• A Corrigendum to this article was published on 07 April 2015
  • A Corrigendum to this article was published on 07 May 2015

This article has been updated

Abstract

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.

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Change history

  • 25 August 2014

     In the version of this article initially published online, the nucleotide numbering of the AS (–3 to + 16), B (+17 to +40) and C (+49 to –10) targeted sequences in Figure 3b was incorrect, and thus the schematic of the sequences was incorrectly drawn. The correct numbering is as follows: AS, –3 to + 18; B, +17 to +44; and C, +49 to –8. The correction has no impact on the results of the study or its conclusions. The error has been corrected for all versions of this article.

  • 13 March 2015

     In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been "Fabri"). The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work has been supported by the US National Institutes of Health National Institute of Neurological Disorders and Stroke (R01 NS043264 (K.M.F., M.T.H. and R.B.W.)), the National Institute of General Medical Sciences (R01 GM038277 and R01 GM084177 (D.R.S.)), and CureDuchenne (K.M.F.). N.W. has received fellowship support from the Ohio State University/Nationwide Children's Hospital Muscle Group and the Philippe Foundation. We wish to acknowledge the technical assistance of A. Rutherford, Y. Kaminoh and L. Taylor and are grateful for the reagents provided by P. Sarnow (pRDEF; Stanford University) and G. Morris (hybridoma cell lines for antibodies MANEX1A, MANCHO3 and MANDAG2; MDA Monoclonal Antibody Resource at the Wolfson Centre for Inherited Neuromuscular Disease). The EU BIO-NMD project (no. 241665) provided support to A. Ferlini and C.A.-K.S., and the Duchenne Parent Project Italy (DMD Diagnostics Project) provided support to A. Ferlini. Thanks are also due to C. Rodolico and M. Fabris for their technical support. The Clinical Proteomics Mass Spectrometry core facility at Karolinska University Hospital and Science for Life Laboratory provided assistance in mass spectrometry and data analysis.

Author information

Author notes

    • Nicolas Wein
    •  & Adeline Vulin

    These authors contributed equally to this work.

Affiliations

  1. The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio State University, Columbus, Ohio, USA.

    • Nicolas Wein
    • , Adeline Vulin
    • , Andrew Findlay
    • , Kristin N Heller
    • , Louise R Rodino-Klapac
    •  & Kevin M Flanigan
  2. Section of Microbiology and Medical Genetics, Department of Medical Sciences, University of Ferrara, Ferrara, Italy.

    • Maria S Falzarano
    • , Simona Passarelli
    • , Matteo Bovolenta
    • , Marcella Neri
    • , Francesca Gualandi
    •  & Alessandra Ferlini
  3. Department of Proteomics and Nanobiotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

    • Christina Al-Khalili Szigyarto
    •  & Mathias Uhlén
  4. Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA.

    • Baijayanta Maiti
  5. Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.

    • Baskar Bakthavachalu
    •  & Daniel R Schoenberg
  6. Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA.

    • Baskar Bakthavachalu
    •  & Daniel R Schoenberg
  7. Department of Neuroscience, University of Messina and Centro Clinico Nemo Sud, Messina, Italy.

    • Sonia Messina
    •  & Giuseppe Vita
  8. Bambino Gesù Children's Hospital, Rome, Italy.

    • Chiara Passarelli
  9. Centre for Comparative Genomics, Murdoch University, Perth, Western Australia, Australia.

    • Steve D Wilton
  10. Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA.

    • Louise R Rodino-Klapac
    •  & Kevin M Flanigan
  11. Division of Biomedical Informatics, Department of Computer Science, University of Kentucky Lexington, Kentucky, USA.

    • Lin Yang
  12. Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, Utah, USA.

    • Diane M Dunn
    • , Robert B Weiss
    •  & Michael T Howard
  13. Department of Neurology, The Ohio State University, Columbus, Ohio, USA.

    • Kevin M Flanigan

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Contributions

N.W. performed the immunostaining staining on human and mouse muscle, generated reagents for and performed all luciferase experiments, immortalized and transdifferentiated human cell lines, performed immunoblot and RT-PCR experiments on C2C12 and FibroMyoD cells, and performed RT-PCR on mouse tissues. A.V. generated the Dup2 mouse model, injected all mice and performed all immublotting on mouse tissue and on muscle biopsies from the subjects with Dup2 and frameshift mutations. B.M. generated the master exon 1–5 construct and performed the initial dual luciferase assays. K.N.H. and L.R.R.-K. performed or analyzed the specific force and eccentric contraction–induced damage tests. L.Y. developed and performed the H&E and EBD quantification. A. Ferlini, C.A.-K.S. and M.U. designed and performed analysis of the exon 2–deleted muscle. S.M., G.V. and C.P. clinically characterized the subject with exon 2 deletion, and S.B., M.B., and M.N. performed the genetic characterization of the patient with exon 2 deletion. M.S.F. performed cell and AON studies on a subject with exon 2 deletion. S.D.W. provided critical reagents (AONs) for cell-based experiments. B.B. and D.R.S. designed and performed the formaldehyde RNA electrophoresis and northern blotting. D.M.D. and R.B.W. designed and performed the ribosome profiling experiments. A. Findlay generated all the U7 constructs. N.W., M.T.H. and K.M.F. designed the experiments and analyzed and interpreted the data. N.W., R.B.W. and K.M.F. wrote the manuscript and the compiled the figures, with contributions from A.V., F.G., R.B.W., A. Ferlini and D.R.S.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kevin M Flanigan.

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https://doi.org/10.1038/nm.3628

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