Transcriptomic Repositioning Analysis Identifies mTOR Inhibitor as Potential Therapy for Epidermolysis Bullosa Simplex.

Expression-based systematic drug repositioning has been explored to predict novel treatments for a number of skin disorders. In this study, we utilize this approach to identify, to our knowledge, previously unreported therapies for epidermolysis bullosa simplex (EBS). RNA sequencing analysis was performed on skin biopsies of acute blisters (<1 week old) (n = 9) and nonblistered epidermis (n = 11) obtained from 11 patients with EBS. Transcriptomic analysis of blistered epidermis in patients with EBS revealed a set of 1,276 genes dysregulated in EBS blisters. The IL-6, IL-8, and IL-10 pathways were upregulated in the epidermis from EBS. Consistent with this, predicted upstream regulators included TNF-α, IL-1ß, IL-2, IL-6, phosphatidylinositol 3-kinase, and mTOR. The 1,276 gene EBS blister signature was integrated with molecular signatures from cell lines treated with 2,423 drugs using the Connectivity Map CLUE platform. The mTOR inhibitors and phosphatidylinositol 3-kinase inhibitors most opposed the EBS signature. To determine whether mTOR inhibitors could be used clinically in EBS, we conducted an independent pilot study of two patients with EBS treated with topical sirolimus for painful plantar keratoderma due to chronic blistering. Both individuals experienced marked clinical improvement and a notable reduction of keratoderma. In summary, a computational drug repositioning analysis successfully identified, to our knowledge, previously unreported targets in the treatment of EBS.

Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is a group of inherited keratinopathies that, in most cases, arise due to mutations in keratins and lead to intraepidermal ruptures. The cellular pathology of most EBS subtypes is associated with the fragility of the intermediate filament network, cytolysis of the basal layer of the epidermis, or attenuation of hemidesmosomal/desmosomal components. Mutations in keratins 5/14 or in other genes that encode associated proteins induce structural disarrangements of different strengths depending on their locations in the genes. Keratin aggregates display impaired dynamics of assembly and diminished solubility and appear to be the trigger for endoplasmic reticulum (ER) stress upon being phosphorylated by MAPKs. Global changes in cellular signaling mainly occur in cases of severe dominant EBS mutations. The spectrum of changes initiated by phosphorylation includes the inhibition of proteasome degradation, TNF-α signaling activation, deregulated proliferation, abnormal cell migration, and impaired adherence of keratinocytes. ER stress also leads to the release of proinflammatory danger-associated molecular pattern (DAMP) molecules, which enhance avalanche-like inflammation. Many instances of positive feedback in the course of cellular stress and the development of sterile inflammation led to systemic chronic inflammation in EBS. This highlights the role of keratin in the maintenance of epidermal and immune homeostasis.

Locally advanced tongue squamous cell carcinoma in epidermolysis simplex bullosa patient: a therapeutic conundrum.

Epidermolysis bullosa simplex (EBS) is a debilitating condition affecting the skin and mucous membranes that is characterised by frequent ulceration and blistering on trivial trauma. In EBS, oral cavity mucosal injuries lead to a high propensity for developing squamous cell carcinomas. Locally advanced tongue carcinoma arising in this background presents a challenging therapeutic conundrum. To our knowledge, this is the first case of aggressive locally advanced tongue carcinoma that has developed sporadically in a patient with EBS and no family history. Routine screening of oral mucosal lesions will lead to early detection and timely management of this debilitating condition.

Slac2-b Coordinates Extracellular Vesicle Secretion to Regulate Keratinocyte Adhesion and Migration.

Slac2-b, also known as exophilin-5, is a Rab27b effector protein with a role in exosome transport and is encoded by the EXPH5 gene. We previously described biallelic loss-of-function mutations in EXPH5 in an autosomal recessive form of epidermolysis bullosa simplex. However, how the loss of Slac2-b expression leads to skin fragility and erosions is unknown. In this study, we demonstrate that keratinocytes (KCs) isolated from two different individuals with mutations in EXPH5 have significant defects in cell‒matrix adhesion. EXPH5-mutant KCs also showed increased perinuclear accumulation and significantly reduced trafficking of CD63+ vesicles. These phenotypes were also seen in Slac2-b‒deficient KCs. This was coincident with a reduction in Rab27a protein expression in Slac2-b‒mutant KCs as well as reduced secretion of extracellular vesicles containing extracellular matrix proteins. Live imaging analysis revealed a strong correlation between CD63+ vesicle trafficking to the plasma membrane and focal adhesion dynamics. These findings support a role for Slac2-b in regulating local focal adhesion dynamics to support effective KC adhesion and provide insight into the underlying pathophysiology of inherited skin blistering.

Severe epidermolysis bullosa simplex phenotype caused by codominant mutations p.Ile377Thr in keratin 14 and p.Gly138Glu in keratin 5.

Epidermolysis bullosa simplex (EBS) is a rare skin disease usually inherited in an autosomal dominant pattern. EBS is resulting from mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes encoding the keratins 5 and 14 proteins expressed in the keratinocytes of the basal layer of the epidermis. To date, seven pathogenic mutations have been reported to be responsible for EBS in the Canadian population from the province of Quebec: p.Pro25Leu, p.Leu150Pro, p.Met327Thr and p.Arg559X in KRT5; p.Arg125Ser, p.Ile377Thr and p.Ile412Phe in KRT14. Here, we present a novel French-Canadian patient diagnosed with EBS confined to the soles but presenting a severe complication form including blisters, hyperkeratosis, skin erosions and toenail abnormalities. Mutation screening was performed by direct sequencing of the entire coding regions of KRT5 and KRT14 genes and revealed the previously reported missense heterozygous mutation c. 1130T > C in KRT14 (p.Ile377Thr). Furthermore, this patient is carrying a second mutation in KRT5, c.413G > A (p.Gly138Glu), which has been linked to an increased risk of basal cell carcinoma in the literature. We suspect an impact of the p.Gly138Glu variant on the EBS phenotype severity of the studied patient. The pathogenicity and consequences of both genetic variations were simulated by in silico tools.

