Neurology Genetics

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.

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.

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

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.

Background: LRRK2 variants are major contributors to Parkinsons disease (PD). Many pathogenic variants increase kinase activity, underscoring the value of functional assays in nominating therapeutic targets and kinase inhibitors as potential disease-modifying therapies. Objective: To develop an interactive resource that provides functional context and ancestry-specific variant frequencies. Methods: Genotyping and short-read sequencing data were analyzed for 101,678 individuals (61,709 PD, 39,969 controls) from the Global Parkinsons Genetics Program (GP2) and integrated with clinical and in-vitro biochemical kinase activity information. Results: The LRRK2 Browser (http://gp2.org/lrrk2browser) displays ancestry-specific genetic data for 19,596 LRRK2 variants (968 exonic, 14 disease-associated) across 11 populations, and functional data for 171 variants. Clinical annotations include age, age at onset, and family history of PD. Discussion: The publicly available LRRK2 Browser represents an open-access, multi-ancestry resource to support LRRK2 variant interpretation. It aims to enhance the translational potential of genetic and functional data for precision medicine and the implementation of gene-targeted therapies in diverse populations.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2) 1 are among the most frequent monogenic causes of Parkinson's disease (PD) and act through a gain-of-function mechanism of increased kinase activity. LRRK2-targeted therapies are in clinical development, but interpretation of the rapidly expanding catalogue of rare LRRK2 variants remains a barrier to translation. Here, we present functionally annotated data on more than 350 LRRK2 variants using a standardized cellular assay with Rab10 phosphorylation as a readout of kinase activity and integrated these data with curated genetic and clinical annotations from the Movement Disorders Society Genetic Mutation Database (MDSGene). Variants differed in activation magnitude, ranging from modest increases (e.g., p.G2019S) to strongly activating substitutions such as p.Y1699C or p.L1795F. Activating variants occurred across the full length of LRRK2, although the largest effects clustered within the ROC-COR regulatory hub, where structural analysis identified subdomains forming an allosteric scaffold controlling kinase output. All known/established pathogenic variants showed increased activity, whereas benign and likely benign variants remained within the wild-type range. Functional effect sizes correlated with pathway activation in patient-derived immune cells, altogether providing a framework for ACMG-based variant interpretation in which kinase activation can support PS3 functional evidence for reclassification of variants.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

Abstract: Background: Lacunar stroke is a common and disabling cerebrovascular disease. Small-vessel vasculopathy is thought to be the most common underlying cause, but this has only been identified on histopathology. 7T MRI allows small vessels to be seen in vivo. This study aimed to investigate rates of small vessel vasculopathy in lacunar stroke using 7T MRI. Methods: Patients with lacunar stroke at an Australian tertiary stroke centre were prospectively screened and recruited to the study. Patients underwent 7T MRI with T1, T2, time-of-flight (TOF), diffusion-weighted imaging (DWI) and susceptibility-weighted imaging (SWI) sequences. Images were interpreted by two blinded neuroradiologists. Results: The likely symptomatic perforator could be identified in 16/19 (84%) of cases. Amongst cases where the symptomatic perforator was observed, 14/16 (88%) of the symptomatic perforator vessels had focal stenosis consistent with steno-occlusive vasculopathy. There were 3/19 (16%) of cases with associated large artery vasculopathy. There were 7/16 (44%) cases where an occluded perforator was seen. The majority of patients had at least one vascular risk factor (15/19, 79%) and there were no cases where non-atherosclerotic vasculopathy was suspected. Conclusions: Lacunar stroke is commonly associated with small vessel vasculopathy, likely due to atherosclerosis, which can be identified in vivo with 7T MRI time-of-flight imaging.

