Vet Dermatol 2015; 26: 3–e2

DOI: 10.1111/vde.12176

Epidermolysis bullosa in animals: a review Gildenor X. Medeiros and Franklin Riet-Correa Postgraduate Program in Veterinary Medicine, Veterinary Hospital, Federal University of Campina Grande, Patos, Para
ıba, CEP 58708-110, Brazil Correspondence: Franklin Riet-Correa, Postgraduate Program in Veterinary Medicine, Veterinary Hospital, Federal University of Campina Grande, Patos, Para
ıba, CEP 58708-110, Brazil. E-mail: [email protected]

Epidermolysis bullosa (EB) is a hereditary mechanobullous disease of animals and humans, characterized by an extreme fragility of the skin and mucous membranes. The main feature of EB in humans and animals is the formation of blisters and erosions in response to minor mechanical trauma. Epidermolysis bullosa is caused by mutations in the genes that code for structural proteins of the cytoskeleton of the basal keratinocytes or of the basement membrane zone. Based on the ultrastructural levels of tissue separation, EB is divided into the following three broad categories: epidermolysis bullosa simplex, junctional epidermolysis bullosa and dystrophic epidermolysis bullosa. Human types of EB are divided into several subtypes based on their ultrastructural changes and the mode of inheritance; subtypes are not fully established in animals. In humans, it is estimated that EB affects one in 17,000 live births; the frequency of EB in different animals species is not known. In all animal species, except in buffalo with epidermolysis bullosa simplex, multifocal ulcers are observed on the gums, hard and soft palates, mucosa of the lips, cheek mucosa and dorsum of the tongue. Dystrophic or absent nails, a frequent sign seen in human patients with EB, corresponds to the deformities and sloughing of the hooves in ungulates and to dystrophy or atrophy of the claws in dogs and cats. This review covers aspects of the molecular biology, diagnosis, classification, clinical signs and pathology of EB reported in animals.

Introduction Epidermolysis bullosa (EB) is a hereditary mechanobullous disease of animals and humans, characterized by an extreme fragility of the skin and mucous membranes. The main feature of EB in humans and animals is the formation of blisters and erosions in response to minor mechanical trauma, especially in areas subject to frictional stress, such as the oral cavity and the limbs.1,2 The fragility of the skin and mucous membranes results from abnormalities in the cytoskeleton of the basal keratinocytes or of the basement membrane zone (BMZ).3,4 Epidermolysis bullosa is caused by mutations in the genes that code for structural proteins of the cytoskeleton of basal keratinocytes and of the BMZ.3–5 Advances in analytical techniques of molecular genetics have allowed the identification of mutations in the genes encoding the structural proteins of the cytoskeleton of keratinocytes and BMZ, namely, PKP1 (plakophilin 1), DSP (desmoplakin), KRT5 (keratin 5), KRT14 (keratin 14), PLEC1 (plectin), ITGA6, ITGB4 (a6b4-integrin), LAMA3, LAMB3, LAMC2 (laminin 332), COL17A1 (type XVII collagen) and COL7A1 (type VII collagen).6 The cytoskeleton comprises a cytoplasmic network of intermediate filaments composed primarily of keratin 5 and keratin 14. Through their connections to the desmo-

Accepted 1 July 2014 Sources of Funding: This work was supported by the National Council of Technological and Scientific Development, CNPq, grant 471386/2010-3. Conflict of Interest: No conflicts of interest have been declared. © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

somes and hemidesmosomes, these filaments play a role in the organization of the shape of the cell and in maintenance of the structural integrity of the epidermis. Desmosomes are specialized complexes that form tight intercellular junctions between adjacent epithelial cells. The main proteins of these intercellular junctions are plakoglobin, plakophilin and desmoplakin and the cadherins desmocollin and desmoglein (Figure 1).4 The BMZ is located at the junction between the dermis and the epidermis. Its function is to maintain the adhesion between these two structurally different tissues by means of a complex network of adhesion molecules intricately related to one another. The BMZ is divided into three areas (hemidesmosomes, lamina lucida and lamina densa), which are attached to the dermis by the anchoring fibrils. The hemidesmosomes are composed of an inner and an outer plaque. The inner plaque is composed of the cytoplasmic hemidesmosomal proteins HD1/plectin and BP230 and is connected to the intermediate filaments within the cytoplasm of the basal-layer keratinocytes. The outer plaque contains the proteins a6b4-integrin and BP180. The lamina lucida is formed by the plasma membrane, the sub-basal dense plate and the anchoring filaments composed of BP180 (also known as collagen XVII) and laminin 332 (formerly known as laminin 5). The lamina densa is composed mainly of type IV collagen. Beneath the lamina densa are anchoring fibrils, cross-banded structures composed of type VII collagen that extend into the papillary dermis (Figure 1).7,8 Epidermolysis bullosa in humans was first described by Koebner in 18866 and has since been widely studied; recent advances have led to the identification of 3

Medeiros and Riet-Correa

Figure 1. Schematic representation of the molecular organization of the cytoskeleton of basal keratinocytes and the basement membrane zone. Abbreviations: E, epidermis; H, hemidesmosome; LD, lamina densa; LL, lamina lucida; and SD, sublamina densa. Proteins of the desmosome and of the intercellular junction are abbreviated as follows: C, cadherins; Dp, desmoplakin; Pg, plakoglobin; and Pp, plakophilin.

