Figure 3.

Functional analyses of the p.Asp398Asn variant. (a) The results of DNA methylation analysis. DNA methylation statuses of 52 CpG sites in the DUX4 promoter region are shown¹⁶. The red and blue boxes depict methylated and unmethylated CpG sites, respectively. The average methylation rate of patient 1 was 25.8%, which is close to the cut off value of the diagnostic criteria of fascioscapulohumeral muscular dystrophy type 2 (25%)¹⁹. The average methylation rates of patient 2 and a control individual without SMCHD1 variants were 58.2% and 47.4%, respectively. (b) Representative results of in vitro functional assays. HEK293 cells transfected with the expression vectors of SMCHD1 or an empty vector were stained with anti-FLAG M2 for SMCHD1 (green) and Hoechst 33342 for DNA (blue). WT, full-length wildtype SMCHD1; p.Glu147Ala, an artificially created variant lacking the GHKL ATPase activity; p.Asp398Asn, a variant identified in patient 1. Scale bar, 20 μm.