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testing for PSSM which were tested for the GYS1 mutationup until February 2009. Information obtained includedthe breed of horse and genotype. The number of horsespositive for the mutation out of the total number testedwas calculated for each breed.

RESULTSGYS1 testing was performed on 1,714 individuals of which588 or 34% were positive for the GYS1 mutation. Breedsaffected by the GYS1 mutation in descending order ofthe prortion of positive tests were Belgian (69/92), Per-cheron (39/55), Mustang (3/5), Appaloosa (21/48),mixed breed (67/155), Rocky Mountain Spotted Horse(2/5), Paints (54/144), Quarter Horses (285/769), Ap-pendix Quarter horses (7/22), no breed given (17/63),Haflinger (5/19), Shire (1/5), Hannoverian (3/18), Hol-steiner (1/7), Tennessee Walking Horse (1/7), Morgan(2/17) and Warmblood unspecified (4/85). Additionalbreeds with the GYS1 mutation where< 5 horses weretested included American Cream (1/1), British ShowPony (1/1), Tori (1/1), Gypsy Vanner (1/2), Rhein-lander (1/3) and Irish Sport Horse (1/3). Breeds with 5or more horses tested without finding the GYS1 mutationincluded 30 Friesians, 18 Dutch Warmbloods, 10 Clydes-dales, 9 Swedish Warmbloods, 8 Arabians, 6 Oldenburgsand 5 Irish Drafts. While the majority of horses positivefor the GYS1 mutation were heterozygotes, homozygoteswere identified in the Quarter Horse (18), Percheron (7),Belgian (7), mixed breed (4), Appaloosa (2), Paint (2),Haflinger (1), Tori (1) and no breed given (1).

DISCUSSIONBased on the samples submitted for testing, PSSM appears tobe most common in Draft breeds, Quarter Horse relatedbreeds, mixed breeds, Mustangs and Morgan horses. Sometypes of Warmbloods such as the Hannoverian, Holsteinerand unspecified Warmbloods also have the GYS1 mutationwhereas it was not found in the Friesians, Swedish and DutchWarmblood horses tested. These results are similar to thebreed distribution found in the retrospective study describ-ing the prevalence of the GYS1 mutation in 831 horses orig-inally diagnosed with PSSM by muscle biopsy.3 Additionalbreeds identified in the present study include the Gypsy Van-ner, British Sport Pony and Tori breeds. In agreement withthe biopsy study the GYS1 mutation was not found in lighterbreeds such as Arabians and Standardbreds where a total of12 and 7 horses have been tested respectively.

This study design was not appropriate to determine thefrequency of the GYS1 mutation within breeds. Sucha study would require random sampling of at least 200 -300 horses of each breed to determine the allele frequencyof GYS1 with 99% confidence. To date this has been re-ported only in Quarter Horses and Paint Horses where

approximately 10% of horses in these breeds possess theGYS1 mutation.4 The breeds of horses sampled in the pres-ent study were likely highly influenced by the recommen-dations provided for testing based on the previousretrospective study of horses originally diagnosed withPSSM by muscle biopsy. Future studies with samplesfrom large numbers of horses provided by cooperativebreed associations are required to determine the overall fre-quency of the GYS1 mutation and thus the incidence ofPSSM susceptibility in a variety of equine breeds.

Keywords: Myopathy; Genetics; Horse; Glycogenpolysaccharide

REFERENCES

1. Valberg SJ, Cardinet GH III, Carlson GP, DiMauro S. Polysaccharide

storage myopathy associated with recurrent exertional rhabdomyolysis

in horses. Neuromuscul Disord. 1992;2:351-359.

2. McCue ME, Valberg SJ, Miller MB, Wade C, DiMauro S, AkmandHO, Mickelson JR. Glycogen synthase (GYS1) mutation causes a novel

skeletal muscle glycogenosis. Genomics 2008 May;91(5):458-66.

3. McCue ME, Valberg SJ, Lucio M, Mickelson JR. Glycogen Synthase 1

(GYS1) Mutation in Diverse Breeds with Polysaccharide Storage My-opathy. J Vet Int Med 2008;22:1228-1233.

4. Tryon RC, Penedo MC, McCue ME, Valberg SJ, Mickelson JR, Fa-

mula TR, Wagner ML, Jackson M, Hamilton MJ, Nooteboom S, Ban-

nasch DL. Evaluation of allele frequencies of inherited disease genes insubgroups of American Quarter Horses. J Am Vet Med Assoc 2009 Jan

1;234(1):120-5.

ACKNOWLEDGEMENTS

Funded by the American Quarter Horse Assocation andMorris Animal Foundation

31520 Light:dark, circadian, andultradian regulation of motor activityand skeletal muscle gene expression inthe horse

B.A. Murphy,*1 A.M. Martin,1 and J.A. Elliott2,1University College Dublin, School of Agriculture, FoodScience and Veterinary Medicine, Belfield, Dublin 4,Ireland, 2University of California, San Diego,CA, USA

INTRODUCTIONCircadian rhythms are approximately 24 h cycles in thephysiological, biochemical and behavioural processes oforganisms that are entrained by the prevailing photope-riod. The master mammalian biological clock, located inthe brain, receives light information from the retina andregulates diverse physiological processes by synchronizingmolecular clockwork mechanisms that consist of a core

