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.

Abstract

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. ( J. Clin. Invest. 1997. 100:2204-2210.) Key words: dilated cardiomyopathy • reading frame • in vitro splicing • pre-mRNA • polypurine

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

“…In some cases, muscle biopsy was conducted for immunohistochemical examination and dystrophin complementary DNA (cDNA) was analyzed as previously reported. [16][17][18] Informed consent was obtained for molecular analysis and this study was approved by the ethics committees of Kobe University School of Medicine (approval no. 28 in 1998).…”

“…16,17,19,20 In brief, genomic DNA (gDNA) was first analyzed to detect deletion or duplication of one or more exons. If no responsible gross mutation was identified, muscle biopsies were obtained for dystrophin immunostaining to confirm the diagnosis.…”

“…18 Dystrophin mRNA extracted from lymphocytes or muscle tissue was analyzed using reverse transcription PCR. 16,17 Analysis of gDNA Peripheral blood samples were obtained from the patients and their family members. gDNA was isolated using standard phenol-chloroform extraction methods.…”

“…Although nonsense mutations usually cause DMD, three nonsense mutations were identified in the milder, BMD cases (4%) ( Table 1). Two were found to produce in-frame dystrophin mRNA with skipping of the nonsense-containing exon, 17,30 and the other was present in the C-terminal region. 31 …”

Aberrant HS1 molecule in a patient with systemic lupus erythematosus

“…Aberrant splicing is mostly caused by the mutation at the splicing consensus sequence of exon-intron boundary, and, to a lesser extent, by a point mutation within the skipped exon. [33][34][35][52][53][54][55] However, there are several reports of exon skipping without any mutations in the sequence around the skipped exon. [56][57][58][59][60] Besides, a mutation in an intron away from an exon-intron boundary results in a defect in splicing.…”