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

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

The Horse Genome Sequence http://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”.

The Equine Genome – EquCab2Wade et al., Science vol 326, 2009

24 individuals from 11 breeds were sequenced to find SNP:s

> 1 million distinct SNP:s found

1 SNP/2000 bp

Hrafnhetta is an Icelandic horse mare.  She was chosen for random sequencing  for comparison to the DNA sequence of Twlight. 

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

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

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

SNP density required for genome-wide mapping in the horse

Wade 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.

Power of gene mapping usingthe EqCab2 SNP chip

Wade et al., Science vol 326, 2009

Number of SNPs needed to differentiate horse haplotypes for within-breed gene mapping (by simulation)

Desired mean r2max

0.7 0.8 0.9 1.0

High LD breed 30,000 100,000 175,000 245,000

Moderate LD breed 155,000 225,000 300,000 370,000

Low LD breed 250,000 320,000 390,000 460,000

Estimated from LD, number of haplotypes within haplotype blocks, and the polymorphism rate.

Tools and strategies when studying hereditary conditions

1) Pattern of inheritance

2) Population and family structure

3) Study design

4) Choice of marker

5) Sample collection

6) DNA preparation

7) Genotyping

8) Analysis of results

9) Functional analysis

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

Population and family structure

Related individuals• Large half-sib groups are often

available in farm animals

• Risk of ”deflated ” p-values

• Population stratification

Unrelated individuals• Could be difficult to find in

inbred populations

• Risk of ”inflated” p-values

• Less population stratification

QQ-plot MDS-plot

Study designs

Linkage analysis Half-sib families

Traits cosegregating with chromosome regions

Large chromosomal areas are covered

Association study Unrelated individuals

Cases vs controls

Smaller chromosomal areas are covered

Figure from Zhu et al 2008

Choice of DNA marker

SNPs 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

Microsatellites Low level of automation

Time consuming

High cost / marker

High PIC / marker

Lower coverage of the genome

No bias on breed level

E02 Eq020912_2 308088 Q Score : 5.3 Allele 1 : 97.5 ( M )

80 90 100 1100

1000

2000

Sample handling

Sample collection Blood, tissue, hair

Blood, and tissue samples are more expensive to collect

Sample collections/Biobanks

Breeders and horse owners are helpful

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

Silver Dapple Coat Color Brunberg et al. BMC Genet 2006

Autosomal dominant trait

Only eumelanin is diluted

Pheomelanin is not affected => chestnut carriers

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

M-, merle M-+H-, harlekin dun

fading vision

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

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

”Identical by Decent” (IBD) mapping of MCOA

Andersson 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?

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

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

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.

Prevalence and heratibility study of Equine IBH

Eriksson 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)

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

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.

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

Performance Traits In Swedish Trotters

The Standardbred trotter (S) The North Swedish horse (NS)

Nei: 0.524Fst: 0.134

Nei: 0.503Fst: 0.130

Nei: 0.367Fst: 0.081

The North Swedish trotter (NST)

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

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

Funding

Formas

ATG

SSH

Carl Trygger

Helge Axelsson-Johnsson

Strömsholm Animal Hospital

SLU:s Animal Hospital (UDS)

Mälarkliniken Animal Hospital

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