Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia

Rodden, Layne N.; Chutake, Yogesh; Gilliam, Kaitlyn M; Lam, Christina; Soragni, Elisabetta; Hauser, Lauren; Gilliam, Matthew; Wiley, Graham B.; Anderson, Michael P.; Gottesfeld, Joel M.; Lynch, David R.; Bidichandani, Sanjay I.

doi:10.1093/hmg/ddaa267

Cited by 34 publications

(57 citation statements)

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“…Bisulfite deep sequencing to assay DNA methylation at the FXN locus was previously described in detail ( Rodden et al, 2021 ). Briefly, genomic DNA (0.5 μg), isolated from freshly isolated PBMCs, was bisulfite converted and prepared for targeted deep sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…A prominent epigenetic signature located in intron 1 of the FXN gene is an FRDA-specific differentially methylated region (FRDA-DMR), which shows >90% methylation in FRDA versus <10% in non-FRDA controls ( Rodden et al, 2021 ). Bisulfite deep sequencing of the FRDA-DMR in patients show individual FXN strands that are variably methylated, providing a quantitative measure of epigenetic heterogeneity in somatic cells.…”

Section: Introductionmentioning

confidence: 99%

“…Hypermethylation of the FRDA-DMR in FRDA, which is stable and reproducible, is broadly comprised of three types of somatic FXN epialleles: fully methylated, partially methylated, and unmethylated. The proportion of unmethylated epialleles accurately predicts FXN transcriptional deficiency and age of onset in patients ( Rodden et al, 2021 ), suggesting that variably methylated FXN epialleles reflect different functional states of the FXN gene in somatic cells. Reasoning that fully methylated epialleles may represent completely silenced FXN genes that are relatively resistant to reactivation, we hypothesized that partially methylated and unmethylated epialleles likely represent potential targets for gene reactivation.…”

Section: Introductionmentioning

confidence: 99%

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Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

et al. 2021

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

et al. 2021

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“…Although primary fibroblasts have been widely used for studying transcription of the FXN gene ( 9 , 29 , 39 , 41 , 48 ), it is essential to also determine the efficacy of ON treatment in human cells relevant to disease pathology. We tested ON pairs using FRDA NPCs differentiated from iPSCs.…”

Section: Resultsmentioning

confidence: 99%

Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

Wang

et al. 2021

Nucleic Acids Research

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Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin ( FXN ) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.

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“…Essentially, all children have GAA1 lengths greater than 500 repeats, the threshold at which additional increases in GAA1 length do not substantially decrease the level FXN silencing. 19 …”

Section: Discussionmentioning

confidence: 99%

Body Mass Index and Height in the Friedreich Ataxia Clinical Outcome Measures Study

et al. 2021

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Background and ObjectivesBody mass index (BMI) and height are important indices of health. We tested the association between these outcomes and clinical characteristics in Friedreich ataxia (FRDA), a progressive neuromuscular disorder.MethodsParticipants (N = 961) were enrolled in a prospective natural history study (Friedreich Ataxia Clinical Outcome Measure Study). Age- and sex-specific BMI and height Z-scores were calculated using CDC 2000 references for participants younger than 18 years. For adults aged 18 years or older, height Z-scores were also calculated, and absolute BMI was reported. Univariate and multivariate linear regression analyses tested the associations between exposures, covariates, and BMI or height measured at the baseline visit. In children, the superimposition by translation and rotation analysis method was used to compare linear growth trajectories between FRDA and a healthy reference cohort, the Bone Mineral Density in Childhood Study (n = 1,535 used for analysis).ResultsMedian age at the baseline was 20 years (IQR, 13–33 years); 49% (n = 475) were women. A substantial proportion of children (17%) were underweight (BMI-Z < fifth percentile), and female sex was associated with lower BMI-Z (β = −0.34, p < 0.05). In adults, older age was associated with higher BMI (β = 0.09, p < 0.05). Regarding height, in children, older age (β −0.06, p < 0.05) and worse modified Friedreich Ataxia Rating Scale (mFARS) scores (β = −1.05 for fourth quartile vs first quartile, p < 0.01) were associated with shorter stature. In girls, the magnitude of the pubertal growth spurt was less, and in boys, the pubertal growth spurt occurred later (p < 0.001 for both) than in a healthy reference cohort. In adults, in unadjusted analyses, both earlier age of FRDA symptom onset (=0.09, p < 0.05) and longer guanine-adenine-adenine repeat length (shorter of the 2 GAA repeats, β = −0.12, p < 0.01) were associated with shorter stature. Both adults and children with higher mFARS scores and/or who were nonambulatory were less likely to have height and weight measurements recorded at clinical visits.DiscussionFRDA affects both weight gain and linear growth. These insights will inform assessments of affected individuals in both research and clinical settings.

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Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia

Cited by 34 publications

References 50 publications

Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

Body Mass Index and Height in the Friedreich Ataxia Clinical Outcome Measures Study

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