Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.