A Milestone in the Treatment of Ataxias: Approval of Omaveloxolone for Friedreich Ataxia.

The exciting news about the US FDA approval of omaveloxolone as the first-ever drug to be approved for an inherited ataxia is welcome news for patients and families that deal with this devastating disease as well as for health care providers and investigators with an interest in this and other rare diseases. This event is the culmination of long and fruitful collaboration between patients, their families, clinicians, laboratory researchers, patient advocacy organizations, industry, and regulatory agencies. The process has generated intense discussion about outcome measures, biomarkers, trial design, and the nature of approval process for such diseases. It also has brought hope and enthusiasm for increasingly better therapies for genetic diseases in general.

Clinically Meaningful Magnetic Resonance Endpoints Sensitive to Preataxic Spinocerebellar Ataxia Types 1 and 3.

OBJECTIVE: This study was undertaken to identify magnetic resonance (MR) metrics that are most sensitive to early changes in the brain in spinocerebellar ataxia type 1 (SCA1) and type 3 (SCA3) using an advanced multimodal MR imaging (MRI) protocol in the multisite trial setting. METHODS: SCA1 or SCA3 mutation carriers and controls (n = 107) underwent MR scanning in the US-European READISCA study to obtain structural, diffusion MRI, and MR spectroscopy data using an advanced protocol at 3T. Morphometric, microstructural, and neurochemical metrics were analyzed blinded to diagnosis and compared between preataxic SCA (n = 11 SCA1, n = 28 SCA3), ataxic SCA (n = 14 SCA1, n = 37 SCA3), and control (n = 17) groups using nonparametric testing accounting for multiple comparisons. MR metrics that were most sensitive to preataxic abnormalities were identified using receiver operating characteristic (ROC) analyses. RESULTS: Atrophy and microstructural damage in the brainstem and cerebellar peduncles and neurochemical abnormalities in the pons were prominent in both preataxic groups, when patients did not differ from controls clinically. MR metrics were strongly associated with ataxia symptoms, activities of daily living, and estimated ataxia duration. A neurochemical measure was the most sensitive metric to preataxic changes in SCA1 (ROC area under the curve [AUC] = 0.95), and a microstructural metric was the most sensitive metric to preataxic changes in SCA3 (AUC = 0.92). INTERPRETATION: Changes in cerebellar afferent and efferent pathways underlie the earliest symptoms of both SCAs. MR metrics collected with a harmonized advanced protocol in the multisite trial setting allow detection of disease effects in individuals before ataxia onset with potential clinical trial utility for subject stratification. ANN NEUROL 2022.

Efficacy of Omaveloxolone in Friedreich's Ataxia: Delayed-Start Analysis of the MOXIe Extension.

BACKGROUND: MOXIe was a two-part study evaluating the safety and efficacy of omaveloxolone in patients with Friedreich's ataxia, a rare, progressive neurological disease with no proven therapy. MOXIe part 2, a randomized double-blind placebo-controlled trial, showed omaveloxolone significantly improved modified Friedreich's Ataxia Rating Scale (mFARS) scores relative to placebo. Patients who completed part 1 or 2 were eligible to receive omaveloxolone in an open-label extension study. OBJECTIVE: The delayed-start study compared mFARS scores at the end of MOXIe part 2 with those at 72 weeks in the open-label extension period (up to 144 weeks) for patients initially randomized to omaveloxolone versus those initially randomized to placebo. METHODS: We performed a noninferiority test to compare the difference between treatment groups (placebo to omaveloxolone versus omaveloxolone to omaveloxolone) using a single mixed model repeated measures (MMRM) model. In addition, slopes of the change in mFARS scores were compared between both groups in the open-label extension. RESULTS: The noninferiority testing demonstrated that the difference in mFARS between omaveloxolone and placebo observed at the end of placebo-controlled MOXIe part 2 (-2.17 ± 1.09 points) was preserved after 72 weeks in the extension (-2.91 ± 1.44 points). In addition, patients previously randomized to omaveloxolone in MOXIe part 2 continued to show no worsening in mFARS relative to their extension baseline through 144 weeks. CONCLUSIONS: These results support the positive results of MOXIe part 2 and indicate a persistent benefit of omaveloxolone treatment on disease course in Friedreich's ataxia. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F.

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG-initiated polyGln and a polyAla protein expressed by repeat-associated non-ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady-state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.

Safety and Efficacy of Omaveloxolone in Friedreich Ataxia (MOXIe Study).

