Association of 4qA-Specific Distal D4Z4 Hypomethylation With Disease Severity and Progression in Facioscapulohumeral Muscular Dystrophy

Objective:To examine whether the regional methylation levels at the most distal D4Z4 repeat units (RU) in the 4qA-permissive haplotype were associated with disease severity and progression in facioscapulohumeral muscular dystrophy type 1 (FSHD1).Methods:This was a 21-year, retrospective, and observational cohort study conducted at the Fujian Neuromedical Centre (FNMC) in China. Methylation levels of the most distal D4Z4 RU, including 10 CpGs, were assessed in all participants by bisulfite sequencing. FSHD1 patients were stratified into 4 groups based on methylation percentage quartiles, including LM1 (low methylation), LM2 (low to intermediate methylation), LM3 (intermediate to high methylation), and highest methylation levels (HM). Patients received evaluations of motor function focusing on lower extremity (LE) progression at baseline and in follow-ups. FSHD clinical score (CS), age-corrected clinical severity scale (ACSS), and modified Rankin Scale were used to assess motor function.Results:The methylation levels of the 10 CpGs were significantly lower in all 823 genetically confirmed FSHD1 patients than in 341 healthy controls (HCs). CpG6 methylation levels could distinguish: 1) FSHD1 patients from HCs; 2) symptomatic from asymptomatic/unaffected patients; 3) patients with LE involvement from those without LE involvement, with AUCs (95% CI) of 0.9684 (0.9584–0.9785), 0.7417 (0.6903–0.7931), and 0.6386 (0.5816–0.6956), respectively. CpG6 methylation levels were negatively correlated with both CS (r = -0.392) and ACSS (r = -0.432), and positively correlated with onset age of first-ever muscle weakness (r = 0.297). For the LM1, LM2, LM3, and HM groups, the respective proportions of LE involvement were 52.9%, 44.2%, 36.9%, and 23.4%; and onset ages of LE involvement were 20, 26.5, 25, and 26.5 years. Cox regression analysis—adjusted for sex, age at examination, D4Z4 RU, and 4qA/B haplotype—showed that the LM1, LM2, and LM3 groups (i.e., groups with lower methylation levels) had a higher risk for independent ambulation loss, with HRs (95% CI) of 3.523 (1.565–7.930), 3.356 (1.458–7.727), and 2.956 (1.245–7.020), respectively.Conclusion:4q35 distal D4Z4 hypomethylation is correlated with disease severity and progression to lower extremity involvement.