Pseudoexon Activation Caused by Intronic Double‐deletion Events in the DMD Gene

Abstract

Gross genomic rearrangements (GGRs) including copy number variations are increasingly being recognized as an important source of the “missing heritability” for both monogenic and complex diseases. The functional consequences of GGRs involving coding sequences or splice sites are often self-evident. By contrast, the functional consequences of GGRs involving deep intronic sequences often need to be sought by laborious experimental testing. In this issue, Khelifi et al. (Hum Mutat 32:467–475, 2011) characterized two unusual and complex intronic GGRs in the DMD gene at the nucleotide level through array-CGH and direct genomic sequencing. Both events comprise two deletions within single introns, the intervening sequence in the first event being also inverted. Each event was shown to give rise to pseudoexon activation by RT-PCR analysis using total RNA isolated from frozen muscle biopsy samples from the patients, suggesting pathogenic relevance. The authors performed a series of deletion experiments using a splicing reporter minigene assay, providing compelling evidence that an upstream intronic 592-bp sequence plays a crucial role in repressing pseudoexon activation in the wild-type allele. However, a combination of in silico analysis and experimental validation failed to identify any splicing regulatory elements that could have accounted for the inclusion/exclusion of the 166-bp pseudoexon. In addition, MFold prediction analysis of possible RNA secondary structure involvement in splicing regulation was inconclusive. Other factors such as nucleosome positioning and specific histone modifications may well turn out to play a role in regulating exon recognition. In reporting these two cases of DMD pseudoexon activation resulting from pure intronic double-deletion events, Khelifi et al. remind us that we still have some way to go before we are able to decode all the regulatory elements lurking in our own genome.