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

Abstract

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.

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Figure 1: An SVA retrotransposal insertion induces abnormal splicing in FCMD.
Figure 2: Abnormal fukutin protein in FCMD.
Figure 3: AON cocktail rescues normal fukutin mRNA.
Figure 4: AON cocktail treatment rescues normal fukutin protein and functional α-DG.

Accession codes

Data deposits

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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Acknowledgements

We thank S. Nakagawa, K. Ohno, S. Tsujino, N. Taniguchi, and I. Nonaka for comments; M. Okabe and A. Kawai for generating the ES cell line from knock-in model mice; Y. Motoyoshi and J. C. Cohen for providing patients’ samples; W. Sako and Y. Izumi for sending patients’ samples; I. Mizuta, T. Mure, M. Furukawa, K. Kaneshiro, Y. Dainin and all laboratory members for technical support; and J. Logan for editing the manuscript. We thank the GAIN for providing chimpanzee brain samples. This work was supported by an Intramural Research Grant (20B-13) for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (to T.T.), the Global COE Program (Frontier Biomedical Science Underlying Organelle Network Biology) (to T.T., M.T.-I. and M.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Scientific Research (A) (23249049 to T.T.), and Young Scientists (A) (21689030 to K.K.) and (B) (20790980 to M.T.-I.) from the Japan Society for the Promotion of Science, and the Takeda Science Foundation (to K.K.).

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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.

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Correspondence to Tatsushi Toda.

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The authors declare no competing financial interests.

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Taniguchi-Ikeda, M., Kobayashi, K., Kanagawa, M. et al. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature 478, 127–131 (2011). https://doi.org/10.1038/nature10456

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