Neurology Genetics

Bryant-Li-Bhoj Syndrome (BLBS; OMIM: 619720, 619721) is a Mendelian neurogenetic condition, first described in 2020, with a mixed neurodevelopmental/neurodegenerative phenotype and variable systemic features. To date, 100 affected individuals with 74 unique causative variants have been published. Clinical data and prior functional work in multiple model systems have emphasized the utility of interrogating the pathogenesis of multiple causal variants to identify a convergent, therapeutically targetable mechanism. Additionally, the ability to evaluate the efficacy of future therapeutics relies on the availability of a robustly validated preclinical model. Here, we characterize the developmental and neurobehavioral phenotypes of a novel BLBS mouse model harboring one of the most recurrent causative variants (h3-3a p.T45I). H3.3T45I mice recapitulate the BLBS natural history: perinatal growth restriction, delayed developmental milestones, and progressive motor and gait impairments. Adult mice additionally display craniofacial differences, impaired nest building, hyperactivity in a social context, and male-specific elevated aggression. The non-invasive, clinically translatable endpoints established here provide a validated preclinical platform for evaluating therapeutics for a community whose current standard of care is symptom management. Summary StatementA new mouse model mirrors the developmental delays, motor decline, and behavioral changes seen in individuals with this rare, progressive genetic brain disorder, providing a foundation for testing future therapies.

Background: Neurological, neuropsychiatric, and neurodevelopmental disorders cluster in ALS families, sharing a common genetic architecture with ALS. Pathogenic variants in genes associated with other neurological, neurodevelopmental, or neuropsychiatric disorders may also co-occur in ALS and modify phenotype. We have sought to determine the prevalence and clinical pattern of likely-pathogenic/pathogenic (LP/P) non-ALS neurological, neurodevelopmental, and neuropsychiatric variants, alone and in combination with ALS-gene variants, in two large ALS cohorts. Methods: Whole-genome sequencing (WGS) of 469 Irish and 774 Answer ALS people with ALS (pwALS) was analysed for ClinVar LP/P variants associated with other neurological (n = 15541), neurodevelopmental (n = 9761), and neuropsychiatric (n = 321) phenotypes. Inheritance patterns for associated genes (autosomal recessive/autosomal dominant) along with the associated phenotype were validated using OMIM. Standardised clinical data included family history, site and age of onset, El Escorial category, survival, motor decline, and cognitive and behavioural assessments. Known ALS-gene variants and C9orf72 repeat expansion status were included for each cohort. Results: Non-ALS neurological variants were identified in 47/469 (10.0%) Irish and 69/774 (8.9%) Answer ALS participants, most frequently in hereditary spastic paraplegia-associated genes (3.2% Irish; 2.8% Answer ALS). Irish neurological variant carriers showed higher frequency of respiratory onset (10.6% vs 1.2%, Fisher's exact p = 0.002, {Phi} = 0.20) and fewer premorbid behavioural symptoms (0.92 +/- 0.56 vs 3.08 +/- 0.97, Cohen's d = -0.40). Neurodevelopmental variants occurred in 12/469 (2.6%) Irish and 20/774 (2.6%) Answer ALS participants. In the Irish cohort, neurodevelopmental variant carriers had significantly shorter survival in Cox proportional hazards model (log-rank p = 0.005), corresponding to a more than two-fold increased hazard of death (HR = 2.25, 95% CI 1.26-4.00), and had significantly increased familial burden of neuropsychiatric disorders among first- and second-degree relatives (negative binomial IRR for carriers = 2.41, 95% CI: 1.12-5.18, p = 0.025). Across combined cohorts, 18 individuals (Irish n = 8; Answer ALS n = 10) carried [≥]2 LP/P variants spanning ALS and non-ALS genes. Conclusion: Rare LP/P variants in genes associated with other neurological and neurodevelopmental disorders occur in up to 12% of pwALS across two independent cohorts. Carriers show distinct phenotypes, shorter survival, and characteristic family history patterns. These findings suggest that extended pleiotropic and oligogenic architectures may contribute to ALS heterogeneity.

