Supplementary materials online

Page(s) Descriptions

2 Supplementary Table S1

3 Supplementary Table S2

4 Supplementary Table S3

5 Supplementary Table S4

6 Supplementary Figure S1

8 Supplementary Figure S2

9 Supplementary Figure S3

10 SUPPLEMENTARY METHODS

13 References

14 GERAD1 author list

Supplementary Table S1. Association with AD risk of SNPs in the 6.7kb LD block locus.

Genotypes were imputed in our French EADI1 cohort using the 1000 Genomes data set. P-

values and ORs with the associated 95% CI have been calculated under an additive model

using logistic regression models adjusted for age, gender and centers when necessary. MAF,

minor allele frequency. R², imputed quality.

SNPs MAF R² OR, CI.95% P-valuers11680911 0.32 0.96 1.15 [1.07;1.24] 2.3x10-4

rs11678744 0.17 0.93 1.10 [1.00;1.21] 5.9x10-2

rs7561528 0.33 0.98 1.15 [1.06;1.23] 3.2x10-4

rs60692476 0.18 0.98 1.16 [1.01;1.21] 3.2x10-2

chr2:127890462 0.05 0.29 1.21 [0.89;1.65] 2.3x10-1

rs4663104 0.18 0.96 1.10 [1.01;1.21] 3.3x10-2

rs6714558 0.18 0.95 1.10 [1.01;1.21] 3.6x10-2

rs35968837 0.13 0.94 1.13 [1.02;1.26] 2.1x10-2

rs34546266 0.14 0.90 1.16 [1.04;1.29] 6.5x10-3

rs60572885 0.17 0.95 1.10 [1.01;1.21] 3.5x10-2

rs56193035 0.18 0.97 1.10 [1.01;1.21] 3.2x10-2

rs61308109 0.18 0.95 1.10 [1.01;1.21] 3.6x10-2

rs57109420 0.18 0.95 1.10 [1.01;1.21] 3.6x10-2

rs4663105 0.40 0.93 1.21 [1.13;1.30] 3.2x10-7

rs59253801 0.18 0.94 1.12 [1.02;1.23] 2.1x10-2

rs59176034 0.18 0.97 1.10 [1.01;1.21] 3.2x10-2

chr2:127891663 0.17 0.92 1.11 [1.01;1.22] 3.7x10-2

chr2:127891795 0.18 0.96 1.10 [1.01;1.21] 3.5x10-2

rs6733839 0.39 0.91 1.20 [1.11;1.29] 2.0x10-6

chr2:127892956 0.18 0.95 1.10 [1.01;1.21] 3.5x10-2

chr2:127893025 0.18 0.97 1.10 [1.01;1.21] 3.2x10-2

rs745717 0.10 0.90 1.20 [1.06;1.35] 3.4x10-3

rs56368748 0.29 0.89 1.14 [1.05;1.23] 1.9x10-3

rs730482 0.29 0.98 1.15 [1.07;1.25] 2.5x10-4

rs744373 0.29 0.99 1.16 [1.07;1.25] 2.1x10-4

rs6431222 0.01 0.34 0.84 [0.50;1.41] 5.1x10-1

rs6431223 0.28 0.76 0.92 [0.84;1.00] 5.9x10-2

Supplementary Table S2. Association with AD risk of polymorphisms in the 6.7kb LD block

locus not included in 1000 Genomes data set. Genotypes were imputed in our French EADI1

cohort using the 350 controls data set. P values and ORs with the associated 95% CI have

been calculated under an additive model using logistic regression models adjusted for age,

gender and centers when necessary. MAF, minor allele frequency. Info, imputed quality. *

denotes insertion/deletion polymorphisms.

