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Introduction/aims: Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is caused by RFC1 expansions. Sensory neuronopathy, polyneuropathy, and involvement of motor, autonomic, and cranial nerves have all been described with RFC1 expansions. We aimed to describe the electrodiagnostic features of patients with RFC1 expansions through multimodal electrophysiological investigations., Methods: Thirty-five patients, with a median age of 70 years, and pathologic biallelic repeat expansions in the RFC1 gene, were tested for motor and sensory nerve conduction, flexor carpi radialis (FCR) and soleus H-reflexes, blink reflex, electrochemical skin conductance, sympathetic skin response (SSR), and heart rate variability with deep breathing (HRV)., Results: Only 16 patients (46%) exhibited the full clinical CANVAS spectrum. Distal motor amplitudes were normal in 30 patients and reduced in the legs of five patients. Distal sensory amplitudes were bilaterally reduced in a non-length dependent manner in 30 patients. Conduction velocities were normal. Soleus H-reflexes were abnormal in 19/20 patients of whom seven had preserved Achilles reflexes. FCR H-reflexes were absent or decreased in amplitude in 13/14 patients. Blink reflex was abnormal in 4/19 patients: R1 latencies for two patients and R2 latencies for two others. Fourteen out of 31 patients (45%) had abnormal results in at least one autonomic nervous system test, either for ESC (12/31), SSR (5/14), or HRV (6/19)., Discussion: Less than half of the patients with RFC1 expansions exhibited the full clinical CANVAS spectrum, but nearly all exhibited typical sensory neuronopathy and abnormal H-reflexes. Involvement of small nerve fibers and brainstem neurons was less common., (© 2024 The Author(s). Muscle & Nerve published by Wiley Periodicals LLC.)

Objective : To examine the relationship between body mass index (BMI) and genotype among pre-symptomatic carriers of different pathogenic variants associated with amyotrophic lateral sclerosis. Methods : C9orf72+ carriers, SOD1+ carriers, and pathogenic variant negative controls (Gene-Negatives) were included from 3 largely independent cohorts: ALS Families Project (ALS-Families); Dominantly inherited ALS (DIALS); and Pre-symptomatic Familial ALS ( Pre-fALS ). First reported ( ALS-Families ) or measured ( DIALS and Pre-fALS ) weight and height were used to calculate BMI. Age at weight measurement, self-reported sex (male vs. female), and highest education (high school or below vs. college education vs . graduate school or above) were extracted. The associations between BMI and genotype in each cohort were examined with multivariable linear regression models, adjusted for age, sex, and education. Results : A total of 223 C9orf72+ carriers, 135 SOD1+ carriers, and 191 Gene-Negatives were included, deriving from ALS-Families ( n  = 114, median age 46, 37% male), DIALS ( n  = 221, median age 46, 30% male), and Pre-fALS ( n  = 214, median age 44, 39% male). Adjusting for age, sex, and education, the mean BMI of C9orf72+ carriers was lower than Gene-Negatives by 2.4 units (95% confidence interval [CI] = 0.3-4.6, p  = 0.02) in ALS-Families ; 2.7 units (95% CI = 0.9-4.4, p  = 0.003) in DIALS ; and 1.9 units (95% CI = 0.5-4.2, p  = 0.12) in Pre-fALS . There were no significant differences in BMI between SOD1+ carriers and Gene-Negatives in any of the 3 cohorts. Conclusions : Compared to Gene-Negatives, average BMI is lower in asymptomatic C9orf72+ carriers across 3 cohorts while no significant difference was found between Gene-Negatives and SOD1+ carriers.

In this issue of Neuron, Wu et al. 1 show that nuclear speckle proteins are sequestered by both nuclear RNA foci and cytoplasmic dipeptide repeat aggregates in C9ORF72-ALS/FTD. Consequently, dysregulation of splicing induces widespread splicing alterations and contributes to neurodegeneration., Competing Interests: Declaration of interests The author declares no competing interests., (Published by Elsevier Inc.)

