[PPT] - Loosening the G Loosening the G Gordian Knot: Gordian Knot: PowerPoint Presentation

SLIDE 1

Loosening the G Loosening the G Unraveling Alzheimer D Finding The Finding The Targets Gene Gene Approa

Lindsay A. y

Division of Bio Boston University No Di

Gordian Knot: Gordian Knot: Disease Biology and erapeutic erapeutic Using etic etic aches

Farrer, Ph.D. ,

medical Genetics

y School of Medicine

1

y isclosures

SLIDE 2

ALZHEIMER ALZHEIMER

 Progressive loss of me  Onset in most cases aft  Onset in most cases aft but can occur as early  No effective treatment

R DISEASE R DISEASE

emory and cognition fter age 65 years fter age 65 years, as age 30 t or cure

SLIDE 3

DIAGNO ALZHEIMER

 Pathologic confirmatio  Neuropsychological an  Rule out other organic i f l  Rule out other organic  Progression for at least

OSIS OF R DISEASE

n (“Gold Standard”)

nd brain imaging tests causes (e g stroke) causes (e.g., stroke) t one year

SLIDE 4

NEUROPATH ALZHEIMER

 $-amyloid deposition

L parenchymal seni L cerebral blood ve

 Neurofibrillary tangle y g cerebral cortex and

HOLOGY OF R DISEASE

n in ile plaques essel walls es in neurons of d hippocampus

SLIDE 5

Neuropathology of Neuropathology of Alzheimer Disease Alzheimer Disease

SLIDE 6

Alzheimer Disease is a

 Affects 13% of people  7th leading cause of de  More than 5.4 million  AD patients fill more t home beds and consum billion per year in hea

Public Health Menace

e >age 65; 43% ages 85+ eath in US (5th among >65) Americans afflicted than 50% of all nursing me an estimated $172 alth care resources

SLIDE 7

Established Gene Loci for Alzheimer Disease

Deterministic Mutations: Amyloid Precurser Protein (APP) Presenilin-1 (PS-1) Presenilin 2 (PS 2) Presenilin-2 (PS-2) Susceptibility Polymorphism: Susceptibility Polymorphism: Apolipoprotein E (APOE)

SLIDE 8

SLIDE 9

Sisodia et al 2001; 24(11):S2-6

SLIDE 10

Gene Defects C D i t Al Dominant Alz

Numbe Gene Chromosome

f Mutat

Gene Chromosome

f Mutat

A 21 33 APP 21 33 PS1 14 185 PS2 1 13 ? ? ? * These effects account for <<1%

Causing Autosomal h i Di zheimer Disease

er Onset Age Relative tions (Range) Frequency* tions (Range) Frequency* 3 6 % 37 – 65 5% 29 – 60 70% 40 – 82 5% – 10% ? 15% – 20%

f all AD cases

SLIDE 11

Established Gene Loci for Alzheimer Disease

Deterministic Mutations: Amyloid Precurser Protein (APP) Presenilin-1 (PS-1) Presenilin 2 (PS 2) Presenilin-2 (PS-2) Susceptibility Polymorphism: Susceptibility Polymorphism: Apolipoprotein E (APOE)

SLIDE 12

APOLIPOPROTE

 Plasma protein involved in choles p  Produced in liver and in astrocyte system  Encoded by gene on chromosome  3 alleles (2, 3, and 4) resulting  2 4 associated with hyperlipide positions 112 or 158  2, 4 associated with hyperlipide and hypertriglyceridemia (decreas

EIN E (APOE)

sterol transport p es in central and peripheral nervous e 19 g from amino acid substitutions at emia type III hyperlipoproteinemia emia, type III hyperlipoproteinemia sed binding to lipoprotein receptors)

SLIDE 13

Myers et al, Neurology 1996; 46:673-677 Myers et al, Neurology 1996; 46:673 677

SLIDE 14

Odds of Alzheim APOE and Age g mer Disease by in Caucasians

Farrer et al. JAMA 1997; 278:1349-1356

SLIDE 15

Relative Odds of Alzheim Genotypes, Age and yp , g mer’s Disease by APOE d Sex in Caucasians

