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.

Repeat expansions in NOP56 are a cause of spinocerebellar ataxia Type 36 in the British population.

Spinocerebellar ataxias form a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia. Their prevalence varies among populations and ethnicities. Spinocerebellar ataxia 36 is caused by a GGCCTG repeat expansion in the first intron of the NOP56 gene and is characterized by late-onset ataxia, sensorineural hearing loss and upper and lower motor neuron signs, including tongue fasciculations. Spinocerebellar ataxia 36 has been described mainly in East Asian and Western European patients and was thought to be absent in the British population. Leveraging novel bioinformatic tools to detect repeat expansions from whole-genome sequencing, we analyse the NOP56 repeat in 1257 British patients with hereditary ataxia and in 7506 unrelated controls. We identify pathogenic repeat expansions in five families (seven patients), representing the first cohort of White British descent patients with spinocerebellar ataxia 36. Employing in silico approaches using whole-genome sequencing data, we found an 87 kb shared haplotype in among the affected individuals from five families around the NOP56 repeat region, although this block was also shared between several controls, suggesting that the repeat arises on a permissive haplotype. Clinically, the patients presented with slowly progressive cerebellar ataxia with a low rate of hearing loss and variable rates of motor neuron impairment. Our findings show that the NOP56 expansion causes ataxia in the British population and that spinocerebellar ataxia 36 can be suspected in patients with a late-onset, slowly progressive ataxia, even without the findings of hearing loss and tongue fasciculation.

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.

POPULATION FREQUENCY OF REPEAT EXPANSIONS INDICATES INCREASED DISEASE PREVALENCE ESTIMATES ACROSS DIFFERENT POPULATIONS.

Repeat expansion disorders (REDs) are a devastating group of predominantly neurological diseases. Together they are common, affecting 1 in 3,000 people worldwide with population-specific differences. However, prevalence estimates of REDs are hampered by heterogeneous clinical presentation, variable geographic distributions, and technological limitations leading to under-ascertainment. Here, leveraging whole genome sequencing data from 82,176 individuals from different populations we found an overall carrier frequency of REDs of 1 in 340 individuals. Modelling disease prevalence using genetic data, age at onset and survival, we show that REDs are up to 3-fold more prevalent than currently reported figures. While some REDs are population-specific, e.g. Huntington's disease type 2, most REDs are represented in all broad genetic ancestries, including Africans and Asians, challenging the notion that some REDs are found only in European populations. These results have worldwide implications for local and global health communities in the diagnosis and management of REDs both at local and global levels.

A phenome-wide association study of methylated GC-rich repeats identifies a GCC repeat expansion in AFF3 as a significant cause of intellectual disability.

GC-rich tandem repeat expansions (TREs) are often associated with DNA methylation, gene silencing and folate-sensitive fragile sites and underlie several congenital and late-onset disorders. Through a combination of DNA methylation profiling and tandem repeat genotyping, we identified 24 methylated TREs and investigated their effects on human traits using PheWAS in 168,641 individuals from the UK Biobank, identifying 156 significant TRE:trait associations involving 17 different TREs. Of these, a GCC expansion in the promoter of AFF3 was linked with a 2.4-fold reduced probability of completing secondary education, an effect size comparable to several recurrent pathogenic microdeletions. In a cohort of 6,371 probands with neurodevelopmental problems of suspected genetic etiology, we observed a significant enrichment of AFF3 expansions compared to controls. With a population prevalence that is at least 5-fold higher than the TRE that causes fragile X syndrome, AFF3 expansions represent a significant cause of neurodevelopmental delay.

Unexpected frequency of the pathogenic AR CAG repeat expansion in the general population.

CAG repeat expansions in exon 1 of the AR gene on the X chromosome cause spinal and bulbar muscular atrophy, a male-specific progressive neuromuscular disorder associated with a variety of extra-neurological symptoms. The disease has a reported male prevalence of approximately 1:30 000 or less, but the AR repeat expansion frequency is unknown. We established a pipeline, which combines the use of the ExpansionHunter tool and visual validation, to detect AR CAG expansion on whole-genome sequencing data, benchmarked it to fragment PCR sizing, and applied it to 74 277 unrelated individuals from four large cohorts. Our pipeline showed sensitivity of 100% [95% confidence interval (CI) 90.8-100%], specificity of 99% (95% CI 94.2-99.7%), and a positive predictive value of 97.4% (95% CI 84.4-99.6%). We found the mutation frequency to be 1:3182 (95% CI 1:2309-1:4386, n = 117 734) X chromosomes-10 times more frequent than the reported disease prevalence. Modelling using the novel mutation frequency led to estimate disease prevalence of 1:6887 males, more than four times more frequent than the reported disease prevalence. This discrepancy is possibly due to underdiagnosis of this neuromuscular condition, reduced penetrance, and/or pleomorphic clinical manifestations.

Functional genomics provide key insights to improve the diagnostic yield of hereditary ataxia.