mutations in different genes, which account for the clinical heterogeneity of EB.2,5,6 The term ‘epidermolysis bullosa’ was first used in veterinary medicine in 1974,9 although it was also suggested for other similar diseases reported in animals, including red foot disease in sheep,10 hereditary mechanobullous diseases in horses11,12 and cattle13 and epitheliogenesis imperfecta in horses.14 It is estimated that in humans the disease affects one in 17,000 live births in the world population.15 In animals, the frequency of EB in different species is not known. In our diagnostic laboratory, in the Brazilian semi-arid region, one of 861 goat specimens for postmortem examination received between 1983 and 2012 was diagnosed with EB. During the same period, in a total of 1144 cattle specimens, two Gir calves from different farms were diagnosed with EB. Genetically modified mice have been used as animal models for understanding the pathogenesis of EB in humans. Molecular studies are rarely reported in domestic animals; however, some mutations have been identified (Table 1).1,16–28 Given the rarity of the disease and the growing volume of literature we assembled this review, which covers the molecular biology, diagnosis, classification, clinical signs and pathology of EB; it is hoped that this review will assist in the diagnosis and future research of the disease in animals.

Diagnosis and classification Epidermolysis bullosa is suspected when newborn animals have blisters and erosions in the skin and mucous membranes. These typically manifest in response to 4

minor trauma or they can be identified by applying the Nikolskiy test,29 performed by gently twisting a finger or an object back and forth on the surrounding normalappearing skin (marginal Nikolskiy test) and on the normal-appearing skin distant from the lesions (direct Nikolskiy test; Figure 2a). In a positive test, the epidermis will be detached easily from the skin. It is important to conduct a complete anamnesis, to determine whether the disease could be hereditary, and a thorough physical examination, including an assessment of the type and distribution of the lesions. Laboratory diagnosis is suggested by typical histological lesions of detachment of the epidermis from the dermis (Figure 2b). Confirmation requires transmission electron microscopy (Figure 3), immunofluorescence mapping and mutation analysis. Electron microscopy and immunofluorescence mapping allow the determination of the level of skin cleavage, which may be intraepidermal, intralamina lucida or sublamina densa. Electron microscopy permits visualization and semi-quantitative assessment of the specific structures (keratin filaments, desmosomes, hemidesmosomes, sub-basal dense plates, anchoring filaments and anchoring fibrils), which can be altered in number and/or appearance in selected EB subtypes.30,31 Immunofluorescence mapping, when coupled with the use of specific monoclonal antibodies, can provide considerable insight into the major type of EB present and also the structural protein most likely to be altered. Immunofluorescence mapping has relied traditionally on the staining of cryopreserved EB skin specimens with antibodies to bullous pemphigoid antigen, laminin-1, type IV collagen and keratin 14, in order to determine the level of blistering.31,32 Genetic © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Epidermolysis bullosa in animals

Table 1. Summary of epidermolysis bullosa in animals Epidermolysis bullosa type and animal species

Mode of inheritance

Epidermolysis bullosa simplex (EBS) Cattle Autosomal dominant Cattle Autosomal dominant Cattle Autosomal recessive Buffalo Autosomal recessive Dogs Autosomal recessive Herlitz junctional epidermolysis bullosa (JEB) Cattle Undetermined Horses Autosomal recessive

Ultrastructural site of separation

Target gene (protein)

Types of mutation known

References

Basal layer Basal layer Basal layer Suprabasal Suprabasal

KRT5 (keratin 5) Undetermined Undetermined Undetermined PKP1 (plakophilin 1)

Missense, G?A at 4164 Undetermined Undetermined Undetermined Splice site, IVS1 + G?C

23 40,44,45 43 39 28

Lamina lucida Lamina lucida

Undetermined LAMA3 (laminin 332)

60 24

Horses

Autosomal recessive

Lamina lucida

LAMC2 (laminin 332)

Horses Sheep

Autosomal recessive Autosomal recessive

Lamina lucida Lamina lucida

Undetermined LAMC2 (laminin 332)

Sheep Dogs

Autosomal recessive Autosomal recessive

Lamina lucida Lamina lucida

Undetermined LAMA3 (laminin 332)

Dogs Undetermined Rats Autosomal recessive Mice Autosomal recessive Non-Herlitz JEB Dogs Autosomal recessive Cats Undetermined Dystrophic epidermolysis bullosa (DEB) Cattle Autosomal recessive Cattle Autosomal recessive Sheep Autosomal recessive Goats Autosomal recessive Dogs Autosomal recessive Dogs Autosomal recessive Cats Autosomal recessive Ostriches Undetermined

Lamina lucida Lamina lucida Lamina lucida

Undetermined Undetermined Undetermined

Undetermined 6589 bp deletion, exons 24–27 Nonsense, insertion of C after 1368 Undetermined Frameshift, deletion of CA after 2746 Undetermined Nonsense, insertion of 227 bp at 4818 Undetermined Undetermined Undetermined

Lamina lucida Lamina lucida

Undetermined Undetermined

Undetermined Undetermined

61,74–79 63

Sublamina densa Sublamina densa Sublamina densa Sublamina densa Sublamina densa Sublamina densa Sublamina densa Sublamina densa

COL7A1 (type VII collagen) Undetermined Undetermined Undetermined COL7A1 (type VII collagen) Undetermined Undetermined Undetermined

Nonsense, C?T at 4756 Undetermined Undetermined Undetermined Missense, G?A at 5716 Undetermined Undetermined Undetermined

