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PubMed2025

Generation of C9orf72 repeat knock-in iPSC lines for modelling ALS and FTD.

Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72-associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.

Journal Article↗

PubMed2026

Two new cases of fragile X syndrome without CGG triplet expansion. Clinical-molecular characterization and review of the literature.

Fragile X syndrome (FXS) is classically caused by CGG repeat expansion in the FMR1 gene leading to gene silencing. We describe two unrelated patients with clinical features consistent with FXS but harbouring distinct molecular mechanisms. The first patient had a pathogenic intronic variant in FMR1, predicted to impair protein function, and no repeat expansion. The second patient carried a complete deletion of the FMR1 gene, resulting in loss of gene expression. Both individuals presented with developmental delay, intellectual disability, and behavioural manifestations typical of FXS. These cases expand the causal heterogeneity underlying a clinically recognizable phenotype. They reinforce the concept that comprehensive molecular testing beyond repeat expansion analysis is mandatory in individuals with a strong clinical suspicion of FXS, in absence of a typical CGG triplet expansion.

Child, Preschool↗

PubMed2026

Altered neuronal start codon stringency favors cap-independent repeat-associated non-AUG translation.

Intronic GGGGCC repeat expansions in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This expansion supports a non-canonical form of translational initiation known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat proteins that contribute to neurodegeneration. Here, we find that the efficiency of RAN translation and its dependency on the 5' 7-methylguanosine mRNA cap are variable across cell types, with both rodent neurons and human iNeurons favoring cap-independent RAN translation from two distinct repeats (CGG and GGGGCC) across multiple reading frames. Treatment with an eIF4E inhibitor that blocks cap-dependent translation enhances RAN translation specifically in neurons. Intriguingly, cap-independent RAN translation exhibits less reliance on near-cognate codons for initiation than cap-dependent RAN translation. This finding led us to identify a surprising global alteration in neuronal start codon stringency as a contributor to the relatively higher cap-independent RAN translation in this cell type. This effect correlates with cytoplasmic redistribution of eIF1 in neurons and is reversed with overexpression of the eukaryotic initiation factor eIF5, which relaxes start codon stringency and preferentially enhances cap-dependent RAN translation. Together, these findings reveal several neuron-specific features of translational regulation that favor cap-independent RAN translation with implications for nucleotide repeat expansion disorder pathogenesis.

Neurons↗

PubMed2025

Delineating the pathogenic threshold and phenotypic spectrum of SCA27B: findings from a large French-Canadian cohort.

BACKGROUND: Autosomal dominant spinocerebellar ataxia 27B (SCA27B), caused by an intronic (GAA•TTC) repeat expansion in FGF14, is a common cause of late-onset cerebellar ataxia, but its genotypic and phenotypic spectrum remains to be fully established. METHODS: We analysed the FGF14 (GAA•TTC) repeat expansion in a cohort of 134 patients with ataxia and 822 controls from Quebec. We conducted segregation study in large families to further characterize intergenerational repeat instability. RESULTS: We found a significant enrichment of (GAA•TTC)≥200 alleles in the ataxia cohort compared to controls (53.0%, 71/134, vs 3.6%, 30/822, p < 0.0001), including for (GAA•TTC)200-249 alleles (8.2% vs 2.6%, p = 0.0026). We identified 12 ataxic patients with a phenotype compatible with SCA27B carrying a (GAA•TTC)200-249 expansion supporting the pathogenicity of these alleles in some patients. We further delineated the phenotype of 125 symptomatic individuals from 69 families who carried an FGF14 (GAA•TTC)≥200 repeat expansion. Patients with (GAA•TTC)200-249, (GAA•TTC)250-299, and (GAA•TTC)≥300 had a similar phenotype. We observed that 14% of patients with episodic symptoms (13/92) had severe episodes that were initially misdiagnosed as stroke, vestibular neuritis, Wernicke's encephalopathy, or seizures. DISCUSSION AND CONCLUSION: This large cohort demonstrates that (GAA•TTC)200-249 alleles are enriched in patients with ataxia compared to controls and can be pathogenic for SCA27B, supporting the need to define a lower pathogenic threshold in the presence of specific clinical criteria.

