The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.

The blistering skin disorder epidermolysis bullosa simplex (EBS) results from dominant mutations in keratin 5 (K5) or keratin 14 (K14) genes, encoding the intermediate filament (IF) network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic IFs, providing a 'null' environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14(R125C) mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14(R125C)/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to a lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for the development of EBS therapies.

Epidermolysis bullosa (EB) is a rare genetic disease with widely different clinical manifestations, but the relationship between genotype and phenotype is not fully understood. In the present study, we recruited a Chinese family in which two members had been diagnosed with localized EB simplex (EBS), with clinical manifestation, including blisters and erosions on the soles of the feet since infancy.

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.

Epidermolysis bullosa simplex (EBS) is a rare genetic condition typified by superficial bullous lesions following incident frictional trauma to the skin. Most cases of EBS are due to dominantly acting mutations in keratin 14 (K14) or K5, the type I and II intermediate filament (IF) proteins that copolymerize to form a pancytoplasmic network of 10 nm filaments in basal keratinocytes of epidermis and related epithelia. Defects in K5-K14 filament network architecture cause basal keratinocytes to become fragile, and account for their rupture upon exposure to mechanical trauma. The discovery of the etiology and pathophysiology of EBS was intimately linked to the quest for an understanding of the properties and function of keratin filaments in skin epithelia. Since then, continued cross-fertilization between basic science efforts and clinical endeavors has highlighted several additional functional roles for keratin proteins in the skin, suggested new avenues for effective therapies for keratin-based diseases, and expanded our understanding of the remarkable properties of the skin as an organ system.

Epidermolysis bullosa refers to a group of genodermatoses that affects the integrity of epithelial layers, phenotypically resulting in severe skin blistering. Dowling-Meara, the major subtype of epidermolysis bullosa simplex, is inherited in an autosomal dominant manner and can be caused by mutations in either the keratin-5 (K5) or the keratin-14 (K14) gene. Currently, no therapeutic approach is known, and the main objective of this study was to identify novel therapeutic targets. We used microarray analysis, semi-quantitative real-time PCR, western blot and ELISA to identify differentially regulated genes in two K14 mutant cell lines carrying the mutations K14 R125P and K14 R125H, respectively. We found kallikrein-related peptidases and matrix metalloproteinases to be upregulated. We also found elevated expression of chemokines, and we observed deregulation of the Cdc42 pathway as well as aberrant expression of cytokeratins and junction proteins. We further demonstrated, that expression of these genes is dependent on interleukin-1 β signaling. To evaluate these data in vivo we analysed the blister fluids of epidermolysis bullosa simplex patients vs. healthy controls and identified matrix metalloproteinase-9 and the chemokine CXCL8/IL-8 as potential therapeutic targets.

Epidermolysis bullosa simplex is a disease that belongs to a group of genodermatoses characterised by the formation of superficial bullous lesions caused by minor mechanical trauma to the skin. The skin fragility observed in the EBS is mainly caused by pathogenic variants in the KRT5 and KRT14 genes that compromise the mechanical stability of epithelial cells. By performing DNA sequencing in a female patient with EBS, we found the pathogenic variant c.967G>A (p.Val323Met) in the KRT5 gene. This variant co-segregated with EBS in the family pedigree and was transmitted in an autosomal dominant inheritance manner. This is the first report showing a familial form of EBS due to this pathogenic variant.

Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype.

A 6-day-old Belgian Blue-Holstein calf was referred because of a syndrome resembling epidermolysis bullosa simplex (EBS). The clinical phenotype included irregular and differently sized erosions and ulcerations spread over the body, in particular on the limbs and over bone prominences, as well as in the nasal planum and oral mucosa. Blisters were easily induced by rubbing the skin. The skin lesions displayed a clear dermal-epidermal separation at the level of the basal cell layer. Post mortem examination revealed erosions in the pharynx, proximal esophagus, and rumen. Whole-genome sequencing revealed a heterozygous disruptive in-frame deletion variant in KRT5 (c.534_536delCAA). Genotyping of both parents confirmed the variant as de novo mutation. Clinicopathological and genetic findings were consistent with the diagnosis of KRT5-related EBS providing the second example of a spontaneous mutation causing epidermolysis bullosa in cattle.

