Genomic studies of schizophrenia: mapping madness?Genomic studies of schizophrenia: mapping madness?

Mike Owen, Department of Psychological Medicine, Wales College of Medicine, Cardiff University, UK.

Mike Owen, Department of Psychological Medicine, Wales College of Medicine, Cardiff University, UK.

Genetic epidemiology of schizophreniaGenetic epidemiology of schizophrenia

Familial, λs =10 Individual differences in liability are largely genetic

– Heritability 0.6-0.9

Non-genetic factors also important Multi-locus model consistently supported by analysis of

family data Genes with population λs > 3 unlikely Number of susceptibility loci, degree of risk conferred by

each and degree of interaction all unknown

Familial, λs =10 Individual differences in liability are largely genetic

– Heritability 0.6-0.9

Non-genetic factors also important Multi-locus model consistently supported by analysis of

family data Genes with population λs > 3 unlikely Number of susceptibility loci, degree of risk conferred by

each and degree of interaction all unknown

Finding genes for schizophreniaFinding genes for schizophrenia

Linkage

Chromosomal abnormalities

Association

Convergent genomics

Linkage

Chromosomal abnormalities

Association

Convergent genomics

Disease geneD18S21

Published linkagedata 2006

Genomewidesignificant

Multiplepositives

8p22-p21

5q21-q336q21-25

6p24-p22

1q21-q22

1 2 3 4 5 6 7 8

1q42

10 11 12 13 14 15 169

13q32-q34

10p15-p11

10q 25-26

22q11-q22

17 18 19 20 21 22 Y X

17p11-q25

Cardiff schizophrenia sib-pair genome screenCardiff schizophrenia sib-pair genome screen

351 microsatellite markers (10.3 cM) in 354 affected sib-pairs179 (UK), 134 (Sweden), 41 (USA)

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LO

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Chromosome

Williams et al. Am J Hum Genet 2003

CNPVCFS

Linkage studies of schizophrenia 2006Linkage studies of schizophrenia 2006

Data consistent with observed recurrence risks in relatives

Meta-analysis suggests some consensus

Many underpowered studies on uncertain value

Data consistent with observed recurrence risks in relatives

Meta-analysis suggests some consensus

Many underpowered studies on uncertain value

Larger studies needed for more genes, stronger evidence, interactions and better location

800-1000 ASPs optimal

A number of groups are now seeking genes in currently positive regions

Larger studies needed for more genes, stronger evidence, interactions and better location

800-1000 ASPs optimal

A number of groups are now seeking genes in currently positive regions

Positional genetics and schizophreniaPositional genetics and schizophrenia

Positional Candidates – NRG1, 8p 1

– DTNBP1, 6p 2 – G72 (DAOA), 13q 3

Multiple positive studies as well as negative

No clear pattern of associated alleles or haplotypes

Risk variants not identified

Positional Candidates – NRG1, 8p 1

– DTNBP1, 6p 2 – G72 (DAOA), 13q 3

Multiple positive studies as well as negative

No clear pattern of associated alleles or haplotypes

Risk variants not identified

1. Stefansson et al. 2002; 2. Straub et al. 2002; 3. Chumakov et al. 2002;

G72/G30

22q11-q22

17 18 19 20 21 22 Y X

17p11-q25

13q32-q34

10p15-p11

HTR2A

10 11 12 13 14 15 169

DAO10q 25-26

DRD3

5q21-q336q21-25

DTNBP1

6p24-p22

RGS4

1q21-q22

8p22-p21

1 2 3 4 5 6 7 8

1q42

NRG1

Positional candidate genes for schizophrenia

NRG1 association with schizophreniaNRG1 association with schizophrenia

Tosato, Dazzan and Collier, Schiz. Bull. 2005.

DTNBP1: complex pattern of association findings in schizophrenia

DTNBP1: complex pattern of association findings in schizophrenia

Williams, O’Donovan and Owen, Schiz Bull, 2005,

DTNBP1 and schizophreniaDTNBP1 and schizophrenia

Multiple studies suggest that variation in DTNBP is associated with schizophrenia (11/14 positive).

Protection may be mediated by IQ. No individual SNPs or haplotypes have consistently been

implicated in susceptibility. No systematic study aimed at detecting all common genetic

variation. More studies needed to identify risk variants. Reduced expression of message and protein in schizophrenia –

cause or compensation? Cis-acting elements regulate DTNBP expression.

