Cardiff Neurosciences Centre(CNC)

Alzheimer’s Disease Network

Cardiff Neurosciences Centre Manager www.cardiff.ac.uk/cnc

Dr Vanessa Davies cnc@cardiff.ac.uk

Aims of AD Network

• Scope research interests

• Facilitate communication between research groups: meetings, website

• Establish strengths, weaknesses and obstacles to success

• Act strategically

AD Research NetworkThemes

• Complex Genetics• Nutrition• Neuropsychology and Imaging• Epidemiology: Caerphilly Cohort• Clinical Trials• Animal Models• Stem Cells• Therapeutic Antibodies

Dementia

• 700,000 people have dementia in the UK• 1 million by 2025• 1.7 million by 2050 - 150% up

• Dementia: main cause of burden in later life• Dementia - 11% of years with disability• Cardiovascular disease 5.4%• Cancer 2.4%

Large Scale Genetic Association Studies

in Alzheimer’s Disease

Julie Williams

Why find genes

• Alzheimer’s disease is heritable- 79%

• Pinpoint biological pathways contributing to disease development

• Test for gene x environment relationships

• Use knowledge to develop new treatments, preventative therapies

• To predict disease susceptibility

Alzheimer’s Disease:Causal and Susceptibility Genes

APOE APPPS1

PS2

PS1: > 150PS2: < 20APP: < 20

APOE

• Acts to reduce age at which disease starts

• Effect observed from 40 diminishes after 70

• Neither necessary nor sufficient to cause AD

• Association stronger in Japanese, weaker in African American & Hispanic samples

• Stronger in females• Accounts for 15% genetic

variance

E3 E3

E3 E2

E4 E4

Reduced risk of AD

Increased risk of AD

Large Scale Genetic Association Studies in Alzheimer’s Disease

↑ Markers tested = ↓ Power

↑ Power = ↑ Sample size

Disease susceptibility

allele

MRC Genetic Resource for Late Onset Alzheimer’s Disease

▲

• 1400 AD cases• 1400 matched controls• Validated case

interview- 92%ppv• Controls screened• Longitudinal follow-up:

600• Breadth phenotypic

data: AAO, ROD, symptoms

• MRI imaging: 150• Brain banking

Identifying Alzheimer’s Disease Susceptibility Genes Through

Genetic Association

17353 markers 1800 AD 2000 controls

Hum. Mol. Genet., 2007

Expected

Accession No. Gene Symbol Allelic p-value Odds Ratio (95% CI) number of falseUK1 WU SD UK2 UK3 Full Sample Full Sample positive

1 rs157581** TOMM40 6.86E-14 3.38E-14 5.1836E-22 2.9394E-15 4.8225E-24# <1.0E-8 2.73 (2.46-3.05) <1.0E-82 rs405509** APOE 0.000081 0.0035 0.00010 0.000300 0.002# <1.0E-8 1.43 (1.3-1.57) <1.0E-83 rs3745833 GALP 0.008 0.036 0.512 0.261 0.061# 0.000050 1.2 (1.09-1.32) 0.874 rs1554948 TNK1 0.130 0.0090 0.699*** 0.054 0.021# 0.000063 1.19 (1.08-1.3) 1.105 rs1132899** APOC2 0.137 0.0075 0.056 0.013 0.975*** 0.000076 1.19 (1.08-1.3) 1.326 rs11622883 0.144 0.010 0.979 0.033 0.091# 0.000094 1.19 (1.08-1.3) 1.637 rs8192708 PCK1 0.0033 0.026 0.396 0.791*** 0.051# 0.000099 1.29 (1.12-1.49) 1.728 rs505058 LMNA 0.0119 0.150 0.357 0.171 0.261 0.00020 1.35 (1.13-1.63) 3.419 rs3800324 PGBD1 0.204 0.026 0.502 0.317 0.146 0.00032 1.43 (1.13-1.8) 5.59

