as part of collaboration between INSERM, Victor Segalen Bordeaux II University, and Sanofi-Synthelabo. The Fondation pour la Recherche Médicale funded the preparation and initiation of the study. The 3C Study was also funded by the Caisse Nationale Maladie des Travailleurs Salaries, Direction Générale de la Santé, MGEN, Institut de la Longévité, Agence Française de Sécurité Sanitaire des Produits de Santé, the Aquitaine and Bourgogne regional councils, Agence Nationale de la Recherche (ANR supported the COGINUT and COVADIS projects), Fondation de France, and the joint French Ministry of Research/INSERM 'Cohortes et Collections de Données Biologiques' program. The Lille Genopole received an unconditional grant from Eisai and was supported by the European Joint Programme for Neurodegenerative Disease (JPND: grant MR/L501517/1). The Three-City Biological Bank was developed and maintained by the Laboratory for Genomic Analysis LAG-BRC, Institut Pasteur de Lille.

Belgium sample collection. Research at the Antwerp site is funded in part by the Interuniversity Attraction Poles program of the Belgian Science Policy Office, the Foundation for Alzheimer Research (SAO-FRA), a Methusalem Excellence Grant of the Flemish Government, the Research Foundation Flanders (FWO), and the Special Research Fund of the University of Antwerp, Belgium. Authors from the Antwerp site thank the personnel of the VIB Genetic Service Facility, the Biobank of the Institute Born-Bunge, and the Departments of Neurology and Memory Clinics at the Hospital Network Antwerp and University Hospitals Leuven.

Finnish sample collection. Financial support for this project was provided by the Health Research Council of the Academy of Finland, EVO grant 5772708 of Kuopio University Hospital, and the Nordic Center of Excellence in Neurodegeneration.

Swedish sample collection. Sample collection was financially supported in part by the Swedish Brain Power network, the Marianne and Marcus Wallenberg Foundation, the Swedish Research Council (521-2010-3134), King Gustaf V and Queen Victoria's Foundation of Freemasons, the Regional Agreement on Medical Training and Clinical Research (ALF) between the Stockholm County Council and the Karolinska Institutet, the Swedish Brain Foundation, and the Swedish Alzheimer Foundation.

AMP AD University of Florida/Mayo Clinic/Institutes of Systems Biology. For human brain donations, we thank all patients and their families, without whom this work would not have been possible. This work was supported by NIH/NIA AG046139-01 (T.E.G., N.E.-T., N.P., S.G.Y.). We thank T.G. Beach (Banner Sun Health Institute) for sharing human tissue.

The Mayo Clinic Brain Bank. Data collection was supported through funding by NIA grants P50 AG016574, R01 AG032990, U01 AG046139, R01 AG018023, U01 AG006576, U01 AG006786, R01 AG025711, R01 AG017216, and R01 AG003949, NINDS grant R01 NS080820, the CurePSP Foundation, and support from the Mayo Foundation.

Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer's Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer's Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901, and 1001 to the Arizona Parkinson's Disease Consortium), and the Michael J. Fox Foundation for Parkinson's Research.

Author information

Authors and Affiliations

Authors

Consortia

Contributions

GERAD/PERADES. Study design or conception: R. Sims, V.E.-P., M.C.O'D., M.J.O., P.A.H., J. Williams. Sample contribution: M.M.N., W.M., S. Herms, A.J.F., J. Williams, A. Ramirez, M.K.L., C. Medway, K. Brown, B.M., P. Proitsi, P. Pastor, A.F.-G., I.G., H. Hampel, P.M., V.B., M. Scherer, M.L., S.R.-H., A. Braae, C. Masullo, G. Spalletta, P. Bossù, E. Sacchinelli, P.S.-J., F.J., J. Morris, C. Corcoran, J. Tschanz, M.N., R. Munger, M.J.B., E.C., V.A., M. Gallo, A.C.B., M. Dichgans, D.G., E. Scarpini, M. Mancuso, U.B., A.D., O.P., B.N., M. Riemenschneider, R.H., C. Brayne, D.C.R., A.A., C.E.S., J. Collinge, D.M., M.T., J. Clarimón, R. Sussams, S. Lovestone, S. Mead, C.H., J.P., K.M., P. Passmore, D.R., S.O.-C., J. Kauwe, M. Dunstan, A. Braddel, C. Thomas, A.M., R. Marshall, M.D.F., A. Hodges, B.V., H. Soininen, I.K., M. Daniilidou, J.U., Y.P., J.T.H., J.L., J. Turton, A.M.H., M. Aguilar, R.C., C. Fenoglio, M. Serpente, M. Arcaro, C. Caltagirone, M.D.O., A.C., S.P., M. Mayhaus, W.G., A. Lleó, J.F., R. Blesa, I.S.B., K. Brookes, C. Cupidi, R.G.M., D. Carrell, S. Sorbi, S. Moebus, M.U., A. Pilotto, J. Kornhuber, P. Bosco, S. Todd, D. Craig, J. Johnston, M. Gill, B.L., A. Lynch, N.C.F., D.S., ARUK Consortium. Data generation: R. Sims, R.R., S.H.-H., P.H., R.T., T.M., N.D., A. Ramirez, J. Williams, J. B., R.G., J. Hardy, A.G., J. Chapman, M. Hill. Analysis: R. Sims, N.B., M.V., D. Harold, E. Majounie, P.A.H. Manuscript preparation: R. Sims, S. Taylor, L.J., P.A.H., J. Williams. Study supervision/management: R. Sims, A. Ramirez, J. Williams.

