The power of gene expression profiling to unravel behaviour

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The power of gene expression profiling to unravel
behaviour
Cathy Fernandes, Jose Paya-Cano, Frans Sluyter,
Ursula D'Souza, Robert Plomin, Leonard C Schalkwyk
Social, Genetic and Developmental Psychiatry
Centre Institute of Psychiatry Kings College
London
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Outline

• Background

• Gene expression using the Affymetrix GeneChip
  system

• Hippocampal gene expression profiles across
  eight
• different inbred mouse strains

• Hippocampal gene expression and cognitive
  ability

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Microarrays, Mice Behavioural Genetics

• the interaction of multiple genes and their
  products
• a snapshot of the simultaneous gene expression
  across
• thousands of genes 

• mice are excellent models
• genetic overlap with humans
• differences in behaviour and gene expression
• genomic information
• access to fresh brain tissue

nominate new candidate genes for behaviour
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• Gene expression studies using microarrays
• Sandberg et al (2000)
• six brain regions in 129SvEv and C57BL/6
• 24 genes strain-specific expression across all
  brain regions
• (240 genes regional gene expression
  differences)
• re-analysis by Pavlidis and Noble (2001)
  identified many more
• genes with strain-specific (63 genes) and/or
  region-specific
• (600 genes) expression

 

• Gene expression profiles
• during development (Mody et al, 2001)
• resulting from ageing (Jiang et al 2001, Lee et
  al, 2000)
• behavioural manipulations (Leil et al, 2002)
• environmental manipulations (Rampon et al, 2000)
  

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Hippocampal gene expression profiling across
eight inbred mouse strains

•  
• AIMS
• determine how much gene expression is due to
  genetic variation
• to expand on the currently available gene
  expression data by
• increasing the number of mouse strains studied
• to find biologically relevant strain differences
  in gene
• expression, filtering out random individual
  differences
• to produce tightly controlled, replicated data
• reliable pattern of gene expression
• maximise detection of relatively small
  differences in expression

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• Selection of inbred strains
• Selected from Group A of the Mouse Phenome
  Database
• commonly used strains with available genetic and
  phenotypic
• information
• progenitors in transgenesis and mutagenesis
  studies
• progenitors of recombinant inbred, consomic and
  congenic
• strains

A/J BALB/cByJ C3H/HeJ DBA/2J 129S1/SvImJ

C57BL/6J
FVB/NJ SJL/J
Celera Mouse Genome Sequencing Projects
Public Mouse Genome Sequencing Projects

• differ in activity, exploration, anxiety,
  learning, aggression

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The role of the hippocampus

• key area of the brain involved in behaviours
  such as
• learning/memory and anxiety
•  
• discrete area and is of a sufficient size in the
  mouse to
• allow a precise and highly reproducible
  dissection
• - yield sufficient quantities of mRNA for
  microarray work
• strain-specific gene expression (Sandberg et al,
  2000)

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• Procedure
• male mice (6 per strain, 48 mice in total) from
  Jackson Laboratories (USA) aged 5-6 weeks
• acclimatised in our barrier facility for 8 weeks
  (singly housed)
• killed by cervical dislocation, in a randomised
  order,
• aged 13-14 weeks (over 3 days to minimise any
  effect of time of day)
• hippocampus was immediately dissected out, snap
  frozen on dry ice and stored at 80 0C
• dissections done by the same operator and
  completed within 1 minute for each mouse 

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• Procedure
• (contd)
• The following procedures were carried out to
  minimise stress to the mouse prior to killing
• minimal handling of mice
• transported to the procedure room in their home
• cage and killed within 3 minutes of transport
• method of kill
• killed by the same operator

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Analysis

• The data was analysed in parallel using
  Affymetrix
• MAS5 and Li and Wong PM-only model (dChip
  v1.2,
• Li and Wong 2001a)
• differ in methods used to summarise the probesets
  and for normalisation of the arrays

• 2. Signal values analysed in R
• (http//www.r-project.org/, Ihaka
  Gentleman 1996, Neuwirth Baier 2001)
• one-way ANOVA (results were filtered using a p
  value
• cut-off of 4 x 10-6 (p 0.05 following
  Bonferroni
• correction for 12,488 probesets)

3. Hierarchical clustering (Eisen, 1998) was
carried on the ANOVA filtered (p lt 4 x 10-6)
gene expression levels
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Results

