Retinal hypoplasia and degeneration result in vision loss in Friedreich ataxia.

OBJECTIVE: Friedreich ataxia (FRDA) is an inherited condition caused by a GAA triplet repeat (GAA-TR) expansion in the FXN gene. Clinical features of FRDA include ataxia, cardiomyopathy, and in some, vision loss. In this study, we characterize features of vision loss in a large cohort of adults and children with FRDA. METHODS: Using optical coherence tomography (OCT), we measured peripapillary retinal nerve fiber layer (RNFL) thickness in 198 people with FRDA, and 77 controls. Sloan letter charts were used to determine visual acuity. RNFL thickness and visual acuity were compared to measures of disease severity obtained from the Friedreich Ataxia Clinical Outcomes Measures Study (FACOMS). RESULTS: The majority of patients, including children, had pathologically thin RNFLs (mean = 73 ± 13 µm in FRDA; 98 ± 9 µm in controls) and low-contrast vision deficits early in the disease course. Variability in RNFL thickness in FRDA (range: 36 to 107 µm) was best predicted by disease burden (GAA-TR length X disease duration). Significant deficits in high-contrast visual acuity were apparent in patients with an RNFL thickness of ≤68 µm. RNFL thickness decreased at a rate of -1.2 ± 1.4 µm/year and reached 68 µm at a disease burden of approximately 12,000 GAA years, equivalent to disease duration of 17 years for participants with 700 GAAs. INTERPRETATION: These data suggest that both hypoplasia and subsequent degeneration of the RNFL may be responsible for the optic nerve dysfunction in FRDA and support the development of a vision-directed treatment for selected patients early in the disease to prevent RNFL loss from reaching the critical threshold.

A Novel Metric for Predicting Severity of Disease Features in Friedreich's Ataxia.

BACKGROUND: Friedreich's ataxia (FRDA), most commonly caused by a GAA triplet repeat (GAA-TR) expansion in intron 1 of the FXN gene, is characterized by deficiency of frataxin protein and clinical features such as progressive ataxia, dysarthria, impaired proprioception and vibration, abolished deep tendon reflexes, Babinski sign, and vision loss in association with non-neurological features such as skeletal anomalies, hearing loss, cardiomyopathy, and diabetes. Pathogenic GAA-TRs range in size from 60 to 1500 triplets and negatively correlate with age of onset. Clinical severity is predicted by a combination of GAA-TR length and disease duration (DD) via multivariable regressions, which cannot typically be used for the small sample sizes in most studies on this rare disease. OBJECTIVE: We aimed to develop a single metric, which we call "disease burden" (DB), that encompasses both GAA-TR length and DD for predicting disease features of FRDA in small sample sizes. METHODS: Linear regression and multivariable regression analysis was used to determine correlation coefficients between different disease features of FRDA. RESULTS: Using large datasets for validation, we found that DB predicts measures of neurological dysfunction in FRDA better than GAA-TR length or DD. Analogous results were found using small datasets. CONCLUSIONS: FRDA DB is a novel metric of disease severity that has utility in small datasets to demonstrate correlations that would not otherwise be evident with either GAA-TR or DD alone. This is important for discovering new biomarkers, as well as improving the prediction of severity of disease features in FRDA. © 2023 International Parkinson and Movement Disorder Society.

FXN gene methylation determines carrier status in Friedreich ataxia.

BACKGROUND: Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat (GAA-TRE) in intron 1 of the FXN gene. Some patients are compound heterozygous for the GAA-TRE and another FXN pathogenic variant. Detection of the GAA-TRE in the heterozygous state, occasionally technically challenging, is essential for diagnosing compound heterozygotes and asymptomatic carriers. OBJECTIVE: We explored if the FRDA differentially methylated region (FRDA-DMR) in intron 1, which is hypermethylated in cis with the GAA-TRE, effectively detects heterozygous GAA-TRE. METHODS: FXN DNA methylation was assayed by targeted bisulfite deep sequencing using the Illumina platform. RESULTS: FRDA-DMR methylation effectively identified a cohort of known heterozygous carriers of the GAA-TRE. In an individual with clinical features of FRDA, commercial testing showed a paternally inherited pathogenic FXN initiation codon variant but no GAA-TRE. Methylation in the FRDA-DMR effectively identified the proband, his mother and various maternal relatives as heterozygous carriers of the GAA-TRE, thus confirming the diagnosis of FRDA. CONCLUSION: FXN DNA methylation reliably detects the GAA-TRE in the heterozygous state and offers a robust alternative strategy to diagnose FRDA due to compound heterozygosity and to identify asymptomatic heterozygous carriers of the GAA-TRE.