ADGC. Study design or conception: L.A.F., J. Haines, R. Mayeux, M.A.P.-V., L.-S.W., G.D.S. Sample contribution: A.R.W., S. Mukherjee, P.K.C., R.C.B., P.M.A., M.S.A., D. Blacker, R.S.D., T.J.F., M.P.F., B.G., R.M.H., M.I.K., M.J.K., C.D.K., E.B.L., R.B.L., T.J.M., R.C.P., E.M.R., J.S.R., D.R.R., M. Sano, P.S.G.-H., D.W.T., C.-K.W., A.M.G., C. Cruchaga, S.G.Y., D.W.D., W.A.K., N.E.-T., R.L.A., L.G.A., S.E.A., 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. 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.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., D.W.F., M.R.F., S.F., T.M.F., D.R.G., M. Gearing, D.H.G., N.R.G.-R., R.C.G., J.H.G., R.L.H., L.E.H., L.S.H., M.J.H., C.M.H., B.T.H., G.P.J., E.A., L.-W.J., A. K., J.A.K., R.K., N.W.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, F.M., E. Masliah, W.C.M., S.M.M., A.N.M., A.C.M., M. Mesulam, B.L.M., C.A.M., 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., A. Pierce, W.W.P., H.P., J.F.Q., A. Raj, M. Raskind, B.R., J.M.R., E.D.R., E.R., H.J.R., R.N.R., M.A.S., A.J.S., J.A. Schneider, L.S.S., W.W.S., A.G.S., J.A. Sonnen, S. Spina, R.A.S., R.H.S., R.E.T., J.Q.T., J.C.T., V.M.V.D., L.J.V.E., H.V.V., J.P.V., S.W., K.A.W.-B., K.C.W., J. Williamson, T.S.W., R.L.W., C.-E.Y., L.Y. Data generation: O.V., L.Q., Y.Z., J. Malamon, C.C.F., H. Li, J.D.Burgess, M. Allen, N.D.P., P. Chakrabarty, X.W., T.E.G., H. Hakonarson, T.W.B., B. Dombroski, W.A.K., N.E.-T., L.B.C., D. Beekly, P.W., C.T.B., R.M.C., C.C.D., E.A.C., J.R.G., D.C.Marsh, W.P., T.A.T.-W., C.B.W. Analysis: A.C.N., B.W.K., E.R.M., A.B.K., R.R.G., B.N.V., K.L.H.-N., G.W.B., S.B., G.J., K.L.L., C.R. Manuscript preparation: A.C.N., G.D.S. Study supervision/management: L.A.F., J.L.H., R. Mayeux, M.A.P.-V., L.-S.W., G.D.S.

CHARGE. Study design or conception: S.J.v.d.L., J.C.B., P.L.D.J., V. Gudnason, A.L.D., L.J.L., N.A., C.M.v.D., S. Seshadri. Sample contribution: J.C.B., A. Ruiz, 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., 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.A.I., L.J.L. Data generation: S.J.v.d.L., A. Ruiz, F.R., A.G.U., J.C.B., M.L.G., 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., 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., Analysis: S.J.v.d.L., A. Ruiz, J.C.B., M.L.G., H. Schmidt, J. Jakobsdottir, A.V.S., S.M.-G., N.A., V.C., C.C.W., S.-H.C., J.D., Y.C., S. Li, A.L.D. Manuscript preparation: S.J.v.d.L., A. Ruiz, J.C.B., J. Jakobsdottir, V.C., C.C.W., C.M.v.D., S. Seshadri. Study supervision/management: C.M.v.D., S. Seshadri, M.A.I.