IMPUTED GENOTYPED

SNPs MAF Info OR, CI.95% P-value MAF OR, CI.95% P-value

rs13009551 0.15 0.96 1.13 [1.02;1.25] 1.9x10-2

rs35579178* 0.15 0.96 1.13 [1.02;1.25] 1.7x10-2

rs10537122* 0.18 0.98 1.11 [1.01;1.21] 3.1x10-2

rs60447541 0.25 0.67 1.10 [1.00;1.22] 5.2x10-2

rs11268249* 0.24 0.87 0.92 [0.84;1.00] 6.1x10-2

rs59335482* 0.27 0.93 1.17 [1.07;1.26] 2.2x10-4 0.27 1.21 [1.11;1.31] 1.2x10-6

rs34779859 0.30 0.91 1.14 [1.05;1.24] 1.1x10-3

rs893432 0.21 0.89 1.10 [1.01;1.21] 3.3x10-2

Supplementary Table S3. Genotypic distribution of rs59335482 in EADI1 and in the Flanders-

Belgium sample.

EADI 1 Flanders-Belgium study

           

DD ID II DD ID II

rs59335482AD cases 1087 948 204 507 434 100

Controls 3132 2344 412 400 384 55

                 

Supplementary Table S4. Human BIN1 is the best predicted Drosophila Amph ortholog DRSC

Integrative Ortholog Prediction Tool (http://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl)

Fly Symbol

Human Symbol

Score(max 9)

Prediction Derived From

Amph BIN1 7 Compara, Homologene, Inparanoid, orthoMCL, Phylome, RoundUp, TreeFam

Amph BIN2 5 Compara, Inparanoid, OMA, Phylome, TreeFamAmph AMPH1 4 Compara, Phylome, RoundUp, TreeFamAmph BIN3 1 Isobase

Supplementary Figure S1.

(a) Western blot showing Amph protein levels in fly. Canton S, wild-type control; Amph5E3/+,

heterozygous null mutant; Amph5E3, homozygous null mutant.

(b) qRT-PCR results showing Amph transcript levels in fly heads of the genotypes Act5c/+

(control, outcrossed ubiquitous Act5c-GAL4 driver), AmphKD/+ (control, outcrossed UAS-

AmphKD responder), Act5c>AmphKD (ubiquitous RNAi-mediated Amph knockdown),

Amph5E3/+ (removal of one genomic copy of Amph).

(c) Altering Amph expression in Drosophila eyes does not modify human Tau transgene

expression levels. Western blots of fly heads showing Amph and human Tau proteins.

Amph5E3, homozygous null mutant; Canton S, wild-type control; GMR>Amph, eye-specific

Amph overexpression; GMR>AmphKD, eye-specific Amph knockdown; GMR>Tau, eye-specific

Tau overexpression; GMR>Tau>AmphKD, eye-specific Tau overexpression and Amph

knockdown; GMR>Tau>Amph, eye-specific Tau overexpression and Amph knockdown.

Quantification of the blot was performed by normalizing to total protein loads stained by the

Ponceau S method10.

(d) Eye morphology is not affected by eye-specific Amph knockdown (GMR>AmphKD) or in

homozygous Amph null flies (Amph5E3) compared to controls (GMR/+, outcrossed GMR-GAL4

driver ; UAS-Amph/+, outcrossed UAS-Amph responder construct).

(e and f) Eye size (e) and notal bristle numbers (f) in non-Tau overexpressing control flies.

Although statistically significant differences were seen for some genotypes these can not

explain the suppressive effect of decreased Amph expression on Tau-toxicity (Fig. 3). Canton

S, wild-type control; GMR/+, outcrossed GMR-GAL4 driver; AmphKD/+, outcrossed Amph

RNAi line; GMR>AmphKD, eye-specific Amph knockdown; Amph5E3/+, heterozygous null

mutant; Eq/+, outcrossed Eq-GAL4 driver; Eq>AmphKD, bristle-specific Amph knockdown.

***p<0.001, *p<0.05. a.u. arbitrary unit.

Supplementary Figure S2.

(a) Eye-specific overexpression of human A42 peptide (GMR>A42) gives rise to a subtle

rough eye phenotype which is not altered by either Amph overexpression

(GMR>A42>Amph) or knockdown (GMR>A42>AmphKD).