Farrer et al. JAMA 1997; 278:1349-1356

SLIDE 16

Association Studies (1995 (1995

B l 2007 968 i ti

By early 2007, 968 association

genes reported on AlzGene (http://www.alzforum.org/res/

None other than APOE with ro
Reasons include:
Initial results false positive
Lack of power in replication s
Lack of power in replication s
Locus heterogeneity
Clinical heterogeneity
Lack of informative markers
Intralocus (non-allelic) hetero

in Alzheimer Disease 2006)

2006)

t di i 398 did t n studies in 398 candidate /com/gen/alzgene

bust confirmation

studies (false negatives) studies (false negatives) geneity

SLIDE 17

Mutations Causing Alz mis-process mis-process

PS1/PS2 mutations APP mutations -secretase -secretase

Extracellular TM domain

  

t

Intracellular

APP -secretase

zheimer Disease cause sing of APP sing of APP

Citron et al. Nature Med. 3: 67-72, 1997 A APOE 4

AICD

↓ Uptake, chaperoning, &

degradation of Aβ

X

AICD

(?Signalling)

Aaggregates into neurotoxic

ligomers

protofibrils A accumulates

SLIDE 18

APP is cleaved at s subcellular traf

ADAM10/17 PPsα APP APP APP

Golgi

APP APP

Endoplasmic Reticulum

sites which require fficking of APP

BACE1

g

BACE1 PS1 γ-sec

Cell surface

PS1 γ-sec APP BACE1 BACE1 APP

Endosome

APP BACE1 APP BACE1 BACE1 APP AICD APPsβ

Recycling Endosome

RC

Late Endosome

Aβ PS1 γ-sec

SLIDE 19

Generation of Aβ req selected subcellul

ADAM10/17 APPsα Cell Surface APP APP-CTFα APP CTFα

Sorting switch

ER- Golgi Secretory Pathway

quires trafficking into lar compartments

BACE1 APPsβ Late endosomal pathways APP-CTFβ AICD Aβ PS1 APP

Recycling

RC APP VPS26 VPS35

Recycling Endosomes

VPS35

Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 20

Study D Study D

Retromer Retromer complex: complex: VPS26 (10q21) VPS26 (10q21)

Candidate G Retromer

VPS35 (16q12). VPS35 (16q12). VPS10 VPS10-

containing

containing sorting receptors: sorting receptors:

Gene(s) associ

SORT1 (1p21 SORT1 (1p21-

p13)

p13) SORCS1 (10q23 SORCS1 (10q23-

q25)

q25) SORCS3 (10q23 SORCS3 (10q23-

q25)

q25) SORCS2 (4p16) SORCS2 (4p16)

More S

SORL1 (11q23 SORL1 (11q23-

q24)

q24)

Functional Assays

3 independent cohorts: Mayo Clinic Mayo Clinic

Design Design

Genes In Pathway

 2 SNPs/gene in 2 family cohorts: N th E 124 North European: 124 Hispanic: 228

iated with AD SNPs Replicate in independent datasets

4 independent cohorts: North European case-controls: 178/142 MIRAGE Caucasian: 276 MIRAGE African American: 238 Israeli-Arab: 111/114

SLIDE 21

1 250 500 750 1000

VPS10 YWT D EG F

rs12364988

7

rs668387

8 9

rs4935775

11 12

rs11600231

14

r

1

rs4935774

2

rs578506

3

rs661057

4

rs11218304

5

rs560573

6

rs689021 rs641120

10

rs12285364

12

rs2298813

13 3

rs582446 5’

L O C283155

SO RL 1

(177.43 Kb) SC5DL

LOC390256

120.68 120.84 121.00

Amino Acid #s 1250 1500 1750 2000 2214 Domains SNPS

F N3 T M L DL a

20

rs2276346

15

T833T

16 17

2070045

19

rs3824966

20

18 Ex26

21 22

rs2282649

24

rs1784933

26 27 25

rs1010159 rs556349

17

rs11218340

18

rs1699102 rs3824968

23

rs1614735 rs1133174

28

rs1131497

29

3’

L O C399959 121.48 121.16 121.32

Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 22

= Hispanic = Arab Ethnic

rigin

Carib Hisp FAD ** Israeli Arab NE FAD ** NE spAD # AD 605 111 321 178 samples Haplo p- value 0.005 0.0085 0.005 0.045/ 0.005

