Downloaded from veterinaryrecord.bmj.com on July 19, 2012 - Published by group.bmj.com Short Communications Short Communications Prevalence of inherited junctional epidermolysis bullosa in German shorthaired pointers bred in Italy G. Pertica, J. Riva, M. G. Strillacci, M. C. Cozzi, M. Longeri, M. Polli EPIDERMOLYSIS bullosa (EB) is a genetic disease involving the skin and mucous membranes in human beings, horses, cattle, sheep, cats and dogs (Bruckner-Tuderman and others 1991, Olivry and others 1999, Jiang and Uitto 2005, Magnol and others 2005, Fine and others 2008, Schumann 2009). Fine and others (2008) have classified the disease in human beings into four major types and several subtypes, based on distinguishing the ultrastructural sites of blister formation: epidermolysis bullosa simplex (EBS suprabasal and EBS basal), which involves the basal epidermal cells; dermolytic epidermolysis bullosa (EBD dominant and EBD recessive), which involves the dermis; junctional epidermolysis bullosa (JEB Herlitz and other forms of JEB), which involves the lamina lucida; and Kindler syndrome, which involves multiple cleavage planes (intraepidermal, junctional or sublamina densa). There are many reports of individual cases of EB in dogs of various breeds (Scott and Schultz 1977, Dunstan and others 1988, Nagata and others 1997, Palazzi and others 2000, Scott and others 2001). Although JEB is a rarely reported disease in dogs, it has been suggested that German shorthaired pointers (GSHPs) could provide an animal model for this disease in human beings (Capt and others 2005). In GSHPs, JEB has been established as an autosomal recessive inherited disease, expressed as a skin blistering disorder in various body regions (Capt and others 2005, Polli and others 2007a). JEB causes separation within the lamina lucida of the basement membrane zone at the dermal-epidermal junction. The defective adhesion of the epithelium results in spontaneous or traumatically induced blisters and ulcers on the footpads, pinnae, tail tip and on pressure points on the distal limbs (Scott and others 2001, Capt and others 2005). In human beings, the four major types of EB are associated with mutations in several genes: plakophilin-1 (PKP-1) and desmoplakin Veterinary Record (2010) 167, 751-752 G. Pertica, DVM, M. G. Strillacci, MSc, M. C. Cozzi, DVM, M. Longeri, DVM, PhD, M. Polli, DVM, PhD, Department of Animal Science, Università degli Studi di Milano, via Celoria 10, 20133 Milano, Italy J. Riva, DVM, PhD, MSc, Vetogene Lab, Viale Ortles 24/A, 20100 Milano, Italy (DSP) for EBS suprabasal; plectin (PLEC1), keratin-5 and -14 (KERT-5, KERT-14), and α6β4 integrin (ITGA6, ITGb4) for EBS basal; laminin 332 (LAMA3, LAMB3, LAMC2) for Herlitz JEB, and collagen type XVII (COL17A1), laminin 332 (LAMA3, LAMB3, LAMC2, α6β4 integrin (ITGA6, ITGb4) for other forms of JEB; collagen type VII (COL7A1) for dominant and recessive EBD; and kindling-1 (KIND-1) for Kindler syndrome (Fine and others 2008). The protein laminin 332, produced by basal keratinocytes, is the major adhesion ligand of epithelial cells. It is composed of α3, β3, γ2 chains, encoded by the genes LAMA3, LAMB3 and LAMC2, respectively. The form of canine JEB studied in GSHPs by Capt and others (2005) is associated with reduced expression of the LAMA3 gene, caused by a homozygous insertion of a repetitive satellite DNA at position 207 of intron 35. Canine keratinocytes expressing the JEBassociated mutation produce a small quantity of laminin 332, which is insufficient to sustain epithelial cell adhesion (Capt and others 2005). This short communication describes a study to evaluate the prevalence of JEB in Italian GSHPs. Since January 2006, in collaboration with the Italian GSHP Club, the authors’ laboratory (Vetogene Lab, a spin-off of the University of Milano) has been allowed to perform a diagnostic test for JEB in this breed in Italy. According to the rules of the Italian GSHP Club, the JEB test and identification and/or parentage verification are obligatory for all dogs chosen for breeding. Serum samples from these dogs were sent to the laboratory by their owners (either with EDTH into a vacutainer or spotted on to FTA cards). Three hundred and nine unrelated GSHPs tested for JEB were chosen from among the approximately 500 dogs registered at the laboratory. The dogs were tested using an allele-specific PCR method for molecular detection of the causative mutation in the LAMA3 gene, developed by Polli and others (2007b). The PCR was performed using genomic DNA extracted either from peripheral blood (Illustra blood genomicPrep Mini Spin Kit; GE Healthcare) or FTA cards (Vet Kard System; Prion Diagnostica), according to the manufacturer’s instructions. PCR was performed in a single reaction using a common forward primer and two allele-specific (wild-type and insertion mutation) reverse primers. The PCR products were separated by electrophoresis in 2 per cent agarose gel and stained using ethidium bromide (Fig 1). The prevalence estimated as a percentage by dividing the number of wild-type, carrier and affected dogs by the total number of dogs analysed. The calculated prevalences were 238 (77 per cent) dogs for wild-type (133 male and 105 female), 69 (22 per cent) dogs for carrier status (31 male and 38 female) and two (1 per cent) for affected (both female). There were no remarkable differences between the sexes, doi: 10.1136/vr.c5178 Correspondence to Dr Cozzi, e-mail: cristina.cozzi@unimi.it Provenance: not commissioned; externally