Ectodermal dysplasia‐skin fragility syndrome: Two new cases and review of this desmosomal genodermatosis

Desmosomes are intercellular cadherin‐mediated adhesion complexes that anchor intermediate filaments to the cell membrane and are required for strong adhesion for tissues under mechanical stress. One specific component of desmosomes is plakophilin 1 (PKP1), which is mainly expressed in the spinous layer of the epidermis. Loss‐of‐function autosomal recessive mutations in PKP1 result in ectodermal dysplasia‐skin fragility (EDSF) syndrome, the initial inherited Mendelian disorder of desmosomes first reported in 1997.


| INTRODUC TI ON
Desmosomes are specialised cadherin-mediated adhesion complexes that anchor intermediate filaments to the cell membrane, providing stability and rigidity to tissues under mechanical stress. [1,2] Attachment of desmosomal cadherins (desmogleins and desmocollins) to intermediate filaments is facilitated by proteins from the armadillo (plakoglobin, plakophilin 1 and plakophilin 2) and plakin (desmoplakin, periplakin and envoplakin) superfamilies. [1] Understanding the exact role of each component within these complexes has been difficult due to their intricate interactions and the heterogeneity of their molecular structure. The discovery of monogenic disorders involving dysfunction or absence of desmosomal components, however, has provided great insight into both the underlying loss of cell cohesiveness and importantly, the clinical consequences to skin and other tissues. [3] Plakophilin 1 (PKP1) is an armadillo protein that is found in desmosomes of stratified epithelia and localises primarily to the spinous layer of the epidermis. [4] PKP1 has been shown to be a multifunctional protein that is inherently involved in the Wnt/β-catenin signalling cascade and promotes desmosomal cadherin stability and clustering. [1,5] It enhances the recruitment of desmosomal proteins to the plasma membrane in cultured keratinocytes through direct and indirect interaction with plakoglobin and desmoplakin. [4] Studies have shown that PKP1 interferes with plakoglobin binding to desmoplakin and promotes desmosomal clustering when bound to plakoglobin. [4,5] PKP1 also has a role in increasing cell survival following cell damage [6] and in regulating keratinocyte proliferation and migration. [1,7] There are two isoforms, PKP1a and PKP1b, which exist due to alternative splicing of the PKP1 gene. PKP1a is expressed in both desmosomes and nuclei, whilst PKP1b is exclusively nuclear. [4,8] Both the desmosomal and nuclear isoforms are degraded by caspases during keratinocytic apoptosis, suggesting that PKP1 may be involved in remodelling of the cytoskeleton and cell integrity. [9] The clinical importance of PKP1 is demonstrated by the human disorder caused by autosomal recessive homozygous null and splice site mutations in the PKP1 gene. In 1997, McGrath et al, first reported that mutations in PKP1 result in the loss of connection between epidermal cells with skin fragility and blistering, and congenital ectodermal dysplasia (MIM #604536). [10] The clinical features of the affected individual were termed "ectodermal dysplasia-skin fragility (EDSF) syndrome," reflecting the combination of skin fragility, hair and nail pathologies that were present. Representing the first human inherited disorder of desmosomes, EDSF syndrome now is classified as a specific form of skin fragility disorder within the spectrum of epidermolysis bullosa. [11] Following the initial report of EDSF syndrome, other cases involving pathogenic mutations in the PKP1 gene have subsequently been described. Nevertheless, clinical diversity in EDSF syndrome has been noted prompting a need to review the clinical manifestations of the disorder. This article therefore reviews genotype-phenotype correlation for EDSF syndrome and PKP1 mutations.

| ME THODS
Two unrelated cases of possible EDSF syndrome were investigated using Sanger sequencing of genomic DNA for PKP1 mutations and immunostaining of non-lesional skin, as described previously. [12] A comprehensive review of the PubMed database was undertaken for reports in English from October 1997 to November 2019.
We used the (a) MeSH terms: 'plakophilins/genetics' AND 'desmosomes/pathology' OR 'ectodermal dysplasia/genetics', 'mutation' and (b) keywords "plakophilin 1", "PKP1", "ectodermal dysplasia-skin fragility syndrome". Titles and abstracts were reviewed, and both references and citations of relevant studies were also examined for additional cases. The available data from each publication were investigated to identify patients with a genetic diagnosis involving a mutation in PKP1. Cases without a genetic diagnosis were excluded.
For each case, demographics, mutation and protein analysis, and phenotypic features were recorded. For new, original cases that underwent genetic analysis by our laboratory, informed consent was obtained and in accordance with the Declaration of Helsinki principles, we performed Sanger sequencing on all coding exons and flanking intronic regions of PKP1 (GenBank NM_001005337.3) using genomic DNA extracted from peripheral blood.

