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 hemid esmosomal protein encoded by COL17A1. This report documents a novel sp lice-site mutation in COL17A1 in a patient with generalized atrophic b enign epidermolysis bullosa, and applies a new methodology to define a nd characterize the resulting mRNA splice variants. Mutational analysi s 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 COL1 7A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effects of the splice-site mutation on splicing of COL17A1 transc ripts were determined using reverse transcriptase polymerase chain rea ction of total RNA from keratinocytes incubated for 2.5 h in the prese nce or absence of 10 mu g cycloheximide per ml. Using this approach, a n abnormally spliced transcript was identified that contains an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splic e variants, including one derived from the skipping of exon 32, were a lso identified. These results indicate the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice -site mutations, because: (i) aberrant splicing often generates a prem ature termination codon, (ii) transcripts with premature termination c odons can occur at low or undetectable levels due to nonsense-mediated mRNA decay, and (iii) the levels of these transcripts can be increase d by cycloheximide.