Mutations in PLOD3, encoding lysyl hydroxylase 3, cause a complex connective tissue disorder including recessive dystrophic epidermolysis bullosa-like blistering phenotype with abnormal anchoring fibrils and type VII collagen deficiency.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

Murine type VII collagen distorts outcome in human skin graft mouse model for dystrophic epidermolysis bullosa.

Human skin graft mouse models are widely used to investigate and develop therapeutic strategies for the severe generalized form of recessive dystrophic epidermolysis bullosa (RDEB), which is caused by biallelic null mutations in COL7A1 and the complete absence of type VII collagen (C7). Most therapeutic approaches are focused on reintroducing C7. Therefore, C7 and anchoring fibrils are widely used as readouts in therapeutic research with skin graft models. In this study, we investigated the expression pattern of human and murine C7 in a grafting model, in which human skin is reconstituted out of in vitro cultured keratinocytes and fibroblasts. The model revealed that murine C7 was deposited in both human healthy control and RDEB skin grafts. Moreover, we found that murine C7 is able to form anchoring fibrils in human grafts. Therefore, we advocate the use of human-specific antibodies when assessing the reintroduction of C7 using RDEB skin graft mouse models.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

BACKGROUND: Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. METHODS: Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. RESULTS: IHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-ß1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. CONCLUSIONS: The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-ß1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

Efficacy of Human Placental-Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal-epidermal junction (DEJ). We recently demonstrated that human cord blood-derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1-/- ) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental-derived stem cells (HPDSCs), currently being used as human leukocyte antigen-independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full-term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1-/- mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short-term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1-/- mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti-C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018;7:530-542.

An RNA-targeted therapy for dystrophic epidermolysis bullosa.

Functional impairment or complete loss of type VII collagen, caused by mutations within COL7A1, lead to the severe recessive form of the skin blistering disease dystrophic epidermolysis bullosa (RDEB). Here, we successfully demonstrate RNA trans-splicing as an auspicious repair option for mutations located in a wide range of exons by fully converting an RDEB phenotype in an ex vivo pre-clinical mouse model based on xenotransplantation. Via a self-inactivating (SIN) lentiviral vector a 3' RNA trans-splicing molecule, capable of replacing COL7A1 exons 65-118, was delivered into type VII collagen deficient patient keratinocytes, carrying a homozygous mutation in exon 80 (c.6527insC). Following vector integration, protein analysis of an isolated corrected single cell clone showed secretion of the corrected type VII collagen at similar levels compared to normal keratinocytes. To confirm full phenotypic and long-term correction in vivo, patches of skin equivalents expanded from the corrected cell clone were grafted onto immunodeficient mice. Immunolabelling of 12 weeks old skin specimens showed strong expression of human type VII collagen restricted to the basement membrane zone. We demonstrate that the RNA trans-splicing technology combined with a SIN lentiviral vector is suitable for an ex vivo molecular therapy approach and thus adaptable for clinical application.

Epidermolysis Bullosa Acquisita (Brunsting-Perry Pemphigoid Variant) Localized to the Face and Diagnosed With Antigen Identification Using Skin Deficient in Type VII Collagen.

Brunsting-Perry pemphigoid is defined as an autoimmune vesiculobullous eruption typically localized on the head and neck region with minimal or no mucosal involvement. The disease tends to run a chronic and recurrent course with residual scarring. Histological features are characterized by subepidermal bullae and linear IgG deposits at the dermo-epidermal junction. We report a case of a 46-year-old lady who presented with typical features of Brunsting-Perry pemphigoid. Autoantibodies to type VII collagen were identified by using recessive dystrophic epidermolysis bullosa skin which lacks type VII collagen in an indirect immunofluorescence assay. As a result, we diagnosed our patient as having the Brunsting-Perry pemphigoid variant of epidermolysis bullosa acquisita (EBA). This finding led us to review the literature on target antigens in Brunsting-Perry pemphigoid. Only 11 out of the 58 cases reported to date had target antigens identified. Interestingly, type VII collagen was the second most common target antigen/autoantibody (4 cases) detected after BP180 (5 cases). However, 2 further cases of EBA localized to the face with typical features of Brunsting-Perry pemphigoid were found in the literature. Although the target antigens are heterogeneous in Brunsting-Perry pemphigoid, a significant number of cases represent a clinical presentation of localized EBA.

Laboratory Diagnosis and Clinical Profile of Anti-p200 Pemphigoid.

