Digital Gait Outcomes for Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS): Discriminative, Convergent, and Ecological Validity in a Multicenter Study (PROSPAX).

BACKGROUND: With treatment trials on the horizon, this study aimed to identify candidate digital-motor gait outcomes for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), capturable by wearable sensors with multicenter validity, and ideally also ecological validity during free walking outside laboratory settings. METHODS: Cross-sectional multicenter study (four centers), with gait assessments in 36 subjects (18 ARSACS patients; 18 controls) using three body-worn sensors (Opal, APDM) in laboratory settings and free walking in public spaces. Sensor gait measures were analyzed for discriminative validity from controls, and for convergent (ie, clinical and patient relevance) validity by correlations with SPRSmobility (primary outcome) and Scale for the Assessment and Rating of Ataxia (SARA), Spastic Paraplegia Rating Scale (SPRS), and activities of daily living subscore of the Friedreich Ataxia Rating Scale (FARS-ADL) (exploratory outcomes). RESULTS: Of 30 hypothesis-based digital gait measures, 14 measures discriminated ARSACS patients from controls with large effect sizes (|Cliff's δ| > 0.8) in laboratory settings, with strongest discrimination by measures of spatiotemporal variability Lateral Step Deviation (δ = 0.98), SPcmp (δ = 0.94), and Swing CV (δ = 0.93). Large correlations with the SPRSmobility were observed for Swing CV (Spearman's ρ = 0.84), Speed (ρ = -0.63), and Harmonic Ratio V (ρ = -0.62). During supervised free walking in a public space, 11/30 gait measures discriminated ARSACS from controls with large effect sizes. Large correlations with SPRSmobility were here observed for Swing CV (ρ = 0.78) and Speed (ρ = -0.69), without reductions in effect sizes compared with laboratory settings. CONCLUSIONS: We identified a promising set of digital-motor candidate gait outcomes for ARSACS, applicable in multicenter settings, correlating with patient-relevant health aspects, and with high validity also outside laboratory settings, thus simulating real-life walking with higher ecological validity. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Repeat expansions nested within tandem CNVs: a unique structural change in GLS exemplifies the diagnostic challenges of non-coding pathogenic variation.

Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~3000 genomes and ~1500 exomes with optical genome mapping and long-read sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping and long-read sequencing)-to effectively flag complex repeat expansions using short-read datasets and thus facilitate diagnosis of ultra-rare disorders.

Stratifying the Presymptomatic Phase of Genetic Frontotemporal Dementia by Serum NfL and pNfH: A Longitudinal Multicentre Study.

OBJECTIVE: Although the presymptomatic stages of frontotemporal dementia (FTD) provide a unique chance to delay or even prevent neurodegeneration by early intervention, they remain poorly defined. Leveraging a large multicenter cohort of genetic FTD mutation carriers, we provide a biomarker-based stratification and biomarker cascade of the likely most treatment-relevant stage within the presymptomatic phase: the conversion stage. METHODS: We longitudinally assessed serum levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in the Genetic FTD Initiative (GENFI) cohort (n = 444), using single-molecule array technique. Subjects comprised 91 symptomatic and 179 presymptomatic subjects with mutations in the FTD genes C9orf72, GRN, or MAPT, and 174 mutation-negative within-family controls. RESULTS: In a biomarker cascade, NfL increase preceded the hypothetical clinical onset by 15 years and concurred with brain atrophy onset, whereas pNfH increase started close to clinical onset. The conversion stage was marked by increased NfL, but still normal pNfH levels, while both were increased at the symptomatic stage. Intra-individual change rates were increased for NfL at the conversion stage and for pNfH at the symptomatic stage, highlighting their respective potential as stage-dependent dynamic biomarkers within the biomarker cascade. Increased NfL levels and NfL change rates allowed identification of presymptomatic subjects converting to symptomatic disease and capture of proximity-to-onset. We estimate stage-dependent sample sizes for trials aiming to decrease neurofilament levels or change rates. INTERPRETATION: Blood NfL and pNfH provide dynamic stage-dependent stratification and, potentially, treatment response biomarkers in presymptomatic FTD, allowing demarcation of the conversion stage. The proposed biomarker cascade might pave the way towards a biomarker-based precision medicine approach to genetic FTD. ANN NEUROL 2022;91:33-47.

The ARCA Registry: A Collaborative Global Platform for Advancing Trial Readiness in Autosomal Recessive Cerebellar Ataxias.

