genetic dissection of human diseases ariel darvasi the hebrew university of jerusalem
Post on 20-Dec-2015
214 views
TRANSCRIPT
Genetic Dissection of
Human Diseases
Ariel Darvasi
The Hebrew University of Jerusalem
Genetic strategies for gene discovery
Genetic associationLinkage analysis
Candidate genes
Whole-genome scans
Linkage analysis
A1A2 A3A4
A1A3 A1A3
IBD=2
Affected sib pairs
(Identical by descent)
Association Analysis
Cases Controls
AA
AA
AA
AA
AA
AA
aa
aa
aa
aa
aa
aa
Linkage Disequilibrium (LD) Mapping
SNPs
Functional SNP
Genes
Genotypic Relative Risk (GRR)
dddd DDDD
dia
gnog
enom
icis
t
Disease penetrance: 1% 4%
GRR=4.0
Sample Size Required With the Case-Control Design:
c
ccca
aaa
N
PP
ZZ
PP
PPN
2
)1(
2/)1(2
2/1
K
LmPa
K
LmPc
1
Na= number of casesNc= number of controlsPa,Pc= disease allele frequency in the affected and control population respectively.
Sample Size Required: Numerical Example
Gene effect
(in terms of GRR)
2 3
LD (/max ) 0.5 0.75
Sample size required
Case-control
(Association)
4,575 820
Sib pairs
(Linkage Analysis)
14,317 6,010
Genetic strategies for gene discovery
Linkage analysis
Candidate genes
Whole-genome scans
Genetic association
* Population selection (outbred/inbred)?* How many SNPs?* How to select the SNPs?* How to genotype the SNPs (DNA pooling)?
A random sample of different chromosomal areas
1 Mb - 16 SNPs
300 Kb 150 Kb 300 Kb150 Kb
5 or 15 Kb
Each SNP was genotyped on:- 90 Caucasians- 90 Afro-Americans- 90 Ashkenazi Jews
Average LD
0.0
0.1
0.2
0.3
0.4
0.5
0.6
5 10 20 40 80 160 320 640 More
Distance (Kb) between SNPs
Ave
rag
e r2
Ashkenazi JewsCaucasiansAfrican American
Proportion of high LD SNP pairs
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
5 50 250 500 1000
Distance (Kb) between SNPs
Pro
po
rtio
n o
f p
airs
0.0
0.1
0.2
0.3
5 50 250
Ashkenazi Jews
Caucasians
African American
D’=1 r2=1
The Advantage of Homogeneity
The Advantage of Homogeneity
LD calculated within ‘haplotype blocks’
0.30
0.40
0.50
0.60
0.70
0.80
0.90
5 10 15 20 25 30 35 40 45 50 55 60
Average distance (Kb)
Ave
rag
e r2
ASHCCAA
LD in 10 selected regions (r2)
ASH
CC
AA
105kb with at least 7 SNPs in 3 populations.
123456789
101112
1 2 3 4 5 6 7 8 9 10 11 12
Combinations testedSelecting 1,2,3,4 or 5 SNPs/100kb with:
• Single SNP analysis:– Random SNP selection– Optimal SNP selection
• Haplotype analysis:– Random SNP selection– Optimal SNP selection
Random and optimal selection for single SNP and haplotype analysis
1
2
3
4
1 2 3 4 5
number of SNPs in the set
1/r2
Random SNPs
Optimal SNPs
Random haplotypes
Optimal haplotypes
1
2
3
4
1 2 3 4 5
number of SNPs in the set
1/r2
Random SNPs
Optimal SNPs
Random haplotypes
Optimal haplotypes
Optimal haplotypes70,000 SNPs
(2.4 SNPs per 105kb)
Random haplotypes80,000 SNPs
(2.8 SNPs per 105kb)
Optimal SNPs100,000 SNPs
(3.5 SNPs per 105kb)
Random SNPs120,000 SNPs
(4.2 SNPs per 105kb)
1/r2=2
Number of SNPs required for whole-genome scan
Common Disease Allele Rare Disease Allele
1
2
3
4
1 2 3 4 5
number of SNPs in the set
1/r2
Random SNPs
Best SNPs
Random haplotypes
Best haplotypes
1
6
11
16
1 2 3 4 5
number of SNPs in the set
1/r2
Random SNPs
Best SNPs
Random haplotypes
Best haplotypes
Rare Variants (<10%) Affecting Complex Traits
DNA Pooling for SNP allelotyping
Cases Controls
• RFLP, Restriction fragment length Polymorphism
• Pyrosequencing
• Single base-pair extension (SBE)
• Mass Spectrometry (MS)
• TaqMan
Evaluating DNA Pooling Technologies
0. 20
0. 40
0. 60
0. 80
1. 00
0. 20 0. 40 0. 60 0. 80 1. 00
RFLP
SE=0.04
0. 20
0. 40
0. 60
0. 80
1. 00
0. 20 0. 40 0. 60 0. 80 1. 00
Pyrosequencing
SE=0.02
0. 20
0. 40
0. 60
0. 80
1. 00
0. 20 0. 40 0. 60 0. 80 1. 00
SBE
SE=0.03
0. 20
0. 40
0. 60
0. 80
1. 00
0. 20 0. 40 0. 60 0. 80 1. 00
MS
SE=0.02
0.5
1
1.5
2
2.5
0.6 1.1 1.6 2.1
Allele 6 MGB (VIC)
All
ele
5MG
B
(FA
M)
0. 20
0. 40
0. 60
0. 80
1. 00
0. 20 0. 40 0. 60 0. 80 1. 00
TaqMan
SE=0.02
An Example:
Dissecting the genetic basis of schizophrenia
Main Symptoms of Schizophrenia
Positive Symptoms: Delusions, hallucinations, disorganized speech, unusual behavior, agitation.
