Genomic Tools And Strategies For Studying Hereditary Conditions In Horses

1. Genomic Tools And Strategies For Studying Hereditary Conditions In Horses Sofia Mikko, L.S. Andersson, S. Eriksson, J. Axelsson, and G. Lindgren Dept. of Animal Breeding and Genetics, SLU, Uppsala, Sweden

2. The Horse Genome Project http://www.uky.edu/Ag/Horsemap/welcome.html • Started in Kentucky 1995 • Approx.100 researchers from more than 20 countries • Meetings are sponsered by Dorothy Russell Havemeyer Foundation • Strong collaboration to make a genetic map, and to sequence the horse genome • "Twilight" was selected as the representative horse • The sequence was publicly available in January 2007 (http://genome.ucsc.edu/) "Twilight" - Equus caballusPhoto courtesy of NHGRI

3. The Horse Genome Sequencehttp://www.broadinstitute.org/mammals/horse • EquCab2 is a Whole Genome Shotgun (WGS) assembly at 6.8X • The assembly is 2.68 Gb • The final gene-set comprises • 20,436 protein-coding genes • 4400 pseudogenes (including retrotranposed genes) Bravo is a male Thoroughbred, closely related to Twilight. DNA from his blood cells was cut into large fragments to make a “BAC Library”.

4. The Equine Genome – EquCab2Wade et al., Science vol 326, 2009 Hrafnhetta is an Icelandic horse mare.  She was chosen for random sequencing  for comparison to the DNA sequence of Twlight.  • 24 individuals from 11 breeds were sequenced to find SNP:s • > 1 million distinct SNP:s found • 1 SNP/2000 bp

5. EqCab2 50K SNP chipWade et al., Science vol 326, 2009 • 54,602 SNP:s were selected for the EqCab 50K SNP chip • Majority of SNPs are polymorphic within breeds • 90% of SNPs are ≤ 110 kb apart • 99.9% of SNPs are ≤ 1000 kb apart • Illumina will no longer provide this version of the chip

6. The Equine Genome – EquCab2Wade et al., Science vol 326, 2009 • The within breed LD in horse is moderate (100-300 kb) • Slightly shorter (50-70 kb) across breeds • Absence of strong bottleneck during breed formation • Many mares are used to maintain population size • LD shortest for ancient breeds, and longest for Thoroughbred

7. The Equine Genome – EquCab2Wade et al., Science vol 326, 2009 • LD is 5X shorter than in dog, but 5X longer than in human. • LD in Thoroughbred is comparable to the dog • Strong conserved synteny between human and horse

8. SNP density required for genome-wide mapping in the horseWade et al., Science vol 326, 2009 A sampling density of 100,000 SNPs were able to obtain mean maximum r2 values of >0.5 for tested SNPs in all breeds as well as the across breed groups.

9. Power of gene mapping usingthe EqCab2 SNP chipWade et al., Science vol 326, 2009 Number of SNPs needed to differentiate horse haplotypes for within-breed gene mapping (by simulation) Estimated from LD, number of haplotypes within haplotype blocks, and the polymorphism rate.

10. Tools and strategies when studying hereditary conditions • Pattern of inheritance • Population and family structure • Study design • Choice of marker • Sample collection • DNA preparation • Genotyping • Analysis of results • Functional analysis

11. Molecular studies of mono-, and multifactorial traits • Until now the focus has been on monogenic traits • SCID, OLWS, HYPP, GBED, JEB, HERDA, PSSM, etc • Now the focus has shifted towards multigenic traits • Allergies, Osteochondrosis, Bone spavin, etc

12. Population and family structure Related individuals Unrelated individuals Could be difficult to find in inbred populations Risk of ”inflated” p-values Less population stratification • Large half-sib groups are often available in farm animals • Risk of ”deflated ” p-values • Population stratification QQ-plot MDS-plot

13. Study designs Linkage analysis Association study Unrelated individuals Cases vs controls Smaller chromosomal areas are covered • Half-sib families • Traits cosegregating with chromosome regions • Large chromosomal areas are covered Figure from Zhu et al 2008

14. Choice of DNA marker SNPs Microsatellites Low level of automation Time consuming High cost / marker High PIC / marker Lower coverage of the genome No bias on breed level • High level of automation • Less bench-time • Low cost / marker • Low PIC / marker • High coverage of the genome • Bias towards the breed used for selecting the SNPs

15. Sample handling Sample collection DNA preparation Good DNA quality from blood samples and ”clean” tissue DNA in hair samples has lower quality Manual / automatic Cases & controls should be prepared in the same way • Blood, tissue, hair • Blood, and tissue samples are more expensive to collect • Sample collections/Biobanks • Breeders and horse owners are helpful

16. Silver DappleCoat ColorBrunberg et al. BMC Genet 2006 • Autosomal dominant trait • Only eumelanin is diluted • Pheomelanin is not affected => chestnut carriers

17. Candidate genes/Comparative approach Brunberg et al. BMC Genet 2006 • Candidate genes – dilution genes • Comparative approach • Silver in mouse • Merle in dogs • Dun in chicken • Fading vision in zebra fish fading vision M-, merle dun M-+H-, harlekin