Autosomal recessive cerebellar ataxias (ARCAs) form an ultrarare yet expanding group of neurodegenerative multisystemic diseases affecting the cerebellum and other neurological or non-neurological systems. With the advent of targeted therapies for ARCAs, disease registries have become a precious source of real-world quantitative and qualitative data complementing knowledge from preclinical studies and clinical trials. Here, we review the ARCA Registry, a global collaborative multicenter platform (>15 countries, >30 sites) with the overarching goal to advance trial readiness in ARCAs. It presents a good clinical practice (GCP)- and general data protection regulation (GDPR)-compliant professional-reported registry for multicenter web-based capture of cross-center standardized longitudinal data. Modular electronic case report forms (eCRFs) with core, extended, and optional datasets allow data capture tailored to the participating site's variable interests and resources. The eCRFs cover all key data elements required by regulatory authorities [European Medicines Agency (EMA)] and the European Rare Disease (ERD) platform. They capture genotype, phenotype, and progression and include demographic data, biomarkers, comorbidity, medication, magnetic resonance imaging (MRI), and longitudinal clinician- or patient-reported ratings of ataxia severity, non-ataxia features, disease stage, activities of daily living, and (mental) health status. Moreover, they are aligned to major autosomal-dominant spinocerebellar ataxia (SCA) and sporadic ataxia (SPORTAX) registries in the field, thus allowing for joint and comparative analyses not only across ARCAs but also with SCAs and sporadic ataxias. The registry is at the core of a systematic multi-component ARCA database cluster with a linked biobank and an evolving study database for digital outcome measures. Currently, the registry contains more than 800 patients with almost 1,500 visits representing all ages and disease stages; 65% of patients with established genetic diagnoses capture all the main ARCA genes, and 35% with unsolved diagnoses are targets for advanced next-generation sequencing. The ARCA Registry serves as the backbone of many major European and transatlantic consortia, such as PREPARE, PROSPAX, and the Ataxia Global Initiative, with additional data input from SPORTAX. It has thus become the largest global trial-readiness registry in the ARCA field.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

A de novo STUB1 variant associated with an early adult-onset multisystemic ataxia phenotype.

BACKGROUND: Biallelic STUB1 variants are a well-established cause of autosomal-recessive early-onset multisystemic ataxia (SCAR16). Evidence for STUB1 variants causing autosomal-dominant ataxia (SCA48) so far largely relies on segregation data in larger families. Presenting the first de novo occurrence of a heterozygous STUB1 variant, we here present additional qualitative evidence for STUB1-disease as an autosomal-dominant disorder. METHODS: Whole exome sequencing on an index patient with sporadic early-onset ataxia, followed by Sanger sequencing in all family members, was used to identify causative variants as well as to rule out alternative genetic hits and intronic STUB1 variants. STUB1 mRNA and protein levels in PBMCs in all family members were analysed using qRT-PCR and Western Blot. RESULTS: A previously unreported start-lost loss-of-function variant c.3G>A in the start codon of STUB1 was identified in the index case, occurring de novo and without evidence for a second (potentially missed) variant (e.g., intronic or copy number) in STUB1. The patient showed an early adult-onset multisystemic ataxia complicated by spastic gait disorder, distal myoclonus and cognitive dysfunction, thus closely mirroring the systems affected in autosomal-recessive STUB1-associated disease. In line with the predicted start-lost effect of the variant, functional investigations demonstrated markedly reduced STUB1 protein expression in PBMCs, whereas mRNA levels were intact. CONCLUSION: De novo occurrence of the loss-of-function STUB1 variant in our case with multisystemic ataxia provides a qualitatively additional line of evidence for STUB1-disease as an autosomal-dominant disorder, in which the same neurological systems are affected as in its autosomal-recessive counterpart. Moreover, this finding adds support for loss-of-function as a mechanism underlying autosomal-dominant STUB1-disease, thus mirroring its autosomal-recessive counterpart also in terms of the underlying mutational mechanism.

Natural History, Phenotypic Spectrum, and Discriminative Features of Multisystemic RFC1 Disease.