27 13,92 84,85 80 20,25 90,91 88 93

analysis of an animal’s ascendents and decendents by mutational analysis through DNA sequencing is the ultimate means of determining the mode of inheritance and the precise site(s) and type(s) of molecular mutation present in an animal with EB. This also plays a key role in identification of EB carriers and provides an excellent research tool.5–7,33 In addition, the eventual application of gene therapy to EB cases will be dependent on the determination of the specific mutation(s) present.20,25,34 The classification of the types of EB in animals has traditionally been based on the human classification, which is based on the ultrastructural level of the tissue separation. Thus, EB can be divided into the following three broad categories: epidermolysis bullosa simplex (EBS), characterized by basal or suprabasal intercellular clefting with or without cytolysis of basal keratinocytes; junctional epidermolysis bullosa (JEB), in which separation occurs in the lamina lucida; and dystrophic epidermolysis bullosa (DEB), with a cleavage within or below the lamina densa. Other human disorders, such as Kindler’s syndrome, which show clinical and biological aspects similar to EB but differ in the presence of other systemic symptoms and usually resolve with age, can be included within the EB group.6,7 Based on the ultrastructural changes and the mode of inheritance, human types of EB are divided into several subtypes (Table 2).6 There is also an acquired form of EB called epidermolysis bullosa acquisita, which is a rare, autoimmune, blister© 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

18,19,21 11,12,14,69,70 26 66 22 62 64 65

ing skin disease, first reported in humans in 1895. Epidermolysis bullosa acquisita was classified as an EB because clinically, it resembles hereditary DEB.35 It is caused by the production of immunoglobulin G autoantibodies to collagen VII, the major component of anchoring fibrils.36 Epidermolysis bullosa acquisita was reported in dogs in 1998,37 but because it can mimic the clinical, histological and immunohistological features of bullous pemphigoid, it is likely that it was misdiagnosed before 1998.38 Epidermolysis bullosa acquisita is included in the group of autoimmune subepidermal bullous diseases (including bullous pemphigoid and mucous membrane pemphigoid) and will therefore not be discussed further in this review. The clinical and pathological findings are similar in all types of EB, varying only in severity. Given that the descriptions are based on findings observed in a small number of animals, the complete range of clinical signs may be more diverse (Table 3). It is interesting to note that skin fragility resulting in the formation of blisters is common in humans, whereas in animals, especially in dogs and cats, the integument is less prone to such lesions. One possible explanation for this phenomenon is that in animals hair prevents the detachment of the epithelium, not only by acting as a mechanical barrier but also by deeply anchoring the epidermis into the dermis at the level of the invagination of the hair follicles. It can be speculated that in animals the severity of lesions is 5

Medeiros and Riet-Correa

(a)

(b)

Figure 2. (a) Skin of the inner face of the pinna of a calf affected by junctional epidermolysis bullosa, showing friction-induced detachment of the epidermis after the Nikolskiy test. (b) Photomicrograph of histopathology of the skin of a calf affected with junctional epidermolysis bullosa. The epidermis is detached from the dermis, forming a subepidermal cleft containing erythrocytes and neutrophils. Haematoxylin and eosin stain. The scale bar represents 50 lm.

directly proportional to the risk of trauma associated with the mode of life of each species. In farm animals, lesions are more severe because they live in herds and in environments more often favourable to trauma. In EBaffected animals, sloughing of the hooves is also common. In dogs and cats, the lesions are milder than in farm animals and are most predominant in hairless areas, such as the muzzle and footpads. Oral lesions in herbivores are more extensive than in other species, possibly because of their fibrous food, except in buffalo with EBS. Most cases of EB in humans lead to death in infancy. Likewise, animals are euthanized or die during the first months of life. Reports of survival for months or years in animals with EB are rare.

Epidermolysis bullosa simplex Epidermolysis bullosa simplex is characterized by suprabasal separation without cytolysis of basal cells, as it occurs in dogs28 and buffalo,39 or by basal separation with cytolysis of basal cells (cytolytic), as seen in cattle.23,40,41 6

Clinical signs occur immediately after birth, with superficial epidermal layers sloughing upon pressure. The signs tend to be less severe than in JEB and DEB (Figure 4). Dystrophic or absent nails are frequently seen in human patients,42 and this corresponds to the deformities and sloughing of the hooves in buffalo (Figure 4c)39 and cattle23,40,41,43–45 and to dystrophic or atrophic claws in dogs.28 Multifocal ulcers on the gums, hard and soft palates, mucosa of the lips, buccal mucosa and dorsum of the tongue are observed in cattle with cytolytic basal layer separation23,40,41 and dogs with noncytolytic suprabasal separation.28 In contrast, lesions of the oral cavity are not observed in buffalo with noncytolytic suprabasal separation.39 Generally, cattle affected by EBS with basal layer separation die at 1–4 weeks old,23,40,41 whereas dogs28 and buffalo39 with suprabasal separation have been reported to die at 1 and 4 years of age, respectively. Histopathological findings in the skin and oral mucosa are similar, but they vary in degree depending on the clinical manifestation period, the distribution of the lesions and the presence of secondary complications, such as bacterial infections. The initial lesion is the subepidermal separation with a minimal neutrophilic inflammation. In more advanced skin lesions, extensive areas of intact fullthickness epidermis are separated from the dermis, forming large clefts containing eosinophilic fluid, extravasated erythrocytes and occasional neutrophils.23,41 Periodic acid Schiff staining of histological skin sections from calves demonstrated cleft formation involving the epidermis, with the basement membrane attached to the floor of the cleft.41 An apparent loss of the intercellular junction and acantholysis are observed in EBS with suprabasal separation (Figure 3a).28,39,43 Detachment of the corneal epithelium with minimal inflammation was observed in cattle.23 In humans46,47 and in cattle,23 EBS is generally caused by mutations in the genes encoding keratins 5 and 14 (KRT5 and KRT14) and is transmitted by autosomal dominant inheritance. In cattle, suprabasal clefts arise from lysis of the basal keratinocytes and the clumping of the intermediate filaments.23,41 The clumping of the intermediate filaments has been described in the Dowling–Meara form of EBS in humans.47,48 In cattle, a missense mutation was identified in the KRT5 gene that revealed a one base change (G-to-A substitution at position 4164 of the genomic sequence). This resulted in a codon change from GAG (glutamic acid, E, acidic) to AAG (lysine, K, basic) at position 478. It is possible that this amino acid change (acidic to basic) altered the formation of the keratin heterodimers and the intermediate filaments, leading to keratinocyte fragility and a separation of the epidermis. In these cattle with EBS, only one mutant allele was identified, present at a frequency of ~20% in the sire’s blood and semen samples. This suggested a mosaic distribution in these tissues and a dominant mode of inheritance; however, the sire was unaffected.23 In some human EBS cases, the disease presents revertant mosaicism, which refers to a situation in which a second mutation attenuates or abolishes the deleterious effect of the original mutation in certain areas of the skin.49 Revertant mosaicism was also demonstrated in humans patients with mutations in the JEB genes COL17A1 and LAMB3,50 in DEB51,52 and in Kindler’s syndrome.53,54 © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Epidermolysis bullosa in animals