A non-synonymous single nucleotide polymorphism in SIRT6 predicts neurological severity in Friedreich ataxia.

Introduction: Friedreich ataxia (FRDA) is a recessive neurodegenerative disease characterized by progressive ataxia, dyscoordination, and loss of vision. The variable length of the pathogenic GAA triplet repeat expansion in the FXN gene in part explains the interindividual variability in the severity of disease. The GAA repeat expansion leads to epigenetic silencing of FXN; therefore, variability in properties of epigenetic effector proteins could also regulate the severity of FRDA. Methods: In an exploratory analysis, DNA from 88 individuals with FRDA was analyzed to determine if any of five non-synonymous SNPs in HDACs/SIRTs predicted FRDA disease severity. Results suggested the need for a full analysis at the rs352493 locus in SIRT6 (p.Asn46Ser). In a cohort of 569 subjects with FRDA, disease features were compared between subjects homozygous for the common thymine SIRT6 variant (TT) and those with the less common cytosine variant on one allele and thymine on the other (CT). The biochemical properties of both variants of SIRT6 were analyzed and compared. Results: Linear regression in the exploratory cohort suggested that an SNP (rs352493) in SIRT6 correlated with neurological severity in FRDA. The follow-up analysis in a larger cohort agreed with the initial result that the genotype of SIRT6 at the locus rs352493 predicted the severity of disease features of FRDA. Those in the CT SIRT6 group performed better on measures of neurological and visual function over time than those in the more common TT SIRT6 group. The Asn to Ser amino acid change resulting from the SNP in SIRT6 did not alter the expression or enzymatic activity of SIRT6 or frataxin, but iPSC-derived neurons from people with FRDA in the CT SIRT6 group showed whole transcriptome differences compared to those in the TT SIRT6 group. Conclusion: People with FRDA in the CT SIRT6 group have less severe neurological and visual dysfunction than those in the TT SIRT6 group. Biochemical analyses indicate that the benefit conferred by T to C SNP in SIRT6 does not come from altered expression or enzymatic activity of SIRT6 or frataxin but is associated with changes in the transcriptome.

Frataxin controls ketone body metabolism through regulation of OXCT1.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of mitochondrial protein frataxin, which plays a crucial role in iron-sulphur cluster formation and ATP production. The cellular function of frataxin is not entirely known. Here, we demonstrate that frataxin controls ketone body metabolism through regulation of 3-Oxoacid CoA-Transferase 1 (OXCT1), a rate limiting enzyme catalyzing the conversion of ketone bodies to acetoacetyl-CoA that is then fed into the Krebs cycle. Biochemical studies show a physical interaction between frataxin and OXCT1 both in vivo and in vitro. Frataxin overexpression also increases OXCT1 protein levels in human skin fibroblasts while frataxin deficiency decreases OXCT1 in multiple cell types including cerebellum and skeletal muscle both acutely and chronically, suggesting that frataxin directly regulates OXCT1. This regulation is mediated by frataxin-dependent suppression of ubiquitin-proteasome system (UPS)-dependent OXCT1 degradation. Concomitantly, plasma ketone bodies are significantly elevated in frataxin deficient knock-in/knockout (KIKO) mice with no change in the levels of other enzymes involved in ketone body production. In addition, ketone bodies fail to be metabolized to acetyl-CoA accompanied by increased succinyl-CoA in vitro in frataxin deficient cells, suggesting that ketone body elevation is caused by frataxin-dependent reduction of OXCT1 leading to deficits in tissue utilization of ketone bodies. Considering the potential role of metabolic abnormalities and deficiency of ATP production in FRDA, our results suggest a new role for frataxin in ketone body metabolism and also suggest modulation of OXCT1 may be a potential therapeutic approach for FRDA.

