Epigenetics of Friedreich’s Disease: Methylation of the (GAA)n-Repeats Region in FXN Gene

Abstract

Background: Friedreich’s disease (FD) is the most common hereditary ataxia. It is associated, most frequently, with homozygous GAA repeats expansion in intron 1 of the FXN gene. Methylation of the FXN gene can play an important role in the pathogenesis of FD. Aims: to study methylation pattern in CpG sites flanking GAA-expansion in intron 1 of the FXN gene in patients with FD and their heterozygous relatives as well as its relationship with clinical features. Materials and methods: We studied DNA samples from patients with FD (n=18), their relatives carrying heterozygous GAA expansion (n=12), and control group (n=15). Pattern of methylation was studied by direct sequencing of DNA regions after bisulphide processing. Results: We analyzed 18 CpG sites in the UP-GAA region of the gene (before GAA-repeats) and 12 CpG sites in the DOWN-GAA region (after GAA-repeats). In the UP-region, the mean methylation level of CpG sites in FD patients was higher compared to controls (n=15) (р0.05), while in the DOWN-region there was a decrease of mean methylation level in FD compared to controls (р0.05). Analysis of methylation level in different CpG sites in the UP-GAA region revealed hypermethylation for 15 of 18 CpG-sites as compared to controls (р0.05). The most significant differences in methylation level in the UP-GAA region were seen for CpG sites 50−54, 57 and 58. In contrast, in the DOWN-GAA region almost all CpG sites were fully methylated in the control group, while in FD patients methylation was significantly lower (р0.05). We revealed positive correlation of mean methylation level and more expanded allele length for the UP-GAA region in FD (r=0.63; p=0.03), and no correlations for the DOWN-GAA region. In heterozygous carriers we observed an analogous positive correlations in the UP-GAA region for CpG site 50 (r=0.77; p=0.04), while in the DOWN-GAA region there was inverse correlation of methylation with GAA repeat number in the expanded allele (r=-0.83, p=0.02). Negative correlation was found between the hypermethylation of some CpG-sites in the UP-GAA region and age of the disease onset (p0.05). Conclusion: We revealed hypermethylation in the UP-GAA region and hypomethylation in the DOWN-GAA region in patients with FD compared to controls and correlations of methylation level with the GAA expansion length and age of disease onset.