Laura Ivete Rudaks

Objective: Biallelic variants in genes encoding the RNA polymerase III complex (Pol III) cause a spectrum of neurological disorders primarily affecting the central nervous system. Monoallelic variants have been reported in the POLR3B subunit only, associated with neurodevelopmental disorder, epilepsy, and peripheral neuropathy. We describe a novel Pol III‐related disorder caused by monoallelic variants in POLR3A and presenting primarily with peripheral neuropathy. Methods: We performed clinical and genetic investigation of the affected families. Biophysical characterization of mutant proteins was performed in silico. Immunoblotting, interactomics, and transcriptomics characterization were done in cellular models. Results: We identified 11 patients across 8 unrelated families harboring heterozygous missense variants in POLR3A, occurring de novo or segregating in an autosomal‐dominant fashion. The patients presented with an early‐onset, progressive sensorimotor peripheral polyneuropathy with intermediate to demyelinating ranges of nerve conduction slowing, occasionally accompanied with neurological or non‐neurological features. White matter abnormalities, characteristic for the biallelic Pol III‐related disorders, where not observed in the brain magnetic resonance imaging (MRI) evaluations. The neuropathy‐associated variants cluster in regions critical for Pol III activity. We observed mis‐regulation of individual Pol III targets and global downregulation of tRNA pools in patient‐derived cells. This Pol III dysfunction was not due to impaired POLR3A expression, subcellular localization, or subunit interactions. Interpretation: Our findings expand the Pol III‐related disease spectrum beyond the known biallelic phenotypes and establish POLR3A as a novel peripheral neuropathy‐associated gene. This work highlights a central role of Pol III dysfunction in disease and reinforces the link between tRNA metabolism and peripheral neurodegeneration. ANN NEUROL 2026

Background: Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder characterized by progressive spasticity and lower limb weakness. The most common forms of autosomal dominant HSP are caused by pathogenic variants in SPAST (SPG4 or HSP‐ SPAST ), ATL1 (SPG3A or HSP‐ ATL1 ), and REEP1 (SPG31 or HSP‐ REEP1 ). Objectives: We performed an MDSGene Systematic Review to determine in‐depth genotype–phenotype associations and to estimate longitudinal progression, to inform prognostication and clinical trial stratification. Methods: We systematically collected demographic, phenotypic, and genetic data from published reports of individuals affected by these forms of HSP using the MDSGene protocol. Results: We reviewed 2177 affected individuals, including 1670 individuals with HSP‐ SPAST , 356 with HSP‐ ATL1 and 151 with HSP‐ REEP1 . HSP‐ ATL1 was associated with an earlier age at onset compared to HSP‐ SPAST and HSP‐ REEP1 . Toe‐walking was more frequently reported in HSP‐ ATL1 (10.4%) and HSP‐ REEP1 (3.3%) than HSP‐ SPAST (0.3%). Upper limb hyperreflexia and abnormalities of bladder function were more frequent in HSP‐ SPAST than HSP‐ ATL1 or HSP‐ REEP1 . Sufficient data was available to estimate disease progression for HSP‐ SPAST ; this showed that Spastic Paraplegia Rating Scale (SPRS) scores increased with increasing age at examination after the age of 40 years. Truncating variants were more frequent in HSP‐ SPAST and HSP‐ REEP1 than HSP‐ ATL1 . Conclusion: Overall, HSP‐ ATL1 , HSP‐ SPAST and HSP‐ REEP1 demonstrated differences in clinical phenotypes. To our knowledge, this is the first systematic review to model longitudinal progression using SPRS scores in HSP. Missing data is a limiting factor in all these comparisons, highlighting the need for uniform data collection. Online resources can be found at https://www.mdsgene.org/ .

Inherited myopathies are a group of disorders with diverse and complex genetic aetiologies. The causative genetic variants vary widely in type, size and sequence context, encompassing small sequence variants and large structural variants in protein-coding genes, mitochondrial variants, repeat expansions, and more complex events, such as the chromosome 4 D4Z4 macrosatellite contraction and hypomethylation that causes facioscapulohumeral muscular dystrophy (FSHD). This poses a challenge for analysis with next generation sequencing and other molecular methods. This is further compounded by phenotypic variability between patients and phenotypic overlap of different forms of genetic myopathy. To accelerate myopathy research and improve diagnosis we have developed a targeted long-read sequencing assay and an integrated bioinformatics analysis framework that captures the full suite of genes, variants and epigenetic signatures currently implicated in all forms of inherited myopathy. Applying this to a cohort of 53 myopathy patients with and without previous genetic diagnoses, we demonstrate the analytical validity of our approach, as well as its improved accuracy and resolution compared to existing methods - especially for FSHD. Our LRS assay identified an array of new information about the genetic and epigenetic landscape of inherited myopathies and provided new diagnoses for 29% of patients who had remained undiagnosed following clinical genetic testing. In our cohort, FSHD and oculopharyngodistal myopathies were the most common new diagnoses that were missed or mis-diagnosed by standard clinical genetic testing. Our new method constitutes a single streamlined assay for comprehensive genetic and epigenetic characterisation of inherited myopathies.