OBJECTIVE: Friedreich ataxia (FA) is a progressive genetic neurodegenerative disorder with no approved treatment. Omaveloxolone, an Nrf2 activator, improves mitochondrial function, restores redox balance, and reduces inflammation in models of FA. We investigated the safety and efficacy of omaveloxolone in patients with FA. METHODS: We conducted an international, double-blind, randomized, placebo-controlled, parallel-group, registrational phase 2 trial at 11 institutions in the United States, Europe, and Australia (NCT02255435, EudraCT2015-002762-23). Eligible patients, 16 to 40 years of age with genetically confirmed FA and baseline modified Friedreich's Ataxia Rating Scale (mFARS) scores between 20 and 80, were randomized 1:1 to placebo or 150mg per day of omaveloxolone. The primary outcome was change from baseline in the mFARS score in those treated with omaveloxolone compared with those on placebo at 48 weeks. RESULTS: One hundred fifty-five patients were screened, and 103 were randomly assigned to receive omaveloxolone (n = 51) or placebo (n = 52), with 40 omaveloxolone patients and 42 placebo patients analyzed in the full analysis set. Changes from baseline in mFARS scores in omaveloxolone (-1.55 ± 0.69) and placebo (0.85 ± 0.64) patients showed a difference between treatment groups of -2.40 ± 0.96 (p = 0.014). Transient reversible increases in aminotransferase levels were observed with omaveloxolone without increases in total bilirubin or other signs of liver injury. Headache, nausea, and fatigue were also more common among patients receiving omaveloxolone. INTERPRETATION: In the MOXIe trial, omaveloxolone significantly improved neurological function compared to placebo and was generally safe and well tolerated. It represents a potential therapeutic agent in FA. ANN NEUROL 2021;89:212-225.

Temporal Invariance in SCA6 Is Related to Smaller Cerebellar Lobule VI and Greater Disease Severity.

Regulating muscle force and timing are fundamental for accurate motor performance. In spinocerebellar ataxia type 6 (SCA6), there is evidence that individuals have greater force dysmetria but display better temporal accuracy during fast goal directed contractions. Here, we test whether greater temporal accuracy occurs in all individuals with SCA6, and can be explained by lesser temporal variability. Further we examine whether it is linked to disease severity and specific degenerative changes in the cerebellum. Nineteen human participants with SCA6 (13 woman) and 18 healthy controls performed fast goal-directed ankle dorsiflexion contractions aiming at a spatiotemporal target. We quantified the endpoint control of these contractions, gray matter (GM) integrity of the cerebellum, and disease severity using the International Cooperative Ataxia Rating Scale (ICARS). SCA6 individuals exhibited lower temporal endpoint error and variability than the healthy controls (p = 0.008). Statistically, SCA6 clustered into two distinct groups for temporal variability. A group with low temporal variability ranging from 10 to 19% (SCA6a) and a group with temporal variability similar to healthy controls (SCA6b; 19-40%).SCA6a exhibited greater disease severity than SCA6b, as assessed with ICARS (p < 0.001). Lower temporal variability, which was not associated with disease duration (R2 = 0.1, p > 0.2), did correlate with both greater ICARS (R2 = 0.3) and reduced GM volume in cerebellar lobule VI (R2 = 0.35). Other cerebellar lobules did not relate to temporal variability. We provide new evidence that a subset of SCA6 with greater loss of GM in cerebellum lobule VI exhibit temporal invariance and more severe ataxia than other SCA6 individuals.SIGNIFICANCE STATEMENT Variability is an inherent feature of voluntary movement, and traditionally more variability in the targeted output infers impaired performance. For example, cerebellar patients present exacerbated temporal variability during multijoint movements, which is thought to contribute to their motor deficits. In the current work, we show that in a subgroup of spinocerebellar ataxia type 6 individuals, temporal variability is lower than that of healthy controls when performing single-joint fast-goal directed movements. This invariance related to exacerbated atrophy of lobule VI of the cerebellum and exacerbated disease severity. The relation between invariance and disease severity suggests that pathological motor variability can manifest not only as an exacerbation but also as a reduction relative to healthy controls.

Improving the efficacy of exome sequencing at a quaternary care referral centre: novel mutations, clinical presentations and diagnostic challenges in rare neurogenetic diseases.