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: C9orf72 gene repeat expansion (C9RE) is the most frequent gene variant associated with amyotrophic lateral sclerosis (ALS). We aimed to study the phenotype of motor neurone disease (MND) patients with C9RE in a Portuguese cohort., Methods: Demographical and clinical data of MND patients with (C9RE+) and without C9RE were compared. ALS al Rating Scale-Revised (ALSFRS-R) and Edinburgh Cognitive and Behavioural ALS Screen (ECAS) were used to evaluate functional and cognitive performance, respectively. Survival analysis was performed using Kaplan Meier log-rank test and Cox proportional hazards model., Results: We included 761 patients of whom 61 (8.0 %) were C9RE+. C9RE+ patients had a higher frequency of ALS (95.1 vs 78.4 %, p = 0.002), and lower frequency of progressive muscular atrophy (3.3 vs 16.7 %, p = 0.006). C9RE+ was associated with earlier age of onset (58.1 vs 62.6 years, p = 0.003) and more frequent MND family history (65.5 vs 11.4 %, p < 0.001). Gender, ethnicity, onset site, diagnostic delay, disease progression rate until diagnosis (ΔF), ALSFRS-R and time until non-invasive ventilation did not differ between groups. Cognitive/behavioural symptoms and ECAS did not differ between groups, except a worse visuospatial score in C9RE+ group (p = 0.035). Death rate was 1.8 and 1.6 times higher in C9RE+ patients with MND and ALS, respectively. Significant survival prognostic factors in C9RE+ group were diagnosis delay (HR = 0.96, 95 %CI 0.92-0.99, p = 0.008) and ΔF (HR = 1.93, 95 %CI 1.26-2.96, p = 0.002)., Conclusion: Our study corroborates most previous cohorts' findings, but harbours some singularities regarding onset site, phenotype, and cognitive profile, that contribute to a better understanding of C9RE epidemiology., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Myotonic dystrophy type 2 (DM2) is a neurogenerative disease caused by caprylic/capric triglyceride (CCTG) tetranucleotide repeat expansions in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene. Non-B DNA structures formed by CCTG repeats can promote genetic instability, whereas interrupting motifs of NCTG (N = A/T/G) within CCTG repeats help to maintain genomic stability. However, whether the interrupting motifs can affect DNA structures of CCTG repeats remains unclear. Here, we report that four CCTG repeats with an interrupting 3'-A/T/G residue formed dumbbell structures, whereas a non-interrupting 3'-C residue resulted in a multi-loop structure exhibiting conformational dynamics that may contribute to a higher tendency of escaping from DNA mismatch repair and causing repeat expansions. The results provide new structural insights into the genetic instability of CCTG repeats in DM2., (© 2024 Federation of European Biochemical Societies.)

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies., (© 2024. The Author(s).)

A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR/Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR/Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR/Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within RFC1 . To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by RFC1 knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.

Repeat expansions in FGF14 cause autosomal dominant late-onset cerebellar ataxia (SCA27B) with estimated pathogenic thresholds of 250 (incomplete penetrance) and 300 AAG repeats (full penetrance), but the sequence of pathogenic and non-pathogenic expansions remains unexplored. Here, we demonstrate that STRling and ExpansionHunter accurately detect FGF14 expansions from short-read genome data using outlier approaches. By combining long-range PCR and nanopore sequencing in 169 patients with cerebellar ataxia and 802 controls, we compare FGF14 expansion alleles, including interruptions and flanking regions. Uninterrupted AAG expansions are significantly enriched in patients with ataxia from a lower threshold (180-200 repeats) than previously reported based on expansion size alone. Conversely, AAGGAG hexameric expansions are equally frequent in patients and controls. Distinct 5' flanking regions, interruptions and pre-repeat sequences correlate with repeat size. Furthermore, pure AAG (pathogenic) and AAGGAG (non-pathogenic) repeats form different secondary structures. Regardless of expansion size, SCA27B is a recognizable clinical entity characterized by frequent episodic ataxia and downbeat nystagmus, similar to the presentation observed in a family with a previously unreported nonsense variant (SCA27A). Overall, this study suggests that SCA27B is a major overlooked cause of adult-onset ataxia, accounting for 23-31% of unsolved patients. We strongly recommend re-evaluating pathogenic thresholds and integrating expansion sequencing into the molecular diagnostic process., (© 2024. The Author(s).)

