Figure 8

Flow chart of epigenetic diagnosis of FSHD1 and FSHD2 by BSS. Clinical samples, including saliva, blood, muscle tissue, or cells, from patients with a clinical diagnosis of neuromuscular disease consistent with FSHD can be used for genomic DNA isolation and an epigenetic diagnosis of FSHD1 or FSHD2. The first level BSS assays, namely the 4qA and 4qA-L BSS assays, identify FSHD. The second level assay, namely the DUX4 5’ assay, distinguishes between FSHD1 and FSHD2. *Sequence analysis can be performed by subcloning and Sanger sequencing of a minimum of 10 independent clones; alternatively, a NGS approach can be used. Sequences are screened for 10A, 10A176T, and 4A166 and, if present, those sequences are removed from the analysis. The lower quartile (Q1) of the percent methylation is computed for the remaining sequences, to improve sensitivity for detecting hypomethylation on a contracted allele when roughly half the sequences are from a non-contracted allele and are hypermethylated. **If no BS PCR product is generated then the subject likely lacks a permissive 4A haplotype. Genomic PCRs for A- and B-type subtelomeres and sequencing can be used to confirm the results. ***Sequence analysis of the BS PCR product, which is derived from both 4q and 10q arrays and thus present in all samples, can be performed by subcloning and Sanger sequencing of a minimum of 10 independent clones; alternatively, a NGS approach can be used. The mean DNA methylation of 10 sequences is not expected to identify strong changes in FSHD1 patients since the vast majority of sequences are likely derived from either the non-contracted 4q or either of the 10q D4Z4 arrays; however, FSHD2 shows hypomethylation (<25% methylation mean) on both 4q and both 10q D4Z4 arrays. Precise cutoffs may need to be adjusted as more samples are examined.