Methylation of the Region Distal to the D4Z4 Array is Lower Than Predicted in FSHD1

Background: Epigenetic changes in FSHD can be investigated using methylation assays specific for permissive chromosomes 4qA (PAS+). Objectives: Taking advantage of the ability of this assay to directly measure methylation levels of a single allele, we deeply investigated the correlation between methylation and the repeats number. To minimize methylation variability due to sequence variations, we restricted the analysis to a large homogeneous subgroup of individuals carrying a single permissive haplotype 4A161. Results: Analysis of single CpGs in the region distal to the D4Z4 array showed that methylation levels correlate with the D4Z4 units number. Despite this correlation, the methylations levels detected in FSHD1 and FSHD2 patients are significantly lower than those predicted by control individuals analysis. Conclusions: Although obvious for SMCHD1 mutation carriers, this observation suggests that, also in FSHD1, methylation levels are affected by factors other than D4Z4 units