Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

Abstract

Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene. The expanded repeat induces repressive histone changes and DNA hypermethylation, which result in epigenetic silencing and FXN transcriptional deficiency. A class I histone deacetylase inhibitor (HDACi-109) reactivates the silenced FXN gene, although with considerable inter-individual variability, which remains etiologically unexplained. Because HDAC inhibitors work by reversing epigenetic silencing, we reasoned that epigenetic heterogeneity among patients may help to explain this inter-individual variability. As a surrogate measure for epigenetic heterogeneity, a highly quantitative measurement of DNA hypermethylation via bisulfite deep sequencing, with single molecule resolution, was used to assess the prevalence of unmethylated, partially methylated, and fully methylated somatic FXN molecules in PBMCs from a prospective cohort of 50 FRDA patients. Treatment of the same PBMCs from this cohort with HDACi-109 significantly increased FXN transcript to levels seen in asymptomatic heterozygous carriers, albeit with the expected inter-individual variability. Response to HDACi-109 correlated significantly with the prevalence of unmethylated and partially methylated FXN molecules, supporting the model that FXN reactivation involves a proportion of genes that are amenable to correction in non-dividing somatic cells, and that heavily methylated FXN molecules are relatively resistant to reactivation. FXN reactivation is a promising therapeutic strategy in FRDA, and inter-individual variability is explained, at least in part, by somatic epigenetic heterogeneity.

Clinical Evidence for Variegated Silencing in Patients With Friedreich Ataxia

“…While the present data support variegated silencing as a mechanism for the observed ceiling effects in clinical features, there are both alternative explanations and differences between this study and the previous ex vivo study. Previous ex vivo data on variegated silencing in FRDA 8 found the GAA-TR ceiling cutoff length to be 500 triplets, which differs slightly from our finding of 700 triplets. The discrepancy could reflect differences in sample sizes between the 2 studies (approximately 70 patients vs ∼1,000 patients), differences in GAA repeat lengths or mechanisms of silencing between different tissues, or the relative granularity of data between the studies.…”

“…The primary cause of FRDA is frataxin deficiency, which varies in severity based on the GAA-TR length; the severity of FRDA also reflects variation in downstream events or unknown variables among patients. Based on the present results and the molecular concept of variegated silencing, 8 agents that alter gene silencing should produce the greatest effects on clinical status or frataxin levels in participants with GAA-TR lengths of less than 700. By contrast, participants with GAA-TRs of greater than 700 might prove most useful in assessing agents acting on disease aspects other than gene silencing because this group has the greatest homogeneity in frataxin levels.…”

“…In this study, we have tested the variegated silencing model in a clinical data set from a large natural history study in FRDA. [7][8][9][10]…”

“… 6 Recently, a study based on ex vivo analysis of peripheral blood mononuclear cells (PBMCs) from patients with FRDA further supported the PEV model of silencing in FRDA. 7 , 8 High-resolution epigenetic mapping revealed that nearly all FXN genes in cells of patients with FRDA are silenced and express <5% FXN . Only a small subpopulation of FXN genes escape silencing (and express approximately 100% FXN ), giving an overall apparent average of 5%–30% residual FXN levels in a given population of cells.…”

DNA methylation in Friedreich ataxia silences expression of frataxin isoform E

“…We focused our study on frataxin-E and FXN-E precisely because of its origin in intron 1, i.e., in close proximity to the site of maximal DNA methylation in FRDA. The FRDA-DMR, which shows GAA repeat-dependent hypermethylation, is predictive of age of onset in FRDA 22 , and FXN gene reactivation 46 , and here we show that it is also involved in regulating expression of frataxin-E. A caveat of our study is that in patient-derived blood samples we measured frataxin-E protein in erythrocytes, but DNA methylation (and GAA repeat length) was necessarily assayed in nucleated blood cells (PBMCs). How erythrocytes in FRDA patients end up with deficiency of frataxin-E is unclear.…”