Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy.

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients' keratinocyte clonogenicity impairment.

KRT5 in-frame deletion in a family of German Shepherd dogs with split paw pad disease resembling localized epidermolysis bullosa simplex in human patients.

Split paw pad disease is a scarcely defined phenotype characterized by skin lesions on the paw pads of dogs. We studied a family of German Shepherd dogs, in which four dogs developed intermittent paw pad lesions and lameness. The paw pads of two of the affected dogs were biopsied and demonstrated cleft formation in the stratum spinosum and stratum corneum, the outermost layers of the epidermis. Whole genome sequencing data from an affected dog revealed a private heterozygous 18 bp in frame deletion in the KRT5 gene. The deletion NM_001346035.1:c.988_1005del or NP_001332964.1:p.(Asn330_Asp335del) is predicted to lead to a loss of six amino acids in the L12 linker domain of the encoded keratin 5. KRT5 variants in human patients lead to various subtypes of epidermolysis bullosa simplex (EBS). Localized EBS is the mildest of the KRT5-related human diseases and may be caused by variants affecting the L12 linker domain of keratin 5. We therefore think that the detected KRT5 deletion in dogs represents a candidate causal variant for the observed skin lesions in dogs. However, while the clinical phenotype of KRT5-mutant dogs of this study closely resembles human patients with localized EBS, there are differences in the histopathology. EBS is defined by cleft formation within the basal layer of the epidermis while the cleft formation in the dogs described herein occurred in the outermost layers, a hallmark of split paw pad disease. Our study provides a basis for further studies into the exact relation of split paw pad disease and EBS.

Epidermolysis Bullosa: Two rare case reports of COL7A1 and EBS-GEN SEV KRT14 variants with review of literature.

EPIDERMOLYSIS: Bullosa is a rare hereditary skin condition that causes blisters. Genes encoding structural proteins at or near the dermal-epidermal junction are mutated recessively or dominantly, and this is the primary cause of EB. Herein, two Chinese boys were diagnosed with the condition, each with a different variant in a gene that serves as a reference for EB genetic counseling. Skincare significantly impacted their prognosis and quality of life. CASE PRESENTATION: Two Chinese boys, with phenotypically normal parents, have been diagnosed with distinct blister symptoms, one with Dominant Dystrophic Epidermolysis Bullosa and the other with a severe form of Epidermolysis Bullosa Simplex. The first patient had a G-to-A variant in the COL7A1 allele, at nucleotide position 6163 which was named "G2055A". The proband is heterozygous for Dystrophic Epidermolysis Bullosa due to a COL7A1 allele with a glycine substitution at the triple helix domain. A similar variant has been discovered in his mother, indicating its potential transmission to future generations. Another patient had severe Epidermolysis Bullosa Simplex with a rare c.377T > A  variant resulting in substitution of amino acid p.Leu126Arg (NM_000526.5 (c.377T > G, p.Leu126Arg) in the Keratin 14 gene. In prior literature, Keratin 14 has been associated with an excellent prognosis. However, our patient with this infrequent variant tragically died from sepsis at 21 days old. There has been a reported occurrence of the variant only once. CONCLUSION: Our study reveals that Epidermolysis Bullosa patients with COL7A1 c.6163G > A and KRT14 c.377T>A variants have different clinical presentations, with dominant forms of Dystrophic EB having milder phenotypes than recessive ones. Thus, the better prognosis in the c.6163G > A patient. Furthermore, c.377T>A patient was more prone to infection than the patient with c.6163G>A gene variant. Genetic testing is crucial for identifying the specific variant responsible and improving treatment options.

