a multi-phenotype protocol for fine scale mapping of qtl in outbred heterogeneous stock mice lc...
TRANSCRIPT
![Page 1: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/1.jpg)
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
![Page 2: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/2.jpg)
![Page 3: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/3.jpg)
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)
![Page 4: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/4.jpg)
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
![Page 5: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/5.jpg)
Multiple Phenotypes
3-6000 molecular markers x 2000 mice = 6-12 million genotypes
How can we make this project cost-effective?
![Page 6: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/6.jpg)
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
![Page 7: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/7.jpg)
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
![Page 8: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/8.jpg)
For several of these phenotypes there are:
• Known phenotypic differences between progenitor strains of HS mice
• Previously identified QTL using HS progenitor inbred crosses
![Page 9: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/9.jpg)
![Page 10: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/10.jpg)
Time Spent in Open Arms of EPM
0
1
2
3
4
5
6
7
8
10 20 30 40 50 60 70 80 90 100 110
Time (sec)
Nu
mb
er o
f A
nim
als
HS
Increased Variation in HS
0
1
2
3
4
5
10 20 30 40 50 60 70 80 90 100 110
Time (sec)
Nu
mb
er
of
An
ima
ls
DBA
C57
Inbred Differences
![Page 11: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/11.jpg)
![Page 12: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/12.jpg)
![Page 13: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/13.jpg)
Response to Metacholine in Plethysmograph
Inbred Differences
0
1
2
3
4
2 2.5 3 3.5 4 4.5 5 More
PenH Response
Nu
mb
er o
f A
nim
als
DBA
C57
Increased Variation in HS
0
1
2
3
4
2 2.5 3 3.5 4 4.5 5 More
PenH Response
Nu
mb
er o
f A
nim
als
HS
![Page 14: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/14.jpg)
Glucose Tolerance TestInbred Differences
5
10
15
20
25
30
35
40
45
0 10 20 30 40 50
time (min)
glu
co
se
(m
g/d
l)
C57
DBA
Increased Variation in HS
5
10
15
20
25
30
35
40
45
0 10 20 30 40 50
time (min)
glu
co
se
(m
g/d
l)
HS
![Page 15: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/15.jpg)
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
![Page 16: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/16.jpg)
We Need a Database!
![Page 17: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/17.jpg)
Integrated Genotyping System
• Subjects (pedigrees)
• Phenotypes (multivariate, covariates)
• Markers (SNPs, microsatellites)
• Genotypes (multiple observations, editing)
(see poster)
![Page 18: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/18.jpg)
IGS: Phenotypes
![Page 19: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/19.jpg)
IGS: Phenotypes
![Page 20: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/20.jpg)
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
![Page 21: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/21.jpg)
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
![Page 22: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/22.jpg)
![Page 23: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/23.jpg)
![Page 24: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/24.jpg)
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
![Page 25: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/25.jpg)
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
![Page 26: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/26.jpg)
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
![Page 27: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/27.jpg)
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
35
40
45
0 10 20 30 40 50
time (min)
glu
cose
(m
g/d
l)
![Page 28: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/28.jpg)
Post-Stress Plasma Corticosterone
Inbred Differences
0
1
2
3
4
5
6
40 60 80 100 120 140 160 180
Corticosterone (ng/ml)
Nu
mb
er
of
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
er
of
an
ima
ls
HS
![Page 29: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/29.jpg)
Phenotypes
![Page 30: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/30.jpg)
![Page 31: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/31.jpg)
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)
![Page 32: 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,](https://reader038.vdocuments.us/reader038/viewer/2022110322/56649d125503460f949e59cd/html5/thumbnails/32.jpg)
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