Humans↗

PubMed2026

Targeted long-read genomic and epigenomic profiling enhances timely comprehensive variant discovery in hypotonia and muscle weakness.

BACKGROUND: Identifying the genetic basis of hypotonia and muscle weakness is critical for patient management and family counseling. However, diagnosis is often hindered by diverse genomic alterations, including repeat expansions, structural variants (SVs), and methylation defects. Standard-of-care testing, largely based on short-read sequencing, is limited in its ability to detect this heterogeneous variation landscape, leaving many patients undiagnosed or requiring lengthy sequential testing. Long-read sequencing represents a promising solution. However, its application as a first-tier diagnostic assay for hypotonia remains unexplored. METHODS: We retrospectively analyzed 227 patients with hypotonia to assess diagnostic yield, time-to-diagnosis, and costs associated with standard-of-care testing. A long-read whole-genome sequencing (LR-WGS) workflow with targeted analysis of hypotonia-associated genes was developed to detect and prioritize pathogenic SNVs, SVs, and CNVs, repeat expansions, and methylation changes at key disease loci. The workflow was validated in a reference-positive cohort with known diagnoses (n = 15) and applied to an unsolved cohort (n = 14). Variant interpretation followed ACMG guidelines and was confirmed with orthogonal methods. RESULTS: Standard-of-care testing achieved a diagnostic yield of 42% with an average time-to-diagnosis of 68.7 days; however, 30% of diagnosed patients experienced significant delays (average 169 days) due to sequential testing. The LR-WGS based approach identified all known pathogenic variants in the positive cohort, including SMN1 deletions, methylation defects at 15q11.2/Prader-Willi locus, FMR1 repeat expansions, and sequence and copy-number variants in > 100 genes underlying myopathies and muscular dystrophies. The targeted long-read pipeline reduced prioritized variant calls by 97.9-99.9% and, in the unsolved cohort, yielded one definitive diagnosis (de novo COL6A3 deletion) and one possible diagnosis (aberrant methylation and copy number at POMK), for an additional 14% yield. Among patients diagnosed after sequential testing (n = 29), LR-WGS is expected to reduce time-to-diagnosis by ~ 85% and decrease cumulative diagnostic delays, with projected healthcare cost savings of $396,000-439,000. Across the entire 227 patient cohort, LR-WGS is anticipated to reduce testing costs by 6.5%, yielding an average savings of $105 per patient. CONCLUSIONS: LR-WGS enables comprehensive discovery of genomic and epigenomic variants in hypotonia and muscle weakness, improving diagnostic yield, shortening diagnostic timelines, and reducing costs compared with current standard-of-care testing.

Humans↗

PubMed2026

The HTT1a protein initiates HTT aggregation in a knock-in mouse model of Huntington's disease.

The mutation that causes Huntington's disease is a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that leads to an abnormally long polyglutamine tract in the huntingtin protein (HTT). Mutant CAG repeats are unstable and increase in size in specific neurons and brain regions with age, a phenomenon that constitutes the first step in the pathogenesis of the disease. In the presence of an expanded CAG repeat, cryptic polyadenylation (polyA) sites in intron 1 of the HTT pre-mRNA can become activated leading to the polyadenylation of a prematurely terminated transcript, HTT1a. This encodes the HTT1a protein, which is known to be very aggregation-prone and highly pathogenic. Given that the longer the CAG repeat the more HTT1a is generated, could the production of HTT1a be the mechanism through which somatic CAG repeat expansion exerts its pathogenic consequences? Resolving this issue is very important for the design of therapeutic approaches to lower huntingtin levels. We have used a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 approach to prevent the production of HTT1a in a knock-in mouse model of Huntington's disease. All potential cryptic polyA sites were deleted from Htt intron 1 in HdhQ150 mice and colonies were established that were heterozygous for the intron 1 deletion on a mutant allele (HdhQ150ΔI) and heterozygous for the deletion on a wild-type allele (WTΔI). The CAG repeat sizes in the HdhQ150 and HdhQ150ΔI colonies were well-matched at approximately 195 CAGs. As predicted, the deletion of the cryptic polyA sites from Htt intron 1 prevented the generation of the Htt1a transcript in the HdhQ150ΔI mice. However, very low levels of the HTT1a protein were detected, which resulted from a Htt readthrough product of exon 1 and exon 2, that had retained the deleted intron and terminated at a cryptic polyA site in intron 2. HdhQ150, HdhQ150ΔI, wild-type and WTΔI mice were studied until 17 months of age. Immunohistochemical and homogeneous time-resolved fluorescence analysis showed that HTT aggregation in both HdhQ150 and HdhQ150ΔI brains contained HTT1a, but the dramatic decrease in soluble HTT1a levels in HdhQ150ΔI brains delayed the appearance of aggregated HTT1a by several months. Although this delay in aggregate pathology only partially reversed transcriptional dysregulation, the biomarkers neurofilament light polypeptide (NEFL) and breast regression protein 39 (BRP39) (YKL40) remained at wild-type levels in HdhQ150ΔI mice at 17 months of age. These data demonstrate that the production of HTT1a initiates HTT aggregation and that it is important to target HTT1a in huntingtin-lowering therapeutic strategies.

