Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

Journal name:
Nature
Volume:
478,
Pages:
127–131
Date published:
DOI:
doi:10.1038/nature10456
Received
Accepted
Published online

Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene1, 2, 3. In FCMD, the SVA insertion occurs in the 3′ untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3′ end of the fukutin coding region, a proximal part of the 3′ UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia4, neutral lipid storage disease5) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known6, 7, 8, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

At a glance

Figures

  1. An SVA retrotransposal insertion induces abnormal splicing in FCMD.
    Figure 1: An SVA retrotransposal insertion induces abnormal splicing in FCMD.

    a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Grey bar, the ratio of RT–PCR product in patients with FCMD relative to the normal control; numbers on the x axis, nucleotide positions of both forward and reverse primers in fukutin. Error bars, s.e.m. b, Long-range PCR using primers flanking the expression-decreasing area (nucleotide position 1,061–5,941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and an 8-kb product in FCMD genomic DNA (filled arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak, probably because VNTR region of SVA is GC-rich (82%). c, Representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is coloured red, and the new stop codon is coloured blue. d, e, Northern blot analysis of fukutin in human lymphoblasts (d) and model mice (e); F, FCMD; N, normal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands in Hp/Hp mice. WT, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Representation of genomic DNA and cDNA in ARH (LDLRAP1, left), NLSDM (PNPLA2, middle) and human (AB627340, right).

  2. Abnormal fukutin protein in FCMD.
    Figure 2: Abnormal fukutin protein in FCMD.

    a–c, Immunoprecipitation analysis of fukutin protein in human lymphoblasts (a), both skeletal muscle and brain tissues from Hp/Hp mice (b) and brain tissue from patients with FCMD (c); filled arrow, abnormal fukutin; N, normal sample; F, sample from patient with FCMD; Hn, Hn/Hn mice; Hp, Hp/Hp mice; PI, pre-immune serum; D, patient with Duchenne muscular dystrophy. d, The subcellular localization of fukutin. Top, normal fukutin; middle, mis-spliced fukutin; bottom, truncated fukutin. Stained with anti-FLAG (left, to detect fukutin), anti-GM130 (middle, Golgi marker, top) and anti-KDEL (endoplasmic reticulum marker, middle and bottom), and merge (right, with DAPI stain). Scale bar, 10μm.

  3. AON cocktail rescues normal fukutin mRNA.
    Figure 3: AON cocktail rescues normal fukutin mRNA.

    a, RT–PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black arrow, a common forward primer located on fukutin coding region; dark grey arrow, a reverse primer to detect the abnormal RT–PCR product (161bp); light grey arrow, the other reverse primer to detect the restored normal RT–PCR product (129bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated Hp/Hp and Hp/− mice. Local injection of AED cocktail into tibialis anterior (n = 3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n = 2) and myotubes (right, n = 2). The y axis shows the percentage recovery of normal mRNA (*P<0.01 by Student’s t-test). TA, tibialis anterior. Error bars, s.e.m.

  4. AON cocktail treatment rescues normal fukutin protein and functional [agr]-DG.
    Figure 4: AON cocktail treatment rescues normal fukutin protein and functional α-DG.

    a, d, Immunoprecipitation analysis of fukutin protein after local treatment with VMO (AED) in FCMD model mice (a) and human FCMD lymphoblasts (d). Arrow, normal fukutin protein. L, left tibialis anterior; R, right tibialis anterior; Dys, negative control. b, c, e, Tibialis anterior muscle after local (b) or systemic (c) treatment with AED and human FCMD lymphoblasts treated with the AED (e) were analysed by western blot using antibodies against α-DG core protein (top panel) and glycosylated α-DG (second), and by a laminin overlay assay (third). Bottom, β-DG (internal control). f, Laminin clustering assay. Left, anti-laminin; middle, anti-glycosylated α-DG; right, merged images. Upper, normal myotubes treated with control VMO; middle, FCMD patient myotubes treated with control VMO; bottom, FCMD patient myotubes treated with AED.