Hereditary spinocerebellar ataxia (SCA) is a group of clinically and genetically heterogeneous inherited disorders characterized by slowly progressive cerebellar ataxia. We ascertained a Japanese pedigree with autosomal dominant SCA comprising four family members, including two patients. We identified a GGCCTG repeat expansion of intron 1 in the NOP56 gene by Southern blotting, resulting in a molecular diagnosis of SCA36. RNA sequencing using peripheral blood revealed that the expression of genes involved in ribosomal organization and translation was decreased in patients carrying the GGCCTG repeat expansion. Genes involved in pathways associated with ribosomal organization and translation were enriched and differentially expressed in the patients. We propose a novel hypothesis that the GGCCTG repeat expansion contributes to the pathogenesis of SCA36 by causing a global disruption of translation resulting from ribosomal dysfunction., (© 2024. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion results in multiple dipeptide repeat proteins, among which arginine-rich poly-GR proteins are highly toxic to neurons and decrease the rate of protein synthesis. We investigated whether the effect on protein synthesis contributes to neuronal dysfunction and degeneration. We found that the expression of poly-GR proteins inhibited global translation by perturbing translation elongation. In iPSC-differentiated neurons, the translation of transcripts with relatively slow elongation rates was further slowed, and stalled, by poly-GR. Elongation stalling increased ribosome collisions and induced a ribotoxic stress response (RSR) mediated by ZAKα that increased the phosphorylation of the kinase p38 and promoted cell death. Knockdown of ZAKα or pharmacological inhibition of p38 ameliorated poly-GR-induced toxicity and improved the survival of iPSC-derived neurons from patients with C9ORF72 -ALS/FTD. Our findings suggest that targeting the RSR may be neuroprotective in patients with ALS/FTD caused by repeat expansion in C9ORF72 .

Background: Using the Clinical Audit Research and Evaluation of Motor Neuron Disease (CARE-MND) database and the Scottish Regenerative Neurology Tissue Bank, we aimed to outline the genetic epidemiology and phenotypes of an incident cohort of people with MND (pwMND) to gain a realistic impression of the genetic landscape and genotype-phenotype associations., Methods: Phenotypic markers were identified from the CARE-MND platform. Sequence analysis of 48 genes was undertaken. Variants were classified using a structured evidence-based approach. Samples were also tested for C9orf72 hexanucleotide expansions using repeat-prime PCR methodology., Results: 339 pwMND donated a DNA sample: 44 (13.0%) fulfilled criteria for having a pathogenic variant/repeat expansion, 53.5% of those with a family history of MND and 9.3% of those without. The majority (30 (8.8%)) had a pathogenic C9orf72 repeat expansion, including two with intermediate expansions. Having a C9orf72 expansion was associated with a significantly lower Edinburgh Cognitive and Behavioural ALS Screen ALS-Specific score (p = 0.0005). The known pathogenic SOD1 variant p.(Ile114Thr), frequently observed in the Scottish population, was detected in 9 (2.7%) of total cases but in 17.9% of familial cases. Rare variants were detected in FUS and NEK1. One individual carried both a C9orf72 expansion and SOD1 variant., Conclusions: Our results provide an accurate summary of MND demographics and genetic epidemiology. We recommend early genetic testing of people with cognitive impairment to ensure that C9orf72 carriers are given the best opportunity for informed treatment planning. Scotland is enriched for the SOD1 p.(Ile114Thr) variant and this has significant implications with regards to future genetically-targeted treatments., (© 2024. The Author(s).)

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive multisystem neurologic disorder caused by biallelic intronic repeats in RFC1. Although the phenotype of CANVAS has been expanding via diagnostic case accumulation, there are scant pedigree analyses to reveal disease penetrance, intergenerational fluctuations in repeat length, or clinical phenomena (including heterozygous carriers). We identified biallelic RFC1 ACAGG expansions of 1000 ~ repeats in three affected siblings having sensorimotor neuronopathy with spinocerebellar atrophy initially presenting with painful muscle cramps and paroxysmal dry cough. They exhibit almost homogeneous clinical and histopathological features, indicating motor neuronopathy. Over 10 years of follow-up, painful intractable muscle cramps ascended from legs to trunks and hands, followed by amyotrophy and subsequent leg pyramidal signs. The disease course combined with the electrophysical and imagery data suggest initial and prolonged hyperexcitability and the ensuing spinal motor neuron loss, which may progress from the lumbar to the rostral anterior horns and later expand to the corticospinal tract. Genetically, heterozygous ACAGG expansions of similar length were transmitted in unaffected family members of three successive generations, and some of them experienced muscle cramps. Leukocyte telomere length assays revealed comparatively shorter telomeres in affected individuals. This comprehensive pedigree analysis demonstrated a non-anticipating ACAGG transmission and high penetrance of manifestations with a biallelic state, especially motor neuronopathy in which muscle cramps serve as a prodromal and disease progress marker. CANVAS and RFC1 spectrum disorder should be considered when diagnosing lower dominant motor neuron disease, idiopathic muscle cramps, or neuromuscular hyperexcitability syndromes., (© 2024. The Author(s).)

