Multiple exon skipping and RNA circularisation contribute to the severe phenotypic expression of exon 5 dystrophin deletion

The “frame rule”, linking protein expression and clinical severity to disruption of the coding frame, represents the largely accepted mechanism explaining genotype-phenotype correlations in dystrophinopathies. Nevertheless, exceptions to this model account for at least 8% of patients with deletions and duplications in the dystrophin gene. We have defined the intronic breakpoint regions in two patients with Duchenne muscular dystrophy carrying the in frame isolated deletion of dystrophin exon 5, representing a known example of “exception to the rule” dystrophin mutation. Transcription analysis in skeletal muscle from one patient showed a complex RNA configuration, combining an unfavourable exon skipping event (involving exon 6 and leading to an out of frame transcript) with the production of scrambled, circular RNA molecules. Circularisation of RNA specifically involved the in frame transcripts (retaining exon 6) with the consequence of depletion of the functional messenger. We also documented abundant in frame exon 9 skipping. This peculiar splicing behaviour leading to a defect in the in frame messenger RNA and in critical protein domain, might represent the pathogenic background underlying the severe clinical impact of the rare exon 5 deletion. The circular molecule formation focuses attention on the role that RNA scrambling might have in contributing to the clinical severity in dystrophin deletions.