Three homozygous PTC mutations in the collagen type VII gene of patients affected by recessive dystrophic epidermolysis bullosa: Analysis of transcript levels in dermal fibroblasts

Revertant Mosaicism Due to a Second-Site Mutation in COL7A1 in a Patient with Recessive Dystrophic Epidermolysis Bullosa

“…The promoter region and all 118 exons and their flanking intronic COL7A1 sequences were amplified by touchdown PCR (Gardella et al, 1999(Gardella et al, , 2000. In exon 80, a homozygous insertion of a cytosine nucleotide was identified at position 6,527.…”

Different phenotypes in recessive dystrophic epidermolysis bullosa patients sharing the same mutation in compound heterozygosity with two novel mutations in the type VII collagen gene

“…The search for mutations was performed on amplified COLVII mRNA by heteroduplex analysis. For this purpose, 3 µg of total RNA purified from in vitro cultured fibroblasts were retrotranscribed and the cDNA was amplified with several sets of primers covering the full‐length COLVII cDNA sequence, as previously described 9 . The reverse transcriptase–polymerase chain products (RT–PCR) were run on mutation detection enhancement (MDE) gels (FMC Bioproducts, Rockland, ME, U.S.A.) and the products showing heteroduplex bands were subjected to direct automated sequencing using the Thermo Sequenase dye terminator cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.) and an ABI 373 A sequencer (PE Biosystems , Foster City, CA, U.S.A.).…”

“…For this purpose, 3 lg of total RNA purified from in vitro cultured fibroblasts were retrotranscribed and the cDNA was amplified with several sets of primers covering the full-length COLVII cDNA sequence, as previously described. 9 The reverse transcriptase-polymerase chain products (RT-PCR) were run on mutation detection enhancement (MDE) gels (FMC Bioproducts, Rockland, ME, U.S.A.) and the products showing heteroduplex bands were subjected to direct automated sequencing using the Thermo Sequenase dye terminator cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A.) and an ABI 373 A sequencer (PE Biosystems, Foster City, CA, U.S.A.). The sets of primers used for the amplification of the cDNA regions containing the mutations were: 5¢-GACAGAGGCGGGG-GTCCTAGCT-3¢ (exon 1) and 5¢-CCCGTCTGTGATCA-GGATGCAG-3¢ (exon 4); GGTGACTCAGGGCCTCCTG-GAG-3¢ (exon 51) and 5¢-CTTTTCTCCTGCTGGGCC-TCGG-3¢ (exon 60); 5¢-AGTTGGAGAGAAAGGTGAC-GAGG-3¢ (exon 52) and 5¢-GTCACCCTTGGGTCCAGAT-GATC-3¢ (exon 55); 5¢-CTGGTCTGGCCCTTGGGG-AGAG-3¢ (exon 73) and 5¢-GGCCTCTTGGACCCTGCA-GACC-3¢ (exon 80).…”

Characterization of mutations leading to recessive dystrophic epidermolysis bullosa and Marfan syndrome in a single patient

“…8 The RT±PCR products were screened by heteroduplex analysis on MDE gels (FMC). 8 The RT±PCR products were screened by heteroduplex analysis on MDE gels (FMC).…”

“…In this report we have investigated an Italian child affected with recessive DEB (RDEB) and demonstrated that he was homozygous for the mutation R226X in the type VII collagen gene (COL7A1), leading to absence of type VII collagen at the dermal±epidermal junction. 8 The RT±PCR products were screened by heteroduplex analysis on MDE gels (FMC). Analysis of this gene showed that the RDEB patient and his father were both heterozygous for a novel FBN1 mutation, C1971Y.…”