Background and purpose: Generalized recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited disease, in which patients suffer from blistering and scarring of the skin and mucous membranes after minor mechanical trauma. Tight genetic linkage has been established to the type VII collagen gene (COL7A1) at 3p21. The purpose of this study was to identify mutations in COL7A1 in one Taiwanese pedigree with generalized RDEB.
Methods: Genomic DNA was used as the template for polymerase chain reaction (PCR) amplification of all 118 COL7A1 exons and the flanking splice junctions. PCR was followed by heteroduplex analysis of the products by single-strand conformation polymorphism (SSCP) studies, and direct nucleotide sequencing was used to search for mutations, which were verified by restriction endonuclease digestion.
Results: We identified a homozygous intronic splice-site at the +1 position of intron 5 (682 + 1G-->A) of COL7A1 in the affected individual. His parents, who were cousins, were not affected by this disease. The mother was heterozygous for the mutation; the father had died before the study, of unrelated causes. This mutation results in a frameshift and downstream stop codons on both alleles, indicating an absence of functional protein. Restriction endonuclease BspHI can be used to verify this mutation and screen other members in the same family.
Conclusions: These molecular findings offer a genetic explanation for the skin fragility in this Taiwanese patient with RDEB. The immediate benefits gained by elucidating mutations in family members include the ability to assess whether they are carriers of this disease and the ability to use this DNA-based method for prenatal testing in subsequent pregnancies.