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Severe mental retardation is a rare complication of Duchenne muscular dystrophy (DMD). Here we report that two DMD cases showing severe mental retardation exhibit the same exon skipping event induced by different intron mutations. In the two Japanese DMD patients studied, the complete sequence of exon 66 of the dystrophin gene was found to be absent from the dystrophin mRNA, creating a premature stop codon in exon 67. Novel point mutations at the consensus sequence of the splice donor site of intron 66 (T9857(+2) to C in one case and G9857(+5) to T in the other case) were found to be the cause of complete exon skipping. Remarkably, severe mental retardation cosegregated with an exon 66-skipping event in their families. Furthermore, pachygyria was disclosed by magnetic resonance imaging (MRI) examination of the brain of one case. Our results suggested that exon 66 skipping should be examined in DMD cases with a severe form of mental retardation.

Exon skipping by alternative splicing and circular RNA formation are proposed to be interrelated events. Since multiple patterns of alternative splicing have been demonstrated in both the 5' and 3' regions of the dystrophin gene, the dystrophin transcript in skeletal muscle cells provides a model system in which this idea is tested. Nine circular RNAs that were expected to result from known exon skipping patterns in the 5' region of this gene were in fact identified, but three other circular RNAs expected to result from other known exon skipping reactions in this region could not be detected. The identification of two unexpected circular RNAs led to the discovery of two novel alternative splicing reactions. One circular RNA originating in the 3' region of the gene was identified but it lacked one small sized exon compared with the expected exon structure. Circular RNAs from the 5' region of the dystrophin gene could not be detected in Duchenne muscular dystrophy patients who have deletions of one or more exons in this segment of the gene, even though alternative splicing products were identified. These results showed that circular RNA formation is not necessarily linked to exon skipping and suggest that an undetermined factor regulates circular RNA formation.

The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, comprises 79 exons and is thus the largest known human gene. A full spectrum of splicing of dystrophin transcript has not been elucidated yet. In this study, 6 novel alternative splicing reactions were discovered in the 5' region by amplifying the cDNA corresponding to exons M1 through 18. Two of these novel transcripts maintain the translational reading frame and are presumed to produce truncated dystrophin, while the other four have disrupted reading frames. The physical distance between splice donor and acceptor sites ranged from 250 kb to 800 kb. Furthermore, the same six alternative splicing products were obtained from mouse skeletal muscle cDNA. This indicated that these novel alternative splicing events are conserved in humans and mice.