Cycloheximide facilitates the identification of aberrant transcripts resulting from a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa

Patients with generalized atrophic benign epidermolysis bullosa often show decreased expression of type XVII collagen, a transmembrane hemidesmosomal protein encoded by COL17A1. This report documents a novel generalized atrophic benign epidermolysis bullosa, and applies a new methodology to define and characterize the resulting mRNA splice variants. Mutational analysis of COL17A1 identified a maternally inherited G-to-T transversion at the -1 position of exon 32. This acceptor splice-site mutation led to the formation of aberrant transcripts present at extremely low levels. Based on our recent finding that cycloheximide stabilized mutant COL17A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effect of the splice-site mutation on splicing of COL17A1 transcripts were determined using reverse transcriptase polymerase chain reaction of total RNA from keratinocytes incubated for 2.5 h in the presence or absence of 10 μg cycloheximide per ml. Using this approach, an abnormally spliced transcript was identified that contain an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splice variants, including one derived from the skipping of exon 32, were also identified. These results indicated the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice-site mutations, because: (i) aberrant splicing often generates a premature termination codon, (ii) transcripts with premature termination codons can occur at low or undetectable levels due to nonsense- mediated mRNA decay, and (iii) the levels of these transcripts can be increased by cycloheximide.