BACKGROUND: We used a multimodal approach including detailed phenotyping, whole exome sequencing (WES) and candidate gene filters to diagnose rare neurological diseases in individuals referred by tertiary neurology centres. METHODS: WES was performed on 66 individuals with neurogenetic diseases using candidate gene filters and stringent algorithms for assessing sequence variants. Pathogenic or likely pathogenic missense variants were interpreted using in silico prediction tools, family segregation analysis, previous publications of disease association and relevant biological assays. RESULTS: Molecular diagnosis was achieved in 39% (n=26) including 59% of childhood-onset cases and 27% of late-onset cases. Overall, 37% (10/27) of myopathy, 41% (9/22) of neuropathy, 22% (2/9) of MND and 63% (5/8) of complex phenotypes were given genetic diagnosis. Twenty-seven disease-associated variants were identified including ten novel variants in FBXO38, LAMA2, MFN2, MYH7, PNPLA6, SH3TC2 and SPTLC1. Single-nucleotide variants (n=10) affected conserved residues within functional domains and previously identified mutation hot-spots. Established pathogenic variants (n=16) presented with atypical features, such as optic neuropathy in adult polyglucosan body disease, facial dysmorphism and skeletal anomalies in cerebrotendinous xanthomatosis, steroid-responsive weakness in congenital myasthenia syndrome 10. Potentially treatable rare diseases were diagnosed, improving the quality of life in some patients. CONCLUSIONS: Integrating deep phenotyping, gene filter algorithms and biological assays increased diagnostic yield of exome sequencing, identified novel pathogenic variants and extended phenotypes of difficult to diagnose rare neurogenetic disorders in an outpatient clinic setting.

Tremor in the Degenerative Cerebellum: Towards the Understanding of Brain Circuitry for Tremor.

Cerebellar degenerative pathology has been identified in tremor patients; however, how the degenerative pathology could contribute to tremor remains unclear. If the cerebellar degenerative pathology can directly drive tremor, one would hypothesize that tremor is likely to occur in the diseases of cerebellar ataxia and follows the disease progression in such disorders. To further test this hypothesis, we studied the occurrence of tremor in different disease stages of classical cerebellar degenerative disorders: spinocerebellar ataxias (SCAs). We further separately analyzed postural tremor and rest tremor, two forms of tremor that both involve the cerebellum. We also explored tremor in different subtypes of SCAs. We found that 18.1% of SCA patients have tremor. Interestingly, SCA patients with tremor have worse ataxia than those without tremor. When stratifying patients into mild, moderate, and severe disease stages according to the severity of ataxia, moderate and severe SCA patients more commonly have tremor than those with mild ataxia, the effect most prominently observed in postural tremor of SCA3 and SCA6 patients. Finally, tremor can independently contribute to worse functional status in SCA2 patients, even after adjusting for ataxia severity. Tremor is more likely to occur in the severe stage of cerebellar degeneration when compared to mild stages. Our results partially support the cerebellar degenerative model of tremor.

C-terminal proline deletions in KCNC3 cause delayed channel inactivation and an adult-onset progressive SCA13 with spasticity.

Mutations in the potassium channel gene KCNC3 (Kv3.3) cause the autosomal dominant neurological disease, spinocerebellar ataxia 13 (SCA13). In this study, we expand the genotype-phenotype repertoire of SCA13 by describing the novel KCNC3 deletion p.Pro583_Pro585del highlighting the allelic heterogeneity observed in SCA13 patients. We characterize adult-onset, progressive clinical symptoms of two afflicted kindred and introduce the symptom of profound spasticity not previously associated with the SCA13 phenotype. We also present molecular and electrophysiological characterizations of the mutant protein in mammalian cell culture. Mechanistically, the p.Pro583_Pro585del protein showed normal membrane trafficking with an altered electrophysiological profile, including slower inactivation and decreased sensitivity to the inactivation-accelerating effects of the actin depolymerizer latrunculin B. Taken together, our results highlight the clinical importance of the intracellular C-terminal portion of Kv3.3 and its association with ion channel function.

Correction to: SPG7 and Impaired Emotional Communication.

The original version of this article unfortunately contained an incorrect assignment of affiliations of Linwei Zhang and Tetsuo Ashizawa.

SPG7 and Impaired Emotional Communication.

The goal of this report is to describe the genetic mutations of a patient with cerebellar degeneration who had ataxia and impaired emotional communication that led to damage of family relationships. We extracted genomic DNA from peripheral blood lymphocytes and performed whole exome sequencing (WES) in this patient and his unaffected parents and siblings. Found mutations were confirmed by Sanger sequencing in each individual. We found compound heterozygous mutations in the paraplegin (SPG7) gene. One mutated allele has been previously described as a disease-causing missense mutation for spastic paraplegia type 7 (SPG7) (c.1529C > T, p.Ala510Val). The second mutated allele involved a single nucleotide deletion which results in a frameshift in the coding sequence (c.2271delG, p.Met757fs*65). The second allele is similar to, but unique from, other described, SPG7-linked truncation mutations. The abnormal emotional communication in this patient broadens the phenotypic boundary of SPG7.

