The inherited mechanobullous disease, dystrophic epidermolysis bullosa, is caused by type VII collagen gene (COL7A1) mutations. We studied six unrelated patients with a distinct clinical subtype of this disease, epidermolysis bullosa pruriginosa, characterized by pruritus, excoriated prurigo nodules, and skin fragility. Mutation analysis using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis and direct nucleotide sequencing demonstrated pathogenetic COL7A1 mutations in each case. Four patients had a glycine substitution mutation on one COL7A1 allele (G1791E, G2242R, G2369S, and G2713R), a fifth was a compound heterozygote for a splice site mutation (5532 + 1G-to-A) and a single base pair deletion (7786delG), and a sixth patient was heterozygous for an out-of-frame deletion mutation (6863del16). This study shows that the molecular pathology in patients with the distinctive clinical features of epidermolysis bullosa pruriginosa is heterogeneous and suggests that other factors, in addition to the inherent COL7A1 mutation(s), may be responsible for an epidermolysis bullosa pruriginosa phenotype.

Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation test that amplified the entire coding region in only four overlapping nested reverse transcriptase-polymerase chain reaction assays. These fragments were transcribed and translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessive dystrophic epidermolysis bullosa yielding a detection sensitivity of 58%. The second strategy was a DNA-based fluorescent chemical cleavage of mismatch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase chain reaction assays. Mismatches, formed between patient and control DNA, were identified using chemical modification and cleavage of the DNA. We have compared fl-CCM with conformational sensitive gel electrophoresis by screening a total of 50 dominant and recessive dystrophic epidermolysis bullosa patients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi2-test). Using a combination of the three techniques we have screened 93 dystrophic epidermolysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comparing all three approaches, we believe that no single method is consistently better than the others, but that the fl-CCM procedure is a sensitive, semiautomated, high throughput system that can be recommended for COL7A1 mutation detection.

In the absence of a positive family history, it is often difficult to determine whether a single case of mild-to-moderately severe dystrophic epidermolysis bullosa (DEB) represents autosomal recessive or de novo dominant disease. Recent molecular analyses of the type VII collagen gene, COL7A1, have established that the vast majority of such cases are recessive in nature. Nevertheless, a small number of de novo dominant patients have been documented. In this report, we describe three further examples of de novo dominant disease. In each case the COL7A1 mutation comprised the same glycine substitution, G2043R. This mutation has previously been reported in both dominant DEB pedigrees and as a de novo phenomenon and is the most common COL7A1 mutation in dominant DEB throughout the world. These cases emphasize the importance of molecular analysis in providing accurate genetic counselling in this genodermatosis.

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). In this study, we determined the molecular basis of autosomal recessive DEB in a 19-year-old Hispanic Mexican woman by PCR amplification of genomic DNA, heteroduplex analysis, and automated sequencing of heteroduplex bandshifts. This approach revealed a homozygous frameshift mutation, 2470insG, in exon 19 of COL7A1 and resulted in attenuated basement membrane zone expression of type VII collagen, a reduced number of anchoring fibrils at the dermal-epidermal junction, and a sub-lamina densa level of blister formation. Clinically, the patient had widespread trauma-induced skin fragility and complete loss of the nails, but had less pseudosyndactyly of the fingers and toes and milder mucosal involvement compared to most patients with the generalized form of this genodermatosis. We also screened 7 other Hispanic-Mexican patients with recessive DEB, none of whom were known to be related to this individual, for the mutation 2470insG using heteroduplex analysis and direct sequencing and detected this mutation on 7/14 alleles. Haplotype analysis using intragenic COL7A1 and flanking polymorphisms and microsatellite markers revealed that all the mutant alleles had arisen on similar allelic backgrounds, consistent with propagation of a common Hispanic Mexican ancestral haplotype. In view of the high allelic frequency of the mutation 2470insG in the patients studied, we recommend initial screening for this mutation when attempting to identify the molecular pathology of recessive DEB in Hispanic Mexican patients.