C G C C G C C G C

8 9 10

C C C

10 12

C T T C T T T T

22 23 24

T T T C

24 25 = Caucasian = Afro-American

MIRAGE Caucasian MIRAGE African American Mayo Jack Mayo Roch Mayo Aut All Cauc 279 244 549 433 423 1583

0.0025

<0.003

0.003

<0.02

T A C C T T T T C T T T T C T T T C TT C

Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 23

1 250 500 750 1000

VPS10 YWT D EG F

rs12364988

7

rs668387

8 9

rs4935775

11 12

rs11600231

14

r

1

rs4935774

2

rs578506

3

rs661057

4

rs11218304

5

rs560573

6

rs689021 rs641120

10

rs12285364

12

rs2298813

13 3

rs582446 5’

Identical alleles are associated with AD in Israeli Arabs, Hispanics and

L O C283155

SO RL 1

Israeli Arabs, Hispanics and some European Caucasians

(177.43 Kb) SC5DL

LOC390256

120.68 120.84 121.00

Amino Acid #s 1250 1500 1750 2000 2214 Domains SNPS

F N3 T M L DL a

20

rs2276346

15

T833T

16 17

2070045

19

rs3824966

20

18 Ex26

21 22

rs2282649

24

rs1784933

26 27 25

rs1010159 rs556349

17

rs11218340

18

rs1699102 rs3824968

23

rs1614735 rs1133174

28

rs1131497

29

3’

Identical alleles associated with AD in European Caucasians; Different haplotype associated

L O C399959

Different haplotype associated with AD in African Americans

121.48 121.16 121.32

Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 24

SORL1 is reduced specifically in

Control AD AD

neocortex neocortical (bar =100μm) pyramidal neuron

Sc

(bar =10μm)

cortical neurons in late onset AD

neocortical dendate astrocyte granule (bar =10μm) neuron

cherzer, C. R. et al. Arch Neurol 61, 1200-1205, 2004.

( μ ) (bar =10μm)

SLIDE 25

How are sequence f i ll functionally asso

Do not affect coding sequence or splicing

g q p g

Intronic variants may affect tissue-specifi

regulation of transcription

CTT22-24 haplotype associated with reduce

transcription in lymphoblasts (not very robust); );

Genotype accounts for 14% of variance in

expression level;

Corollary: modifiers of SORL1

expression could be other causes of AD o potential therapies. p p

variants in SORL1 i d i h A ?

ciated with AD?

g; g; ic NA

20,000

p < 0.05

ed RL1 mRN

,

n SOR

10,000

r

Non- carrier Risk Allele carrier carrier (n=8) carrier (n=8)

Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 26

Suppressing SORL1 gene does no but increases BACE and γ (more Aβ pepti

#1 #2 222 222 318 318 806 806

siRNA

SORL1 siRNA

(more Aβ pepti

Con Con LR12 LR12 LR13 LR13 LR58 LR58 SORL1

ligo

APP  APP APPs APPs APP-CT PS1-NT APPs

No change

X

E

X

RE

LE

t alter expression of APP or PS1
secretase cleavage of APP

ide and APPsβ)

6000 8000 10000

pg/ml)

*** *** **

ide and APPsβ)

2000 4000 Con LR1222 LR1318 LR5806

A (p

1000 1500

 (pg/ml)

*** ** **

500 Con LR1222 LR1318 LR5806

A F TF

1.0 1.5 2.0

PPsβ

*** *** ***

0.0 0.5 Con LR1222 LR1318 LR5806

. AP

SLIDE 27

SORL1 is a sorting

SORL1 dependent swit

SORL1 is a sorting

APP ADAM17 APPsα SORL1-dependent swit APP-CTFα SORL1 ER ER- Golgi Secretory Pathway

g receptor for APP

tch

g receptor for APP

BACE1 APPsβ tch Late endosomal pathways APP-CTFβ AICD Aβ PS1 APP

Recycling

RC VPS26 VPS35 SORL1

y g Endosomes Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 28

SORL1 is a sorting

SORL1 dependent swit

SORL1 is a sorting

APP ADAM17 APPsα SORL1-dependent swit APP-CTFα SORL1 ER ER- Golgi Secretory Pathway

