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, 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

3. AJ AKRBALBC3H C57 DBA CBA LP HS Random 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)

4. Power Calculation in HS: Percent Success for QTL Detection Percent variance explained by QTL 2.5 555 Significance level5%1%0.1%5%1%0.1% Population Size5001285655441 Population Size1000665245969087 Population Size2000907765100

5. Multiple Phenotypes 3-6000 molecular markers x 2000 mice = 6-12 million genotypes How can we make this project cost-effective?

6. Multiple Phenotypes Behavioral 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

7. WeekDayTest(s) 5MMicrochip, Ear Punch, Imm. Sample 6MOpen Field Test TElevated Plus Maze WFood Neophagia Th, FHome Cage Activity, Burrowing 7M-WFear Potentiated Startle ThContext Freezing FCue Conditioning, Plasma for CORT 8FPlethysmograph 9T, WGlucose Tolerance Test Th, FTissue Harvest Testing Order

8. For several of these phenotypes there are: Known phenotypic differences between progenitor strains of HS mice Previously identified QTL using HS progenitor inbred crosses

10. Time Spent in Open Arms of EPM Increased Variation in HS Inbred Differences

13. Response to Metacholine in Plethysmograph Inbred Differences Increased Variation in HS

14. Glucose Tolerance Test Inbred Differences Increased Variation in HS

15. 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

16. We Need a Database!

17. Integrated Genotyping System Subjects (pedigrees) Phenotypes (multivariate, covariates) Markers (SNPs, microsatellites) Genotypes (multiple observations, editing) (see poster)

18. IGS: Phenotypes

20. 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

21. 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

24. 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

25. 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

26. Fine-resolution mapping using HS Each chromosome is a random mosaic of the founders

27. Glucose Tolerance Test Inbred Differences Increased Variation in HS

28. Post-Stress Plasma Corticosterone Inbred Differences Increased Variation in HS

29. Phenotypes

31. Bioinformatics: Analysis HAPPY (http://www.well.ox.ac.uk/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)

32. Need for a Database TestNumber of Phenotype Elements Number of Mice Total Data Points EPM10200020,000 PG8200016,000 Glucose420008,000 In TOTAL: 21 Phenotypes, 87 Phenotype elements 174,000 Data Points