Negative Symptoms: Lack of emotion, inability to speak, lack of motivation, no pleasure from fun activities, slow movements.
Symptoms Involving Thoughts: Decreased attention span and memory, difficulty making decisions.
Mood:Depression, unpleasant feelings, hopelessness, low self-esteem.
Average lifetime morbid risks for developing schizophrenia (Riley & McGuffin, 2000)
Schizophrenia Family Studies
48
46
17
17
13
9
6
6
5
4
2
2
1
0 10 20 30 40 50 60
MZ twins
Offspring of dual matings
DZ twins
Siblings with one affected parent
Children
Siblings
Parents
Half siblings
Grand children
Nephews/Nieces
Uncles/Aunts
First cousins
Baseline
Gene Discovery in Schizophrenia
Choosing a genomic interval and creating a high density SNP map in that region
High throughput scanning of all SNPs in DNA pools
0
0.5
1
1.5
2
2.5
3
3.5
4
Chromosomal Location
Z S
co
re
100 KB
Pooled genotyping of additional
SNPs at the COMT locus
COMT gene
N P-value
SNP Sex Con Scz Genotype Allele GG
F 706 262 0.25 0.30 0.90
M 2264 458 0.027 0.041 0.0074
rs165688
All 0.041 0.024 0.023
F 1380 263 1.0x10-5 9.1x10-6 6.8x10-6
M 3519 461 0.203 0.104 0.090
rs165599
All 3.0x10-5 8.8x10-5 7.2x10-5
F 685 250 0.012 0.014 0.43
M 2164 464 0.0011 0.0083 2.3x10-4
rs737865
All 1.6x10-4 3.6x10-4 4.8x10-4
Individual Genotyping Results
G-----------G-----------G
Haplotype P-value = 9.5 x 10-8
SNPs: rs165688 rs737865 rs16599
Haplotype Analysis
FemalesMales
PAR32.2%13.5%
The G-G-G risk haplotype also has the strongest effect on mRNA expression levels (Bray et al. 2003)
Candidate Functional SNPs
Point deletion (C/-) 3’ UTR
SNP (C/T) near
Estrogen Response Element in promoter
• Is COMT a reasonable susceptibility gene for schizophrenia?
• Is it reasonable to expect a sex-specific effect of COMT?
COMT and Schizophrenia
Few facts on COMT
COMT catalyzes one of the major degradation pathways of dopamine (and other catecholamine transmitters)
COMT is involved in the metabolism of catechol estrogens.
Estrogen down regulates COMT transcription
Gender Differences in Schizophrenia
Age of Onset
Men: early 20sWomen: mid to late 20s
Symptoms
Men: have more negative symptomsWomen: have more depressive symptoms and paranoia
Course of illness
Woman schizophrenic patients have a more benign course of illness than men
COMT Knockout Mice
In homozygous males: 2-3 fold increase in the amount of dopamine in the frontal cortex
Homozygous females demonstrate increased anxiety in a dark/light exploratory model
Heterozygous males exhibit increased aggressive behavior
The Estrogen Connection
According to the estrogen theory, women are protected to some extent against schizophrenia between puberty and menopause by their relatively high physiological estrogen production during this phase.
Estrogen Theory
Estrogen levels have significant effects on the mental state of schizophrenic women
Women have a second peak of illness onset after age 45, with a more severe course of illness
Estrogen was tested with success as a therapeutic agent in schizophrenia
The protective effect of estrogen seems to contribute to some of the gender differences in schizophrenia: age of onset, severity and response to neuroleptic treatment
Science has reached today the point where complete dissection of the genetic basis of common diseases is an achievable challenge
This will lead to the identification of disease related biochemical pathways which in turn will lead to novel and efficient therapies
Summary
Naomi ZakMichal BornsteinEfrat Lev-LehmanBecky HouryGalit HershkoGuy AmitIlana BlechIrina BarskyMichal MilloSvetlana LobovskySvetlana ShpiudlezSari LubinVardit Ben-DorErella KenoshiDvora Rubinow
Sagiv ShifmanEster InbarMeira SternfeldTami MendelbaumAnat HorowitzShoshi BergerGal RomanoAnne Pisante
Benjamin Yakir
Mira Korner
Acknowledgements
Clinical Collaborations Avraham WeizmanHaim Y. KnoblerNimrod GrisaruLeon KarpMoshe KotlerIlya ReznikRichard SchifferEilat ShinarBaruch SpivakRael D. StrousMarnina Swartz- Vanetik StanfordNeil Risch
Technology CompaniesQiagen Genomics Inc.
IDgeneHUJI