OBJECTIVE: To delineate the full phenotypic spectrum, discriminative features, piloting longitudinal progression data, and sample size calculations of replication factor complex subunit 1 (RFC1) repeat expansions, recently identified as causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). METHODS: Multimodal RFC1 repeat screening (PCR, Southern blot, whole-exome/genome sequencing-based approaches) combined with cross-sectional and longitudinal deep phenotyping in (1) cross-European cohort A (70 families) with ≥2 features of CANVAS or ataxia with chronic cough (ACC) and (2) Turkish cohort B (105 families) with unselected late-onset ataxia. RESULTS: Prevalence of RFC1 disease was 67% in cohort A, 14% in unselected cohort B, 68% in clinical CANVAS, and 100% in ACC. RFC1 disease was also identified in Western and Eastern Asian individuals and even by whole-exome sequencing. Visual compensation, sensory symptoms, and cough were strong positive discriminative predictors (>90%) against RFC1-negative patients. The phenotype across 70 RFC1-positive patients was mostly multisystemic (69%), including dysautonomia (62%) and bradykinesia (28%) (overlap with cerebellar-type multiple system atrophy [MSA-C]), postural instability (49%), slow vertical saccades (17%), and chorea or dystonia (11%). Ataxia progression was ≈1.3 Scale for the Assessment and Rating of Ataxia points per year (32 cross-sectional, 17 longitudinal assessments, follow-up ≤9 years [mean 3.1 years]) but also included early falls, variable nonlinear phases of MSA-C-like progression (SARA points 2.5-5.5 per year), and premature death. Treatment trials require 330 (1-year trial) and 132 (2-year trial) patients in total to detect 50% reduced progression. CONCLUSIONS: RFC1 disease is frequent and occurs across continents, with CANVAS and ACC as highly diagnostic phenotypes yet as variable, overlapping clusters along a continuous multisystemic disease spectrum, including MSA-C-overlap. Our natural history data help to inform future RFC1 treatment trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that RFC1 repeat expansions are associated with CANVAS and ACC.

Clinico-Genetic, Imaging and Molecular Delineation of COQ8A-Ataxia: A Multicenter Study of 59 Patients.

OBJECTIVE: To foster trial-readiness of coenzyme Q8A (COQ8A)-ataxia, we map the clinicogenetic, molecular, and neuroimaging spectrum of COQ8A-ataxia in a large worldwide cohort, and provide first progression data, including treatment response to coenzyme Q10 (CoQ10). METHODS: Cross-modal analysis of a multicenter cohort of 59 COQ8A patients, including genotype-phenotype correlations, 3D-protein modeling, in vitro mutation analyses, magnetic resonance imaging (MRI) markers, disease progression, and CoQ10 response data. RESULTS: Fifty-nine patients (39 novel) with 44 pathogenic COQ8A variants (18 novel) were identified. Missense variants demonstrated a pleiotropic range of detrimental effects upon protein modeling and in vitro analysis of purified variants. COQ8A-ataxia presented as variable multisystemic, early-onset cerebellar ataxia, with complicating features ranging from epilepsy (32%) and cognitive impairment (49%) to exercise intolerance (25%) and hyperkinetic movement disorders (41%), including dystonia and myoclonus as presenting symptoms. Multisystemic involvement was more prevalent in missense than biallelic loss-of-function variants (82-93% vs 53%; p = 0.029). Cerebellar atrophy was universal on MRI (100%), with cerebral atrophy or dentate and pontine T2 hyperintensities observed in 28%. Cross-sectional (n = 34) and longitudinal (n = 7) assessments consistently indicated mild-to-moderate progression of ataxia (SARA: 0.45/year). CoQ10 treatment led to improvement by clinical report in 14 of 30 patients, and by quantitative longitudinal assessments in 8 of 11 patients (SARA: -0.81/year). Explorative sample size calculations indicate that ≥48 patients per arm may suffice to demonstrate efficacy for interventions that reduce progression by 50%. INTERPRETATION: This study provides a deeper understanding of the disease, and paves the way toward large-scale natural history studies and treatment trials in COQ8A-ataxia. ANN NEUROL 2020;88:251-263.

CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells.

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions.

Truncating Mutations in UBAP1 Cause Hereditary Spastic Paraplegia.

The diagnostic gap for rare neurodegenerative diseases is still considerable, despite continuous advances in gene identification. Many novel Mendelian genes have only been identified in a few families worldwide. Here we report the identification of an autosomal-dominant gene for hereditary spastic paraplegia (HSP) in 10 families that are of diverse geographic origin and whose affected members all carry unique truncating changes in a circumscript region of UBAP1 (ubiquitin-associated protein 1). HSP is a neurodegenerative disease characterized by progressive lower-limb spasticity and weakness, as well as frequent bladder dysfunction. At least 40% of affected persons are currently undiagnosed after exome sequencing. We identified pathological truncating variants in UBAP1 in affected persons from Iran, USA, Germany, Canada, Spain, and Bulgarian Roma. The genetic support ranges from linkage in the largest family (LOD = 8.3) to three confirmed de novo mutations. We show that mRNA in the fibroblasts of affected individuals escapes nonsense-mediated decay and thus leads to the expression of truncated proteins; in addition, concentrations of the full-length protein are reduced in comparison to those in controls. This suggests either a dominant-negative effect or haploinsufficiency. UBAP1 links endosomal trafficking to the ubiquitination machinery pathways that have been previously implicated in HSPs, and UBAP1 provides a bridge toward a more unified pathophysiology.

