Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia.

Hereditary Spastic Paraplegia (HSP) is a group of rare inherited disorders characterized by progressive weakness and spasticity of the legs. Recent newly discovered biallelic variants in the gene FICD were found in patients with a highly similar phenotype to early onset HSP. FICD encodes filamentation induced by cAMP domain protein. FICD is involved in the AMPylation and deAMPylation protein modifications of the endoplasmic reticulum (ER) chaperone BIP, a major constituent of the ER that regulates the unfolded protein response. Although several biochemical properties of FICD have been characterized, the neurological function of FICD and the pathological mechanism underlying HSP are unknown. We established a Drosophila model to gain mechanistic understanding of the function of FICD in HSP pathogenesis, and specifically the role of BIP in neuromuscular physiology. Our studies on Drosophila Fic null mutants uncovered that loss of Fic resulted in locomotor impairment and reduced levels of BIP in the motor neuron circuitry, as well as increased reactive oxygen species (ROS) in the ventral nerve cord of Fic null mutants. Finally, feeding Drosophila Fic null mutants with chemical chaperones PBA or TUDCA, or treatment of patient fibroblasts with PBA, reduced the ROS accumulation. The neuronal phenotypes of Fic null mutants recapitulate several clinical features of HSP patients and further reveal cellular patho-mechanisms. By modeling FICD in Drosophila, we provide potential targets for intervention for HSP, and advance fundamental biology that is important for understanding related rare and common neuromuscular diseases.

Customized antisense oligonucleotide-based therapy for neurofilament-associated Charcot-Marie-Tooth disease.

DNA-based therapeutics have emerged as a revolutionary approach for addressing the treatment gap in rare inherited conditions by targeting the fundamental genetic causes of disease. Charcot-Marie-Tooth (CMT) disease, a group of inherited neuropathies, represents one of the most prevalent Mendelian disease groups in neurology and is characterized by diverse genetic etiology. Axonal forms of CMT, known as CMT2, are caused by dominant mutations in over 30 different genes which lead to degeneration of lower motor neuron axons. Recent advances in antisense oligonucleotide (ASO) therapeutics have shown promise in targeting neurodegenerative disorders. Here we elucidate pathomechanistic changes contributing to variant specific molecular phenotypes in CMT2E, caused by a single nucleotide substitution (p.N98S) in the neurofilament light chain gene (NEFL). We used a patient-derived pluripotent stem cell (iPSC)-induced motor neuron model, which recapitulates several cellular and biomarker phenotypes associated with CMT2E. Using an ASO treatment strategy targeting a heterozygous gain-of-function variant, we aimed to resolve molecular phenotypic changes observed in the CMT2E p.N98S subtype. To determine ASO therapeutic potential, we employed our treatment strategy in iPSC-derived motor neurons and used established as well as novel biomarkers of peripheral nervous system axonal degeneration. Our findings have demonstrated a significant decrease in clinically relevant biomarkers of axonal degeneration, presenting the first clinically viable genetic therapeutic for CMT2E. Similar strategies could be used to develop precision medicine approaches for otherwise untreatable gain of function inherited disorders.

Somatic instability of the FGF14 -SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14 . Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14 . We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure and late-onset cerebellar involvement in SCA27B.

Biallelic variants in COX18 cause a mitochondrial disorder primarily manifesting as peripheral neuropathy.

Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygous splice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases, we identified two additional families with rare deleterious biallelic variants in COX18 . All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>G variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. Our study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Our research has important implications for the diagnostic workup of CMT patients.

A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.

Neurological disorders caused by novel non-coding repeat expansions: clinical features and differential diagnosis.

Nucleotide repeat expansions in the human genome are a well-known cause of neurological disease. In the past decade, advances in DNA sequencing technologies have led to a better understanding of the role of non-coding DNA, that is, the DNA that is not transcribed into proteins. These techniques have also enabled the identification of pathogenic non-coding repeat expansions that cause neurological disorders. Mounting evidence shows that adult patients with familial or sporadic presentations of epilepsy, cognitive dysfunction, myopathy, neuropathy, ataxia, or movement disorders can be carriers of non-coding repeat expansions. The description of the clinical, epidemiological, and molecular features of these recently identified non-coding repeat expansion disorders should guide clinicians in the diagnosis and management of these patients, and help in the genetic counselling for patients and their families.