g receptor for APP

tch

g receptor for APP

BACE1 tch Late endosomal pathways PS1 APP

Recycling

RC VPS26 VPS35 SORL1

y g Endosomes Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 29

SORL1 is a sorting

SORL1 dependent swit

SORL1 is a sorting

APP ADAM17 APPsα SORL1-dependent swit APP-CTFα SORL1 ER ER- Golgi Secretory Pathway

g receptor for APP

tch

g receptor for APP

BACE1 APPsβ tch Late endosomal pathways APP-CTFβ AICD Aβ PS1

Recycling

RC VPS26 VPS35

y g Endosomes Rogaeva et al, Nature Genetics 39:168-177, 2007

SLIDE 30

Intracellular Trafficking of APP

SLIDE 31

May You Live in In

Since 2005, about 1400 geno studies have identified robust variants for more than 220 co and traits:

10 Mar 2005: Age-related m
30 Apr 2006: QT interval p
19 Oct 2006: “Wet” AMD
26 Oct 2006: Inflammatory
11 Feb 2007: Type 2 diabe
5 Mar 2007: Crohn’s diseas
12 Apr 2007: Obesity

nteresting Times…

me-wide association

t associations with genetic

mmon, complex diseases

macular degeneration rolongation bowel disease etes se

SLIDE 32

Published Genome‐Wide Associations 1,617 published GWA at p≤5X10‐8 NH ww through 09/2011 for 249 traits HGRI GWA Catalog ww.genome.gov/GWAStudies

SLIDE 33

SLIDE 34

“There have been few, if an discovery in the history of m y y f

Hunter DJ and Kraft P, N Engl J Med 20

ny, similar bursts of medical research…”

007; 357:436-439.

SLIDE 35

Unique Aspects

Permits examination of inh

unprecedented level of res P it " ti "

Permits "agnostic" genome
Most robust associations in

been with genes previously been with genes previously related to the disease

Some associations in regio

g harbor genes “The chief strength of the new a f g f problem: with more than 500,00 potential for false positive result

N Engl J Med 2007; 357:436-439.

f GWA Studies

erited genetic variability at

lution

id i ewide comparison n GWA studies have not y suspected of being y suspected of being

ns not even known to

approach also contains its chief pp f 00 comparisons per study, the ts is unprecedented.”

SLIDE 36

Genome Wide As

Source Population Coon et al, 2007 1411 cases and controls Reiman et al, 2007 (autopsy and clinical series Liu et al, 2007 103 LOAD and 170 1st - degree relatives from large Dutch pedigree Grupe et al, 2007 5 case-control samples; total 1808 AD & 2062 contro Li et al, 2008 753 cases, 736 controls (Ca 418 cases, 249 controls (UK

ssociation Studies

Markers Most Significant Examined Results Affy 500K APOE; GAB2 (among s) APOE ε4 carriers) Affy 500K linkage to 1q21-q25 & 10q22-24 (confirm); and 3q23 17,343 cSNPs APOE; GALP, TNK1,

ls

PCK1 n) Affy 500K APOE; GOLPH2, ch. 9 K) ATP8B4/SLC27A2

SLIDE 37

Genome Wide As (conti

Source Population Bertram et al, 2008 410 NIMH Study families Beecham et al, 2009 492 LOAD cases, 498 controls Carrasquillo et al, 2009 844 LOAD cases, 1,255 controls

ssociation Studies nued)

Markers Most Significant Examined Results Affy 500K APOE; intergenic SNP on chr 14q31 Illumina 550K APOE; chr 12q13 SNP in vitamin D receptor Illumina 317K APOE; PCDH11X (Xq21.3)

SLIDE 38

Genome Wide A

University of Miami Hussman Institute for University of Pen School of Medic Hussman Institute for Human Genomics School of Medic

