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6. Piccione G, Costa A, Gianetto C, Caola G. Daily rhythms of activity in

horses housed in different stabling conditions. Biological Rhythm Re-

search 2007;39:79-84.

7. Murphy BA, Vick MM, Sessions DR, Cook RF, Fitzgerald BP. Evi-dence of an oscillating peripheral clock in an equine fibroblast cell

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31771 Investigation of DwarfismAmong Miniature Horses using theIllumina Horse SNP50 Bead ChipJ. Eberth,* T. Swerczak, and E. Bailey, MH Gluck EquineResearch Center, University of Kentucky, Lexington,KY, USA

INTRODUCTIONPonies and Miniature horses differ from full size horse onlyby their stature. Ponies are often defined as those whoseheight is not greater than 14.2 hands, however the maxi-mum height for Miniature horses is constitutionally de-fined as 8.2 hands. This reduced stature is usually thecumulative effect of hundreds of genes, each having a smallimpact on stature. Unfortunately, there are also dwarfismgenes which greatly reduce statute and may negatively im-pact health and reproduction. This is not considered a de-sirable genetic trait for Miniature horses. Therefore, thefollowing studies were conducted to discover the geneticbasis for dwarfism.

MATERIALS AND METHODSPedigree records suggested that dwarfism occurs as a reces-sive genetic trait among miniature horses. Pathological ex-amination, involving comparison of skeletal developmentand other phenotypic traits, suggested that there may beas many as four distinct types of dwarfism segregatingamong miniature horses. Indeed, among humans thereare over 100 genetic mutations found responsible for dif-ferent forms of dwarfism.

Type I dwarves exhibit cranial abnormalities of a dispro-portionately large head, large bulging eyes and eye sockets,a forehead with relative frontal domed prominence anda relatively short stubbed muzzle. An underbite of variableseverity is commonly seen in this type however some havea normal bite. The midface is often small with a flat nasalbridge and narrow nasal passages and the airway obstruc-tion can be ‘‘central’’ in origin (due to foramen magnumcompression) or ‘‘obstructive’’ in origin (due to narrowednasal passages). Symptoms of airway obstruction includesnoring. Other characteristics are shortened limbs, en-larged joints, malformed or bowed legs with limited exten-sion and flexion and overall disproportionate short stature.Progressive hoof deformities resulting from malformed

limbs as well as progressive arthritis in the limbs becomeworse with age. Spinal abnormalities such as roachbackmay or may not develop later.

To avoid pooling samples from different genetic forms ofdwarfism, horses were selected based on a common pheno-type and belonging to a common family line. This pheno-type was identified as Type I dwarfism of miniature horses.DNA was isolated from blood or tissues of 20 horses exhib-iting type I dwarfism and from 20 relatives that did not ex-hibit the dwarfism traits. The DNA was tested using theIllumina Equine SNP50 bead chip. The results were ana-lyzed using PLINK v 1.04.

RESULTSA single chromosome region was strongly associated withthe trait. EMP2 (empirical P value, corrected for all tests)value of 0.019 was obtained, strongly supporting this can-didate region. Within the candidate region a candidategene was found which causes dwarfism among humans.

DISCUSSIONThe candiate gene is currently being sequenced for horseswith the hope of discovering a mutation responsible forthe trait. Additional work will entail identifying a haplo-type signature for Type I dwarfism that could be usedto estimate its frequency in the population and determinethe phenotypic heterogeneity associated with this haplo-type.

ACKNOWLEDGEMENTS

Morris Animal Foundation and the American MiniatureHorse Association provided funds for this research.

31762 Illumina Equine SNP50 BeadChip Investigation of Adolescentidiopathic lordosis among AmericanSaddlebred HorsesDeborah Cook,* Patrick Gallagher, and Ernest Bailey,MH Gluck Equine Research Center, University ofKentucky, Lexington, KY, USA

INTRODUCTIONAdolescent idiopathic lordosis (AIL) is a heritable traitamong American Saddlebred horses often referred to asswayback, low-backed or soft-backed. Extreme lordosis isa major ventral curvature of the thoracolumbar vertebralcolumn. This trait was characterized in a previous study

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where measurement of back curvature (MBC) was takenfor 305 ASB horses; those with an index greater than 6.5were classified as extreme lordosis. Aged horses can acquirelordosis as a result of foaling, poor conformation, excessivework, etc. In contrast, AIL is present at birth or developswithin the first two years of life.