(a)

(b)

(c)

(d)

Figure 3. Transmission electron photomicrographs. (a) Skin of a buffalo affected with epidermolysis bullosa simplex. The floor of the suprabasal blister (star) is formed by basal cells attached to the dermis (D). Note acantholytic cells (arrows) within the blister. The scale bar represents 2 lm. (b) Skin of a calf affected with junctional epidermolysis bullosa. Basal cells form the roof of the blister (star) after separation from the dermis at the level of the lamina lucida. Note the small hemidesmosomes (box). The scale bar represents 1 lm. (c) Higher magnification of the box in (b). Note the cell plasma membrane (arrow) and small and ill-defined hemidesmosomes within the boxes. The scale bar represents 200 nm. (d) Skin of a goat affected with dystrophic epidermolysis bullosa. The lamina densa (arrows) forms the roof of the bulla (star). The basal lamina, hemidesmosomes and anchoring filaments are well preserved. The scale bar represents 500 nm.

In humans, recessively inherited EBS is caused by mutations in the plectin,55,56 desmoplakin57 and plakophilin-1 (PKP1) genes.46,58 In Chesapeake Bay retriever dogs, an autosomal recessive acantholytic dermatosis with deficiency of plakophilin-1 was reported.28 Electron microscopy demonstrated a reduced number of partly formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Nucleotide sequencing revealed a G-to-C conversion at the IVS1 splice donor site within the first intron of PKP1, resulting in a premature stop codon. As a consequence, the mutated protein is predicted to be truncated and composed of 75 instead of 749 amino acids.28 These changes resemble EBS with a suprabasal separation, associated with a deficiency of plakophilin-1, known as ectodermal dysplasia/skin fragility syndrome in humans.46,58 In buffalo, a hereditary mechanobullous disease called hereditary suprabasilar mechanobullous acantholytic der© 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

matosis was not classified as EB because the ultrastructural site of the separation is suprabasal and oral lesions are not observed.39 However, following the current classification of EB in humans (Table 2), this buffalo disease can be defined as epidermolysis bullosa simplex with a suprabasal separation. The disease is similar to EBS, which also has minimal or absent oral lesions, in its deficiency of plakophilin-1.6 Epidermolysis bullosa simplex in buffalo is inherited through an autosomal recessive mechanism, characterized by a suprabasal separation between the stratum basale and stratum spinosum, with a loss of the desmosomal attachment (Figure 3a).39 Further studies suggested that it is likely the disease in buffalo occurs due to defects in the stability, structure or function of the desmosomal cadherins and not to alterations in the desmosomal plaque proteins.59

Junctional epidermolysis bullosa In JEB, the clinical signs are due to defects and changes in the hemidesmosomes, the plasma membrane, the 7

Medeiros and Riet-Correa

Table 2. Types and subtypes of epidermolysis bullosa based on the ultrastructural level of tissue separation in the basement membrane zone, based on the classification in humans* Epidermolysis bullosa type and subtype

Ultrastructural site separation

Other ultrastructural findings

Epidermolysis bullosa simplex (EBS) Localized EBS Dowling–Meara EBS

Basal layer Basal layer

Split may spread to suprabasilar layer Dense, circumscribed clumps of keratin filaments (most commonly observed within lesional biopsy site) Reduced integration of keratin filaments within hemidesmosomes Absent or reduced keratin filaments within basal keratinocytes –

EBS–muscular dystrophy Autosomal recessive EBS Superficial EBS Lethal acantholytic EBS Plakophilin-1 deficiency EBS† Junctional epidermolysis bullosa (JEB) Herlitz JEB†

Predominantly in basal layer, above the hemidesmosome attachment plaque Basal keratinocytes Suprabasal, usually at interface between granular and cornified cell layers Suprabasal and acantholysis Suprabasal, mid-epidermal cell–cell separation

Lamina lucida

Non-Herlitz JEB†

Lamina lucida

JEB–pyloric atresia

Lamina lucida

Perinuclear retraction of keratin filaments Diminutive suprabasal desmosomes; perinuclear retraction of keratin filaments Markedly reduced or absent hemidesmosomes; absent sub-basal dense plate Hemidesmosomes may be normal or reduced in size and number Small hemidesmosome plaques often with attenuated sub-basal dense plate

