Comparative mutation detection screening of the type VII collagen gene (COL7A1) using the protein truncation test, fluorescent chemical cleavage of mismatch, and conformation sensitive gel electrophoresis

Mutations in the type VII collagen gene, COL7A1, give rise to the blisterin g skin disease, dystrophic epidermolysis bullosa, We have developed two new mutation detection strategies for the screening of COL7A1 mutations in pat ients with dystrophic epidermolysis bullosa and compared them with an estab lished protocol using conformational sensitive gel electrophoresis, The fir st strategy consisted of an RNA based protein truncation test that amplifie d the entire coding region in only four overlapping nested reverse transcri ptase-polymerase chain reaction assays. These fragments were transcribed an d translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylam ide gel electrophoresis, We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessi ve dystrophic epidermolysis bullosa yielding a detection sensitivity of 58% , The second strategy was a DNA-based fluorescent chemical cleavage of mism atch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase cha in reaction assays. Mismatches, formed between patient and control DNA, wer e identified using chemical modification and cleavage of the DNA, We have c ompared fl-CCM with conformational sensitive gel electrophoresis by screeni ng a total of 50 dominant and recessive dystrophic epidermolysis bullosa pa tients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi(2)-test). Using a combination of the three techniques we have screened 93 dystrophic epider molysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comp aring all three approaches, we believe that no single method is consistentl y better than the others, but that the fl-CCM procedure is a sensitive, sem iautomated, high throughput system that can be recommended for COL7A1 mutat ion detection.