10 rs6907175 LOC651924 0.017 0.304 0.042 0.957 0.00047 1.16 (1.04-1.3) 8.1511 rs1859849 0.0047 0.168 0.776 0.031 0.575*** 0.00058 1.15 (1.03-1.28) 10.1012 hCV22274641* THEM5 0.000031 0.109 0.647*** 0.169 0.467*** 0.00061 1.24 (1.05-1.47)## 10.5813 rs2074877 MYH13 0.017 0.029 0.664 0.931*** 0.742 0.00072 1.12 (1.02-1.23) 12.4414 hCV15746640* CTSS 0.0008 0.031 0.595*** 0.116 0.293*** 0.00078 1.21 (1.02-1.42)## 13.4615 rs444013 UBD 0.010 0.042 0.299 0.741*** 0.845*** 0.00079 1.11 (1.01-1.22) 13.7716 rs9608099 BCR 0.261 0.0018 0.651*** 0.134 0.197*** 0.00097 1.09 (0.99-1.21)## 16.8117 rs2882676 AGC1 0.076 0.033 0.358 0.375 0.050*** 0.0010 1.07 (0.97-1.18)## 17.2218 rs13022344 TRAK2 0.0051 0.0077 0.821*** 0.628 0.015*** 0.0010 1.07 (0.97-1.18)## 17.3519 rs11016976 EBF3 0.050 0.024 0.294 0.966 0.033*** 0.0010 1.08 (0.95-1.24)## 17.47

*: denotes SNPs that map to reported AD linkage regions**: SNPs that are in LD with APOE 2/3/4***: opposite allele associated, compared with UK1 and WU#: Evidence of replication in UK3 (p<0.05, 1-tailed)

Genes showing most evidence of association with Alzheimer’s disease

Galanin-like Peptide Precursor-GALP,Tyrosine Kinase, Non-

receptor – TNK1, Phosphoenolpyruvate Carboxykinase

1- PCK1

• Galanin modulates cholinergic pathways & effects TAU phosphorylation-tangles

• TNK1- protein trafficking & signal transduction

• PCK1-risk factor for Type 2 Diabetes

Possible AD Susceptibility Genes

• ALZGENE (Bertram et al, NAT. GENET., 39, 2007)

• ACE, GAPDHS, CHRNB2, CST3,ESR1, IDE, MTHFR, NCSTN, PRNP, PSEN1, TF, TFAM and TNF

• DAPK1 (Li et al, Hum. Mol. Genet., 17, 2006),

RPS3A/SORBS1 (Grupe et al, 78, 2006),

NEDD9 (Hum.Mol.Genet.,17, 2008)0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

Cambridge Cardiff London St Louis San Diego

Frequency of Risk allele of NEDD9 (neural precursor cell expressed, developmenatlly downregulated) in

Late Onset Alzheimer's Disease

Controls

Cases

Joint analysis of Alzheimer's Disease samples (N=3569): OR = 1.22, p-value = 0.00004Joint analysis of Parkinson's Disease samples (N=1464): OR = 1.26, p-value = 0.0032

Combined Dementia analysis (N=5043): OR = 1.24, p-value = 1.54 x 10 -7

131 new susceptibility variants for complex traits identified in the last year

Current Genome Wide Association Studies (GWAS) of AD

• Coon et al, Reiman et al (2007): GAB2 (APOE e4+)

• Li et al (2007): GOLPH2• Beecham et al (2007): AMIGO2 & SENP1• All find association with APOE locus• No genome wide significant gene• All AD case samples between 500-750

(discovery phase)

Pooled GWA Study of Late-onset Alzheimer’s Disease

Bonferroni corrected p-value 9.1 x 10-8

DNA Pools 1,082 LOAD cases and 1,239 controls (x replicates)

Illumina Beadchip 550,000 SNPs genotyped

109 top hits genotyped individually (+70% sig p<0.05)

