Molecular Diagnosis of FSHD1 Protocol

DNA was prepared from isolated lymphocytes according to standard procedures. In brief, restriction endonuclease digestion of DNA was performed in agarose plugs with the appropriate restriction enzyme: EcoRI, EcoRI/BlnI. Digested DNA was separated by pulsed field gel electrophoresis (PFGE) in 1% agarose gels, as previously described^{18 (link)}. Allele sizes were estimated by Southern hybridization with probe p13E-11 of 7 μg of EcoRI-digested, EcoRI/BlnI-digested genomic DNA extracted from peripheral blood lymphocytes, electrophoresed in a 0.4% agarose gel, for 62–64 h at 35 V, alongside an 8–48 kb marker (Bio-Rad). Participants carrying alleles of 36–41 kb (9–10 D4Z4 units) in size were included in the study. Within the European population, pathological D4Z4 contractions usually are associated with the permissive 4qA haplotype in the subtelomeric region of chromosome 4q. The qA polymorphism was assessed by HindIII digestion and hybridization with qA probe. Restriction fragments were detected by autoradiography or by using the Typhoon Trio system (GE Healthcare). To verify that the obtained shortened D4Z4 fragment on chromosome 4 has a causative 4qA haplotype, an additional HindIII Southern blot was performed as suggested for the molecular diagnosis of FSHD1^{33 (link)}.