peer reviewed M 1 2 3 4 5 6 7 8 9 10 FIG 1: Electrophoresis of products of a PCR targeting the canine LAMA3 gene associated with junctional epidermolysis bullosa (JEB), using a common forward primer and reverse primers specific for the wild-type and insertion mutant alleles. Lane M Marker (VC 100 bp Plus DNA Ladder; Vivantis); Lanes 2, 3, 5, 6, 7 and 10 Wild-type (normal) dogs: each sample shows one specific band of 222 bp. Lanes 1, 4 and 9 Carriers (heterozygous in the LAMA3 gene): each sample shows two bands, one of 222 bp and one of 397 bp. Lane 8 Dog showing signs of JEB (homozygous for the mutant allele): this sample shows one specific band of 397 bp November 6, 2010 | Veterinary Record Downloaded from veterinaryrecord.bmj.com on July 19, 2012 - Published by group.bmj.com Short Communications except that the two affected dogs were both female. However, this was not statistically significant, because the dogs were tested in order to confirm a veterinary diagnosis. The prevalence of affected GSHPs in Italy was probably underestimated in this study. In the authors’ experience, affected dogs are usually euthanased without being tested because the footpad lesions make them almost unable to walk, oral lesions render them unable to eat, or because they develop early-onset septicaemia, with a poor prognosis. However, in the authors’ opinion, the estimated prevalence of GSHP carriers found in this study is probably correct and gives a good representation of the situation in Italy. The identification of healthy carrier dogs is the only way to enable control of the presence of JEB in the GSHP breed by applying an appropriate breeding strategy. In conclusion, the quick and clear method of detection used in this study permitted the identification of JEB carriers, providing a suitable tool for disease control and to facilitate mating planning by breeders. Acknowledgements The authors thank to Chiara Noli for providing support and for the descriptions of dermatological lesions. References BRUCKNER-TUDERMAN, L., GUSCETTI, F. & EHRENSPERGER, F. (1991) Animal model for dermolytic mechanobullous disease: sheep with recessive dystrophic epidermolysis bullosa lack collagen VII. Journal of Investigative Dermatology 96, 452458 CAPT, A., SPIRITO, F., GUAGUERE, E., SPADAFORA, A., ORTONNE, J. P. & MENEGUZZI, G. (2005) Inherited junctional epidermolysis bullosa in the German Pointer: establishment of a large animal model. Journal of Investigative Dermatology 124, 530-535 Veterinary Record | November 6, 2010 DUNSTAN, R. W., SILLS, R. C., WILKINSON, J. E., PALLER, A. S. & HASHIMOTO, K. H. (1988) A disease resembling junctional epidermolysis bullosa in a toy poodle. American Journal of Dermatopathology 10, 442-447 FINE, J. D., EADY, R. A., BAUER, E. A., BAUER, J. W., BRUCKNER-TUDERMAN, L., HEAGERTY, A. & OTHERS (2008) The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB. Journal of the American Academy of Dermatology 58, 931950 JIANG, Q. J. & UITTO, J. (2005) Animal models of epidermolysis bullosa – targets for gene therapy. Journal of Investigative Dermatology 124, xi-xiii MAGNOL, J. P., PIN, D., PALAZZI, X., LACOUR, J. P., GACHE, Y. & MENEGUZZI, G. (2005) Characterization of a canine model of dystrophic bullous epidermolysis (DBE). Development of a gene therapy protocol. Bulletin De l’Académie Nationale De Médecine 189, 107-119 [In French] NAGATA, M., IWASAKI, T., MASUDA, H. & SHIMIZU, H. (1997) Non-lethal junctional epidermolysis bullosa in a dog. British Journal of Dermatology 137, 445-449 OLIVRY, T., DUNSTON, S. M. & MARINKOVICH, M. P. (1999) Reduced anchoring fibril formation and collagen VII immunoreactivity in feline dystrophic epidermolysis bullosa. Veterinary Pathology 36, 616-618 PALAZZI, X., MARCHAL, T., CHABANNE, L., SPADAFORA, A., MAGNOL, J. P. & MENEGUZZI, G. (2000) Inherited dystrophic epidermolysis bullosa in inbred dogs: a spontaneous animal model for somatic gene therapy. Journal of Investigative Dermatology 115, 135-137 POLLI, M., BIGHIGNOLI, B., STRILLACCI, M. G., GILARDONI, C., BONASEGALE, C., CAVALCHINI, L. G. & LONGERI, M. (2007a) L’epidermolisi bollosa giunzionale ereditaria nel cane diagnosi molecolare per l’identificazione dei portatori sani della malattia. Veterinaria 21, 21-25 POLLI, M., LONGERI, M., DARDANO, S. & CAVALCHINI, L. G. (2007b) Method for the determination of junctional epidermolysis bullosa in dogs. IT Patent MI2007A000036. University of Milan – VetoGene SCHUMANN, H. (2009) Epidermolysis bullosa. An update. Hautarzt 60, 614-621 [In German] SCOTT, D. W., MILLER, W. H. & GRIFFIN, C. E. (2001) Epidermolysis bullosa. In Small Animal Dermatology. 6th edn. W. B. Saunders. pp 938-940 SCOTT, D. W. & SCHULTZ, R. D. (1977) Epidermolysis bullosa simplex in the Collie dog. Journal of the American Veterinary Medical Association 171, 721-727 Downloaded from veterinaryrecord.bmj.com on July 19, 2012 - Published by group.bmj.com Prevalence of inherited junctional epidermolysis bullosa in German shorthaired pointers bred in Italy G. Pertica, J. Riva, M. G. Strillacci, et al. Veterinary Record 2010 167: 751-752 doi: 10.1136/vr.c5178 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/167/19/751.full.html These include: References This article cites 12 articles, 1 of which can be accessed free at: http://veterinaryrecord.bmj.com/content/167/19/751.full.html#ref-list-1 Email alerting service Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article. Notes To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/ View publication stats