Disadhesion of epidermal keratinocytes: A histologic clue to palmoplantar keratodermas caused by DSG1 mutations

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1 DERMATOPATHOLOGY Disadhesion of epidermal keratinocytes: A histologic clue to palmoplantar keratodermas caused by DSG1 mutations Reuven Bergman, MD, a Dov Hershkovitz, MD, b Dana Fuchs, MSc, c Margarita Indelman, MSc, a Yael Gadot, MD, a and Eli Sprecher, MD c Haifa and Tel Aviv, Israel Background: Recent developments in molecular genetics may lead to re-examination of the histopathology of inherited palmoplantar keratodermas (PPKs) based on more precise groupings of the various entities and syndromes. Objective: We sought to characterize the histopathological findings in PPKs associated with mutations in DSG1, which encodes desmoglein 1. Methods: We studied the histopathology of 3 cases of keratosis palmoplantaris striata type I and one case of diffuse PPK, all associated with autosomal-dominant mutations in DSG1. Our cases for comparison included 4 cases with Mal de Meleda PPK associated with autosomal-recessive SLURP1 mutations, one case with pachyonychia congenita type II PPK associated with an autosomal-dominant KRT17 mutation, and one case with focal PPK associated with an autosomal-dominant KRT16 mutation. Results: The distinguishing histopathological features of the 3 keratosis palmoplantaris striata type I cases and the diffuse PPK case associated with DSG1 mutation were: varying degrees of widening of the intercellular spaces and partial disadhesion of keratinocytes in the mid and upper epidermal spinous cell layers, often extending to the granular cell layer. These findings, which are associated with haploinsufficiency of desmoglein 1, were not observed in any of the other 6 PPK cases. Mild perinuclear eosinophilic condensations and cytoplasmic vacuolizations were observed in the spinous cell layer keratinocytes of the pachyonychia congenita type II PPK and the nonspecified focal PPK cases. Limitations: There were a limited number of patients and control patients with hereditary PPKs. Conclusion: Widening of the intercellular spaces and disadhesion of epidermal keratinocytes may serve as a histologic clue to PPKs caused by DSG1 mutations. ( J Am Acad Dermatol 2010;62: ) Key words: desmoglein 1; histopathology; keratoderma. Histopathological features of inherited palmoplantar keratodermas (PPKs) are mostly nonspecific. There are a few exceptions From the Departments of Dermatology a and Pathology, b Rambam Medical Center, Haifa; and Department of Dermatology, Tel Aviv Sourasky Medical Center. c Funding sources: None. Conflicts of interest: None declared. Accepted for publication May 14, Reprints not available from the authors. Correspondence to: Reuven Bergman, MD, Department of Dermatology, Rambam Medical Center, Haifa, 31096, Israel. r_bergman@rambam.health.gov.il /$36.00 ª 2009 by the American Academy of Dermatology, Inc. doi: /j.jaad Abbreviations used: EDSFS: ectodermal dysplasia/skin fragility syndrome KPS: keratosis palmoplantaris striata PPK: palmoplantaris keratoderma such as epidermolytic PPK caused by mutations in KRT9 encoding keratin 9, in which the characteristic epidermolytic changes in the malpighian cell layer are prominent and may lead to a specific diagnosis. 1-3 Recent developments in molecular genetics have enabled regrouping of various entities and syndromes leading to more precise histopathologic classifications. 4 Keratosis palmoplantaris striata (KPS) is a rare autosomal-dominant form of PPK 107

2 108 Bergman et al JAM ACAD DERMATOL JANUARY 2010 characterized by linear hyperkeratotic streaks along the volar surface of the fingers, and focal keratoderma of the soles. Non-sense and frame-shift mutations in DSG1, DSP, and KRT1 genes encoding desmoglein 1, desmoplakin, and keratin 1, respectively, have been shown to be the underlying causes of KPS types I, II, and III, respectively. 5-8 Ultrastructural studies of the involved epidermis of KPS types I and II have demonstrated abnormalities of the desmosomes, loosening of the intercellular connections, and widening of the intercellular spaces. 