Gross motor development in children with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a group of rare, congenital skin disorders, characterized by skin fragility and formation of blisters. The gross motor outcomes of children with EB are not known. OBJECTIVES: The primary objective of the study was to measure the proportion of gross motor delay in children with EB. The secondary objectives were to measure the difference in gross motor outcomes between EB sub-types and change in gross motor outcomes over time. METHODS: Children with EB, aged between one month and five and a half years of age, attending the Sydney Children's Hospital, Epidermolysis Bullosa Clinic, were eligible. Carers completed Ages and Stages Questionnaires, Third Edition, on behalf of their children. Questionnaires were scored, and outcomes were compared to age-expected norms. RESULTS: There were 24 participants to complete a questionnaire. Eleven participants completed additional questionnaires over the 24 month study duration. The proportion of children with EB with gross motor delay was greater than age-expected norms (29.17% vs. 2.5%). The delay occurred in children with recessive dystrophic (80%) and epidermolysis bullosa simplex (33.33%) sub-types, but not dominant dystrophic (0%). No children with Junctional EB or Kindler EB joined this study. CONCLUSIONS: This study demonstrates a difference in gross motor outcomes in children with EB. Children with recessive dystrophic and epidermolysis bullosa simplex should be prioritized for monitoring of, and intervention for, gross motor outcomes through multidisciplinary care. Further research investigating long-term outcomes for children with EB and the effectiveness of interventions would be beneficial.

EBS in Children with De Novo Pathogenic Variants Disturbing Krt14.

Epidermolysis bullosa simplex (EBS) is a dermatological condition marked by skin fragility and blister formation resulting from separation within the basal layer of the epidermis, which can be attributed to various genetic etiologies. This study presents three pathogenic de novo variants in young children, with clinical manifestations appearing as early as the neonatal period. The variants contribute to the EBS phenotype through two distinct mechanisms: direct keratin abnormalities due to pathogenic variants in the Krt14 gene, and indirect effects via pathogenic mutation in the KLHL24 gene, which interfere with the natural proteasome-mediated degradation pathway of KRT14. We report one severe case of EBS with mottled pigmentation arising from the Met119Thr pathogenic variant in KRT14, another case involving a pathogenic KLHL24 Met1Val variant, and a third case featuring the hot spot mutation Arg125His in KRT14, all manifesting within the first few weeks of life. This research underscores the complexity of genetic influences in EBS and highlights the importance of early genetic screening for accurate diagnosis and management.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

IgA nephropathy in adults with epidermolysis bullosa.

Epidermolysis bullosa (EB) is a devastating genetic condition caused by mutations in genes that give rise to aberrant proteins. There are 16 different such proteins implicated in EB that are important in maintaining the integrity of the dermoepidermal junction. It is classified into four major subtypes: (i) EB simplex; (ii) junctional EB (JEB); (iii) dystrophic EB (DEB); and (iv) Kindler EB. Renal disease is a recognized complication of EB and the aetiology is complex. We describe our experience of managing five patients with EB and IgA nephropathy. We recommend that patients with recessive DEB and JEB routinely have the following monitored: renal function, urinary albumin/creatinine ratio, urine analysis, serum albumin levels and immunoglobulins; specifically serum IgA. Management of IgA nephropathy in the context of EB should be tailored to the individual and be carried out within a specialist multidisciplinary team. Our case series provides important insights into the treatment of IgA nephropathy in patients with EB and will help inform treatment in this rare genetic disease. Case series and reports like ours are key in gaining real-life data to quantify the actual risk of morbidity and mortality from each of the treatment modalities discussed.

Epidemiology of inherited epidermolysis bullosa in Germany.

BACKGROUND: Epidermolysis bullosa (EB) is a rare genetic disorder manifesting with skin and mucosal membrane blistering in different degrees of severity. OBJECTIVE: Epidemiological data from different countries have been published, but none are available from Germany. METHODS: In this population-based cross-sectional study, people living with EB in Germany were identified using the following sources: academic hospitals, diagnostic laboratories and patient organization. RESULTS: Our study indicates an overall EB incidence of 45 per million live births in Germany. With 14.23 per million live births for junctional EB, the incidence is higher than in other countries, possibly reflecting the availability of early molecular genetic diagnostics in severely affected neonates. Dystrophic EB was assessed at 15.58 cases per million live births. The relatively low incidence found for EB simplex, 14.93 per million live births, could be explained by late or missed diagnosis, but also by 33% of cases remaining not otherwise specified. Using log-linear models, we estimated a prevalence of 54 per million for all EB types, 2.44 for junctional EB, 12.16 for dystrophic EB and 28.44 per million for EB simplex. These figures are comparable to previously reported data from other countries. CONCLUSIONS: Altogether, there are at least 2000 patients with EB in the German population. These results should support national policies and pharmaceutical companies in decision-making, allow more precise planning of drug development and clinical trials, and aid patient advocacy groups in their effort to improve quality of life of people with this orphan disease.

