Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia

Abstract

Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene, which results in transcriptional deficiency via epigenetic silencing. Most patients are homozygous for alleles containing > 500 triplets, but a subset (~20%) have at least one expanded allele with < 500 triplets and a distinctly milder phenotype. We show that in FRDA DNA methylation spreads upstream from the expanded repeat, further than previously recognized, and establishes an FRDA-specific region of hypermethylation in intron 1 (~90% in FRDA versus < 10% in non-FRDA) as a novel epigenetic signature. The hypermethylation of this differentially methylated region (FRDA-DMR) was observed in a variety of patient-derived cells; it significantly correlated with FXN transcriptional deficiency and age of onset, and it reverted to the non-disease state in isogenically corrected induced pluripotent stem cell (iPSC)-derived neurons. Bisulfite deep sequencing of the FRDA-DMR in peripheral blood mononuclear cells from 73 FRDA patients revealed considerable intra-individual epiallelic variability, including fully methylated, partially methylated, and unmethylated epialleles. Although unmethylated epialleles were rare (median = 0.33%) in typical patients homozygous for long GAA alleles with > 500 triplets, a significantly higher prevalence of unmethylated epialleles (median = 9.8%) was observed in patients with at least one allele containing < 500 triplets, less severe FXN deficiency (>20%) and later onset (>15 years). The higher prevalence in mild FRDA of somatic FXN epialleles devoid of DNA methylation is consistent with variegated epigenetic silencing mediated by expanded triplet-repeats. The proportion of unsilenced somatic FXN genes is an unrecognized phenotypic determinant in FRDA and has implications for the deployment of effective therapies.

Figure 1

DNA methylation spreads upstream from the expanded GAA triplet-repeat to establish the FRDA-DMR. (A) Schematic of the FXN gene shows the locations of the five amplicons (amp1-amp5) used for DNA methylation analysis, relative to CpG sites (vertical black lines), transcriptional start site (TSS, at −59), CpG island (light blue box), exon 1 (Ex 1) and the GAA repeat in intron 1 (triangle). The FRDA-DMR, represented by amp3, is indicated by a black dashed box. The numbering of CpG sites starts in the promoter region and continues through to 95 at the end of amp5. The three CpGs downstream of 95, located within the Alu element (orange box), were not analyzed in this study. The exact positions of amps 1–5, and the CpGs contained within them, are listed in Supplementary Material, Table S1. (B) DNA methylation (%), calculated from n = 1000 reads at the CpGs in amp2-amp5, is shown for FRDA (white bars; red trendline) and non-FRDA (black bars and trendline) in both PBMCs (n = 3 each) and LBCLs (n = 2 each). Upstream spreading of DNA methylation in FRDA tapers off at the 3′ end of amp2 (3′ end of the CpG island). Amp3 is uniformly hypermethylated in FRDA and almost devoid of methylation in non-FRDA, thus establishing the FRDA-DMR just downstream of the CpG island. (C) Bisulfite sequencing reads (n = 300) of the 11 CpGs contained in the FRDA-DMR (amp3), assayed for methylation in cis, are stacked vertically and sorted with highest methylation at the bottom (black dash = methylated CpG). Representative results shown for non-FRDA, FRDA and heterozygous carriers (HET) from PBMCs (left) and LBCLs (right) show that the FRDA-DMR is hypermethylated in FRDA (>90% versus < 10% in non-FRDA), and heterozygous carriers have approximately half methylated and half unmethylated FXN strands.

Figure 2

The FRDA-DMR is variably hypermethylated in FRDA, and patients with shorter expanded repeats have a higher prevalence of unmethylated FXN epialleles. (A) Size distribution of triplet-repeats (GAA1 and GAA2 represent the shorter and longer allele, respectively) and (B) the range of methylation in the FRDA-DMR in our cohort of 73 FRDA patients homozygous for the expanded GAA repeat (the heavy dotted line and the two light dotted lines in the violin plots represent the median and 25th/75th percentiles, respectively). Average methylation values for three heterozygous carriers (HET) and three non-FRDA controls (and standard deviation) are indicated by horizontal lines. (C) Hypermethylation and epiallelic variability of the FRDA-DMR in patients with a GAA1 of > 500 triplets showing a higher proportion of fully methylated epialleles (red bracket) and few unmethylated epialleles (blue bracket). (D) Hypermethylation and epiallelic variability of the FRDA-DMR in FRDA patients with a GAA1 of ≤ 500 triplets showing a higher proportion of unmethylated epialleles (blue bracket) and fewer fully methylated epialleles (red bracket). Both groups of patients show similar levels of partially methylated epialleles (gray bracket). (E) When FRDA-DMR methylation is relatively low (<80%), the epiallelic variability is mostly determined by the prevalence of unmethylated epialleles, and with high levels of methylation (>80%) this is mostly driven by the prevalence of fully methylated epialleles. R² values are from Pearson correlation and in both cases P < 0.0001 (95% confidence intervals are indicated). (F) The correlation of FRDA-DMR methylation with total repeat length (GAA1 + GAA2) indicates that most of the correlation is due to patients with a GAA1 of ≤ 500 triplets. R² value is from second order polynomial correlation, and the 95% confidence interval is indicated. (G) Patients with a GAA1 of ≤ 500 triplets have a significantly higher prevalence of unmethylated epialleles compared with patients with a GAA1 of > 500 triplets (median = 9.8% vs. 0.33%; Mann–Whitney test P < 0.0001).

