Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe.

Abstract

Recent molecular studies have shown that in a patient with Duchenne muscular dystrophy (DMD) Kobe, the size of exon 19 of the dystrophin gene was reduced to 36 bp due to the deletion of 52 bp out of 88 bp of the exon. The consensus sequences at the 5' and 3' splice sites of exon 19 were unaltered (Matsuo, M., et al. 1990. Biochem. Biophys. Res. Commun. 170:963-967). To further elucidate the molecular nature of the defect, we examined the primary structure of cytoplasmic dystrophin mRNA of the DMD Kobe patient across the junctions of exons 18, 19, and 20 by gel electrophoresis and sequencing of polymerase chain reaction-amplified cDNA. The mRNA coding for dystrophin was reverse transcribed using random primers, and the cDNA was then enzymatically amplified in vitro. The targeted fragment was smaller than expected from the genomic DNA analysis. By sequencing of the amplified product, we found that exon 18 was joined directly to exon 20, so that exon 19 was completely absent, suggesting that this exon was skipped during processing of the dystrophin mRNA precursor. All other bases in the amplified product were unaltered. Therefore, the data strongly suggest that the internal exon deletion generates an abnormally spliced mRNA in which the sequence of exon 18 is joined to the sequence of exon 20. We propose that the deletion is responsible for abnormal processing of the DMD Kobe allele. This finding has important implications regarding the determinants of a functional splice site. (J. Clin.

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.…”

Twenty seven nucleotide deletion within exon 11 of the erythrocyte band 3 gene in Indonesian ovalocytosis

“…Only one band corresponding to 175 bp was visualized after amplification of normal DNA (lane 1) while the Indonesian ovalocytosis DNA gave this product as well as the smaller 148 bp product. The PCR was carried out as described before (Matsuo et al, 1991). The amplified DNA fragment was separated by 3 % agarose gel electrophoresis and photographed after ethidium bromide staining.…”

Le saut d’exon thérapeutique : un espoir pour les dystrophinopathies

“…Ces sauts d'exon peuvent être induits par des oligonucléotides antisens (modifiés pour résister aux RNases), complémen-taires de séquences-clés qui président à l'épissage du transcrit primaire. Dès 1991, M. Matsuo [3] avait insisté sur l'importance des phénomènes d'épis-sage dans le déterminisme et la théra-peutique des dystrophinopathies. Plus récemment, d'autres groupes se sont engagés dans la voie de l'épissothéra-pie, comme alternative à la thérapie génique classique qui n'a pas encore remporté les succès escomptés [4][5][6].…”