Objective: The hereditary spastic‐ataxia spectrum disorders are a group of disabling neurological diseases. The traditional genetic testing pathway is complex, multistep and leaves many cases unsolved. We aim to streamline and improve this process using long‐read sequencing.Methods: We developed a targeted long‐read sequencing strategy with the capacity to characterise the genetic variation of all types and sizes within 469 disease‐associated genes, in a single assay. We applied this to a cohort of 34 individuals with unsolved spastic‐ataxia. An additional five individuals with a known genetic diagnosis were included as positive controls.Results: We identified causative pathogenic variants that would be sufficient for genetic diagnosis in 14/34 (41%) unsolved participants. The success rate was 5/11 (45%) in those who were naïve to genetic testing and 9/23 (39%) in those who were undiagnosed after prior genetic testing, completed on a clinical basis. Short tandem repeat expansions in FGF14 were the most common (7/34, 21%). Two individuals (2/34, 6%) had biallelic pathogenic expansions in RFC1 and one individual had a monoallelic pathogenic expansion in ATXN8OS/ATXN8. Causative pathogenic sequence variants other than short tandem repeat expansions were found in four individuals, including in VCP, STUB1, ANO10 and SPG7. Furthermore, all five positive controls were identified.Interpretation: Our results demonstrate the utility of targeted long‐read sequencing in the genetic evaluation of patients with spastic‐ataxia spectrum disorders, highlighting both the capacity to increase overall diagnostic yield and to streamline the testing pathway by capturing all known genetic causes in a single assay.

Genetic myopathies are caused by pathogenic variants in >300 genes across the nuclear and mitochondrial genomes. Although short-read next-generation sequencing (NGS) has revolutionised the diagnosis of genetic disorders, large and/or complex genetic variants, which are over-represented in the genetic myopathies, are not well characterised using this approach. Long-read sequencing (LRS) is a newer genetic testing technology that overcomes many of the limitations of NGS. In particular, LRS provides improved detection of challenging variant types, including short tandem repeat (STR) expansions, copy number variants and structural variants, as well as improved variant phasing and concurrent assessment of epigenetic changes, including DNA methylation. The ability to concurrently detect multiple STR expansions is particularly relevant given the growing number of recently described genetic myopathies associated with STR expansions. LRS will also aid in the identification of new myopathy genes and molecular mechanisms. However, use of LRS technology is currently limited by high cost, low accessibility, the need for specialised DNA extraction procedures, limited availability of LRS bioinformatic tools and pipelines, and the relative lack of healthy control LRS variant databases. Once these barriers are addressed, the implementation of LRS into clinical diagnostic pipelines will undoubtedly streamline the diagnostic algorithm and increase the diagnostic rate for genetic myopathies. In this review, we discuss the utility and critical impact of LRS in this field.

The hereditary spastic-ataxia spectrum disorders are a group of rare disabling neurological diseases. The genetic testing process is complex, and often requires multiple different assays to evaluate the many potential causative genes and variant types, including short tandem repeat expansions, single nucleotide variants, insertions/deletions, structural variants and copy number variants. This can be a protracted process and, even after all avenues are exhausted, many individuals do not receive a genetic diagnosis. Aiming to streamline and improve this process, we developed a targeted long-read sequencing strategy with capacity to characterise genetic variation of all types and sizes within 469 disease-associated genes, in a single assay. We applied this to a cohort of 34 individuals with genetically undiagnosed spastic-ataxia spectrum disorders. An additional five individuals with a known genetic diagnosis were included as positive controls. We identified causative pathogenic variants that would be sufficient for genetic diagnosis in 14/34 (41%) unsolved participants. The success rate was 5/11 (45%) in those who were naïve to genetic testing and 9/23 (39%) in those who were undiagnosed after prior genetic testing, completed on a clinical basis. Short tandem repeat expansions in FGF14 were the most common cause, present in 7/34 (21%). Two individuals (2/34, 6%) had biallelic pathogenic short tandem repeat expansions in RFC1 and one individual had a monoallelic pathogenic short tandem repeat expansion in ATXN8OS / ATXN8 . Causative pathogenic sequence variants other than short tandem repeat expansions were found in four individuals, including a heterozygous missense variant in VCP , a heterozygous in-frame deletion in STUB1 , a homozygous splicing variant in ANO10 , and compound heterozygous missense and nonsense variants in SPG7 . In addition to these solved cases, a pathogenic or likely-pathogenic variant with uncertain clinical implications was identified in a further three individuals, including a single individual who was found to have a short tandem repeat expansion in BEAN1 in addition to biallelic expansions in FGF14 within the range of 200-249 repeats. Our results demonstrate the utility of targeted long-read sequencing in the genetic evaluation of patients with spastic-ataxia spectrum disorders, highlighting both the capacity to increase overall diagnostic yield and to streamline the testing pathway by capturing all known genetic causes in a single assay.

The hereditary cerebellar ataxias (HCAs) are rare, progressive neurologic disorders caused by variants in many different genes. Inheritance may follow autosomal dominant, autosomal recessive, X-linked or mitochondrial patterns. The list of genes associated with adult-onset cerebellar ataxia is continuously growing, with several new genes discovered in the last few years. This includes short-tandem repeat (STR) expansions in RFC1, causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), FGF14-GAA causing spinocerebellar ataxia type 27B (SCA27B), and THAP11. In addition, the genetic basis for SCA4, has recently been identified as a STR expansion in ZFHX3. Given the large and growing number of genes, and different gene variant types, the approach to diagnostic testing for adult-onset HCA can be complex. Testing methods include targeted evaluation of STR expansions (e.g. SCAs, Friedreich ataxia, fragile X-associated tremor/ataxia syndrome, dentatorubral-pallidoluysian atrophy), next generation sequencing for conventional variants, which may include targeted gene panels, whole exome, or whole genome sequencing, followed by various potential additional tests. This review proposes a diagnostic approach for clinical testing, highlights the challenges with current testing technologies, and discusses future advances which may overcome these limitations. Implementing long-read sequencing has the potential to transform the diagnostic approach in HCA, with the overall aim to improve the diagnostic yield.