A common flanking variant is associated with enhanced stability of the FGF14-SCA27B repeat locus.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Spinocerebellar ataxia 27B: a frequent and slowly progressive autosomal-dominant cerebellar ataxia-experience from an Italian cohort.

BACKGROUND: Autosomal-dominant spinocerebellar ataxia (ADCA) due to intronic GAA repeat expansion in FGF14 (SCA27B) is a recent, relatively common form of late-onset ataxia. OBJECTIVE: Here, we aimed to: (1) investigate the relative frequency of SCA27B in different clinically defined disease subgroups with late-onset ataxia collected among 16 tertiary Italian centers; (2) characterize phenotype and diagnostic findings of patients with SCA27B; (3) compare the Italian cohort with other cohorts reported in recent studies. METHODS: We screened 396 clinically diagnosed late-onset cerebellar ataxias of unknown cause, subdivided in sporadic cerebellar ataxia, ADCA, and multisystem atrophy cerebellar type. We identified 72 new genetically defined subjects with SCA27B. Then, we analyzed the clinical, neurophysiological, and imaging features of 64 symptomatic cases. RESULTS: In our cohort, the prevalence of SCA27B was 13.4% (53/396) with as high as 38.5% (22/57) in ADCA. The median age of onset of SCA27B patients was 62 years. All symptomatic individuals showed evidence of impaired balance and gait; cerebellar ocular motor signs were also frequent. Episodic manifestations at onset occurred in 31% of patients. Extrapyramidal features (17%) and cognitive impairment (25%) were also reported. Brain magnetic resonance imaging showed cerebellar atrophy in most cases (78%). Pseudo-longitudinal assessments indicated slow progression of ataxia and minimal functional impairment. CONCLUSION: Patients with SCA27B in Italy present as an adult-onset, slowly progressive cerebellar ataxia with predominant axial involvement and frequent cerebellar ocular motor signs. The high consistency of clinical features in SCA27B cohorts in multiple populations paves the way toward large-scale, multicenter studies.

Generation of 3 patient induced Pluripotent stem cell lines containing SORD mutations linked to a recessive neuropathy.

The SORD neuropathy has been identified as the most common autosomal recessive inherited neuropathy, occurring in thousands of patients worldwide. Fibroblast lines from 3 different patients containing the c.753delG; p.Ala253GlnfsTer27 SORD mutations were reprogrammed into induced Pluripotent Stem Cell (iPSC) lines. These iPSC lines demonstrate an apparent normal karyotype and have positive expression of pluripotency markers. These iPSC lines also stain positively for Ectoderm, Endoderm and Mesoderm markers following Embryoid body differentiation. These lines pose to serve as a valuable disease modeling resource for studying the SORD neuropathy, including studying disease phenotype and treatment efficacy.

A study concept of expeditious clinical enrollment for genetic modifier studies in Charcot-Marie-Tooth neuropathy 1A.

BACKGROUND: Caused by duplications of the gene encoding peripheral myelin protein 22 (PMP22), Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common hereditary neuropathy. Despite this shared genetic origin, there is considerable variability in clinical severity. It is hypothesized that genetic modifiers contribute to this heterogeneity, the identification of which may reveal novel therapeutic targets. In this study, we present a comprehensive analysis of clinical examination results from 1564 CMT1A patients sourced from a prospective natural history study conducted by the RDCRN-INC (Inherited Neuropathy Consortium). Our primary objective is to delineate extreme phenotype profiles (mild and severe) within this patient cohort, thereby enhancing our ability to detect genetic modifiers with large effects. METHODS: We have conducted large-scale statistical analyses of the RDCRN-INC database to characterize CMT1A severity across multiple metrics. RESULTS: We defined patients below the 10th (mild) and above the 90th (severe) percentiles of age-normalized disease severity based on the CMT Examination Score V2 and foot dorsiflexion strength (MRC scale). Based on extreme phenotype categories, we defined a statistically justified recruitment strategy, which we propose to use in future modifier studies. INTERPRETATION: Leveraging whole genome sequencing with base pair resolution, a future genetic modifier evaluation will include single nucleotide association, gene burden tests, and structural variant analysis. The present work not only provides insight into the severity and course of CMT1A, but also elucidates the statistical foundation and practical considerations for a cost-efficient and straightforward patient enrollment strategy that we intend to conduct on additional patients recruited globally.