• strain means for the probesets fit a normal
  distribution
• - 252 (MAS5) and 200 (dChip) probesets with p
  values for
• difference of lt 4x 10-6
• 100 probesets were identified in both analysis
  programs
• - discrepant probes most commonly are those of
  low signal
•  
• many of the strain differences due to up or
  down-regulation,
• rather than presence or absence, of the
  transcript

- the bulk of the probesets expression profiles
are very similar (pairwise correlations between
chips MAS5 0.894 - 0.997, dChip 0.901 - 0.997)
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Clustering
- numerous and clear strain differences in gene
expression
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Clustering(contd)
- strains cluster together
(in 10 different random permutation runs of the
strain factor to assess the false positive rate,
only two p-values lt 4 x 10-6 were found (i.e.
1000 fold fewer than with the real factor)
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Clustering(contd)

• among clusters of probesets, several reunite
  multiple probesets
• representing the same transcript
• for example, four caspase 9 probesets cluster
  together (more highly
• expressed in BALB/cByJ and C3H/HeJ)

• many can be identified which are biologically
  plausible
• for example, one striking cluster includes 5
  loci from the H2 region of chromosome 17 H2-d
  (3 probesets), H2-k, and Qa, which are expressed
  above the mean in FVB/NJ and DBA/2J (BUT does not
  correlate with the H2 haplotypes of the strains)

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Effect of gene mutation on expression

• increased expression of Alad in DBA/2J compared
  to
• C57BL/6J strain (gene is present in two copies
  in DBA
• and one in C57BL/6J), Claudio et al (1997)

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Effect of gene mutation on expression
- Gas5 gene is known to harbour mutations that
affect the stability of its mRNA transcript
in the 129 substrains (Muller et al 1998)
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Some potential candidates

• microtubule-associated protein tau has key
  structural functions and is essential to
  beta-amyloid-induced neurotoxicity
• preliminary data on protein levels (Western
  blots) support the
• expression RNA data (D'Alcontres and Hanger,
  Neuroscience, IoP)

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Some potential candidates

• a key bifunctional enzyme in the activation of
  neuropeptides
• gene maps to chromosome 1 at 57.5 cM (an
  ethanol-induced loss of
• righting reflex locus at chr 1, 43 and 59 cM)

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Some potential candidates
- Camk2a is implicated in the establishment of
long-term potentiation (Bejar et al 2002)
and spatial learning (Silva et al 1992, Giese et
al 1998)  - BUT does not correlate with learning
in these strains ?
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Correlation

• more and more phenotype data for inbred strains
  is available
• it may be possible to find meaningful
  correlations with expression data (WebQTL)
• similar to Grupe et al in silico genetic mapping
• shortcomings also resemble Grupe

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Aggression

• Consensus aggression ranking (intermale offensive
  aggression), Sluyter
• FVB/NJgt SJL/Jgt BALB/cByJgt C3H/HeJgt DBA/2Jgt
  C57BL/6Jgt 129S1/SvImJgt A/J
• Spearman correlation with our chip data

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COMT expression correlation
550
500
COMT
450
400
350
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2
3
4
5
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7
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Strain ranks

• link between low COMT activity and increased
  aggression in mice
• and humans (Gogos et al, 1998 Lachman et al,
  1998 Jones et al,
• 2001)

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Limitations
- biased towards detection of abundantly
expressed, well- characterised genes  - rare
transcripts, short half-life, alternative
splicing  
BUT low-abundance mRNAs or those expressed only
at very specific times in development and/or
processes may be key to determining the
behavioural phenotype

• - cellular heterogeneity
• polymorphisms may obscure differences or create
• spurious ones

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Multiple probesets
Results (contd)

• one third of the highly significant probesets
  have one or more additional probesets
  representing the same transcript
• compare or combine multiple probesets

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Schalkwyk et al 1999
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History of inbred strains - analysis of CIDR
data (http//www.cidr.jhmi.edu/) by Schalkwyk et
al (1999)
HS progenitor strains
Wagner parsimony analysis using MIX (Felsenstein
1988b) of microsatellite data (298 loci from all
19 autosomes and X) on 48 strains, transformed
into binary characters according to Schalkwyk et
al 1999, and using SPRET as outgroup. Internal
figures are the number of bootstrap replicates
out of 100 supporting each group. The overall
topology agrees with Schalkwyk (1999) except that
the C57 and 129 groups are reversed.
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Cheveruds take
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Witmer et al 2003
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New microsatellites
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