GGGGCC (G 4 C 2 ) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded G 4 C 2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G 4 C 2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G 4 C 2 repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these in vitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) and Frontotemporal lobar degeneration (FTLD) represent the most common forms of neurodegenerative dementias with a highly phenotypic variability. Herein, we investigated the role of genetic variants related to the immune system and inflammation as genetic modulators in AD and related dementias. In patients with sporadic AD/FTLD (n = 300) and GRN / C9orf72 mutation carriers (n = 80), we performed a targeted sequencing of 50 genes belonging to the immune system and inflammation, selected based on their high expression in brain regions and low tolerance to genetic variation. The linear regression analyses revealed two genetic variants: (i) the rs1049296 in the transferrin ( TF ) gene, shown to be significantly associated with age at onset in the sporadic AD group, anticipating the disease onset of 4 years for each SNP allele with respect to the wild-type allele, and (ii) the rs7550295 in the calsyntenin-1 ( CLSTN1 ) gene, which was significantly associated with age at onset in the C9orf72 group, delaying the disease onset of 17 years in patients carrying the SNP allele. In conclusion, our data support the role of genetic variants in iron metabolism ( TF ) and in the modulation of the calcium signalling/axonal anterograde transport of vesicles ( CLSTN1 ) as genetic modulators in AD and FTLD due to C9orf72 expansions.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Aim: Short tandem repeats (STRs) are repetitive DNA sequences and highly mutable in various human disorders. While the involvement of STRs in various genetic disorders has been extensively studied, their role in autism spectrum disorder (ASD) remains largely unexplored. In this study, we aimed to investigate genetic association of STR expansions with ASD using whole genome sequencing (WGS) and identify risk loci associated with ASD phenotypes., Methods: We analyzed WGS data of 634 ASD families and performed genome-wide evaluation for 12,929 STR loci. We found rare STR expansions that exceeded normal repeat lengths in autism cases compared to unaffected controls. By integrating single cell RNA and ATAC sequencing datasets of human postmortem brains, we prioritized STR loci in genes specifically expressed in cortical development stages. A deep learning method was used to predict functionality of ASD-associated STR loci., Results: In ASD cases, rare STR expansions predominantly occurred in early cortical layer-specific genes involved in neurodevelopment, highlighting the cellular specificity of STR-associated genes in ASD risk. Leveraging deep learning prediction models, we demonstrated that these STR expansions disrupted the regulatory activity of enhancers and promoters, suggesting a potential mechanism through which they contribute to ASD pathogenesis. We found that individuals with ASD-associated STR expansions exhibited more severe ASD phenotypes and diminished adaptability compared to non-carriers., Conclusion: Short tandem repeat expansions in cortical layer-specific genes are associated with ASD and could potentially be a risk genetic factor for ASD. Our study is the first to show evidence of STR expansion associated with ASD in an under-investigated population., (© 2024 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