EADI. Study design or conception: P.A., J.-C.L. Sample contribution: J.E., D.W., D. Hannequin, F. Pasquier, C. Berr, J.-F. Dartigues, D. Campion, C. Tzourio, P.A., J.-C.L., V.D., N.F., O.H., C. Dufouil, A. Brice, K.R., B. Dubois, K.S., M. Hiltunen, F.S.G., M.C.D.N., L.F., L. Keller, F. Panza, P. Caffarra, L. Lannfelt, M.I., C.G., O.C., C.V.B., S.E., R.V., P.P.D.D., A.S., P.S.-J., C.M.F., Y.A.B., H.T., C. Forsell, L. Lilius, A.K.-S., V. Giedraitis, L. Kilander, R. Brundin, L.C., S. Helisalmi, A.M.K., A. Haapasalo, V.S. Data generation: A. Boland, C. Dulary, C. Derbois, D. Bacq, J.-F. Deleuze, F. Garzia, F. Golamaully, G. Septier, R.O. Analysis: C. Bellenguez, B.G.-B., P.A., J.-C.L. Manuscript preparation: C. Bellenguez, J.-C.L. Study supervision/management: P.A., J.-C.L.

Corresponding authors

Correspondence to Sven J van der Lee, Julie Williams or Gerard D Schellenberg.

Ethics declarations

Competing interests

R.G. and T.W.B. are full-time employees of Genentech, Inc. D. Blacker is a consultant for Biogen, Inc. R.C.P. is a consultant for Roche, Inc., Merck, Inc., Genentech, Inc., Biogen, Inc., and Eli Lilly. A.R.W. is a former employee and stockholder of Pfizer, Inc., and a current employee of the Perelman School of Medicine at the University of Pennsylvania Orphan Disease Center in partnership with the Loulou. A.M.G. is a member of the scientific advisory board for Denali Therapeutics. N.E.-T. is a consultant for Cytox. J. Hardy holds a collaborative grant with Cytox cofunded by the Department of Business (Biz). F.J. acts as a consultant for Novartis, Eli Lilly, Nutricia, MSD, Roche, and Piramal. Neither J. Morris nor his family own stock or have equity interest (outside of mutual funds or other externally directed accounts) in any pharmaceutical or biotechnology company. J. Morris is currently participating in clinical trials of antidementia drugs from Eli Lilly and Company, Biogen, and Janssen. J. Morris serves as a consultant for Lilly USA. He receives research support from Eli Lilly/Avid Radiopharmaceuticals and is funded by NIH grants P50AG005681, P01AG003991, P01AG026276, and UF01AG032438.

Integrated supplementary information

Supplementary Figure 1 IGAP study design.

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

Supplementary Figure 2 Quantile–quantile plots for the minimally adjusted (‘unadjusted’) model single-variant association analyses in SeqMeta.

(a) Plot showing all variants with minor allele count (MAC) ≥4, genomic inflation factor (λ) = 1.188. (b) Plot showing variants with MAC ≥4 and MAF <0.05, genomic inflation factor (λ) = 1.066. (c) Plot showing variants with MAC ≥4 and excluding all associations in the APOE region, genomic inflation factor (λ) = 1.102. (d) Plot showing variants with MAC ≥4, with MAF <0.05, and excluding all associations in the APOE region, genomic inflation factor (λ) = 1.054. Although there is some evidence of inflated association statistics, this inflation is consistent with inflation observed in previous exome array studies (Jeroen et al. Nat. Genet. 45, 197–201, 2013) and has been shown in multiple studies to be dramatically reduced with the exclusion of common variants, which on the exome array are mostly previously reported variants with strong associations in GWAS empaneled on the exome array, and regions of strong association, such as the APOE region in Alzheimer’s disease.

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

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

Supplementary Figure 4 PLCG2 conservation at human position 522 (p.P522R) across human, chimpanzee, rhesus monkey, mouse, rat, rabbit, horse, dog, and elephant.

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

Supplementary Figure 5 Regional plot of the conditional analysis performed at the PLCG2 locus in the stage 1 data set.

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

Supplementary Figure 6 Forest plot of rs616338 (ABI3) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

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

Supplementary Figure 7 ABI3 conservation at human position 209 (p.S209F) across multiple species.

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

Supplementary Figure 8 Regional plot of the conditional analysis performed at the ABI3 locus in the stage 1 data set.

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

Supplementary Figure 9 Forest plot of rs143332484 (TREM2) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

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

Supplementary Figure 10 Forest plot of rs75932628 (TREM2) association with LOAD in combined (stage 1 + stage 2 + stage 3) analysis, shown by each cohort independently analyzed.

