Premature Termination Codons Are Present on Both Alleles of the Bullous Pemphigoid Antigen 2/Type XVII Collagen Gene in Five Austrian Families with Generalized Atrophic Benign Epidermolysis Bullosa

Abstract

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.

Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a frame-restoring mutation

“…The proband, a 56-year-old woman, is a member of a large Austrian GABEB kindred that includes 5 affected and 5 unaffected siblings in 1 generation, as well as a pedigree that signifies propagation of the mutant allele through at least 6 generations (18,21). All 4 living affected siblings (1 affected having died in infancy because of complications of this inherited blistering disease) are homozygous for a 2-bp deletion in COL17A1, 4003delTC, and show the same homozygous haplotype for 5 intragenic COL17A1 polymorphisms (17,18,22). The proband's skin shows the same extent and character of blistering as that of her affected siblings; i.e., regions of nonfragile skin are not present.…”

“…In the largest GABEB kindred identified to date, affected individuals are homozygous for a 2-bp deletion, 4003delTC, which results in a frameshift and a premature termination codon (PTC) 86 bp downstream (17,18). This mutation results in nonsense-mediated mRNA decay that abolishes synthesis of type XVII collagen and accounts for the absence of this adhesion molecule in the epidermal BM of these patients (18). Interestingly, 1 affected individual in this large kindred showed focal areas of epidermal BM that stained positive for type XVII collagen by IF microscopy (9), suggesting revertant mosaicism as described above.…”

Treatment decision-making for patients with the Herlitz subtype of junctional epidermolysis bullosa

“…5 JEB-nH is caused by less deleterious mutations in the laminin 5 genes or by mutations in the gene encoding another protein, type 17 collagen. 5,6 JEB-H has also been called JEB-letalis, and as this moniker suggests, it is a lethal disease. According to the National Epidermolysis Bullosa Registry (NEBR), as of 1 December 1995, 9/22 (40.9%) enrolled JEB-H patients had died, with nearly 90% of deaths occurring before 1 year of age; and 51/148 (34.5%) enrolled JEB-nH patients had died.…”

Truncated Type XVII Collagen Expression in a Patient with Non-Herlitz Junctional Epidermolysis Bullosa Caused by a Homozygous Splice-Site Mutation

“…Most COL17A1 mutations described to date are nonsense and deletion mutations leading to premature termination of translation and absence of type XVII collagen from the skin (Darling et al, 1997;Floeth et al, 1998;Gatalica et al, 1997;Jonkman et al, 1997;McGrath et al, 1995McGrath et al, , 1996aMcGrath et al, , 1996bScheffer et al, 1997;Schumann et al, 1997;Shimizu et al, 1998 887 acceptor splice-site mutations (Chavanas et al, 1997;Darling et al, 1998;Pulkkinen et al, 1999), one donor splice-site mutation (Pulkkinen et al, 1999), and four missense mutations (Floeth et al, 1998;McGrath et al, 1996a;Schumann et al, 1997;Tasanen et al, 2000) have been reported. In only one (Schumann et al, 1997) of the patients could type XVII collagen be immunologically detected in skin or cultured keratinocytes.…”