BACKGROUNDThe persistent puzzle of how GBA1 coding variants underlying monogenic Gaucher disease confer such strong risk for polygenic Parkinsons disease (PD) remains unresolved. Here, we sought to resolve this long-standing paradox. METHODSUsing the Parkinsons Progression Markers Initiative resource, we integrated whole-genome sequencing from sporadic PD cases and controls with matched transcriptomic, epigenomic and cerebrospinal fluid proteomic data across Northern European and Ashkenazi Jewish populations. Ancestry-specific genetic maps enabled precise integration of disease localisation with eQTL, pQTL and chromatin architecture, linking genetic association to molecular mechanism, while evolutionary genetic analyses resolved the regional haplotypic architecture. RESULTSWe identified a PD-associated regulatory signal located 150 kilobases distal to GBA1 that robustly replicated across all molecular datasets and both ancestries. Integrative mapping identified rs77268551-G as the candidate regulatory variant, which we show resides on an extended haplotype also carrying the Gaucher disease-causing N370S allele. Evolutionary analyses reveal a clear signature of recent positive selection acting on the Gaucher disease-causing N370S allele, giving rise to this extended shared haplotype that likely concealed the underlying distal PD-associated regulatory signal. rs77268551-G resides within a neuronal enhancer with features consistent with super-enhancer activity that orchestrates hierarchical enhancer-to-enhancer-to-promoter interactions with eleven cis-target genes, including ADAR, IL6R and GBA1, driving a coordinated transcriptional programme. Proteomic profiling identified genotype-specific cerebrospinal fluid protein signatures, including PARK7, BIN1 and SAA1, consistent with a neuroinflammatory programme. CONCLUSIONSThe findings support a dual-hit model in which a distal regulatory element is the primary driver of PD-associated molecular programmes, while co-inherited GBA1 protein-coding variation on the same haplotype modifies and amplifies the risk established by the regulatory variant, giving rise to the clinically distinct GBA1-associated PD phenotype. Regulatory activation and GBA1-related functional effects converge on neuroinflammatory and lysosomal pathways, and the resulting molecular signatures suggest genotype-linked biomarkers with potential clinical relevance.

A major obstacle for clinical trials is the lack of objective, sensitive, and reliable measures that can detect modest changes in disease progression. Here, we determine whether acoustic and linguistic digital speech measures automatically obtained during a functionally relevant passage-reading task capture multiple dimensions of disease in ataxia, including functional communication impairment, subclinical cerebellar dysfunction and disease progression. A total of 157 individuals with ataxia and 84 controls contributed cross-sectional data, and 54 individuals with ataxia and 43 controls contributed longitudinal data within the ongoing Neurobooth natural history study. Participants completed standardized speech recordings, patient-reported outcome measures (PROMs) and neurologist-rated clinical evaluations. A novel speech processing pipeline was developed to automatically transcribe audio recordings, identify word boundaries and extract a predefined set of linguistic and within-word acoustic features. Individuals with ataxia exhibited marked disruption of speech timing, coordination and articulatory control, including slowed speech (d=1.23), prolonged inter-word pauses (d=-0.91), higher/more variable vocal intensity (|d|=0.43-0.51) and altered spectral content (|d|=0.43-0.79) compared to healthy controls. Linguistic features (e.g. speaking rate and within-word pause duration) showed strong associations with clinician-rated severity and PROMs (|r|=0.23-68), indicating alignment with functional communication impairment and patient-perceived disease burden. In contrast, acoustic features derived from cepstral measures captured subtle abnormalities in speech motor control, differentiating not only individuals with ataxia (d=0.65) but also pre-ataxic individuals (d=0.56), and those without clinically evident dysarthria (d=0.45), from controls. These findings indicate that acoustic features reflect subclinical cerebellar motor dysfunction involving impaired temporal coordination and vocal control before overt clinical speech impairment emerges. Longitudinally, several acoustic measures were sensitive to disease progression (MSDR=0.19-0.68), even in cases where clinical scales showed no detectable change. Speech-derived changes correlated with changes in clinical scales and PROMs. Both acoustic and linguistic features exhibited strong intra-session reliability. During passage reading, acoustic and linguistic measures provide complementary but different clinical information in ataxias. Linguistic measures primarily reflect downstream functional consequences of ataxic dysarthria, whereas acoustic measures provide sensitive indicators of subclinical cerebellar motor dysfunction and progression. These findings demonstrate that natural speech analysis can produce digital measures for detecting subclinical disease, quantifying functional impairment, monitoring progression in ataxia, with strong potential for application in clinical trials and remote monitoring.

