Journal of Equine Veterinary Science 32 (2012) 844-847

Journal of Equine Veterinary Science

journal homepage: www.j -evs.com

Case Report

Digital Agenesia in Martina Franca Donkey Foal: A Case Report

Domenico Robbe DVM, PhD a, Augusto Carluccio DVM, PhD a, Alessia Gloria DVM, PhD a,Alberto Contri DVM, PhD a, Mario Ventura BS, PhD b, Claudia Rita Catacchio BS, PhD c,Paola Straticò DVM, PhD a, Lucio Petrizzi DVM, PhD a

aDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Teramo, Teramo, ItalybDepartment of Genome Sciences, University of Washington, Seattle, WAcDepartment of Genetics and Microbiology, University of Bari, Bari, Italy

a r t i c l e i n f o

Article history:Received 27 January 2012Received in revised form14 March 2012Accepted 21 March 2012Available online 26 April 2012

Keywords:Digital agenesiaDonkey foalRadiographyKaryotype

Corresponding author at: Alessia Gloria, DVM, PVeterinary Clinical Sciences, College of Veterinary MedTeramo, Viale Crispi 212, Teramo, Italy.

E-mail address: gloriaalessia@libero.it (A. Gloria).

0737-0806/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.jevs.2012.03.009

a b s t r a c t

A 4-day-old male Martina Franca donkey foal was evaluated for a forelimb alteration.Clinical examination and radiographs revealed the agenesia of the distal digit.Biochemical parameters were normal, and ultrasonographic evaluation did not identifyany relievable organ alteration. Karyotype study revealed an abnormality on chromo-some 1. The foal was discharged with a distal limb bandage in which a palmar splint wasapplied. A poor prognosis for the functionality of the limb was given. In endangeredspecies, such as the Martina Franca donkey, the excessive inbreeding could result in anincrease in genetic disorders. These findings shed new light on the possible pathogenesisof the digital dysgenesia. The study of the karyotype could be a useful approach to detectgenetic alterations that could or could not be expressed in the animal, especially inendangered species in which a risk of an excessive inbreeding is considerable. Thesedefects should be considered in the choice and selection of the breeders.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Malformation of the phalanges occurs uncommonly inall species, including the horse [1]. In a 13-year surveystudy of equine congenital defects conducted in centralKentucky, contracted foal syndrome (33.2%), miscellaneouslimb contraction (20%), multiple defects (5.3%), micro-phthalmia (4.6%), craniofacial malformations (4.3%), cleftpalate (4%), heart defects (3.5%), umbilical defects (3.5%),and hydrocephalus (3%) were observed [2]. Few authorsdescribed dysgenesia of the limbs in the horse; thus, onlycomplete unilateral agenesia of the navicular bone, patella,and all bones distal to the radius were reported [3]. Etiologyof the digital dysgenesia is not known, but genetic causesare hypothesized, although not demonstrated. In some

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orthopedic pathologies, such as the lateral luxation of thepatella, the homozygosity of a simple autosomal recessivegene was suggested to be involved [4].

2. The Clinical Case

2.1. Animal and History

The patientwas a 4-day-oldmaleMartina Franca donkeyfoal. This breed is an endangered species that numbers justfew exemplars. The foal was referred to the VeterinaryTeaching Hospital of the University of Teramo, Italy, fora forelimb disorder. The general condition was excellent,and delivery at birth was normal; standing on four limbsand nursing occurred within physiological time. The jennyhad not been exposed to medications other than routinevaccinations during pregnancy. The owner referred thatclinically normal foals had been previously delivered by thedam and that there were no other instances of anomalousfoals in the respective parents’ blood lines.

Fig. 1. Photograph of the 4-day-old foal (A), and the left digit abnormality, consisting of pastern shortening, distal palmaromedial luxation of the hoof, and hoofdysplasia (B, C).

D. Robbe et al. / Journal of Equine Veterinary Science 32 (2012) 844-847 845

2.2. Physical Examination

At the time of admission, the foal was in good bodycondition, and organic functions were normal. At exami-nation, the foal was severely lame at a walk. The onlyphysical finding was a left fore digit abnormality, consistingof pastern shortening, distal palmaromedial luxation of thehoof, and hoof dysplasia (Fig. 1). The left hoof appearedsmaller than the contralateral one; in standing position, thehoof was deviated palmaromedially, and the skin of thelateral distal pastern touched the ground. When the foalmoved at walk, trot, or gallop, the affected limb was sparedin a three-limb gait (lameness of 5/5 degree). No pain orcrepitus could be elicited with manipulation. The hoofcould be freely moved around the distal limb, as it wascompletely disconnected from the bone. At the time ofadmission, no skinwound could be detected on the pastern.

