Large-scale methylation analysis in facioscapulohumeral muscular dystrophy (FSHD)

FSHD forms are associated to D4Z4 DNA hypomethylation on 4qA allelic chromosomes. These epigenetic changes provide a transcriptionally permissive chromatin environment leading to the production of a pathogenic protein DUX4. We have shown that the epigenetic changes in FSHD, can be investigated using methylation assays specific for permissive chromosomes 4qA (PAS+). Here, we report the analysis of a large cohort of individuals consisting of 287 FSHD1, 53 FSHD2 and 165 control subjects. This analysis showed highly significant difference of methylation levels between affected and control subjects further supported by strong correlation with the number of D4Z4 repeats both in FSHD1 and FSHD2. The different haplotype distribution between affected and control groups as well as particular methylation levels of some haplotypes suggested that diagnosis by methylation analysis can be improved by developing haplotype specific assays. To this aim, we developed haplotype specific assays and additional assays that allow precise genotyping. Furthermore, we set up bisulfite methylation assays to discriminate FSHD1 from FSHD2. Finally, assessment of methylation of intra-D4Z4 region and another SMCHD1 target sequence allowed evaluation of the effect of SMCHD1 mutations. Our results can allow the development of an integrate platform of methylation and sequencing assays to accelerate and improve the diagnostic procedure of FSHD.