Most of the heritability in frontotemporal dementia (FTD) is accounted for by autosomal dominant hexanucleotide expansion in the chromosome 9 open reading frame 72 (C9orf72), pathogenic/likely pathogenic variants in progranulin (GRN), and microtubule-associated protein tau (MAPT) genes. Until now, there has been no systematic analysis of these genes in the Serbian population. Herein, we assessed the frequency of the C9orf72 expansion, pathogenic/likely pathogenic variants in GRN and MAPT in a well-characterized group of 472 subjects (FTD, Alzheimer's disease - AD, mild cognitive impairment - MCI, and unspecified dementia - UnD), recruited in the Memory Center, Neurology Clinic, University Clinical Center of Serbia. The C9orf72 repeat expansion was detected in 6.98% of FTD cases (13.46% familial; 2.6% sporadic). In the UnD subgroup, C9orf72 repeat expansions were detected in 4.08% (8% familial) individuals. Pathogenic variants in the GRN were found in 2.85% of familial FTD cases. Interestingly, no MAPT pathogenic/likely pathogenic variants were detected, suggesting possible geographical specificity. Our findings highlight the importance of wider implementation of genetic testing in neurological and psychiatric practice managing patients with cognitive-behavioral and motor symptoms., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. It is mostly sporadic, with the C9orf72 repeat expansion being the most common genetic cause. While the prevalence of C9orf72-ALS in patients from different populations has been studied, data regarding the yield of C9orf72 compared to an ALS gene panel testing is limited.We aimed to explore the application of C9orf72 versus a gene panel in the general Israeli population. A total of 140 ALS patients attended our Neurogenetics Clinic throughout 2018-2023. Disease onset was between ages 60 and 69 years for most patients (34%); however, a quarter had an early-onset disease (< 50 years). Overall, 119 patients (85%) were genetically evaluated: 116 (97%) were tested for the C9orf72 repeat expansion and 64 (54%) underwent gene panel testing. The C9orf72 repeat expansion had a prevalence of 21% among Ashkenazi Jewish patients compared to 5.7% in non-Ashkenazi patients, while the gene panel had a higher yield in non-Ashkenazi patients with 14% disease-causing variants compared to 5.7% in Ashkenazi Jews. Among early-onset ALS patients, panel testing was positive in 12% compared to 2.9% for C9orf72.We suggest a testing strategy for the Israeli ALS patients: C9orf72 should be the first-tier test in Ashkenazi Jewish patients, while a gene panel should be considered as the first step in non-Ashkenazi and early-onset patients. Tiered testing has important implications for patient management, including prognosis, ongoing clinical trials, and prevention in future generations. Similar studies should be implemented worldwide to uncover the diverse ALS genetic architecture and facilitate tailored care., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms., (© 2024. Springer Nature Limited.)

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., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

The abnormal expansion of GGGGCC/GGCCCC hexanucleotide repeats (HR) in C9orf72 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Structural polymorphisms of HR result in the multifactorial pathomechanism of ALS/FTD. Consequently, many ongoing studies are focused at developing therapies targeting pathogenic HR RNA. One of them involves small molecules blocking sequestration of important proteins, preventing formation of toxic nuclear foci. However, rational design of potential therapeutics is hindered by limited number of structural studies of RNA-ligand complexes. We determined the crystal structure of antisense HR RNA in complex with ANP77 ligand (1.1 Å resolution) and in the free form (0.92 and 1.5 Å resolution). HR RNA folds into a triplex structure composed of four RNA chains. ANP77 interacted with two neighboring single-stranded cytosines to form pseudo-canonical base pairs by adopting sandwich-like conformation and adjusting the position of its naphthyridine units to the helical twist of the RNA. In the unliganded structure, the cytosines formed a peculiar triplex i-motif, assembled by trans C•C+ pair and a third cytosine located at the Hoogsteen edge of the C•C+ pair. These results extend our knowledge of the structural polymorphisms of HR and can be used for rational design of small molecules targeting disease-related RNAs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The newly discovered intronic repeat expansions in the genes encoding replication factor C subunit 1 (RFC1) and fibroblast growth factor 14 (FGF14) frequently cause late-onset cerebellar ataxia., Objectives: To investigate the presence of RFC1 and FGF14 pathogenic repeat expansions in Serbian patients with adult-onset cerebellar ataxia., Methods: The study included 167 unrelated patients with sporadic or familial cerebellar ataxia. The RFC1 repeat expansion analysis was performed by duplex PCR and Sanger sequencing, while the FGF14 repeat expansion was tested for by long-range PCR, repeat-primed PCR, and Sanger sequencing., Results: We identified pathogenic repeat expansions in RFC1 in seven patients (7/167; 4.2%) with late-onset sporadic ataxia with neuropathy and chronic cough. Two patients also had bilateral vestibulopathy. Repeat expansions in FGF14 were found in nine unrelated patients (9/167; 5.4%) with ataxia, less than half of whom presented with neuropathy and two-thirds with global brain atrophy. Tremor and episodic features were the most frequent additional characteristics in carriers of uninterrupted FGF14 repeat expansions. Among the 122 sporadic cases, 12 (9.8%) carried an expansion in either RFC1 or FGF14, comparable to 4/45 (8.9%) among the patients with a positive family history., Conclusions: Pathogenic repeat expansions in RFC1 and FGF14 are relatively frequent causes of adult-onset cerebellar ataxia, especially among sporadic patients, indicating that family history should not be considered when prioritizing ataxia patients for testing of RFC1 or FGF14 repeat expansions., (© 2024 The Authors. Movement Disorders Clinical Practice published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of the proposed pathogenic mechanisms is the neurotoxicity arising from dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation. Therefore, reducing DPR levels emerges as a potential therapeutic strategy for C9ORF72-ALS/FTD. We previously identified an RNA helicase, DEAD-box helicase 3 X-linked (DDX3X), modulates RAN translation. DDX3X overexpression decreases poly-GP accumulation in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons (iPSNs) and reduces the glutamate-induced neurotoxicity. In this study, we examined the in vivo efficacy of DDX3X overexpression using a mouse model. We expressed exogenous DDX3X or GFP in the central nervous system (CNS) of the C9-500 ALS/FTD BAC transgenic or non-transgenic control mice using adeno-associated virus serotype 9 (AAV9). The DPR levels were significantly reduced in the brains of DDX3X-expressing C9-BAC mice compared to the GFP control even twelve months after virus delivery. Additionally, p62 aggregation was also decreased. No neuronal loss or neuroinflammatory response were detected in the DDX3X overexpressing C9-BAC mice. This work demonstrates that DDX3X overexpression effectively reduces DPR levels in vivo without provoking neuroinflammation or neurotoxicity, suggesting the potential of increasing DDX3X expression as a therapeutic strategy for C9ORF72-ALS/FTD., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V β = -1.06, P < 0.001; lobules VI-VII β = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me 2/3 . Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me 3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC) n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention., (© 2024. The Author(s).)