Dominant dystrophic epidermolysis bullosa (DDEB) Generalized DDEB Sublamina densa DDEB–bullous dermolysis Sublamina densa of the newborn Recessive dystrophic epidermolysis bullosa (RDEB) Severe generalized RDEB† Sublamina densa Other generalized RDEB Sublamina densa RDEB–bullous dermolysis Sublamina densa of the newborn Kindler’s syndrome Variable; intraepidermal, intralamina lucida or sublamina densa

Normal or decreased numbers of anchoring fibrils Electron-dense stellate bodies within basal layer, reduced anchoring fibrils Absent or rudimentary anchoring fibrils Reduced or rudimentary-appearing anchoring fibrils Electron-dense stellate bodies within basal layer, reduced anchoring fibrils Reduplication of lamina densa; presence of upper dermal colloid bodies and melanophages

*Adapted from Fine et al. (2008)6 and Bruckner-Tuderman and Has (2014).7 Subtypes described in animals.

†

sub-basal dense plate and the anchoring fibrils. In cattle (Figure 5),60 horses,18 and sheep26 affected by Herlitz type of JEB, the skin lesions are more severe than in the other types of EB, are distributed throughout the body with all hooves affected at the same time (Figure 5a,b), and most animals die in the first week of life. The same pattern is observed in dogs,61,62 cats (Figure 6),63 rats64 and mice65 with JEB, although the lesions are somewhat less severe. In all animal species with JEB, oral multifocal ulcers are observed (Figure 6a).18,26,60,63,66 Enamel hypoplasia is a common finding in humans67 with JEB; in animals, it has only been sporadically observed in horses18,19 and dogs.22 Sporadically, ulcers are observed elsewhere, such as on the anus, vagina14 and rumen (Figure 5c). Histopathological findings in skin (Figure 2b) and oral mucosa are similar to those observed in EBS with separation in the basal layer. In cattle, areas of a total loss of the oral and lingual epithelium are observed, leading to necrosis, extensive ulceration, infiltration by neutrophils and haemorrhages.60 Similar changes are observed in the vaginal and anal mucosa in horses.14 Junctional epidermolysis bullosa in humans is inherited through autosomal dominant68 and recessive genes.2,6,46 In animals, JEB is inherited through autosomal recessive genes.1,11,12,14,18,19,21,22,24,26,64–66,69,70 The disease is characterized by blister formation in the lamina lucida (Figure 3b) and defects in the proteins of the hemidesmo8

some-anchoring filament complex. The hemidesmosomes are the principal structures of adhesion at the BMZ. Abnormalities in the hemidesmosome–anchoring filament complex can cause extreme fragility in the dermo-epidermal junction.4,15 In cattle,60 horses14,70 and rats,64 abnormalities in this complex include reduced anchoring filaments, small hemidesmosomes without clear demarcation (Figure 3c) and an attenuated or absent sub-basal dense plate. In contrast, dogs affected by JEB, with severe fragility of the skin and mucosa caused by mutations in the LAMA3 gene, do not present abnormalities in hemidesmosomes.22 Most mutations causing JEB in humans are located in one of three genes encoding the a3, b3, or c2 laminin 332 heterotrimer chains, the LAMA3, LAMB3 and LAMC2 genes, respectively.2 These mutations result in a lack of expression of the respective protein chain. The laminin 332 heterotrimer forms the anchoring filaments that attach the a6b4-integrin of the hemidesmosome to the collagen VII of the anchoring fibrils.8 In Belgian horses, a nonsense mutation in the LAMC2 gene, designated 1368insC, caused a shift in the open reading frame of the c2 messenger RNA, resulting in a downstream premature termination codon (TGA).18,19 The same mutation was associated with JEB in trait Breton and trait Comtois horses.21 Mutations in the gene LAMA3 due to a deletion of 6589 bp in the region that © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Epidermolysis bullosa in animals

Table 3. Summary of the clinical and pathological findings of epidermolysis bullosa in animals Clinical and pathological findings

Epidermolysis bullosa simplex

Junctional epidermolysis bullosa

Dystrophic epidermolysis bullosa

Onset of the clinical signs Skin findings Predominant distribution

First week

At birth

First week

Generalized

Limbs, pressure points, glabrous areas

Blisters, erosions and crust

Limbs, pressure points, glabrous areas +++ (cattle), ++ (buffalo, dogs)*

Alopecia

+++ (cattle), ++ (buffalo, dogs)

+++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) +++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) – – –

++ (cattle, sheep, goats, ostriches), + (dogs, cats) ++ (cattle, sheep, goats, ostriches), + (dogs, cats) ++ (goats) + (dogs, cats) ++ (cattle)

+++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) ++ (cattle, horses, sheep), + (dogs, cats, rats, mice) +++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice)

++ (cattle, sheep, goats, ostriches), + (dogs, cats) +++ (cattle, sheep, goats), + (dogs, cats)

Atrophic scarring Milia Ear deformities Hoof and claw findings Erythema of the coronary band Deformities Exungulation Extracutaneous involvement Growth retardation Oral cavity lesions Corneal ulcer Postmortem examination Ulcers in the oral cavity

– – – +++ (cattle), ++ (buffalo, dogs) +++ (cattle), ++ (buffalo, dogs) +++ (cattle), ++ (buffalo, dogs)

++ +++ (cattle), ++ (dogs) + (cattle) +++ (cattle), ++ (dogs)

Ulcers on the tongue

+++ (cattle), ++ (dogs)

