BackgroundOver 15 inherited diseases are caused by expansion of triplet-repeats. Friedreich ataxia (FRDA) patients are homozygous for an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene. The expanded GAA triplet-repeat results in deficiency of FXN gene transcription, which is reversed via administration of histone deacetylase inhibitors indicating that transcriptional silencing is at least partially due to an epigenetic abnormality.Methodology/Principal FindingsWe found a severe depletion of the chromatin insulator protein CTCF (CCCTC-binding factor) in the 5′UTR of the FXN gene in FRDA, and coincident heterochromatin formation involving the +1 nucleosome via enrichment of H3K9me3 and recruitment of heterochromatin protein 1. We identified FAST-1 (FXN Antisense Transcript – 1), a novel antisense transcript that overlaps the CTCF binding site in the 5′UTR, which was expressed at higher levels in FRDA. The reciprocal relationship of deficient FXN transcript and higher levels of FAST-1 seen in FRDA was reproduced in normal cells via knockdown of CTCF.Conclusions/SignificanceCTCF depletion constitutes an epigenetic switch that results in increased antisense transcription, heterochromatin formation and transcriptional deficiency in FRDA. These findings provide a mechanistic basis for the transcriptional silencing of the FXN gene in FRDA, and broaden our understanding of disease pathogenesis in triplet-repeat diseases.

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.

Background: Friedreich ataxia (FRDA) is caused by an expanded GAA triplet repeat (GAA-TR) mutation that results in FXN transcriptional deficiency.Results: Repressive chromatin spreads from the expanded GAA-TR mutation, switching off the FXN gene promoter.Conclusion: Reduced transcriptional initiation is the major cause of FXN transcriptional deficiency in FRDA.Significance: Reactivation of FXN transcriptional initiation is a potential therapeutic strategy in FRDA.

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.

Objective Friedreich ataxia (FRDA) is caused by an expanded GAA triplet-repeat (GAA-TR) mutation in the FXN gene. Patients are typically homozygous for expanded alleles containing 100–1300 triplets, and phenotypic severity is significantly correlated with the length of the shorter of the two expanded alleles. Patients have a severe deficiency of FXN transcript, which is predominantly caused by epigenetic silencing of the FXN promoter. We sought to determine if the severity of FXN promoter silencing is related to the length of the expanded GAA-TR mutation in FRDA. Methods Patient-derived lymphoblastoid cell lines bearing a range of expanded alleles (200–1122 triplets) were evaluated for FXN transcript levels by quantitative RT-PCR. FXN promoter function was directly measured by quantitative analysis of transcriptional initiation via metabolic labeling of newly synthesized transcripts in living cells. Results FXN transcriptional deficiency was significantly correlated with the length of the shorter of the two expanded alleles, which was noted both upstream (R2=0.84; p=0.014) and downstream (R2=0.89; p=0.002) of the expanded GAA-TR mutation, suggesting that FXN promoter silencing in FRDA is related to repeat length. A bilinear regression model revealed that length-dependence was strongest when the shorter of the two expanded alleles contained <400 triplets. Direct measurement of FXN promoter activity in patients with expanded alleles containing <400 versus >400 triplets in the shorter of the two expanded alleles revealed a significantly greater deficiency in individuals with longer GAA-TR alleles (p<0.05). Interpretation FXN promoter silencing in FRDA is dependent on the length of the expanded GAA-TR mutation.