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

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Increased Variation in HS

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Inbred Differences

Response to Metacholine in Plethysmograph

Inbred Differences

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PenH Response

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Glucose Tolerance TestInbred Differences

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Increased Variation in HS

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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

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Post-Stress Plasma Corticosterone

Inbred Differences

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Increased Variation in HS

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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