The Initial Symptom and Motor Progression in Spinocerebellar Ataxias.

The aim of this study is to determine whether the initial symptom associates with motor progression in spinocerebellar ataxias (SCAs). SCAs are clinically heterogeneous and the initial presentation may represent different subtypes of SCA with different motor progression. We studied 317 participants with SCAs1, 2, 3, and 6 from the Clinical Research Consortium for SCAs (CRC-SCA) and repeatedly measured the severity of ataxia for 2 years. SCA patients were divided into gait-onset and non-gait-onset (speech, vision, and hand dexterity) groups based on the initial presentation. In addition to demographic comparison, we employed regression models to study ataxia progression in these two groups after adjusting for age, sex, and pathological CAG repeats. The majority of SCA patients had gait abnormality as an initial presentation. The pathological CAG repeat expansions were similar between the gait-onset and non-gait-onset groups. In SCA1, gait-onset group progressed slower than non-gait-onset group, while gait-onset SCA6 group progressed faster than their counterpart. In addition, the disease presented 9 years later for SCA2 gait-onset group than non-gait-onset group. Initial symptoms of SCA3 did not influence age of onset or disease progression. The initial symptom in each SCA has a different influence on age of onset and motor progression. Therefore, gait and non-gait-onset groups of SCAs might represent different subtypes of the diseases.

Apraxia in anti-glutamic acid decarboxylase-associated stiff person syndrome: link to corticobasal degeneration?

Corticobasal syndrome (CBS) is associated with asymmetrical rigidity as well as asymmetrical limb-kinetic and ideomotor apraxia. Stiff person syndrome (SPS) is characterized by muscle stiffness and gait difficulties. Whereas patients with CBS have several forms of pathology, many patients with SPS have glutamic acid decarboxylase antibodies (GAD-ab), but these 2 disorders have not been reported to coexist. We report 2 patients with GAD-ab-positive SPS who also had signs suggestive of CBS, including asymmetrical limb rigidity associated with both asymmetrical limb-kinetic and ideomotor apraxia. Future studies should evaluate patients with CBS for GAD-ab and people with SPS for signs of CBS.

Genome modification leads to phenotype reversal in human myotonic dystrophy type 1 induced pluripotent stem cell-derived neural stem cells.

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3' UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step toward autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 induced pluripotent stem (iPS) cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci, the molecular hallmarks of DM1, using RNA fluorescence in situ hybridization. Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1, 2 aberrant splicing in DM1 NSCs were reversed to normal pattern in genome-modified NSCs. Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1.

RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.

The finding that a GGGGCC (G4C2) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G2C4) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G2C4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.

Coenzyme Q10 and spinocerebellar ataxias.

The aim of this study was to investigate the association between drug exposure and disease severity in SCA types 1, 2, 3 and 6. The Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA) enrolled 319 participants with SCA1, 2, 3, and 6 from 12 medical centers in the United States and repeatedly measured clinical severity by the Scale for Assessment and Rating of Ataxia (SARA), the Unified Huntington's Disease Rating Scale part IV (UHDRS-IV), and the 9-item Patient Health Questionnaire during July 2009 to May 2012. We employed generalized estimating equations in regression models to study the longitudinal effects of coenzyme Q10 (CoQ10), statin, and vitamin E on clinical severity of ataxia after adjusting for age, sex, and pathological CAG repeat number. Cross-sectionally, exposure to CoQ10 was associated with lower SARA and higher UHDRS-IV scores in SCA1 and 3. No association was found between statins, vitamin E, and clinical outcome. Longitudinally, CoQ10, statins, and vitamin E did not change the rates of clinical deterioration indexed by SARA and UHDRS-IV scores within 2 years. CoQ10 is associated with better clinical outcome in SCA1 and 3. These drug exposures did not appear to influence clinical progression within 2 years. Further studies are warranted to confirm the association.

Altered activation of the tibialis anterior in individuals with Pompe disease: Implications for motor unit dysfunction.

