STRATEGIES FOR GENE DISCOVERY IN SCHIZOPHRENIA

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Genetics and Neuroimaging Current Findings and
Future Strategies
James L Kennedy MD, FRCPC
IAnson Professor of Psychiatry and Medical
Science Head, Neurogenetics Section, Clarke
Division, Director, Department of Neuroscience
Research Centre for Addiction and Mental Health
(CAMH), University of Toronto SG Potkin, D
Mueller, M Masellis, N Potapova, F Macciardi
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How do genes determine brain characteristics?
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Molecular Genetic Approach
Pharmacogenetics
Gene Expression
Pharmacology
Neurobiology
Phenotype
Endophenotype
-Psychophysiology Neuroimaging
Sub-pheno
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Candidate gene selection for schizophrenia

• Neurotransmitter system genes
• e.g. dopamine receptors, transporter
• Neurodevelopmental genes
• Cortical development, patterning
• Neuronal differentiation, migration
• Synaptic protein genes
• Human post-mortem expression studies (Mirnics et
  al, 2000 Vawter et al, 2001)
• Animal model expression studies (Wong et al,
  2002 Barrett et al, 2003)

Dissect the neurotransmitter, synaptic machinery,
myelin system, etc, with molecular genetics
Neuroimaging will help both hypothesis generation
and validation of new genetic findings
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Cytoarchitectural abnormalities
Control
Comparison of hippocampal pyramids at the CA1 and
CA2 interface between control and
schizophrenic. Cresyl violet stain,
original magnification X250 Conrad et
al. (1991) Arch Gen
Psychiatry
Schizophrenia
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TARDIVE DYSKINESIA
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Symptoms of TD
TD is characterized by abnormal involuntary
movements of the lip, jaw and tongue. Choreoathet
oid movements of the extremities and/or trunk may
occur as well.
- Symptoms are measured using scales such as
the Abnormal Involuntary Movement Scale
(AIMS) and the Rockland Simpson Scale (RSS).
- The AIMS scale ranges from 0 - 40 and
patients with higher AIMS scores have more
severe symptoms.
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Motor effects
Cognitive effects
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Why DRD3 ?

• D3 mRNA and protein have been localized to the
  ventral side of the striatum and the ventral
  putamen (motor control) (Joyce MeadorWoodruff,
  1997)
• D3 receptors have been shown to have an
  inhibitory effect on locomotor activity in rats.
  (Kling-Peterson et al, 1995)
• Evidence that the Msc I polymorphism of DRD3 is
  functional allelic differences display different
  affinities for dopamine in vitro. (Lundstrom
  Turpin, 1996)

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Mean AIMS Scores for DRD3 Msc I Polymorphism
after Typical Neuroleptic Treatment
Mean 14.20
Corrected Mean AIMS score
Mean 3.920
Mean 3.470
n34
n53
n25
DRD3 Genotype
F2,95 8.25, p lt 0.0005 ( n 112 ), Power
0.568, r-square0.297 (Bonferroni p lt
0.0015) (Basile et al, 1999)
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Ethnically Stratified Means
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16
43
33
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F2,833.85, p 0.026 (Bonferroni p 0.078)
F1,238.10, p 0.0091 (Bonferroni p 0.009)
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Brain Metabolism Following Haloperidol Treatment
by D3 Genotype (FDG, n14)
Gly-Ser Ser-Ser (n9)
Haloperidol (5wks)
Baseline
Baseline
Gly-Gly (n5)
(UCI Brain Imaging Centre Potkin, Kennedy
Basile, 2003)
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CYP2D6 vs 1A2
(12.7)
- Metabolism is determined by both affinity and
abundance relative to the total liver P450
content. - 2D6 has a higher affinity for most
typicals, but it accounts for only 2 of total
liver content. ? 2D6 is high affinity-low
capacity -2D6 is not inducible 1A2 is
(Shimoda et al, 1994)
(1.5)
Note CYP2D6 also expressed in brain
neuroimaging may capture some of this variance
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Mean AIMS Scale Scores for DRD3 by CYP1A2 Genotype
Mean AIMS Score
CYP1A2 Genotype
DRD3 Genotype
Additive recessive model strongly supported
compared to alternative models
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Tardive Dyskinesia Summary
- Given the numerous replications of the DRD3-TD
finding, and the PET neuroimaging validation, it
appears that the dopamine D3 receptor is involved
in TD susceptibility. - The interaction between
DRD3 and CYP1A2 genes fits a recessive -
recessive model with each gene interacting
additively. - The DRD3 and CYP1A2 results
account for 55 of the variance in TD other
genes and environment may account for the
rest. ??Clinical genetic test for TD risk in the
future??
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DOPAMINE D1 RECEPTOR
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Dopamine D1 Receptor
D1 concentration in PFC 10X greater than D2
(Lidow 1991) Important for antipsychotic action?

(Missale 1998)
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Antipsychotic Binding Profiles
(Kerwin 1996, in vitro tissue analyses)
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Tauscher et al., 2004
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D1 Receptor Blockade

• D1 blockade by antipsychotics may potentiate
  activity in the PFC by disinhibiting NMDA
  receptor (Williams 1995)

D1
NMDA
Cognition?
Improvement of Symptoms
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Dopamine D1 Gene (DRD1) (5q35.1)
Markers in 5 upstream region may be implicated
in the regulation of D1 gene expression.
(-1251)
(-800)
(-48)
(1403)
P1
P2
ERE
Coding Region
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DRD1, PET FDG, Clozapine Response
Genotype 2/2 BPRS 30 Improvement
Genotype 1/2 BPRS 7 Worsening
Genotype 1/2
(Potkin et al, 2002)
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Clozapine Response Prediction Algorithm?