– Can we relate specific haplotypes to gene expression?

Multiple studies suggest that variation in DTNBP is associated with schizophrenia (11/14 positive).

Protection may be mediated by IQ. No individual SNPs or haplotypes have consistently been

implicated in susceptibility. No systematic study aimed at detecting all common genetic

variation. More studies needed to identify risk variants. Reduced expression of message and protein in schizophrenia –

cause or compensation? Cis-acting elements regulate DTNBP expression.

– Can we relate specific haplotypes to gene expression?

DTNBP1 risk haplotype is associated with reduced expression

DTNBP1 risk haplotype is associated with reduced expression

Allele ratios at SNP rs1047631, stratified by heterozygosity for the defined schizophrenia risk haplotype.

Schizophrenia risk haplotype tags one or more cis-acting variants that result in a relative reduction in DTNBP1 mRNA expression in human cerebral cortex.

Further analyses suggest that risk haplotypes identified in other Caucasian samples also index lower DTNBP1 expression.

Ties risk haplotypes to altered function

Suggests explanation for some of the discrepancies between studies

Allele ratios at SNP rs1047631, stratified by heterozygosity for the defined schizophrenia risk haplotype.

Schizophrenia risk haplotype tags one or more cis-acting variants that result in a relative reduction in DTNBP1 mRNA expression in human cerebral cortex.

Further analyses suggest that risk haplotypes identified in other Caucasian samples also index lower DTNBP1 expression.

Ties risk haplotypes to altered function

Suggests explanation for some of the discrepancies between studies

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io A

/G

Genomic cDNA: risk het

cDNA:non risk

Bray et al, Human Molecular Genetics, 2005.

G72/G30

COMT

PRODH

22q11-q22

17 18 19 20 21 22 Y X

13q32-q34

10p15-p11

HTR2A

10 11 12 13 14 15 169

DAO

DRD3

5q21-q336q21-25

DTNBP1

6p24-p22

RGS4

1q21-q22

8p22-p21

1 2 3 4 5 6 7 8

1q42

NRG1

Chromosomal abnormalities and schizophrenia

VCFS and Schizophrenia

25+% of adults with VCFS develop schizophrenia (Murphy et al 2000)

Does a gene on 22q11 predispose to schizophrenia in the general population?

Some claims (COMT, PRODH, ZDHHC8, ARVCF) but none convincing

DISC1 and schizophrenia (Porteous, Millar, Blackwood, St Clair et al)

DISC1 and schizophrenia (Porteous, Millar, Blackwood, St Clair et al)

• DISC1: unknown function; N globular domain; C -helical domain with coiled coil domains; widely expressed, adult > fetal

• DISC2: unknown function; RNA >15kb but no ORF; putative anti-sense RNA regulator of DISC1; widely expressed, heart >> other

• TRAX: translin related protein X; putative role in single strand DNA repair and RNA translocation; intergenic splicing to DISC1

Schizophrenia Associated t(1:11) Translocation

High density of genes around ch#1 breakpoint

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6

1 2I

II

II

VI

V

ASSOCIATION OF A BALANCED AUTOSOMAL TRANSLOCATION t(1;11)

WITH MAJOR MENTAL ILLNESS

Schizophrenia Bipolar

Disorder

Recurrent

Major

Depression

Adolescent

Conduct

Disorder

Anxiety, Alcoholism,

Minor Depression

Translation

t(1;11) Carrier

Possibility of position effect Evidence for haplotype association

with SZ Associations with visual and

working memory deficits t (1,16) disrupts PDE4B (binding

partner of DISC1) in family with psychosis (Millar et al 2005)

DISC1 complex protein associated with numerous cytoskeletal proteins involved in centrosomal and microtubule function, and with cell migration, neurite outgrowth, and membrane trafficking of receptors and possibly mitochondrial function.

Possibility of position effect Evidence for haplotype association

with SZ Associations with visual and

working memory deficits t (1,16) disrupts PDE4B (binding

partner of DISC1) in family with psychosis (Millar et al 2005)

DISC1 complex protein associated with numerous cytoskeletal proteins involved in centrosomal and microtubule function, and with cell migration, neurite outgrowth, and membrane trafficking of receptors and possibly mitochondrial function.

