The FSHD atrophic myotube phenotype is caused by DUX4 expression.

Laboratory of Molecular Biology, University of Mons, Mons, Belgium.
PLoS ONE (Impact Factor: 3.53). 10/2011; 6(10):e26820. DOI: 10.1371/journal.pone.0026820
Source: PubMed

ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is linked to deletions in 4q35 within the D4Z4 repeat array in which we identified the double homeobox 4 (DUX4) gene. We found stable DUX4 mRNAs only derived from the most distal D4Z4 unit and unexpectedly extended to the flanking pLAM region that provided an intron and a polyadenylation signal. DUX4 encodes a transcription factor expressed in FSHD but not control primary myoblasts or muscle biopsies. The DUX4 protein initiates a large transcription deregulation cascade leading to muscle atrophy and oxidative stress, which are FSHD key features.
We now show that transfection of myoblasts with a DUX4 expression vector leads to atrophic myotube formation associated with the induction of E3 ubiquitin ligases (MuRF1 and Atrogin1/MAFbx) typical of muscle atrophy. DUX4 induces expression of downstream targets deregulated in FSHD such as mu-crystallin and TP53. We developed specific siRNAs and antisense oligonucleotides (AOs) targeting the DUX4 mRNA. Addition of these antisense agents to primary FSHD myoblast cultures suppressed DUX4 protein expression and affected expression of the above-mentioned markers.
These results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.

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Available from: Alexandra Belayew, Jul 31, 2015
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    • "This main form of FSHD, referred to as FSHD1, accounts for approximately 95% of cases [Richards et al., 2012]. Such a heterozygous D4Z4-array reduction is associated with chromatin relaxation on specific permissive haplotypes [Lemmers et al., 2002], which might result in stabilization of the DUX4 transcript , encoded by the retrogene located in D4Z4, and expression of this transcription factor [Gabriëls et al., 1999; Dixit et al., 2007; Lemmers et al., 2010; Spurlock et al., 2010; Vanderplanck et al., 2011]. A form of FSHD not linked to D4Z4 contraction accounts for 5% of patients (contraction-independent FSHD) [van Overveld et al., 2003]. "
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    ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is linked to copy number reduction (n<10) of the 4q D4Z4 subtelomeric array, in association with DUX4-permissive haplotypes. This main form is indicated as FSHD1. FSHD-like phenotypes may also appear in the absence of D4Z4 copy number reduction. Variants of the SMCHD1 gene have been reported to associate with D4Z4 hypomethylation in DUX4-compatible haplotypes, thus defining FSHD2. Recently, mice carrying a muscle-specific knock-out of the protocadherin gene Fat1 or its constitutive hypomorphic allele were shown to develop muscular and non-muscular defects mimicking human FSHD. Here we report FAT1 variants in a group of patients presenting with neuromuscular symptoms reminiscent of FSHD. The patients do not carry D4Z4 copy number reduction, 4q hypomethylation or SMCHD1 variants. However, abnormal splicing of the FAT1 transcript is predicted for all identified variants. To determine their pathogenicity, we elaborated a minigene approach coupled to an antisense oligonucleotide (AON) assay. In vitro, four out of five selected variants induced partial or complete alteration of splicing by creating new splice sites or modifying splicing regulators. AONs confirmed these effects. Altered transcripts may affect FAT1 protein interactions or stability. Altogether, our data suggest that defective FAT1 is associated with an FSHD-like phenotype.This article is protected by copyright. All rights reserved
    Human Mutation 01/2015; 36(4). DOI:10.1002/humu.22760 · 5.05 Impact Factor
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    • "DUX4 regulated targets also include genes involved in RNA splicing, developmentally regulated components of the Pol II transcription complex, and ubiquitin-mediated protein degradation pathways, all of which may have pathophysiological consequences. A recent study indicated that induction of E3 ubiquitin ligases by DUX4 might cause muscle atrophy in FSHD (Vanderplanck et al., 2011), consistent with our findings that multiple ubiquitin ligase family members are induced by DUX4. In addition, myostatin induces some of these ubiquitin ligases in skeletal muscle (Lokireddy et al., 2011) and it is therefore possible that both DUX4 induction of ubiquitin ligases and the modest upregulation of myostatin by DEFB103 that we observed in this study can both contribute to muscle atrophy. "
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    ABSTRACT: Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.
    Developmental Cell 12/2011; 22(1):38-51. DOI:10.1016/j.devcel.2011.11.013 · 10.37 Impact Factor
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    ABSTRACT: No treatment exists for facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle diseases. Although FSHD can be debilitating, little effort has been made to develop targeted therapies. This lack of focus on targeted FSHD therapy perpetuated because the genes and pathways involved in the disorder were not understood. Now, more than 2 decades after efforts to decipher the root cause of FSHD began, this barrier to translation is finally lowering. Specifically, several recent studies support an FSHD pathogenesis model involving overexpression of the myopathic DUX4 gene. DUX4 inhibition has therefore emerged as a promising therapeutic strategy for FSHD. In this study, we tested a preclinical RNA interference (RNAi)-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of-principle for RNAi therapy of FSHD through DUX4 inhibition.
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