Patients suffering from dystrophic epidermolysis bullosa are prone to developing autoantibodies against skin proteins: A longitudinal confirmational study.

Epidermolysis bullosa (EB) is a heritable skin blistering disease caused by variants in genes coding for proteins that secure cell-cell adhesion and attachment of the epidermis to the dermis. Interestingly, several proteins involved in inherited EB are also associated with autoimmune blistering diseases (AIBD). In this study, we present a long-term follow-up of 15 patients suffering from recessive dystrophic or junctional EB. From these patients, 62 sera were analysed for the presence of autoantibodies associated with AIBD. We show that patients suffering from recessive dystrophic EB (RDEB) are more susceptible to developing autoantibodies against skin proteins than patients suffering from junctional EB (70% vs. 20%, respectively). Interestingly, no correlation with age was observed. Most patients showed reactivity to Type XVII collagen/linear IgA bullous dermatosis autoantigen (n = 5; 33%), followed by BP230 (n = 4; 27%), Type VII collagen (n = 4; 27%) and laminin-332 (n = 1; 7%). The pathogenicity of these autoantibodies remains a subject for future experiments.

Genotype-Phenotype Correlation in Junctional Epidermolysis Bullosa: Signposts to Severity.

Junctional epidermolysis bullosa (JEB) is a rare autosomal recessive genodermatosis with a broad spectrum of phenotypes. Current genotype-phenotype paradigms are insufficient to accurately predict JEB subtype and characteristics from genotype, particularly for splice site variants, which account for over a fifth of disease-causing variants in JEB. This study evaluated the genetic and clinical findings from a JEB cohort, investigating genotype-phenotype correlations through bioinformatic analyses and comparison with previously reported variants. Eighteen unique variants in LAMB3, LAMA3, LAMC2, or COL17A1 were identified from 17 individuals. Seven had severe JEB, 9 had intermediate JEB, and 1 had laryngo-onycho-cutaneous syndrome. Seven variants were previously unreported. Deep phenotyping was completed for all intermediate JEB cases and demonstrated substantial variation between individuals. Splice site variants underwent analysis with SpliceAI, a state-of-the-art artificial intelligence tool, to predict resultant transcripts. Predicted functional effects included exon skipping and cryptic splice site activation, which provided potential explanations for disease severity and in most cases correlated with laminin-332 immunofluorescence. RT-PCR was performed for 1 case to investigate resultant transcripts produced from the splice site variant. This study expands the JEB genomic and phenotypic landscape. Artificial intelligence tools show potential for predicting the functional effects of splice site variants and may identify candidates for confirmatory laboratory investigation. Investigation of RNA transcripts will help to further elucidate genotype-phenotype correlations for novel variants.

METTL14-mediated N6-methyladenosine modification of Col17a1/Itgα6/Itgß4 governs epidermal homeostasis.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant and reversible modification occurring in eukaryotic mRNAs, however, its functions in mammalian epidermal development are still not fully elucidated. OBJECTIVE: To explore the role of METTL14 (Methyltransferase like 14), one of the m6A methyltransferases, in maintaining epidermal homeostasis. METHODS: We constructed mice with Mettl14-inactivation in the epidermal basal cells. The phenotype was explored by H&E staining and immunofluorescence staining. To explore the underlying mechanisms, we performed RNA-seq, Ribosome profiling and MeRIP-seq on wild-type and Mettl14-inactivation epidermal keratinocytes. Moreover, HaCaT cells were used for in vitro validation. RESULTS: Inactivation of Mettl14 in murine epidermis led to transient thicker epidermis and exhaustion of the epidermal stem cell pool. Interestingly, we found that the mRNA of type XVII collagen (Col17a1), integrin ß4 (Itgß4) and α6 (Itgα6) had m6A modifications, and the proteins expression were decreased in Mettl14-inactivated epidermis. Furthermore, in epidermis-specific Mettl4-inactivated mice, the epidermis was detached from the dermis and presented a phenotype similar to junctional epidermolysis bullosa (JEB), which may result from hemidesmosomes damage (decrease of COL17A1, ITGB4 and ITGA6). Knockdown of Mettl14 in HaCaT cells impaired the self-renewal and decreased the protein level of COL17A1, ITGB4 and ITGA6 and Itgß4 knockdown inhibited colony formation. CONCLUSION: Our study highlighted the role of METTL14 in the maintenance of epidermal homeostasis and identified its critical role through m6A-mediated translational inhibition of Col17a1, Itgß4 and Itgα6. Our study suggested that METTL14 may be a potential therapeutic target for the treatment of hemidesmosomes-deficient diseases, such as JEB.

A case of junctional epidermolysis bullosa intermediate with collagen XVII deficiency treated with dupilumab.