6,8 Light microscopy of semithin sections and routinely stained sections have shown widening of the intercellular spaces between the keratinocytes in the spinous cell layer of the epidermis in KPS type II. 6,8,9 The widening of the intercellular spaces was less prominent in the semithin sections of a KPS type I case than in a case with CAPSULE SUMMARY KPS type II. 8 We have recently observed disadhesion and widening of the intercellular spaces in the involved epidermis of KPS type I, and in a case with diffuse keratoderma associated with DSG1 mutation. 10,11 Although several authors did not report this finding, 12,14 we sought to determine the consistency of our finding, which may constitute a novel histologic clue to KPS caused by DSG1 mutations. We studied the histopathology of 4 cases of PPK associated with DSG1 mutations and compared them with the histologic findings in 6 cases of PPK associated with mutations in other genes. d d Varying degrees of widening of the intercellular spaces and partial disadhesion of keratinocytes in the suprabasal epidermis were found in 4 palmoplantar keratodermas associated with mutations in DSG1 encoding desmoglein 1, but not in 6 palmoplantar keratodermas associated with mutations in other genes. Widening of the intercellular spaces and disadhesion of epidermal keratinocytes may serve as a histologic clue to palmoplantar keratodermas associated with DSG1 mutations. METHODS We included PPK cases in which the individual clinical diagnoses were supported by molecularly deciphered underlying mutations. The mutations are presented in Table I. In 6 patients (Nos. 5-10) (Table I) the mutations were novel and not published previously. There were 10 patients, 6 female and 4 male, aged 1 to 42 years (Table I). Three patients had KPS type I and one patient had diffuse PPK, all with autosomal-dominant DSG1 mutations. Four patients had Mal de Meleda PPK associated with autosomalrecessive SLURP1 mutations, one patient had pachyonychia congenita type II PPK associated with autosomal-dominant KRT17 mutation, and one patient had focal PPK associated with autosomaldominant KRT16 mutation without signs of pachyonychia congenita type II (Table I). Biopsy specimens were obtained from the palms or soles. None of the patients received systemic therapy or local therapy at the time of the biopsy. The specimens were processed according to the routine methods and stained with hematoxylin and eosin. The slides were examined by one of us (R. B.) in a blinded fashion. RESULTS The results are summarized in Table I. In the 3 KPS type I cases, and in the diffuse PPK case associated with DSG1 mutation, the epidermis showed acanthosis, marked papillomatosis, compact orthohyperkeratosis, and hypergranulosis (Fig 1). Foci of widened intercellular spaces could be occasionally detected under low-power magnification (Fig 1). Higher magnifications revealed conspicuous widening of the intercellular spaces and partial dehiscence of keratinocytes in the mid and upper spinous cell layers of 3 of the 4 cases associated with DSG1 mutations (Nos. 1, 2, and 4) (Table I) (Figs 2 to 5). In two cases (Nos. 2 and 4) (Table I), the conspicuous widening of the intercellular spaces and partial dehiscence of keratinocytes extended into the granular cell layer (Figs 4 and 5). In the fourth case (No. 3) (Table I), the widening of the intercellular spaces in the spinous and granular cell layers were more subtle. In the 4 Mal de Meleda PPK cases, the epidermis demonstrated acanthosis, marked compact orthohyperkeratosis, and hypergranulosis without papillomatosis (Fig 6). One of the 4 Mal de Meleda PPK cases (No. 8) (Table I) showed also small foci of parakeratosis, spongiosis, mild exocytosis of small lymphocytes, and mild perivascular mononuclear cell infiltrates in the papillary dermis. A periodic acideschiff stain demonstrated hyphae in the corneal layer. This patient was known to be afflicted by recurrent fungal superinfections in her involved PPK skin. The focal PPK case associated with KRT16 mutation (No. 10) and the pachyonychia congenita type II case (No. 9) demonstrated acanthosis, papillomatosis, marked compact orthohyperkeratosis, and hypergranulosis (Table I). Mild perinuclear eosinophilic condensations and intracytoplasmic

