Figure 1

The molecular signatures of FSHD are complex, as illustrated by healthy and FSHD-type chromosomes. In the general healthy population, each chromosome 4q arm has a large polymorphic array of D4Z4 repeats containing more than 10 RUs. In FSHD1, there is a dominant contraction of one 4q array to between 1 and 10 D4Z4 repeat units, whereas FSHD2 is contraction-independent. There are two main allelic variants in the subtelomere distal to the array, termed A and B. A rare third classification of subtelomere, termed C, is used for subtelomeres that do not hybridize with probes for A or B due to distal sequence changes [18]. In some instances, the distal-most repeat fragment of the 4q D4Z4 array contains additional ~2 kb of D4Z4 sequence, resulting in a longer terminal RU in cis with a 4qA subtelomere; this type of 4qA allele is referred to as 4qA-L [15]. Both FSHD1 and FSHD2 are exclusively linked to the 4qA subtelomere allelic variants containing a PAS for the DUX4-fl mRNA [12, 15]. In addition, both FSHD1 and FSHD2 require the epigenetic disruption of the D4Z4 array to a less methylated and more relaxed chromatin state. Results of the described bisulfite sequencing assays are indicated by “+” if a bisulfite (BS) PCR product is produced and “–” if no BS PCR product is produced. *On rare occasions, due to primer degradation, a 10qA BS PCR product is detected; however, sequencing eliminates these from analysis. **Diagnosis of this healthy chromosome requires genomic PCR and sequencing of the 4qA subtelomere to identify a non-permissive 4qA PAS.