Abstract
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.
Highlights
Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite
We studied our well-characterized FSHD1 family cohorts of myogenic cells derived from muscle biopsies [33,45,46], minimizing differences related to genetic background and allowing the analysis of multiple cohorts of FSHD1-affected subjects and nonmanifesting carriers containing the same D4Z4 contraction
FSHD is a myopathy, and double homeobox 4 (DUX4)-fl expression is induced in differentiated myogenic cells [47]; the use of these cells, as opposed to the lymphocytes used in most other studies, allowed analysis of epigenetic status and pathogenic gene expression in the most affected cell type
Summary
Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Genetically unaffected individuals are defined as having more than 10 D4Z4 repeat units (RUs) on both 4q chromosome arms (generally 25 to 35 RUs and as high as 120 RUs per array [13,14]), whereas individuals with genetic FSHD1 have between 1 and 10 D4Z4 RUs on one 4q chromosome arm, classifying FSHD as an autosomal dominant disease These polymorphic FSHD1-sized D4Z4 contractions by themselves are not pathogenic, and development of FSHD requires a disease-permissive allele of the chromosome 4q subtelomere (4A) in cis with the contracted array [14,15,16,17]. FSHD2 is, still genetically linked to the 4q35 region by the requirement of at least one permissive 4A-type subtelomere in order to develop disease [4,17]
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