3 JAM ACAD DERMATOL VOLUME 62, NUMBER 1 Bergman et al 109 Table I. Results of histologic findings in palmoplantar keratoderma No./sex/ age, y 1/F/42 Keratoderma type Diffuse, nonspecified Mutation type References Biopsy site Histologic findings. Hematoxylin-eosin staining DSG1 (p.r26x) 10 Sole Acanthosis, papillomatosis, compact hypergranulosis; partial dehiscence of keratinocytes and widening of intercellular spaces in mid and upper spinous cell layers 2/M/19 Striate DSG1 (c.1931dela) 11 Sole Acanthosis, papillomatosis, compact hypergranulosis; partial dehiscence of keratinocyte and widening of intercellular spaces in mid and upper spinous cell and granular cell layers 3/M/18 Striate DSG1 (p.r219x) 11 Palm Acanthosis, papillomatosis, compact mild hypergranulosis; subtle partial dehiscence of keratinocytes and widening of intercellular spaces in mid and upper spinous cell and granular cell layers 4/F/42 Striate DSG1 (p.q201x) 11 Sole Acanthosis, papillomatosis, compact hypergranulosis; partial dehiscence of keratinocytes and widening of intercellular spaces in mid and upper spinous cell and granular cell layers 5/M/16 Mal de Meleda SLURP1 (p.g86r) Unpublished Palm Acanthosis, compact hyperkeratosis, and hypergranulosis 6/M16 Mal de Meleda SLURP1 (p.g86r) Unpublished Palm Acanthosis, compact hyperkeratosis, and hypergranulosis 7/F/1 Mal de Meleda SLURP1 (p.g86r) Unpublished Sole Acanthosis, compact hyperkeratosis, and hypergranulosis 8/F/15 Mal de Meleda SLURP1 (c.82delt) Unpublished Palm Acanthosis, hypergranulosis, and compact hyperkeratosis with a few foci of parakeratosis, spongiosis, and exocytosis of a few lymphocytes; superficial perivascular mononuclear cell infiltrates in papillary dermis; hyphae in corneal layer 9/M/18 Pachyonychia congenita type II KRT17 (p.n92s) Unpublished Sole Acanthosis, papillomatosis, compact hypergranulosis; mild perinuclear eosinophilic condensations and cytoplasmic vacuolizations in spinous layer keratinocytes 10/F/10 Focal, nonspecified KRT16 (p.r127c) Unpublished Sole Acanthosis, papillomatosis, compact hypergranulosis; mild perinuclear eosinophilic condensations and cytoplasmic vacuolizations in spinous layer keratinocytes F, Female; M, male.

4 110 Bergman et al JAM ACAD DERMATOL JANUARY 2010 Fig 1. Case No. 2. Striate keratoderma type I. Biopsy specimen from involved sole demonstrates acanthosis, papillomatosis, ortho hypergranulosis. Widening of intercellular spaces may be appreciated at low magnification. (Hematoxylin-eosin stain; original magnification 3100.) Fig 4. Case No. 4. Striate keratoderma type I. Biopsy specimen from involved sole shows widening of intercellular spaces and partial dehiscence of keratinocytes in spinous and granular cell layers. (Hematoxylin-eosin stain; original magnification 3400.) Fig 2. Case No. 2. Higher magnification of Fig 1 shows widening of intercellular spaces. (Hematoxylin-eosin stain; original magnification 3400.) Fig 5. Case No. 1. Diffuse keratoderma associated with DSG1 mutation. Biopsy specimen taken from sole demonstrates widening of intercellular spaces and partial dehiscence of keratinocytes in spinous and granular cell layers. (Hematoxylin-eosin stain; original magnification 3400.) keratinocytes were observed in the PPK cases associated with DSG1 mutations only. Absence of papillomatosis characterized the 4 Mal de Meleda PPKs, and mild perinuclear eosinophilic condensations and intracytoplasmic vacuolizations were observed in the two PPKs associated with heterozygous mis-sense mutations in KRT16 and KRT17. Fig 3. Case No. 2. Further magnification of Fig 2 demonstrates widening of intercellular spaces with disruption of spinous processes and partial dehiscence of keratinocytes. (Hematoxylin-eosin stain; original magnification ) vacuolizations were observed in the spinous cell layer keratinocytes of both cases (Fig 7). In summary, varying degrees of widening of the intercellular spaces and partial dehiscence of DISCUSSION PPKs are a diverse group of skin diseases characterized by hyperkeratosis of the palmoplantar epidermis. The majority of PPKs have been attributed to mutations in structural proteins such as keratins, desmosomal proteins, and connexins. 15 Desmosomes are cell-cell junctions found predominantly in the heart and skin. They link intermediate filament networks to the cell membrane and function in maintaining tissue architecture and integrity. 15 Desmosomes consist of a number of membrane proteins, two

