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

• Hippocampal gene expression and cognitive ability

• Background

• Gene expression using the Affymetrix GeneChip system

• Hippocampal gene expression profiles across eight different inbred mouse strains

Outline

• the interaction of multiple genes and their products

• a snapshot of the simultaneous gene expression across thousands of genes 

Microarrays, Mice & Behavioural Genetics

• 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 strainsSelected 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

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

* Celera Mouse Genome Sequencing Projects# Public Mouse Genome Sequencing Projects

- 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)

The role of the hippocampus

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

- 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)

Results

Clustering

- numerous and clear strain differences in gene expression

Clustering(contd)

(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)

- strains cluster together

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

- 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)

Alad (aminolevulinate, delta-, dehydratase)

0

50

100

150

200

250

300

129 A BALB C3H C57BL DBA FVB SJL

Strains

MAS

5 ex

pres

sion

leve

l

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)

Gas5(growth arrest specific 5)

0

200

400

600

800

1000

129 A BALB C3H C57 DBA FVB SJL

Strains

Mas

5 ex

pres

sion

leve

l

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)

Mapt(microtubule-associated protein tau)

0

200

400

600

800

1000

1200

129 A BALB C3H C57 DBA FVB SJL

Strains

MAS

5 ex

pres

sion

leve

l

Pam (peptidylglycine alpha-amidating monooxygenase)

0.0

50.0

100.0

150.0

200.0

129 A BALB C3H C57BL DBA FVB SJL

Strains

MAS

5 ex

pres

sion

leve

l

- 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)

Some potential candidates

Camk2a( calcium/calmodulin-dependent protein kinase II alpha)

0100200300400500600700

129 A BALB C3H C57 DBA FVB SJL

Strains

MAS

5 ex

pres

sion

leve

l

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 ?

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 rho pval probesetcatechol-O-methyltransferase 0.90 0.0020 98535_atfibroblast growth factor 1 0.93 0.0009 100494_at

COMT expression correlation

1 2 3 4 5 6 7 8

350

400

450

500

550

Strain ranks

CO

MT

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

- 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

Limitations

- cellular heterogeneity

- polymorphisms may obscure differences or create spurious ones

Results (contd)

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

• compare or combine multiple probesetsMicrotubule-associated protein tau

02004006008001000120014001600

129 A BALB C3H C57 DBA FVB SJL

Strains

DCHI

P ex

pres

sion

leve

l

Caspase 9

020406080100120140160

129 A BALB C3H C57 DBA FVB SJL

Strains

MAS

5 ex

pres

sion

leve

l

Carbonic anhydrase 14

0

100

200

300

400

500

600

700

129 A BALB C3H C57 DBA FVB SJL

Strains

DCHI

P ex

pres

sion

leve

l

Multiple probesets

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

SKIVE/Ei

MOLF/Ei

MOLG/Dn100

9855

PERC/Ei

PERA/Rk100

C58/J

C57BR/cdJ

C57L/J100

C57BL/10J

RF/J

C57BL/6J100

100

80

100

BTBR+Ttf/t

LP/J

129T2/SvEm

129X1/SvJ51

129P3/J

129S2/SvPa

129S6/SvEv98

94

100

100

100

KK/HlJ

RIIIS/J30

NZW/LacJ

NZB/BlNJ99

MRL/MpJ

AKR/J74

BUB/BnJ

NOD/LtJ78

14

ST/bJ

NON/LtJ

SJL/J

SWR/J

FVB/NJ85

7558

34

9

A/J

BALB/cJ96

SM/J

P/J

BDP/J100

69

I/LnJ

DBA/2J

DBA/1J100

34

23

CE/J

CBA/J

CBA/CaJ88

SF/CamEi

C3H/HeJ

C3HeB/FeJ100

57

84

17

9

22

21

11

13

26

46

100

100

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

10

SM/J

NOD/LtJ74 ISS/IbgC57BL/6ByJ

C57BL/6J

100

35

ILS/Ibg

P/J

I/LnJ

78

27

DBA/2J

CBA/JC3H/HeJ

94

96

4513

NZB/BINJ

129X1/SvJBTBR

367 5

PL/J

AKR/J

RF/J

99

77

FVB/NJBUB/BnJ

81

23

RIIIS/JKK/HIJ

2341648

BALB/cByJ

BALB/cJ

100LP/J

A/J100

97

MI6i/Pomp

NON/LtJ

45

LG/J

NZW/Wehi

NZO/Wehi

94

30

SJL/JSWR/J

100

28

WSB/Ei

PERA/EiPERC/Ei

ZALENDE/Ei

TIRANO/Ei

100

CZECHII/Ei

PWK/Ph SKIVE/Ei

76

73

MOLF/Ei

JF/1

MSM

100

99

99

CASA/Ei

CAST/Ei

99

77

SPRET/Ei

PANCEVO/Ei

100

10045

3537

100

1884

PANCEVO/EiSPRET/EiCZECHII/EiPWK/PhSKIVE/Ei

7673

MOLF/EiJF/1MSM

10099

99

CASA/EiCAST/Ei

99

77

PERC/EiPERA/EiWSB/EiSM/JNOD/LtJ

74

ISS/IbgC57BL/6ByJC57BL/6J

10035

ILS/IbgP/JI/LnJ

7827

DBA/2JCBA/JC3H/HeJ

9496

45

13

NZB/BINJ129X1/SvJBTBR

367

5

PL/JAKR/JRF/J

9977

FVB/NJBUB/BnJ

81

23

RIIIS/JKK/HIJ

23

4

16

48

BALB/cByJBALB/cJ

100

LP/JA/J

10097

84

MI6i/PompNON/LtJ

45

18

LG/JNZW/WehiNZO/Wehi

9430

SJL/JSWR/J

100

28

100

37

35

45

ZALENDE/EiTIRANO/Ei

100

100

100

100

100

PANCEVO/EiSPRET/Ei

CZECHII/EiPWK/PhSKIVE/Ei

7673

MOLF/EiJF/1MSM

10099

99

CASA/EiCAST/Ei

99

77

PERC/EiPERA/Ei

WSB/EiSM/JNOD/LtJ

74

ISS/IbgC57BL/6ByJC57BL/6J

10035

ILS/IbgP/JI/LnJ

7827

DBA/2JCBA/JC3H/HeJ

9496

45

13

NZB/BINJ129X1/SvJBTBR

367

5

PL/JAKR/JRF/J

9977

FVB/NJBUB/BnJ

81

23

RIIIS/JKK/HIJ

23

4

16

48

BALB/cByJBALB/cJ

100

LP/JA/J

10097

84

MI6i/PompNON/LtJ

45

18

LG/JNZW/WehiNZO/Wehi

9430

SJL/JSWR/J

100

28

100

37

35

45

ZALENDE/EiTIRANO/Ei

100

100

100

100