Spinocerebellar Ataxia Type 3 Pathophysiology-Implications for Translational Research and Clinical Studies.

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.

Phenotypic analysis of ataxia in spinocerebellar ataxia type 6 mice using DeepLabCut.

This study emphasizes the benefits of open-source software such as DeepLabCut (DLC) and R to automate, customize and enhance data analysis of motor behavior. We recorded 2 different spinocerebellar ataxia type 6 mouse models while performing the classic beamwalk test, tracked multiple body parts using the markerless pose-estimation software DLC and analyzed the tracked data using self-written scripts in the programming language R. The beamwalk analysis script (BAS) counts and classifies minor and major hindpaw slips with an 83% accuracy compared to manual scoring. Nose, belly and tail positions relative to the beam, as well as the angle at the tail base relative to the nose and tail tip were determined to characterize motor deficits in greater detail. Our results found distinct ataxic abnormalities such as an increase in major left hindpaw slips and a lower belly and tail position in both SCA6 ataxic mouse models compared to control mice at 18 months of age. Furthermore, a more detailed analysis of various body parts relative to the beam revealed an overall lower body position in the SCA684Q compared to the CT-longQ27PC mouse line at 18 months of age, indicating a more severe ataxic deficit in the SCA684Q group.

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage.

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

[Abnormalities in signal transduction of Purkinje cells in spinocerebellar ataxias: a review].

Spinocerebellar ataxias (SCAs) are a group of autosomal dominant neurodegenerative diseases that have been currently identified with numerous subtypes exhibiting genetic heterogeneity and clinical variability. Purkinje neuronal degeneration and cerebellar atrophy are common pathological features among most SCA subtypes. The physiological functions of Purkinje cells are regulated by multiple factors, and their dysfunction in signal transduction may lead to abnormal cerebellar motor control. This review summarizes the abnormalities in voltage-gated ionic channels, intracellular calcium signaling, and glutamate signaling transduction of Purkinje cells in SCAs, aiming to provide a theoretical basis for further understanding the common pathogenesis of SCAs and developing specific treatments.

An iPSC model for POLR3A-associated spastic ataxia: Generation of three unrelated patient cell lines.

Spastic Ataxias (SA) are a group of neurodegenerative disorders with combined pyramidal and cerebellar system affection, leading to an overlap phenotype between Hereditary Spastic Paraplegias (HSP) and Cerebellar Ataxias (CA). Here we describe the generation of iPSCs from three unrelated patients with an ultra-rare subtype of SA caused by compound heterozygous mutations in POLR3A, that encodes the largest subunit of RNA polymerase III. iPSCs were reprogrammed from normal human dermal fibroblasts (NHDFs) using episomal reprogramming with integration-free plasmid vectors: HIHRSi004-A, derived from a 44 year-old male carrying the mutations c.1909 + 22G > A/c.3944_3945delTG, HIHRSi005-A obtained from a 66 year-old male carrying the mutations c.1909 + 22G > A/c.1531C > T, and HIHRSi006-A from a 27 year-old male carrying the mutations c.1909 + 22G > A/c.2472_2472delC (ENST00000372371.8).

Spinocerebellar ataxia 27B: A novel, frequent and potentially treatable ataxia.

Hereditary ataxias, especially when presenting sporadically in adulthood, present a particular diagnostic challenge owing to their great clinical and genetic heterogeneity. Currently, up to 75% of such patients remain without a genetic diagnosis. In an era of emerging disease-modifying gene-stratified therapies, the identification of causative alleles has become increasingly important. Over the past few years, the implementation of advanced bioinformatics tools and long-read sequencing has allowed the identification of a number of novel repeat expansion disorders, such as the recently described spinocerebellar ataxia 27B (SCA27B) caused by a (GAA)•(TTC) repeat expansion in intron 1 of the fibroblast growth factor 14 (FGF14) gene. SCA27B is rapidly gaining recognition as one of the most common forms of adult-onset hereditary ataxia, with several studies showing that it accounts for a substantial number (9-61%) of previously undiagnosed cases from different cohorts. First natural history studies and multiple reports have already outlined the progression and core phenotype of this novel disease, which consists of a late-onset slowly progressive pan-cerebellar syndrome that is frequently associated with cerebellar oculomotor signs, such as downbeat nystagmus, and episodic symptoms. Furthermore, preliminary studies in patients with SCA27B have shown promising symptomatic benefits of 4-aminopyridine, an already marketed drug. This review describes the current knowledge of the genetic and molecular basis, epidemiology, clinical features and prospective treatment strategies in SCA27B.

The FGF14 GAA repeat expansion in Greek patients with late-onset cerebellar ataxia and an overview of the SCA27B phenotype across populations.