Animals↗

PubMed2026

Establishment and characterization of three human pluripotent stem cell lines from patients with spinocerebellar ataxia 27B (SCA27B).

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant cerebellar ataxia caused by an intronic GAA•TTC repeat expansion in the FGF14 gene. Here, we report the generation and validation of three human induced pluripotent stem cell (iPSC) lines derived from unrelated individuals with SCA27B, including two lines carrying a monoallelic pathogenic GAA•TTC repeat expansion in FGF14 and one line with biallelic expansions. These iPSC lines constitute a valuable resource, particularly given the predominantly neuronal expression of FGF14, and enable the investigation of disease mechanisms in relevant cell types following neuronal differentiation.

Humans↗

PubMed2026

DNA Methylation and Proteomic Profiling of Postmortem Brain Tissue Reveals Epigenetic Dysregulation and Neuroinflammatory in Fragile X-associated Tremor/Ataxia Syndrome (FXTAS).

BACKGROUND: Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by FMR1 premutation CGG repeat expansions (55-200 repeats). The epigenetic landscape of the FXTAS brain remains uncharacterized. We performed genome-wide DNA methylation profiling of postmortem prefrontal cortex tissue to identify differentially methylated positions (DMPs) and candidate genes, and sought protein-level support for a neuroinflammatory signal. METHODS: DNA methylation was profiled in postmortem prefrontal cortex (Brodmann area 9) from 27 male FXTAS cases and 29 male controls using the Illumina MethylationEPIC array (EPICv1 and EPICv2 platforms), merging 721,802 common probes. Surrogate variable analysis (SVA) controlled for confounders. DMPs were defined by |Δβ| > 0.10 and FDR < 0.05; exploratory Reactome 2024 pathway analysis was performed on the DMP-associated gene list. Targeted proteomic profiling was performed in the same brain region using the Olink (proximity extension assay) Inflammation panel in 9 FXTAS cases and 12 controls, with SVA-adjusted differential abundance analysis, and concordance assessment against a prior mass spectrometry dataset. RESULTS: We identified 108 significant cg-type DMPs mapping to 80 genes (50 hypermethylated, 58 hypomethylated in FXTAS). The strongest signal was CYP2E1 (7 concordant hypomethylated DMPs, mean Δβ = -0.143), an oxidative stress gene also implicated in Parkinson's disease. FTCD, a one-carbon cycle enzyme, carried 5 hypermethylated DMPs (mean Δβ = +0.210). A cluster of DMP-associated genes with established roles in innate immune and NF-κB signaling, TRAF3 (the single most significant DMP among the inflammation genes, hypermethylated), BATF, RCOR1, and MSI2; they pointed toward neuroinflammatory dysregulation. Additional genes included LINGO1 (myelination inhibitor), SYT3 (synaptic vesicle), and SLC39A4 (zinc transporter). Exploratory Reactome enrichment using the DMP-associated gene set nominated themes including neuroinflammation resolution, axonal growth inhibition, zinc homeostasis, and CYP2E1 metabolism at nominal significance (p<0.05); however, the gene-to-pathway mapping rate was low and no pathway survived correction for multiple testing. Olink proteomic analysis independently identified 60 significantly altered inflammation proteins (59 downregulated), including CXCL8, CXCL10, IL6, IL15, IL18, TLR3, IRAK1/4, and complement C1QA, which were directionally concordant with prior mass spectrometry data. CONCLUSIONS: This integrated study reveals a genome-wide epigenetic signature in the FXTAS prefrontal cortex implicating oxidative stress, myelination failure, zinc dysregulation, one-carbon cycle disruption, and most notably a coordinated set of epigenetically altered genes governing innate immune and NF-κB signaling. Convergence of TRAF3 hypermethylation with independent downregulation of TLR3 and NF-κB-pathway proteins at the protein level supports a coherent, cross-platform model of dysregulated neuroinflammatory signaling in FXTAS, identified here through individual gene- and protein-level convergence rather than formal pathway enrichment. FTCD hypermethylation proposes a self-reinforcing epigenetic loop via SAM depletion. These multi-omic findings establish FXTAS as a disorder of pervasive epigenetic reprogramming and nominate candidate genes for future mechanistic and therapeutic investigation.

