Point mutations in the dystrophin gene: evidence for frequent use of cryptic splice sites as a result of splicing defects

Abstract

Ten different mutations have been identified in patients with Becker (n = 1) or Duchenne (n = 9) muscular dystrophy using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and protein truncation test (PTT) analysis. Seven mutations had not been reported previously, and these consist in three nonsense mutations (Q2522X, E2726X, R3381X), three frameshifting deletions (3686-3687delGT, 5126delA, 5759delC), and four splicing defects of which the effects on the muscle dystrophin mRNA transcripts have been analyzed. In one case, a 3' splice-site mutation (IVS74-2A-->G) resulted in a complex pattern of exon skipping involving exons of the C-terminal domain. In the three other cases, nucleotide substitutions in splice donor (IVS26+2T-->A, IVS65+1G-->A) or acceptor (IVS8-15A-->G) recognition sequences led to the use of cryptic splice sites, with consequent insertions of intronic sequences in the processed mRNA. Up to 34% (70/203) of the point mutations reported to date in the dystrophin database (http://www.dmd.nl) affect splice sites of the dystrophin gene. However, altered mRNA splicing has been confirmed experimentally in only 23% of cases (16/70). Combined with PTT, the transcript analysis protocol defined in this study permits direct determination of the impact of intronic variations on the structure of dystrophin mRNA and of the resulting consequences on the translational reading frame. We present evidence for a frequent use of cryptic splice sites as a result of splicing defects.