Patients with generalized atrophic benign epidermolysis bullosa (GABEB), an inherited subepidermal blistering disease, often have no immunologically detectable bullous pemphigoid antigen 2 (BPAG2) in their epidermal basement membrane. Recently, we analyzed the BPAG2 gene (GenBank no. M91669) in an Austrian family with GABEB and identified a homozygous deletion mutation, 4003delTC, that results in a downstream premature termination codon (PTC). This mutation has now been identified in additional descendants, suggesting transmission of this mutant allele through at least six generations. Screening of four other Austrian GABEB families revealed that affected members were homozygous for 4003delTC in two cases and heterozygous in two others. In the latter, mutational analysis identified two novel nonsense mutations, Q1403X and G803X, that were confirmed by restriction endonuclease digestions. Thus, PTCs on both alleles of BPAG2 are present in all of these GABEB families. Immunoprecipitation and northern blot studies of cultured keratinocytes from homozygous GABEB patients show that 4003delTC results in undetectable levels of BPAG2 protein and mRNA-findings consistent with the process of nonsense-mediated mRNA decay. Incubating keratinocytes with cycloheximide increased BPAG2 mRNA to a level detectable by northern analysis. When the latter was used in reverse transcription-PCR studies, the mutation was demonstrated, suggesting that cycloheximide may allow mutational analysis in cases where low transcript levels have previously thwarted RT-PCR studies. These findings account for the absence of BPAG2 in GABEB patients and attest to the importance of this protein in adhesion of epidermis to epidermal basement membrane.

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

We report the sixth case of a human keratin 14 'knockout' mutation resulting in recessive epidermolysis bullosa simplex (EBS). A novel, homozygous nonsense mutation resulting from a deletion/insertion mutation (744delC/insAG) leads to a premature termination codon in the KRT14 gene (Y248X). The patient suffers from generalized cutaneous blistering since birth, mild nail dystrophy, involvement of mucous membranes and multiple epidermolysis bullosa naevi. The clinical variability noted in K14-deficient EBS patients suggests phenotypic modulation by additional genetic and/or epigenetic factors.

We report a 72-year-old male patient with a nodular malignant melanoma that was associated with focal suprabasal acantholysis (FSA). This phenomenon, which is regarded as an incidental finding by dermatopathologists, may be associated with inflammatory and also neoplastic skin diseases. Haematoxylin and eosin stained sections from an erythematous plaque surrounding the patient's tumour showed FSA, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) on normal human skin, monkey oesophagus and rat urinary bladder were negative. On electron microscopy few desmosomes could be detected in the basal cell layer of the acantholytic areas and there was a nearly complete loss of these structures in the spinous cell layer. Only remnants of cytoplasmic plaques and keratin filaments could be observed in those areas. In contrast, adherens junctions appeared to be well preserved. An enzyme-linked immunosorbent assay (ELISA) using recombinant fusion proteins as antigens did not show circulating autoantibodies against desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). In contrast, immunoblotting revealed autoantibodies directed against keratinocyte antigens with a molecular weight of 85 kDa and 250 kDa, the first band corresponding to the molecular weight of comigrating plakoglobin. Immunoprecipitation with patient serum also revealed a 85-kDa band. We conclude that these autoantibodies, probably in conjunction with cofactors produced by the tumour, could play a part in the pathogenesis of this variant of FSA, for which we propose the term 'localized paraneoplastic pemphigus.'

Epidermolysis bullosa acquisita (EBA) is an autoimmune bullous disease with frequent ocular involvement, but visual loss is rare. In contrast, EBA patients with predominant IgA autoantibodies more frequently develop severe ocular involvement, which tends to be refractory to therapy. We report two patients with 'IgA-EBA' with ocular involvement. Both initially presented with a generalized bullous disease, and direct immunofluorescence microscopy demonstrated IgA in the basement membrane zone of the skin, and in the conjunctiva and cornea of patient 1. On salt-split patient skin, IgA was found predominantly on the dermal side of the artificial split in both patients. Direct immunoelectron microscopy demonstrated IgA below the lamina densa in close association with the anchoring fibrils in both patients. In patient 1, who had a prolonged course of the disease, the skin disorder responded well to treatment with cyclosporin, but the ocular involvement ended in bilateral blindness despite repeated surgical treatment. In patient 2, the blister formation and scarring conjunctivitis was stopped by a combination of prednisolone and colchicine. These patients show that in subepithelial blistering diseases, early delineation of disease nosology is critical to detect subtypes with severe ocular involvement such as 'IgA-EBA'. In addition, colchicine may be a valuable alternative in the treatment of EBA with ocular involvement.