CYP2E1↗

PubMed2026

C9orf72-associated poly-GR in skeletal muscle leads to neuromuscular junction deficits and muscle atrophy.

Hexanucleotide repeat expansions in C9orf72 produce dipeptide repeat (DPR) proteins that are widely expressed, including in the nervous system and skeletal muscle. Among these DPRs, arginine-containing proteins, poly-GR and poly-PR, are toxic in the nervous system, but whether DPRs in skeletal muscle contribute to amyotrophic lateral sclerosis (ALS) pathogenesis is unclear. Here, we show that muscle-restricted expression of poly-GR drives motor deficits in mice, including muscle atrophy and neuromuscular junction (NMJ) deficits. Poly-GR in muscle interacted with the NMJ key organizer MuSK and promoted MuSK degradation, disrupting postsynaptic structure and impairing neuromuscular transmission. Importantly, a MuSK agonist antibody (X-17) stabilized NMJs and rescued neuromuscular transmission. Moreover, poly-GR in muscle activated the integrated stress response (ISR), elevating eIF2α phosphorylation and broadly suppressing protein translation. ISR inhibition with ISRIB restored translation and MuSK protein levels and ameliorated both muscle atrophy and NMJ deficits. These findings demonstrate that skeletal muscle actively contributes to C9orf72-ALS pathology. Targeting muscle with ISRIB offers a therapeutic strategy to preserve motor function in C9orf72-ALS.

Animals↗

PubMed2026

Frataxin deficiency drives cardiac dysfunction and transcriptional dysregulation in Friedreich ataxia iPSC model.

Friedreich ataxia (FRDA) is a progressive neuromuscular degenerative disorder caused by GAA repeat expansions in the FXN gene, leading to frataxin deficiency and multisystem pathology. Cardiomyopathy is the leading cause of mortality in individuals with FRDA. To investigate the cellular and molecular mechanisms underlying FRDA-associated cardiac dysfunction, we employed induced pluripotent stem cell (iPSC) lines derived from three individuals with FRDA, each paired with an isogenic control line generated through CRISPR/Cas9-mediated excision of the pathogenic GAA repeat expansion. Correction of the mutation restored FXN expression to levels comparable to healthy donor iPSCs, and all lines differentiated efficiently into cardiomyocytes. Functional analysis revealed significant contractile abnormalities in FRDA cardiomyocytes and multicellular cardiac microtissues, including prolonged contraction and relaxation times and faster beating rates, consistent with clinical observations of cardiac contractile dysfunction. FRDA cardiomyocytes also exhibited pathological features such as increased cell size, irregular calcium transients, elevated mitochondrial reactive oxygen species levels, increased mitochondrial fission and increased cell death. These phenotypes were exacerbated by pathological levels of iron supplementation in culture media, highlighting the heightened sensitivity of frataxin-deficient cardiomyocytes to iron-induced metabolic stress. RNA sequencing revealed a distinct transcriptional profile associated with frataxin deficiency. MEG3 and PCDHGA10 were consistently dysregulated across all three FRDA-iPSC lines and may represent early molecular markers of FRDA cardiomyopathy. Functional interrogation of these candidates demonstrated that targeted silencing of MEG3 or PCDHGA10 in FRDA cardiomyocytes significantly reduced disease‑associated cell death without affecting FXN expression. Notably, PCDHGA10 silencing also normalized elevated mitochondrial reactive oxygen species, whereas MEG3 silencing did not, highlighting gene‑specific contributions to FRDA cardiomyocyte survival. Collectively, these findings identify MEG3 and PCDHGA10 as functionally relevant regulators of FRDA cardiomyocyte pathology.