(b) Eye specific Amph overexpression (GMR>Tau>Amph) does not affect Tau-induced

reduced eye size.

(c) SEM image showing that eye-specific expression of human Tau (GMR>Tau) gives rise to a

severe rough eye phenotype although ommatidial structures can still be discerned. Eye-

specific co-expression of human Tau and fly Amph (GMR>Tau>Amph) enhances the rough

eye phenotype as illustrated by a decreased number of ommatidia. ***p<0,0001 (Mann

Whitney test)

8

Supplementary Figure S3. Association of rs744373 with AD risk in EADI1, GERAD1 and the

Flanders-Belgian population. P-values and ORs with the associated 95% CI have been

calculated under an additive model using logistic regression models adjusted for age, gender

and centers when necessary.

9

SUPPLEMENTARY METHODS

Population description

Written informed consent was obtained from study participants or, for those with

substantial cognitive impairment, from a caregiver, legal guardian, or other proxy, and the

study protocols for all populations were reviewed and approved by the appropriate

Institutional review boards.

EADI1.1 Controls were selected from the 3C Study (n=6,017, mean age 74.0±5.4 years, 61.7%

women).2 This cohort is a population-based, prospective (7-years follow-up) study of the

relationship between vascular factors and dementia. It has been carried out in three French

cities: Bordeaux (southwest France), Montpellier (southeast France) and Dijon (central

eastern France). A sample of non-institutionalised, over-65 subjects was randomly selected

from the electoral rolls of each city. Prevalent and incident AD cases were included as cases.

Patients with other types of dementia, and individuals for whom information on their

dementia status during the 7-year follow-up was missing were excluded. All AD cases

(n=2,243, mean age 74.1±8.6 years, mean age at onset 68.5±8.9 years, 64.9% women) were

ascertained by neurologists from Bordeaux, Dijon, Lille, Montpellier, Paris, Rouen, and were

identified as French Caucasian. Clinical diagnosis of probable AD was established according

to the DSM-III-R and NINCDS-ADRDA criteria.3

GERAD1.4 The GERAD sample included 3785 cases (mean age 78.6±8.3 years, mean age at

onset 74.0±8.0 years, 53.7% women) and 8130 controls. Cases and elderly screened

controls were recruited by the Medical Research Council (MRC) Genetic Resource for AD

(Cardiff University; Institute of Psychiatry, London; Cambridge University; Trinity College

Dublin), the Alzheimer’s Research Trust (ART) Collaboration (University of Nottingham;

University of Manchester; University of Southampton; University of Bristol; Queen’s

University Belfast; the Oxford Project to Investigate Memory and Ageing (OPTIMA), Oxford

University); Washington University, St Louis, United States; MRC PRION Unit, University

College London; London and the South East Region AD project (LASER-AD), University

College London; Competence Network of Dementia (CND) and Department of Psychiatry,

University of Bonn, Germany; the National Institute of Mental Health (NIMH)AD Genetics

Initiative; the Mayo Clinic, Jacksonville, Florida; Mayo Clinic, Rochester, Minnesota; and the

Mayo Brain Bank. Data from the Mayo Clinic and Brain Bank was included in a previous AD

GWAS5. Population controls were drawn from large existing cohorts with available GWAS

10Results

EADI :7359Cases/Controls GWA

582.892 SNPs genotyped

11.572.523 SNPs imputed

Association studyAssociation study

ImputationImputation

p<10-3

-log 1

0(p-v

alue

)

8

6

4

2

0

127.52 127.54 127.56 127.58

data, including the 1958 British Birth Cohort (1958BC) (http://www.b58cgene.sgul.ac.uk),

the KORA F4 Study and the Heinz Nixdorf Recall Study. All AD cases met criteria for either

probable (NINCDS-ADRDA, DSM-IV) or definite (CERAD) AD. All elderly controls were

screened for dementia using the MMSE or ADAS-cog, were determined to be free from

dementia at neuropathological examination or had a Braak score of 2.5 or lower. Genotypes

from all cases and 5470 controls were previously included in the AD GWAS by Harold and

colleagues (2009). Genotypes for the remaining 2660 population controls were obtained

from WTCCC2.