Funded by The Alzheimer’s Research Trust

rs chrom PositionGene

SymbolMinor Allele

Minor Allele Freq

Cases

Minor Allele Freq

Controls

Minor Allele Freq 1958

p Cases vs MRC

Cons

p Cases vs CombCons

rs8106922α 19 50093506 TOMM40 G 0.30 0.40 0.40 1.21E-10 3.96E-14rs727153* 4 156012026 intergenic C 0.52 0.46 0.47 0.00002 3.37E-06rs3754675 2 101008361 NPAS2 C 0.06 0.10 0.06 0.00003 0.0144rs4699852 4 95699967 intergenic A 0.43 0.49 0.45 0.00004 0.0032rs2905990 5 11160513 CTNND2 T 0.30 0.36 0.33 0.00009 0.0014rs9600764 13 76208794 intergenic G 0.03 0.05 0.04 0.00019 0.0027rs1032412 2 163464614 KCNH7 G 0.27 0.33 0.29 0.00019 0.0097

rs12510838 4 73326573 NPFFR2 G 0.17 0.22 0.19 0.00024 0.0047rs7140253 14 106305044 intergenic G 0.10 0.14 0.13 0.00024 0.0007rs1455280 4 61600197 intergenic G 0.10 0.14 0.12 0.00027 0.0026

rs13397414 2 154056922 intergenic G 0.11 0.14 0.13 0.00028 0.0003rs587259 19 39348246 LSM14A T 0.40 0.34 0.36 0.00028 0.0009

rs13115107 4 183493069 intergenic G 0.31 0.26 0.28 0.00032 0.0005rs1373900 2 193561888 intergenic T 0.33 0.38 0.35 0.00037 0.0087rs1911014 4 127131703 intergenic A 0.12 0.16 0.15 0.00037 0.0003

rs17228994 5 14095376 intergenic C 0.15 0.12 0.13 0.00060 0.0025rs407146 16 13223156 LOC729993 T 0.42 0.36 0.40 0.00060 0.0137

rs7937776 11 94336790 HSPC148 C 0.20 0.16 0.17 0.00067 0.0007rs3819902 21 42973084 PDE9A C 0.09 0.12 0.11 0.00068 0.0004

rs16916856 8 52901703 PCMTD1 C 0.11 0.15 0.13 0.00069 0.0036rs393195 19 49184982 ZNF155 G 0.24 0.29 0.25 0.00071 0.0147

rs12146414 10 17108257 CUBN G 0.07 0.09 0.08 0.00086 0.0067rs7798395 7 70326814 WBSCR17 C 0.08 0.05 0.06 0.00090 0.0011rs741477* 2 64977962 intergenic G 0.10 0.13 0.14 0.00124 3.05E-05

rs1016138* 12 114328023 intergenic C 0.10 0.13 0.12 0.00176 0.0009rs10406335* 19 49325793 ZNF225 G 0.10 0.12 0.12 0.00390 0.0009rs9324088* 14 106041164 intergenic G 0.25 0.28 0.30 0.01295 0.0002

POOLED GWAS – INDIVIDUAL GENOTYPING RESULTS

Pooled GWAS – Lecithin Retinol Acyltransferase (LRAT) LD Structure

* *

POOLED GWAS – Additional genotyping of Lecithin Retinol Acyltransferase (LRAT) TAG

SNPsrs Position Gene Symbol Possible Functional Effects

Minor Allele

Minor Allele Freq

Cases

Minor Allele Freq

Controls

Minor Allele

Freq 1958

p Cases vs MRC

Cons

p Cases vs CombCons

rs6536024 155,900,974 Q6ZUB9_HUMAN Promoter/regulatory region T 0.46 0.44 0.44490

rs727153* 156,012,026 intergenic C 0.52 0.46 0.47 0.00002 3.37E-06

rs12501328 156,019,936 LRAT Intronic G 0.10 0.08 0.07 0.02661 0.0032

rs201825 156,024,540 LRAT Promoter/regulatory region C 0.52 0.44 0.46 1.65E-06 6.12E-07