Candidate genes for schizophreniaCandidate genes for schizophrenia

NRG1 and DTNBP1: multiple positive studies. DAOA(G72): some positives for both schizophrenia and bipolar disorder DISC1: highly promising. Effect sizes small-moderate (OR 1.3-2) RGS4: some positives but support weakening. COMT, PRODH, ZDHHC8, PPP3CC, CAPON, CHRNA7, TRAR4 and

others: not yet convincing. Cannot be explained by variants with manifest functional consequences.

– Presumably effects on expression, splicing etc. Remain cautious until risk variants identified. Inconsistencies between studies.

– Lack of power– Genotyping error– Stratification– Incomplete genetic information from indirect studies

» NB dysbindin– Susceptibility variants on different backgrounds??– Allelic heterogeneity/complexity– Heterogeneity: differences in case definition and ascertainment

It’s epidemiology now!

NRG1 and DTNBP1: multiple positive studies. DAOA(G72): some positives for both schizophrenia and bipolar disorder DISC1: highly promising. Effect sizes small-moderate (OR 1.3-2) RGS4: some positives but support weakening. COMT, PRODH, ZDHHC8, PPP3CC, CAPON, CHRNA7, TRAR4 and

others: not yet convincing. Cannot be explained by variants with manifest functional consequences.

– Presumably effects on expression, splicing etc. Remain cautious until risk variants identified. Inconsistencies between studies.

– Lack of power– Genotyping error– Stratification– Incomplete genetic information from indirect studies

» NB dysbindin– Susceptibility variants on different backgrounds??– Allelic heterogeneity/complexity– Heterogeneity: differences in case definition and ascertainment

It’s epidemiology now!

Emil Kraepelin, 1896, splits psychosis.Emil Kraepelin, 1896, splits psychosis.

“crystallized dementia praecox and manic-depressive illness from an amorphous mass of madness” (Brockington & Leff, 1979).

Organic Functional

– Dementia Praecox– Manic-depressive insanity

“crystallized dementia praecox and manic-depressive illness from an amorphous mass of madness” (Brockington & Leff, 1979).

Organic Functional

– Dementia Praecox– Manic-depressive insanity

Deconstructing Psychosis: Challenges to the Kraepelinian Dichotomy.

Deconstructing Psychosis: Challenges to the Kraepelinian Dichotomy.

• No “point of rarity”• Risk factors in common (Murray et al 2004)• Familial co-occurrence of SZP, SA and BP• Cardno et al twin study• Overlapping linkage regions

• 13q, 22q, 6q

New genetic studies confirm this and suggest association with clinical syndromes.

• No “point of rarity”• Risk factors in common (Murray et al 2004)• Familial co-occurrence of SZP, SA and BP• Cardno et al twin study• Overlapping linkage regions

• 13q, 22q, 6q

New genetic studies confirm this and suggest association with clinical syndromes.

Studying candidate genes across the Kraepelinian divide: Dysbindin.

Studying candidate genes across the Kraepelinian divide: Dysbindin.

No association between BP and the Cardiff haplotype in DTNBP1.

Suggestive evidence for association with BP with predominant psychosis.

No association between BP and the Cardiff haplotype in DTNBP1.

Suggestive evidence for association with BP with predominant psychosis.

Raybould et al, Biological Psychiatry, 57: 696-701, 2005.

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Upward trend: p = 0.014

Studying candidate genes across the Kraepelinian divide: Neuregulin.

Studying candidate genes across the Kraepelinian divide: Neuregulin.

NRG1 HAPICE confers risk to illness with both schizophrenia and mood features.

Effect size of NRG1 HAPICE increases with preponderance of mood-incongruent psychotic symptoms (sign test p=0.002).

NRG1 HAPICE confers risk to illness with both schizophrenia and mood features.

Effect size of NRG1 HAPICE increases with preponderance of mood-incongruent psychotic symptoms (sign test p=0.002).

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Green et al, Archives of General Psychiatry, 2005.

Studying candidate genes across the Kraepelinian divide: G72 (DAOA).

Studying candidate genes across the Kraepelinian divide: G72 (DAOA).

Some positive replications of G72 but no clear associated alleles or haplotypes.

Independent support for association with Bipolar disorder in several studies.– G72 probably strongest candidate gene for BP.

Is this a gene for both disorders?

Some positive replications of G72 but no clear associated alleles or haplotypes.

Independent support for association with Bipolar disorder in several studies.– G72 probably strongest candidate gene for BP.