Belgium-Flanders case-control study.6 Belgian AD patients (n=1039, mean onset age 74.1±9.1

years, 65.4% women) were selected from a larger collection of dementia patients of which

the majority was ascertained at the neurology policlinic at ZNA Middelheim and the memory

clinic at ZNA Hoge Beuken, Antwerpen, Belgium (P.P.D.D. and S.E.) as part of a prospective

study of neurodegenerative and vascular dementia in Flanders, the Dutch-speaking region of

Belgium. The remaining group of the patients was recruited at the memory clinic of the

University Hospitals Leuven (UHL), Leuven, Belgium (R.V.) as part of a prospective study on

the molecular genetics of cognitive impairment3. Each patient underwent a diagnostic

neuropsychological examination, including Mini Mental State Examination (MMSE),

structural neuroimaging consisting of CT or MRI, and functional neuroimaging (brain

perfusion SPECT). Consensus diagnosis of possible or probable AD was given by at least two

neurologists based on the NINCDS/ADRDA criteria.

The Belgian control group consisted of 844 unrelated individuals from Flanders (mean age at

inclusion 65.4±14.8 years, 57.0% women). The control group included 334 subjects without

neurological or psychiatric antecedents or neurological complaints without organic disease

involving the central nervous system examined at the Memory Clinic of ZNA Hoge Beuken,

Antwerpen, Belgium (P.P.D.D. and S.E.). An additional 510 individuals were community

control individuals, included after interview enquiring medical and family history of

dementia and a Mini Mental State Examination (MMSE>26).

mRNA quantification

The quality of total RNA was assessed using Agilent 2100 bionalyser and the ratio of

ribosomal RNA 28S/18S systematically estimated using the Agilent 2100 bionalyser bio-sizing

software. The quantigene technology is well adapted to our purpose for several reasons7-9: (i)

this one allows for the direct quantification of a target mRNA without retro-transcription

step and PCR amplification; (ii) it limits biases due to RNA degradation.

11

Briefly, capture and label extender probe sets specific for BIN1, β-actin or b-glucuronidase

mRNA (as furnished by the supplier) were combined and diluted to 100 fmol/µl in a lysis

buffer supplied in the QuantiGene bDNA Signal Amplification Kit (Bayer Diagnostics, East

Walpole, MA). Total RNA (0.6 µg for IL33, 0.1 µg for β-actin and 0.2 µg for b-glucuronidase in

a final volume of 10 µl) was added to each well of a 96-well plate with 40 µl of capture

buffer, 40 µl of lysis buffer and 10 µl of each diluted probe set. RNA was allowed to hybridize

for at least 16 h at 53°C. Plates were then washed at room temperature (600 µl of a wash

buffer). Samples were then hybridized for 60 min at 46°C with the bDNA amplifier molecules

(100 µl/well) diluted in a amplifier/label probe buffer (1:100). At room temperature, plates

were then rinsed with the wash Buffer. Label probe (1:100 in a amplifier/label probe buffer)

was added to each well (100 µl/well) and hybridized to the bDNA-RNA complex for 60 min at

46°C. Plates were again rinsed with wash buffer at room temperature. Alkaline phosphatase-

mediated luminescence was triggered by the addition of a dioxetane substrate solution

(100 µl /well). The enzymatic reaction was allowed to proceed for 30 min at 46°C, and

luminescence was measured with the 1420 Victor light luminometer (Perkin Elmer).

Scanning electron microscopy of Drosophila eyes.

Fresh fly heads were mounted on 25 mm stubs and sputtered with platina for 120s with an

Agar auto sputter coater. Pictures were taken using a field emission scanning electron

microscope JSM-7401F (JEOL) at the Katholieke Universiteit Leuven Electron Microscopy

Core Facility of the Department of Human Genetics (Leuven, Belgium).