IMPORTANCE: Anti-p200 pemphigoid is a rare subepidermal autoimmune blistering disease characterized by autoantibodies against a 200-kDa protein in the basement membrane zone. Anti-p200 pemphigoid is probably often misdiagnosed because of low availability of diagnostic assays and expertise and classified as bullous pemphigoid or epidermolysis bullosa acquisita. OBJECTIVE: To clinically characterize patients with anti-p200 pemphigoid, identified by using indirect immunofluorescence microscopy on skin substrates deficient in type VII collagen and laminin-332 (knockout analysis), to validate this technique by immunoblot with dermal extract, and to incorporate direct immunofluorescence serration pattern analysis in the diagnostic algorithm. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective study performed from January 2014 to June 2015 with biobank patient materials and clinical data for the period 1998 to 2015 from the single national referral center on autoimmune bullous diseases. Patients were selected based on a dermal side binding on 1-mol/L salt (sodium chloride)-split human skin substrate by indirect immunofluorescence microscopy, not diagnosed epidermolysis bullosa acquisita or anti-laminin-332 mucous membrane pemphigoid. MAIN OUTCOMES AND MEASURES: Indirect immunofluorescence microscopy knockout analysis was performed and diagnosis of anti-p200 confirmed by immunoblot with dermal extract. Clinical, histological, and immunological findings were registered. Autoantibodies against laminin γ1 were determined by immunoblot. RESULTS: Twelve patients with anti-p200 pemphigoid (7 male and 5 female; mean age, 66.6 years) were identified using the indirect immunofluorescence microscopy knockout analysis. Direct immunofluorescence microscopy showed a linear n-serrated IgG deposition pattern along the basement membrane zone in 9 of 11 patients. The diagnosis was confirmed by immunoblot showing autoantibodies against 200-kDa protein in dermal extract in 12 of 12 patients. Autoantibodies against recombinant laminin γ1 were detected by immunoblot in 8 of 12 patients. Remarkable similarities were seen in clinical features with predominantly tense blisters on hands and feet, resembling dyshidrosiform pemphigoid. Mucosal involvement was seen in 6 (50%) of the patients. CONCLUSIONS AND RELEVANCE: Predominance of blisters on hands and feet may be a clinical clue to the diagnosis of anti-p200 pemphigoid. Direct immunofluorescence microscopy serration pattern analysis and indirect immunofluorescence microscopy knockout analysis are valuable additional techniques to facilitate the diagnosis of anti-p200 pemphigoid.

Estrategias terapéuticas innovadoras para la epidermólisis bullosa distrófica recesiva / Innovative Therapeutic Strategies for Recessive Dystrophic Epidermolysis Bullosa

La epidermólisis bullosa distrófica recesiva (EBDR) es una de las enfermedades raras (ER) de la piel más graves y que mayor interés ha suscitado en cuanto al desarrollo de estrategias avanzadas de intervención terapéutica. La EBDR es debida a la deficiencia de colágeno vii como consecuencia de mutaciones en el gen COL7A1, y los distintos abordajes terapéuticos buscan la reposición de colágeno vii para restituir la adhesión dermo-epidérmica. La variedad de terapias en evaluación incluyen tanto la proteica como diversas estrategias celulares y génicas. Algunas de estas estrategias tienen un potencial terapéutico que va más allá del defecto cutáneo, pudiendo corregir el problema también a nivel de las mucosas internas. En los próximos años se espera que estos nuevos abordajes brinden una mejora sustancial en la calidad de vida de los pacientes con EBDR (AU)

Recessive dystrophic epidermolysis bullosa (RDEB) is among the most serious rare skin diseases. It is also the rare skin disease for which most effort has been expended in developing advanced therapeutic interventions. RDEB is caused by collagen VII deficiency resulting from COL7A1 mutations. Therapeutic approaches seek to replenish collagen VII and thus restore dermal-epidermal adhesion. Therapeutic options under development include protein therapy and different cell-based and gene-based therapies. In addition to treating skin defects, some of these therapies may also target internal mucosa. In the coming years, these novel therapeutic approaches should substantially improve the quality of life of patients with RDEB (AU)

Innovative therapeutic strategies for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is among the most serious rare skin diseases. It is also the rare skin disease for which most effort has been expended in developing advanced therapeutic interventions. RDEB is caused by collagen VII deficiency resulting from COL7A1 mutations. Therapeutic approaches seek to replenish collagen VII and thus restore dermal-epidermal adhesion. Therapeutic options under development include protein therapy and different cell-based and gene-based therapies. In addition to treating skin defects, some of these therapies may also target internal mucosa. In the coming years, these novel therapeutic approaches should substantially improve the quality of life of patients with RDEB.

