Expanded [CCTG]n repetitions are not associated with abnormal methylation at the CNBP locus in myotonic dystrophy type 2 (DM2) patients

Abstract

Myotonic Dystrophy type 2 (DM2) is a multisystemic disorder associated with an expanded [CCTG]n repeat in intron 1 of the CNBP gene. Epigenetic modifications have been reported in many repeat expansion disorders, including myotonic dystrophy type 1 (DM1), either as a mechanism to explain somatic repeat instability or transcriptional alterations in disease genes. The purpose of our work was to determine the effect of DM2 mutation on the methylation status of CpG islands localized in the 5' promoter region and in the 3' end of the [CCTG]n expansion of the CNBP gene. By bisulfite pyrosequencing, we characterized the methylation profile of two different CpG islands within these regions, either in whole blood and skeletal muscle tissues of DM2 patients (n=72 and n=7, respectively) and controls (n=50 and n=7, respectively). Moreover, we compared the relative mRNA transcript levels of CNBP gene in leukocytes and in skeletal muscle tissues from controls and DM2 patients. We found that CpG sites located in the promoter region showed hypomethylation, whereas CpG sites at 3' end of the CCTG array are hypermethylated. Statistical analyses did not demonstrate any significant differences in the methylation profile between DM2 patients and controls in both tissues analyzed. According to the methylation analysis, CNBP gene expression levels are not significantly altered in DM2 patients. These results show that [CCTG]n repeat expansion, differently from the DM1 mutation, does not influence the methylation status of the CNBP gene and suggest that other molecular mechanisms are involved in the pathogenesis of DM2.

248th ENMC International Workshop: Myotonic dystrophies: Molecular approaches for clinical purposes, framing a European molecular research network, Hoofddorp, the Netherlands, 11–13 October 2019

“…She then showed the results of a methylation study of DNA regions flanking the (CCTG)n expansion carried out in blood and muscle samples from a large cohort of DM2 patients. Pyrosequencing analysis of two CpG islands, upstream and downstream of the DM2 mutation, did not demonstrate any significant difference in the methylation profile between DM2 patients and controls in both tissues analyzed [20][21][22][23] .…”

Epigenetics of Myotonic Dystrophies: A Minireview

“…Moreover, CNBP gene expression seems to be independent from the methylation pattern of the DNA regions analyzed. These preliminary evidences suggest the existence of molecular mechanisms other than epigenetics involved in the pathogenesis of DM2 [45].…”

“…All the studies described so far are strictly focused on DM1, and the in cis effect of (CCTG)n expansion on the DM2 locus is still largely unknown. To date, only one study analyzed the methylation status of CNBP gene in whole blood and skeletal muscle tissues from DM2 patients [45]. The hypomethylation of CpG sites upstream and hypermethylation of CpG sites downstream of the (CCTG) n expansion was observed either in DM2 patients and healthy individuals (Figure 2A), with no significant differences.…”

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

“…This can provide additional information concerning the impact of expansions on the functionality of the CNBP gene. The methylation of the CNBP gene has been analyzed using a pyrosequencing method, revealing hypomethylation of CpG sites upstream and hypermethylation of CpG sites downstream of the (CCTG) n expansion in DM2 patients and healthy individuals, with no significant differences between these groups(Massimo Santoro et al 2018). However, it remains possible that the DM2 mutation could have epigenetic effects in other regulatory regions of the CNBP gene and/or in different tissues.…”