1. Regions of the Canine Genome Associated with Osteosarcoma Identified by a Whole Genome Case-Control Association Study Kenine E Comstock, Elinor Karlsson, Claire Wade, Evan T Keller, Eric Lander, Laurence Baker, Kerstin Lindblad-Toh University of Michigan, Ann Arbor & Broad Institute, Boston, USA

2. Osteosarcoma in pet dogs and humans Common Features 1. Primary tumor occurs on weight bearing limbs 2. Tumor growth with an intact immune system 3. Similarities in histology 4. Location of metastases 5. Common environment Differences 1.Age of onset 2.Incidence in population

3. Dogs and humans: Similarities in cancer genetics The first example: Renal cancer syndrome Canine Hereditary Multifocal Renal Cystadenocarcinoma and Nodular Dermatofibrosis (RCND) German Shepherds Autosomal dominant Similar in phenotype to human Birt-Hogg Dube syndrome. We showed that mutations in the same gene in human and dogs were associated with both diseases. RCND-affected RCND-unaffected Lingaas and Comstock, et al. Hum Mol Genet. 2003 12(23):3043-53

4. Cancer in dogs is highly breed-specific CancerBreedIncreased risk HemangiosarcomaGolden Retriever5 Lymphoma, T-cellBoxer4 MelanomaScottish Terrier6 Mammary TumorSpringer Spaniel5 OsteosarcomaGreyhound Rottweiler 10 5

5. Advantages of canine models for mapping cancer genes Large families, inbred, good genealogical records, relatively short generation times Disease progression accelerated. Genetic similarity within breeds: cancer is associated with fewer mutations than in the species as a whole.

6. Methods for mapping genes 1. Linkage mapping using families Best for rare, high penetrance diseases 2. Association mapping For complex traits Compares cases vs controls No need for families Markers: SNPs 100,000 SNPs or more for humans Only ~30,000 SNPs in dogs! Can narrow associated regions to kilobases

7. Resources available for Canine Disease Gene Mapping 7x Canine Genome Sequence-released July 2005* Comparative maps of human and dog chromosomes SNP mapping set *Lindblad-Toh et al. Nature (2005) 438:803-19

8. 2.5 million SNPs discovered (1 SNP/kb) Boxer Poodle SNP discovery in 11 breeds and 5 wild canids Alaskan Malamute English Shepherd Bedlington Terrier Beagle German Shepherd Italian Greyhound Labrador Retriever Portuguese Waterdog Rottweiler China gray wolf Alaska gray wolf India gray wolf Spanish gray wolf California Coyote

9. Dog SNP Chip for whole genome scans Designed and tested by Michigan/Broad group in collaboration with Affymetrix ~26,500 optimized SNP Mapping Set Now commercially available Coverage Chromosome1Mb ≥ 5 SNPs Autosomes97% Chrom X42%

10. Power analysis: Prediction of sample size needed *In humans, would need approx 800 + 800 *

11. Can we map real traits using this SNP marker set? Yes! Power calculations appear correct Predicted low sample numbers work One significant associated region * Elinor Karlsson and Claire Wade, Broad Institute Mendelian Trait Co-DominantRecessiveDominant Cases+Controls10+97+1127+40 LOD535 Region Size (kb)800 300 Genes173

12. Objectives: 1.To identify genes involved with osteosarcoma susceptibility in dogs 2.To map a complex trait using the new SNP set.

13. Large and giant breed dogs have a higher risk of osteosarcoma Greyhound-5 to 10 X Irish Wolfhound Great Dane Scottish Deerhound Rhodesian Ridgeback Great Pyrenees Borzoi Saint Bernard Rottweiler-5 X increased risk Labrador Retriever Flat-coated Retriever Golden Retriever Mastiff Bullmastiff Newfoundland 1) Mastiff-type breeds 2) Long-limbed breeds

14. Eligibility Criteria Cases: Dogs diagnosed with osteosarcoma Cases were confirmed by either radiography and/or by biopsy. All cases are being confirmed by a single independent pathologist. Controls: Healthy, cancer-free dogs 10 years and older for Greyhounds, 8 years and older for all other breeds. Owners of control dogs are contacted regularly. All dogs of a single breed in the study are unrelated through the grandparents.

15. Study participants enrolled Table 1: Samples collected as of May 1, 2006 BreedBreed groupOSA cases Controls (8 years and older) RottweilerMastiff9174 Golden retrieverMastiff29370 LeonbergerMastiff2651 GreyhoundLong-limbed82151 (*10 yrs and older) Irish WolfhoundLong-limbed1211 Great DaneLong-limbed2233 All other breedsNA49189

16. Haplotype The series of alleles along a chromosome inherited by an individual from one parent. Haplotype  The series of alleles along a chromosome inherited by an individual from one parent. Number of generations since common ancestor Number of founders Shared Haplotypes Within a breed (Corgi-Corgi) few large Between breeds (Corgi-Irish wolfhound many small Relationship between haplotype size and breed Irish wolfhound Corgi

17. Stage 1: OSA susceptibility gene mapping in each breed separately We’ve started with Rottweilers: Searched genome for 0.5-1 Mb disease-associated haplotypes 1 / 2 Mb-1Mb OSA+ xxxx

18. Stage 2: Fine mapping across dog breeds within a breed group Add related breeds to narrow the region to a smaller, shared ancestral haplotype (~10-20kb). We are just getting started with this!  10-20kb {{ Golden retriever (OSA+) Leonberger (OSA+) “OSA” gene

19. Whole Genome Scan in Rottweilers: Whole Genome Scan in Rottweilers: Likelihood of an association Location in the genome

20. Regions associated with osteosarcoma in Rottweilers -log(10) of the p-value Localized to three haplotype blocks Association performed using Haploview p< 0.01 with 100,000 permutations. ACB SNPsSNPs grouped into 4 gamete “haplotype” blocks

21. Currently ongoing… Screening for mutations Genome scan in greyhounds Fine mapping in related breeds

22. Acknowledgements Broad Institute Elinor Karlsson Claire Wade Tara Biagi Sara Fryc Noriko Tonomura Kerstin Lindblad-Toh Collaborators Frode Lingaas (Oslo) Guillermo Couto (Ohio) Jaime Modiano (Denver) Elaine Ostrander (NHGRI) Bill Kisseberth (Ohio) Cheryl London (Ohio) Elizabeth McNiel (Minnesota) Michigan Sarah Mandelbaum Evan Keller Larry Baker This study was funded in part by the Canine Health Foundation. Dog photos were provided by Warren Photographic. Barbara Kitchell (Michigan State) Karen Michalski (Serenity Animal Hosp) Chieko Azuma (Tufts) David Vail (Colorado) Chand Khanna (NCI) Uppsala Nicolette Hillbertz Leif Andersson Göran Andersson Åke Hedhammar Olle Kämpe Henrik von Euler Gerli Pielberg