SPLICING MUTATIONS IN DMD BMD DETECTED BY RT-PCR/PTT - DETECTION OF A19AA INSERTION IN THE CYSTEINE-RICH DOMAIN OF DYSTROPHIN COMPATIBLE WITH BMD/

We have used an RNA based mutation detection method to screen the tota l coding region of the dystrophin gene of a Duchenne and a Becker musc ular dystrophy patient in whom DNA based mutation detection methods ha ve so far failed to detect mutations. By RT-PCR and the protein trunca tion test (PTT) we could identify point mutations in both cases. DMD p atient DL184.3 has a T-->A mutation in intron 59 at position -9, creat ing a novel splice acceptor site for exon 60. As a result seven intron ic bases are spliced into the mRNA, causing a frameshift and premature translation termination 20 codons downstream. Since this patient had died and only fibroblasts were available, we applied MyoD induced myod ifferentiation of stored fibroblasts to enhance muscle specific gene e xpression. With the results of this mutation analysis, prenatal diagno sis could subsequently be performed in this family. BMD patient BL207. 1 carries a G-->C mutation at position +5 of intron 64, disrupting the splice donor consensus sequence and activating a cryptic splice donor site 57 bp downstream. The inclusion of these 57 intronic bases in th e mRNA leaves the reading frame open and results in the insertion of 1 9 amino acids into the cysteine rich domain of dystrophin. Interesting ly, this insertion in a part of the dystrophin considered to interact with the dystrophin binding complex of the sarcolemma is apparently co mpatible with mild BMD-like clinical features. Both mutations reported are Netherlands missed by analysis of multiplex PCR products designed for deletion screening of the coding region. Extrapolation from exist ing point mutation detection efficiencies by DNA and RNA based methods emphasises that RNA based methods are more sensitive and that most of the remaining undetected mutations may affect splice or branch sites or create cryptic splice sites.