Friedreich ataxia: clinical features and new developments.

Friedreich's ataxia (FRDA), a neurodegenerative disease characterized by ataxia and other neurological features, affects 1 in 50,000-100,000 individuals in the USA. However, FRDA also includes cardiac, orthopedic and endocrine dysfunction, giving rise to many secondary disease characteristics. The multifaceted approach for clinical care has necessitated the development of disease-specific clinical care guidelines. New developments in FRDA include the advancement of clinical drug trials targeting the NRF2 pathway and frataxin restoration. Additionally, a novel understanding of gene silencing in FRDA, reflecting a variegated silencing pattern, will have applications to current and future therapeutic interventions. Finally, new perspectives on the neuroanatomy of FRDA and its developmental features will refine the time course and anatomical targeting of novel approaches.

Friedreich's ataxia (FRDA), mainly referred to as a disorder of balance, is characterized by loss of coordination (ataxia) in the arms and legs and other neurological features, affecting about 1 in 50,000 people in the USA. FRDA also includes serious heart disease, aggressive scoliosis, diabetes and many other disease characteristics. Due to various clinical care needs, disease-specific clinical care guidelines have been created. New developments in FRDA include the advancement of clinical drug trials targeting cell signaling pathways and restoration of the deficient protein found in individuals with FRDA. Additionally, a new understanding of the role of the various genetic factors that contribute to the development of FRDA could affect current and future therapies. Finally, new perspectives on the early developmental features of FRDA will help refine the time course and accelerate new treatments.

Clinical Evidence for Variegated Silencing in Patients With Friedreich Ataxia.

Background and Objectives: Friedreich ataxia (FRDA) is a neurodegenerative disease caused by a GAA triplet repeat (GAA-TR) expansion in intron 1 of the FXN gene. Patients have 100-1,300 GAA triplets compared with less than 30 in healthy controls. The GAA-TR expansion leads to FXN silencing, and consequent frataxin protein deficiency results in progressive ataxia, scoliosis, cardiomyopathy, and diabetes. The overt heterogeneity in age at onset and disease severity is explained partly by the length of the GAA-TR, in which shorter repeats correlate with milder disease. Evidence of variegated silencing in FRDA suggests that patients with shorter repeats retain a significant proportion of cells with FXN genes that have escaped GAA-TR expansion-induced silencing, explaining the less severe frataxin deficiency in this subpopulation. In ex vivo experiments, the proportion of spared cells negatively correlates with GAA-TR length until it plateaus at 500 triplets, an indication that the maximal number of silenced cells has been reached. In this study, we assessed whether an analogous ceiling effect occurs in severity of clinical features of FRDA by analyzing clinical outcome data. Methods: The FRDA Clinical Outcome Measures Study database was used for a cross-sectional analysis of 1,000 patients with FRDA. Frataxin levels were determined by lateral flow immunoassays. Results: The length of the GAA-TR in our cohort predicted frataxin level (R2 = 0.38, p < 0.0001) and age at onset (R2 = 0.46, p < 0.0001) but only with GAA-TRs with ≤700 triplets. Age and disease duration predicted performance on clinical outcome measures, and such predictions in linear regression models statistically improved in the subcohort of patients with >700 GAA triplets. The prevalence of cardiomyopathy and scoliosis increased as GAA-TR length increased up to 700 GAA triplets where prevalence plateaued. Discussion: Our data suggest that there is a ceiling effect on the clinical consequences of GAA-TR length in FRDA, as would be predicted by variegated silencing. Patients with GAA-TRs of >700 triplets represent a subgroup in which the severity of clinical manifestations based on GAA-TR length have reached maximal levels and therefore display limited clinical variability in disease progression.