BackgroundHuntington disease (HD) is a neurological disorder caused by an aberrant CAG expansion in the HTT gene, producing a mutant protein (mHTT). Although HD is classically characterized by adult-onset cortical and striatal degeneration, accumulating evidence suggests that altered cortical development may also contribute to disease pathogenesis. ObjectiveWe sought to investigate the impact of mHTT on neocortical patterning, which is a largely unexplored aspect of HD. MethodsUsing the HdhQ140 HD knock-in mouse model, we performed immunofluorescence and in situ hybridization to analyze the patterning of the cortex from embryonic day 10 to postnatal day 7. ResultsDuring embryogenesis, HTT expression exhibited a high medial-to-low lateral gradient in the neocortex, like that observed for key transcription factors involved in cortical patterning. Notably, HTT expression was absent from the cortical hem, a critical patterning center. In HD, the protein gradient remained unchanged whereas the expression in medial pallium seemed increased. During the early development of the cerebral hemispheres, the expression of morphogens and signaling pathways, including Shh, Fgf8, and Wnt/BMP genes, were disrupted in organizing centers, leading to altered expression of major neocortical transcription factors. At postnatal stages, the motor and somatosensory cortical areas were misplaced. These developmental alterations were associated with postnatal sensorimotor deficits relevant to HD. ConclusionsOur findings demonstrate that HD-related neurodevelopmental alterations arise as early as embryonic day 10 in mice. This supports previous work suggesting that defects in brain development contribute to HD pathogenesis prior to clinical onset.

CLN3 disease is an inherited neurodegenerative disease, typically with childhood onset, and characterized by vision loss, seizures, cognitive decline, and difficulties. The CLN3 Staging System (CLN3SS) characterizes disease progression. Our aim was to assess differences in cognitive test scores in relation to CLN3SS among individuals with CLN3 disease. We evaluated the relationship between cognitive test performance and the CLN3SS in individuals with genetically confirmed CLN3 disease. Participants completed tasks of verbal reasoning, vocabulary knowledge, attention, fund of information, and ability to recite the alphabet. One-way ANOVA testing assessed differences in mean cognitive test score among CLN3SS score groups, and Chi-square testing was used to compare the proportion in each CLN3SS group that could recite the alphabet. Data were evaluated from a sample of 85 individuals with a total 245 CLN3SS assessments conducted within 6 months of their cognitive testing, A significant decrease in test scores was found between CLN3SS Stages 1 (vision loss present) and 2 (vision loss and seizures present) for each of the cognitive tests. The proportion of participants able to recite the alphabet also decreased from Stage 1 to Stage 2 (X2=12.1, p<.01). Cognitive ability declines with advanced disease severity in CLN3 disease, though motor disability in Stage 3 likely contributes to difficulty participating in cognitive assessment at this later disease stage. Understanding the relationship between cognition and CLN3 disease stage may help guide decision making, i.e., determining who could or should undergo cognitive assessment for clinical care or for group stratification in disease modifying clinical trials.

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 p-value 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), an oxidative stress gene also implicated in Parkinsons disease. FTCD, a one-carbon cycle enzyme, carried 5 hypermethylated DMPs. A cluster of DMP-associated genes with established roles in innate immune and NF-kB 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-kB signaling. Convergence of TRAF3 hypermethylation with independent downregulation of TLR3 and NF-kB-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.

Lewy body diseases, including Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are defined by neuronal accumulation of misfolded -synuclein (-Syn), yet the ultrastructural diversity of these inclusions across brain regions and disease contexts remains unclear. Here, we applied large-scale correlative light and electron microscopy (CLEM) to map -Syn pathology across cortical regions (entorhinal cortex, ENT; anterior cingulate cortex, AC; hippocampal CA2 region) and substantia nigra (SN) in clinically and pathologically confirmed PD and DLB donors. We identified pronounced regional heterogeneity in Lewy pathology, with cortical inclusions showing diverse maturation stages at the ultrastructural level, ranging from low-density fibrils interspersed with organelles to highly compact fibrillar inclusions. In the SN of DLB donors, we observed the full range of classical nigral LB morphologies previously described in PD. We additionally characterized diverse neuritic -Syn pathologies in DLB and identified a distinct population of electron-dense, degenerating, -Syn-positive cortical neurons not previously reported. Importantly, we found no significant difference in LB ultrastructure between PD and DLB in either cortical or nigral pathology. In contrast, quantitative analysis of >10,000 mitochondria revealed disease- and region-specific signatures of altered mitochondrial homeostasis. PD showed increased mitochondrial density and enlargement in the SN, whereas DLB showed increased mitochondrial density only in the ENT. Mitochondrial enlargement was exclusive to PD. These findings indicate that LB ultrastructure alone does not distinguish PD from DLB; instead, region-specific mitochondrial phenotypes may better reflect disease identity and regional susceptibility. Overall, we provide a high-resolution framework for human Lewy pathology in PD and DLB, revealing that ultrastructural responses to -Syn pathology are driven primarily by neuronal identity and regional vulnerability. Our results highlight the need for disease- and region-specific models that capture human phenotypes to advance mechanistic understanding and therapeutic targeting of synucleinopathies.