A nonsense variant in the KRT14 gene in a domestic shorthair cat with epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a hereditary blistering disease affecting the skin and mucous membranes. It has been reported in humans, cattle, buffaloes and dogs, but so far not in cats. In humans, EBS is most frequently caused by variants in the KRT5 or KRT14 genes. Here, we report a case of feline epidermolysis bullosa simplex and describe the causative genetic variant. An 11-month-old male domestic shorthair cat presented with a history of sloughed paw pads and ulcerations in the oral cavity and inner aspect of the pinnae, starting a few weeks after birth. Clinical and histopathological findings suggested a congenital blistering disease with a split formation within the basal cell layer of the epidermis and oral mucous epithelium. The genetic investigation revealed a homozygous nonsense variant in the KRT14 gene (c.979C>T, p.Gln327*). Immunohistochemistry showed a complete absence of keratin 14 staining in all epithelia present in the biopsy. To the best of our knowledge, this is the first report of feline EBS, and the first report of a spontaneous pathogenic KRT14 variant in a non-human species. The homozygous genotype in the affected cat suggests an autosomal recessive mode of inheritance.

Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype.

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis.

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

Downstream effects of plectin mutations in epidermolysis bullosa simplex with muscular dystrophy.

Mutations of the human plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In the present study we analyzed the downstream effects of PLEC mutations on plectin protein expression and localization, the structure of the extrasarcomeric desmin cytoskeleton, protein aggregate formation and mitochondrial distribution in skeletal muscle tissue from three EBS-MD patients. PLEC gene analysis in a not previously reported 35-year-old EBS-MD patient with additional disease features of cardiomyopathy and malignant arrhythmias revealed novel compound heterozygous (p.(Phe755del) and p.(Lys1040Argfs*139)) mutations resulting in complete abolition of plectin protein expression. In contrast, the other two patients with different homozygous PLEC mutations showed preserved plectin protein expression with one only expressing rodless plectin variants, and the other markedly reduced protein levels. Analysis of skeletal muscle tissue from all three patients revealed severe disruption of the extrasarcomeric intermediate filament cytoskeleton, protein aggregates positive for desmin, syncoilin, and synemin, degenerative myofibrillar changes, and mitochondrial abnormalities comprising respiratory chain dysfunction and an altered organelle distribution and amount.Our study demonstrates that EBS-MD causing PLEC mutations universally result in a desmin protein aggregate myopathy phenotype despite marked differences in individual plectin protein expression patterns. Since plectin is the key cytolinker protein that regulates the structural and functional organization of desmin filaments, the defective anchorage and spacing of assembled desmin filaments is the key pathogenetic event that triggers the formation of desmin protein aggregates as well as secondary mitochondrial pathology.

Distinct Impact of Two Keratin Mutations Causing Epidermolysis Bullosa Simplex on Keratinocyte Adhesion and Stiffness.

Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and type II keratin subunits. Missense mutations in keratin 5 or keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS) in humans by rendering the keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe generalized EBS, by stable expression in keratin-free keratinocytes. This revealed distinctly different effects on keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with keratin-free keratinocytes. Thus, a keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS therapy approaches.

Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways.

Spontaneous regression of an epidermolysis bullosa nevus on the sole.

Epidermolysis bullosa (EB) is a group of inherited rare diseases characterized by fragility and blistering of the skin and mucous membranes. An EB nevus (EBN) is a very rare acquired nevus that occurs only in patients with EB. An EBN usually arises in an area of previous blistering, and frequently has an atypical appearance that mimics malignant melanoma. We describe an older man with an EBN on the sole. Physical examination revealed irregularly pigmented band-like black macules on his right sole, suggestive of malignant melanoma. Dermoscopic examination showed a parallel furrow pattern with a monotonous area. The histopathological findings were consistent with a benign acquired nevus. Interestingly, the lesion regressed spontaneously within a year. To the best of our knowledge, this is the first reported case of EBN on the sole. Dermoscopy may be a useful addition to histopathological examination to ensure correct diagnosis of EBN.

Malignant melanoma arising in the setting of epidermolysis bullosa simplex: an important distinction from epidermolysis bullosa nevus.

IMPORTANCE: Patients with epidermolysis bullosa (EB) do not carry a significantly increased risk of melanoma but are prone to developing large, markedly atypical melanocytic nevi (EB nevi), which may mimic melanoma clinically and histologically. Many authors now favor a conservative approach in managing atypical pigmented lesions in patients with EB. OBSERVATIONS: We present the case of a 30-year-old woman with severe EB simplex who sought care for a large red and black ulcerated plaque. The clinical differential diagnosis included EB nevus and melanoma. An incisional punch biopsy specimen revealed an atypical melanocytic proliferation with focal florid pagetoid spread and involving elongated rete ridges, consistent with invasive acral lentiginous melanoma. The subsequent amputation was confirmatory. Micrometastasis was detected in 1 of 5 sentinel lymph nodes. CONCLUSIONS AND RELEVANCE: To our knowledge, this is the first reported case of melanoma arising in EB simplex-affected skin. It highlights the difficulty in differentiating melanoma from an EB nevus. Despite the increasing awareness of EB nevi, a high index of suspicion for melanoma should be maintained, and early biopsy is recommended when evaluating large pigmented lesions in patients with EB.