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 b een well elucidated, We now report results of in vitro splicing studie s 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 n ucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Pecker muscular dystrophy case, Partial skipping of the ex on resulted in the production of truncated dystrophin mRNA, although t he consensus sequences for splicing at both ends of exon 27 were unalt ered, To determine how E1211X induced exon 27 skipping, the splicing e nhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. Th e mutant sequence containing G3839T abolished splicing enhancer activi ty of the wild-type purine-rich sequence for the upstream intron in th is chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicki ng the purine-rich sequence of exon 27 also showed enhancer activity t hat was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a non sense codon was created by the inserted T residue, This is the first e vidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.