| Case 1
A 1-year-old Egyptian boy, born to consanguineous parents presented with a history of trauma-induced skin fragility since birth. On examination, he had hypotrichosis with scalp erosions, and erosions and crusts over the face and limbs ( Figure 1A). He also displayed finger and toenail subungual hyperkeratosis with dystrophy and mild palmoplantar keratoderma (PPK). There was no perioral fissuring, hypohidrosis or mucous membrane involvement. There was no family history of skin fragility ( Figure 1B). Histopathology and immunofluorescence microscopy showed widened intercellular spaces, hyperkeratosis and acantholysis, and staining for PKP1 showed complete absence compared to control skin (see Figure S1). Sanger sequencing revealed a homozygous insertion in exon 3 of the PKP1 gene (c.409_410insAC; p.Thr137Thrfs*61) in the proband, whereas both parents were found to be heterozygous carriers of the mutation ( Figure 1C). The c.409_410insAC mutation maps upstream of the arm repeat domains of PKP1 (head domain) and therefore is predicted to result in severely truncated polypeptides lacking these arm-repeats.

| Case 2
An 11-month-old Egyptian boy, born to non-consanguineous parents presented similarly to case 1, with trauma-induced erosions and perioral bullae developing from 5 days of age and spreading to the upper and lower limbs (Figure 2A). He was noted to have hypotrichosis with subungual hyperkeratosis and nail dystrophy.
He had normal dentition, without hearing abnormalities or hypohidrosis. There was no family history of skin fragility ( Figure 2B).
Histopathology showed widened intercellular spaces, hyperkeratosis and acantholysis. Immunostaining for PKP1 showed almost complete absence compared to control skin (see Figure S1). Sanger sequencing revealed a homozygous deletion in exon 6 of the PKP1 gene (c.1213delA, p.Arg411Glufs*22) in the proband, whereas his unaffected brother and mother were found to be heterozygous   [24] (Continues) carriers for that mutation ( Figure 2C). The c.1213delA mutation maps on the armadillo arm domain 4.
Both PKP1 mutations result in frameshift and premature protein termination. Neither variant identified has been previously reported in the Human Gene Mutation Database, gnomAD (v3.0) or the 1000 Genomes Database (internationalgenome.org).
Of the cases that reported PKP1 protein expression, all had either reduced skin labelling or a complete absence of immunostaining.
All germline mutation cases reported skin fragility (18/18)  although these pathologies may well have an unrelated aetiology. [4] Additional observations included moderate levels of pruritus, failure to thrive with low height/weight centiles, follicular hyperkeratosis, walking difficulties, dysplastic dentition and recurrent chest infections.

| Clinical phenotype
EDSF was the first inherited desmosomal disorder to be identified. [ Note: An additional case involving a frameshift mutation (c.638delT, p.Val213Glyfs*33) along with a post-zygotic somatic loss of PKP1 was also noted. This case presented with a reduced PKP1 protein expression only on lesional skin. Clinically, the presentation was mild with unilateral superficial erosions, hypopigmented plaques and segmental hyperkeratosis. Sweating, teeth and scalp hair were normal. (Vázquez-Osorio et al., 2017 [27] ). This studycase 2

Abbreviations
Note: An additional case involving a frameshift mutation (c.638delT, p.Val213Glyfs*33) along with a post-zygotic somatic loss of PKP1 was also noted. This case presented with a reduced PKP1 protein expression only on lesional skin. Clinically, the presentation was mild with unilateral superficial erosions, hypopigmented plaques and segmental hyperkeratosis. Sweating, teeth and scalp hair were normal. (Vázquez-Osorio et al., 2017 [27] ).