Background: PD GENEration (NCT04057794, NCT04994015), sponsored by the Parkinson's Foundation in partnership with Aligning Science Across Parkinson's (ASAP) through the Global Parkinson's Genetics Program (GP2), is an international, observational, clinical research study that offers genetic testing and counseling to people living with Parkinson's disease (PwP) at no cost. PD GENEration has aimed to empower PwP and their clinicians with knowledge of their genetic status, to accelerate recruitment into precision medicine trials, and to advance research through data sharing. Since its launch in 2019, the study has expanded to enroll over 32,000 PwP (as of March 31, 2026), from 10 countries across North, Central, and South America, the Caribbean, and Israel. Methods: Over the course of 6 years, PD GENEration has evolved to accommodate the growing scientific and research needs of the Parkinson's community while also increasing the ability to return genetic test results to PwP at a greater scale. Participants with a diagnosis of Parkinson's disease (PD) may enroll in-person or virtually where informed consent and blood sample collection can occur. Samples are analyzed at a College of American Pathologists/Clinical Laboratory Improvement Amendments (CAP/CLIA)-certified laboratory using whole genome sequencing, with variants curated for a primary panel of seven PD-associated genes. Results are disclosed during a genetic counseling visit, where further testing is offered for two optional additional gene panels. Those who consent undergo analysis of additional genes, and results are returned during a genetic counseling visit for those that test positive for a variant. In addition to returning genetic results to PwP, a central pillar of the study design has been the open sharing of genomic data to advance discovery in PD research in partnership with ASAP and GP2. Discussion: PD GENEration applies a flexible framework, allowing for country specific considerations and the integration of multiple site models, evolving based on participant needs and the prioritization of equity and accessibility. We summarize PD GENEration's implementation and scaling, highlight key accomplishments and lessons learned, and provide guidance for those interested in implementing large-scale clinical genetic testing studies across other diseases and therapeutic domains.

ObjectiveSpinocerebellar ataxia type 1 (SCA1) is a rare, inherited neurodegenerative disease characterised by progressive deterioration of motor and cognitive function. Here, we illustrate the pattern and evolution of brain atrophy in people with SCA1 using a large multisite dataset. MethodsStructural magnetic resonance imaging data from SCA1 (n=152) and healthy control (n=131) participants from seven sites and two consortia were analyzed using voxel-based morphometry. Cross-sectional stratification and correlations were undertaken with ataxia severity and duration to profile disease evolution. Cerebrocerebellar structural covariance analysis was used to understand the relationship between cerebral and cerebellar tissue atrophy. ResultsAtrophy in SCA1 first manifests in the lower brainstem and cerebellar white matter (WM), before progressing to the pons, anterior cerebellum, and cerebellar lobule IX. The midbrain and peri-thalamic WM and the remainder of the cerebellar cortex are then affected, with preferential involvement of specific motor and cognitive areas. Finally, degeneration in the striatum and cerebral WM corresponding to the corticospinal tract become apparent. Atrophy and correlations with ataxia severity are most pronounced in the cerebellar WM and pons. Structural covariance analysis showed reduced correlations between cerebellar and cerebral WM volume in SCA1 participants. InterpretationCross-sectional stratification of a large SCA1 cohort by ataxia severity indicates a pattern of atrophy spread across the brainstem, cerebellum, and subcortical grey and white matter. Ongoing volume loss throughout the disease course is most evident in a core set of infra-tentorial brain regions. Atrophy of cerebellum spans both motor and cognitive functional zones. Cerebellar degeneration is not directly mirrored by downstream effects in the cerebrum.