Defects in tooth enamel

–

Gastrointestinal tract lesions

–

Genitourinary tract lesions Death

– Third week (cattle) First year (dogs) and fourth year (buffalo)

++ (cattle, sheep, goats, ostriches), + (dogs, cats)

+++ +++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) + (cats)

++ +++ (cattle, sheep, goats), ++ (dogs, cats)

+++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) +++ (cattle, horses, sheep), ++ (dogs, cats, rats, mice) Enamel hypoplasia, + (horses, dogs) Rumenal ulcers, + (cattle); anal ulcers, + (horses) Vaginal ulcers, + (horses) Herlitz: first week Non-Herlitz: first year (cats) and fourth year (dogs)

+++ (cattle, sheep, goats), ++ (dogs, cats)

+ (sheep, goats)

+++ (cattle, sheep, goats), ++ (dogs, cats) – Oesophageal ulcers, + (sheep, goats, dogs) Vulvar ulcers, + (goats) First at third month (cattle, sheep, goats, ostriches) First year (dogs, cats)

Scale: –, absent; +, sporadically present; ++, frequently present; and +++, always present. *In buffalo and dogs, in which the separation is suprabasal without cytolysis, the lesions are less severe than in cattle, in which separation is basal with cytolysis.

includes exons 24–27 have been identified in American saddlebred horses with JEB.24 A 2 bp deletion within exon 18 of LAMC2 (c.2746delCA) was identified as responsible for Herlitz-type JEB in black-headed mutton sheep, causing a frameshift with a premature stop codon and an alternative splicing of exon 18.26 In German shorthaired pointer dogs, the mutation is due to an abnormal mRNA transcript with an insertion of an out-of-frame 227 nucleotides in position 4818 at the junction between exons 35 and 36 of the LAMA3 gene. This 227 bp insertion carries a nonsense (TAA) codon 33 bp downstream of the insertion site.22 In animals, mutations located in one of the three genes encoding laminin 33218,19,21,22,24,26 are genetically similar to mutations causing Herlitz-type JEB in humans.71,72 In humans, JEB with less severe clinical findings, known as non-Herlitz JEB, is caused by mutations in the COL17A1 (encoding type XVII collagen), ITGA6 and ITGB4 genes (encoding a6b4-integrin). In these cases, the hemidesmosomes are either normal or reduced in size and number.6,73 Clinical and ultrastructural findings similar to the non-Herlitz form of JEB in humans have been observed in dogs.74–79 © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Dystrophic epidermolysis bullosa In DEB, lesions of the skin and mucous membranes can heal with scar formation, and this has been reported in goats80 and humans.81 Healing with scar formation occurs because blisters below the lamina densa will lead to a mesenchymal wound-healing response in the dermis.81,82 As reported in humans,67,83 healing in goats with DEB80 led to changes in the architecture of some of the anatomical structures of the oral mucosa, including the palatal rugae and tongue papillae, which may reduce in length or disappear after healing, leaving a flat surface (Figure 7a). This finding is not observed in ruminants with EBS23,40,41,43,44 or JEB (Figure 7b).60 Ulcers in the oesophagus,80,84,85 vulva80 and cornea80,84 are described. Histopathological findings in the skin and oral mucosa are similar to those found in EBS and JEB; however, periodic acid Schiff staining of histological sections skin demonstrated that the basement membrane was attached to the roof of the cleft.80,85 Dystrophic epidermolysis bullosa comprises dominant and recessive forms caused by mutations in the COL7A1 gene that codes for type VII collagen, which 9

Medeiros and Riet-Correa

(a)

(b)

(c)

Figure 4. Buffalo with epidermolysis bullosa simplex. (a) Numerous areas of chronic lesions with alopecia, erosions, crusts and scars are observed on the skin. Note that the buffalo is an adult and nearly 4 years old. (b) Skin with recent detachment of the epidermis caused by occasional friction. (c) Palmar aspect of the digits of the thoracic limb showing loss of the hoof of a paradigit and deformities in the other hooves. Courtesy of Cristina Ghever Fernandes.

(a)

(b)

(c)

Figure 5. Calf (2 months old) with junctional epidermolysis bullosa. (a) Large areas of erosions and crusts are observed on the skin, especially at the areas of friction, mainly on the limbs. Note exungulation of all hooves. (b) Lateral aspect of the digits of the thoracic limb showing skin detachment and exungulation of the hooves. (c) Rumenal mucosa showing several ulcers, mainly in the ruminal pillars.

10

is the major component of the anchoring fibrils of the dermo-epidermal junction.81,86,87 Ultrastructurally, the site of blistering is the sublamina densa zone (Figure 3d). In humans,81,86 cats,88,89 dogs,90,91 sheep,84 cattle13,92 and goats80 with recessive DEB, the anchoring fibrils are scarce and rudimentary. Dystrophic epidermolysis bullosa with similar ultrastructural alterations has also been reported in ostriches, although the mode of inheritance was not determined.93 These changes in the anchoring fibrils decrease their adhesion to the collagen fibres of the dermis, leaving the dermo-epidermal junction susceptible to separation.94 In dominant DEB, recorded in humans but not in animals, the anchoring fibrils are normal but decreased in number.95 Other structures of the BMZ, such as the basal keratinocytes, the lamina lucida, the lamina densa and the hemidesmosomes, appear to be normal in DEB. Over 300 mutations have been reported in humans, and DEB is strongly associated with the glycine substitution by different amino acids in one allele of the gene encoding the collagenous domain of type VII collagen. Glycine substitutions probably have a dominant negative effect on the formation of type VII collagen.95–97 A missense mutation in the COL7A1 gene (5716G-to-A) with the substitution of glycine by a serine has also been observed in golden retriever dogs with recessive DEB. This mutation results in reduced secretion of abnormal type VII colla© 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Epidermolysis bullosa in animals

the COL7A1 gene (c.4756C.T), resulting in a stop codon at amino acid residue 1586 in the bovine COL7A1 protein sequence, which probably prevents anchoring fibril formation in the homozygous animals.27