Association Study

nnsylvania ine Boston University School

f Medicine

ine

f Medicine

SLIDE 39

Discovery Meta-Analysis

SLIDE 40

Strongest A

SNP CH:MB Nearest Gene MA MAF # rs6701713 1:207 8 CR1* A 0 20 rs6701713 1:207.8 CR1* A 0.20 rs7561528 2:127.9 BIN1* A 0.35 rs9349407 6 47 5 CD2AP C 0 27 rs9349407 6:47.5 CD2AP C 0.27 rs11767557 7:143.1 EPHA1‡ C 0.19 1532278 8 27 5 CLU* T 0 36 rs1532278 8:27.5 CLU* T 0.36 rs4938933 11:60.0 MS4A4A C 0.39 561655 11 85 8 PICALM* G 0 34 rs561655 11:85.8 PICALM* G 0.34 rs3752246 19:1.1 ABCA7% G 0.19

#

rs3865444 19:51.7 CD33# A 0.30

Associations

# SNPs ADGC Meta‐analysis Orstage 1+2 (95% CI) Pstage 1+2 Orstage 1+2+3 (95% CI) Pstage 1+2+3 7

1.16

4 6×10‐10

ND

ND 7

1.11‐1.22

4.6×10 10

ND

ND 10

1.17 1.13‐1.22

4.2×10‐14

ND

ND 1

1.12

1 0×10‐6

1.11

8 6 10 9 1

1.07‐1.18

1.0×10‐6

1.07‐1.15

8.6x10‐9 1

0.87 0.83‐0.92

2.4×10‐7

0.90 0.86‐0.93

6.0x10‐10 2

0.93

8 3×10 8

ND

ND 2

0.89‐0.97

8.3×10‐8

ND

ND 22

0.88 0.84‐0.92

1.7×10‐9

0.89 0.87‐0.92

8.2x10‐12 36

0.87

7 0 10 11 ND 36

0.84‐0.91

7.0×10‐11

ND

ND 2

1.15 1.09‐1.21

5.8×10‐7

ND

0.89

7

0 91

9

1

0.89 0.86‐0.93

1.1×10‐7

0.91 0.88‐0.93

1.6x10‐9

SLIDE 41

New Gene Loci for

Ge

Ph pro Cl Cl Br

BIN1 CLU

Co

(Ha Se

PICAM CR1

Pa

So

SORL1

co

(Ro

r Alzheimer Disease

enome Wide Association Study:

hosphatidylinositol binding clathrin assembly

tein (PICALM)

usterin (CLU) usterin (CLU) ridging Integrator 1 (BIN1)

mplement component (3b/4b) receptor 1 (CR1)

arold et al. & Lambert et al. Nat Genet 2009; shadri et al. JAMA 2010)

athway Candidate Gene Analysis:

rtilin-related receptor, LDLR class A repeats-
ntaining (SORL1)
gaev et al. Nat Genet 2007)

SLIDE 42

New Gene Loci for

G

C

CD2AP

C M (

MS4A

( E C

CD33 EPHA1

C

r Alzheimer Disease

Genome Wide Association Study:

CD33 antigen (CD33) CD33 antigen (CD33) Membrane-spanning 4-domains, subfamily A (MS4A) ( ) Ephrin type-A receptor 1 (EPHA1) CD2 associated protein (CD2AP) CD2-associated protein (CD2AP)

(Naj et al. Nat Genet 2011)

SLIDE 43

Genetics AD : Whe Genetics AD : Whe

Genomic convergence

Genomic convergence

Compare results from GW sources:

expression arrays
siRNA knockdown expe
proteomic databases
Identify Functional Varia
Biomarkers and Endoph

ere do we go next? ere do we go next?

WAS with results from other eriments

ants (may be rare) henotypes

SLIDE 44

The Endopheno The Endopheno

APOE + 10 GWAS loci accou

variance for AD

Where is the “missing herita

Where is the missing herita

Answer: Small(er) effect loci, rare gene & gene-environment interac g g

Requires extremely large

AD complex phenotype
Endophenotypes (MRI cogn
Endophenotypes (MRI, cogn

signal-to-noise ratio

type Advantage type Advantage

unt for ~ 35% of genetic ability?” ability?

e variants, structural variants, gene- ctions

samples to address nitive biomarker) can increase nitive, biomarker) can increase

SLIDE 45

Study Populations f

Multi Institutional R Multi Institutional Resear search in ch in E i E id i l g ( (MIRAGE) S St d Epid idem emiology (MIRA (MIRAGE) St E) Stud

