Splicing biomarkers of disease severity in myotonic dystrophy

Authors

  • Masayuki Nakamori MD, PhD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    2. Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY
    Current affiliation:
    1. Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
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  • Krzysztof Sobczak PhD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    2. Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY
    Current affiliation:
    1. Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland
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  • Araya Puwanant MD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Steve Welle PhD,

    1. Department of Medicine, University of Rochester Medical Center, Rochester, NY
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  • Katy Eichinger DPT,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Shree Pandya DPT,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Jeannne Dekdebrun MS,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Chad R. Heatwole MD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Michael P. McDermott PhD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    2. Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY
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  • Tian Chen MA,

    1. Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY
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  • Melissa Cline PhD,

    1. RNA Center, Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, Santa Cruz, CA
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  • Rabi Tawil MD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Robert J. Osborne PhD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    Current affiliation:
    1. Population Genetics Technologies, Babraham Institute, Babraham, Cambridgeshire, United Kingdom
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  • Thurman M. Wheeler MD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    2. Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY
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  • Maurice S. Swanson PhD,

    1. Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, FL
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  • Richard T. Moxley III MD,

    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
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  • Charles A. Thornton MD

    Corresponding author
    1. Department of Neurology, University of Rochester Medical Center, Rochester, NY
    2. Center for Neural Development and Disease, University of Rochester Medical Center, Rochester, NY
    3. Center for RNA Biology, University of Rochester Medical Center, Rochester, NY
    • Address correspondence to Dr Thornton, Department of Neurology, Box 673, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail: charles_thornton@urmc.rochester.edu

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Abstract

Objective

To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Methods

In a discovery cohort, we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects, we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides to reduce levels of toxic RNA.

Results

Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue.

Interpretation

Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy. Ann Neurol 2013;74:862–872

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