Rescue of the mucocutaneous manifestations by human cord blood derived nonhematopoietic stem cells in a mouse model of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disease caused by mutations in COL7A1-encoding type VII collagen (C7). Currently, there is no curative therapy for patients with RDEB. Our previous studies demonstrated that human umbilical cord blood (HUCB) derived unrestricted somatic stem cells (USSCs) express C7 and facilitate wound healing in a murine wounding model. The primary objective of this study is to investigate the therapeutic functions of USSCs in the C7 null (Col7a1(-/-) ) C57BL6/J mice, a murine model of RDEB. We demonstrated that intrahepatic administration of USSCs significantly improved the blistering phenotype and enhanced the life span in the recipients. The injected USSCs trafficked to the sites of blistering and were incorporated in short-term in the recipients' skin and gastrointestinal tract. Consistent with an overall histological improvement in the epidermal-dermal adherence following USSC treatment, the expression of C7 at the basement membrane zone was detected and the previously disorganized integrin α6 distribution was normalized. We also demonstrated that USSCs treatment induced an infiltration of macrophages with a regenerative "M2" phenotype. Our data suggest that HUCB-derived USSCs improved the RDEB phenotype through multiple mechanisms. This study has warranted future clinical investigation of USSCs as a novel and universal allogeneic stem cell donor source in selected patients with RDEB.

Gene therapy: pursuing restoration of dermal adhesion in recessive dystrophic epidermolysis bullosa.

The replacement of a defective gene with a fully functional copy is the goal of the most basic gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is characterised by a lack of adhesion of the epidermis to the dermis. It is an ideal target for gene therapy as all variants of hereditary RDEB are caused by mutations in a single gene, COL7A1, coding for type VII collagen, a key component of anchoring fibrils that secure attachment of the epidermis to the dermis. RDEB is one of the most severe variants in the epidermolysis bullosa (EB) group of heritable skin diseases. Epidermolysis bullosa is defined by chronic fragility and blistering of the skin and mucous membranes due to mutations in the genes responsible for production of the basement membrane proteins. This condition has a high personal, medical and socio-economic impact. People with RDEB require a broad spectrum of medications and specialised care. Due to this being a systemic condition, most research focus is in the area of gene therapy. Recently, preclinical works have begun to show promise. They focus on the virally mediated ex vivo correction of autologous epithelium. These corrected cells are then to be expanded and grafted onto the patient following the lead of the first successful gene therapy in dermatology being a grafting of corrected tissue for junctional EB treatment. Current progress, outstanding challenges and future directions in translating these approaches in clinics are reviewed in this article.

Global remodelling of cellular microenvironment due to loss of collagen VII.

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post-translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF-ß and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.

Type VII collagen deficiency causes defective tooth enamel formation due to poor differentiation of ameloblasts.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen (COL7), a major component of anchoring fibrils in the epidermal basement membrane zone. Patients with RDEB present a low oral hygiene index and prevalent tooth abnormalities with caries. We examined the tooth enamel structure of an RDEB patient by scanning electron microscopy. It showed irregular enamel prisms, indicating structural enamel defects. To elucidate the pathomechanisms of enamel defects due to COL7 deficiency, we investigated tooth formation in Col7a1(-/-) and COL7-rescued humanized mice that we have established. The enamel from Col7a1(-/-) mice had normal surface structure. The enamel calcification and chemical composition of Col7a1(-/-) mice were similar to those of the wild type. However, transverse sections of teeth from the Col7a1(-/-) mice showed irregular enamel prisms, which were also observed in the RDEB patient. Furthermore, the Col7a1(-/-) mice teeth had poorly differentiated ameloblasts, lacking normal enamel protein-secreting Tomes' processes, and showed reduced mRNA expression of amelogenin and other enamel-related molecules. These enamel abnormalities were corrected in the COL7-rescued humanized mice expressing a human COL7A1 transgene. These findings suggest that COL7 regulates ameloblast differentiation and is essential for the formation of Tomes' processes. Collectively, COL7 deficiency is thought to disrupt epithelial-mesenchymal interactions, leading to defective ameloblast differentiation and enamel malformation in RDEB patients.