314 Abstracts � Vol 29, No 5 (2009)

group of clock genes in each cell. Rodent studies have re-vealed that up to 10% of all genes in peripheral tissues areexpressed with a 24 h rhythm1 and a subset of circadianexpressed genes has recently been identified in mouseskeletal muscle.2 Robust diurnal (daily) variations inmany equine physiological parameters have been re-ported.3 A circadian rhythm in athletic performance hasrecently been demonstrated in humans4,5 and is sup-ported by findings of daily variation in muscle contractionstrength. Understanding the relationship between loco-motor activity rhythms and circadian gene expression pat-terns in skeletal muscle is key to improving athleticperformance for humans and horses alike. Horses housedin stabled conditions are reported to display diurnal (day-time) patterns of activity.6 However, a self-sustained cir-cadian rhythm is identified by persistence underconstant conditions in the absence of time cues thatmay mask as well as entrain endogenous rhythms. Theaims of this study are 1) to determine the activity patternsof horses in their natural environment, 2) to determinefor the first time whether equine activity persists with a cir-cadian rhythm under constant conditions, and 3) to in-vestigate circadian regulation of gene expression inequine skeletal muscle.

MATERIALS AND METHODSSix mares of lightweight breed were fitted with halter-mounted Actiwatch-L� data-loggers that record a digitallyintegrated measure of motor activity and light exposure.Mares were maintained outdoors as a group on pasturefor 48 h, subsequently stabled in individual stalls ina light-proofed barn for 48 h under a light-dark (LD) cycle,and finally maintained in the light-proofed barn for a further48 h under continuous darkness (DD) (<5Lux). While sta-bled, mares were fed at 4 h intervals to avoid a conspicuous24 h cue. For the second 24 h period under LD and subse-quent 24 h under DD, blood samples were collected viaa jugular catheter at 2 h intervals for serum melatonin anal-ysis. For the first 24 h period under DD, muscle biopsieswere collected by punch biopsy from the mid-gluteal mus-cle at 4 h intervals. Total RNA was isolated from musclesamples using the Trizol method. A panel of 20 geneswere selected for expression analysis. These included previ-ously identified core clock genes,7 and muscle metabolism2

and exercise-associated genes. Gene expression results fromquantitative RT-PCR assays are pending.

RESULTSAt pasture, mares demonstrated intense bouts of activitythat were synchronous across individuals, numbered ~ 6-9/24 h, generally lasted 1-4 h and were not clustered toform an overall pattern that was circadian. In the barn, un-der LD, activity levels were greatly reduced. Nonetheless,

activity bouts continued to show an ultradian (<24 h)rhythm, with a higher frequency than at pasture, andwith the emergence of diurnal modulation, evidenced bybouts displaying increased amplitude (more activity) inday (L), compared with night (D). When the barn LD cyclewas discontinued and mares were housed in continuousdim light (DD), activity levels remained low while activitybouts persisted with an ultradian rhythm that in some an-imals displayed no obvious 24 h circadian periodicity.These differences in activity patterns between field andbarn recordings were replicated in two separate trials andare being analyzed quantitatively to compare with gene ex-pression and blood melatonin data.

DISCUSSIONThe emergence of synchronous ultradian bouts of activity inhorses maintained at pasture implies a strong influence ofsocial cues. This suggests that temporal organization of ac-tivity and perhaps other aspects of equine physiology andbehaviour may show a reduced dependency on environmen-tal time cues or endogenous circadian regulation whenhorses are in their natural environment. It is noteworthythat a circadian pattern of behaviour emerges in a stabled en-vironment under an LD cycle, and to a lesser extent in DD.Stabling limits a horse’s ability to exhibit its natural herd be-haviours of movement, feeding and grooming. It is possibletherefore that the absence of social cues in a confined spacepermits the unmasking of an endogenous activity rhythm.Results from this study will advance our understanding ofequine circadian rhythms by correlating information frommuscle gene expression analysis with activity and melatoninrhythms, thereby providing crucial information for optimaltiming of daily exercise to ultimately improve performanceand potentially minimize training-associated injury.

Keywords: Horse; Circadian; Muscle; Activity rhythm;Gene expression; RT-PCR; Melatonin

REFERENCES

1. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH,

Weitz CJ. Extensive and divergent circadian gene expression in liverand heart. Nature 2002;417:78-83.

2. McCarthy JJ, Andrews JL, McDearmon EL, Campbell KS, Barber BK,

Miller BH, Walker JR, Hogenesch JB, Takahashi JS, Esser KA. Identi-

fication of the circadian transcriptome in adult mouse skeletal muscle.Physiol Genomics 2007;31:86-95.

3. Piccione G, Caola G, Refinetti R. Temporal relationships of 21 physi-

ological variables in horse and sheep. Comp Biochem Physiol A Mol

Integr Physiol 2005;142:389-96.4. Kline CE, Durstine JL, Davis JM, Moore TA, Devlin TM, Zielinski

MR, Youngstedt SD. Circadian variation in swim performance.

J Appl Physiol 2007;102:641-9.5. Martin A, Carpentier A, Guissard N, van Hoecke J, Duchateau J. Effect

of time of day on force variation in a human muscle. Muscle Nerve

1999;22:1380-7.

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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

line and adipose tissue but not in peripheral blood. J Comp Physiol

A Neuroethol Sens Neural Behav Physiol 2006;192:743-51.

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