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

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 histology4. Location of metastases5. Common environment

Differences

1. Age of onset2. Incidence in population

Dogs and humans: Similarities in cancer genetics

The first example: Renal cancer syndromeCanine 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

Cancer in dogs is highly breed-specific

Cancer Breed Increased risk

Hemangiosarcoma Golden Retriever 5

Lymphoma, T-cell Boxer 4

Melanoma Scottish Terrier 6

Mammary Tumor Springer Spaniel 5

Osteosarcoma Greyhound

Rottweiler

10

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.

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

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

2.5 million SNPs discovered (1 SNP/kb)

Boxer

Poodle

• SNP discovery in 11 breeds and 5 wild canids

Alaskan Malamute

English Shepherd

Bedlington TerrierBeagle

German Shepherd Italian Greyhound

Labrador Retriever Portuguese Waterdog Rottweiler

China gray wolfAlaska gray wolfIndia gray wolf

Spanish gray wolf

California Coyote

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

CoverageChromosome 1Mb ≥ 5 SNPs

Autosomes 97%

Chrom X 42%

Power analysis: Prediction of sample size needed

Trait Dogs Power (LOD>5)

Recessive 20 + 20 100%

Dominant 50 + 50 100%

5-fold risk 100 + 100 98%

*In humans, would need approx 800 + 800

*

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-Dominant Recessive Dominant

Cases+Controls 10+9 7+11 27+40

LOD 5 3 5

Region Size (kb) 800 800 300

Genes 1 7 3

Objectives:

1. To identify genes involved with osteosarcoma susceptibility in dogs

2. To map a complex trait using the new SNP set.

Large and giant breed dogs have a higher risk of osteosarcoma

Greyhound-5 to 10 XIrish WolfhoundGreat DaneScottish DeerhoundRhodesian RidgebackGreat PyreneesBorzoiSaint Bernard

Rottweiler-5 X increased riskLabrador RetrieverFlat-coated RetrieverGolden RetrieverMastiffBullmastiffNewfoundland

1) Mastiff-type breeds 2) Long-limbed breeds

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.

Study participants enrolled

Table 1: Samples collected as of May 1, 2006

Breed Breed group OSA cases

Controls(8 years and older)

Rottweiler Mastiff 91 74

Golden retriever Mastiff 29 370

Leonberger Mastiff 26 51

Greyhound Long-limbed 82 151 (*10 yrs and older)

Irish Wolfhound Long-limbed 12 11

Great Dane Long-limbed 22 33

All other breeds NA 49 189

Haplotype Haplotype The series of alleles The series of alleles along a chromosome inherited by an along a chromosome inherited by an individual from one parent.individual from one parent.

Number of generations since common ancestor

Number of founders

Shared

Haplotypes

Within a breed

(Corgi-Corgi)

few few large

Between breeds

(Corgi-Irish wolfhound

many many small

Relationship between haplotype size and breed

Irish wolfhound

Corgi

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 /2M

b-1M

b

OSA+ OSA+

xx xx

Stage 2: Fine mapping across dog breeds within a breed groupAdd related breeds to narrow the region to a smaller, shared ancestral haplotype (~10-20kb). We are just getting started with this!

10-2

0kb

{ {Golden retriever

(OSA+)Leonberger

(OSA+)

“OSA”gene

Whole Genome Scan in Rottweilers:Whole Genome Scan in Rottweilers:

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an

asso

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Location in the genome

Regions associated with osteosarcoma in RottweilersRegions associated with osteosarcoma in Rottweilers-lo

g(1

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• Localized to three haplotype blocks

• Association performed using Haploview

• p< 0.01 with 100,000 permutations.

A CB

SNPs SNPs grouped into 4 gamete “haplotype” blocks

Currently ongoing…

•Screening for mutations•Genome scan in greyhounds•Fine mapping in related breeds

Acknowledgements

Broad InstituteElinor KarlssonClaire WadeTara BiagiSara FrycNoriko TonomuraKerstin Lindblad-Toh

CollaboratorsFrode Lingaas (Oslo)Guillermo Couto (Ohio)Jaime Modiano (Denver)Elaine Ostrander (NHGRI)Bill Kisseberth (Ohio)Cheryl London (Ohio)Elizabeth McNiel (Minnesota)

MichiganSarah Mandelbaum Evan KellerLarry 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)

UppsalaNicolette HillbertzLeif AnderssonGöran AnderssonÅke HedhammarOlle KämpeHenrik von EulerGerli Pielberg