The genetic basis of early-onset hereditary ataxia in Iran: results of a national registry of a heterogeneous population.

BACKGROUND: To investigate the genetics of early-onset progressive cerebellar ataxia in Iran, we conducted a study at the Children's Medical Center (CMC), the primary referral center for pediatric disorders in the country, over a three-year period from 2019 to 2022. In this report, we provide the initial findings from the national registry. METHODS: We selected all early-onset patients with an autosomal recessive mode of inheritance to assess their phenotype, paraclinical tests, and genotypes. The clinical data encompassed clinical features, the Scale for the Assessment and Rating of Ataxia (SARA) scores, Magnetic Resonance Imaging (MRI) results, Electrodiagnostic exams (EDX), and biomarker features. Our genetic investigations included single-gene testing, Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS). RESULTS: Our study enrolled 162 patients from various geographic regions of our country. Among our subpopulations, we identified known and novel pathogenic variants in 42 genes in 97 families. The overall genetic diagnostic rate was 59.9%. Notably, we observed PLA2G6, ATM, SACS, and SCA variants in 19, 14, 12, and 10 families, respectively. Remarkably, more than 59% of the cases were attributed to pathogenic variants in these genes. CONCLUSIONS: Iran, being at the crossroad of the Middle East, exhibits a highly diverse genetic etiology for autosomal recessive hereditary ataxia. In light of this heterogeneity, the development of preventive strategies and targeted molecular therapeutics becomes crucial. A national guideline for the diagnosis and management of patients with these conditions could significantly aid in advancing healthcare approaches and improving patient outcomes.

Integration of transcriptomics and long-read genomics prioritizes structural variants in rare disease.

Rare structural variants (SVs) - insertions, deletions, and complex rearrangements - can cause Mendelian disease, yet they remain difficult to accurately detect and interpret. We sequenced and analyzed Oxford Nanopore long-read genomes of 68 individuals from the Undiagnosed Disease Network (UDN) with no previously identified diagnostic mutations from short-read sequencing. Using our optimized SV detection pipelines and 571 control long-read genomes, we detected 716 long-read rare (MAF < 0.01) SV alleles per genome on average, achieving a 2.4x increase from short-reads. To characterize the functional effects of rare SVs, we assessed their relationship with gene expression from blood or fibroblasts from the same individuals, and found that rare SVs overlapping enhancers were enriched (LOR = 0.46) near expression outliers. We also evaluated tandem repeat expansions (TREs) and found 14 rare TREs per genome; notably these TREs were also enriched near overexpression outliers. To prioritize candidate functional SVs, we developed Watershed-SV, a probabilistic model that integrates expression data with SV-specific genomic annotations, which significantly outperforms baseline models that don't incorporate expression data. Watershed-SV identified a median of eight high-confidence functional SVs per UDN genome. Notably, this included compound heterozygous deletions in FAM177A1 shared by two siblings, which were likely causal for a rare neurodevelopmental disorder. Our observations demonstrate the promise of integrating long-read sequencing with gene expression towards improving the prioritization of functional SVs and TREs in rare disease patients.

Rare homozygous disease-associated sequence variants in children with spinal muscular atrophy: a phenotypic description and review of the literature.

