Does DNA Methylation Matter in FSHD?

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the genetic and epigenetic molecular features of the CpG-rich D4Z4 repeat tandem array at 4q35. Reduced DNA methylation of D4Z4 repeats is considered part of the FSHD mechanism and has been proposed as a reliable marker in the FSHD diagnostic procedure. We considered the assessment of D4Z4 DNA methylation status conducted on distinct cohorts using different methodologies. On the basis of the reported results we conclude that the percentage of DNA methylation detected at D4Z4 does not correlate with the disease status. Overall, data suggest that in the case of FSHD1, D4Z4 hypomethylation is a consequence of the chromatin structure present in the contracted allele, rather than a proxy of its function. Besides, CpG methylation at D4Z4 DNA is reduced in patients presenting diseases unrelated to muscle progressive wasting, like Bosma Arhinia and Microphthalmia syndrome, a developmental disorder, as well as ICF syndrome. Consistent with these observations, the analysis of epigenetic reprogramming at the D4Z4 locus in human embryonic and induced pluripotent stem cells indicate that other mechanisms, independent from the repeat number, are involved in the control of the epigenetic structure at D4Z4.

Keywords: D4Z4 macrosatellite; DNA methylation; FSHD1; FSHD2; epigenetics.

Figure 1

Molecular complexity in facioscapulohumeral muscular dystrophy (FSHD). D4Z4 contractions are associated with a permissive 4qA genotype that involves the aberrant expression of the DUX4 retrogene and is responsible for FSHD1, but also occurs in 1.3–2% of the normal population. 4qA is also found in cases presenting complex phenotypes and as well as in 30–50% of healthy relatives. Mutations in SMCHD1 or DNMT3B genes have been associated with FSHD2 and are responsible for ICF and BAMS syndromes. Parents of ICF patients are heterozygous for DNMT3B pathogenic variants but do not show any sign of muscular dystrophy. Other myopathic patients carry SMCHD1 mutations. Grey dot = methyl CpG; white dot = unmethylated CpG.

Figure 2

Overview of the D4Z4 repeats and of the region selected in methylation analyses. The D4Z4 array at 4q35 with an enlarged schematic representation of the D4Z4 distal most repeat (from position 1 to 3303 given relative to the two flanking KpnI sites). The different regions within D4Z4 are indicated: LSau repeat (position 1–340), Region A (position 869–1071), hhspm3 (position 1313–1780), DUX4 ORF (position 1792–3063), plus the 3′ pLAM region. The figure highlights the proximal BsaAI and FseI methylation-sensitive restriction sites analyzed by Southern blotting, and the location of bisulfite (BS) PCR products used in the selected literature reports, each represented with a differently colored line. The position of each region within the array, starting form the first KpnI site, is indicated together with the assayed number of CpGs. The white lines within these regions indicate the presence of CpGs in PCR primers.

Figure 3

The lack of a unique molecular signature in FSHD complicates genotype–phenotype correlation in clinical practice. The figure illustrates the complex molecular scenario observed in individuals with clinical features of FSHD. Hypomethylation is observed in a large subset of them, including in patients who carry a mutation in SMCHD1 or DNMT3B. This scheme also highlights that hypomethylation of D4Z4 is observed in other rare diseases linked to mutations in SMCHD1 (BAMS), DNMT3B (ICF) or carrying a deletion of the 18p locus that contains the SMCHD1 gene.