(a)

Conclusion Knowledge about EB in animals has increased in recent years; however, more extensive use of molecular techniques is needed to identify further gene mutations responsible for the different clinical and pathological pictures of the diseases. In animals, the three basic types of EB have been reported; subtypes remain poorly defined. In animals, the severity of oral, cutaneous and hoof lesions may be related to the type of EB and to the mode of life of each species. At present, there is no specific treatment for EB. Clinical management is mainly supportive, aiming at protection of the skin from friction, prevention of infections and loss of body fluids, analgesia and optimal nutritional status. Progress in understanding the molecular basis of recessive DEB will provide the basis for development of novel genetic and cellular therapies targeted at restoring the defective anchoring fibrils. The availability of animal models to study human EB may provide a basis for gene therapy approaches as a potential definitive treatment for this devastating disease.

(b)

Acknowledgements We would like to thank Severo Sales de Barros and Cristina Gevher from the Federal University of Pelotas, Brazil
te
rinaire Roquefort and Zeneib Alhaidari from Clinique Ve les Pins, France, for providing photographs. Figure 6. Cat with junctional epidermolysis bullosa. (a) Oral lesions at 6 months of age. (b) Superficial ulcer on the anterior aspect of the left pinna at initial presentation. Reproduced with permission from Alhaidari et al.9 Copyright © 2005, John Wiley and Sons.

(a)

(b)

Figure 7. Oral vestibule. (a) Goat with dystrophic epidermolysis bullosa. (b) Calf with junctional epidermolysis bullosa. In the goat, there are ulcers and scars on the cheek mucosa with a loss of papillae (arrow). In the calf, an ulcer is observed on the cheek mucosa (arrow) but without scarring and loss of cheek papillae.

gen and assembly of anomalous anchoring fibrils abut€henvieh catting the lamina densa.20,25,91 In Rotes Ho tle, DNA sequencing revealed a nonsense mutation in © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

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sume
– L’e
pidermolyse bulleuse (EB) est une maladie he
re
ditaire bulleuse des animaux et des humains, Re
rise par une extre ^me fragilite
de la peau et des muqueuses. La principale caracte
ristique de qui se caracte
rosions en re
ponse 
canique l’EB animale et humaine est la formation de cloques et d’e a un traumatisme me
pidermolyse bulleuse est cause
e par des mutations des ge nes codant pour les prote
ines mineur.L’e
ratinocytes basaux ou de la membrane basale. Compte tenu des nivestructurales du cytosquelette des ke © 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