Friedreich Ataxia↗

PubMed2023

Dynamics and variability of transcriptomic dysregulation in congenital myotonic dystrophy during pediatric development.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by expansion of CTG microsatellite repeats within DMPK. The most severe form, congenital myotonic dystrophy (CDM), has symptom onset at birth due to large intergenerational repeat expansions. Despite a common mutation, CDM individuals present with a distinct clinical phenotype and absence of common DM1 symptoms. Given the clinical divergence, it is unknown if the hallmark of DM1 pathology, dysregulation of alternative splicing (AS) due to sequestration of MBNL proteins within toxic CUG repeat RNAs, contributes to disease throughout pediatric development. To evaluate global transcriptomic dysregulation, RNA-seq was performed on 36 CDM skeletal muscle biopsies ages 2 weeks to 16 years, including two longitudinal samples. Fifty DM1 and adult/pediatric controls were also sequenced as comparative groups. Despite a large CTG expansion and shared age of onset, CDM individuals presented with a heterogenous, MBNL-dependent mis-splicing signature. Estimation of intracellular MBNL concentrations from splicing responses of select events correlated with total spliceopathy and revealed a distinct, triphasic pattern of AS dysregulation across pediatric development. CDM infants (< 2 years) possess severe mis-splicing that significantly improves in early childhood (2-8 years) independent of sex or CTG repeat load. Adolescent individuals (8-16 years) stratified into two populations with a full range of global splicing dysregulation. DMPK expression changes correlated with alterations in splicing severity during development. This study reveals the complex dynamics of the CDM muscle transcriptome and provides insights into new therapeutic strategies, timing of therapeutic intervention, and biomarker development.

Child, Preschool↗

PubMed2026

Population-scale detection of methylation outliers from long-read genome sequencing.

BACKGROUND: Aberrant DNA methylation can mediate the functional effects of rare genetic variation and contribute to imprinting disorders, repeat expansion diseases, and other pathogenic regulatory mechanisms. Long-read sequencing technologies now enable genome-wide detection of CpG methylation alongside genetic variation from a single assay. However, methods for systematic identification and interpretation of methylation outliers from long-read sequencing data remain limited. METHODS: We developed METAFORA, a computational workflow for detecting methylation outlier regions from PacBio and Oxford Nanopore long-read sequencing data. METAFORA constructs population-level methylation references, segments the genome into correlated CpG blocks, infers technical and biological sources of variation through hidden factor estimation, models uncertainty due to variable depth sequencing, and computes covariate-adjusted methylation outlier scores for individual samples. We applied METAFORA across large long-read sequencing cohorts and integrated methylation outliers with multi-omic data. METAFORA is implemented as a snakemake workflow available at https://github.com/tjense25/METAFORA. RESULTS: METAFORA identified methylation outlier regions associated with rare structural variants, tandem repeat expansions, and imprinting abnormalities. We found outlier regions were enriched for molecular outliers across transcriptomic and chromatin accessibility datasets, supporting their functional relevance in gene regulation. In a representative case, METAFORA identified an imprinting defect affecting the GNAS locus associated with an STX16 deletion. CONCLUSIONS: METAFORA enables scalable detection and interpretation of methylation outliers from long-read sequencing data and provides a framework for integrating epigenetic outliers with genomic and multi-omic analyses. These approaches may improve interpretation of rare regulatory variation and support discovery of clinically relevant epigenetic abnormalities in genomic medicine.