Is this a gene for both disorders?

DAOA (G72) in schizophrenia and bipolar disorder

DAOA (G72) in schizophrenia and bipolar disorder

Significant whole gene association in BP (n=706, p=0.045) but not SZ (n=709) vs controls (n=1416).

Significant whole gene association in “Mood” (n= 1153, p=0.0086) and in schizophrenia-mood (n=112, p=0.02) but not psychosis (n=818).

DAOA is probably a susceptibility locus for mood disorder rather than schizophrenia per se.

Extent to which association seen in schizophrenia depends upon clinical characteristics of sample.

Significant whole gene association in BP (n=706, p=0.045) but not SZ (n=709) vs controls (n=1416).

Significant whole gene association in “Mood” (n= 1153, p=0.0086) and in schizophrenia-mood (n=112, p=0.02) but not psychosis (n=818).

DAOA is probably a susceptibility locus for mood disorder rather than schizophrenia per se.

Extent to which association seen in schizophrenia depends upon clinical characteristics of sample.

(Williams et al Archives of General Psychiatry, 2006).

DISC1 is associated with broad psychosis and mood phenotype.

DISC1 is associated with broad psychosis and mood phenotype.

t(1:11) segregates with Sz, BP and UP.

Linkage to SA– Hamshere et al 2005.

Evidence for haplotype association– Hennah et al 2003– Hodgkinson et al 2004

» Schizophrenia, SA and BP– Thomson et al 2005

» SZ and BP.

t(1:11) segregates with Sz, BP and UP.

Linkage to SA– Hamshere et al 2005.

Evidence for haplotype association– Hennah et al 2003– Hodgkinson et al 2004

» Schizophrenia, SA and BP– Thomson et al 2005

» SZ and BP.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6

1 2I

II

II

VI

V

ASSOCIATION OF A BALANCED AUTOSOMAL TRANSLOCATION t(1;11)

WITH MAJOR MENTAL ILLNESS

Schizophrenia Bipolar Disorder

RecurrentMajor

Depression

AdolescentConduct Disorder

Anxiety, Alcoholism,Minor Depression

Translationt(1;11) Carrier

Using genetics to dissect psychosisUsing genetics to dissect psychosis

Craddock, O’Donovan and Owen, Schizophrenia Bulletin, 2006.

Do genetic findings in psychosis point to a common mechanism?Do genetic findings in psychosis point to a common mechanism?

The genes most clearly implicated (NRG1, DTNBP1, G72) all code for proteins that potentially impact, directly or indirectly, on the function of glutamate synapses. Harrison and Owen, Lancet, 2003.

But caution required!– proteins implicated poorly

understood – multiple processes implicated for

NRG1 and DTNBP1

The genes most clearly implicated (NRG1, DTNBP1, G72) all code for proteins that potentially impact, directly or indirectly, on the function of glutamate synapses. Harrison and Owen, Lancet, 2003.

But caution required!– proteins implicated poorly

understood – multiple processes implicated for

NRG1 and DTNBP1

White matter abnormalities in schizophrenia (Mt. Sinai Conte Center)

White matter abnormalities in schizophrenia (Mt. Sinai Conte Center)

Imaging studies– Defects of connectivity– DTI

Multiple gene expression studies in postmortem schizophrenia brains have found significant reduction of expression of myelin and oligodendrocyte related genes (e.g. Hakak et al., 2001)

Quantitative anatomical analyses have demonstrated decreased oligodendrocyte numbers in prefrontal cortex (Hof et al 2002, 2003).

Cause or effect?

Imaging studies– Defects of connectivity– DTI

Multiple gene expression studies in postmortem schizophrenia brains have found significant reduction of expression of myelin and oligodendrocyte related genes (e.g. Hakak et al., 2001)

Quantitative anatomical analyses have demonstrated decreased oligodendrocyte numbers in prefrontal cortex (Hof et al 2002, 2003).

Cause or effect?

CNP is marker for oligodendrocytes

Message and protein show reduced brain expression in schizophrenia

Located at 17q21.2 rs2070106 is associated with CNP

expression (P=0.001). the lower-expressing allele was

significantly associated with schizophrenia (P=.04) in a case-control sample.

All affected individuals in a linked pedigree were homozygous for the lower-expression allele (P=.03).