Caucasian and African American

discordant sib pairs discordant sib pairs

Cross-sectional, single time-point
Semi-quantitative MRI measures

Semi quantitative MRI measures and cerebrovascular disease (WM

Al Al h i ’ Di Di N N i Al Alzheimer mer’s Di Disease sease N Neur euroima

Unrelated subjects

Q tit ti l t i

Quantitative volumetric measures
Baseline + Longitudinal follow-up

for MRI Trait GWAS

Alzheimer’s Genetic Alzheimer’s Genetic dy

families containing primarily t

f neurodegeneration (HV, TCV)
f neurodegeneration (HV, TCV)

MH) included in study

gi gi g I g I iti ti ti ti (ADNI) (ADNI) agi aging I g Initi itiati tive ve (ADNI) (ADNI)

AD : MCI : CON 1 2 1 s 1 : 2 : 1 p

SLIDE 46

Subject Cha Subject Cha

PHASE PHASE ONE ONE ( Genome ( Genome Wide ) Wide ) ADNI Class AD MCI CON S l Sample Size 168 336 188 Age 75.4 75.2 75.0 (S.D.) (7.6) (7.1) (4.9) Freq APOE 4 0.420 0.342 0.144

aracteristics aracteristics

SLIDE 47

Subject Cha Subject Cha

PHASE PHASE ON ONE ( Genome ( Genome Wi Wid ADNI Class AD MCI CON S l Sample Size 168 336 188 Age 75.4 75.2 75.0 (S.D.) (7.6) (7.1) (4.9) Freq APOE 4 0.420 0.342 0.144

aracteristics aracteristics

E de ) e ) MIRAGE Caucasian AD CON 454 537 73.2 69.0 (8.3) (8.7) 0.291 0.194

SLIDE 48

Subject Cha Subject Cha

PHASE PHASE ON ONE ( Genome ( Genome Wi Wid ADNI Class AD MCI CON S l Sample Size 168 336 188 Age 75.4 75.2 75.0 (S.D.) (7.6) (7.1) (4.9) Freq APOE 4 0.420 0.342 0.144

aracteristics aracteristics

E PHASE PHASE T TWO de ) e ) ( R Regions gions ) MIRAGE Caucasian MIRAGE African American AD CON AD CON 454 537 188 231 73.2 69.0 74.7 68.4 (8.3) (8.7) (9.4) (10.2) 0.291 0.194 0.335 0.205

SLIDE 49

Phase 1: GWAS in Tw

ADNI MET A A P- VAL UE (MET

P-VALUE (E

MO ST SIG NIF IC EACH REG IO EACH REG IO CANDIDAT E EVAL UAT IO

wo Caucasian datasets

MIRAG E CAUCASIAN ANAL YSIS T A) < = 1 X 10- 5

EACH) < 0.05

CANT SNP F RO M O N SEL ECT ED O N SEL ECT ED E REG IO NS F O R O N IN PHASE 2

SLIDE 50

GWAS (Phase 1) R ( )

Tr Trait Tr Trait

Hippocampal Volume Hippocampal Volume Total Cerebral Volume White Matter Hyperintensi yp

ROI’s Tested in African Am

Regions of Interest g

Nu Number Nu Number 14 14 3 ities 5

mericans in Phase 2

SLIDE 51

Hippocamp Hippocamp

Cauc asian Afric an Americ an Co mbined

pal Volume pal Volume

rs6703865 P = 1.1 x 10-9

SLIDE 52

Hippocampal V

Factor V

essential co-factor of blood coa
Leiden variant associated with

and perhaps AD in Rotterdam S

P-Selectin P Selectin

Granule membrane protein that m

activated endothelial cells or plat

Stellos et al. J Cereb Blood Flow
Higher levels – AD fast cognitive d
Lower levels – AD slow cognitive d

Volume Genes

agulation cascade risk of vascular dementia Study

mediates interaction of telets with leukocytes

w Metab 2010

decline decline

SLIDE 53

Whole Genome The New

First human genome to be sequ

l b t i d billi f d ll

The New

laboratories and billions of dolla

Today, whole genome can be s

$5 000 $5,000

Still technical issues in capturin

aligning all of the fragments aligning all of the fragments

Biological relevance of most of
Most researchers use whole ex

genome) or targeted gene re-se per exome or sequencing lane per exome or sequencing lane.