Mitochondrial diseases (MDs) are genetically and phenotypically diverse and can be difficult to diagnose. Prevalence estimates derive largely from diagnosed cases and may underestimate population MD risk. Population-based studies are limited in scope and number but indicate MD variants are common. As genomic sequencing advances have made comprehensive population-based evaluation feasible, we sought to evaluate nuclear MD variation in a population cohort to understand variant prevalence and differences in MD risk estimates We identified disease-associated nuclear gene variants in 270 nuclear MD genes across 2,845 healthy older individuals in the Medical Genome Reference Bank. From Pathogenic or Likely Pathogenic Variants (PLPVs) we estimated autosomal recessive (AR) and autosomal dominant (AD) MD risk for individual genes and all nuclear variant-associated MDs. We identified 554 PLPV alleles representing 357 unique variants in 145 genes. Combined AR MD risk was estimated at 25.8 per 100,000 (95% CI 18.7 to 32.9), or 1 in 3,880 individuals. SPG7 (12.65 per 100,000; 95% CI 7.52-20.6) and POLG (4.23 per 100,000; 95% CI 2.10-8.01) contributed the greatest single gene AR MD risks and OPA1 variants posed the greatest AD MD risk. We observed a high rate of MD-associated nuclear gene variation in this healthy older cohort. The estimated lifetime AR MD risk was higher than commonly quoted prevalence estimates for all MDs, and the presence of common AD variants suggests variant penetrance may be lower than previously understood. These data help contextualise population MD risk and may inform clinical counselling and care.

Background: Motor neuron disease (MND) is a fatal neurodegenerative condition with significant clinical heterogeneity that is incompletely captured by existing phenotype classifications based on onset site. Electronic health records (EHRs) contain detailed symptom documentation in clinical narratives that may enable data-driven discovery of clinically meaningful patient subgroups. Methods: We developed a natural language processing (NLP) pipeline using MedCAT to extract symptoms from clinical notes of 2,361 people with a confirmed diagnosis of MND at a tertiary neurology center. MND cohort confirmation used three complementary methods: clinic attendance records, text-based diagnosis detection, and NLP extraction with negation detection. Extracted symptoms were filtered to Unified Medical Language System semantic type T184 (Sign or Symptom) with removal of negated concepts. Patients were clustered using latent class analysis on binary symptom profiles. Survival differences were assessed using Kaplan-Meier analysis, log-rank tests, and Cox proportional hazards regression. Results: From the first clinical notes, we identified four clusters of symptoms among 872 patients and 76 symptoms: Motor-Bulbar (n=373), Motor-Tremor (n=154), Sensory-Pain (n=222), and Motor-Respiratory (n=123). When extended to all clinical notes (n=2,065; 184 symptoms), these reorganized into three clusters: Autonomic-Respiratory (n=472), Nocturnal-Respiratory (n=338), and Classic Motor (n=1,255). Survival differences were significant across all clusters in both the first notes and all notes analyses (log-rank p < 0.001). Conclusions: NLP-based symptom extraction from EHRs identifies clinically meaningful MND subgroups that extend beyond traditional onset-site classifications. Autonomic-respiratory symptom burden is associated with poorer survival while a newly identified Sensory-Pain subtype with a better prognosis. These data-driven phenotypes may improve prognostication and inform targeted supportive care.

A molecular diagnosis remains out of reach for a substantial subset of patients with clinically recognizable Mendelian disorders, even after comprehensive next-generation sequencing. Causal variants in non-coding regions are difficult to detect and interpret using standard pipelines. Deep intronic variants that disrupt splicing are a known but underexplored source of pathogenic alleles, and systematic tools to evaluate them at scale have only recently emerged. We aimed to resolve an incomplete genetic diagnosis in two siblings with early-onset parkinsonism, prominent neuropsychiatric features, and autonomic dysfunction consistent with PLA2G6-associated neurodegeneration (PLAN), an autosomal recessive condition. Prior clinical exome sequencing, genome sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA), and long-read sequencing had identified only a single heterozygous PLA2G6 missense variant, c.2132C>G (p.Pro711Arg). We used AlphaGenome to score 91 non-coding variants shared among the affected siblings and their father within 1 megabase of the PLA2G6 locus. The deep-learning model identified an intronic variant (c.2034+355G>A) that was predicted to create a cryptic splice acceptor site that could result in inclusion of a 160-bp cryptic exon. Tissue-specific predictions indicated the aberrant splicing would be detectable in blood, confirmed by junction-spanning RNA-seq reads from an unrelated carrier. This analysis completed a compound heterozygous PLAN diagnosis nearly two decades after symptom onset and demonstrates the utility of sequence-to-function models. Systematic integration of tools like AlphaGenome into rare disease workflows offers a practical, low-barrier route to closing the diagnostic gap for patients with compelling Mendelian phenotypes and incomplete genetic diagnoses.