rs201824 156,026,315 LRAT Intronic T 0.40 0.35 0.00066

rs201823 156,026,490 LRAT Intronic G 0.40 0.35 0.37 0.00042 0.0004

rs156500 156,034,920 LRAT Intronic C 0.13 0.11 0.09457

rs156501 156,035,950 LRAT Intronic A 0.22 0.19 0.19 0.01635 0.0058

rs149225 156,040,821 LRAT transcription factor bind site C 0.42 0.40 0.34900

Mutations in LRAT cause severe early-onset retinal dystrophy

Plays prominent role in Vitamin A (retinoid) cascade

Retinoid levels in plasma, serum and brain lowered in LOAD

*

*

Powerful Genome-wide Association Screen For AD Susceptibility Genes

610,000 markers Stage 1: 4,283 AD & 8995 CON; Top Hits: 2278 AD & 3,797 CON

19,000+ Subjects

Cases ControlsStage 1 - Discovery SampleMRC

Cardiff University 549 344Institute of Psychiatry 361 262Cambridge University 348 387Trinity College, Dublin 137 0

ARTUniversity of Nottingham 118 3University of Manchester 219 0University of Bristol 280 50University of Southampton 132 0Queen University, Belfast 779 99

OPTIMA Sample, Oxford University 249 0John Hardy, University College London 354 0LASER-AD, University College London 95 0Washington University, St Loius, US 500 500NIMH Cases Linked to Chromosome 10 162 0

1958 Controls (1M Chip) 0 3000Blood Transfusion Controls (1M Chip) 0 3000ALS Controls, (250UK 1100 US, Aged) 0 1350

TOTAL Samples 4283 8995Stage 2 - Samples Available for Meta analysisMayo Clinics Jacksonville/Rocester 800 1200GSK Canadian Sample 753 736Hardy, Reiman Genome Wide Association Study 664 442Additional 'In house' samples for gentoypying 61 1419

TOTAL Samples 2278 3797

Total Samples = 13278

Total Samples = 6075

Genome Wide Association Study of Alzheimer’s Disease

Secondary Analysis• APOE, age at onset, rate of decline• Relationship with symptoms: psychosis, depression, • Copy number variation• TOP hits: functional analysis - animal models• Biological pathways

• Meta-analyses (+ imputation): other AD GWAS GWAS of neurodegenerative disorders GWAS vascular diseases GWAS of psychosis, depression

AD Pathways

• Amyloid β clearance

• Oxidative stress

• Tau

• Insulin signalling

• Apoptosis

• Cholesterol

• 1178 AD pathway genes included to date

Conclusions

• AD susceptibility & disease modifying genes remain to be identified.

• Powerful GWA studies are likely to succeed in discovering new susceptibility genes.

• New AD genes on the horizon!

School of Medicine, Cardiff UniversityJulie Williams Michael J. OwenMichael O’DonovanRichard AbrahamPaul Hollingworth Peter HolmansAngharad MorganRebecca SimmsFunded by Medical Research CouncilAlzheimer’s Research Trust, Wellcome Trust, Alzheimer’s Society &Welsh Assembly Government.

Cardiff AD Genetics Group

Valentina Moskvina Marian HamshereIvan NikolovLeslie JonesKimberley DowzellGeorge Kirov Alexandra StrettonRicardo SeguradoGary Hill

williamsj@cardiff.ac.uk

Cardiff UniversityJulie Williams, Mike OwenRichard Abraham, Paul Hollingworth, Angharad Morgan, Peter Holmans, Mick O’Donovan