2.3. Radiographic Examination

Lateromedial, dorsopalmar, dorsomedial-palmarolateral,and dorsolateral-palmaromedial oblique radiographs of theleft front digit were taken (Fig. 2). The short pastern bone,the distal phalanx, and the navicular bone could not bedetected in any view. The distal physis of the proximalphalanx was almost closed, with the presence of a thinradiolucent area in the centrodistal part of the bone. Thedistal border of the proximal phalanx appeared asymmetric,wider, and longer on the lateral side than the medial side.The hoof was small and hypoconic. All the other limbs wereevaluated radiographically, but no bone and joint abnor-malities were found.

Fig. 2. Left lateral (A), dorsopalmar (B), and right lateral (C) radio

2.4. Hematobiochemical Analysis and Ultrasonography

To rule out alteration or anomalies in other organs,hematobiochemical analysis and ultrasonographic exami-nationwere carried out, and no alterations were found. Thefoal was discharged with a distal limb bandage, in whicha palmar splint was applied. A poor prognosis for the func-tionalityof the limbwasgiven.At1-year follow-up, the foal isreported to be alive and healthy, despite the limb deformity.

2.5. Laboratory Evaluation

A study of the karyotype of the jenny and the foal wasperformed on peripheral blood lymphocyte samples toidentify a possible genetic cause of the disease. Metaphasespreads andnucleiwere obtained fromphytohemagglutinin-stimulated lymphocytes by standard procedures. Karyotypeanalysis of donkeyswas performed according to the standardprocedures of Q-banding. Briefly, the cell line was processedwith colcemid (0.1 mg/mL in culture medium; D1925, Sigma-Aldrich, Milan, Italy) for 4 hours followed by three washes ina fixative (3 methanol:1 acetic acid). Chromosomes werebanded by immersion in quinacrinemustard (Q2876, Sigma-Aldrich), fully washed with tap water, and mounted inphosphate-buffered saline (1X). Metaphases were acquiredby a charge-coupled device (CCD) using Leica fluorescencemicroscope (Leica Microsystems, Wetzlar, Germany). Chro-mosomes were counted, and banding patterns of the jennyand the foal were compared with standard Equus asinuskaryotype [1]. In the foal, chromosome 1 appeared abnormaland a different size of the pericentromeric region was found(Fig. 3).

graphs of the 4-day-old Martina Franca male donkey foal.

Fig. 3. Quinacrine band karyogram showing chromosomes of the 4-day-oldmale foal (A) and its mother (B).

D. Robbe et al. / Journal of Equine Veterinary Science 32 (2012) 844-847846

3. Discussion and Conclusions

Etiology of the digital dysgenesia is not known;however, genetic causes have been hypothesized but havenot been demonstrated. In some orthopedic pathologies,such as the lateral luxation of the patella, the homozygosityof a simple autosomal recessive gene was suggested to bethe involved [5]. In human medicine, a series of geneticsyndromes have been reported to cause digital anomalies[6,7]. In this case report, an aberrant karyotype was

hypothesized as a cause of the digital dysgenesia. Kar-yotyping is a test that allows the evaluation of the size,shape, and number of chromosomes in a sample of bodycells. This test helps to rule out the presence of geneticabnormalities that could result in diseases of the foal. Inboth the jenny and the foal, same abnormality in chromo-some 1was observed and this can be interpreted by neutralpolymorphism. Chromosomes 1, 9, and Y are known to behighly polymorphic, largely because of the expansiondifferences existing in classical satellite DNA families [8]. Avariety of molecular and cytogenetic evidence supports thehypothesis that polymorphisms result from illegitimaterecombination during DNA replication, leading to quanti-tative variability in constitutive heterochromatin and mostsatellite DNA sequences [9,10]. Previous data have reportedthis genomic region as highly unstable [11]. Polymorphismsdue to the accretion of duplicated genomic segments arecommon in mammalian genomes, in particular at centro-meric and telomeric regions where they are usuallysilenced and show no effect on the phenotype [12-15]. Inmammalian species, unexplored polymorphisms wereidentified in swine, sheep, stallions, and donkeys [16-18],and the technique also provided evidence for the presenceof highly conserved repetitive DNA.