A pathogenic GAA repeat expansion in the first intron of the fibroblast growth factor 14 gene (FGF14) has been recently identified as the cause of spinocerebellar ataxia 27B (SCA27B). We herein screened 160 Greek index cases with late-onset cerebellar ataxia (LOCA) for FGF14 repeat expansions using a combination of long-range PCR and bidirectional repeat-primed PCRs. We identified 19 index cases (12%) carrying a pathogenic FGF14 GAA expansion, a diagnostic yield higher than that of previously screened repeat-expansion ataxias in Greek LOCA patients. The age at onset of SCA27B patients was 60.5 ± 12.3 years (range, 34-80). Episodic onset (37%), downbeat nystagmus (32%) and vertigo (26%) were significantly more frequent in FGF14 expansion-positive cases compared to expansion-negative cases. Beyond typical cerebellar signs, SCA27B patients often displayed hyperreflexia (47%) and reduced vibration sense in the lower extremities (42%). The frequency and phenotypic profile of SCA27B in Greek patients was similar to most other previously studied populations. We conclude that FGF14 GAA repeat expansions are the commonest known genetic cause of LOCA in the Greek population and recommend prioritizing testing for FGF14 expansions in the diagnostic algorithm of patients with LOCA.

Adaptive Long-Read Sequencing Reveals GGC Repeat Expansion in ZFHX3 Associated with Spinocerebellar Ataxia Type 4.

BACKGROUND: Spinocerebellar ataxia type 4 (SCA4) is an autosomal dominant ataxia with invariable sensory neuropathy originally described in a family with Swedish ancestry residing in Utah more than 25 years ago. Despite tight linkage to the 16q22 region, the molecular diagnosis has since remained elusive. OBJECTIVES: Inspired by pathogenic structural variation implicated in other 16q-ataxias with linkage to the same locus, we revisited the index SCA4 cases from the Utah family using novel technologies to investigate structural variation within the candidate region. METHODS: We adopted a targeted long-read sequencing approach with adaptive sampling on the Oxford Nanopore Technologies (ONT) platform that enables the detection of segregating structural variants within a genomic region without a priori assumptions about any variant features. RESULTS: Using this approach, we found a heterozygous (GGC)n repeat expansion in the last coding exon of the zinc finger homeobox 3 (ZFHX3) gene that segregates with disease, ranging between 48 and 57 GGC repeats in affected probands. This finding was replicated in a separate family with SCA4. Furthermore, the estimation of this GGC repeat size in short-read whole genome sequencing (WGS) data of 21,836 individuals recruited to the 100,000 Genomes Project in the UK and our in-house dataset of 11,258 exomes did not reveal any pathogenic repeats, indicating that the variant is ultrarare. CONCLUSIONS: These findings support the utility of adaptive long-read sequencing as a powerful tool to decipher causative structural variation in unsolved cases of inherited neurological disease. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Mitochondrial damage and impaired mitophagy contribute to disease progression in SCA6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that manifests in midlife and progressively worsens with age. SCA6 is rare, and many patients are not diagnosed until long after disease onset. Whether disease-causing cellular alterations differ at different disease stages is currently unknown, but it is important to answer this question in order to identify appropriate therapeutic targets across disease duration. We used transcriptomics to identify changes in gene expression at disease onset in a well-established mouse model of SCA6 that recapitulates key disease features. We observed both up- and down-regulated genes with the major down-regulated gene ontology terms suggesting mitochondrial dysfunction. We explored mitochondrial function and structure and observed that changes in mitochondrial structure preceded changes in function, and that mitochondrial function was not significantly altered at disease onset but was impaired later during disease progression. We also detected elevated oxidative stress in cells at the same disease stage. In addition, we observed impairment in mitophagy that exacerbates mitochondrial dysfunction at late disease stages. In post-mortem SCA6 patient cerebellar tissue, we observed metabolic changes that are consistent with mitochondrial impairments, supporting our results from animal models being translatable to human disease. Our study reveals that mitochondrial dysfunction and impaired mitochondrial degradation likely contribute to disease progression in SCA6 and suggests that these could be promising targets for therapeutic interventions in particular for patients diagnosed after disease onset.

Cognitive, Emotional, and Other Non-motor Symptoms of Spinocerebellar Ataxias.

PURPOSE OF REVIEW: Spinocerebellar ataxias (SCAs) are autosomal dominant degenerative syndromes that present with ataxia and brain stem abnormalities. This review describes the cognitive and behavioral symptoms of SCAs in the context of recent knowledge of the role of the cerebellum in higher intellectual function. RECENT FINDINGS: Recent studies suggest that patients with spinocerebellar ataxia can display cognitive deficits even early in the disease. These have been given the term cerebellar cognitive affective syndrome (CCAS). CCAS can be tracked using newly developed rating scales. In addition, patients with spinocerebellar ataxia also display impulsive and compulsive behavior, depression, anxiety, fatigue, and sleep disturbances. This review stresses the importance of recognizing non-motor symptoms in SCAs. There is a pressing need for novel therapeutic interventions to address these symptoms given their deleterious impact on patients' quality of life.