DNA methylation↗

PubMed2026

Hypothesis-free evaluation of circulating metabolome provides cell-specific insights regarding the role of energy substrate availability in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with limited therapeutic options. The circulating metabolome comprises small molecules present in plasma/serum which are the intermediates and end-products of cellular metabolism, and is linked to ALS pathogenesis. METHODS: We conducted hypothesis-free two-sample Mendelian randomisation (MR) analysis of the concentration of 575 plasma/serum metabolites, to determine which are causally linked to risk of ALS. Significant metabolites were validated in an independent GWAS of plasma/serum metabolite concentrations and evaluated for sex-specific effects. Correlations between directly measured patient biofluid metabolite concentrations and ALS risk/severity were examined in 94 ALS patients and 40 controls. We experimentally assessed metabolic function in a murine neurons and human astrocytes carrying an ALS-associated G4C2-repeat expansion within C9orf72. RESULTS: MR causally associated five metabolites with ALS risk after multiple-testing correction. Higher serum concentration of glycoprotein acetyls (P = 9.7e - 9, β = 0.21) and the peptide DSGEGDFXAEGGGVR (P = 8.0e - 6, β = 0.22) was associated with increased ALS risk, whereas higher plasma concentration of phenylalanylserine, isobutyrylcarnitine, and acetylcarnitine was protective (P < 5e - 5, β = - 0.29 to - 0.72). DSGEGDFXAEGGGVR has been linked to glucose metabolism but we have used genetic fine-mapping to link DSGEGDFXAEGGGVR, neuronal glucose uptake through GLUT3, and ALS risk. Direct measurement of metabolite concentrations in patient biofluids revealed elevated acetylcarnitine levels in patients with ALS, which were associated with delayed symptom onset (Cox regression, P = 0.02, HR = 0.4). Similarly, lactate is elevated in ALS patient CSF (ANOVA, P = 1.3e - 3) and in patients with longer survival time (Cox regression, P = 0.03, HR = 0.3). Plasma fructose is elevated in ALS patients with shorter survival time (Cox regression, P = 0.02, HR = 1.1). In vitro, neurons and astrocytes carrying an ALS-associated G4C2-repeat expansion within C9orf72 demonstrated reduced metabolic flexibility. CONCLUSIONS: We provide evidence that impaired energy substrate availability contributes to ALS risk and severity. CNS cell types differ in their use of energy substrates and therefore we postulate the relative importance of different cell types for different stages of disease. Our findings support further investigation of metabolic interventions to treat or prevent ALS.

Amyotrophic Lateral Sclerosis↗

PubMed2026

Longitudinal progression, metrics, age-dependence, and modifiers of ataxia severity in SCA27B: a multicentre study of 219 patients.

BACKGROUND: Spinocerebellar Ataxia 27B (SCA27B) is a novel, frequent and likely treatable late-onset autosomal-dominant ataxia caused by GAA repeat-expansions in FGF14. For understanding disease evolution and imminent trial planning, metrics of the most widely used clinical outcome assessment (Scale for the Assessment and Rating of Ataxia/SARA), longitudinal progression and modifiers thereof are warranted. METHODS: Multicentre intercontinental observational study (2015-2024) of 661 assessments from 219 patients with SCA27B (age: 68 ± 10 years; SARA: 9 ± 6 points) with item-level distribution-based analyses to characterise SARA metrics relative to ageing-related impairment in 390 healthy controls; and linear mixed-effects modelling to determine longitudinal progression and demographic or genetic modifiers. FINDINGS: Ataxia severity in SCA27B as assessed by SARA was primarily attributable to gait, stance, and lower-limb impairment; other ataxia domains scored ≤1 SARA point in 79-94% of patients. Discrimination of SCA27B motor performance from controls decreased with age due to ageing-related motor variability captured by SARA, thus limiting potential metric response windows for symptomatic treatments. Disease progression was faster in the presence of interfering ageing-related comorbidities in 14 (6%) patients. Overall longitudinal progression of SCA27B was 0.54 SARA points/year [95% CI: 0.37-0.71]. Expansions of (GAA)> 180 repeats were frequent also on the shorter allele (n = 18 (8%), range: 196-348 repeats), and associated with faster progression (+1.6 SARA points/year, [95% CI: 0.9-2.2]), including also otherwise less affected ataxia domains speech and sitting. INTERPRETATION: Disease progression in SCA27B is characterised by mild progression, ageing-related motor variabilities and comorbidities, and associated with repeat size on both alleles. FUNDING: Else-Kröner-Fresenius-Stiftung, EU, DFG, BMBF, CIHR, NAF, Ataxia-UK, CSC.