5 JAM ACAD DERMATOL VOLUME 62, NUMBER 1 Bergman et al 111 Fig 6. Case No. 7. Mal de Meleda keratoderma. Biopsy specimen from sole shows acanthosis and marked compact orthohyperkeratosis and hypergranulosis. Note absence of papillomatosis. (Hematoxylin-eosin stain; original magnification 3100.) Fig 7. Case No. 9. Pachyonychia congenita type II. Biopsy specimen from sole showing mild perinuclear eosinophilic condensations and cytoplasmic vacuolizations in spinous cell layer. (Hematoxylin-eosin stain; original magnification 3400.) of which (desmoglein 1 and desmoplakin) have been shown to underlie autosomal-dominant nonsyndromic striate PPK. 5,6,8,9,11 We have described also a family with autosomal-dominant diffuse keratoderma associated with DSG1 mutation. 10 Autosomal-recessive mutations in the DSP gene have been shown to disrupt the desmoplakin-intermediate filament interactions, causing syndromic diseases affecting the hair, skin, and heart, whose manifestations include wooly hair, dilated cardiomyopathy, and PPK. 15,16 Histologic studies of involved PPK skin in these cases demonstrates large intercellular spaces between suprabasal keratinocytes, suggesting a cell adhesion defect, and electron microscopy shows clumping of desmosomes at the infrequent sites of cell-cell adhesion. 15 Disadhesion of keratinocytes and widened intercellular spaces were also observed in the involved epidermis in skin fragility/wooly hair syndrome caused by compound heterozygosity for non-sense and mis-sense mutations in DSP. 17 Heterozygous mutations in DSP do not result in cardiomyopathy or wooly hair, but cause nonsyndromic KPS type II in which carriers present with a less extensive pattern of keratoderma. 6,9 Light microscopy of KPS type II has demonstrated widening of the intercellular spaces between keratinocytes throughout the spinous cell layer. 7 Semithin sections of KPS type II have demonstrated acanthosis and hyperkeratosis with darkly staining granular or filamentous material within the cytoplasm of spinous cells associated with loosening of the intercellular connections. 6,8 Electron microscopy confirms abnormal assembly of the keratin filament network associated with loss of connections to the desmosomes, which were often small and rudimentary, and widened intercellular spaces. 6,8,9 Therefore, disadhesion of keratinocytes and widening of the intercellular spaces might be observed in syndromic and nonsyndromic PPKs associated with DSP mutations. Wan et al 8 used electron microscopy to study skin sections from 4 patients with KPS with DSP mutations and one patient with DSG1 mutation. They found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layer of the epidermis of both types of KPS. 8 The semithin sections showed widening of the intercellular spaces. These changes, however, were more advanced in the DSP mutationsassociated cases than in the DSG1 mutations-associated case. Three other studies of KPS type I failed to detect light microscopic abnormalities of cell adhesion in the involved epidermis, except for acanthosis and marked orthohyperkeratosis and hypergranulosis In the current series, we studied 3 KPS type I cases and one diffuse PPK case associated with autosomal-dominant DSG1 mutations leading to haploinsufficiency of desmoglein 1. 10,11 Varying degrees of widening of the intercellular spaces and partial disadhesion of keratinocytes were demonstrated in the spinous cell layer of all 4 cases. These changes were conspicuous in 3 cases and more subtle in the fourth. In 3 cases, the abnormalities extended to the granular cell layer. The partial disadhesion and widening of the intercellular spaces were more intense in the upper spinous and granular cell layers, where desmoglein 1 is maximally expressed. 18 These are also the sites in which the split in pemphigus foliaceus occurs and is believed to be caused by anti-desmoglein 1 antibodies. Haploinsufficiency of desmoglein 1 as a result of DSG1 mutations might be the underlying cause of the intercellular spaces widening and the partial disadhesion of keratinocytes, as also evident from the lack of transcription of the DSG1 mutant