Different Purkinje cell pathologies cause specific patterns of progressive gait ataxia in mice.

Gait ataxia is one of the most common and impactful consequences of cerebellar dysfunction. Purkinje cells, the sole output neurons of the cerebellar cortex, are often involved in the underlying pathology, but their specific functions during locomotor control in health and disease remain obfuscated. We aimed to describe the effect of gradual adult-onset Purkinje cell degeneration on gaiting patterns in mice, and to determine whether two different mechanisms that both lead to Purkinje cell degeneration cause different patterns in the development of gait ataxia. Using the ErasmusLadder together with a newly developed limb detection algorithm and machine learning-based classification, we subjected mice to a challenging locomotor task with detailed analysis of single limb parameters, intralimb coordination and whole-body movement. We tested two Purkinje cell-specific mouse models, one involving stochastic cell death due to impaired DNA repair mechanisms (Pcp2-Ercc1-/-), the other carrying the mutation that causes spinocerebellar ataxia type 1 (Pcp2-ATXN1[82Q]). Both mouse models showed progressive gaiting deficits, but the sequence with which gaiting parameters deteriorated was different between mouse lines. Our longitudinal approach revealed that gradual loss of Purkinje cell function can lead to a complex pattern of loss of function over time, and that this pattern depends on the specifics of the pathological mechanisms involved. We hypothesize that this variability will also be present in disease progression in patients, and that our findings will facilitate the study of therapeutic interventions in mice, as subtle changes in locomotor abilities can be quantified by our methods.

Dimeric structures of DNA ATTTC repeats promoted by divalent cations.

Structural studies of repetitive DNA sequences may provide insights why and how certain repeat instabilities in their number and nucleotide sequence are managed or even required for normal cell physiology, while genomic variability associated with repeat expansions may also be disease-causing. The pentanucleotide ATTTC repeats occur in hundreds of genes important for various cellular processes, while their insertion and expansion in noncoding regions are associated with neurodegeneration, particularly with subtypes of spinocerebellar ataxia and familial adult myoclonic epilepsy. We describe a new striking domain-swapped DNA-DNA interaction triggered by the addition of divalent cations, including Mg2+ and Ca2+. The results of NMR characterization of d(ATTTC)3 in solution show that the oligonucleotide folds into a novel 3D architecture with two central C:C+ base pairs sandwiched between a couple of T:T base pairs. This structural element, referred to here as the TCCTzip, is characterized by intercalative hydrogen-bonding, while the nucleobase moieties are poorly stacked. The 5'- and 3'-ends of TCCTzip motif are connected by stem-loop segments characterized by A:T base pairs and stacking interactions. Insights embodied in the non-canonical DNA structure are expected to advance our understanding of why only certain pyrimidine-rich DNA repeats appear to be pathogenic, while others can occur in the human genome without any harmful consequences.

Subcellular localization and ER-mediated cytotoxic function of α1A and α1ACT in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a polyglutamine (polyQ) disease, which is caused by the elongation of CAG repeats encoding polyQ in the CACNA1A gene. The CACNA1A gene encodes two proteins, namely, α1A (a subunit of the plasma membrane calcium channel), which is translated in its entire length, and α1ACT, which is translated from the second cistron, and both proteins have a polyQ tract. The α1A-polyQ and α1ACT-polyQ proteins with an elongated polyQ stretch have been reported to form aggregates in cells and induce neuronal cell death, but the subcellular localization of these proteins and their cytotoxic properties remain unclear. In this study, we first analyzed SCA6 model mice and found that α1A-polyQlong localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQlong localized to the nucleus. Analysis using Neuro2a cells also showed similar subcellular localizations of these proteins, and a proportion of both proteins localized to the endoplasmic reticulum (ER). Cytotoxic studies demonstrated that both proteins induce both the ER stress response and apoptosis, indicating that they are able to induce ER stress-induced apoptosis.

Spinocerebellar ataxia type 2 has multiple ancestral origins.