Inherited epidermolysis bullosa is a heterogeneous group of hereditary skin diseases characterized by skin (mucosa) fragility, which leads to blistering. Junctional epidermolysis bullosa is associated with mutations in genes expressing proteins of the dermo-epidermal junction. Dupilumab, an antibody that directly targets interleukin (IL)-4 receptor alpha, may be an effective treatment for dystrophic epidermolysis bullosa. We describe a case of junctional epidermolysis bullosa that improved with dupilumab.

Independent COL17A1 Variants in Cats with Junctional Epidermolysis Bullosa.

Epidermolysis bullosa (EB), characterized by defective adhesion of the epidermis to the dermis, is a heterogeneous disease with many subtypes in human patients and domestic animals. We investigated two unrelated cats with recurring erosions and ulcers on ear pinnae, oral mucosa, and paw pads that were suggestive of EB. Histopathology confirmed the diagnosis of EB in both cats. Case 1 was severe and had to be euthanized at 5 months of age. Case 2 had a milder course and was alive at 11 years of age at the time of writing. Whole genome sequencing of both affected cats revealed independent homozygous variants in COL17A1 encoding the collagen type XVII alpha 1 chain. Loss of function variants in COL17A1 lead to junctional epidermolysis bullosa (JEB) in human patients. The identified splice site variant in case 1, c.3019+1del, was predicted to lead to a complete deficiency in collagen type XVII. Case 2 had a splice region variant, c.769+5G>A. Assessment of the functional impact of this variant on the transcript level demonstrated partial aberrant splicing with residual expression of wildtype transcript. Thus, the molecular analyses provided a plausible explanation of the difference in clinical severity between the two cases and allowed the refinement of the diagnosis in the affected cats to JEB. This study highlights the complexity of EB in animals and contributes to a better understanding of the genotype-phenotype correlation in COL17A1-related JEB.

Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum.

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.

Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice.

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes.

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.

Trametinib-Induced Epidermal Thinning Accelerates a Mouse Model of Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) patients experience skin and epithelial fragility due to a pathological deficiency in genes associated with epidermal adhesion. Disease severity ranges from post-natal lethality to localized skin involvement with persistent blistering followed by granulation tissue formation and atrophic scarring. We evaluated the potential of utilizing Trametinib, an MEK inhibitor previously shown to target fibrosis, with and without the documented EB-anti-fibrotic Losartan for reducing disease severity in a mouse model of JEB; Lamc2jeb mice. We found that Trametinib treatment accelerated disease onset and decreased epidermal thickness, which was in large part ameliorated by Losartan treatment. Interestingly, a range of disease severity was observed in Trametinib-treated animals that tracked with epidermal thickness; those animals grouped with higher disease severity had thinner epidermis. To examine if the difference in severity was related to inflammation, we conducted immunohistochemistry for the immune cell markers CD3, CD4, CD8, and CD45 as well as the fibrotic marker αSMA in mouse ears. We used a positive pixel algorithm to analyze the resulting images and demonstrated that Trametinib caused a non-significant reduction in CD4 expression that inversely tracked with increased fibrotic severity. With the addition of Losartan to Trametinib, CD4 expression was similar to control. Together, these data suggest that Trametinib causes a reduction in both epidermal proliferation and immune cell infiltration/proliferation, with concurrent acceleration of skin fragility, while Losartan counteracts Trametinib's adverse effects in a mouse model of JEB.

A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.

A heterozygous mutation in ITGB4 causing a mild phenotype of junctional epidermolysis bullosa.

Mutations in ITGB4 are known to cause autosomal recessive junctional epidermolysis bullosa (JEB), which is manifested by severe blistering and granulation tissue, usually complicating pyloric atresia and even leading to death. ITGB4-associated autosomal dominant epidermolysis bullosa has rarely been documented. Herein, we identified a heterozygous pathogenic variant (c.433G>T; p.Asp145Tyr) in ITGB4 causing a mild phenotype of JEB in a Chinese family.

A novel splice-site variant of the LAMB3 gene is associated with junctional epidermolysis bullosa

Background: Junctional epidermolysis bullosa (JEB) is a rare inherited genetic disorder in which pathogenic mutations are mostly located within exons of the associated genes. This report describes a novel variant located at a splice site. Objectives: To confirm the diagnosis of the JEB family and identify the pathogenic variant. Materials & Methods: We collected clinical data and DNA from the members of the family. Whole-exome sequencing (WES) and Sanger sequencing were used to detect gene variants. The pMINI minigene system was used to design in vitro experiments, to confirm the pathogenic variants. Results: A novel splice-site variant (c.629-12T>G) of the LAMB3 gene was detected in all patients and was shown to be a pathogenic variant. Conclusion: The diagnosis of JEB should depend on gene sequencing, and variants at splice sites may also cause the disease.

