Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

Mariko Okubo,Masafumi Matsuo,Ichizo Nishino,Hirofumi Komaki,Harumasa Nakamura,Shinichiro Hayashi,Satoru Noguchi

doi:10.1007/s00439-019-02107-4

Abstract

Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.

Highlights

Dystrophinopathy is a group of progressive neuromuscular diseases caused by mutations in Duchenne muscular dystrophy (DMD) (OMIM 300,377) located at Xp21.2
Patient phenotypes were classified into three subgroups, DMD, intermediate muscular dystrophy (IMD), and Becker muscular dystrophy (BMD), based on the age at loss of ambulation [DMD < 13 years, 13 years ≤ IMD ≤ 16 years, and 16 years < BMD (TufferyGiraud et al 2009)] and the results of dystrophin immunostaining in muscles
We included 16 DMD patients with nonsense or frameshift mutations in these exons whose dystrophin was confirmed to be absent on muscle biopsy

Summary

Introduction

Dystrophinopathy is a group of progressive neuromuscular diseases caused by mutations in DMD (OMIM 300,377) located at Xp21.2. It has been proposed that the effects of these single point mutations are dependent on weak intrinsic exon definition elements (Kevin et al 2011). These exon skipping events by nonsense mutations are not specific to skeletal muscle, and they will be responsible for milder phenotype of skeletal muscle and other organs in the patients. The minigene assay could be performed in HeLa cells as in previous reports (Nishida et al 2011; Zhu et al 2019) To understand these events and to evaluate the nonsense/frameshift mutations on the skipping rate of the corresponding exon, we analyzed exon skipping caused by the nonsense/frameshift mutations from a Japanese large cohort in artificial minigene experiments (Okubo et al 2016, 2017)

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