Abstract Background: Parkinson's disease (PD) genetics has been predominantly studied in Western European and North American populations, leaving Central Europe underrepresented. We aimed to characterise the genetic architecture of PD in the V4 countries (Slovakia, Czech Republic, Hungary, and Poland). Methods: The CEGEMOD study combined a systematic review of published PD genetic studies from the V4 region with prospective genetic screening of 1373 patients. All participants underwent genotyping array screening, while genetically unresolved patients with early-onset or familial PD underwent whole-exome sequencing analysis. Variants were interpreted using current clinical standards and validated using Sanger sequencing. Results: The systematic review identified 48 eligible studies reporting pathogenic or risk variants in PD patients from the V4 countries. In the prospective cohort, pathogenic, likely pathogenic, or established risk variants were identified in 157/1373 patients (11.4%). GBA1 variants accounted for the majority of findings, mostly driven by the two GBA1:p.(Thr408Met) and p.(Glu365Lys) mild common risk variants, followed by LRRK2, PRKN, POLG, PLA2G6, and ATP1A3. Whole-exome sequencing provided an additional 5% diagnostic yield in genetically unresolved high-risk patients. Our findings also demonstrate substantial disparities in genetic research across Central Europe. Conclusions: This study provides the largest genetic characterisation of PD in Central Europe to date. The CEGEMOD study expands knowledge of the regional genetic landscape, supports the implementation of genetic testing in clinical practice, and establishes an important resource for future precision medicine and collaborative PD genetics research.

Introduction: Variants in the polymerase gamma (POLG) gene are associated with a wide range of mitochondrial disorders. Emerging evidence suggests a potential link between POLG variants and Parkinson's disease (PD); yet, results remain inconclusive. Objectives: To investigate the genetic spectrum and prevalence of POLG variants in PD across diverse ancestries. Methods: We leveraged multi-ancestry genetic data from the Global Parkinson's Genetics Program (GP2), including genotyping data from 98,589 and short-read sequencing data from 36,022 individuals. We performed a POLG rare variant screen, case-control association, and gene-level burden analyses. Results: Five PD cases carried potentially biallelic rare pathogenic/likely pathogenic POLG variants. Additionally, 228 individuals (<1%; 161 PD cases, 28 individuals with other neurological disorders, and 39 controls) carried 34 distinct rare pathogenic/likely pathogenic heterozygous variants, with no significant frequency differences between cases and controls, except for the p.Ala467Thr variant in the European population. The co-inherited pathogenic variants p.Thr251Ile and p.Pro587Leu were present in <1% of both cases and controls, with no significant group differences. Burden and variant-level association analyses showed no association between rare POLG variant burden or common POLG variant enrichment and PD. Conclusions: POLG variants are overall rare in PD. The identification of rare pathogenic variants among PD cases suggests that POLG-related mitochondrial dysfunction may contribute to PD in isolated instances, particularly under recessive inheritance. Our findings support a role for POLG variants in select cases and underscore the need for larger-scale sequencing and functional studies.

Background: Neurofilament light chain (NfL) gained FDA recognition in amyotrophic lateral sclerosis (ALS) through SIMOA-based validation, where baseline serum NfL predicts ALSFRS-R slope and survival, and through the 2023 tofersen approval for SOD1-ALS. The commercial Roche Elecsys electrochemiluminescence immunoassay (ECLIA) reads 6- to 8-fold lower than SIMOA, and its clinical utility in ALS is uncharacterized. We assessed whether ECLIA NfL retains this correlation in routine care and whether GFAP or S-100B helps. Methods: Retrospective analysis of 58 chart-confirmed ALS patients at Georgetown University Hospital (2022-2026), biomarkers on the LabCorp Roche Elecsys ECLIA. The NfL-ALSFRS-R correlation was assessed where both measures fell within matching windows; serial NfL, in patients with repeat draws. Results: First-per-patient NfL median was 7.06 pg/mL (IQR 4.06-17.30; CV 99%). Among 31 patients with matched NfL and ALSFRS-R decline rates, Spearman r = 0.704; within 90 days (n = 17), r = 0.809 (both p < 0.0001). Fast progressors (n = 8) had mean NfL 17.10 pg/mL versus 4.64 in slow progressors (n = 21), a 3.7-fold separation. Serial NfL captured rising trajectories and stable low values. GFAP rose within patients but tracked neither progression rate, disease stage, nor motor-neuron predominance; S-100B added no value. Conclusions: Commercial ECLIA brings NfL into routine ALS care; its prognostic correlation with progression rate survives real-world fragmentation. The actionable unit is the longitudinal trajectory, not the single value, read against platform-specific reference ranges and clinical context (genotype, onset, stage). GFAP and S-100B add little. Keywords: amyotrophic lateral sclerosis, neurofilament light chain, biomarkers, implementation science, ECLIA, GFAP, monitoring, tofersen, real-world data