Data management and bioinfor

e Sequencing… w Frontier

uenced took 10 years, multiple

w Frontier

ars sequenced in a few days for < ng all of the sequence and sequence still unknown xome sequencing (~ 3% of equencing which costs < $1,200 matic analysis significant

SLIDE 54

NEXT GENERATIO TO IDENTIFY ALZ ON SEQUENCING ZHEIMER’S GENES

With thanks to Dr. Clinton Baldwin

SLIDE 55

Overall Strategy gy

Alzheimer Subjects and Controls j Genetic Association Study

Id

and

SN
Id
Id

High-Throughput Sequencing

Id
Eva

Bio - Informatic Analysis

Eva Tran

Pri

Generate Testable Hypothesis about Disease Mech

entify Genetic SNPs that show a frequency difference between cases d controls NPs used are Linkage Disequilibrium Based entifies Genes, NOT functional SNPS, NOT mechanism entify ALL genetic Variants in Cases and Controls entifies Possible Functional SNPS aluates Potential Functional Impact of SNP (Amino Acid Changes, aluates Potential Functional Impact of SNP (Amino Acid Changes, nscription Factor Binding etc) ioritize SNPs to Evaluate Further.

hanism, Develop Authentic Models

SLIDE 56

Sequencing of Wad q g

 Inbred Arab Community in N  Inbred Arab Community in N

Israel – lower genetic comp

 High Prevalence of AD  High Prevalence of AD  Whole EXOME sequence fo

controls controls

 Candidate gene custom seq

(intron plus exon) for AD rel (intron plus exon) for AD rel including SORL1 in 7 cases a selected based on genotype

Sequencing effort directed by Dr. Cli

di Ara AD samples p

Northern Northern lexity? r 2 cases/2 uencing ated genes ated genes and controls e

int Baldwin

SLIDE 57

Summary of Whole Exome Sequencing in Wadi Ara

SLIDE 58

Protein Structur

The mutation (red circle) is located in the central portio ( ) p binding site. This region of the protein is highly cons shown) and are generally glutamic acid or aspartic acid

re of FZD5

n of the extracellular cysteine rich domain at the WNT

y served among related proteins (gi accession numbers d at that position.

SLIDE 59

LETTER LETTER

A mutation in APP prote p disease and age-related

Thorlakur Jonsson1, Jasvinder K. Atwal2, Stacy Steinberg1, Jon Hreinn Stefansson1 Patrick Sulem1 Daniel Gudbjartsson1 Jan Hreinn Stefansson1, Patrick Sulem1, Daniel Gudbjartsson1, Jan Yanmei Lu2, Tushar Bhangale2, Robert R. Graham2, Johanna Erik G. Jönsson6, Aarno Palotie7, Timothy W. Behrens2, Olafur Ryan J. Watts2 & Kari Stefansson1,8

Table 1 | APP A673T protects against Alzheimer’s disease

Analysis 1/OR OR ______________________________________________________________________________

AD AD

AD versus population controls

4.24 0.236 AD versus population controls aged 85 or greater 5.29 0.189 AD versus cognitively intact controls at age 85 7.52 0.133

The table shows association results, comparing patients with Alzheimer’s disease (AD) to three d Nchip, number of individuals with chip-based genotype information; Nin silico, number of individuals

doi:10.1038/nature11283

ects against Alzheimer’s g d cognitive decline

n Snaedal3, Palmi V. Jonsson3,8, Sigurbjorn Bjornsson3, nice Maloney2 Kwame Hoyte2 Amy Gustafson2 Yichin Liu2 nice Maloney2, Kwame Hoyte2, Amy Gustafson2, Yichin Liu2, Huttenlocher1,4, Gyda Bjornsdottir1, Ole A. Andreassen5, r T. Magnusson1, Augustine Kong1, Unnur Thorsteinsdottir1,8,

.

P value Controls ___________________ Frequency (%) Nchip

Nin silico_____________

0 13 2 199 849

0.13

2,199 849 4.19 x 10-5 0.45 57,174 22,074 4.78 x 10-7 0.62 7,653 1,350 6.92 x 10-6 0.79 827 407________

different control groups (top line gives numbers for patients with Alzheimer’s disease only). with genealogy-based genotype information.

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