A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy

• Published in: Journal of human genetics Vol. 59; no. 8; pp. 423 - 429
• Main Authors: Dwianingsih, Ery Kus, Malueka, Rusdy Ghazali, Nishida, Atsushi, Itoh, Kyoko, Lee, Tomoko, Yagi, Mariko, Iijima, Kazumoto, Takeshima, Yasuhiro, Matsuo, Masafumi
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.08.2014
• Subjects: Antisense oligonucleotides; Base Sequence; Child; Doublesex gene; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Dystrophin - metabolism; Exons; Exons - genetics; Frameshift Mutation; Gene deletion; Humans; Immunohistochemistry; Immunomodulation; Male; Molecular Sequence Data; Muscle, Skeletal - metabolism; Muscular Dystrophy, Duchenne - genetics; Mutation; Nucleotide Motifs; Oligonucleotides, Antisense; Phenotype; Real-Time Polymerase Chain Reaction; Regulatory sequences; Reverse transcription; RNA Precursors - genetics; RNA Splicing - genetics; RNA, Messenger - genetics; Sequence Analysis, DNA; Sequence Deletion; Silencer Elements, Transcriptional - genetics; Splicing
• ISSN: 1434-5161; 1435-232X
• DOI: 10.1038/jhg.2014.36

Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease, is mostly caused by exon deletion mutations in the DMD gene. The reading frame rule explains that out-of-frame deletions lead to muscle dystrophin deficiency in DMD. In outliers to this rule, deletion junction sequences have never previously been explored as splicing modulators. In a Japanese case, we identified a single exon 45 deletion in the patient's DMD gene, indicating out-of-frame mutation. However, immunohistochemical examination disclosed weak dystrophin signals in his muscle. Reverse transcription-PCR amplification of DMD exons 42 to 47 revealed a major normally spliced product with exon 45 deletion and an additional in-frame product with deletion of both exons 44 and 45, indicating upstream exon 44 skipping. We considered the latter to underlie the observed dystrophin expression. Remarkably, the junction sequence cloned by PCR walking abolished the splicing enhancer activity of the upstream intron in a chimeric doublesex gene pre-mRNA in vitro splicing. Furthermore, antisense oligonucleotides directed against the junction site counteracted this effect. These indicated that the junction sequence was a splicing silencer that induced upstream exon 44 skipping. It was strongly suggested that creation of splicing regulator is a modifier of dystrophinopathy.