18. Linkage Analysis of Silver Dapple Brunberg et al. BMC Genet 2006 • Well characterized family material • 1 heterozygous stallion, 34 offspring, and 29 non-silver dams • 41 microsatellites genotyped • Linkage analysis using CRIMAP • Sequencing and SNP analysis to confirm mutation in PMEL17

19. Multiple Congenital Ocular Anomalies (MCOA)Andersson et al. BMC Genet 2008, in prep 2010 • Codominant mode of inheritance • Heterozygotes have cysts in the eye • Homozygotes have multiple abnormalities including cysts, wideopen eyes, deformed pupils • Similar disease is present in mice • Most common in Rocky Mountain ponies, and silver coat colored horses

20. ”Identical by Decent” (IBD) mapping of MCOAAndersson et al. BMC Genet 2008, in prep 2010 • 11 genetic markers on ECA6q • 4.9 Mb interval • The causative mutation must be present within a 421 bp fragment on ECA6q, in the same region as SILV • More individuals that are unrelated or from other breeds could shorten the interval. • Sequencing next step?

21. Skeletal Atavism In Shetland Ponies • Most likely a monogenic trait • Autosomal recessive inheritance • Fully elongated ulna and fibula • Low prevalence • Not always reported by breeders • Carriers may remain undetected Photo: Lisa Andersson Photo: Göran Dalin

22. Association Study of Skeletal Atavism • Old samples from a biobank • 6 affected, 18 carriers, and 24 non-carriers • Average sample success rate of 0.974 • Stratification detected • GWA show no significant peak, but a few ”small” peaks are detected • Homozygosity mapping • Sequencing Photo: Göran Dalin

23. Equine Insect Bite Hypersensitivity(EIBH, Summer Eczema) • Most common equine skin disease • Present in many breeds around the world • Proteins in the saliva of the biting midges Culicoides is the main allergen • Itching dermatitis may cause open wounds, lichenification, crusts, dandruff and alopecia.

24. Prevalence and heratibility study of Equine IBHEriksson et al Animal 2007 • Prevalence of 8% in Swedish born Icelandic Horses • Range 0-30% between different parental half-sib groups. • Phenotypes were graded in four classes • Heritability estimated to 14% (40-50% on the underlying, continuous scale)

25. GWA study of EIBH • 104 cases, and 105 matched half-sib controls • 54602 SNPs analyzed • Genotyping rate was 0.99 • 539 SNPs had >10% missing genotyping • 1014 SNPs were not in HWE • 14651 SNPs with MAF<0.05 • Left 38398 SNPs to be analyzed • Average spacing between markers is 59.8 kb (1 bp - 1.3 Mb) • Average maximum LD (r2)=0.3 at ≈14.4 kb

26. GWA study of EIBH • No stratification detected • Matched cases and controls • Conservative phenotype inclusion for cases • Deflation of p-values due to family structure • No GWA found using allelic case/control, as well as logistic regression.

27. GWA study of EIBH • No SNP reached genome wide significance • With risk ratio of 2 we need > 5X the cases • Power calculations show that we can only detect loci with very strong effect (RR>7) • 173,000 markers needed to cover the Icelandic horse genome

28. Performance Traits In Swedish Trotters Nei: 0.524 Fst: 0.134 The Standardbred trotter (S) The North Swedish horse (NS) Nei: 0.503 Fst: 0.130 Nei: 0.367 Fst: 0.081 The North Swedish trotter (NST)

29. Population structure • The STRUCTURE analysis of the microsatellite dataset (n=122) could not separate NS and NST • “Identity By State” cluster analysis of the SNP dataset revealed three breeds separated into three distinct clusters. NS S NST

30. Breed diversity in ECA regions • Regions of positive or balancing slection have been detected by Ewens Watterson test • Fst values of the microsatellite data analysis reveal chromosome areas where the NST may be closer related to S than to NS. • Fst values on SNP data will be next analysis

31. Funding • Formas • ATG • SSH • Carl Trygger • Helge Axelsson-Johnsson • Strömsholm Animal Hospital • SLU:s Animal Hospital (UDS) • Mälarkliniken Animal Hospital

32. Collaborators • Swedish Univ of Agricultural Sciences (SLU): • Leif Andersson • Lisa Andersson • Jeanette Axelsson • Hans Broström • Emma Brunberg • Göran Dalin • Björn Ekesten • Susanne Eriksson • Freddy Fikse • Katja Grandinsson • Ingrid Jacobsson • Gabriella Lindgren • Jennifer Meadows • Sofia Mikko • Aneta Ringholm • Kaj Sandberg • Hanna Smedstad • Gunilla Thyreen • Norsholms Animal Hospital: • Rebecka Frey • Östra Greda Research Group: • Marie Sundquist • Texas A&M: • Gus Cothran • Rytis Juras • Michigan State Univ.: • Jessica Eason-Butler • Susan Ewart • David Ramsey • Norwegian School of Veterinary Science: • Knut Röed