Figure 3

FRDA-DMR methylation is predictive of FXN transcriptional deficiency in FRDA. (A) The range of FXN transcript levels in 50 FRDA patients, relative to three heterozygous carriers (HET; set to 0.5). (B) The range of FRDA-DMR methylation level in the cohort of 50 patients, relative to three heterozygous carriers (HET) and three non-FRDA controls (the heavy dotted line, and the two light dotted lines in the violin plots represent the median and 25th/75th percentiles, respectively). (C) Bar graph showing R² values (Pearson) for the correlation of residual FXN transcript levels in the 50 patients with methylation at each CpG site in amps 2–5. Note the clustering of CpG sites showing significant correlation (blue bars), with R² values ~ 0.5, in the FRDA-DMR (dashed box). ^†Evans-Galea et al. (37) reported significant correlation of FXN transcript deficiency with methylation at CpG #90. (D) FXN transcript deficiency in FRDA is inversely correlated with FRDA-DMR methylation and (E) directly correlated with the prevalence of unmethylated epialleles, and in both cases this correlation is mostly driven by patients with a GAA1 of ≤ 500 triplets. (F) LASSO regression analysis determined that FRDA-DMR methylation and repeat lengths contribute equally to FXN deficiency in FRDA. Multiple regression considered GAA1, GAA2, GAA1 + GAA2 and DNA methylation in and around the FRDA-DMR (see boxes indicating M1–5 in C; the CpGs included were M1 = 68–82, M2 = 68–71, M3 = 72–82, M4 = 72–78, M5 = 79–82). Methylation in M4 and the total repeat length (GAA1 + GAA2) were the strongest drivers of FXN transcript deficiency.

Figure 4

FRDA-DMR methylation is predictive of age of onset in FRDA. (A) The range of age of onset is shown for 71 FRDA patients (the heavy dotted line, and the two light dotted lines in the violin plot represent the median and 25th/75th percentiles, respectively). (B) Bar graph showing R² values (Pearson) for the correlation of age of onset in the 71 patients with methylation at each CpG site in amps 2–5. Note the clustering of CpG sites showing significant correlation (blue bars), with R² values ~ 0.5, in the FRDA-DMR (dashed box). ^†Evans-Galea et al. (37) reported significant correlation of age of onset and methylation at CpGs 90 and 94. ^‡Castaldo et al. (36) reported significant correlation of age of onset and methylation CpGs 88 and 89. (C) Age of onset in FRDA is inversely correlated with FRDA-DMR methylation and (D) directly correlated with the prevalence of unmethylated epialleles, and in both cases this correlation is mostly driven by patients with a GAA1 of ≤ 500 triplets. R² values are from Pearson correlations.

Figure 5

FRDA-specific and expansion-dependent hypermethylation of the FRDA-DMR in neurons. (A) The FRDA-DMR is hypermethylated in iPSC-derived neurons from three FRDA patients (FA1, FA2, FA3), as compared with (B) three non-FRDA controls (N1, N2, N3). The expanded GAA alleles in the three patient-derived neurons are as follows: FA1 = 550/720; FA2 = 620/620; FA3 = 510/720. (C) The methylation pattern in neurons from a seamlessly corrected, isogenic iPSC control (FA3^*) reverted to the non-FRDA state. FA3^* is homozygous for GAA-6, and otherwise genetically identical to FA3. (D) FRDA neurons have no unmethylated epialleles and a high prevalence of fully methylated epialleles. Neurons from non-FRDA controls and from the isogenically corrected control showed the reciprocal pattern of absence of fully methylated epialleles and a higher prevalence of unmethylated epialleles.

Figure 6

Model of variegated silencing in FRDA. Ten cells per hypothetical person are shown (circles), with each semicircle representing a somatic FXN epiallele (methylated = shaded). (A) A non-FRDA control (GAA-8/8), heterozygous carrier (GAA-8/800), and a typical FRDA patient with both expanded alleles containing > 500 triplets (GAA-800/800) show homogeneous distributions of FXN epialleles, based on the normal allele (GAA-8) remaining unmethylated and the expanded allele (GAA-800) becoming fully methylated. This is consistent with FRDA-DMR methylation of < 10% in non-FRDA, ~ 50% in heterozygotes and > 90% in typical FRDA patients, who also have very few, if any, unmethylated epialleles. (B) FRDA patients with at least one allele containing ≤ 500 triplets have FRDA-DMR methylation levels of 50–80% and show a variable proportion of cells with unmethylated FXN epialleles. This model predicts that the proportion of cells spared from epigenetic silencing, i.e. variegated silencing, is a determinant of the milder FRDA phenotype seen in FRDA patients with relatively short expanded repeats.