Epidermolysis bullosa simplex (EBS) is a blistering dermatosis that is mostly caused by dominant mutations in KRT5 and KRT14. In this study, we investigated one patient with localized recessive EBS caused by novel homozygous c.1474T > C mutations in KRT5. Biochemical experiments showed a mutation-induced alteration in the keratin 5 structure, intraepidermal blisters, and collapsed keratin intermediate filaments, but no quantitative change at the protein levels and interaction between keratin 5 and keratin 14. Moreover, we found that MAPK signaling was inhibited, while desmosomal protein desmoglein 1 (DSG1) was upregulated upon KRT5 mutation. Inhibition of EGFR phosphorylation upregulated DSG1 levels in an in vitro model. Collectively, our findings suggest that this mutation leads to localized recessive EBS and that keratin 5 is involved in maintaining DSG1 via activating MAPK signaling.

Epidermolysis bullosa simplex, generalized severe (EBS-gen sev) is one of the major forms of EBS, caused by mutations of the keratin 5 (KRT5) or keratin 14 (KRT14). However, it is rarely reported in the Chinese population. The current study was performed on three unrelated Chinese families with five patients clinically suspicious for distinct stages of EBS. Mutation screening was performed by direct sequencing of the entire coding regions of KRT5 and KRT14 genes. A diagnosis of EBS-gen sev for patients in these three families was confirmed by revealing missense mutations c.373C>T (p.Arg125Cys), c.374G>T (p.Arg125Leu), and a novel frameshift mutation c.1231delG (p.Glu411Argfs*31) in KRT14. Considering two previously reported cases and the results of the current report, amino acid residue 125 is likely the most frequent hotspot of EBS-gen sev in the Chinese population. The current study further indicated that the symptoms of EBS-gen sev patients decline with age.

Plectin is a cytoskeletal linker protein which has a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS-MD), and EBS with pyloric atresia (EBS-PA). Previous studies have demonstrated that loss of full-length plectin with residual expression of the rodless isoform leads to EBS-MD, whereas complete loss or marked attenuation of expression of full-length and rodless plectin underlies the more severe EBS-PA phenotype. However, muscular dystrophy has never been identified in EBS-PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon-causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient.

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

Background Epidermolysis bullosa simplex (EBS) is a rare genodermatosis resulting from multiple gene mutations, including KRT5 and KRT14. The clinical expression of the mechanobullous skin fragility disease has not been fully explained by the genotype. Case Description An 11-day-old Japanese newborn infant was hospitalized because of herpetiform skin blistering on the feet, which expanded systemically after birth. There was no evidence of virus infection. The biopsied skin lesion showed a blister on the lamina densa without keratin clumps, indicating a diagnosis of EBS-generalized intermediate. We punctured the blisters to remove the contents daily, which led to no exacerbation or infection. The genetic study determined that the patient carried double substitutions of KRT5 c.1424A > G (p.E475G) and KRT14 c.1237G > A (p.A413T). The asymptomatic mother and sister carried the KRT14 substitution, but the healthy father had no substitution of the KRT gene. Conclusion This is the first report of EBS-generalized intermediate in a newborn with de novo KRT5 gene mutation and KRT14 gene polymorphism, and no familial history of epidermolysis. Neonatal blistering due to EBS requires optimal skin management after excluding infectious and immunobullous diseases.

Epidermolysis bullosa simplex (EBS) is usually inherited as an autosomal dominant disease due to monoallelic gain-of-function mutations in KRT5 or KRT14. Although autosomal recessive forms of EBS have been associated with mutations in at least 10 genes, recessive EBS due to homozygous biallelic KRT5 mutations has not been reported previously; it has been hypothesized that it would result in prenatal lethality. We sought the genetic causes of EB in a cohort of 512 distinct EB families by performing whole exome sequencing (WES) and using an EB-targeting next-generation sequencing (NGS) panel of 21 genes. The pathogenicity and consequences of the mutations were determined by expression profiling and at tissue and ultrastructural levels. Two pathogenic, homozygous missense variants of KRT5 in two patients with generalized EBS and a homozygous null mutation in a patient who died as a neonate from complications of EB were found. The two missense mutations disrupted keratin 5 expression on immunofluorescence microscopy, and the human "knock-out" of KRT5 showed no RNA and protein expression. Collectively, these findings identify biallelic KRT5 mutations with a phenotypic spectrum varying from mild, localized and generalized to perinatal lethal, expanding the genotypic profile of autosomal recessive EBS.