INTRODUCTION: Spinocerebellar ataxia type 2 (SCA2) is a dominant neurodegenerative disorder due to expansions of a CAG repeat tract (CAGexp) at the ATXN2 gene. Previous studies found only one ancestral haplotype worldwide, with a C allele at rs695871. This homogeneity was unexpected, given the severe anticipations related to SCA2. We aimed to describe informative ancestral haplotypes found in South American SCA2 families. METHODS: Seventy-seven SCA2 index cases were recruited from Brazil, Peru, and Uruguay; 263 normal chromosomes were used as controls. The SNPs rs9300319, rs3809274, rs695871, rs1236900 and rs593226, and the STRs D12S1329, D12S1333, D12S1672 and D12S1332, were used to reconstruct haplotypes. RESULTS: Eleven ancestral haplotypes were found in SCA2 families. The most frequent ones were A-G-C-C-C (46.7 % of families), G-C-C-C-C (24.6 %) and A-C-C-C-C (10.3 %) and their mean (sd) CAGexp were 41.68 (3.55), 40.42 (4.11) and 45.67 (9.70) (p = 0.055), respectively. In contrast, the mean (sd) CAG lengths at normal alleles grouped per haplotypes G-C-G-A-T, A-G-C-C-C and G-C-C-C-C were 22.97 (3.93), 23.85 (3.59), and 30.81 (4.27) (p < 0.001), respectively. The other SCA2 haplotypes were rare: among them, a G-C-G-A-T lineage was found, evidencing a G allele in rs695871. CONCLUSION: We identified several distinct ancestral haplotypes in SCA2 families, including an unexpected lineage with a G allele at rs695871, a variation never found in hundreds of SCA2 patients studied worldwide. SCA2 has multiple origins in South America, and more studies should be done in other regions of the world.

Longitudinal MRI and 1H-MRS study of SCA7 mouse forebrain reveals progressive multiregional atrophy and early brain metabolite changes indicating early neuronal and glial dysfunction.

SpinoCerebellar Ataxia type 7 (SCA7) is an inherited disorder caused by CAG triplet repeats encoding polyglutamine expansion in the ATXN7 protein, which is part of the transcriptional coactivator complex SAGA. The mutation primarily causes neurodegeneration in the cerebellum and retina, as well as several forebrain structures. The SCA7140Q/5Q knock-in mouse model recapitulates key disease features, including loss of vision and motor performance. To characterize the temporal progression of brain degeneration of this model, we performed a longitudinal study spanning from early to late symptomatic stages using high-resolution magnetic resonance imaging (MRI) and in vivo 1H-magnetic resonance spectroscopy (1H-MRS). Compared to wild-type mouse littermates, MRI analysis of SCA7 mice shows progressive atrophy of defined brain structures, with the striatum, thalamus and cortex being the first and most severely affected. The volume loss of these structures coincided with increased motor impairments in SCA7 mice, suggesting an alteration of the sensory-motor network, as observed in SCA7 patients. MRI also reveals atrophy of the hippocampus and anterior commissure at mid-symptomatic stage and the midbrain and brain stem at late stage. 1H-MRS of hippocampus, a brain region previously shown to be dysfunctional in patients, reveals early and progressive metabolic alterations in SCA7 mice. Interestingly, abnormal glutamine accumulation precedes the hippocampal atrophy and the reduction in myo-inositol and total N-acetyl-aspartate concentrations, two markers of glial and neuronal damage, respectively. Together, our results indicate that non-cerebellar alterations and glial and neuronal metabolic impairments may play a crucial role in the development of SCA7 mouse pathology, particularly at early stages of the disease. Degenerative features of forebrain structures in SCA7 mice correspond to current observations made in patients. Our study thus provides potential biomarkers that could be used for the evaluation of future therapeutic trials using the SCA7140Q/5Q model.

Early-onset familial essential tremor is associated with nucleotide expansions of spinocerebellar ataxia in China.

BACKGROUND: Essential tremor (ET) is a neurological disease characterized by action tremor in upper arms. Although its high heritability and prevalence worldwide, its etiology and association with other diseases are still unknown. METHOD: We investigated 10 common spinocerebellar ataxias (SCAs), including SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, SCA17, SCA36, dentatorubral-pallidoluysian atrophy (DRPLA) in 92 early-onset familial ET pedigrees in China collected from 2016 to 2022. RESULT: We found one SCA12 proband carried 51 CAG repeats within PPP2R2B gene and one SCA3 proband with intermediate CAG repeats (55) with ATXN3 gene. The other 90 ET probands all had normal repeat expansions. CONCLUSION: Tremor can be the initial phenotype of certain SCA. For early-onset, familial ET patients, careful physical examinations are needed before genetic SCA screening.

Dysregulation of alternative splicing in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. We performed RNA sequencing in mouse models of spinocerebellar ataxia type 1 and identified that mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. We found that the diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing us to identify overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. We also provide evidence that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1.

Exonic trinucleotide repeat expansions in ZFHX3 cause spinocerebellar ataxia type 4: A poly-glycine disease.

Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.

Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions.

Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r = 0.98; P < 0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.