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing., Competing Interests: Competing interests statement:B.R.C. is a founder of Tenaya Therapeutics, a company focused on finding treatments for heart failure, including genetic cardiomyopathies. B.R.C. holds equity in Tenaya. C.D.C. is a founder, with equity, in Ciznor Co., a CNS therapeutics company. B.R.C. and C.D.C. are inventors on a patent application for this work. Y.-C.T. is a full-time employee at PacBio, a company developing single-molecule sequencing technologies. The other authors declare no competing interests.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients., (© 2024. The Author(s).)

The discovery of hexanucleotide repeats expansion (RE) in Chromosome 9 Open Reading frame 72 ( C9orf72) as the major genetic cause of amyotrophic lateral sclerosis (ALS) and the association between intermediate repeats in Ataxin-2 ( ATXN2) with the disorder suggest that repetitive sequences in the human genome play a significant role in ALS pathophysiology. Investigating the frequency of repeat expansions in ALS in different populations and ethnic groups is therefore of great importance. Based on these premises, this study aimed to define the frequency of REs in the NIPA1 , NOP56, and NOTCH2NLC genes and the possible associations between phenotypes and the size of REs in the Italian population. Using repeat-primed-PCR and PCR-fragment analyses, we screened 302 El-Escorial-diagnosed ALS patients and compared the RE distribution to 167 age-, gender-, and ethnicity-matched healthy controls. While the REs distribution was similar between the ALS and control groups, a moderate association was observed between longer RE lengths and clinical features such as age at onset, gender, site of onset, and family history. In conclusion, this is the first study to screen ALS patients from southern Italy for REs in NIPA1 , NOP56 , and NOTCH2NLC genes, contributing to our understanding of ALS genetics. Our results highlighted that the extremely rare pathogenic REs in these genes do not allow an association with the disease.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD., (© 2024. The Author(s).)

To conduct a meta-analysis investigating the relationship between the chromosome 9 open reading frame 72 ( C9orf72 ) GGGGCC (G4C2) and neurodegenerative diseases (NDs), including Alzheimer's disease (AD), Parkinson's disease (PD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We searched the EMBASE, PubMed, Web of Science, and Cochrane databases. Twenty-seven case-control studies were included, comprising 7202 AD, 5856 PD, 644 MSA, 439 PSP, and 477 CBD cases. This study demonstrated that C9orf72 repeat expansions (>30) were associated with AD, MSA, PSP, and CBD (AD: OR = 4.88, 95% CI = 2.71-8.78; MSA: OR = 6.98, 95% CI = 1.48-33.01; PSP: OR =10.04, 95% CI = 2.72-37.10; CBD: OR = 28.04, 95% CI = 10.17-77.31). C9orf72 intermediate repeat expansions (20-30) were not associated with AD and MSA (AD: OR = 1.16, 95% CI = 0.39-3.45; MSA: OR = 5.65, 95% CI = 0.69-46.19), while C9orf72 repeat expansions (>30) were not associated with the risk of PD (OR = 1.51, 95% CI = 0.55-4.17), C9orf72 intermediate repeat expansions (20-30) were indeed associated with PD (OR = 2.43, 95% CI = 1.20-4.9). The pathological mechanism of C9orf72 G4C2 repeat expansions differs across various NDs due to the varying number of pathogenic expansions. Measuring the number of C9orf72 G4C2 repeats may be useful in the early-stage differential diagnosis of various NDs.