• Arranz et al (2000) have reported a multi-gene
  (n 10 markers) model for clozapine response in
  one sample. Includes D2, D4 5HT1A, 2A, 2C, 4,
  6 H1, H3,
• Model must be replicated in a new sample to be
  meaningful
• Neuroimaging as a augmenting phenotype in each
  patient may provide valuable intermediary
  information from the brain, allowing more
  biologically meaningful subtyping.

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Serotonin Transporter
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5HTT specific ligand in PET

• 5HTT specific ligand for PET 11CDASB optimized
  in Toronto by radiochemist Alan Wilson
• N 20 medication-free depressed patients, 20
  anxiety disorder, and 20 normal controls
  underwent PET imaging with 11CDASB ligand
  blood for genetics
• 5HTT gene typed for ins/del in promoter and the
  VNTR in intron II

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11C DASB Binding to 5HTT in Depressives vs
Normals by Genotype Frontal Region
nM
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PET Ligand (DASB) 5HTT Binding Potential
correlates with Dysfunctional Attitude
Meyer et al, Toronto PET Group, Arch Gen
Psych, 2004 5HTTLPR genetic marker does not
predict 5HTT Binding (Kennedy et al, in prep)
Serotonin Transporter Ligand Binding Potential
N20 Major Depressives P lt .001
Dysfunctional Attitude Scale Score
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Will the Brain Derived Neurotrophic Factor (BDNF)
Gene Predict Grey Matter Volume?
BDNF-1 SNP BDNF-2 BDNF-3 BDNF-4
Exon 11
Val-66-met
(GT)n repeat (function? mRNA stability)
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BDNF val66met MRI functional brain imaging (Egan
et al, Cell 2003)
The red/yellow areas indicate brain regions
(primarily hippocampus) that function differently
between val/val (n8) and val/met (n5) subjects
while performing a working memory task. Subjects
with the met allele had more abnormal function.
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Haplotype TDT BDNF (GT)n repeat val66met in
schizophrenia

HTDT for 170-val66 c2 7.11 1 df p
0.007 Muglia et al, (2002)
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Neurotrophic Mechanisms in Depression Nestler et
al, 2002
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Bipolar Disorder TDT studies show robust effect
of BDNF gene

• Combined Toronto sample (Neves-Pereira et al,
  2002 N300) plus MIT (Sklar et al, 2003) (N200
  150 NIMH) for val66met yields p .0000001
• Phenotype dissection of our Toronto bipolar
  sample shows association with rapid cycling,
  non-suicidal, non-psychotic, earlier age at onset
  subjects (Mueller et al, in preparation)

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BDNF polymorphisms in Childhood Onset Depression
Pittsburgh Sample
N104 pairs ?24.7df1 p0.03
Haplotype p 0.001
N104 pairs ?2 17.8 df5 p 0.0032 172 bp
allele OR 0.55 168 bp allele OR 3.94
GT repeat alleles
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Hippocampal shape as a phenotype for genetic
studies
Figure 1d Principal deformation for the right
hippocampus for normal controls (top) and
schizophrenia patients (bottom). Four views
(front, lateral, back, medial) of each shape are
shown. The color indicates the direction and the
magnitude of the deformation, changing from blue
(inwards) to green (no deformation) to red
(outwards).
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Will MOG gene variants predict white matter
abnormalities?
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Prefrontal fMRI activity and myelin reduced in
schizophrenia
Figure 31-4 Statistical parametric maps of the
fractional anisotropy (FA) (left) and Magnetic
Transfer Ratio (MTR) (myelin) (right) group
comparison. Similar areas in yellow on both maps
correspond to the location of both the internal
capsule and prefrontal white matter, and indicate
smaller values of FA and myelin in schizophrenia
patients (n14) compared with controls (n15).
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UNC
Fractional Anisotropy
Hypothesis MOG, MAG, MBP genes will predict
quantity or distribution of myelinated tracts
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DTI New MRI Imaging Technique Reveals Brain
Circuits
Cingulum
Corpus callosum
Dorsal stream
Frontal striatial projections
Fornix
Actual white matter tracks in schizophrenic
patient revealed by DTI (colors and location by
J. Fallon)
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Complexities in Genetics Neuroimaging

• Genetic variants express themselves in many ways
  singularly, or combined (haplotypes, epistasis,
  partial penetrance)
• What are the appropriate phenotypes to use from
  brain imaging data?
• How to control massive multiple testing of genome
  scan x brain voxels (millions x millions)?

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Summary

• D3 gene link to tardive dyskinesia validated by
  PET imaging
• D1 role in schizophrenia and clozapine response
  supported by genetic variants and PET activity
  pre/post clozapine
• BDNF gene candidate for grey matter measures?
• MOG gene candidate for white matter?
• Vast expanses of quality data await us we only
  need to develop our informatics sophistication
• National Alliance for Medical Imaging and
  Computing
• NAMIC
• www.na-mic.org

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