References

  1. Toda, T. et al. Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31–33. Nature Genet. 5, 283286 (1993)
  2. Kobayashi, K. et al. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 394, 388392 (1998)
  3. Watanabe, M. et al. Founder SVA retrotransposal insertion in Fukuyama-type congenital muscular dystrophy and its origin in Japanese and Northeast Asian populations. Am. J. Med. Genet. A. 138, 344348 (2005)
  4. Wilund, K. R. et al. Molecular mechanisms of autosomal recessive hypercholesterolemia. Hum. Mol. Genet. 11, 30193030 (2002)
  5. Akman, H. O. et al. Neutral lipid storage disease with subclinical myopathy due to a retrotransposal insertion in the PNPLA2 gene. Neuromuscul. Disord. 20, 397402 (2010)
  6. Hancks, D. C. et al. Exon-trapping mediated by the human retrotransposon SVA. Genome Res. 19, 19831991 (2009)
  7. Damert, A. et al. 5′-Transducing SVA retrotransposon groups spread efficiently throughout the human genome. Genome Res. 19, 19922008 (2009)
  8. Bantysh, O. B. & Buzdin, A. A. Novel family of human transposable elements formed due to fusion of the first exon of gene MAST2 with retrotransposon SVA. Biochemistry (Mosc.) 74, 13931399 (2009)
  9. Michele, D. E. et al. Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature 418, 417422 (2002)
  10. Barresi, R. & Campbell, K. P. Dystroglycan: from biosynthesis to pathogenesis of human disease. J. Cell Sci. 119, 199207 (2006)
  11. Strichman-Almashanu, L. Z. et al. Retroposed copies of the HMG genes: a window to genome dynamics. Genome Res. 13, 800812 (2003)
  12. Ostertag, E. M. et al. SVA elements are nonautonomous retrotransposons that cause disease in humans. Am. J. Hum. Genet. 73, 14441451 (2003)
  13. Bennett, E. A. et al. Natural genetic variation caused by transposable elements in humans. Genetics 168, 933951 (2004)
  14. Wang, H. et al. SVA elements: a hominid-specific retroposon family. J. Mol. Biol. 354, 9941007 (2005)
  15. Hancks, D. C. et al. Retrotransposition of marked SVA elements by human L1s in cultured cells. Hum. Mol. Genet. 20, 33863400 (2011)
  16. Kanagawa, M. et al. Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy. Hum. Mol. Genet. 18, 621631 (2009)
  17. Hancks, D. C. & Kazazian, H. H., Jr SVA retrotransposons: evolution and genetic instability. Semin. Cancer Biol. 20, 234245 (2010)
  18. Wu, B. et al. Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice. Mol. Ther. 17, 864871 (2009)
  19. Barresi, R. et al. LARGE can functionally bypass α-dystroglycan glycosylation defects in distinct congenital muscular dystrophies. Nature Med. 10, 696703 (2004)
  20. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860921 (2001)
  21. Kazazian, H. H., Jr Mobile elements: drivers of genome evolution. Science 303, 16261632 (2004)
  22. Cordaux, R. & Batzer, M. A. The impact of retrotransposons on human genome evolution. Nature Rev. Genet. 10, 691703 (2009)
  23. Hassoun, H. et al. A novel mobile element inserted in the α spectrin gene: spectrin dayton. A truncated α spectrin associated with hereditary elliptocytosis. J. Clin. Invest. 94, 643648 (1994)
  24. Rohrer, J. et al. Unusual mutations in Btk: an insertion, a duplication, an inversion, and four large deletions. Clin. Immunol. 90, 2837 (1999)
  25. Legoix, P. et al. Molecular characterization of germline NF2 gene rearrangements. Genomics 65, 6266 (2000)
  26. Makino, S. et al. Reduced neuron-specific expression of the TAF1 gene is associated with X-linked dystonia-parkinsonism. Am. J. Hum. Genet. 80, 393406 (2007)
  27. O’Brien, S. et al. Randomized phase III trial of fludarabine plus cyclophosphamide with or without oblimersen sodium (Bcl-2 antisense) in patients with relapsed or refractory chronic lymphocytic leukemia. J. Clin. Oncol. 25, 11141120 (2007)
  28. Crooke, S. T. et al. Vitravene—another piece in the mosaic. Antisense Nucleic Acid Drug Dev. 8, viiviii. (1998)
  29. Lu, Q. L. et al. Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nature Med. 9, 10091014 (2003)
  30. Alter, J. et al. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nature Med. 12, 175177 (2006)
  31. Yokota, T. et al. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann. Neurol. 65, 667676 (2009)
  32. Takeda, S. et al. Fukutin is required for maintenance of muscle integrity, cortical histiogenesis and normal eye development. Hum. Mol. Genet. 12, 14491459 (2003)
  33. Jurka, J. Repbase Update: a database and an electronic journal of repetitive elements. Trends Genet. 9, 418420 (2000)

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Author information

  1. These authors contributed equally to this work.

    • Mariko Taniguchi-Ikeda &
    • Kazuhiro Kobayashi

Affiliations

  1. Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

    • Mariko Taniguchi-Ikeda,
    • Kazuhiro Kobayashi,
    • Motoi Kanagawa,
    • Chih-chieh Yu,
    • Kouhei Mori,
    • Tetsuya Oda,
    • Atsushi Kuga &
    • Tatsushi Toda
  2. Division of General Pediatrics, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

    • Mariko Taniguchi-Ikeda
  3. Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi 470-1192, Japan

    • Hiroki Kurahashi
  4. Department of Neurology, Columbia University Medical Center, New York, NY 10032, USA

    • Hasan O. Akman &
    • Salvatore DiMauro
  5. Department of Clinical Neuroscience, The University of Tokushima Graduate School, Tokushima 770-8503, Japan

    • Ryuji Kaji
  6. Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada

    • Toshifumi Yokota
  7. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan

    • Shin’ichi Takeda

Contributions

M.T.-I., K.K., M.K. and T.T. designed the study. M.T.-I. performed most of the experiments. K.K. developed a system to detect endogenous fukutin protein. M.K. performed biochemical analysis of VMO-injected mice. C.Y. produced the fukutin cDNA constructs for transfection experiments. K.M., T.O., and A.K. performed analyses of AON treatment in mice and various cell types. H.K., T.Y. and S.T. provided intellectual input. H.O.A., S.D. and R.K., provided patients’ samples. M.T.-I., K.K. and T.T. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The patient fukutin and a chimpanzee mRNA sequences are deposited in GenBank/European Molecular Biology Laboratory/DNA Data Bank of Japan under accession numbers AB609007 and AB627340, respectively.

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