12

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baseline characteristics of the study population. Neuroepidemiology 2003. 22, 316-325.

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Alzheimer’s disease: report of the NINCDS-ADRDA work group under the auspices of

department of Health and Human services task force on Alzheimer’s disease. Neurology

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4 Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML et al. Genome-wide

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6 Brouwers N, Van Cauwenberghe C, Engelborghs S, Lambert JC, Bettens K, Bastard NL et al.

Alzheimer risk associated with a copy number variation in the complement receptor 1

increasing C3b/C4b binding sites. Mol Psychiatry 2012. 17, 223-33.

7 Canales, R.D. et al. Evaluation of DNA microarray results with quantitative gene expression

platforms. Nat. Biotechnol., 2006. 24, 1115-1122.

8 Chapuis, J. et al. Transcriptomic and genetic studies identify IL-33 as a candidate gene for

Alzheimer's disease. Mol Psychiatry 2009. 14, 1004-1016.

9 Hansmannel F, et al. Is the urea cycle involved in Alzheimer’s disease? J of Alzheimers dis

2010. 21, 1013-21.

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10 Cieniewski-Bernard C, Mulder P, Henry JP, Drobecq H, Dubois E, Pottiez G, Thuillez C,

Amouyel P, Richard V, Pinet F. Proteomic analysis of left ventricular remodeling in an

experimental model of heart failure. J Proteome Res 2008. 7, 5004-16.

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Genetic and Environmental Risk for Alzheimer's disease Consortium (GERAD1) Collaborators

Richard Abraham1, Paul Hollingworth1, Giancarlo Russo1, Marian Hamshere1, Jaspreet Singh Pahwa1,

Valentina Moskvina1, Kimberley Dowzell1, Amy Williams1, Nicola Jones1, Charlene Thomas1, Alexandra

Stretton1, Angharad Morgan1, Simon Lovestone2, John Powell2, Petroula Proitsi2, Michelle K Lupton2,

Carol Brayne3, David C. Rubinsztein4, Michael Gill5, Brian Lawlor5, Aoibhinn Lynch5, Kevin Morgan6,

Kristelle Brown6, Peter Passmore7, David Craig7, Bernadette McGuinness7, Stephen Todd7, Janet

Johnston7, Clive Holmes8, David Mann9, A. David Smith10, Seth Love11, Patrick G. Kehoe11, John Hardy12,

Simon Mead13, Nick Fox14, Martin Rossor14, John Collinge13, Wolfgang Maier15, Frank Jessen15, Reiner

Heun15, Heike Kölsch15, Britta Schürmann15, Hendrik van den Bussche16, Isabella Heuser17, Johannes

Kornhuber18, Jens Wiltfang19, Martin Dichgans20,21, Lutz Frölich22, Harald Hampel23,24, Michael Hüll25,

Dan Rujescu25, Alison Goate26, John S.K. Kauwe27, Carlos Cruchaga26, Petra Nowotny26, John C.

Morris26, Kevin Mayo26, Gill Livingston31, Nicholas J. Bass31, Hugh Gurling31, Andrew McQuillin31, Rhian

Gwilliam32, Panagiotis Deloukas32, Ammar Al-Chalabi33, Christopher E. Shaw33, Andrew B. Singleton34,

Rita Guerreiro34, Thomas W. Mühleisen35,36, Markus M. Nöthen35,36, Susanne Moebus37, Karl-Heinz

Jöckel37, Minerva M. Carrasquillo38, V. Shane Pankratz39, Steven G. Younkin40, Peter Holmans1

1 Medical Research Council (MRC) Centre for Neuropsychiatric Genetics and Genomics,

Neurosciences and Mental Health Research Institute, Department of Psychological Medicine and

Neurology, School of Medicine, Cardiff University, Cardiff, UK.