PurposeAlthough most developmental and epileptic encephalopathies (DEEs) have a monogenic aetiology, routine clinical genetic testing is negative for 50% of patients. We hypothesized that the diagnostic yield could be increased in a large cohort of individuals with unsolved DEEs by applying genome sequencing along with enhanced variant analyses outside of coding regions. MethodsWe performed genome sequencing for 242 participants with DEEs negative on prior genetic testing. We interrogated single nucleotide variants (SNVs), indels, and structural variants in both established and candidate DEE genes. All variants of interest were reviewed, classified, and validated by a multidisciplinary team. ResultsA molecular diagnosis was discovered for 36/242 (15%) participants. The pathogenic or likely pathogenic variants comprised 26 SNVs and indels within coding regions, 9 structural variants, and 5 SNVs and indels in introns or non-coding genes. Variants of uncertain significance were detected in a further 10/242 (4%) participants. ConclusionGenetic diagnostic yield for individuals with unsolved DEEs improves with genome sequencing analysis. This increase reflects both the identification of structural and non-coding variants not detectable on exome or gene panel analysis, and the detection of variants in genes newly associated with DEEs.

Huntingtons disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HTT) gene, with longer repeats linked to earlier onset. Somatic CAG expansion, particularly in the striatum, contributes to disease progression and is influenced by HTT biology and genetic modifiers. Modulating somatic expansion is emerging as a promising approach to slow or prevent HD, and mouse models have been crucial for preclinical testing of different therapeutic strategies. The BAC-CAG model, developed on the FVB strain, has been used to study somatic expansion of human expanded HTT. However, comparisons with other key HD mouse models have been limited by differences in genetic background, as many other models are on the C57BL/6 strain. The BAC-CAG model has now been developed on a C57BL/6 background. To determine whether the C57BL/6 BAC-CAG model can be used to study and modulate somatic expansion, we compared CAG expansion in mice on C57BL/6 or FVB backgrounds, with and without intraventricular divalent small interfering RNAs (siRNA) targeting HD modifiers MutS homolog 3 (MSH3) and HTT. Both strains exhibited robust, comparable somatic expansion over two months, which was blocked by MSH3-, but not HTT-, targeted siRNA. RNA sequencing identified gene expression differences primarily in pseudogenes, with no differences in endogenous Htt, human HTT, or mismatch repair genes. These results demonstrate that BAC-CAG mice on a C57BL/6 background exhibit somatic CAG expansion comparable to the validated FVB strain, providing a model to study and preclinically test therapies targeting somatic expansion in HD.

Background and Objectives: Leukodystrophies are rare genetic diseases affecting the central nervous system white matter, leading to progressive disabilities and death. Although early diagnosis is critical for therapies, the penetrance and phenotypic spectrum of many leukodystrophies remain poorly defined. Here, we integrate sequencing population screening with longitudinal electronic health record (EHR) data. Our goals were to assess the prevalence of undiagnosed leukodystrophy, characterize phenotypic variability among genotype-positive individuals, and estimate penetrance across multiple leukodystrophies. Methods: We analyzed 19 genes associated with 13 leukodystrophies in pediatric and adult individuals recruited via the HerediGene Population Study, a 5-year study conducted primarily of healthy individuals in the U.S. intermountain west. Sequencing was performed on 210,983 individuals, consisting of genome sequencing for 34,033 and SNP panel imputation for 176,950. Variant results were cross-referenced to comprehensive and longitudinal (20+ years) clinical data in the Intermountain Health Enterprise Data Warehouse and to the Utah Leukodystrophy Program. Results: Pathogenic variants were identified in 4 genes (CSF1R, PLP1, POLR3A, SNORD118) in 9 individuals, none of whom had a clinical leukodystrophy diagnosis or characteristic MRI findings. These findings suggest that missed clinical diagnoses of most leukodystrophies are uncommon in a centralized healthcare system, but also demonstrate that for some leukodystrophies there may be variable or reduced penetrance, or broader phenotypic spectra than recognized. We used published incidence estimates and the observed leukodystrophy-associated genotypes to infer penetrance ranges that varied from wide for ultra-rare leukodystrophies, to tightly bounded for more prevalent conditions. Discussion: In this predominantly healthy population, we did not find any patients with leukodystrophy who had been genetically undiagnosed but then identified by sequencing. However, we identified 9 individuals with genotypes previously reported to result in leukodystrophy, but none of whom had clinical symptoms or MRI features associated with the specific leukodystrophy. Our results support a revised model in which leukodystrophies exist along a continuum of penetrance and expressivity, with implications for newborn screening, variant interpretation, and risk stratification.