TA B L E 2 (Continued)
features of skin fragility, nail dystrophy, alopecia/hypotrichosis, perioral fissuring and PPK. These features are consistent with the known cutaneous pathology of PKP1 deficiency due to a reduction in desmosomal stability. [1,4,26] The EDSF syndrome extended phenotype now includes perianal and perineal erosions/fissuring and pruritus.
Unlike the original report, no further cases of ocular abnormalities were observed, providing evidence that this original case presented an unrelated congenital astigmatism. Additionally, no ocular defects were noted in Pkp1 knockout mice, confirming a lack of ocular involvement with PKP1 mutations. [27] Extracutaneous features such as failure to thrive/ low height or weight centiles were a shared feature in some patients. A similar outcome was noted in Pkp1 knockout mice who were born at 25% of the Mendelian ratio with reduced subcutaneous adipose tissue. [26] PKP1 deficient dogs were also noted to grow to only one-third the size and weight of others in the same litter. [26,28] The exact mechanism is unknown, but it is hypothesised that this is due to alterations during in utero adipogenic signalling from the epidermis, reducing Wnt/β-catenin signalling, which is needed for initiation of proadipogenic pathways including insulin/growth factor signalling. [26] Dental abnormalities were only observed in 3/18 cases, but all three cases showed significant dysplastic dentition. It has been shown that PKP1 is highly expressed in the process of tooth development and is required for dynamic Wnt/β-catenin signalling. [27] In mouse models, it was noted that PKP1 translocates and localises to the cell nucleus and contributes to inner dental epithelial cell differentiation, forming ameloblasts that secrete enamel matrix proteins for tooth development. [27] Thus, even though the reported prevalence in this cohort is small, the risk of severe dental abnormalities should be considered significant. Although a patent foramen ovale and a right aortic arch were observed in our review, no major cardiovascular abnormality was identified, probably reflecting the tissue-specific pattern of expression of PKP1, which is not expressed in the heart or vasculature. [29] Hypohidrosis was noted in one-third of cases, although the link between loss of PKP1 expression and reduced sweating may be an indirect one. It has been shown that the Wnt/β-catenin-Eda/ NF-kB-Shh cascade functions in a genetic relay for eccrine gland development. [30] Sweat gland induction was noted to completely fail when canonical WNT signalling was blocked in skin epithelium. [31] Thus, complete knockout of PKP1 protein expression may result in hypohidrosis, whilst a more mild form that produces a reduced PKP1 protein may result in partial function, allowing sweat production.

| Histopathological findings
Histologically, the lesional skin of patients with EDSF syndrome is characterised by widening of the epidermal intercellular spaces in the suprabasal cell layer and detachment of keratinocytes in the mid and upper spinous-cell layer. [10,13,32] These hallmark features usually result in intraepidermal clefts and blisters with a few acantholytic keratinocytes in the mid-epidermis or in a complete detachment of the epidermis above the upper spinous layers. [33] Dyskeratotic keratinocytes were also noted particularly in the plantar keratoderma of patients. [33] Electron microscopy has demonstrated loss of keratinocyte-keratinocyte adhesion, and desmosomes that are small, poorly formed and reduced in number. [1,32] Lack of connections to the desmosomes is apparent due to disruption of keratin Findings from hypotrichotic scalp skin demonstrated a mild decrease in the total number of hair follicles, without an increase in the number of vellus hair. [33] It was also noted that there were no histological signs of inflammatory or scarring alopecia, but there was an increase in the number of catagen-telogen hair follicles as observed in chronic telogen effluvium. [33]

| Mutational analysis
It is noteworthy that 57.1% of PKP1 mutations affect splicing. In general, only about 10%-15% of genetic diseases described in humans are caused by splice-site mutations at exon-intron junctions, with most splice-site mutations occurring at the +1 or +2 positions for donor sites or the −1 or −2 positions for acceptor sites. [28] Almost all splice-site mutations resulted in a premature termination codon and had similar clinical features to cases resulting from nonsense mutation. One exception was case 7, in which there was a partially functional protein expression due to a non-consensus cryptic splicesite mutation generating in-frame transcripts. This case presented with a relatively mild phenotype with plantar hyperkeratosis and nail involvement, but instead of alopecia or hypotrichosis, scalp hair was dark, thick and curly. [16] Another case of interest (Tables 1 and 2-footnote) involved both a frameshift deletion mutation and a postzygotic somatic mutation, yet only presented with mild unilateral superficial erosions in a mosaic pattern, PPK and nail dystrophy. [25] Immunofluorescence analysis in this case showed negative staining for anti-PKP1 antibodies in a mosaic pattern, but reduced immunoreactivity in surrounding, unaffected skin compared to control skin.
The molecular pathology involved a heterozygous germline mutation that appeared to be homozygous in affected skin, possibly due to a loss of heterozygosity. Regarding the benefits of molecular characterisation of EDSF syndrome, the discovery of PKP1 mutations also formed the basis of the first successful preimplantation genetic skin disease testing procedure for an inherited skin disease. [34] In summary, we have reviewed and expanded on the current literature of PKP1 mutations causing EDSF syndrome. We expand specifically on the extracutaneous effects of PKP1 mutation including, hypohidrosis, pruritus, perianal erosions and dysplastic dentition.

ACK N OWLED G EM ENTS
We would like to acknowledge all the patients and their families who have kindly contributed their samples, and Dr Nehal El Shebiny from the Dermatology Department, Faculty of Medicine, Tanta University, Tanta, Egypt for her clinical assistance.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicting interests. All authors contributed significantly, and all authors are in agreement with the contents of the manuscript.