Although our results ruled out macro-chromosomalrearrangements as the cause for the observed phenotype,micro-chromosomal rearrangements and point mutationscan also be present. The presence of a defect in the karyotypecould be expressed (foal) or stay silent (jenny). The donkeyspecies is mainly distributed in small and often consan-guine and endangered breeds in Europe. In Martina Francadonkey, a population of 25 jackasses and 280 jennies wasreported. For this reason, a high grade of consanguinitycould result in an overexpression of genetic abnormalities.The increase of consanguinity could carry to a higher risk ofkaryotype defects.

For the first time, this study shed a new light on thepossible pathogenesis of digital dysgenesis. Karyotype testscould be critically important to rule out some chromosomalrearrangement usually not analyzed by the standard tech-nique to detect point mutations. Specific studies should becarried out to verify the risk of genetic defect expression inendangered species.

References

[1] Huston R, Saperstein G, Leipold HW. Congenital defects in foals.J Equine Med Surg 1977;1:146-61.

[2] Crowe MW, Swerczek TW. Equine congenital defects. Am J Vet Res1985;46:353-8.

[3] Leipold LW, McDonald KR. Adactylia and polydactylia in a Welshfoal. Vet Med 1971;66:928-30.

[4] Hermans WA, Kersjes AW, van der Mey GJ, Dik KJ. Investigation intothe heredity of congenital lateral patellar (sub) luxation in theShetland pony. Vet Q 1987;9:1-8.

[5] Nguyen ML, Jones NF. Undergrowth: brachydactyly. Hand Clin2009;25:247-55.

[6] Di Meo GP, Perucatti A, Peretti V, Incarnato D, Ciotola F, Liotta L, et al.The 450-band resolution G- and R-banded standard karyotype ofthe donkey (Equus asinus, 2n ¼ 62). Cytogenet Genome Res2009;125:266-71.

[7] Byrnes AM, Racacho L, Nikkel SM, Xiao F, MacDonald H,Underhill TM, et al. Mutations in GDF5 presenting as semidominantbrachydactyly A1. Hum Mutat 2010;31:1155-62.

[8] Pita M, Fernandez JL, Gosalvez J. Whole comparative genomichybridization (WCGH): the quick overview of repetitive DNAsequences on a genome. Cromosome Res 2003;11:673-9.

D. Robbe et al. / Journal of Equine Veterinary Science 32 (2012) 844-847 847

[9] Levison G, Gutman GA. Slipped-strand mispairing: a major mecha-nism for DNA sequence evolution. Mol Biol Evol 1987;4:203-21.

[10] Charlesworth B, Sniegowski P, Stephan W. The evolutionarydynamics of repetitive DNA in eukaryotes. Nature 1994;371:215-20.

[11] Raimondi E, Piras FM, Nergadze SG, Di Meo GP, Ruiz-Herrera A,Ponsà M, et al. Polymorphic organization of constitutive hetero-chromatin in Equus asinus (2n ¼ 62) chromosome 1. Hereditas 2011;148:110-3.

[12] Locke DP, Archidiacono N, Misceo D, Cardone MF, Deschamps S,Roe B, et al. Refinement of a chimpanzee pericentric inversionbreakpoint to a segmental duplication cluster. Genome Biol 2003;4:R50.

[13] De Gregori M, Pramparo T, Memo L, Gimelli G, Messa J, Rocchi M,et al. Direct duplication 12p11.21-p13.31 mediated by segmentalduplications: a new recurrent rearrangement? Hum Genet 2005;118:207-13.

[14] Zhang L, Lu HH, Chung WY, Yang J, Li WH. Patterns of segmentalduplication in the human genome. Mol Biol Evol 2005;22:135-41.

[15] She X, Cheng Z, Zollner S, Church DM, Eichler EE. Mouse segmentalduplication and copy number variation. Nat Genet 2008;40:909-14.

[16] Cortes-Gutierrez EI, Davila-Rodriguez MI, Lopez-Fernandez C,Fernandez JL, Gosalvez J. Alkalilable sites in sperm cells from Sus andOvis species. Int J Androl 2008;31:354-63.

[17] Devila-Rodriguez MI, Cortes-Gutierrez EI, Lopez-Fernandez C,Pita M, Mezzanotte R, Gosalvez J. Whole-comparative genomichybridization in domestic sheep (Ovis aries) breeds. CytogenetGenome Res 2009;124:19-26.

[18] Gosalvez J, Crespo F, Vega-Pla JL, Lopez-Fernandez C, Cortes-Gutierrez EI, Devila-Rodriguez MI, et al. Shared Y chromosomerepetitive DNA sequences in stallion and donkey as visualized usingwhole-genomic comparative hybridization. Eur J Histochem 2010;54:e2.