Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy with a relatively late age of onset (usually in the late teens) compared with Duchenne and many other muscular dystrophies. The current FSHD disease model postulates that contraction of the D4Z4 array at chromosome 4q35 leads to a more open chromatin conformation in that region and allows transcription of the DUX4 gene. DUX4 mRNA is stable only when transcribed from certain haplotypes that contain a polyadenylation signal. DUX4 protein is hypothesized to cause FSHD by mediating cytotoxicity and impairing skeletal muscle differentiation. We recently showed in a cell culture model that DUX4 expression is regulated by telomere length, suggesting that telomere shortening during aging may be partially responsible for the delayed onset and progressive nature of FSHD. We here put our data in the context of other recent findings arguing that progressive telomere shortening may play a critical role in FSHD but is not the whole story and that the current disease model needs additional refinement.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is linked to chromatin relaxation due to epigenetic changes at the 4q35 D4Z4 macrosatellite array. Molecular diagnostic criteria for FSHD are complex and involve analysis of high molecular weight (HMW) genomic DNA isolated from lymphocytes, followed by multiple restriction digestions, pulse-field gel electrophoresis (PFGE), and Southern blotting. A subject is genetically diagnosed as FSHD1 if one of the 4q alleles shows a contraction in the D4Z4 array to below 11 repeats, while maintaining at least 1 repeat, and the contraction is in cis with a disease-permissive A-type subtelomere. FSHD2 is contraction-independent and cannot be diagnosed or excluded by this common genetic diagnostic procedure. However, FSHD1 and FSHD2 are linked by epigenetic deregulation, assayed as DNA hypomethylation, of the D4Z4 array on FSHD-permissive alleles. We have developed a PCR-based assay that identifies the epigenetic signature for both types of FSHD, distinguishing FSHD1 from FSHD2, and can be performed on genomic DNA isolated from blood, saliva, or cultured cells. RESULTS: Samples were obtained from healthy controls or patients clinically diagnosed with FSHD, and include both FSHD1 and FSHD2. The genomic DNAs were subjected to bisulfite sequencing analysis for the distal 4q D4Z4 repeat with an A-type subtelomere and the DUX4 5' promoter region. We compared genomic DNA isolated from saliva and blood from the same individuals and found similar epigenetic signatures. DNA hypomethylation was restricted to the contracted 4qA chromosome in FSHD1 patients while healthy control subjects were hypermethylated. Candidates for FSHD2 showed extreme DNA hypomethylation on the 4qA DUX4 gene body as well as all analyzed DUX4 5' sequences. Importantly, our assay does not amplify the D4Z4 arrays with non-permissive B-type subtelomeres and accurately excludes the arrays with non-permissive A-type subtelomeres. CONCLUSIONS: We have developed an assay to identify changes in DNA methylation on the pathogenic distal 4q D4Z4 repeat. We show that the DNA methylation profile of saliva reflects FSHD status. This assay can distinguish FSHD from healthy controls, differentiate FSHD1 from FSHD2, does not require HMW genomic DNA or PFGE, and can be performed on either cultured cells, tissue, blood, or saliva samples.