Kidney-Urinary Tract Involvement in Intermediate Junctional Epidermolysis Bullosa.

Importance: Kidney-urinary tract (KUT) manifestations cause substantial morbidity in patients with junctional epidermolysis bullosa (JEB), but the spectrum of disease severity and the clinical course have been poorly characterized. Objective: To examine in a large cohort of patients with intermediate JEB the KUT manifestations, diagnostic and therapeutic procedures, genotype-phenotype correlations, and outcomes as a basis for recommendations, prognosis, and management. Design, Setting, and Participants: In this retrospective, longitudinal case series study, 99 patients with a diagnosis of JEB based on clinical and genetic findings who were treated in a single dermatology department in Freiburg, Germany, were assessed during an 18-year period (January 1, 2003, to December 31, 2021). Clinical, laboratory, and molecular genetic parameters were extracted from patients' medical records. Main Outcomes and Measures: Clinical characteristics, natural history, management of KUT manifestations, and genotype-phenotype correlations of intermediate JEB. Results: Of the 183 patients with JEB, 99 (54%) had intermediate JEB and were included in this cohort. The cohort included 49 female patients and 50 male patients. None of 49 female patients and 15 of 50 male patients had KUT involvement affecting different levels of the urinary tract, resulting in a prevalence of 30% for males; thus, the overall prevalence was 15%. The mean age at onset of KUT manifestations was 6.9 years (range, first weeks of life to 20 years; age was not available for 1 patient). Median follow-up after diagnosis of KUT involvement was 13 years (range, 3 months to 54 years). Patients with laminin 332 or integrin ß4 deficiency had at least 1 missense or splice site genetic variant, leading to residual expression of laminin 332 or integrin α6ß4, respectively. Severity of KUT complications did not correlate with the extent of skin involvement but with the affected protein. Conclusions and Relevance: Physicians and patients with JEB should be aware of the risk for KUT involvement in intermediate JEB, and physicians should apply interdisciplinary and individualized diagnostic and therapeutic procedures for management of these complications. Because this disorder is so rare, multicenter studies are required to make general recommendations.

First case report of complete paternal isodisomy of chromosome 10 harbouring a novel variant in COL17A1 that causes junctional epidermolysis bullosa intermediate.

BACKGROUND: Uniparental disomy (UPD) is a condition in which both chromosomes are inherited from the same parent, except for imprinting disorders. Uniparental isodisomy (UPiD) may result in a homozygous variant contributing to an autosomal recessive disorder in the offspring of a heterozygous carrier. Junctional epidermolysis bullosa intermediate (JEB intermediate) is an autosomal recessive inherited disease that is associated with a series of gene variants, including those of COL17A1. CASE PRESENTATION: We report the first case of complete paternal UPiD of chromosome 10 harbouring a novel homozygous variant in COL17A1: c.1880(exon23)delG (p.G627Afs*56). This variant led to the clinical phenotype of junctional epidermolysis bullosa intermediate in a 5-year-old child. Trio-whole exome sequencing (Trio-WES) and in silico data analysis were used for variant identification, Sanger sequencing was performed for variant validation, and pathological examination was performed as the gold standard for phenotype confirmation. CONCLUSIONS: We recommend the use of WES as a first-tier test for the diagnosis of epidermolysis bullosa, especially for paediatric patients. Moreover, UPD events should be detected and analysed routinely through WES data in the future.

Oral Alterations in Heritable Epidermolysis Bullosa: A Clinical Study and Literature Review.

Epidermolysis bullosa (EB) is a group of skin disorders with skin fragility characterized by blistering from minimal mechanical trauma with rupture at the dermoepidermal junction. There are four major classical heritable EB types, due to mutations in as many as 20 distinct genes: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB). This study is aimed at reporting case series on patients (N = 8; males, n = 5 and females, n = 3, age range 12-68 years) affected by EB and performs a review of the literature on this topic. This group of disorders can affect oral soft and hard tissues in various ways, resulting in various effects including enamel hypoplasia, dental caries, microstomia, ankyloglossia, oral blistering, and ulcerations early-onset periodontal disease. From the sample results, it can be concluded that the clinical manifestation of EB patients is highly variable and very different in prognosis. Oral health deeply influences the quality of life of EB patients. Dental management is essential to prevent the aggravation of soft tissue damage and tooth loss and to improve the quality of life through prosthetic and restorative therapies. Dentists should consider the oral alterations of EB subtypes to perform a personalized approach to the patients' needs in a preventive and therapeutic point of view.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.