INTRODUCTION: Pompe disease is a progressive disease that affects skeletal muscles and leads to loss of ambulation. We investigated the activation of the tibialis anterior (TA) in late-onset Pompe disease (LOPD) individuals during maximal voluntary contraction (MVC) and evoked involuntary responses. METHODS: Four LOPD patients and matched control subjects performed MVC of the TA using dorsiflexion and TA evoked responses. Activation of the TA was recorded with surface electromyography. RESULTS: The Pompe patients exhibited greater power at frequencies below 60 Hz and reduced power above 100 Hz. They also exhibited a reduced increase in M-wave and prolonged M-wave latency and duration in response to stimulation. CONCLUSIONS: These results provide evidence that LOPD individuals have an altered activation pattern of the TA during maximal contractions. The observed activation pattern may reflect impairments in voluntary command, neuromuscular junction pathology, or compensatory drive due to a reduced number of functional motoneurons.

Clinical evaluation of eye movements in spinocerebellar ataxias: a prospective multicenter study.

BACKGROUND: Ocular motor abnormalities reflect the varied neuropathology of spinocerebellar ataxias (SCAs) and may serve to clinically distinguish the different SCAs. We analyzed the various eye movement abnormalities detected prospectively at the baseline visit during a large multicenter natural history study of SCAs 1, 2, 3, and 6. METHODS: The data were prospectively collected from 12 centers in the United States in patients with SCAs 1, 2, 3, and 6, as part of the Clinical Research Consortium for Spinocerebellar Ataxias (NIH-CRC-SCA). Patient characteristics, ataxia rating scales, the Unified Huntington Disease Rating Scale functional examination, and clinical staging were used. Eye movement abnormalities including nystagmus, disorders of saccades and pursuit, and ophthalmoparesis were recorded, and factors influencing their occurrence were examined. RESULTS: A total of 301 patients participated in this study, including 52 patients with SCA 1, 64 with SCA 2, 117 with SCA 3, and 68 with SCA 6. Although no specific ocular motor abnormality was pathognomonic to any SCA, significant differences were noted in their occurrence among different disorders. SCA 6 was characterized by frequent occurrence of nystagmus and abnormal pursuit and rarity of slow saccades and ophthalmoparesis and SCA 2 by the frequent occurrence of slow saccades and infrequent nystagmus and dysmetric saccades. SCA 1 and SCA 3 subjects had a more even distribution of eye movement abnormalities. CONCLUSIONS: Prospective data from a large cohort of patients with SCAs 1, 2, 3, and 6 provide statistical validation that the SCAs exhibit distinct eye movement abnormalities that are useful in identifying the genotypes. Many of the abnormalities correlate with greater disease severity measures.

Affective communication deficits associated with cerebellar degeneration.

The cerebellum has extensive connections with the frontal lobes. Cerebellar injury has been reported to induce frontal-executive cognitive dysfunction and blunting of affect. We examined a patient with idiopathic cerebellar degeneration with impaired family relationships attributed to an "emotional disconnection." Examination revealed ataxia, dysmetria, and adiadochokinesia more severe on the left and frontal-executive dysfunction; memory and cognitive functions were otherwise normal. Testing of emotional communication included assessments of emotional semantic knowledge, emotional prosody, and emotional facial expressions. Comprehension was normal but expression was severely impaired. Cerebellar dysfunction can cause a defect in facial and prosodic emotional communication.

Frataxin analysis using triple quadrupole mass spectrometry: application to a large heterogeneous clinical cohort.

BACKGROUND: Friedreich ataxia is a progressive multisystem disorder caused by deficiency of the protein frataxin; a small mitochondrial protein involved in iron sulfur cluster synthesis. Two types of frataxin exist: FXN-M, found in most cells, and FXN-E, found almost exclusively in red blood cells. Treatments in clinical trials include frataxin restoration by gene therapy, protein replacement, and epigenetic therapies, all of which necessitate sensitive assays for assessing frataxin levels. METHODS: In the present study, we have used a triple quadrupole mass spectrometry-based assay to examine the features of both types of frataxin levels in blood in a large heterogenous cohort of 106 patients with FRDA. RESULTS: Frataxin levels (FXN-E and FXN M) were predicted by GAA repeat length in regression models (R2 values = 0.51 and 0.27, respectively), and conversely frataxin levels predicted clinical status as determined by modified Friedreich Ataxia Rating scale scores and by disability status (R2 values = 0.13-0.16). There was no significant change in frataxin levels in individual subjects over time, and apart from start codon mutations, FXN-E and FXN-M levels were roughly equal. Accounting for hemoglobin levels in a smaller sub-cohort improved prediction of both FXN-E and FXN-M levels from R2 values of (0.3-0.38 to 0.20-0.51). CONCLUSION: The present data show that assay of FXN-M and FXN-E levels in blood provides an appropriate biofluid for assessing their repletion in particular clinical contexts.