Intracellular Accumulation of Collagen VII in Cultured Keratinocytes from a Patient with Dominant Dystrophic Epidermolysis Bullosa

Expression of collagen VII, a candidate molecule for dystrophic epidermolysis bullosa, was analyzed in cultured keratinocytes from a patient with generalized dominant dystrophic epidermolysis bullosa (DEBD) of the Pasini subtype. Double immunofluorescence revealed an increased intracellular staining of collagen VII that co-localized with protein disulfide isomerase, a marker of the rough endoplasmic reticulum. Ultrastructural analysis of cultured DEBD cells showed dilated cisternae of the rough endoplasmic reticulum and numerous residual bodies, both of which contained abundant collagen VII as detected by immunoelectron microscopy. Immunoblotting of keratinocyte extracts indicated an increased ratio of cell-associated versus secreted soluble collagen VII in DEBD cells. Collagen VII mRNA was of normal size in the DEBD cells, but present in excessive amounts. The data suggest a mutation in the collagen VII gene that leads to intracellular accumulation and degradation of this collagen, and thus to a reduced number of anchoring fibrils at the dermo-epidermal junction, and subsequently to blistering of the skin in this family

Molecular Mechanisms of Junctional Epidermolysis Bullosa: Col15 Domain Mutations Decrease the Thermal Stability of Collagen XVII

Mutations in the collagen XVII gene, COL17A1, are associated with junctional epidermolysis bullosa. Most COL17A1 mutations lead to a premature termination codon (PTC), whereas only a few mutations result in amino acid substitutions or deletions. We describe here two novel glycine substitutions, G609D and G612R, and a splice site mutation resulting in a deletion of three Gly–X–Y amino acid triplets. In order to investigate the molecular pathomechanisms of non-PTC mutations, G609D and G612R and two previously known substitutions, G627V and G633, and deletion of the amino acids 779–787 were introduced into recombinant collagen XVII. The thermal stability of the mutated collagens was assessed using trypsin digestions at incremental temperatures. All the four glycine substitutions significantly destabilized the ectodomain of collagen XVII, which manifested as 16°C–20°C lower Tm (midpoint of the helix-to-coil transition). These results were supported by secondary structure predictions, which suggested interruptions of the collagenous triple helix within the largest collagenous domain, Col15. In contrast, deletion of the three full Gly–X–Y triplets, amino acids 779–787, had no overall effect on the stability of the ectodomain, as the deletion was in register with the triplet structure and also generated compensatory changes in the NC15 domain

Disruption of ERBB2IP Is not Associated with Dystrophic Epidermolysis Bullosa in Both Father and Son Carrying a Balanced 5;13 Translocation

Mutations in the type VII collagen gene (COL7A1) cause autosomal recessive and autosomal dominant inherited dystrophic epidermolysis bullosa (DEB). We report a family with three individuals who present blistering, scarring, hypo- and hyperpigmentation, and nail dystrophy suggestive for DEB. Whereas father and son carry a 5;13 translocation, the daughter shows a normal karyotype. Segregation analysis revealed that all affected family members inherited the same COL7A1 allele. Mutation analysis disclosed a heterozygous missense mutation, c.6227G>A (p.G2076D), in COL7A1 in all affected individuals. Delineation of the translocation breakpoints showed that the ERBB2IP (erbb2 interacting protein or Erbin) gene is disrupted in 5q13.1 and GPC6 in 13q32. GPC6 encodes glypican 6 belonging to a family of cell surface heparan sulfate proteoglycans. The binding partners of Erbin, BP230 (BPAG1) and the integrin β4 subunit, both involved in hemidesmosome (HD) function, and the presence of Erbin in HD suggested that it plays a role in establishment and maintenance of cell-basement membrane adhesions. However, loss of function of one ERBB2IP copy or expression of a putative novel ERBB2IP fusion protein did not apparently modulate the DEB phenotype in both translocation patients. Nonetheless, one cannot yet exclude that ERBB2IP is a candidate for human blistering disorders such as epidermolysis bullosa

Epidermolysis Bullosa Simplex Associated with Severe Mucous Membrane Involvement and Novel Mutations in the Plectin Gene

