Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.

BackgroundBoth forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.ResultsFamily cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.ConclusionsThe epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0072-6) contains supplementary material, which is available to authorized users.

Telomeres may regulate human disease by at least two independent mechanisms. 1) Replicative senescence occurs once short telomeres generate DNA damage signals that produce a barrier to tumor progression. 2) Telomere Position Effect (TPE) can change gene expression at intermediate telomere lengths in cultured human cells. We here report a human disease, facioscapulohumeral muscular dystrophy (FSHD) where telomere length may well contribute to its pathogenesis. FSHD is age-related and genetically only 25-60 kb from the end of chromosome 4q. We used a floxable telomerase to generate isogenic clones with different telomere lengths from patients and their unaffected siblings. DUX4, the primary candidate for FSHD pathogenesis, is upregulated >10-fold in FSHD myoblasts-myotubes with short versus long telomeres, and its expression is inversely proportional to telomere length. FSHD may represent a human disease in which TPE contributes to its age-related phenotype.