CNP is marker for oligodendrocytes

Message and protein show reduced brain expression in schizophrenia

Located at 17q21.2 rs2070106 is associated with CNP

expression (P=0.001). the lower-expressing allele was

significantly associated with schizophrenia (P=.04) in a case-control sample.

All affected individuals in a linked pedigree were homozygous for the lower-expression allele (P=.03).

Archives of General Psychiatry (2006) 63: 18-24.

Oligodendrocyte Lineage Transcription Factor 2 (OLIG2): a master regulator of all stages of oligodendrocyte lineage.

Oligodendrocyte Lineage Transcription Factor 2 (OLIG2): a master regulator of all stages of oligodendrocyte lineage.

Basic helix–loop–helix transcription factor central to oligodendrocyte development

Down-regulated in schizophrenia (Tchakev et al, 2003; Katsel et al, 2005; Iwamoto et al, 2005)

Centrality in OL allows for a primary change responsible for several others (parsimony)

Basic helix–loop–helix transcription factor central to oligodendrocyte development

Down-regulated in schizophrenia (Tchakev et al, 2003; Katsel et al, 2005; Iwamoto et al, 2005)

Centrality in OL allows for a primary change responsible for several others (parsimony)

OLIG2 associated with schizophrenia: non redundant (r2<0.9) positives (submitted).

OLIG2 associated with schizophrenia: non redundant (r2<0.9) positives (submitted).

Meff = 9 (all 16 pooled SNPs) gene corrected p = 0.0009 Experiment-wide corrections p = 0.013 (primary 14 genes)

p = 0.038 (all 44 genes)

CONSERVATIVE

ConclusionsConclusions

Some highly promising findings (NRG1, DTNBP1, G72/DAOA, DISC1)

Need to establish risk nucleotides/mechanisms » And this might not be easy without functional readout

(endophenotypic, animal, cellular)» Needs more detailed studies, collaboration, re-sequencing» Expect allelic heterogeneity, effects of ascertainment

Refining the associated phenotype by iteration (Symptoms, Course and outcome, Cognitive function, Imaging). NB samples.

Curation of association data (it’s epidemiology)– Quality standards– Meta-analysis

Need to support genetic associations with biology– DTNBP1 and NRG1 expression studies

Some highly promising findings (NRG1, DTNBP1, G72/DAOA, DISC1)

Need to establish risk nucleotides/mechanisms » And this might not be easy without functional readout

(endophenotypic, animal, cellular)» Needs more detailed studies, collaboration, re-sequencing» Expect allelic heterogeneity, effects of ascertainment

Refining the associated phenotype by iteration (Symptoms, Course and outcome, Cognitive function, Imaging). NB samples.

Curation of association data (it’s epidemiology)– Quality standards– Meta-analysis

Need to support genetic associations with biology– DTNBP1 and NRG1 expression studies

FutureFuture

There are more significant linkages to account for.– Few if any exhaustive fine mapping studies

WGAs– Depend on CDCV hypothesis– Need very large samples– Sample characteristics will be crucial– Data handling and statistical challenges are huge– WTCCC

CNV analysis Candidate pathway analysis (GxG) Inclusion of E in genetic studies (GxE) WGS (CDRV) Understanding gene regulation (identifying regulatory sequences,

miRNA, chromatin effects etc)

There are more significant linkages to account for.– Few if any exhaustive fine mapping studies

WGAs– Depend on CDCV hypothesis– Need very large samples– Sample characteristics will be crucial– Data handling and statistical challenges are huge– WTCCC

CNV analysis Candidate pathway analysis (GxG) Inclusion of E in genetic studies (GxE) WGS (CDRV) Understanding gene regulation (identifying regulatory sequences,

miRNA, chromatin effects etc)

AcknowledgementsAcknowledgements

N Williams N Norton H Williams N Bray A Preece J Wilkinson S Dwyer Elaine Green Rachel Raybould Detelina Grozeva T Peirce B Glaser L Carroll

N Williams N Norton H Williams N Bray A Preece J Wilkinson S Dwyer Elaine Green Rachel Raybould Detelina Grozeva T Peirce B Glaser L Carroll

M O’Donovan N Craddock G Kirov I Jones

M Hamshere, P Holmans. S Macgregor, V Moskvina,

M O’Donovan N Craddock G Kirov I Jones

M Hamshere, P Holmans. S Macgregor, V Moskvina,