A multi-phenotype protocol for fine scale mapping of QTL in outbred
heterogeneous stock mice
LC Solberg, C Arboledas, P Burns, S Davidson, G Nunez, A Taylor, W Valdar, R Deacon, D Bannerman, W
Cookson, D Gauguier, JNP Rawlins, R Mott, J Flint
University of Oxford, Wellcome Trust Centre for Human Genetics
AJ AKR BALB C3H C57 DBA CBA LP
HSRandom Breeding>40 Generations
Each chromosome is a random mosaic of the founders
Heterogeneous Stock (HS) mice
Northport HS founded by Robert Hitzemann (Demarest et al., 2001)
Power Calculation in HS: Percent Success for QTL Detection
Percent variance explained by QTL
2.5 2.5 2.5 5 5 5
Significance level 5% 1% 0.1% 5% 1% 0.1%
Population Size 500 12 8 5 65 54 41
Population Size 1000 66 52 45 96 90 87
Population Size 2000 90 77 65 100 100 100
Multiple Phenotypes
3-6000 molecular markers x 2000 mice = 6-12 million genotypes
How can we make this project cost-effective?
Multiple PhenotypesBehavioral Anxiety•Open Field Test•Elevated Plus Maze•Food Neophagia•Fear Potentiated Startle
Lung Function (Asthma)•Plethysmograph
Metabolic Function (Diabetes)•Glucose Tolerance•Insulin Sensitivity•Adiposity Index
Other•Corticosterone (post-stress)•Electrolyte measurements•Haematology•Immunology•Mandible shape•Wound Healing•Tissue Collection
Week Day Test(s)
5 M Microchip, Ear Punch, Imm. Sample
6 M Open Field Test
T Elevated Plus Maze
W Food Neophagia
Th, F Home Cage Activity, Burrowing
7 M-W Fear Potentiated Startle
Th Context Freezing
F Cue Conditioning, Plasma for CORT
8 F Plethysmograph
9 T, W Glucose Tolerance Test
Th, F Tissue Harvest
Testing Order
For several of these phenotypes there are:
• Known phenotypic differences between progenitor strains of HS mice
• Previously identified QTL using HS progenitor inbred crosses
Time Spent in Open Arms of EPM
0
1
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10 20 30 40 50 60 70 80 90 100 110
Time (sec)
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HS
Increased Variation in HS
0
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10 20 30 40 50 60 70 80 90 100 110
Time (sec)
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An
ima
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DBA
C57
Inbred Differences
Response to Metacholine in Plethysmograph
Inbred Differences
0
1
2
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2 2.5 3 3.5 4 4.5 5 More
PenH Response
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Increased Variation in HS
0
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2 2.5 3 3.5 4 4.5 5 More
PenH Response
Nu
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HS
Glucose Tolerance TestInbred Differences
5
10
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20
25
30
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40
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0 10 20 30 40 50
time (min)
glu
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se
(m
g/d
l)
C57
DBA
Increased Variation in HS
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0 10 20 30 40 50
time (min)
glu
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g/d
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HS
21 Phenotypes, 90 Phenotype Elements
200 Phenotype Elements and Covariates * 2000 mice = 400,000 Data Points
3-6,000 Molecular Markers * 2000 mice =6-12 Million Genotypes
We Need a Database!
Integrated Genotyping System
• Subjects (pedigrees)
• Phenotypes (multivariate, covariates)
• Markers (SNPs, microsatellites)
• Genotypes (multiple observations, editing)
(see poster)
IGS: Phenotypes
IGS: Phenotypes
Future Work
• Genotyping– 3-6,000 SNPs and microsatellites – sub-centimorgan spacing across entire genome
• Statistical Analysis– Dynamic programming using ancestral
haplotypes (HAPPY)– Statistical modeling in R
• Gene Identification
Conclusions
• Genetic heterogeneity of HS mice make them ideal for fine mapping QTL
• We are able to collect data accurately for multiple phenotypes from a large number of HS mice
• We have developed a database to store all phenotypic and genotypic information
• Data collected from this study will be used to search for genes involved in all phenotypes measured
The mouse is an ideal animal model
• Genetically well-defined strains
• Control of environmental factors
• Similarity with the human genome
• Inexpensive to test
• Validated mouse models of human quantitative traits
How do we measure anxiety in mice?
Open field arena
• Emotionality in rodents is a good measure for susceptibility to human anxiety (Green and Hodges, 1991; Ramos and Mormede, 1998)
Low Ambulation + High defecation = Anxious Mouse
• Phenotypic correlation between open field activity and defecation defines emotionality
Fine-resolution mapping using HS
AJ AKR Balb C3H C57 DBA IS RIII
HS
Random Breeding
HS Generation > 60
Each chromosome is a random mosaic of the founders
Glucose Tolerance Test
Inbred Differences
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25 30 35 40 45 50
time (min)
glu
cose
(m
g/d
l)
Increased Variation in HS
0
5
10
15
20
25
30
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0 10 20 30 40 50
time (min)
glu
cose
(m
g/d
l)
Post-Stress Plasma Corticosterone
Inbred Differences
0
1
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40 60 80 100 120 140 160 180
Corticosterone (ng/ml)
Nu
mb
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an
ima
ls
DBA
C57
Increased Variation in HS
0
12
34
56
78
9
40 60 80 100 120 140 160 180
Corticosterone (ng/ml)
Nu
mb
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an
ima
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HS
Phenotypes
Bioinformatics: Analysis
• HAPPY (http://www.well.ox.ac.uk/happy)• for each mouse, calculates the probability of
descent from each HS founder at each locus by dynamic programming
• test for QTL = test for differences between HS founder effects
• HAPPY now integrated into R:• dynamic-programming in C to compute probabilities• full range of R analyses available (multivariate,logistic
regression etc)
Need for a Database
Test Number of Phenotype Elements
Number of Mice
Total Data
Points
EPM 10 2000 20,000
PG 8 2000 16,000
Glucose 4 2000 8,000
In TOTAL:21 Phenotypes, 87 Phenotype elements
174,000 Data Points