Background and Objectives To investigate the associations of blood-based tau biomarkers with clinical, electrophysiologic and prognostic measures in amyotrophic lateral sclerosis (ALS), and to determine whether they reflect distinct disease-related processes. Methods We studied 119 patients with ALS from a longitudinal observational cohort. Plasma and serum p-tau181, p-tau217, p-tau231, brain-derived tau (BD-tau), NfL and GFAP were measured using Lumipulse and Simoa assays. Associations with demographic variables, disease severity (ALSFRS-R and slow vital capacity), lower motor neuron (LMN), muscle involvement (creatine kinase [CK] and high-sensitivity cardiac troponin T [hs-cTnT]), disease progression and survival were assessed using multivariable models. Results Tau-related biomarkers, specifically p-tau217 and BD-tau, were associated with greater cross-sectional disease severity, reflected by lower ALSFRS-R scores. Plasma and serum p-tau181, p-tau217, p-tau231, and BD-tau were associated with higher CK and hs-cTnT, whereas p-tau181 and p-tau231 were also associated with greater LMN involvement. In contrast, NfL and GFAP were not associated with muscle or LMN involvement. Across analytical platforms, plasma and serum NfL were associated with faster ALSFRS-R decline and shorter survival. NfL was the only biomarker independently associated with both disease progression and survival. Discussion Blood biomarkers capture distinct dimensions of ALS. Tau-related biomarkers are associated with cross-sectional disease severity, LMN involvement and muscle injury, whereas NfL primarily reflects disease progression and survival. These findings support the complementary use of tau-related biomarkers and NfL for ALS phenotypic characterization and prognosis assessment.

KCNT1-related disorders represent clinically heterogeneous severe epilepsies associated with profound neurodevelopmental impairment. The full phenotypic spectrum and longitudinal disease trajectory remain incompletely characterized, which is a critical gap limiting the establishment of quantifiable endpoints necessary for future clinical trials. Compounding this challenge, identical pathogenic variants result in phenotypically distinct syndromes, including early infantile developmental and epileptic encephalopathy (EIDEE) and autosomal dominant sleep-related hypermotor epilepsy (ADSHE), underscoring unresolved genotype-phenotype relationships. To address these gaps, we performed a comprehensive analysis of 159 individuals with KCNT1-related disorders, including a longitudinally characterized subgroup of 62 individuals across 390 patient years, systematically defining disease progression, seizure trajectories, developmental outcomes, and treatment response across the full spectrum of the disorder. Seizures were nearly universal, affecting 157 of 159 individuals, with 81% (n=126/156) having seizure onset within the first year of life. Stratification by clinical subgroup revealed divergent seizure onset patterns. Recurrent variants did not significantly differ in age of seizure onset yet exhibited variant-specific clinical fingerprints, such as the preponderance of focal clonic seizures (OR=5.03, 95% CI 1.60-15.7, f=0.47) in those with the p.Gly288Ser variant. Comparison with a broader cohort of 14,893 individuals with neurodevelopmental disorders revealed phenotypic features such as migrating focal seizures (OR=21716, 95% CI 2409-Inf, f=0.42) and hypertonia (OR=26.5, 95% CI 18.2-38.3, f=0.45) to be more common in EIDEE, and nocturnal seizures (OR=29787, 95% CI 3062-Inf, f=0.5) and hyperactivity (OR=13.7, 95% CI 4.70-35.9, f=0.32) to be more common in ADSHE. These findings corroborate and extend those reported in the existing literature. Developmental milestones revealed marked delays across all domains. Analysis of longitudinal medication prescription patterns exposed striking therapeutic variability, reflecting the absence of a consistent treatment framework. Several anti-seizure medications frequently cited as beneficial, quinidine and cannabidiol, were not associated with seizure improvement or sustained seizure freedom in our cohort. In contrast, clobazam (OR=1.39, 95% CI 1.12-1.72, f=0.85), ketogenic diet (OR=1.30, 95% CI 1.07-1.57, f=0.75), and lacosamide (OR=2.03, 95% CI 1.54-2.66, f=0.59) demonstrated positive comparative effectiveness. Quantitative EEG analysis distinguished individuals with KCNT1-related disorders from age-matched controls with high accuracy (AUC=0.906), with key discriminating spectral features, including alpha power in the central and parietal regions, demonstrating significant reduction across childhood and adolescence. Collectively, these findings expand the phenotypic and genotypic landscape of KCNT1-related disorders through large-scale real-world clinical data, establish quantifiable longitudinal clinical endpoints, and provide actionable insights into genotype-phenotype relationships and differential treatment response. Together, these findings will help identify outcome measures and biomarkers to inform future clinical trial design.

