Allele-specific DNA hypomethylation characterises FSHD1 and FSHD2

Background Facioscapulohumeral muscular dystrophy is associated with an epigenetic defect on 4q subtelomere. This dystrophy is caused by contraction of the D4Z4 macrosatellite array on chromosome 4qter in FSHD1, or by functional impairment of SMCHD1, a chromatin modifier binding to D4Z4, in FSHD2. Both genetic defects lead to D4Z4 DNA hypomethylation associated with inappropriate expression in skeletal muscle of the D4Z4-encoded DUX4 transcription factor in the presence of a polymorphic polyadenylation signal (PAS) distal to the last D4Z4 unit (4qA). Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and because it does not interrogate the presence of DUX4-PAS. Importantly, the methylation status of the DUX4-PAS critical region has not been thoroughly investigated. Methods We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array critical to FSHD development in PAS-positive alleles. Results Comparison of FSHD1, FSHD2 and control subjects showed highly significant differences of methylation levels in all CpGs tested. Noteworthy, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 in a fully characterized samples, supporting the potential usefulness of this assay for FSHD diagnosis. Moreover, our study evidenced a relationship between PAS-specific methylation and severity of the disease. Conclusions These data point to CpGs distal to the D4Z4 array as a critical region that summarizes multiple factors affecting epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis