Dysregulation of 4q35- and muscle-specific genes in fetuses with a short D4Z4 array linked to facio-scapulo-humeral dystrophy

Abstract

Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array (n = 6) compared with fetuses with a non-pathogenic D4Z4 array (n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with <11 D4Z4. The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene

“…We examined multiple proliferating and differentiating cultures of biceps-derived FSHD (donors 07Abic, 09Abic, and 17Abic) and unaffected (donors 07Ubic, 09Ubic, 17Ubic, and 17Vbic) myogenic cells (see Table S1 in the supplemental material) (28,42) by double immunostaining using MAb E55, which is specific for the C-terminal extension of DUX4-FL that distinguishes it from the shorter DUX4-S (27,43), in combination with MAb F59, which reacts with all human MyHC isoforms (37,44). Consistent with previous work, we found DUX4-FL-positive nuclei in cells from both FSHD and unaffected donors (28,45). In total our analysis identified 826 cells that contained DUX4-FL-positive nuclei, of which 824 (99.76%) also expressed MyHC (see Fig.…”

“…Although the pathogenic expression of DUX4 in FSHD occurs in skeletal muscle, DUX4 is expressed normally in testis and in pluripotent stem cells (27), and a recent report describes expression of DUX4-fl in nonmuscle somatic tissues of both FSHD and normal fetuses (45). Recently, the cell adhesion molecule FAT1 (OMIM 600976), a downstream target of DUX4, has been described as a potential FSHD modifier during development (71).…”

SORBS2transcription is activated by telomere position effect–over long distance upon telomere shortening in muscle cells from patients with facioscapulohumeral dystrophy

“…In summary, the early expression of SORBS2 in FSHD myoblasts with short telomere influences the differentiation program and may contribute to the pathology with other FSHD candidate genes such as the DUX4 toxic protein (Winokur et al 2003;Cheli et al 2011;Tsumagari et al 2011;Broucqsault et al 2013). Due to the complexity of SORBS2 (42 exons, 118 putative transcripts), previous results are often contradictory, with some observing up-regulation (Celegato et al 2006), and others reporting no change in the expression of this gene (Masny et al 2010;Rahimov et al 2012).…”

“…However, in a number of cases, the role of DUX4 remains unclear due to the complexity of its expression pattern (Dixit et al 2007;Snider et al 2010;Tassin et al 2013), low mRNA and protein abundance Ferreboeuf et al 2014), and the presence of DUX4 in unaffected individuals (Jones et al 2012;Broucqsault et al 2013). Thus, DUX4 might not be sufficient to explain the wide variability of the pathology, the muscle specificity and asymmetry, or the age-related onset suggesting that other factors are likely involved (Cabianca et al 2012;Caruso et al 2013;Stadler et al 2013;Mariot et al 2015;Puppo et al 2015).…”

Facioscapulohumeral Muscular Dystrophy As a Model for Epigenetic Regulation and Disease

“…Although generally silent in adult somatic tissues, DUX4-fl is expressed in testis and in pluripotent stem cells (143), and a recent report describes expression of DUX4-fl in both muscle and non-muscle somatic tissues of FSHD1 and healthy fetuses (23). Thus, the DUX4 retrogene has likely evolved to play a normal role strictly during primate development, but on loss of epigenetic silencing in FSHD, abnormal DUX4-fl expression in adult skeletal muscle has pathological consequences.…”