Improvements in functional genomic annotation have led to a critical mass of neurogenetic discoveries. This is exemplified in hereditary ataxia, a heterogeneous group of disorders characterised by incoordination from cerebellar dysfunction. Associated pathogenic variants in more than 300 genes have been described, leading to a detailed genetic classification partitioned by age-of-onset. Despite these advances, up to 75% of patients with ataxia remain molecularly undiagnosed even following whole genome sequencing, as exemplified in the 100 000 Genomes Project. This study aimed to understand whether we can improve our knowledge of the genetic architecture of hereditary ataxia by leveraging functional genomic annotations, and as a result, generate insights and strategies that raise the diagnostic yield. To achieve these aims, we used publicly-available multi-omics data to generate 294 genic features, capturing information relating to a gene's structure, genetic variation, tissue-specific, cell-type-specific and temporal expression, as well as protein products of a gene. We studied these features across genes typically causing childhood-onset, adult-onset or both types of disease first individually, then collectively. This led to the generation of testable hypotheses which we investigated using whole genome sequencing data from up to 2182 individuals presenting with ataxia and 6658 non-neurological probands recruited in the 100 000 Genomes Project. Using this approach, we demonstrated a high short tandem repeat (STR) density within childhood-onset genes suggesting that we may be missing pathogenic repeat expansions within this cohort. This was verified in both childhood- and adult-onset ataxia patients from the 100 000 Genomes Project who were unexpectedly found to have a trend for higher repeat sizes even at naturally-occurring STRs within known ataxia genes, implying a role for STRs in pathogenesis. Using unsupervised analysis, we found significant similarities in genomic annotation across the gene panels, which suggested adult- and childhood-onset patients should be screened using a common diagnostic gene set. We tested this within the 100 000 Genomes Project by assessing the burden of pathogenic variants among childhood-onset genes in adult-onset patients and vice versa. This demonstrated a significantly higher burden of rare, potentially pathogenic variants in conventional childhood-onset genes among individuals with adult-onset ataxia. Our analysis has implications for the current clinical practice in genetic testing for hereditary ataxia. We suggest that the diagnostic rate for hereditary ataxia could be increased by removing the age-of-onset partition, and through a modified screening for repeat expansions in naturally-occurring STRs within known ataxia-associated genes, in effect treating these regions as candidate pathogenic loci.

Truncating Variants in RFC1 in Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome.

BACKGROUND AND OBJECTIVE: Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)n repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition. METHODS: Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)n expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines. RESULTS: We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)n expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein. DISCUSSION: Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)n expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.

Heterozygous UCHL1 loss-of-function variants cause a neurodegenerative disorder with spasticity, ataxia, neuropathy, and optic atrophy.

PURPOSE: Biallelic variants in UCHL1 have been associated with a progressive early-onset neurodegenerative disorder, autosomal recessive spastic paraplegia type 79. In this study, we investigated heterozygous UCHL1 variants on the basis of results from cohort-based burden analyses. METHODS: Gene-burden analyses were performed on exome and genome data of independent cohorts of patients with hereditary ataxia and spastic paraplegia from Germany and the United Kingdom in a total of 3169 patients and 33,141 controls. Clinical data of affected individuals and additional independent families were collected and evaluated. Patients' fibroblasts were used to perform mass spectrometry-based proteomics. RESULTS: UCHL1 was prioritized in both independent cohorts as a candidate gene for an autosomal dominant disorder. We identified a total of 34 cases from 18 unrelated families, carrying 13 heterozygous loss-of-function variants (15 families) and an inframe insertion (3 families). Affected individuals mainly presented with spasticity (24/31), ataxia (28/31), neuropathy (11/21), and optic atrophy (9/17). The mass spectrometry-based proteomics showed approximately 50% reduction of UCHL1 expression in patients' fibroblasts. CONCLUSION: Our bioinformatic analysis, in-depth clinical and genetic workup, and functional studies established haploinsufficiency of UCHL1 as a novel disease mechanism in spastic ataxia.

REViewer: haplotype-resolved visualization of read alignments in and around tandem repeats.

BACKGROUND: Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. RESULTS: We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. CONCLUSIONS: Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Whole genome sequencing for the diagnosis of neurological repeat expansion disorders in the UK: a retrospective diagnostic accuracy and prospective clinical validation study.

