Cryptic splice activation but not exon skipping is observed in minigene assays of dystrophin c.9361+1G>A mutation identified by NGS

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Next-generation sequencing (NGS) discloses nucleotide changes in the genome. Mutations at splicing regulatory elements are expected to cause splicing errors, such as exon skipping, cryptic splice site activation, partial exon loss or intron retention. In dystrophinopathy patients, prediction of splicing outcomes is essential to determine the phenotype: either severe Duchenne or mild Becker muscular dystrophy, based on the reading frame rule. In a Vietnamese patient, NGS identified a c.9361+1G>A mutation in the dystrophin gene and an additional DNA variation of A>G at +117 bases in intron 64. To ascertain the consequences of these DNA changes on dystrophin splicing, minigene constructs were prepared inserting dystrophin exon 64 plus various lengths of intron 64. Exon 64 skipping was observed in the minigene construct with 160 nucleotide (nt) of intron 64 sequence with both c.9361+1A and +117G. In contrast, minigene constructs with larger flanking intronic domains resulted in cryptic splice site activation rather than exon skipping. Meanwhile, the cryptic splice site activation was induced even in +117G when intron 64 was elongated to 272 nt and longer. It was expected that cryptic splice site activation is an in vivo splicing outcome.

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This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI; 24390267 and 26860803), a Research Grant for Research on Psychiatric and Neurological Diseases and Mental Health from Japan Agency for Medical Research and Development, AMED, and an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (NCNP). We are grateful for technical assistance from Mina Murakami.

Ethical standards

The mutation study was approved by ethical committees of National Hospital of Pediatrics, Kobe University and Kobe Gakuin University and the mutation analysis of the dystrophin gene was carried out after obtaining informed consent from the parents of the patient.

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Correspondence to Masafumi Matsuo.

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Competing interests

MM is an advisor for JCR Pharma, Japan and Daiichi Sankyo, Japan. The remaining authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Journal of Human Genetics website

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