Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine- to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.      

Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms

Kindlin-1 is an epithelium-specific phosphoprotein and focal adhesion adaptor component. Mutations in the corresponding gene (KIND1) cause Kindler syndrome (KS), which is manifested by skin blistering, poikiloderma, photosensitivity and carcinogenesis. Some patients also exhibit gastrointestinal symptoms, but it has remained unclear whether these represent a feature of Kindler syndrome or a coincidence. We examined kindlin-1 in human gastrointestinal epithelia and showed that it is involved in the aetiopathology of Kindler syndrome-associated colitis. Kindlin-1 expression was assessed by indirect immunofluorescence, western blot and RT-PCR. Kindlin-1 is expressed in oral mucosa, colon and rectum. Both the full-length 74 kDa kindlin-1 protein and a 43 kDa isoform were detected in CaCo2 cells, the latter resulting from alternative splicing. In the first months of life, patients (homozygous for null mutations) had severe intestinal involvement with haemorrhagic diarrhoea and showed morphological features of severe ulcerative colitis. Later in childhood, histopathology demonstrated focal detachment of the epithelium in all segments of the colon, chronic inflammation and mucosal atrophy. These findings define an intestinal phenotype for Kindler syndrome as a consequence of a primary epithelial barrier defect. The different clinical intestinal manifestations in Kindler syndrome patients may be explained by partial functional compensation of kindlin-1 deficiency by the intestinal isoform or by the presence of truncated mutant kindlin-1.     

Esophageal stenosis in childhood: dystrophic epidermolysis bullosa without skin blistering due to collagen VII mutations.

We report a 9-year-old girl who experienced recurrent dysphagia since infancy. Crohn's disease was suspected because she had aphthous ulcers of the mouth and anal dermatitis with hematochezia. After bougienages of esophageal stenoses and medication for inflammatory bowel disease proved unsuccessful, interdisciplinary re-examination revealed the cause of the symptoms to be an extracutaneous form of dystrophic epidermolysis bullosa, a genetic skin fragility disorder. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII, a protein of the epidermal attachment complex, and typically manifests with trauma-induced skin blistering, scarring, nail dystrophy, and, in some cases, mucosal involvement. The present proband never developed skin blisters but had nail dystrophy and erosions of the oral, esophageal, and genitoanal mucosa, which healed with slight scarring. Mutation analysis disclosed compound heterozygosity for recessive mutations in the COL7A1 gene. The paternal mutation 425 A-->G caused abnormal splicing resulting in a premature stop codon. The maternal mutation G2775S led to the substitution of a glycine by a serine in the triple helical domain of collagen VII. This case shows that mucosal disease and esophageal strictures in childhood are not always acquired, but can also represent a genetic defect of dermal-epidermal adhesion, even in the absence of skin blistering.     

Epidermolysis Bullosa Dystrophica Inversa in a Child

A 4-year-old child with dystrophic epidermolysis bullosa inversa is described. Clinical features were blistering of the skin, erosions, scarring and milia formation. The areas involved included the trunk, with preference for the axillary and inguinal folds, the neck and sacral area, and proximal extremities. Notably, the hands and feet were completely spared, with only mild nail dystrophy. Ultrastructural analysis revealed dermolytic blistering and absent or rudimentary anchoring fibrils. Collagen VII, the main structural protein of these fibrils, was present in the skin, as shown by indirect immunofluorescence. These findings suggest that a mutation that prevents appropriate supramolecular aggregation of collagen VII into anchoring fibrils may underlie this subtype of dystrophic epidermolysis bullosa in some patients.     

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.     

Necrobiosis lipoidica

A 69-year-old patient presented with different skin lesions all of which belonged to group of necrobiosis lipoidica. The initial histologic diagnosis was actinic granuloma O??Brien. A subsequent biopsy was interpreted as granulomatous necrobiosis lipoidica. The history of these necrobiotic variants is reviewed and exemplarily depicted with this case. Necrobiosis lipoidica is part of the spectrum of granulomatous skin disorders. Although its etiology is unclear, an association with diabetes mellitus is often discussed. Multiple therapeutic options exist, but standardized guidelines for treatment are missing.     

A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrophic epidermolysis bullosa keratinocyte cell line

Dystrophic epidermolysis bullosa (DEB) comprises a family of inherited blistering skin disorders for which no corrective therapy currently exists. In the most severe form, the Hallopeau-Siemens subtype (RDEB-HS), the epidermal adhesion protein collagen VII is absent from the skin as a consequence of null mutations in the COL7A1 gene. In order to develop an ex vivo gene therapy approach for DEB, we aimed to restore expression of intact procollagen VII in RDEB-HS keratinocytes. The entire human COL7A1 locus in a P1-derived artificial chromosome (PAC) was transferred to RDEB-HS keratinocytes by microinjection, after which sustained biosynthesis and secretion of procollagen VII was detected for 1 year in vitro. Protein chemical analysis demonstrated that the chain composition, domain structure, N-glycosylation and protein folding of the newly produced procollagen VII were similar, if not identical, to its authentic counterpart, indicating that transgenic procollagen VII was structurally normal. These data demonstrate a "proof of principle" for genomic DNA vectors as a means of restoring collagen VII production in RDEB-HS skin and help develop future gene therapy protocols.     

High Local Concentrations of Intradermal MSCs Restore Skin Integrity and Facilitate Wound Healing in Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an incurable skin fragility disorder caused by mutations in the COL7A1 gene, coding for the anchoring fibril protein collagen VII (C7). Life-long mechanosensitivity of skin and mucosal surfaces is associated with large body surface erosions, chronic wounds, and secondary fibrosis that severely impede functionality. Here, we present the first systematic long-term evaluation of the therapeutic potential of a mesenchymal stromal cell (MSC)-based therapy for DEB. Intradermal administration of MSCs in a DEB mouse model resulted in production and deposition of C7 at the dermal-epidermal junction, the physiological site of function. The effect was dose-dependent with MSCs being up to 10-fold more potent than dermal fibroblasts. MSCs promoted regeneration of DEB wounds via normalization of dermal and epidermal healing and improved skin integrity through de novo formation of functional immature anchoring fibrils. Additional benefits were gained by MSCs'' anti-inflammatory effects, which led to decreased immune cell infiltration into injured DEB skin. In our setting, the clinical benefit of MSC injections lasted for more than 3 months. We conclude that MSCs are viable options for localized DEB therapy. Importantly, however, the cell number needed to achieve therapeutic efficacy excludes the use of systemic administration.