Transgenic Epidermal Cultures for Junctional Epidermolysis Bullosa - 5-Year Outcomes.

Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014-2020 - Regione Emilia-Romagna and others.).

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.

Junctional Epidermolysis Bullosa Linked to Homozygous Mutation in LAMC2 Gene: A Case Report With Eosinophil-Rich Inflammatory Infiltrate.

ABSTRACT: Junctional epidermolysis bullosa (JEB) is a rare, incurable, devastating, and mostly fatal congenital genetic disorder characterized by painful blistering of the skin and mucous membranes in response to minor trauma or pressure. JEB is classified roughly into 2 subtypes: JEB-Herlitz is caused by mutations on genes encoding laminin-332. The authors present a patient consulted with a suspicion of primary immunodeficiency due to skin sores that started at the age of 1 month and a history of 3 siblings who died with similar sores, who was diagnosed with JEB-Herlitz after detecting a homozygous LAMC2 gene mutation in WES analysis. Microscopic evaluation of hematoxylin and eosin-stained sections showed vesicle formation with subepidermal separation, which is accompanied by striking neutrophil and eosinophil leukocyte infiltration both in the vesicle and papillary dermis (eosinophil-rich inflammatory infiltrate). Such a histopathological finding has been rarely reported in this condition.

Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

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.

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.

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.

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.

Junctional epidermolysis bullosa with extensive lung involvement in three patients with a LAMB3 Mutation.

Junctional epidermolysis bullosa (JEB) is characterized by skin and mucous membrane fragility leading to easy blistering. Blistering may be the result of multiple genetic mutations, including the LAMB3 gene encoding a subunit of laminin 332, an important protein in the basement membrane zone. The clinical presentation of JEB includes blistering and granulation tissue forming anywhere on the skin including around oral and nasal cavities, fingers, toes, and within mucous membranes such as the upper respiratory tract. Lung pathology associated with JEB is less commonly reported; we describe three children with LAMB3 pathogenic variants with extensive lung injury contributing to decline in clinical status and likely leading to their demise early in life.

Whole exome sequencing in a sample of Peruvian patients diagnosed with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a complex and heterogeneous dermatological disease. Four main types of EB have been described, each of them with distinct characteristics: EB simplex (EBS), dystrophic EB (DEB), junctional EB (JEB) and Kindler EB (KEB). Each main type varies in its manifestations, severity, and genetic abnormality. METHODS: We sought mutations in 19 genes known to cause EB and 10 genes associated with other dermatologic diseases in 35 Peruvian pediatric patients of a rich Amerindian genetic background. Whole exome sequencing and bioinformatics analysis was performed. RESULTS: Thirty-four of 35 families revealed an EB mutation. Dystrophic EB was the most frequently diagnosed type, with 19 (56%) patients, followed by EBS (35%), JEB (6%), and KEB (3%). We found 37 mutations in seven genes; 27 (73%) were missense mutations; 22 (59%) were novel mutations. Five cases changed their initial diagnosis of EBS. Four were reclassified as DEB and one as JEB. Inspection into other non-EB genes revealed a variant, c.7130C>A, in the gene FLGR2, which was present in 31 of the 34 patients (91%). CONCLUSION: We were able to confirm and identify pathological mutations in 34 of 35 patients.

Successful kidney transplantation in a patient with neonatal-onset ILNEB.

BACKGROUND: ILNEB constitute an autosomal recessive disorder caused by homozygous or compound heterozygous mutation of the gene for the ITGA3. To date, 8 ILNEB patients have been reported, but all 6 neonatal-onset ILNEB patients suffered early death within 2 years. The most common cause of death among previously reported ILNEB patients was exacerbation of the respiratory condition. METHODS: In this study, we describe a case of ILNEB with neonatal onset in a female patient and the genetic and histopathological testing performed. RESULTS: Our patient had a compound heterozygous mutation in ITGA3. Compared to previously reported patients, this patient exhibited milder clinical and histopathological characteristics. After experiencing a life-threatening respiratory infection at 8 months old, the patient started periodic subcutaneous immunoglobulin treatment once every 1-2 weeks for nephrotic-range proteinuria-induced secondary hypogammaglobulinemia. At the age of 3 years, proteinuria gradually increased with severe edema despite strict internal management. Therefore, our patient underwent unilateral nephrectomy and insertion of a peritoneal dialysis catheter followed by another unilateral nephrectomy. One month later, she underwent an ABO-compatible living-donor kidney transplantation at the age of 4 years. CONCLUSIONS: Our patient is a neonatal-onset ILNEB patient who survived for more than 2 years and underwent successful kidney transplantation.

Gentamicin Induces Laminin 332 and Improves Wound Healing in Junctional Epidermolysis Bullosa Patients with Nonsense Mutations.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients' skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients' blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

Immunofluorescence mapping, electron microscopy and genetics in the diagnosis and sub-classification of inherited epidermolysis bullosa: a single-centre retrospective comparative study of 87 cases with long-term follow-up.