Humans↗

PubMed2026

Dissecting the relationship between haplotypes around ATXN2 CAG repeats and the number of CAA interruptions by long-read sequencing.

BACKGROUND: CAG repeat expansions in ATXN2 are implicated as risk factors for several neurological diseases, including spinocerebellar ataxia type 2 (SCA2) when >=33 CAG repeats are present, and amyotrophic lateral sclerosis (ALS) when 27-33 CAG repeats are present. However, how haplotypes around the repeats and CAA interruptions within the repeats are associated with disease phenotypes remains poorly understood. Previous studies on haplotypes around ATXN2 were limited to SNPs very close to the repeats (<5kb) or were based on statistical inference only. METHODS: Here, we used long-read sequencing on the Oxford Nanopore Technologies (ONT) platform to simultaneously infer haplotypes around ATXN2, the number of CAG repeats, and the number of CAA interruptions, along with NYGC ALS Consortium NGS dataset. We further sequenced 41 individuals (EUR = 39) with neurological diseases with intermediate repeats by ONT. RESULTS: We found that haplotypes around ATXN2 and the number of interruptions show ethnicity-specific and ALS-specific distribution. Three CAA interruptions are present at low prevalence (~1%) in control populations in multiple ancestry groups, but high prevalence (~55%) in ALS individuals with intermediate repeats. Furthermore, we examined 159 individuals with ALS (~90% European ancestry) with intermediate ATXN2 repeats and found a unique haplotype in ALS individuals with three CAA interruptions, which can be tagged by an SNV, rs148019457. We also validated that the rs148019457-G allele is only present in haplotypes with three CAA interruptions. CONCLUSIONS: In summary, our study shows that 3 CAA interruptions are rarely seen in healthy controls but are common in those with expanded ATXN2 CAG repeats who have neurological disorders, and that rs148019457 tags a specific haplotype with 3 CAA interruptions within expanded ATXN2 CAG repeats in individuals of European ancestry. These results have implications for the development of precision genomic medicine for neurological disorders, and the tag SNP may help identify those with interruptions from existing population genotyping data.

ATXN2↗

PubMed2025

Rare variants in MIR184 are a novel genetic cause of Fuchs endothelial corneal dystrophy.

PURPOSE: To identify novel genetic causes of Fuchs endothelial corneal dystrophy (FECD) within a genetically unsolved patient cohort lacking repeat expansions in the TCF4 gene (Exp-). METHODS: A rare variant analysis framework (CoCoRV) was applied to exome data, in combination with in silico modeling, luciferase reporter, and RNA-seq analysis to characterize transcriptome-wide consequences of identified variants. RESULTS: A gene burden analysis identified MIR184, a microRNA encoding gene, to be enriched for rare pathogenic variants within the studied Exp- FECD cohort. In total, 2 noncoding rare variants were identified in 4 unrelated FECD probands: NR_029705.1:n.58G>A and n.73G>T. Both variants altered highly conserved mature sequence residues, were predicted to induce hairpin structural changes, and were experimentally determined to disrupt microRNA-mRNA interactions. RNA-seq of transfected human corneal endothelial cells revealed that the mutants elicited distinct transcriptomic profiles. Enriched KEGG pathways included PI3K-Akt signaling, focal adhesion, and immune response, revealing shared pathogenic mechanisms between MIR184-associated FECD and the more common TCF4 repeat expansion-mediated form of disease. CONCLUSION: MIR184 variants are a novel rare genetic cause of FECD, and common pathways of transcriptomic dysregulation are shared across genetically distinct subtypes of the disease. These pathways may serve as future gene agnostic targets for therapeutic interventions.

Humans↗

PubMed2025

Genome reorganisation and expansion shape 3D genome architecture and define a distinct regulatory landscape in coleoid cephalopods.

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.

Journal Article↗

PubMed2026

High-affinity, structure-validated and selective macrocyclic peptide tools for chemical biology studies of Huntingtin.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a Cytosine-Adenosine-Guanine (CAG) repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryoelectron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically copurified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex.

Huntingtin Protein↗