Discovering Disease Genes-Discovering Disease Genes- The Example of Schizophrenia The Example of Schizophrenia

Pippa Thomson, Medical Genetics Section, Dept of Medical Sciences, MMC, University of Edinburgh.

Importance of the illnessImportance of the illness

• Severe psychiatric

• Affects ~1% of the population

• One of the top 10 causes of disability worldwide

• Economic cost (23% Drug, 14% Hospital)

• Pharmacological rationale for treatment weak or absent

• 1/3rd patients unresponsive or experience unacceptable side effects

• Strong genetic component– concordance rate between identical twins of 60%

Schizophrenia Positive symptoms:

visual & auditory hallucinations,

delusions, incoherent speech

Schizophrenia

Negative symptoms: withdrawal & isolation, impaired attention &

blunted emotions

Altered brain structure & Altered brain structure & functionfunction

High Heels Cause High Heels Cause Schizophrenia Schizophrenia and 6 Other and 6 Other Outlandish Medical Theories Outlandish Medical Theories

2. High-heeled shoes cause schizophrenia. You have to wonder where some medical theories originate. Why did Swedish scientist Jarl Flensmark decide to study a connection between heeled shoes and the incidence of schizophrenia? The world may never know. But his initial research seems sound, and he has connected certain brain activity with stimulation of certain points on the feet. The spread of schizophrenia around the globe has closely followed the spread of availability of heeled shoes. Is it an eerie coincidence or a real cause for concern? Look out, men - this theory applies not only to stilettos, but to any shoe with a heel.

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Relative risk of developing Relative risk of developing SchizophreniaSchizophrenia

1st degree relatives

MZ~50% 50

40

30

20

10

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no

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d r

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~10-15%

~1%

general population

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1st degree relatives

MZ~50% 50

40

30

20

10

0

no

aff

ecte

d r

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ive

~10-15%

~1%

general population

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Environment !

Kraepelin, 1896 “As we do not know what causes the illness there cannot be a rational treatment”

Benefits of gene identificationBenefits of gene identification

• Understand aetiology

• Improved drug development & testing

• Development of definitive diagnostic tests

• Understanding of interaction with non-genetic risk factors

• Insight into normal brain development & function

Allelic architecture and Allelic architecture and mapping strategymapping strategy

Mag

nitu

de o

f ef

fect

Frequency in population

Family-based linkage studies

Association studies in populations

Unlikely to exist

Fnct. Studies

Locus Identification-problemsLocus Identification-problems

• Uncertainty in diagnostic boundaries• Non-Mendelian inheritance• Variable age of onset • Genetic heterogeneity

– Many different genes can cause the illness>1% risk world wide>phenotypic variation

• Oligogenic/polygenic causation– More than one mutant gene required to produce

phenotype

Locus identification- reducing Locus identification- reducing the problemsthe problems

• Single large families

• Avoid bilineal descent • rigorous interviews

• family history

Reduce genetic heterogeneity Significant LOD score = gene of major effect

• Reduce uncertainty of diagnosis – classify minor diagnoses as unaffected

– >1 category of affected phenotype

• Marker analysis in multiply affected family or families

• Look for co-segregation of a particular allele with phenotype

• Results expressed as a LOD score (Significant at > 3)

= log (likelihood of data, if locus & disease are linked) ---------------------------------------------------------------- (likelihood of data, if locus & disease are not linked)

• Generally a large region is identified

Linkage AnalysisLinkage Analysis

der1

der11

11

1

1

1q42

11q14

11

A balanced t(1;11)(q42;q14) A balanced t(1;11)(q42;q14) translocationtranslocation

?

recurrent major depression

minor diagnosis

unaffectedschizophrenia

bipolar affective disorder

(1;11)(q42;q14) translocation

translocation increases risk by

50-fold

t(1;11) co-segregates with t(1;11) co-segregates with major mental illnessmajor mental illness

• Genetic association studies seek to relate variation in human DNA sequence with a disease or trait

• Association method provides greater power to detect common genetic variants conferring susceptibility to complex phenotypes

• Estimates population attributable risk (effect size)

• Controls should match cases and be a representative sample of the population.

100’s Individuals = 1% Schizophrenia 100’s Individuals = 100% Schizophrenia

Case-control association Case-control association studiesstudies

• Comparison of frequencies of polymorphisms between populations of cases and controls (usually a simple chi-square test or logistic regression)

• Polymorphism studied can be directly responsible for the defect frequency of cases >>> controls

• Polymorphism studied can be in linkage disequilibrium with the mutation responsible for the disease %T cases >> controls

• Association studies can be conducted for candidate genes, or through a whole region or across the whole genome (WTCCC)

p

Mb

T

A

G

C

SNPs are genotyped in parent-offspring trios, initially in CEPH trios.This can be used to identify SNPs that co-segregate (i.e. are in linkage disequilibrium) versus those that segregate independently.

A subset of SNPs can therefore be chosen that best represent the genetic diversity in a region/gene, reducing the costs of genotyping.