Diacerein 1% Ointment for the Treatment of Epidermolysis Bullosa Simplex: A Randomized, Controlled Trial.

BACKGROUND: In epidermolysis bullosa simplex (EBS), epithelial structural fragility results in blisters and erosions. Diacerein 1% ointment has been shown to reduce this blistering. OBJECTIVE: To evaluate the efficacy and safety of diacerein 1% ointment in the treatment of EBS. METHODS: A double-blind study of 54 patients with EBS were randomized to diacerein 1% or vehicle ointment once daily. The primary endpoint ( ≥60% reduction in body surface area of EBS) and the key secondary endpoint ( ≥2-point reduction in the Investigator’s Global Assessment) were evaluated at 8 weeks. RESULTS: There was no difference in the proportion of patients achieving either key efficacy endpoint between the diacerein 1% and vehicle groups (P>0.05). No difference in treatment emergent adverse events were noted between the groups. In post hoc analysis stratified by EBS subtypes, an IGA score of 0 or 1 was reported in 6 of 13 patients with severe EBS in the diacerein group (46.2%), compared with 2 of 13 patients with severe EBS in the vehicle group (15.4%); (relative risk= 3.08, 95% CI = 0.71, 13.4). CONCLUSIONS: Although there was no significant difference in outcomes between the groups, further study may elucidate the effects of diacerein on EBS lesions, especially in patients with severe EBS. Teng J, Paller AS, Bruckner AL, et al. Diacerein 1% ointment for the treatment of epidermolysis bullosa simplex: a randomized, controlled trial. J Drugs Dermatol. 2023;22(6):599-604. doi:10.36849/JDD.7108.

Clinical and molecular features in a cohort of Middle Eastern patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) features skin and mucosal fragility due to pathogenic variants in genes encoding components of the cutaneous basement membrane. Based on the level of separation within the dermal-epidermal junction, EB is sub-classified into four major types including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB) with 16 EB-associated genes reported to date. METHODS: We ascertained a cohort of 151 EB patients of various Middle Eastern ethnic backgrounds. RESULTS: The cohort was comprised of EBS (64%, 97/151), DEB (21%, 31/151), JEB (12%, 18/151), and KEB (3%, 5/151). KRT14 and KRT5 variants were most common among EBS patients with 43% (42/97) and 46% (45/97) of EBS patients carrying mutations in either of these two genes, respectively. Truncal involvement was more common in KRT14-associated EBS as compared to EBS due to KRT5 mutations (p < .05). Mutations in COL17A1 and laminin 332-encoding genes were identified in 55% (10/18) and 45% (8/18) of JEB patients. Scarring alopecia, caries, and EB nevi were most common among JEB patients carrying COL17A1 mutations as compared to laminin 332-associated JEB (p < .05). Abnormal nails were evident in most DEB and JEB patients while poikiloderma was exclusively observed in KEB (p < .001). CONCLUSIONS: EB patients of Middle Eastern origin were found to feature specific phenotype-genotype correlations of relevance to the diagnosis and genetic counseling of patients in this region.

Mutation update: The spectra of PLEC sequence variants and related plectinopathies.

Plectin, encoded by PLEC, is a cytoskeletal linker of intermediate filaments expressed in many cell types. Plectin consists of three main domains that determine its functionality: the N-terminal domain, the Rod domain, and the C-terminal domain. Molecular defects of PLEC correlating with the functional aspects lead to a group of rare heritable disorders, plectinopathies. These multisystem disorders include an autosomal dominant form of epidermolysis bullosa simplex (EBS-Ogna), limb-girdle muscular dystrophy (LGMD), aplasia cutis congenita (ACC), and an autosomal recessive form of EBS, which may associate with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and/or congenital myasthenic syndrome (EBS-MyS). In this study, genotyping of over 600 Iranian patients with epidermolysis bullosa by next-generation sequencing identified 15 patients with disease-causing PLEC variants. This mutation update analyzes the clinical spectrum of PLEC in our cohort and in the literature and demonstrates the relationship between PLEC genotype and phenotypic manifestations. This study has integrated our seven novel PLEC variants and phenotypic findings with previously published data totaling 116 variants to provide the most complete overview of pathogenic PLEC variants and related disorders.

Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migration.

Keratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell-extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell-ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.

Potential di-genic contribution to guttate leukoderma as the predominant feature of epidermolysis bullosa simplex.

Inherited epidermolysis bullosa (EB) simplex is a heterogeneous group of skin fragility disorders caused by mutations in genes encoding cell-cell or cell-matrix adhesion proteins. A recently identified, rare subtype of EB simplex is due to bi-allelic mutations in the EXPH5 gene, which encodes exophilin5, an effector protein of the Rab27B GTPase involved in intracellular vesicle trafficking and exosome secretion. The EXPH5 EB subtype is characterized by early-onset skin blisters and scars, mainly on extremities, and varying degrees of pigmentary alterations. Here, we present a 31-year-old female with diffuse guttate hypopigmentation on the trunk and extremities since early childhood, with no apparent blisters or scars. We employed whole exome sequencing of germline DNA extracted from the patient's leukocytes to determine the genetic aetiology of the phenotype. A novel homozygous variant in EXPH5, c.1153C>T causing a premature stop codon at amino acid Glutamine 385, was identified. Histologic examination after skin pricking disclosed focal keratinocyte detachment typical to EB. Additionally, we identified a deleterious-predicted variant in ENPP1, a gene associated with disturbed transfer of melanosomes to keratinocytes in Cole disease. Our report expands the clinical spectrum of inherited EB simplex with a possible di-genic synergism contributing to co-presentation with guttate leukoderma.

Pathogenic DST sequence variants result in either epidermolysis bullosa simplex (EBS) or hereditary sensory and autonomic neuropathy type 6 (HSAN-VI).

DST encodes bullous pemphigoid antigen-1 (BPAG1), a protein with eight tissue-specific isoforms expressed in the skin, muscle, brain and nerves. Accordingly, mutations in this gene are associated with epidermolysis bullosa simplex (EBS) and hereditary sensory and autonomic neuropathy type 6 (HSAN-VI). The genotypic spectrum is attested to by 19 distinct mutations but genotype-phenotype correlation for both disorders is not well established. In this study, we performed next-generation sequencing (NGS) on two families with different phenotypic presentations, one foetus (P1) with musculoskeletal and neurological malformations established by prenatal ultrasound and family history, and a 15-year-old female patient (P2) with skin blistering. P1 had a novel homozygous nonsense mutation, DST: NM_001144769, c.3805C>T, p.R1269* within a region of genetic homozygosity (ROH). This mutation resides within the plakin domain of BPAG1 and ablates all isoforms of this protein, leading to novel extracutaneous phenotypes consistent with HSAN-VI in P1. P2 had a recurrent homozygous mutation DST: NM_001723.7, c.3370C>T, p.Gln1124* that presented with giant, trauma-induced skin blisters without extracutaneous involvement. This mutation is located within the coiled-coil domain present in the skin isoform of DST, BPGA1-e, associated with EBS. In summary, we report two families with pathogenic DST variants and expand the spectrum of DST genotype and phenotypes.

Clinical heterogeneity in epidermolysis bullosa simplex with plectin (PLEC) mutations-A study of six unrelated families from India.

