Abstract
We report on two unprecedented cases of pseudoexon activation in the DMD gene resulting from pure intronic double-deletion events that possibly involve microhomology-mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological pseudoexon inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387-bp pseudoexon was originated from an inverted ∼57kb genomic region of intron 44 flanked by two deleted ∼52kb and ∼1kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166-bp pseudoexon located in a very close proximity (134 bp) to exon 57. The key role of the del 1 in pseudoexon activation was established by using splicing reporter minigenes. However the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the pseudoexon and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene that emphasizes the potential role of underdetected intronic rearrangements in human diseases.