Objective: The resting-state functional connectome has not been extensively investigated in amyotrophic lateral sclerosis (ALS) spectrum disease, in particular in relationship with patients' genetic status., Methods: Here we studied the network-to-network connectivity of 19 ALS patients carrying the C9orf72 hexanucleotide repeat expansion (C9orf72+), 19 ALS patients not affected by C9orf72 mutation (C9orf72-), and 19 ALS-mimic patients (ALSm) well-matched for demographic and clinical variables., Results: When compared with ALSm, we observed greater connectivity of the default mode and frontoparietal networks with the visual network for C9orf72+ patients (P = 0.001). Moreover, the whole-connectome showed greater node degree (P < 0.001), while sensorimotor cortices resulted isolated in C9orf72+., Interpretation: Our results suggest a crucial involvement of extra-motor functions in ALS spectrum disease. In particular, alterations of the visual cortex may have a pathogenic role in C9orf72-related ALS. The prominent feature of these patients would be increased visual system connectivity with the networks responsible of the functional balance between internal and external attention., (© 2024 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: We aimed to estimate the age-related risk of ALS in first-degree relatives of patients with ALS carrying the C9orf72 repeat expansion., Methods: We included all patients with ALS carrying a C9orf72 repeat expansion in The Netherlands. Using structured questionnaires, we determined the number of first-degree relatives, their age at death due to ALS or another cause, or age at time of questionnaire. The cumulative incidence of ALS among first-degree relatives was estimated, while accounting for death from other causes. Variability in ALS risk between families was evaluated using a random effects hazards model. We used a second, distinct approach to estimate the risk of ALS and FTD in the general population, using previously published data., Results: In total, 214 of the 2,486 (9.2%) patients with ALS carried the C9orf72 repeat expansion. The mean risk of ALS at age 80 for first-degree relatives carrying the repeat expansion was 24.1%, but ranged between individual families from 16.0 to 60.6%. Using the second approach, we found the risk of ALS and FTD combined was 28.7% (95% CI 17.8%-54.3%) for carriers in the general population., Conclusions: On average, our estimated risk of ALS in the C9orf72 repeat expansion was lower compared to historical estimates. We showed, however, that the risk of ALS likely varies between families and one overall penetrance estimate may not be sufficient to describe ALS risk. This warrants a tailor-made, patient-specific approach in testing. Further studies are needed to assess the risk of FTD in the C9orf72 repeat expansion.

The C9orf72 hexanucleotide repeat expansion (HRE) is a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The inheritance is autosomal dominant, but a high proportion of subjects with the mutation are simplex cases. One possible explanation is de novo expansions of unstable intermediate-length alleles (IAs). Using haplotype sharing trees (HSTs) with the haplotype analysis tool kit (HAPTK), we derived majority-based ancestral haplotypes of HRE samples and discovered that IAs containing ≥18-20 repeats share large haplotypes in common with the HRE. Using HSTs of HRE and IA samples, we demonstrate that the longer IA haplotypes are largely indistinguishable from HRE haplotypes and that several ≥18-20 IA haplotypes share over 5 Mb (>600 markers) haplotypes in common with the HRE haplotypes. These analysis tools allow physical understanding of the haplotype blocks shared with the majority-based ancestral haplotype. Our results demonstrate that the haplotypes with longer IAs belong to the same pool of haplotypes as the HRE and suggest that longer IAs represent potential premutation alleles., Competing Interests: Declaration of interests P.J.T. holds a patent on C9orf72 in diagnostics and treatment of ALS/FTD., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The C9orf72 hexanucleotide repeat (HR) expansion is the main genetic cause of amyotrophic lateral sclerosis (ALS), with expansion size from 30 to >4000 units. Normal C9orf72 HR length is polymorphic (2-23 repeats) with alleles >8 units showing a low frequency in the general population. This study aimed to investigate if the normal C9orf72 HR length influences C9orf72 gene expression and acts as disease modifier in ALS patients negative for C9orf72 mutation (ALS-C9Neg). We found that the distribution of HR alleles was similar in 325 ALS-C9Neg and 303 healthy controls. Gene expression analysis in blood revealed a significant increase of total C9orf72 and V3 mRNA levels in ALS-C9Neg carrying two long alleles (L/L; ≥8 units) compared to patients homozygous for the 2-unit short allele (S/S). However, HR allele genotypes (L/L, S/L, S/S) correlated with no clinical parameters. Our data suggest that normal C9orf72 HR length does not act as disease modifier in ALS-C9Neg despite increasing gene expression.