Epidermolysis bullosa simplex (EBS) with plectin mutations is a very rare subtype of EB usually associated with pyloric atresia (PA) or muscular dystrophy (MD). We report six unrelated children between ages 4 and 14 years from India with varied clinical manifestations. Only one had PA, and none has developed MD to date. All except the one with PA presented with early onset blistering along with laryngeal involvement in the form of hoarseness of voice and nail involvement. Patient with PA presented with aplasia cutis and died in the first week. Two patients had predominantly respiratory and gastrointestinal involvement with varying severity while two had features of myasthenic syndrome but no limb-girdle involvement and one patient phenocopied laryngo-onycho-cutaneous (LOC) syndrome. Using whole-exome sequencing, we identified novel mutations in PLEC. Histopathological analysis (Immunofluorescence antigen mapping) showed absence of staining to plectin antibodies. Our observations propose to append a phenotype of EBS, hoarseness of voice and nail dystrophy or LOC-like phenotype with plectin mutations. Long-term follow up is necessary to monitor for the development of muscular dystrophy.

Detection and characterization of low-level mosaicism among clinically unaffected parents of 'sporadic' epidermolysis bullosa simplex cases.

In this study, two (18.2%) clinically unaffected parents from 11 trios were identified with mosaic KRT14 variants.To our knowledge, this is the first report to study the proportion of low-level mosaicism in the clinically unaffected parents whose children were previously regarded as sporadic EBS cases.

A translation re-initiation variant in KLHL24 also causes epidermolysis bullosa simplex and dilated cardiomyopathy via intermediate filament degradation.

This study shows that gain-of-function variants in KLHL24 causing EBS and DCM, do not only originate in the start-codon and suggest that any nonsense-inducing variant affecting nucleotides c.4_84 will likely cause the same effect on protein level and a similar potential lethal phenotype.

A retrospective analysis of diagnostic testing in a large North American cohort of patients with epidermolysis bullosa.

BACKGROUND: Accurate diagnosis of epidermolysis bullosa (EB) has significant implications for prognosis, management, and genetic counseling. OBJECTIVE: To describe diagnostic testing patterns and assess diagnostic concordance of transmission electron microscopy (TEM), immunofluorescence mapping (IFM), and genetic analysis for EB. METHODS: A retrospective cohort included patients enrolled in the Epidermolysis Bullosa Clinical Characterization and Outcomes Database from January 1, 2004, to July 8, 2019. Tests concluding the same EB type (EB simplex, junctional EB, dominant dystrophic EB, and recessive dystrophic EB) were considered concordant; those concluding different EB types were considered discordant; and those with nonspecific/nondefinitive results were equivocal. RESULTS: A total of 970 diagnostic tests were conducted from 1984 to 2018 in 771 patients. Genetic analyses were performed chronologically later than IFM or TEM (P < .001). The likelihood of undergoing genetic analysis was greater for junctional EB and recessive dystrophic EB, and the same for dominant dystrophic EB as compared with EB simplex. TEM results in 163 patients were equivocal (55%), concordant (42%), and discordant (3%). IFM results in 185 patients were equivocal (54%), concordant (42%), and discordant (4%). LIMITATIONS: Retrospective design. CONCLUSIONS: Diagnostic testing has shifted in favor of genetic analysis. TEM and IFM frequently offer equivocal findings when compared to the specificity afforded by genetic analysis.

KRT5 missense variant in a Cardigan Welsh Corgi with epidermolysis bullosa simplex.

Epidermolysis bullosa (EB) is a group of blistering disorders that includes several subtypes, classified according to their level of cleavage. Typical clinical signs are blisters and erosions resulting from minimal trauma. The disease has been described in many mammalian species and pathogenic variants in at least 18 different genes have been identified. In the present study, we investigated a Cardigan Welsh Corgi with congenital clinical signs consistent with epidermolysis bullosa. The puppy had blisters and erosions on the paw pads, and the oral mucosa. Histologic examination demonstrated the typical clefting between the dermis and epidermis and confirmed the clinical suspicion. We obtained whole genome sequencing data from the affected puppy and searched for variants in candidate genes known to cause EB. This revealed a heterozygous missense variant, KRT5:p.(E476K), affecting the highly conserved KLLEGE motif of keratin 5. The mutant allele in the affected puppy arose owing to a de novo mutation event as it was absent from both unaffected parents. Knowledge of the functional impact of KRT5 variants in other species together with the demonstration of the de novo mutation event establishes KRT5:p.(E476K) as causative variant for the observed EBS.