Molecular diagnosis of FSHD by epigenetic signature

A method of determining whether a human subject has, or is at risk of developing, facioscapulohumeral muscular dystrophy 2 (FSHD2) comprising performing a DNA methylation analysis of deoxycytosine-phosphate-deoxyguanine dinucleotides (CpGs) in a distal D4Z4 repeat unit of a D4Z4 repeat array and a proximal region of an A-type subtelomere of each of a chromosome 4qA allele and a chromosome 4qA-L allele of chromosome 4q, if present, wherein the DNA methylation analysis comprises:

• i) performing polymerase chain reaction (PCR) using genomic DNA from at least one sample from the subject and a forward primer comprising the sequence of SEQ ID NO:
• 8 and a first reverse primer comprising the sequence of SEQ ID NO:
• 9 to obtain at least one first 4qA PCR product if the genomic DNA comprises a 4qA allele;
• ii) performing PCR using the at least one first 4qA PCR product, the forward primer comprising the sequence of SEQ ID NO:
• 8 and a second reverse primer comprising the sequence of SEQ ID NO:
• 10 to obtain at least one second 4qA PCR product if the genomic DNA comprises a 4qA allele;
• iii) performing PCR using genomic DNA from at least one sample from the subject and a forward primer comprising the sequence of SEQ ID NO:
• 11 and the first reverse primer comprising the sequence of SEQ ID NO:
• 9 to obtain at least one first 4qA-L PCR product if the genomic DNA comprises a 4qA-L allele;
• iv) performing PCR using the at least one first 4qA-L PCR product, the forward primer comprising the sequence of SEQ ID NO:
• 11 and the second reverse primer comprising the sequence of SEQ ID NO:
• 10 to obtain at least one second 4qA-L PCR product if the genomic DNA comprises a 4qA-L allele;
• v) performing bisulfite sequencing for each of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product, if the genomic DNA comprises a 4qA allele and/or 4qA-L allele, respectively;
• vi) quantifying methylation of the CpGs in the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere in each of the at least one second 4qA PCR product and the at least one second 4qA-L PCR product to obtain the first quartile of the methylation percent of the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere of each of the second 4qA PCR product and the second 4qA-L PCR product,
• wherein if 25% or less of the CpGs in the distal D4Z4 repeat unit and the proximal region of the A-type subtelomere in the first quartile of the second 4qA PCR product are methylated or if 25% or less of the CpGs in the first quartile of the second 4qA-L PCR product are methylated, then the subject has, or is at risk of developing, FSHD2.