13

Medeiros and Riet-Correa


paration ultrastrcturale tissulaire, l’EB est divise
e en trois grandes cate
gories : l’e
pidermolyse bulaux de se
pidermolyse billeuse jonctionnelle et l’e
pidermolyse bulleuse dystrophique. Les types huleuse simple, l’e
s en plusieurs sous-types base
s sur les modifications ultrastructurales et le mode mains d’EB sont divise
tablis chez les animaux. Chez l’homme, on esde transmission ; les sous-types ne sont pas totalement e
quence de l’EB dans les time que l’EB touche 1 individu sur 17 000 naissances ; on ne conna^ıt pas la fre
rentes espe ces animales.Pour toutes les espe ces animales, 
pidermodiffe a l’exception du bison atteint d’e res multifocaux sont observe
s sur les gencives, les palais mous et durs, les lyse bulleuse simplex, les ulce muqueuses labiales, et les muqueuses des joues et la face dorsale de la langue. Les ongles absents ou dy
quent chez l’homme atteint d’EB, correspond 
formation et 
des strophiques, un signe fre a la de a la friabilite
s et a de la dystrophie  sabots chez les ongule a l’atrophie des griffes chez le chien et le chat.Cette revue
culaire, du diagnostic, de la classification, des signes cliniques et de couvre les aspects de la biologie mole
crite chez l’animal. la pathologie de l’EB de Resumen – La epidermolisis bullosa (EB) es una enfermedad hereditaria mecanovesicular de animales y humanos, caracterizada por una extrema fragilidad de la piel y de las membranas mucosas. La principal car
n de ampollas y erosiones en respuesta a traumas acter
ıstica de EB en humanos y animales es la formacio mec
anicos menores.La epidermis bullosa se caracteriza por mutaciones en los genes que codifican para prote
ınas estructurales del esqueleto de los queratinocitos basales o de la zona de la membrana basal. Ba
n del tejido a nivel ultraestructural, la epidermis bullosa se divide en las tres sado en los niveles de separacio
n, y epidermal siguientes amplias categor
ıas: epidermolisis bullosa simplex, epidermolisis bullosa de la unio
fica. Los tipos humanos de epidermolisis se dividen en varios subtipos basados en sus cambibullosa distro os estructurales y el modo de herencia; Los subtipos no est
an totalmente establecidos en animales. En humanos, se estima que la epidermolisis bullosa a uno en 17.000 nacimientos; la frecuencia de epidermolisis bullosa en diferentes especies animales no se conoce.En todas las especies animales, excepto el bis
lceras multifocales en las enc
ıas, paladares duro y onte con epidermolisis bullosa simplex, se observan u ~as est
blando, mucosa de los labios, mucosa de los carrillos, y superficie dorsal de la lengua. Las un an dis
plasicas o ausentes, un signo frecuente en pacientes humanos con epidermaolisis bullosa, que corre
n y la pe
rdida de pezun ~as de ungulados y la distrofia o atrofia de las un ~as en sponde con la deformacio
n cubre aspectos de la biolog
ıa molecular, diagno
stico, clasificacio
n, signos perros y en gatos.Esta revisio cl
ınicos y patolog
ıa de la epidermolisis bullosa descritos en animales. €re mechanobullo €se Erkrankung von Zusammenfassung – Die Epidermolysis bullosa (EB) ist eine heredita Tieren und Menschen, die charakterisiert wird durch extreme Fragilit€ at der Haut und der Schleimh€ aute. Das Hauptcharakteristikum der EB des Menschen und der Tiere ist die Blasenbildung und die Entstehung von Erosionen als Reaktion auf geringes mechanisches Trauma.Die Epidermolysis bullosa wird durch Mutationen von Genen verursacht, die die Strukturproteine des Zytoskeletts der basalen Keratinozyten oder der Basalmembran kodieren. Basierend auf den ultrastrukturellen Levels der Gewebstrennung, wird die EB in die folgenden drei breiten Kategorien eingeteilt: Epidermolysis bullosa simplex, junktionale Epidermolysis bullosa und dystrophische Epidermolysis bullosa. Die Typen der humanen EB werden basierend auf den ultrastrukturellen Ver€anderungen und der Art der Vererbung in verschiedene Subtypen eingeteilt; bei den Tieren sind die Subtypen nicht g€anzlich bekannt. Beim Menschen wird gesch€ atzt, dass EB eine von 17000 Lebendgeburten betrifft, w€ahrend die Frequenz von EB bei den verschiedenen Tierspezies nicht €ffel mit Epidermolysis bullosa simplex, kommen multifokbekannt ist.Bei allen Tierspezies, außer beim Bu ale Ulzera am Zahnfleisch, sowie am harten und weichen Gaumen, an den Schleimh€ auten der Lippen, der €cken, vor. Dystrophische oder fehlende N€ Wangenmukosa und dem Zungenru agel, ein h€ aufig auftretendes Symptom beim menschlichen Patienten mit EB, korrespondiert mit Deformit€ aten und dem Ausschuhen von Hufen bei Einhufern und mit Dystrophie oder Atrophie der Krallen bei Hunden und Katzen.Diese Review befasst sich mit Aspekten der Molekularbiologie, der Diagnose, Einteilung, den klinischen Symptomen und der Pathologie der EB, die bei Tieren beschrieben sind. 要約 – 表皮水疱症(EB)は皮膚と粘膜の極度の脆弱性を特徴とする、イヌおよびヒトの遺伝性水疱性疾患 である。ヒトと動物におけるEBの主な特徴は水疱の形成と軽度の機械的な外傷による糜爛形成である。 表皮水疱症は基底部ケラチノサイトの細胞骨格あるいは基底膜領域の構造タンパク質をコードする遺伝 子の変異により生じる。微細構造的なレベルの組織分離に基づくと、EBは以下の3つの広いカテゴリー、 単純型表皮水疱症、接合部表皮水疱症、栄養障害型表皮水疱症に分類される。ヒトのEBのタイプはそれ らの微細構造的な変化と遺伝形式に基づいて複数の亜型に分類されている。しかし、亜型は動物では十 分に確立されていない。ヒトでは、EBは17,000人に1人が罹患すると推定されているが、他の動物種にお けるEBの頻度は知られていない。バッファローにおける単純型表皮水疱症以外で、すべての動物種にお いて、歯肉、硬口蓋、軟口蓋、口唇部粘膜、頬粘膜ならびに舌の背側に多病巣性の潰瘍が認められた。 異栄養性あるいは爪の欠損はヒトのEB患者では頻繁に認められるが、有蹄動物における蹄の変形や脱落 およびイヌやネコにおける爪の変形あるいは萎縮と合致する。このレビューは動物で報告されたEBの分 子生物学、診断、分類、臨床症状、および病理的な側面を網羅している。 e1

© 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

Epidermolysis bullosa in animals

摘要 – 大疱性表皮松懈症(EB)是一种人和动物的遗传性机械性大疱性疾病,以皮肤和黏膜极端脆弱为特 点。人和动物EB的主要特征是形成水疱和溃疡,主要源于轻微的机械损伤。大疱性表皮松懈症是基因突变引 起,导致基底膜区的基底角质形成细胞的细胞骨架结构蛋白编码改变。根据组织分离的超微结构等级不同, EB被分为以下三个类别:单纯大疱性表皮松懈症、交接处大疱性表皮松懈症、发育不良性大疱性表皮松懈 症。根据超微结构和遗传类型的不同,人EB类型分为多个亚型;亚型不全适用于动物。人类每17,000人就有 一人患有EB;然而其他物种EB发病率没有统计。单纯大疱性表皮松懈症,所有动物,除了水牛,均表现为牙 龈、硬软颚、唇部黏膜、颊黏膜和舌背部多处溃疡。人EB也会出现指甲发育不良或缺失,对应有蹄动物则是 蹄部畸形和脱落,犬猫则是甲发育不良或萎缩。本综述包含了动物EB分子生物学、诊断、分类、临床症状和 发病机理等方面。

© 2014 ESVD and ACVD, Veterinary Dermatology, 26, 3–e2.

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