BACKGROUND: Repeat expansion disorders affect about 1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in underdiagnosis of people who have atypical clinical presentations, especially in paediatric patients without a previous positive family history. Whole genome sequencing is increasingly used as a first-line test for other rare genetic disorders, and we aimed to assess its performance in the diagnosis of patients with neurological repeat expansion disorders. METHODS: We retrospectively assessed the diagnostic accuracy of whole genome sequencing to detect the most common repeat expansion loci associated with neurological outcomes (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, and TBP) using samples obtained within the National Health Service in England from patients who were suspected of having neurological disorders; previous PCR test results were used as the reference standard. The clinical accuracy of whole genome sequencing to detect repeat expansions was prospectively examined in previously genetically tested and undiagnosed patients recruited in 2013-17 to the 100 000 Genomes Project in the UK, who were suspected of having a genetic neurological disorder (familial or early-onset forms of ataxia, neuropathy, spastic paraplegia, dementia, motor neuron disease, parkinsonian movement disorders, intellectual disability, or neuromuscular disorders). If a repeat expansion call was made using whole genome sequencing, PCR was used to confirm the result. FINDINGS: The diagnostic accuracy of whole genome sequencing to detect repeat expansions was evaluated against 793 PCR tests previously performed within the NHS from 404 patients. Whole genome sequencing correctly classified 215 of 221 expanded alleles and 1316 of 1321 non-expanded alleles, showing 97·3% sensitivity (95% CI 94·2-99·0) and 99·6% specificity (99·1-99·9) across the 13 disease-associated loci when compared with PCR test results. In samples from 11 631 patients in the 100 000 Genomes Project, whole genome sequencing identified 81 repeat expansions, which were also tested by PCR: 68 were confirmed as repeat expansions in the full pathogenic range, 11 were non-pathogenic intermediate expansions or premutations, and two were non-expanded repeats (16% false discovery rate). INTERPRETATION: In our study, whole genome sequencing for the detection of repeat expansions showed high sensitivity and specificity, and it led to identification of neurological repeat expansion disorders in previously undiagnosed patients. These findings support implementation of whole genome sequencing in clinical laboratories for diagnosis of patients who have a neurological presentation consistent with a repeat expansion disorder. FUNDING: Medical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, and Illumina.

Myoclonic status epilepticus and cerebellar hypoplasia associated with a novel variant in the GRIA3 gene.

AMPA-type glutamate receptors (AMPARs) are postsynaptic ionotropic receptors which mediate fast excitatory currents. AMPARs have a heterotetrameric structure, variably composed by the four subunits GluA1-4 which are encoded by genes GRIA1-4. Increasing evidence support the role of pathogenic variants in GRIA1-4 genes as causative for syndromic intellectual disability (ID). We report an Italian pedigree where some male individuals share ID, seizures and facial dysmorphisms. The index subject was referred for severe ID, myoclonic seizures, cerebellar signs and short stature. Whole exome sequencing identified a novel variant in GRIA3, c.2360A > G, p.(Glu787Gly). The GRIA3 gene maps to chromosome Xq25 and the c.2360A > G variant was transmitted by his healthy mother. Subsequent analysis in the family showed a segregation pattern compatible with the causative role of this variant, further supported by preliminary functional insights. We provide a detailed description of the clinical evolution of the index subjects and stress the relevance of myoclonic seizures and cerebellar syndrome as cardinal features of his presentation.

Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders: cohort study.

OBJECTIVE: To determine whether whole genome sequencing can be used to define the molecular basis of suspected mitochondrial disease. DESIGN: Cohort study. SETTING: National Health Service, England, including secondary and tertiary care. PARTICIPANTS: 345 patients with suspected mitochondrial disorders recruited to the 100 000 Genomes Project in England between 2015 and 2018. INTERVENTION: Short read whole genome sequencing was performed. Nuclear variants were prioritised on the basis of gene panels chosen according to phenotypes, ClinVar pathogenic/likely pathogenic variants, and the top 10 prioritised variants from Exomiser. Mitochondrial DNA variants were called using an in-house pipeline and compared with a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. American College of Medical Genetics guidelines were followed for classification of variants. MAIN OUTCOME MEASURE: Definite or probable genetic diagnosis. RESULTS: A definite or probable genetic diagnosis was identified in 98/319 (31%) families, with an additional 6 (2%) possible diagnoses. Fourteen of the diagnoses (4% of the 319 families) explained only part of the clinical features. A total of 95 different genes were implicated. Of 104 families given a diagnosis, 39 (38%) had a mitochondrial diagnosis and 65 (63%) had a non-mitochondrial diagnosis. CONCLUSION: Whole genome sequencing is a useful diagnostic test in patients with suspected mitochondrial disorders, yielding a diagnosis in a further 31% after exclusion of common causes. Most diagnoses were non-mitochondrial disorders and included developmental disorders with intellectual disability, epileptic encephalopathies, other metabolic disorders, cardiomyopathies, and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments.

100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Biallelic variants in TSPOAP1, encoding the active-zone protein RIMBP1, cause autosomal recessive dystonia.

Dystonia is a debilitating hyperkinetic movement disorder, which can be transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense, and missense variants in TSPOAP1, which encodes the active-zone RIM-binding protein 1 (RIMBP1), as a genetic cause of autosomal recessive dystonia in 7 subjects from 3 unrelated families. Subjects carrying loss-of-function variants presented with juvenile-onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between dysfunction of the presynaptic active zone and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis.