Figure 1.
Clinical characteristics of dogs with ectodermal dysplasia-skin fragility syndrome with PKP1 deficiency.
Panel A: silhouette with lesion distribution of a 3-month-old puppy: skin lesions (highlighted in red) are localized at mucocutaneous junctions of the face (periocular, nose, lips, ear openings) and areas of friction on the axillae, groin, joints and footpads. Panel B: erosions of the nasal planum (arrowhead) and dorsal nasal fissure (arrow). Panel C: fissures around the lips (cheilitis, arrowhead). Panel D: footpad erosions due to sloughing of the superficial epithelium. Panel E: erosions and crusts on the extensor aspect of the hock. Panel F: superficial erosions and crusts with vertical fissuring on the right axilla. Inset: rubbing of normal appearing skin with a pencil eraser led to erosion formation (positive direct Nikolskiy sign).
Figure 2.
Histopathology findings for neonatal puppies with ectodermal dysplasia-skin fragility syndrome and PKP1 deficiency.
Panel A: coalescing keratinocyte acantholysis disrupts epidermal architecture in a biopsy from the distal limb with prominent involvement of the stratum spinosum. At higher magnification (insert), individual acantholytic keratinocytes (arrows) are rounded and have retraction and condensation of eosinophilic tonofilaments within the cytoplasm. Hematoxylin and eosin. Panel B: in normal canine skin, the epidermis consists of keratinocytes abutting each other at all layers. Bar = 100 µm.
Figure 3.
Histopathology findings for 3-month-old puppies with ectodermal dysplasia-skin fragility syndrome and PKP1 deficiency.
Panel A: Trunk skin exhibits diffuse epidermal hyperplasia topped by prominent, laminated to compact, orthokeratotic hyperkeratosis. Panel B: Higher magnification photomicrograph (Box insert from Panel A) illustrates mild keratinocyte acantholysis (arrows) in the stratum granulosum as well as condensation and aggregation of eosinophilic tonofilaments in the cytoplasm (arrow head). Panel C: Similar changes of keratinocyte acantholysis (arrows) and condensation of tonofilaments (arrowheads) are observed in the carpal pad epidermis. Hematoxylin and eosin. Bar = 100 µm.
Figure 4.
Ultrastructural findings for 3-month-old puppies with ectodermal dysplasia-skin fragility syndrome and PKP1 deficiency.
Panel A: Transmission electron microscopy of carpal pad epidermis reveals widening of intercellular spaces between keratinocytes and retraction of keratin filaments from the cell membrane. Panel B: Higher magnification photomicrograph (Box insert from Panel A) demonstrates reduced numbers of desmosomes attaching keratinocytes as well as the presence of small, partially formed desmosomes (arrows). Panels C and D: A higher magnification confirms that desmosomes appear small and rudimentary in an affected dog (C) compared to those of a normal dog (D). Bar = 1 µm.
Figure 5.
Expression of selected keratin and desmosomes proteins in normal and affected Chesapeake Bay retriever dogs.
Panels A–D: normal age- and breed-matched control dog footpad E–H: affected dog footpad. Panels A, E: keratin K14 was expressed in the cytoplasm of basal keratinocytes of both affected and normal dogs, but the staining pattern was more heterogeneous in affected dogs; some mid-epidermal keratinocytes expressed K14 in affected but not normal dogs. Panels B, F: in normal dogs, keratin K10 was seen with an homogeneous staining in suprabasal keratinocytes (B); in affected dogs, the staining intensity was heterogeneous within and between keratinocytes. Panels C, G: desmoplakin (DSP) staining revealed a typical fishnet intercellular pattern in normal dog skin (C), while it was cytoplasmic and heterogeneous in affected dogs (G). Panels D, H: Using the monoclonal antibody specific for armadillo-repeats of plakophilin-1 (PKP1), this molecule was detected intercellularly, with a dotted pattern, in the superficial epidermis of the normal control CBR (D); this pattern was not seen in samples from affected dogs (H). All panels were shot at ×20 magnification.
Figure 6.
Mutation in PKP1 causing ED-SFS in Chesapeake Bay retrievers.
Panel A: Transition from G-to-C at intronic splice donor site at the beginning of the first intron ([G/C]). Panel B. Consequence of the mutation described in panel A. The intronic splice donor site is destroyed by the transition from a G to a C resulting in a continual read through to a premature stop codon. Nucleotides in capitals are from exon 1 and those in small letters from intron 1.
Figure 7.
Detection of the G-to-C mutation in a family of purebred Chesapeake Bay retrievers.
DNA was isolated from tissues or whole blood, amplified by PCR, digested with a restriction enzyme that cut if the mutation was present, and visualized on an agarose gel. Symbols indicate the genotype as determined by the DNA results, which are shown beneath the pedigrees. Squares = males, circles = females; open symbols = homozygous normal; filled in symbols = affected with ED-SFS and homozygous for the mutation; half-filled symbols = heterozygous for the mutation; dotted-line symbols = no DNA or clinical information available. Approximate sizes (in bp) of the DNA fragments are indicated by comparison to the marker on the left of the panel.