SERAC1 deficiency causes complicated HSP: evidence from a novel splice mutation in a large family.

OBJECTIVE: To demonstrate that mutations in the phosphatidylglycerol remodelling enzyme SERAC1 can cause juvenile-onset complicated hereditary spastic paraplegia (cHSP) clusters, thus adding SERAC1 to the increasing number of complex lipid cHSP genes. METHODS: Combined genomic and functional validation studies (whole-exome sequencing, mRNA, cDNA and protein), biomarker investigations (3-methyl-glutaconic acid, filipin staining and phosphatidylglycerols PG34:1/PG36:1), and clinical and imaging phenotyping were performed in six affected subjects from two different branches of a large consanguineous family. RESULTS: 5 of 6 affected subjects shared cHSP as a common disease phenotype. Three subjects presented with juvenile-onset oligosystemic cHSP, still able to walk several miles at age >10-20 years. This benign phenotypic cluster and disease progression is strikingly divergent to the severe infantile phenotype of all SERAC1 cases reported so far. Two family members showed a more multisystemic juvenile-onset cHSP, indicating an intermediate phenotype between the benign oligosystemic cHSP and the classic infantile SERAC1 cluster. The homozygous splice mutation led to loss of the full-length SERAC1 protein and impaired phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than in classic infantile-onset SERAC1 cases, which might partially explain the milder SERAC1 phenotype. CONCLUSIONS: Our findings add SERAC1 to the increasing list of complex lipid cHSP genes. At the same time they redefine the phenotypic spectrum of SERAC1 deficiency. It is associated not only with the severe infantile-onset 'Methylglutaconic aciduria, Deafness, Encephalopathy, Leigh-like' syndrome (MEGDEL syndrome), but also with oligosystemic juvenile-onset cHSP as part of the now unfolding SERAC1 deficiency spectrum.

STUB1/CHIP mutations cause Gordon Holmes syndrome as part of a widespread multisystemic neurodegeneration: evidence from four novel mutations.

BACKGROUND: CHIP, the protein encoded by STUB1, is a central component of cellular protein homeostasis and interacts with several key proteins involved in the pathogenesis of manifold neurodegenerative diseases. This gives rise to the hypothesis that mutations in STUB1 might cause a far more multisystemic neurodegenerative phenotype than the previously reported cerebellar ataxia syndrome. METHODS: Whole exome sequencing data-sets from n = 87 index subjects of two ataxia cohorts were screened for individuals with STUB1 mutations. In-depth phenotyping by clinical evaluation and neuroimaging was performed in mutation carriers. RESULTS: We identified four novel STUB1 mutations in three affected subjects from two index families (frequency 2/87 = 2.3%). All three subjects presented with a severe multisystemic phenotype including severe dementia, spastic tetraparesis, epilepsy, and autonomic dysfunction in addition to cerebellar ataxia, plus hypogonadism in one index patient. Diffusion tensor imaging revealed degeneration of manifold supra- and infratentorial tracts. CONCLUSIONS: Our findings provide clinical and imaging support for the notion that CHIP is a crucial converging point of manifold neurodegenerative processes, corresponding with its universal biological function in neurodegeneration. Further, our data reveal the second STUB1 family with ataxia plus hypogonadism reported so far, demonstrating that Gordon Holmes syndrome is indeed a recurrent manifestation of STUB1. However, it does not present in isolation, but as part of a broad multisystemic neurodegenerative process. This supports the notion that STUB1 disease should be conceptualized not by historical or clinical syndromic names, but as a variable multisystemic disease defined by disturbed function of the underlying STUB1 gene, which translates into a multidimensional gradual spectrum of variably associated clinical signs and symptoms.

Prominent Oncogenic Roles of EVI1 in Breast Carcinoma.

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma where its contributions are unexplored. Analyzing a tissue microarray of 608 breast carcinoma patient specimens, we documented EVI1 overexpression in both estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast carcinomas. Here, we report prognostic relevance of EVI1 overexpression in triple-negative breast carcinoma but not in the HER2-positive breast carcinoma subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance, and tumorigenicity, effects rescued by estrogen supplementation in ER+ breast carcinoma cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on constitutive ERK activity in HER2+ breast carcinoma cells. Microarray analyses revealed G-protein-coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in breast carcinoma. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated breast carcinoma cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer. Cancer Res; 77(8); 2148-60. ©2017 AACR.