BackgroundAccurate diagnosis of multiple system atrophy (MSA) is critical for clinical management and efficient trial designs, yet remains challenging, particularly distinguishing MSA (especially cerebellar-subtype [MSA-C]) from sporadic adult-onset ataxia (SAOA). Combining a marker of neuroaxonal degeneration, neurofilament light chain (NfL), with a marker of the pathogenic MSA hallmark, -synuclein seeding activity, may define a mechanistically-informed CSF signature of MSA, enabling sensitive and specific differentiation from SAOA even in early disease. MethodsWe analyzed 60 cross-sectional patient CSF samples (n=32 clinically diagnosed MSA [MSAclin] 22/32 MSA-C; n=28 SAOA) for NfL (Simoa) and -synuclein seeding activity (seed amplification assay [synSAA], Piperazine-N,N-bis(2-ethanesulfonic acid)-based), and assessed diagnostic accuracy, disease-duration correlations, and trial power using biomarker-based stratification. ResultsAge-adjusted NfL was higher in MSAclin than SAOA (3859 vs. 997pg/mL), yielding 96.9% sensitivity and 85.7% specificity. SynSAA was concordant with clinical diagnosis (25/32 MSAclin synSAA-positive; 23/28 SAOA synSAA-negative), with 78.1% sensitivity and 85.2% specificity (all confirmed in MSA-C subgroup). Both biomarkers displayed divergent trajectories with disease duration: NfL peaked early before declining (r=-0.45, p=0.01); whereas synSAA maximum fluorescence intensity increased (r=0.42, p=0.016), suggesting greater synSAA signal with accumulating MSA burden. Integrating both biomarkers in MSA treatment trials allows sample-size reduction by 20% versus NfL alone. ConclusionsCSF NfL and synSAA capture complementary aspects of MSA biology: while NfL provides high diagnostic accuracy for MSAclin, peaking early, synSAA adds mechanistic specificity for -synuclein seeding activity and might allow target engagement assessment. Combined, they might enable biological diagnostic frameworks, molecular trial stratification, and treatment monitoring in MSA. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSWhile highly warranted for clinical management and efficient treatment trial design, accurate diagnosis of multiple system atrophy (MSA) against overlapping and reciprocally mimicking conditions such as sporadic adult-onset ataxia (SAOA) remains clinically challenging, especially in early disease stages. A mechanistically informed biofluid signature of MSA might enable sensitive and specific differentiation from SAOA, even in early disease stage. Recently merging molecular markers reflecting neuroaxonal damage (NfL) and -synuclein seeding activity (measured by the seed amplification assay; synSAA) might here show particular promise. What this study addsThis is the first study to systematically assess the ability of both CSF NfL and CSF -synuclein seeding activity to distinguish clinically diagnosed MSA (MSAclin) from SAOA, thereby offering a window into underlying MSA biology in patients in vivo. Our findings suggest that the rate of axonal degeneration is most pronounced in early MSA disease stages but decreases with longer disease duration; whereas -synuclein seeding signal activity increases as MSA-related disease burden accumulates. Finally, it demonstrates the impact of a combined molecular fluid signature of MSA for improving trial design: a biomarker-based stratification of MSA subjects in future MSA treatment trials combining NfL plus -synuclein seeding activity allows to reduce sample sizes by 20% compared to NfL alone. How this study might affect research, practice or policyThe findings from this study may help to molecularly diagnose patients with MSA against overlapping and reciprocally mimicking conditions such as SAOA, in particular and even in early disease stages. Moreover, they might lay the foundation for a future biologically-informed diagnostic framework of MSA; support trial stratification for more efficient upcoming MSA treatment trials; and might facilitate molecular treatment effect monitoring in MSA, in particular in synuclein-targeted treatment trials.