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Ann Clin Transl Neurol. 2019 Mar 3;6(4):642-654. doi: 10.1002/acn3.738. eCollection 2019 Apr.

Correction of pseudoexon splicing caused by a novel intronic dysferlin mutation.

Author information

1
Department of Neurology University of Massachusetts Medical School Worcester Massachusetts.
2
Department of Human Genetics Emory University School of Medicine Atlanta Georgia.
3
Present address: Perkin Elmer Genomics Waltham Massachusetts.
4
Marseille Medical Genetics - Translational Neuromyology Aix-Marseille Univ INSERM MMG Marseille France.
5
Département de Génétique Médicale APHM Hôpital Timone Enfants Marseille France.
6
Northern Molecular Genetics Service Newcastle upon Tyne United Kingdom.
7
Newcastle University John Walton Centre for Muscular Dystrophy Research MRC Centre for Neuromuscular Diseases Institute of Genetic Medicine Newcastle upon Tyne United Kingdom.
8
Department of Neuropediatrics and Muscle Disorders Faculty of Medicine Medical Center-University of Freiburg Freiburg Germany.
9
Centro Nacional de Análisis Genómico (CNAG-CRG) Center for Genomic Regulation Barcelona Institute of Science and Technology (BIST) Barcelona Catalonia Spain.
10
Children's Hospital of Eastern Ontario Research Institute University of Ottawa Ottawa Canada.
11
Division of Neurology Department of Medicine The Ottawa Hospital Ottawa Canada.
12
Jain Foundation, Inc. Seattle Washington.

Abstract

Objective:

Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin-deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD2B), distal myopathy with anterior tibial onset (DMAT)), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions.

Methods:

We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease-causing variant identified. We designed antisense oligonucleotides (AONs) to bypass the effects of this mutation on RNA splicing.

Results:

We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon (PE, we term PE50.1) within the mature dysferlin mRNA. PE50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON-mediated exon skipping to correct the aberrant PE50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression.

Interpretation:

Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON-based exon-skipping strategies.

Conflict of interest statement

J.A.D. and R.H.B. are employed by the University of Massachusetts Medical School and co‐inventors on a patent application for antisense sequences and exon‐skipping technology targeting dysferlinopathies. M.K., N.L. and M.B. are co‐inventors on a patent for dysferlin exon skipping.

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