The Molecular Basis of Dystrophic Epidermolysis Bullosa: Mutation Detection and Study of Clinical, Biochemical and Molecular Findings in 29 Patients

Dystrophic epidermolysis bullosa (DEB) is a hereditary skin disorder characterized by traumainduced blistering. It is caused by mutations in the collagen VII gene, COL7A1, which consists of 118 small exons. Molecular diagnostics in DEB remain complex due to the gene structure, large variety of mutations, high rate of novel mutations, and the heterogeneity of phenotypes. Using a highly sensitive and efficient strategy for COL7A1 mutation analysis with direct automated DNA sequencing and the implementation of software tools we disclosed mutations in 29 DEB patients, including 18 novel mutations and the first de novo mutation in recessive DEB. Genotype-phenotype correlations were assessed with RT-PCR, immunochemical collagen VII protein analysis, and collagen triple helix stability assays using limited proteinase digestion and temperature gradients. In a very rare DEB form with solely mucosal involvement and no skin blistering, the glycine substitution G2689R led to decreased thermal stability of collagen VII. The mutation E2059G did not influence thermal stability but resulted in reduction of collagen VII levels, and mild clinical affection. Elucidation of the clinical, genetic and biological background of 29 DEB patients contributes to the EB mutation database, the understanding of the mechanisms underlying DEB and lays a basis for novel therapeutic approaches.