5q-associated spinal muscular atrophy (SMA) is the most common autosomal recessive neurological disease. Depletion in functional SMN protein leads to dysfunction and irreversible degeneration of the motor neurons. Over 95 % of individuals with SMA have homozygous exon 7 deletions in the SMN1 gene. Most of the remaining 4-5 % are compound heterozygous for deletion and a disease-associated sequence variant in the non-deleted allele. Individuals with SMA due to bi-allelic SMN1 sequence variants have rarely been reported. Data regarding their clinical phenotype, disease progression, outcome and treatment response are sparse. This study describes six individuals from three families, all with homozygous sequence variants in SMN1, and four of whom received treatment with disease-modifying therapies. We also describe the challenges faced during the diagnostic process and intrafamilial phenotypic variability observed between siblings.

Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009-2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome and whole genome sequencing (WGS), and latterly WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100,000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre, and has benefitted from the use of WGS, particularly access to the raw data. However, almost one quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

Testing SIPA1L2 as a modifier of CMT1A using mouse models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), leading to muscle weakness and loss of sensation in the hands and feet. A recent case-only genome-wide association study of CMT1A patients conducted by the Inherited Neuropathy Consortium identified a strong association between strength of foot dorsiflexion and variants in signal induced proliferation associated 1 like 2 (SIPA1L2), indicating that it may be a genetic modifier of disease. To validate SIPA1L2 as a candidate modifier and to assess its potential as a therapeutic target, we engineered mice with deletion of exon 1 (including the start codon) of the Sipa1l2 gene and crossed them to the C3-PMP22 mouse model of CMT1A. Neuromuscular phenotyping showed that Sipa1l2 deletion in C3-PMP22 mice preserved muscular endurance assayed by inverted wire hang duration and changed femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggest involvement of Sipa1l2 in cholesterol biosynthesis, a pathway that is also implicated in C3-PMP22 mice. Although Sipa1l2 deletion did impact CMT1A-associated phenotypes, thereby validating a genetic interaction, the overall effect on neuropathy was mild.

Dominant NARS1 mutations causing axonal Charcot-Marie-Tooth disease expand NARS1-associated diseases.

Pathogenic variants in six aminoacyl-tRNA synthetase (ARS) genes are implicated in neurological disorders, most notably inherited peripheral neuropathies. ARSs are enzymes that charge tRNA molecules with cognate amino acids. Pathogenic variants in asparaginyl-tRNA synthetase (NARS1) cause a neurological phenotype combining developmental delay, ataxia and demyelinating peripheral neuropathy. NARS1 has not yet been linked to axonal Charcot-Marie-Tooth disease. Exome sequencing of patients with inherited peripheral neuropathies revealed three previously unreported heterozygous NARS1 variants in three families. Clinical and electrophysiological details were assessed. We further characterized all three variants in a yeast complementation model and used a knock-in mouse model to study variant p.Ser461Phe. All three variants (p.Met236del, p.Cys342Tyr and p.Ser461Phe) co-segregate with the sensorimotor axonal neuropathy phenotype. Yeast complementation assays show that none of the three NARS1 variants support wild-type yeast growth when tested in isolation (i.e. in the absence of a wild-type copy of NARS1), consistent with a loss-of-function effect. Similarly, the homozygous knock-in mouse model (p.Ser461Phe/Ser472Phe in mouse) also demonstrated loss-of-function characteristics. We present three previously unreported NARS1 variants segregating with a sensorimotor neuropathy phenotype in three families. Functional studies in yeast and mouse support variant pathogenicity. Thus, NARS1 is the seventh ARS implicated in dominant axonal Charcot-Marie-Tooth disease, further stressing that all dimeric ARSs should be evaluated for Charcot-Marie-Tooth disease.

Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism.

ATP1A1 encodes a sodium-potassium ATPase that has been linked to several neurological diseases. Using exome and genome sequencing, we identified the heterozygous ATP1A1 variant NM_000701.8: c.2707G>A;p.(Gly903Arg) in two unrelated children presenting with delayed motor and speech development and autism. While absent in controls, the variant occurred de novo in one proband and co-segregated in two affected half-siblings, with mosaicism in the healthy mother. Using a specific ouabain resistance assay in mutant transfected HEK cells, we found significantly reduced cell viability. Demonstrating loss of ATPase function, we conclude that this novel variant is pathogenic, expanding the phenotype spectrum of ATP1A1.