Diminished basal parasympathetic nervous system activity is a feature of frontotemporal dementia that relates to left frontoinsula dysfunction and empathy impairment. Individuals with a pathogenic expansion of the hexanucleotide repeat in chromosome 9 open reading frame 72 (C9orf72), the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, provide a unique opportunity to examine whether parasympathetic activity is disrupted in genetic forms of frontotemporal dementia and to investigate when parasympathetic deficits manifest in the pathophysiological cascade. We measured baseline respiratory sinus arrhythmia, a parasympathetic measure of heart rate variability, over two minutes in a sample of 102 participants that included 19 asymptomatic expansion carriers (C9 + asymp), 14 expansion carriers with mild cognitive impairment (C9 + MCI), 16 symptomatic expansion carriers with frontotemporal dementia (C9 + FTD), and 53 expansion-negative healthy controls (C9 - HC) who also underwent structural magnetic resonance imaging. In follow-up analyses, we compared baseline respiratory sinus arrhythmia in the C9 + FTD group with an independent age-, sex-, and clinical severity-matched group of 26 people with sporadic behavioral variant frontotemporal dementia. The Frontotemporal Lobar Degeneration-modified Clinical Dementia Rating-Sum of Boxes score was used to quantify behavioral symptom severity, and informant ratings on the Interpersonal Reactivity Index provided measures of participants' current emotional (empathic concern) and cognitive (perspective-taking) empathy. Results indicated that the C9 + FTD group had lower baseline respiratory sinus arrhythmia than the C9 + MCI, C9 + asymp, and C9 - HC groups, a deficit that was comparable to that of sporadic behavioral variant frontotemporal dementia. Linear regression analyses indicated that lower baseline respiratory sinus arrhythmia was associated with worse behavioral symptom severity and lower empathic concern and perspective-taking across the C9orf72 expansion carrier clinical spectrum. Whole-brain voxel-based morphometry analyses in participants with C9orf72 pathogenic expansions found that lower baseline respiratory sinus arrhythmia correlated with smaller gray matter volume in the left frontoinsula and bilateral thalamus, key structures that support parasympathetic function, and in the bilateral parietal lobes, occipital lobes, and cerebellum, regions that are also vulnerable in individuals with C9orf72 expansions. This study provides novel evidence that basal parasympathetic functioning is diminished in FTD due to C9orf72 expansions and suggests that baseline respiratory sinus arrhythmia may be a potential non-invasive biomarker that is sensitive to behavioral symptoms in the early stages of disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Spinocerebellar ataxias (SCAs) are a heterogenous group of neurodegenerative disorders which commonly inherited in an autosomal dominant manner. They cause muscle incoordination due to degeneration of the cerebellum and other parts of nervous system. Out of all the characterized (>50) SCAs, 14 SCAs are caused due to microsatellite repeat expansion mutations. Repeat expansions can result in toxic protein gain-of-function, protein loss-of-function, and/or RNA gain-of-function effects. The location and the nature of mutation modulate the underlying disease pathophysiology resulting in varying disease manifestations. Potential toxic effects of these mutations likely affect key major cellular processes such as transcriptional regulation, mitochondrial functioning, ion channel dysfunction and synaptic transmission. Involvement of several common pathways suggests interlinked function of genes implicated in the disease pathogenesis. A better understanding of the shared and distinct molecular pathogenic mechanisms in these diseases is required to develop targeted therapeutic tools and interventions for disease management. The prime focus of this review is to elaborate on how expanded 'CAG' repeats contribute to the common modes of neurotoxicity and their possible therapeutic targets in management of such devastating disorders., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC) n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC) n , which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD., (© 2023. The Author(s).)