2 Department of Neuroscience, Institute of Psychiatry, Kings College, London, UK.

3 Institute of Public Health, University of Cambridge, Cambridge, UK.

4 Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

5 Mercer's Institute for Research on Aging, St. James Hospital and Trinity College, Dublin, Ireland.

6 Institute of Genetics, Queen's Medical Centre, University of Nottingham, NG7 2UH, UK.

7 Ageing Group, Centre for Public Health, School of Medicine, Dentistry and Biomedical Sciences,

Queen's University Belfast, UK.

8 Division of Clinical Neurosciences, School of Medicine, University of Southampton,

Southampton, UK.

15

9 Clinical Neuroscience Research Group, Greater Manchester Neurosciences Centre, University of

Manchester, Salford, UK.

10 Oxford Project to Investigate Memory and Ageing (OPTIMA), University of Oxford, Level 4, John

Radcliffe Hospital, Oxford OX3 9DU, UK.

11 Dementia Research Group, University of Bristol Institute of Clinical Neurosciences, Frenchay

Hospital, Bristol, UK.

12 Department of Molecular Neuroscience and Reta Lilla Weston Laboratories, Institute of

Neurology, London, UK.

13 MRC Prion Unit, Department of Neurodegenerative Disease, UCL Institute of Neurology, London,

UK.

14 Dementia Research Centre, Department of Neurodegenerative Diseases, University College

London, Institute of Neurology, London, UK.

15 Department of Psychiatry, University of Bonn, Sigmund-Freud-Straβe 25, 53105 Bonn, Germany.

16 Institute of Primary Medical Care, University Medical Center Hamburg-Eppendorf, Germany.

17 Department of Psychiatry, Charité Berlin, Germany.

18 Department of Psychiatry, University of Erlangen, Nürnberg, Germany.

19 LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, University Duisburg-Essen,

Germany.

20 Institute for Stroke and Dementia Reserach, Klinikum der Universität München, Marchioninistr.

15, 81377, Munich, Germany.

21 Department of Neurology, Klinikum der Universität München, Marchioninistr. 15, 81377,

Munich, Germany.

22 Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg,

Germany.

23 Discipline of Psychiatry, School of Medicine and Trinity College Institute of Neuroscience,

Laboratory of Neuroimaging & Biomarker Research, Trinity College, University of Dublin, Ireland.

24 Alzheimer Memorial Center and Geriatric Psychiatry Branch, Department of Psychiatry, Ludwig-

Maximilian University, Munich, Germany.

16

25 Centre for Geriatric Medicine and Section of Gerontopsychiatry and Neuropsychology,

University of Freiburg, Germany.

26 Departments of Psychiatry, Neurology and Genetics, Washington University School of Medicine,

St Louis, MO 63110, US.

27 Department of Biology, Brigham Young University, Provo, UT, 84602, USA.

28 Neurodegenerative Brain Diseases group, Department of Molecular Genetics, VIB, Antwerpen,

Belgium.

29 Institute Born-Bunge and University of Antwerp; Antwerpen, Belgium.

30 Memory Clinic and Department of Neurology, ZNA Middelheim, Antwerpen, Belgium.

31 Department of Mental Health Sciences, University College London, UK.

32 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge,

UK.

33 MRC Centre for Neurodegeneration Research, Department of Clinical Neuroscience, King’s

College London, Institute of Psychiatry, London, SE5 8AF, UK.

34 Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health,

Bethesda, MD, 20892, USA.

35 Department of Genomics, Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25, D-

53127 Bonn, Germany.

36 Institute of Human Genetics, University of Bonn, Wilhelmstr. 31, D-53111 Bonn, Germany.

37 Institute for Medical Informatics, Biometry and Epidemiology, University Hospital of Essen,

University Duisburg-Essen, Hufelandstr. 55, D-45147 Essen, Germany.

38 Klinikum Grosshadern, Munich, Germany.

39 Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida 32224, USA.

40 Division of Biomedical Statistics and Informatics, Mayo Clinic and Mayo Foundation, Rochester,

Minnesota 55905, USA.

17