Institute of Psychiatry, LondonSimon Lovestone

Cambridge UniversityCarol Brayne, David Rubinsztein

Trinity College, Dublin Brian Lawlor, Mike Gill

University of Nottingham Kevin Morgan

University of Manchester David Mann

University of Southampton Clive Holmes

University of Bristol Pat Kehoe

Queen University, Belfast Peter Passmore David Craig

OPTIMA Study, Oxford University David Smith Donald Lehhman

LASER- AD, University College London Livingston, Hugh Gurling,Katona and Nick Bass

University College LondonJohn Hardy, Martin Rosser, Nick Fox, Simon Mead

Washington University, St Louis, US Alison Goate

Mayo Clinic, Florida, US Steve Younkin

NIH, Bethesda, Washington, US

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1. Mutation detection should be concentrated in clinical genetics laboratories offering a national service.

2. Suitable candidates for mutation detection would need to meet the minimum criteria of:i) Positive family history (more than two affected members in two generations)ii) Diagnosed Alzheimer’s diseaseiii) Family counselling regarding the implications of mutation screening, with particular emphasis being given to the fact that in some families mutations may not be foundiv) Appropriate consent and assent

3. Genetic testing for prediction should always follow the detection of a mutation in an affected family member and accord with the guidelines established for Huntington’s disease (Simpson and Harding, 1993).

International Journal of Geriatric Psychiatry14, 789-791 (1999)

Genetic Testing

Alzheimer’s Disease

• Common: 62% of dementia

1% <65yrs, 40% >90yrs

• Episodic memory loss followed by gradual cognitive decline

• Key biological markers: plaques & tangles

• Genetically complex: many genes involved

RPS3A Homologue, SORBS1 & DAPK

• RPS3A hom- function unknown

• RPS3A acts with Bcl-2 to prevent apoptosis

• SORBS1 involved in insulin signalling & upregulated in hippocampus AD brains

• DAPK affects apoptosis

Glyceraldehyde-3-phosphate dehydrogenase: GAPD

• Key enzyme in cellular energy processes• Neuronal apoptosis• Binds APP & Aβ• GAPD mediated apoptosis inhibited by tacrine &

donepezil• GAPD activity higher in AD brains and over-

expressed in Down’s syndrome brains

APOE 112 158

ApoE-2 GAC GTG TGC TGC CGC CTG……….CTG CAG AAG TGC CTG GCA

ApoE-3 GAC GTG TGC TGC CGC CTG……….CTG CAG AAG CGC CTG GCA

ApoE-4 GAC GTG CGC CGC CGC CTG……….CTG CAG AAG CGC CTG GCA

-491 A/T -219 G/T-419 T/C +1 +113 C/G

1 2 3 4

DRE

EXON1

N.S.E

URE 1

TATA BOX

GC BOX

URE 3

+300

Effects of ApoE4 Generated by CNS Astrocytes and Neurons

Cellular origin of

apoE4

Effects of apoE4 References

Astrocyte

Neuron

+Aß production

+Aß deposition

-Aß clearance

-Neurite outgrowth

+Cognitive decline

+Fragmentation-related neurotoxicity

-Neurite outgrowth

+Tau phosphorylation & aggregation

Aß Apoptosis: lysosomal leakage

+Neurodegeneration

Androgen receptor deficiency

+Cognitive decline

Vincent & Smith (2001)

Bales et al (1999)

Holtzman et al (2000a)

Irizarry et al (2000)

Ji et al (2001)

LaDu et al (1994)

Holtzman et al (2000a)

Holtzman et al (1995)

DeMattos et al (1998)

Sun et al (1998)

Hartman et al (2001)

Huang et al (2001)

Harris et al (2003)

Bellosta et al (1995)

Tesseur et al (2000a, 2000b)

Brecht et al (2004)

Ji et al (2002)

Buttini et al (1999, 2002)

Raber et al (2002)

Raber et al (1998, 2000, 2002)

Huang et al, 2004

APOE

• APOE may affect one or a number of processes implicated in late-onset AD pathogenesis:

• Aβ processing

• Tangle formation

• Neurite outgrowth & branching

• Apoptosis