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Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene1,2. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing5,6,7. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

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Figure 1: Mice overexpressing FRG1 show kyphosis.
Figure 2: Skeletal muscles from FRG1 transgenic mice show dystrophic histological features.
Figure 3: Increased FRG1 transgene expression correlates with a reduction in body weight and muscle atrophy.
Figure 4: Muscles from FRG1 transgenic mice and FSHD patients show aberrant pre-mRNA splicing of selective genes.

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Acknowledgements

We are indebted to all FSHD families and the FSH Society. We thank M. Mora for providing FSHD myoblasts; G. Di Giulio for performing the radiology; C. Ghigna, S. Jones, L. Castilla and members of the M.R.G. laboratory for helpful discussions; and S. Evans for assistance with preparing the manuscript. Space constraints have limited the amount of original work that we have been able to cover in this Letter. This work was supported by the Muscular Dystrophy Association (D.G.); Associazione Amici del Centro Dino Ferrari, Telethon Bank, Eurobiobank and R.F. 2002 Criobanca Automatizzata di Materiale Biologico (M.M.); Telethon (R.B.); and the Cariplo Fundation, NIH-NINDS, the Muscular Dystrophy Association, the Italian National Council for Research Progetto Genomica Funzionale, the Association Francaise contre les Myopaties, the Italian Ministry of Education, University, and Research, Legato Ferrari and Telethon (R.T). M.R.G. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Rossella Tupler.

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Drs Gabellini, Tupler and Green have filed U.S. patent application 20050054012, entitled “Methods of detecting and treating FSHD”.

Supplementary information

Supplementary Methods

Methods for RT-PCR analysis, real time RT–PCR, FRG1 transgene copy number determination, FRG1 transgene northern blotting, histological and histochemical analyses, analysis of sarcolemmal integrity, immunofluorescence analysis of FRG1, overexpression of ASF/SF2 in C2C12 cells.

Supplementary Figures

This file contains Supplementary Figures 1–9.

Supplementary Tables 1 and 2

Supplementary Table 1 details the histopathological and histochemical comparison of ten different skeletal muscles of FRG1 transgenic mice. Supplementary Table 2 details the comparison of the muscles affected and the degree of dystrophy between FSHD patients and FRG1 transgenic mice.

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Gabellini, D., D'Antona, G., Moggio, M. et al. Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature 439, 973–977 (2006). https://doi.org/10.1038/nature04422

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