Inter-individual differences in CpG methylation at D4Z4 correlate with clinical variability in FSHD1 and FSHD2

Abstract

Facioscapulohumeral muscular dystrophy (FSHD: MIM#158900) is a common myopathy with marked but largely unexplained clinical inter- and intra-familial variability. It is caused by contractions of the D4Z4 repeat array on chromosome 4 to 1-10 units (FSHD1), or by mutations in the D4Z4-binding chromatin modifier SMCHD1 (FSHD2). Both situations lead to a partial opening of the D4Z4 chromatin structure and transcription of D4Z4-encoded polyadenylated DUX4 mRNA in muscle. We measured D4Z4 CpG methylation in control, FSHD1 and FSHD2 individuals and found a significant correlation with the D4Z4 repeat array size. After correction for repeat array size, we show that the variability in clinical severity in FSHD1 and FSHD2 individuals is dependent on individual differences in susceptibility to D4Z4 hypomethylation. In FSHD1, for individuals with D4Z4 repeat arrays of 1-6 units, the clinical severity mainly depends on the size of the D4Z4 repeat. However, in individuals with arrays of 7-10 units, the clinical severity also depends on other factors that regulate D4Z4 methylation because affected individuals, but not non-penetrant mutation carriers, have a greater reduction of D4Z4 CpG methylation than can be expected based on the size of the pathogenic D4Z4 repeat array. In FSHD2, this epigenetic susceptibility depends on the nature of the SMCHD1 mutation in combination with D4Z4 repeat array size with dominant negative mutations being more deleterious than haploinsufficiency mutations. Our study thus identifies an epigenetic basis for the striking variability in onset and disease progression that is considered a clinical hallmark of FSHD.

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

“…The signal from the 4061-bp fragment (methylated) was divided by the total amount of hybridizing fragments at 4061 bp and 3387 bp (unmethylated) to yield the average percentage of methylated FseI sites within the most proximal D4Z4 unit on all four D4Z4 arrays. The Delta1 and Delta2 scores were calculated as described in Lemmars et al 21 …”

“…14 Recently, we showed that D4Z4 methylation level at this site is repeat array size dependent. 21 We introduced a new methylation parameter, Delta1, which represents the difference between the experimentally observed methylation and the predicted methylation level based on repeat size in controls. In SMCHD1 mutation carriers, the average Delta1 score is highly negative ranging between − 20 and − 45, suggesting a strong contribution of the variant to D4Z4 hypomethylation.…”

“…In SMCHD1 mutation carriers, the average Delta1 score is highly negative ranging between − 20 and − 45, suggesting a strong contribution of the variant to D4Z4 hypomethylation. 21 Accordingly, a second model was then fitted to predict the methylation in SMCHD1 variant carriers, which resulted in the Delta2 score. For SMCHD1 variants that preserve the open-reading frame (ORF), a mean Delta2 score of − 1.8% was found, which is significantly lower than the mean Delta2 of ORF-disrupting variants (mean 2.7%).…”

“…This suggests that ORF-preserving variants are more deleterious for the maintenance of a repressed D4Z4 chromatin state in somatic cells. 21 Several studies have so far identified disease-causing variants in SMCHD1 in approximately 70 FSHD2 families. Heterozygous diseasecausing SMCHD1 variants are distributed over the entire SMCHD1 locus and include splice site, insertion-deletion, missense and nonsense variants.…”

“…Heterozygous diseasecausing SMCHD1 variants are distributed over the entire SMCHD1 locus and include splice site, insertion-deletion, missense and nonsense variants. 14,[20][21][22][23][24] We have shown that a combination of the size of the permissive D4Z4 array and the type of SMCHD1 variant together determine the epigenetic susceptibility to disease presentation. 21 Until now, only heterozygous SMCHD1 variants, which are dominant in combination with a permissive haplotype, have been reported.…”