Gene expression signatures of mouse bone marrow-derived mesenchymal stem cells in the cutaneous environment and therapeutic implications for blistering skin disorder.

BACKGROUND AIMS: Multiple studies have demonstrated that mesenchymal stromal cells (MSC) can be utilized therapeutically for various congenital and acquired disorders. The involvement of MSC in the maintenance of skin homeostasis and their curative application for the treatment of skin wounds have also been documented. However, it is not known whether MSC can commit to cutaneous lineages, produce structural proteins essential for the skin integrity or be used for hereditary skin disorders. METHODS: To address these questions, we conducted a comparative expression analysis between MSC and potentially adjacent cutaneous cells, fibroblasts and keratinocytes, with specific emphasis on extracellular matrix encoding and related genes. RESULTS: Our data demonstrated that MSC share many features with cutaneous fibroblasts. We also observed that under direct influence of cutaneous fibroblasts in vitro and fibroblast-derived matrix in vivo, MSC acquired a fibroblastic phenotype, suggesting that specific cell-cell interactions play a key regulatory role in the differentiation of MSC. Additionally, the observed fibroblastic transition of MSC was underlined by a significant up-regulation of several cutaneous-specific genes encoding lumican, decorin, type VII collagen, laminin and other structural proteins. As many of the identified genes have considerable therapeutic value for dermatologic afflictions, particularly type VII collagen, we evaluated further the therapeutic potential of congenic MSC in the skin of Col7a1-null mice recapitulating human recessive dystrophic epidermolysis bullosa (RDEB). Remarkably, MSC-derived type VII collagen was sufficient for restoration of the damaged dermal-epidermal junction and partial reversal of the RDEB phenotype. CONCLUSIONS: Collectively, our results suggest that MSC may offer promising therapeutics for the treatment of RDEB and potentially other genodermatoses.

Pretibial epidermolysis bullosa.

A 47-year-old Vietnamese woman presented with dystrophic fingernails and toenails that had been present since infancy. She also had developed, in the third decade, pretibial pruritus with vesicle formation and progressive localized papules and scars. Multiple family members were similarly affected. Physical examination showed lichenoid papules that coalesced into large plaques that were studded with milia over the pretibial areas and 20 nail dystrophy. A biopsy specimen showed milia-like structures and dermal fibrosis. Pretibial epidermolysis bullosa is a rare variant of dystrophic epidermolysis bullosa that shows appreciable clinical overlap with dystrophic epidermolysis bullosa pruginosa. Both disease subsets are characterized by the late age of onset, nail dystrophy, and predominantly pretibial pruritic lichenoid skin lesion; they are associated with glycine substitution mutations in COL7A1.

Muscle MRI in Ullrich congenital muscular dystrophy and Bethlem myopathy.

The aim of this study was to evaluate the spectrum of muscle involvement on Magnetic Resonance Imaging (MRI) in patients with collagen VI related disorders. Nineteen patients with genetically confirmed collagen VI related disorders, 10 with Bethlem myopathy and 9 with Ullrich congenital muscular dystrophy (CMD), had muscle MRI of their legs using T1 sequences through calves and thighs. In patients with Bethlem myopathy the vasti muscles appeared to be the most frequently and most strikingly affected thigh muscles, with a rim of abnormal signal at the periphery of each muscle and relative sparing of the central part. Another frequent finding was the presence of a peculiar involvement of the rectus femoris with a central area of abnormal signal within the muscle. Patients with Ullrich CMD had a more diffuse involvement of the thigh muscles with relative sparing of sartorius, gracilis and adductor longus. In 8 of the 9 patients with Ullrich CMD, we also observed the peripheral rim of the vastus lateralis and the central area in the rectus femoris observed in patients with Bethlem myopathy. At calf level the results were more variable but a significant proportion of patients with both Bethlem myopathy (8/10) and Ullrich CMD (6/9) showed a rim of abnormal signal at the periphery of soleus and gastrocnemii. Bethlem myopathy and Ullrich CMD patients have distinct patterns of muscle involvement on MRI with some overlap between the two forms. Our results suggest that muscle MR may be used, as an additional tool, to identify patients with collagen VI related disorders. This information is even more important in the patients with a typical Ullrich CMD clinical phenotype but with normal collagen expression of VI in muscle and/or skin.