Advances in the development of a methylation assay for FSHD

Facioscapulohumeral muscular dystrophy is a disease linked to an epigenetic defect in the chromosome 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 the 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, diagnosis by methylation analysis has two main limits: the interference of non-pathogenic arrays and the lack of information about the presence of the DUX4-PAS. Importantly, the methylation status of the DUX4-PAS critical region has not been thoroughly investigated. We investigated the DNA methylation levels of the region immediately distal to the D4Z4 array critical to FSHD development and encompassing 10 CpGs in PAS-positive alleles. Comparison of FSHD1, FSHD2 and control subjects showed highly significant differences of methylation levels in all CpGs tested. Noteworthy, one of these CpGs (CpG6) was able to discriminate the affected individuals with a sensitivity 0.95 in a cohort of 112 samples, supporting the potential usefulness of this assay for FSHD diagnosis. Moreover, our study evidenced a relationship between PAS-specific methylation and the severity of the disease. 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 may allow the establishment of a rapid and sensitive tool for FSHD diagnosis.