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