MATERIALS AND METHODSBack measurements (MBC) were made on 749 AmericanSaddlebred horses with hair samples collected from eachindividual during the course of the previous study byDr. Gallagher. For this study, 40 total horses dividedinto groups of 20 cases (MBC of 8.0 or greater) or 20controls (MBC between 2.0 and 5.0) were selected forgenotyping. The 40 individuals were selected mostly onthe basis of being half siblings. DNA was isolated fromhair bulbs and genotyped for nearly 60,000 SNPs usingthe Illumina Equine SNP50 Bead Chip. Genotypes wereanalyzed using PLINK v 1.04.

RESULTSGenome scanning with the Illumina horse SNP chip led toidentification of a haplotype associated with extreme lor-dosis. This haplotype includes 6 SNPs spanninga 489.6 kb region or ECA20. Seventeen of the 20 horseswith extreme lordosis are homozygous for this haplotype(compared to 7 of the 20 controls) suggesting a recessivemode of inheritance with the haplotype predating the con-dition. Using PLINK case/control association, the SNPwith greatest association by P-value had a minor allele fre-quency of 0.95 among affected individuals compared toa minor allele frequency of 0.4545 among unaffected indi-viduals with a P-value of 1.566E-8 and an odds ration of22.8. An adjoining SNP had the highest association byodds ration of 32.5 had a P-value of 1.533E-7 and minorallele frequency of 0.975 among affected individuals and0.5455 among unaffected.

DISCUSSIONFurther issues to be addressed are:

1. Is the trait caused by a single recessive gene or is ita complex trait involving multiple genes.

2. Does this haplotype have an impact on performance andsubject to positive selection by breeders?

3. Is this haplotype associated with extreme lordosis inother horse breeds?

ACKNOWLEDGEMENTS

American Saddlebred Horse Association and the MorrisAnimal Foundation, Inc provided research funds for thisproject.

31755 Assessing ChromosomeAbnormalities in Horses withInfertility or Congenital AbnormalitiesT.L. Lear,* University of Kentucky, Lexington, KY, USA

INTRODUCTIONChromosome abnormalities cause equine infertility andcongenital abnormalities in foals. A recent survey sug-gested the prevalence of chromosome abnormalities inthe overall equine population is between 2-3%.1 Samplesfrom horses with fertility problems or congenital abnor-malities are submitted by equine veterinarians to our clini-cal cytogenetics laboratory for chromosome analysis. Whilethe cytogenetic techniques used for equine karyotypinghave been adapted from human cytogenetic methods, mo-lecular tools that were developed for the Horse GenomeMapping Project have provided high resolution analysisof chromosomal aberrations.

MATERIALS AND METHODSWhole blood from horses suspected to have a chromosomeabnormality was collected in sodium heparin tubes. Lym-phocytes were removed by centrifugation and placed ina special cell culture medium containing various mitogens.The cells were cultured for approximately 70-72 hours.Colcemid, a mitotic spindle apparatus inhibitor, was addedto arrest the cells at metaphase. The cells were centrifugedout of the medium, treated with a hypotonic solution, andfixed prior to being dropped onto microscope slides.

Different chromosome staining and banding methodswere used depending on the suspected chromosome ab-normality. For chromosome number counts slides werestained with Giemsa. C-banding was done to identify thesex chromosomes, X and Y. To identify structural chromo-some rearrangements such as translocations or deletions,G-banding was done. Chromosomes were aligned in a kar-yotype following the accepted standard for the horse.2

To confirm chromosome abnormalities caused by struc-tural rearrangements or cases of aneuploidy or mosaicism,chromosome-specific genes in bacterial artificial chromo-some clones (BAC) were mapped by fluorescent in situ hy-bridization (FISH). BACs were selected from previouslymapped clones or based on their putative map positionalong the Horse Genome Sequence.

RESULTSChromosome abnormalities were identified in 35% of theclinical samples submitted for karyotyping. Chromosometranslocations were seen in four mares that had experiencedrepeated early embryonic loss prior to day 65.3,4 Each marehad a unique chromosome translocation that caused