GAA-FGF14 disease: defining its frequency, molecular basis, and 4-aminopyridine response in a large downbeat nystagmus cohort.

BACKGROUND: GAA-FGF14 disease/spinocerebellar ataxia 27B is a recently described neurodegenerative disease caused by (GAA)≥250 expansions in the fibroblast growth factor 14 (FGF14) gene, but its phenotypic spectrum, pathogenic threshold, and evidence-based treatability remain to be established. We report on the frequency of FGF14 (GAA)≥250 and (GAA)200-249 expansions in a large cohort of patients with idiopathic downbeat nystagmus (DBN) and their response to 4-aminopyridine. METHODS: Retrospective cohort study of 170 patients with idiopathic DBN, comprising in-depth phenotyping and assessment of 4-aminopyridine treatment response, including re-analysis of placebo-controlled video-oculography treatment response data from a previous randomised double-blind 4-aminopyridine trial. FINDINGS: Frequency of FGF14 (GAA)≥250 expansions was 48% (82/170) in patients with idiopathic DBN. Additional cerebellar ocular motor signs were observed in 100% (82/82) and cerebellar ataxia in 43% (35/82) of patients carrying an FGF14 (GAA)≥250 expansion. FGF14 (GAA)200-249 alleles were enriched in patients with DBN (12%; 20/170) compared to controls (0.87%; 19/2191; OR, 15.20; 95% CI, 7.52-30.80; p < 0.0001). The phenotype of patients carrying a (GAA)200-249 allele closely mirrored that of patients carrying a (GAA)≥250 allele. Patients carrying a (GAA)≥250 or a (GAA)200-249 allele had a significantly greater clinician-reported (80%, 33/41 vs 31%, 5/16; RR, 2.58; 95% CI, 1.23-5.41; Fisher's exact test, p = 0.0011) and self-reported (59%, 32/54 vs 11%, 2/19; RR, 5.63; 95% CI, 1.49-21.27; Fisher's exact test, p = 0.00033) response to 4-aminopyridine treatment compared to patients carrying a (GAA)<200 allele. Placebo-controlled video-oculography data, available for four patients carrying an FGF14 (GAA)≥250 expansion, showed a significant decrease in slow phase velocity of DBN with 4-aminopyridine, but not placebo. INTERPRETATION: This study confirms that FGF14 GAA expansions are a frequent cause of DBN syndromes. It provides preliminary evidence that (GAA)200-249 alleles might be pathogenic. Finally, it provides large real-world and preliminary piloting placebo-controlled evidence for the efficacy of 4-aminopyridine in GAA-FGF14 disease. FUNDING: This work was supported by the Clinician Scientist program "PRECISE.net" funded by the Else Kröner-Fresenius-Stiftung (to CW, AT, and MSy), the grant 779257 "Solve-RD" from the European's Union Horizon 2020 research and innovation program (to MSy), and the grant 01EO 1401 by the German Federal Ministry of Education and Research (BMBF) (to MSt). This work was also supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) N° 441409627, as part of the PROSPAX consortium under the frame of EJP RD, the European Joint Programme on Rare Diseases, under the EJP RD COFUND-EJP N° 825575 (to MSy, BB and-as associated partner-SZ), the NIH National Institute of Neurological Disorders and Stroke (grant 2R01NS072248-11A1 to SZ), the Fondation Groupe Monaco (to BB), and the Montreal General Hospital Foundation (grant PT79418 to BB). The Care4Rare Canada Consortium is funded in part by Genome Canada and the Ontario Genomics Institute (OGI-147 to KMB), the Canadian Institutes of Health Research (CIHR GP1-155867 to KMB), Ontario Research Foundation, Genome Quebec, and the Children's Hospital of Eastern Ontario Foundation. The funders had no role in the conduct of this study.