Heatstroke is a life-threatening condition in which heat-shock and unfolded-protein responses are strongly activated but fail to prevent proteostasis disruption and severe cellular injury. Whether post-transcriptional regulation contributes to this mismatch remains unknown. We integrated small RNA sequencing with mRNA profiling in peripheral blood mononuclear cells from patients with classical heatstroke and matched heat-exposed controls recruited during the Hajj pilgrimage. mRNA profiling was performed in 19 cases and 19 controls, and miRNA sequencing in 17 cases and 16 controls from the same cohort. Differentially expressed miRNAs were integrated with 4,462 differentially expressed mRNAs using high-confidence inverse-expression miRNA-mRNA pairs. Twenty-six miRNAs mapped to 376 mRNA targets, forming 414 regulatory pairs and two opposing programmes. Programme A, comprising 16 downregulated miRNAs, was associated with activation of PI3K-mTOR, NRF2 oxidative stress and HIF-1 signalling, consistent with stress-survival signalling. Programme B, comprising 10 upregulated miRNAs, was associated with suppression of stress-granule components and fatty-acid {beta}-oxidation genes, consistent with impaired protein quality control and reduced metabolic flexibility. miR-92a-3p emerged as a central regulatory node, and its target PIK3R3 connected 9 of the 10 enriched pathways. These findings suggest a post-transcriptional regulatory layer that could contribute to the limited protection afforded by activated stress defences in human heatstroke.

Background: The cystine/glutamate antiporter encoded by SLC7A11 maintains cellular redox balances and lysosomal pH. Recent molecular evidence suggested that SLC7A11 may be involved in Parkinson's disease (PD). However, the genetic contribution of SLC7A11 to PD susceptibility remains unclear. Methods: We analysed whole-genome sequencing data from the Accelerating Medicines Partnership-Parkinson's Disease (AMP-PD) and United Kingdom Biobank (UKBB) cohorts, comprising of 5,5375 cases and 35,002 controls. Rare variant burden analyses were performed using SKAT-O and MetaSKAT. Common-variant associations with PD risk and glucocerebrosidase (GCase) enzyme activity were assessed with regional linkage disequilibrium plots using previous GWAS summary statistics. Gene expression effects were examined through brain-specific expression quantitative trait loci (eQTL) data from GTEx v7. Colocalization analyses and regional association plots were generated to visualize and compare GWAS and eQTL signals across the SLC7A11 locus. Results: No rare or common SLC7A11 variants were associated with PD or GCase activity, including variants that had strong effects on SLC7A11 expression in relevant brain regions. Discussion: These findings suggest that genetic variation in SLC7A11 or its expression are unlikely to have a major role in PD susceptibility.

Introduction: Narrative recall is widely used to detect cognitive impairment, but dominant instruments carry proprietary restrictions. The Craft Story 21 (CS), the non-proprietary NACC UDS4 standard, is not available standalone. Here, we validate the freely available Puppy Escape (PE). Methods: 346 participants (153 cognitively normal, 106 subjective cognitive impairment, 87 mild cognitive impairment) completed PE and CS. Analyses evaluated convergent and criterion validity, MCI-vs-control discrimination, and incremental validity. Results: PE and CS converged (r=.43-.47) and were equivalent on 10/12 neuropsychological measures. PE Delayed discriminated MCI from controls (d=1.03; ROC-AUC equal to CS, DeLong p=.510) and added variance beyond CS (R2=+.054, p<.001). Automated subscores revealed MCI deficits in location, action, and name content. PE-18 short form retained discrimination (d=1.02) with 18 items. Discussion: PE matched CS across all validation domains and captured complementary diagnostic information. PE and PE-18 are available via online registration explicitly permitting industry-sponsored research and fee-for-service clinical use.