BACKGROUND: Epidermolysis bullosa (EB) comprises a heterogeneous group of skin fragility disorders, classified in four major types based on skin cleavage level, i.e. EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), Kindler EB, and in more than 30 subtypes defined by the combination of laboratory and clinical data, including disease course. OBJECTIVES: Our aims were to address whether, in the age of genomics, electron microscopy (TEM) has still a role in diagnosing EB, and whether the genotype per se may be sufficient to sub-classify EB. METHODS: A thoroughly characterized single-centre EB case series was retrospectively evaluated to compare the power of TEM with immunofluorescence mapping (IFM) in establishing the EB type, and the ability of TEM, IFM and genetics to predict selected EB subtypes, i.e. severe dominant EBS (DEBS), severe JEB, severe recessive DEB (RDEB) and DEB self-improving, using genetic and final diagnosis, respectively, as gold standard. RESULTS: The series consisted of 87 patients, including 44 newborns, with a median follow-up of 54 months. Ninety-five mutations were identified in EB-associated genes, including 25 novel variants. Both IFM and TEM were diagnostic in about all cases of JEB (21/21 for both) and DEB (43/44 for IFM, 44/44 for TEM). TEM sensitivity was superior to IFM for EBS (19/20 vs. 16/19). As to EB subtyping, IFM performed better than genetics in identifying severe JEB cases due to laminin-332 defect (14/14 vs. 10/14) and severe RDEB (eight/nine vs. seven/nine). Genetics had no role in self-improving DEB diagnosis; it almost equalled TEM in predicting severe DEBS (eight/nine vs. nine/nine) and enabled to discriminate dominant from recessive non-severe DEB phenotypes and to identify special subtypes, e.g. DEBS with KLHL24 mutations. CONCLUSIONS: Transmission electron microscopy remains relevant to the diagnosis of EBS. IFM and genetics are essential and complementary tools in the vast majority of EB cases.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. OBJECTIVES: To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. ANIMALS: Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. METHODS AND MATERIALS: Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. RESULTS: Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

NGS-based expanded carrier screening for genetic disorders in North Indian population reveals unexpected results - a pilot study.

BACKGROUND: To determine the carrier frequency and pathogenic variants of common genetic disorders in the north Indian population by using next generation sequencing (NGS). METHODS: After pre-test counselling, 200 unrelated individuals (including 88 couples) were screened for pathogenic variants in 88 genes by NGS technology. The variants were classified as per American College of Medical Genetics criteria. Pathogenic and likely pathogenic variants were subjected to thorough literature-based curation in addition to the regular filters. Variants of unknown significance were not reported. Individuals were counselled explaining the implications of the results, and cascade screening was advised when necessary. RESULTS: Of the 200 participants, 52 (26%) were found to be carrier of one or more disorders. Twelve individuals were identified to be carriers for congenital deafness, giving a carrier frequency of one in 17 for one of the four genes tested (SLC26A4, GJB2, TMPRSS3 and TMC1 in decreasing order). Nine individuals were observed to be carriers for cystic fibrosis, with a frequency of one in 22. Three individuals were detected to be carriers for Pompe disease (frequency one in 67). None of the 88 couples screened were found to be carriers for the same disorder. The pathogenic variants observed in many disorders (such as deafness, cystic fibrosis, Pompe disease, Canavan disease, primary hyperoxaluria, junctional epidermolysis bullosa, galactosemia, medium chain acyl CoA deficiency etc.) were different from those commonly observed in the West. CONCLUSION: A higher carrier frequency for genetic deafness, cystic fibrosis and Pompe disease was unexpected, and contrary to the generally held view about their prevalence in Asian Indians. In spite of the small sample size, this study would suggest that population-based carrier screening panels for India would differ from those in the West, and need to be selected with due care. Testing should comprise the study of all the coding exons with its boundaries in the genes through NGS, as all the variants are not well characterized. Only study of entire coding regions in the genes will detect carriers with adequate efficiency, in order to reduce the burden of genetic disorders in India and other resource poor countries.

Next-generation sequencing through multigene panel testing for the diagnosis of hereditary epidermolysis bullosa in Chinese population.

Epidermolysis bullosa (EB) is a heritable blistering disorder. We performed a next-generation sequencing-based multigene panel test and successfully predicted 100% of the EB types, including, 36 EB simplex (EBS), 13 junctional EB (JEB), 86 dystrophic EB (DEB), and 3 Kindler EB. Chinese JEB and recessive DEB (RDEB) patients have relatively mild phenotypes; for severe type separately accounts for 45.5% and 23.8%, respectively. We identified 96 novel and 49 recurrent pathogenic variants in 11 genes, although we failed to detect the second mutation in one JEB and five RDEB patients. We identified one novel p.E475K mosaic mutation in the clinically normal mother of one out of 13 EBS patients with KRT5 mutations, one recurrent p.G2034R mosaic mutation, and one novel p.G2043R mosaic mutation in the clinically normal relatives of two out of 19 dominant DEB patients. This study shows that next-generation technology could be an effective tool in diagnosing EB.

Collagen XVII Processing and Blistering Skin Diseases.

Collagen XVII (COL17) is a hemidesmosomal transmembrane protein in the skin, which, in several autoimmune blistering skin diseases, may be targeted by autoantibodies. In addition, loss-of-function mutations in the COL17A1 gene induce a subtype of junctional epidermolysis bullosa. The extracellular domain of COL17 can be physiologically cleaved from the cell surface by ADAM family proteins in a process known as ectodomain shedding. COL17 ectodomain shedding is thought to be associated with the migration and proliferation of keratinocytes. Furthermore, the C-terminal cleavage of COL17 may be associated with basement membrane formation. COL17 can be targeted by various proteases, including MMP9, neutrophil elastase, plasmin and granzyme B, which may be associated with blister formation in pemphigoid diseases. Interestingly, cleavage of COL17 may induce neoepitopes on the proteolysed fragments, and such induction is associated with dynamic structural changes. This review summarizes the current understanding of cleavage of COL17, and how such cleavage relates to blistering skin diseases.