Mutations in keratin 5 (KRT5) or KRT14 genes are responsible for the most severe form of epidermolysis bullosa simplex (EBS), which is EBS generalized severe (EBS-gen sev). To date, only four pathogenic mutations (p.Arg165Ser and p.Lys199Asn in KRT5; p.Arg125Cys and p.Arg125His in KRT14) have been reported to be responsible for EBS-gen sev in the Chinese population. In the present study, a 2-year-old Chinese boy was clinically suspected to suffer from EBS, and thus Sanger sequencing was performed in the extracted genomic DNA samples from the patient, his parents and 100 healthy controls. A novel de novo heterozygous missense mutation c.503A>G (p.Glu168Gly) located at the N-terminal end segment of the 1A domain in KRT5 was identified by molecular analysis. In silico analysis tools were used to predict the pathogenicity of the novel missense mutation. A diagnosis of EBS-gen sev was thus confirmed according to the clinical presentations and molecular results.

We report two Hong Kong children with severe generalized epidermolysis bullosa simplex (EBS), the most severe form of EBS, without a family history of EBS. EBS is a rare genodermatosis usually inherited in an autosomal dominant fashion although rare autosomal recessive cases have been reported. Genetic studies in these patients showed that the first case was due to a novel de novo heterozygous variant, c.377T>G (NM_000526.5 (c.377T>G, p.Leu126Arg)) in the KRT14 gene and the second case was due to a rare de novo heterozygous variant c.527A>G (NM_000424.4, c.527A>G, p.Asn176Ser) in the KRT5 gene. To our knowledge, the c.377T>G variant in the KRT14 gene has not been previously reported, and the c.527A>G variant in the KRT5 gene is a rare cause of severe generalized EBS. In severe generalized EBS, infants exhibit severe symptoms at the onset; however, they tend to improve with time. A precise genetic diagnosis in these two cases aided in counseling the families concerning the prognosis in their affected children and the recurrence risk for future pregnancies.

No abstract available

Background: Epidermolysis bullosa simplex is a hereditary skin disorder caused by mutations in several genes such as KRT5 and KRT14 . Skin fragility in basal keratinocytes presence regions led to the cytolysis of epidermis and blistering. Aim of this study was to detect the molecular defects in KRT5 and KRT14 genes hot spots in patients with clinical suspicion of EBS and investigation of their probable genotype-phenotype correlations. Methods: Exons 1 and 6-7 of KRT5 and exons 1 and 4-7 of KRT14 amplification and mutation detection were performed by polymerase chain reaction and Sanger sequencing, respectively. Novel variants pathogenicity evaluated by bioinformatics tools. Results: Nine important variants detected in seven different patients within 6 Iranian families affected by Epidermolysis bullosa simplex, of which four variants were novel. Three patients had a mottled pigmentation phenotype [G96D (p.Gly96Asp) and F97I (p.Phe97Ile) in KRT5 ]. One of them showed a Dowling-Meara phenotype [A417P (p.Ala417Pro) and E477D (p.Glu477Asp) in KRT5 ] and another had a Koebner type phenotype [R397I (p.Arg397Ile) and Q444* (p.Gln444Ter) in KRT5 ]. A novel variant [G92E (p.Gly92Glu) in KRT5 ] in a double heterozygous state with a challenging variant [A413T (p.Ala413Thr) in KRT14 ] identified in one patient with Koebner type phenotype. Also, a previously reported mutation [I377T (p.Ile377Thr) in KRT14 gene] identified in this study. Conclusion: The results of molecular data analysis showed that the most severe phenotypes were associated with mutations in highly conserved regions. In some cases, different inheritance modes were observed.

Epidermolysis bullosa is a group of severe skin blistering disorders, which currently have no cure. The pathology of epidermolysis bullosa is recognized as having an inflammatory component, but the role of inflammation in different epidermolysis bullosa disorders is unclear. Epidermolysis bullosa simplex (EBS) is primarily caused by sequence variants in keratin genes; its most severe form, EBS generalized severe, is characterized by aggregates of keratin proteins, and cell models of EBS generalized severe show constitutively elevated stress. IFN-γ is a major mediator of inflammation, and we show that the addition of IFN-γ alone to disease model keratinocytes promotes keratin aggregation, decreases cell-cell junctions, delays wound closure, and reduces cell proliferation. IFN-γ exposure weakens the intercellular cohesion of monolayers on mechanical stress, with IFN-γ-treated EBS monolayers more fragmented than IFN-γ-treated wild-type monolayers. A humanized monoclonal antibody to IFN-γ neutralized the detrimental effects on keratinocytes, restoring cell proliferation, increasing cell-cell adhesion, accelerating wound closure in the presence of IFN-γ, and reducing IFN-γ-mediated keratin aggregation in EBS cells. These suggest that treatment with IFN-γ blocking antibodies may constitute a promising new therapeutic strategy for patients with EBS and may also have ameliorating effects on other inflammatory skin diseases.