Cerebellar Pathology in an Inducible Mouse Model of Friedreich Ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by deficiency of the mitochondrial protein frataxin. Lack of frataxin causes neuronal loss in various areas of the CNS and PNS. In particular, cerebellar neuropathology in FRDA patients includes loss of large principal neurons and synaptic terminals in the dentate nucleus (DN), and previous studies have demonstrated early synaptic deficits in the Knockin-Knockout mouse model of FRDA. However, the exact correlation of frataxin deficiency with cerebellar neuropathology remains unclear. Here we report that doxycycline-induced frataxin knockdown in a mouse model of FRDA (FRDAkd) leads to synaptic cerebellar degeneration that can be partially reversed by AAV8-mediated frataxin restoration. Loss of cerebellar Purkinje neurons and large DN principal neurons are observed in the FRDAkd mouse cerebellum. Levels of the climbing fiber-specific glutamatergic synaptic marker VGLUT2 decline starting at 4 weeks after dox induction, whereas levels of the parallel fiber-specific synaptic marker VGLUT1 are reduced by 18-weeks. These findings suggest initial selective degeneration of climbing fiber synapses followed by loss of parallel fiber synapses. The GABAergic synaptic marker GAD65 progressively declined during dox induction in FRDAkd mice, while GAD67 levels remained unaltered, suggesting specific roles for frataxin in maintaining cerebellar synaptic integrity and function during adulthood. Expression of frataxin following AAV8-mediated gene transfer partially restored VGLUT1/2 levels. Taken together, our findings show that frataxin knockdown leads to cerebellar degeneration in the FRDAkd mouse model, suggesting that frataxin helps maintain cerebellar structure and function.

DNA methylation in Friedreich ataxia silences expression of frataxin isoform E.

Epigenetic silencing in Friedreich ataxia (FRDA), induced by an expanded GAA triplet-repeat in intron 1 of the FXN gene, results in deficiency of the mitochondrial protein, frataxin. A lesser known extramitochondrial isoform of frataxin detected in erythrocytes, frataxin-E, is encoded via an alternate transcript (FXN-E) originating in intron 1 that lacks a mitochondrial targeting sequence. We show that FXN-E is deficient in FRDA, including in patient-derived cell lines, iPS-derived proprioceptive neurons, and tissues from a humanized mouse model. In a series of FRDA patients, deficiency of frataxin-E protein correlated with the length of the expanded GAA triplet-repeat, and with repeat-induced DNA hypermethylation that occurs in close proximity to the intronic origin of FXN-E. CRISPR-induced epimodification to mimic DNA hypermethylation seen in FRDA reproduced FXN-E transcriptional deficiency. Deficiency of frataxin E is a consequence of FRDA-specific epigenetic silencing, and therapeutic strategies may need to address this deficiency.

Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation.

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.

Designing phase II clinical trials in Friedreich ataxia.

INTRODUCTION: Friedreich ataxia (FRDA) is an autosomal recessive disorder caused by deficiency of frataxin, an essential mitochondrial protein involved in iron sulfur cluster biogenesis, oxidative phosphorylation and other processes. FRDA most notably affects the heart, sensory neurons, spinal cord, cerebellum, and other brain regions, and manifests clinically as ataxia, sensory loss, dysarthria, spasticity, and hypertrophic cardiomyopathy. Therapeutic approaches in FRDA have consisted of two different approaches: (1) augmenting or restoring frataxin production and (2) modulating a variety of downstream processes related to mitochondrial dysfunction, including reactive oxygen species production, ferroptosis, or Nrf2 activation. AREAS COVERED: In this review, we summarize data from major phase II clinical trials in FRDA published between 2015 and 2020, which includes A0001/EPI743, Omaveloxolone, RT001, and Actimmune. EXPERT OPINION: A growing number of drug candidates are being tested in phase II clinical trials for FRDA; however, most have not met their primary endpoints, and none have received FDA approval. In this review, we aim to summarize completed phase II clinical trials in FRDA, outlining critical lessons that have been learned and that should be incorporated into future trial design to ultimately optimize drug development in FRDA.

Methylated and unmethylated epialleles support variegated epigenetic silencing in Friedreich ataxia.

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.