Transcript Document

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
King’s College London
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
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
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)
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
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
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)
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
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
Analysis
1. 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 < 4 x 10-6) gene expression levels
Results
- strain means for the probesets fit a normal distribution
- 252 (MAS5) and 200 (dChip) probesets with p values for
difference of < 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)
Clustering
- numerous and clear strain differences in gene expression
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 < 4 x 10-6 were found (i.e. 1000 fold fewer than
with the real factor)
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)
Effect of gene mutation on expression
MAS5 expression level
Alad
(aminolevulinate, delta-, dehydratase)
300
250
200
150
100
50
0
129
A
BALB
C3H
C57BL
DBA
FVB
Strains
- 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)
SJL
Effect of gene mutation on expression
Mas5 expression level
Gas5
(growth arrest specific 5)
1000
800
600
400
200
0
129
A
BALB
C3H
C57
DBA
FVB
Strains
- Gas5 gene is known to harbour mutations that affect the
stability of its mRNA transcript in the 129 substrains
(Muller et al 1998)
SJL
Some potential candidates
MAS5 expression level
Mapt
(microtubule-associated protein tau)
1200
1000
800
600
400
200
0
129
A
BALB
C3H
C57
DBA
FVB
SJL
Strains
- 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)
Some potential candidates
MAS5 expression level
Pam
(peptidylglycine alpha-amidating monooxygenase)
200.0
150.0
100.0
50.0
0.0
129
A
BALB
C3H
C57BL
DBA
FVB
Strains
- 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)
SJL
Some potential candidates
Camk2a
MAS5 expression level
( calcium/calmodulin-dependent protein kinase II alpha)
700
600
500
400
300
200
100
0
129
A
BALB
C3H
C57
DBA
FVB
SJL
Strains
- 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 ?
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
Aggression
• Consensus aggression ranking (intermale offensive
aggression), Sluyter:
FVB/NJ> SJL/J> BALB/cByJ> C3H/HeJ> DBA/2J> C57BL/6J> 129S1/SvImJ> A/J
• Spearman correlation with our chip data:
name
catechol-O-methyltransferase
fibroblast growth factor 1
rho
pval
probeset
0.90 0.0020 98535_at
0.93 0.0009 100494_at
500
450
350
400
COMT
550
COMT expression correlation
1
2
3
4
5
6
7
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)
8
Limitations
- biased towards detection of abundantly expressed, wellcharacterised 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
Results
Multiple
probesets
(contd)
• one third of the highly significant probesets have one or more
additional probesets representing the same transcript
• compare or combine multiple probesets
Caspase 9
MAS5 expression level
1600
1400
1200
1000
800
600
400
200
0
129
A
BALB
C3H
C57
DBA
FVB
160
140
120
100
80
60
40
20
0
SJL
129
A
BALB
Strains
C3H
C57
Strains
Carbonic anhydrase 14
DCHIP expression level
DCHIP expression level
Microtubule-associated protein tau
700
600
500
400
300
200
100
0
129
A
BALB
C3H
C57
Strains
DBA
FVB
SJL
DBA
FVB
SJL
Schalkwyk et al 1999
History of inbred strains
- analysis of CIDR data (http://www.cidr.jhmi.edu/) by Schalkwyk et al (1999)
SPRET/Ei
CAST/Ei
55
SKIVE/Ei
98
MOLF/Ei
100
MOLG/Dn
PERC/Ei
100
PERA/Rk
100
C58/J
C57BR/cdJ
100
C57L/J
100
100
80
C57BL/10J
100 RF/J
100
C57BL/6J
BTBR+Ttf/t
46
LP/J
100
100
51
100
129X1/SvJ
129P3/J
94
98
26
30
99
13
74
14
78
11
129T2/SvEm
9
129S2/SvPa
129S6/SvEv
KK/HlJ
RIIIS/J
NZW/LacJ
NZB/BlNJ
MRL/MpJ
AKR/J
BUB/BnJ
NOD/LtJ
ST/bJ
34
NON/LtJ
58
SJL/J
75
85
21
96
SWR/J
FVB/NJ
A/J
BALB/cJ
SM/J
69
P/J
100
BDP/J
34
DBA/2J
100
DBA/1J
22
23
I/LnJ
9
CE/J
17
88
84
CBA/J
CBA/CaJ
SF/CamEi
57
C3H/HeJ
100
C3HeB/FeJ
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.