6 112 Bergman et al JAM ACAD DERMATOL JANUARY 2010 allele in a case of KPS type I. 11 We consequently suggest that widened intercellular spaces and partial disadhesion of keratinocytes may be used as a histologic clue to KPS type I. Ectodermal dysplasia/skin fragility syndrome (EDSFS) is another inherited disease leading to epidermal disadhesion and acantholysis that should be differentiated histologically from PPK associated with DSG1 mutations. EDSFS is characterized by skin fragility and blistering, PPK, abnormal hair growth, nail dystrophy, and occasionally impaired sweating EDSFS is inherited in an autosomal-recessive fashion and it is associated with germline mutations in the plakophilin 1 gene, which is a critical component of the desmosomal plaque. 19,21 The fragile skin in EDSFS is characterized histologically by widening of the epidermal intercellular spaces above the basal cell layer and detachment of keratinocytes in the mid and upper spinous layers The involved keratinocytes may also show variable degrees of dyskeratosis. 24 Electron microscopy has demonstrated loss of keratinocytekeratinocyte adhesion, and desmosomes that are small, poorly formed, and reduced in number. The association of the keratin intermediate filament network with the cell membrane is disrupted, leading to compact perinuclear distribution and lack of connections to the desmosomes We have found that the most severe dyskeratotic changes were present in the involved hyperkeratotic plantar skin of an EDSFS case, along with disadhesion of keratinocytes and acantholysis in the upper spinous cell layer. 24 The finding of dyskeratosis in acantholytic keratinocytes may be used as a clue to differentiate histologically PPK associated with EDSFS from PPK associated with DSG1 mutations. In this study, we also studied biopsy specimens from the involved skin of 4 Mal de Meleda cases associated with autosomal-recessive SLURP1 mutations, a case of pachyonychia congenita type II associated with an autosomal-dominant KRT 17 mutation, and a case of focal PPK associated with an autosomal-dominant KRT16 mutation, without other clinical signs of pachyonychia congenita type I. These 6 PPKs did not display widening of the intercellular spaces and disadhesion of keratinocytes. However, we detected mild perinuclear eosinophilic condensations and intracytoplasmic vacuolizations in the spinous cell layer keratinocytes of the pachyonychia type II case and the focal nonspecified PPK case. The eosinophilic condensations might represent perinuclear clumpings of keratin tonofilaments as a result of a dominantnegative effect of the altered keratin 16 and 17 filaments. This phenomenon has been observed in inherited autosomal keratin disorders such as epidermolysis bullosa Dowling-Meara and epidermolytic hyperkeratosis, 3,25 in which the mutated keratin filament is believed to form an abnormal complex with the wild-type keratin filament. Ultrastructural examination of a family with pachyonychia type I associated with a KRT16 mutation revealed keratin filaments that were greatly condensed but did not form the dense amorphous aggregates seen in a number of other keratin disorders. 26 Shamsher et al 27 described two families with autosomal-dominant disorder of focal nonepidermolytic PPK associated with heterozygous mutations in KRT16. These mutations did not appear to cause epidermolysis on light or electron microscopy. 27 Other authors have also recognized a focal nonepidermolytic form of PPK associated with KRT16 mutations, but without other clinical signs of pachyonychia congenita type I. 28 Terrinoni et al 29 studied a patient who exhibited a localized thickening of the skin in parts of the right palm and right sole, following Blaschko lines. The keratin 16 complementary DNA sequence from the lesional epidermis demonstrated a heterozygous 12 base pair deletion, predicted to delete 4 amino acids, and proved to be a postzygotic mutation. 29 Ultrastructural analysis of the affected palmar epidermis showed marked tonofilament clumping and cytoplasmic vacuolization in the suprabasal cells. 29 Thus, condensed keratin filaments as a result of a dominant-negative effect may have been the cause of the mild perinuclear condensations and cytoplasmic vacuolizations observed in our two PPK cases associated with KRT16 and KRT17 heterozygous mis-sense mutations. In summary, biopsy specimens of inherited PPK may be more informative than previously thought. Disadhesion and widening of the intercellular spaces in the involved epidermis may direct the molecular study toward mutations involving genes that encode desmosomal proteins such as desmoglein 1, desmoplakin, and plakophilin. REFERENCES 1. Ries A, Kuster W, Eckardt R, Sperling K. 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