Summary of genotyped SNPs: Populations CEU CHB+JPT YRITotal Non-Redundant 3,204,709 3,244,897 3,150,433

International HapMap projectInternational HapMap project

http://www.hapmap.org/

Region of interest

HapMap genotyped SNPs

Known SNPs*

Known genes in the regions

Linkage Disequilibrium(LD)

*http://www.ncbi.nlm.nih.gov/SNP/

Tagging SNP selection Tagging SNP selection

Proportion of haplotype diversity explained : SNPs 1-23 - 97% SNPs 24-46 - 98%

DISC2

2 3 4 5 6 7 8 9 10 1112 13

DISC1

TRANSLOCATION

LOD=7.1, SCOTLAND

Genetic evidence implicatingGenetic evidence implicating DISC1DISC1 in psychiatric illness in psychiatric illness

1

LOD=2, BRITAIN & ICELAND (Curtis et al 2003)

D1S251

LOD=1, TAIWAN (Hwu et al 2003)

SCZ

BPAD

HAPLOTYPE

p=0.00024, FINLAND (Hennah et al 2003)

SCZ & SCZAFF

SCZ & BPAD & MDD

LOD=3.21, FINLAND (Ekelund et al 2001)

SCZD1S2709

p=0.000027, North-America (Hodgkinson et al 2004)

rs6675281

SCZAFF

SCOTLAND

SCZ, BPADHAPLOTYPE

p=0.0044, p=0.0016

Table 1. Summary of current evidence supporting several of the more promising genes implicated in schizophrenia, bipolar disorder, and mixed bipolar-psychosis phenotypes

Craddock et al., SCZ Bulletin, 2006

>130 genes implicated

DISC1 interactomeDISC1 interactome protein-protein interactionsprotein-protein interactions

Chris Carter, http://www.polygenicpathways.co.uk/disc11_vml.htm

• ENU generated mouse mutants

• Two independent lines with missense mutations in DISC1 exon 2

– Q31L (Glutamine-Leucine) > Q- hydrophillic; L – hydrophobic

– L100P (Leucine-Proline)> Predicted to cause transition in polypeptide chain direction

• Normal levels of DISC1 protein in brain

• L100P line models schizophrenia; Q31L, depression

Effects of altered DISC1 on Effects of altered DISC1 on gene expressiongene expression

Clapcote et al., Neuron. 2007 May 3;54(3):387-402.

Phenotype 31L/31L 100P/100PAnxiety (elevated plus-maze) = =Horizontal activity = >>Vertical activity = >Prepulse inhibition (PPI) < <<Acoustic startle response = <Startle reactivity = <Latent inhibition (LI) << <<Working memory (T maze) < <<Spatial learning and memory (Morris water maze) = =Forced swim immobility (FST) > =Sociability and social novelty < =Sucrose consumption < =Brain volume < <<PDE4B activity << =

Drug Treatment 31L/31L 100P/100PPPI Clozapine = +

Bupropion +++ =

Schizophrenia

Depression

Effects of Altered DISC1 on Effects of Altered DISC1 on Behaviour (Behaviour (How do you know How do you know if a mouse is schizophrenic?if a mouse is schizophrenic?))

Effects of altered DISC1 on Effects of altered DISC1 on gene expressiongene expression

•Samples collected and microarray study ongoing –Mutated lines vs background strain (C57/BL6)

–47,000 transcripts

–Hippocampus

–Adult and embryonic stage- Microarray

–Confirmation/Investigation of changes

–Series of embryonic; postnatal and adult stages

–Drug treated adult mice

Detect disrupted pathways

Resequencing, SNP detection, genome comparisons, gene expression, transcription factor studies, small RNA analysis

Individual genomes – All SNPs in each individual

Currently :

Using the Illumina 1G to sequence genes in the DISC1 pathway. Total sequence read 3.5megabases in 1200 individuals

• Identify coding and non-coding polymorphisms• Mutation detection• Detection of variants in conserved regions• Detection of variants affecting binding of transcription factors

Whole genome sequencingWhole genome sequencing

Psychiatric Genetics-Psychiatric Genetics-Unanswered QuestionsUnanswered Questions

How many susceptibility genes are there? What is their function? Is function conserved across species? Can we relate gene (dys)function to mental (dis)order? Do gene variants predict risk, course, outcome and response to treatment? Will gene discovery lead to drug discovery? How do genes and environment interact? How and when will the patient benefit?

DISC1

Kirsty MillarShaun MackieFumiaki OgawaJennifer ChubbBecky CarlyleNick BradshawSheila Christie

Steve ClapcoteKathy EvansSarah Brown

William Hennah

Medical Genetics

Prof David PorteousProf Douglas BlackwoodWalter MuirBen Pickard

Other collaborators

DISC1 ConsortiumWellcome Trust CRFIllumina, San DiegoCold Spring Harbor laboratories

AcknowledgementsAcknowledgements