Cheverud’s take
Witmer et al 2003
New microsatellites
SKIVE/Ei
PWK/Ph
MOLF/Ei
CZECHII/Ei
JF/1
76
TIRANO/Ei
100
73
ZALENDE/Ei
99
MSM
PERC/Ei
PERA/Ei
SWR/J
WSB/Ei
SJL/J
99
100
99
100
NZO/Wehi
CASA/Ei
77
45
35
CAST/Ei
100
37
NZW/Wehi
94 100
SPRET/Ei
100
LG/J
NON/LtJ
30
28
4518 84
97
MI6i/Pomp
A/J
PANCEVO/Ei
100
48
16
47 513 35
23
23
74
45
36
100
LP/J
81 77
NOD/LtJ
ISS/Ibg
27
ILS/Ibg C57BL/6J
NZB/BINJ
BALB/cByJ BUB/BnJ
FVB/NJ
99
BTBR 129X1/SvJ
94
PL/J
P/J
I/LnJ
DBA/2J
RF/J
AKR/J
CBA/J
C3H/HeJ
10
C57BL/6ByJ
100
96 78
KK/HIJ
RIIIS/J
BALB/cJ
SM/J
100
45
35
100
48
5
37
16
84
100
18
100
PANCEVO/Ei
SPRET/Ei
CZECHII/Ei
73 PWK/Ph
76
SKIVE/Ei
99
MOLF/Ei
99 JF/1
77
100
MSM
CASA/Ei
99
CAST/Ei
PERC/Ei
PERA/Ei
WSB/Ei
SM/J
74
NOD/LtJ
ISS/Ibg
35 C57BL/6ByJ
100
C57BL/6J
13
ILS/Ibg
27 P/J
78
I/LnJ
45
DBA/2J
96 CBA/J
94
C3H/HeJ
NZB/BINJ
7 129X1/SvJ
36
BTBR
PL/J
77 AKR/J
99
RF/J
23
FVB/NJ
81
4
BUB/BnJ
RIIIS/J
23
KK/HIJ
BALB/cByJ
100
BALB/cJ
97
LP/J
100
A/J
MI6i/Pomp
45
NON/LtJ
LG/J
30 NZW/Wehi
94
NZO/Wehi
28
SJL/J
100
SWR/J
ZALENDE/Ei
100
TIRANO/Ei
PANCEVO/Ei
SPRET/Ei
CZECHII/Ei
PWK/Ph
SKIVE/Ei
MOLF/Ei
99
JF/1
100
MSM
CASA/Ei
CAST/Ei
PERC/Ei
PERA/Ei
WSB/Ei
73
76
99
77
99
100
35
100
37
100
100
100
SM/J
NOD/LtJ
ISS/Ibg
35
C57BL/6ByJ
100
C57BL/6J
13
ILS/Ibg
27
P/J
78
48
I/LnJ
45
DBA/2J
5
96
CBA/J
94
C3H/HeJ
NZB/BINJ
7
129X1/SvJ
36
BTBR
16
PL/J
84
77
AKR/J
99
RF/J
23
FVB/NJ
81
4
BUB/BnJ
RIIIS/J
23
KK/HIJ
18
BALB/cByJ
100
BALB/cJ
97
LP/J
100
A/J
100
MI6i/Pomp
45
NON/LtJ
LG/J
30
NZW/Wehi
94
NZO/Wehi
28
SJL/J
100
SWR/J
ZALENDE/Ei
TIRANO/Ei
74
45