Transcript A multi-phenotype protocol for fine scale mapping of QTL

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
Heterogeneous Stock (HS) mice
AJ
AKR
BALB
C3H
C57
DBA
CBA
LP
HS
Random Breeding
>40 Generations
Each chromosome is a random mosaic of the founders
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
Significance level
5%
1% 0.1% 5%
2.5
5
5
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
3-6000 molecular markers x 2000 mice =
6-12 million genotypes
How can we make this project cost-effective?
Multiple Phenotypes
Multiple Phenotypes
Behavioral Anxiety
•Open Field Test
•Elevated Plus Maze
•Food Neophagia
•Fear Potentiated Startle
Lung Function (Asthma)
•Plethysmograph
Other
•Corticosterone (post-stress)
•Electrolyte measurements
Metabolic Function (Diabetes) •Haematology
•Glucose Tolerance
•Immunology
•Insulin Sensitivity
•Mandible shape
•Adiposity Index
•Wound Healing
•Tissue Collection
Testing Order
Week
5
Day
M
Test(s)
Microchip, Ear Punch, Imm. Sample
6
M
T
W
Th, F
Open Field Test
Elevated Plus Maze
Food Neophagia
Home Cage Activity, Burrowing
7
M-W
Th
F
Fear Potentiated Startle
Context Freezing
Cue Conditioning, Plasma for CORT
8
9
F
T, W
Th, F
Plethysmograph
Glucose Tolerance Test
Tissue Harvest
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
Inbred Differences
Number of Animals
5
4
3
DBA
2
C57
1
0
10
20
30
40
50
60
70
80
90
100
110
Time (sec)
Increased Variation in HS
8
Number of Animals
7
6
5
HS
4
3
2
1
0
10
20
30
40
50
60
Tim e (sec)
70
80
90
100
110
Response to Metacholine in Plethysmograph
Inbred Differences
Number of Animals
4
3
DBA
2
C57
1
0
2
2.5
3
3.5
4
4.5
5
More
PenH Response
Increased Variation in HS
Number of Animals
4
3
HS
2
1
0
2
2.5
3
3.5
4
PenH Response
4.5
5
More
Glucose Tolerance Test
Inbred Differences
45
glucose (mg/dl)
40
35
DBA
30
C57
25
20
15
10
5
0
10
20
30
40
50
time (min)
Increased Variation in HS
45
glucose (mg/dl)
40
35
30
HS
25
20
15
10
5
0
10
20
30
time (min)
40
50
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
• Validated mouse models of human
quantitative traits
• Inexpensive to test
• Similarity with the human genome
How do we measure anxiety in mice?
• Phenotypic correlation between open field
activity and defecation defines emotionality
Open field arena
Low Ambulation
+
High defecation
Anxious Mouse
• Emotionality in rodents is a good measure for susceptibility to human
anxiety (Green and Hodges, 1991; Ramos and Mormede, 1998)
=
Fine-resolution mapping using HS
AJ
AKR
Balb
C3H
C57
DBA
IS
HS
Random Breeding
HS Generation > 60
Each chromosome is a random mosaic of the founders
RIII
Glucose Tolerance Test
Inbred Differences
45
glucose (mg/dl)
40
35
30
25
20
15
10
5
0
0
5
10
15
20
25
30
35
40
45
50
tim e (m in)
Increased Variation in HS
glucose (mg/dl)
45
40
35
30
25
20
15
10
5
0
0
10
20
30
tim e (m in)
40
50
Post-Stress Plasma Corticosterone
Inbred Differences
6
Number of animals
5
DBA
4
C57
3
2
1
0
40
60
80
100
120
140
160
180
Corticosterone (ng/ml)
Number of animals
Increased Variation in HS
9
8
7
6
5
4
3
2
1
0
HS
40
60
80
100
120
Corticosterone (ng/ml)
140
160
180
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 Mice
EPM
Number of
Phenotype
Elements
10
2000
Total
Data
Points
20,000
PG
8
2000
16,000
Glucose
4
2000
8,000
In TOTAL:
21 Phenotypes, 87 Phenotype elements
174,000 Data Points