We report a novel case of epidermolysis bullosa simplex with severe mucous membrane involvement and mutations in the plectin gene (PLEC1). The patient suffered from extensive blistering of the skin and oral and laryngeal mucous membranes. Electron microscopy of a lesional skin biopsy showed cleft formation within the basal cell layer of the epidermis. Antigen mapping displayed entirely negative staining for plectin, a large (>500 kDa) multifunctional adhesion protein present in hemidesmosomes of the basal keratinocytes. Mutation analysis revealed compound heterozygous, previously undisclosed nonsense mutations, Q1713X and R2351X, of paternal and maternal origin, respectively, within exon 32 of PLEC1. Based on earlier reports, plectin deficiency is associated with late onset muscular dystrophy in patients with epidermolysis bullosa. No signs of muscle weakness have been observed during the 4 y follow-up of our patient. This case illustrates the fact that molecular pathological analyses have prognostic implications in identification and evaluation of patients who appear to be at risk for development of muscular dystrophy later in life

Compound Heterozygosity for a Recessive Glycine Substitution and a Splice Site Mutation in the COL7A1 Gene Causes an Unusually Mild Form of Localized Recessive Dystrophic Epidermolysis Bullosa

Type VII collagen is the major component of anchoring fibrils, adhesion structures of stratified epithelia that span the basement membrane region and papillary dermis. Mutations in the gene COL7A1 encoding type VII collagen cause dystrophic epidermolysis bullosa, a clinically heterogeneous autosomal dominant or recessive blistering disorder of the skin and mucous membranes. In this report, we investigate three siblings affected by an unusually mild form of localized recessive dystrophic epidermolysis bullosa who were shown to be compound heterozygotes for novel mutations affecting COL7A1. The maternally inherited mutation is a G→C transversion that converts a codon for glycine to a codon for arginine (G1347R). The paternal mutation is a neutral G→A transition at the last base of exon 70 (5820G→A) that alters the correct splicing of COL7A1 pre-mRNA, giving rise to an aberrant mRNA carrying the in-frame skipping of exon 70 in addition to the full-length RNA transcript carrying the G→A substitution. Consistent with the normal levels of COL7A1 mRNA transcripts detected by northern analysis, immunoblotting and immunofluorescence studies evidenced that the patient keratinocytes synthesize and secrete normal amounts of stable type VII collagen, which is correctly deposited at the dermal–epidermal junction. In addition, mutated type VII collagen molecules assemble to form numerous, normally shaped anchoring fibrils, as shown by electron microscopic examination. The combination of a recessive glycine substitution with a splice site mutation that permits partially correct splicing therefore leads to a normal expression of mutated type VII collagen molecules with marginally altered biologic activity, and to the extremely mild phenotype observed in our patients

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms

Amlexanox Enhances Premature Termination Codon Read-Through in COL7A1 and Expression of Full Length Type VII Collagen: Potential Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare monogenic blistering disorder caused by the lack of functional type VII collagen, leading to skin fragility and subsequent trauma-induced separation of the epidermis from the underlying dermis. A total of 46% of patients with RDEB harbor at least one premature termination codon (PTC) mutation in COL7A1, and previous studies have shown that aminoglycosides are able to overcome RDEB PTC mutations by inducing read-through and incorporation of an amino acid at the PTC site. However, aminoglycoside toxicity will likely prevent widespread clinical application. Here the FDA-approved drug amlexanox was tested for its ability to read-through PTC mutations in cells derived from patients with RDEB. Eight of 12 different PTC alleles responded to treatment and produced full length protein, in some cases more than 50% relative to normal controls. Read-through type VII collagen was readily detectable in cell culture media and also localized to the dermal-epidermal junction in organotypic skin culture. Amlexanox increased COL7A1 transcript and the phosphorylation of UPF-1, an RNA helicase associated with nonsense-mediated mRNA decay, suggesting that amlexanox inhibits nonsense-mediated mRNA decay in cells from patients with RDEB that respond to read-through treatment. This preclinical study demonstrates the potential of repurposing amlexanox for the treatment of patients with RDEB harboring PTC mutation in COL7A1

Development of an ELISA for sensitive and specific detection of IgA autoantibodies against BP180 in pemphigoid diseases

<p>Abstract</p> <p>Background</p> <p>Pemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids.</p> <p>Methods</p> <p>We expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis.</p> <p>Results</p> <p>The optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n = 105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n = 31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value.</p> <p>Conclusions</p> <p>We developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients with pemphigoids. This immunoassay should prove a useful tool for clinical and translational research and should essentially improve the diagnosis and disease monitoring of patients with IgA pemphigoid. Moreover, our findings strongly suggest that IgA pemphigoid and IgG bullous pemphigoid represent two ends of the clinical spectrum of an immunological loss of tolerance against components of hemidesmosomes, which is mediated by both IgG and IgA autoantibodies.</p