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

Supplementary Figure 11 Regional plots of the conditional analyses performed at the TREM2 locus in the stage 1 data set.

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

Supplementary Figure 12 Gene expression profiles (RNA-seq) of PLCG2, ABI3, and TREM2 from transcriptome data from six cell types from human temporal lobe cortex (pink) and transcriptome data from seven cell types from mouse whole cortex (blue).

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

Supplementary Figure 13 Schematic of the PLCG2 protein from the Protein Data Bank.

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

Supplementary Figure 14 Schematic of the ABI3 protein from the Protein Data Bank.

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

Supplementary Figure 15 Schematic of the TREM2 protein from the Protein Data Bank.

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Note. (PDF 3976 kb)

Supplementary Table 1

Full description of the different stage 1 samples from the GERAD/PERADES, ADGC, and CHARGE consortia. (XLSX 48 kb)

Supplementary Table 2

Full description of the different stage 2 and stage 3 samples from the GERAD/PERADES, ADGC, CHARGE, and EADI consortia. (XLSX 17 kb)

Supplementary Table 3

Details of stage 1 calling software(s) and quality control metrics applied across the ADGC, CHARGE, and GERAD/PERADES cohorts. (XLSX 31 kb)

Supplementary Table 4

Table of 43 variants eligible to be taken forward from stage 1, meeting P < 1 × 10−4 before reclustering and P < 0.05 after reclustering. (XLSX 59 kb)

Supplementary Table 5

Observed associations at previously identified genome-wide significant Alzheimer's disease risk loci. (XLSX 46 kb)

Supplementary Table 6

Concordance of alternate allele carrier genotypes for all replicated SNPs among samples with both exome chip genotyping and GWAS imputed to HRC. (XLSX 45 kb)

Supplementary Table 7

Results of unadjusted analysis of the SNVs identified as eligible for replication in stage 1. (XLSX 72 kb)

Supplementary Table 8

Results of adjusted analysis of the SNVs identified as eligible for replication in stage 1. (XLSX 64 kb)

Supplementary Table 9

Unadjusted association with single-nucleotide variation within the PLCG2 gene on chromosome 16. (XLSX 58 kb)

Supplementary Table 10

Results of unadjusted SKAT-O gene-wide analysis of the SNVs in stage 1. (XLSX 43 kb)

Supplementary Table 11

Results of adjusted SKAT-O gene-wide analysis of the SNVs in stage 1. (XLSX 43 kb)

Supplementary Table 12

Unadjusted association with single-nucleotide variation within the ABI3 gene on chromosome 17. (XLSX 52 kb)

Supplementary Table 13

Unadjusted association with single-nucleotide variation within the TREM2 gene on chromosome 6. (XLSX 56 kb)

Supplementary Table 14

Linkage disequilibrium calculations generated for the observed SNV associations at the PLCG2 and TREM2 loci. (XLSX 32 kb)

Supplementary Table 15

Enrichment for the IGAP pathway clusters based on combining gene-wide P values from variants with MAF <0.01 with Fisher's method. (XLSX 46 kb)

Supplementary Table 16

Significant pathways (FDR < 0.01) from an analysis of the rare variant data (MAF < 1%) on all 9,816 pathways originally analyzed in the IGAP GWAS. (XLS 40 kb)

Supplementary Table 17

ALIGATOR enrichment analysis of the 151 genes in the overlap of immune-related gene expression modules in the IGAP GWAS, stratifying by membership of the protein interaction network. (XLSX 39 kb)

Supplementary Table 18

List of the 56 genes in the protein–protein interaction network, with gene-based P values in the IGAP common variant GWAS and in the present rare variant study (unadjusted model). (XLSX 60 kb)

Supplementary Table 19

Differential expression of genes (DEG) in human temporal cortex. (XLSX 38 kb)

Supplementary Table 20

Differential expression of genes (DEG) in brains from the CRND8 transgenic mouse model at 3, 6, and 12 months of age (n = 12, 12, and 14, respectively), the PS1APP model at 12 months of age (n = 11), and wild-type mice at 3, 6, and 12 months of age (n = 12, 12, and 10, respectively). (XLSX 43 kb)

Supplementary Table 21

Functional annotation of the PLCG2 and ABI3 genome-wide significant SNVs and variants in LD (r2 > 0.7). (XLSX 49 kb)

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Sims, R., van der Lee, S., Naj, A. et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease. Nat Genet 49, 1373–1384 (2017). https://doi.org/10.1038/ng.3916

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