Heterogous dystrophin mRNA produced by a novel splice acceptor site mutation in intermediate dystrophinopathy.

Abstract

The molecular background of an intermediate type of dystrophinopathy [Duchenne and Becker muscular dystrophy (DMD/BMD)] remains to be clarified, and out-of -frame and in-frame mutations of the dystrophin gene are shown to be causes of DMD and BMD, respectively. In a boy with this disease, dystrophin mRNA extracted from lymphocytes and muscle were analyzed both qualitatively and quantitatively using reverse transcription PCR. Three different dystrophin mRNA were found to be produced via the use of three cryptic splice acceptor sites resulting from a novel point mutation of 2831-2A>G at the conserved splice acceptor site of intron 20. One of three mRNA showed an insertion of six nucleotides of intron 20 between exons 20 and 21 (dys+6) that encoded two novel amino acids in the rod domain of dystrophin. Two other mRNA species showed an insertion of seven nucleotides of intron 20 between exons 20 and 21 (dys+7) or a seven-nucleotide deletion in exon 21 (dys-7). Quantitative analysis of each dystrophin mRNA expressed in the boy's skeletal muscle disclosed that around 95% and 5% of dystrophin mRNAs were dys-7 and dys+6, respectively, whereas these two mRNA were almost equally expressed in lymphocytes. It is suggested that production of a small fraction of in-frame mRNA in muscle explains the molecular background of the intermediate type of dystrophinopathy in the index case. This finding underlines the potential of genetic therapeutic strategies aimed to modify mRNA in DMD to generate a much milder disease.