Background: Children and young people (CYP) with intellectual disability (ID) frequently have co-occurring neurodevelopmental (ND) and mental health (MH) difficulties. While copy number variants (CNVs) are identified as an important aetiology of ID, it is unclear whether and how CNV risk scores predict ND and MH characteristics within the CNV-associated ID population. Methods: We analysed data from the UK-based IMAGINE-ID cohort of CYP (aged 4-19 years) with ID and clinically-reported CNVs (N = 1,640). CNVs were annotated with Gencode 19 in ENSEMBL to calculate CNV risk scores, including summed probability of loss-of-function intolerance (pLI) and dosage sensitivity. Multivariate regression models examined the prediction of CNV variables and inheritance on ND and MH characteristics, assessed via the Development and Well-Being Assessment (DAWBA). Post-hoc analyses explored CNV variable stratification (lower vs. higher range pLI). Results: Higher summed pLI scores (indexing CNV genes' intolerance to loss of function) unexpectedly predicted fewer MH difficulties and a lower likelihood of ND diagnoses, even after accounting for demographic factors and CNV inheritance. Post-hoc analyses identified a threshold effect. Within the lower pLI range, higher pLI scores were associated with greater MH difficulties, consistent with findings from population-based samples. In contrast, within the higher pLI range, higher pLI scores were associated with fewer MH difficulties (among individuals more likely to have severe ID). Conclusion: These findings challenge the assumption that CNV genomic "risk scores" universally predict ND and MH difficulties. Instead, within CNV-associated ID, complex relationships exist between CNV risk scores, inheritance and phenotypes. These insights emphasise the necessity of integrating genomic results with familial and developmental context to understand individual vulnerabilities and support needs.

Background: Plasma phosphorylated tau-217 (ptau217) has demonstrated accuracy exceeding 90% for Alzheimers disease (AD) diagnosis. While its diagnostic validity has been established, its real-world clinical utility in altering or corroborating clinician diagnoses remains less understood. This study evaluates the impact of plasma ptau217 on diagnostic reclassification in a memory disorders clinic. Methods: We conducted a retrospective chart review of 100 patients evaluated for memory impairment who subsequently underwent plasma biomarker testing. Initial clinical diagnoses were established using the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer Disease and Related Disorders Association criteria, neuropsychological testing, and brain magnetic resonance imaging without knowledge of plasma biomarker results. Follow-up diagnoses were assigned after the availability of plasma ptau217 or Precivity AD2 results. Diagnostic reclassification was evaluated under two p-tau217 classification schemes to determine the AD etiology: a binary cutoff with lower threshold (AD = ptau217 > 0.18) and a three-level cutoff incorporating a higher threshold (AD = ptau217 > 0.325). Reclassification matrices and McNemar's tests were used to assess changes in diagnosis. Subgroup analyses were conducted according to apolipoprotein E (APOE) {epsilon}4 carrier status. Results: The overall diagnosis reclassification rates were 37.8% (chi-square (1) = 10.81, p = 0.001) and 46.9% (chi-square (1) = 29.76; p < 0.001) using lower and higher threshold analyses, respectively. With the higher threshold (ptau217 > 0.325), reclassification among APOE {epsilon}4 carriers occurred in both directions at similar frequencies, without evidence of a net shift toward AD or non-AD (42% from AD to non-AD, 43% from non-AD to AD, chi-square (1) = 2.77 p = 0.096). Among {epsilon}4 non-carriers, reclassification was significantly asymmetric toward non-AD (66% from AD to non-AD, 14% from non-AD to AD, chi-square (1) = 16.41, p < 0.001), suggesting ptau217 may be identifying diagnostically heterogeneous cases in which the clinical presentation resembles AD but instead reflects an alternative or co-morbid etiologies. Conclusion: Plasma ptau217 meaningfully influences diagnostic decisions in a memory clinic setting and may be particularly valuable among APOE {epsilon}4 non-carriers, where biomarker-informed evaluation frequently shifted diagnoses away from AD.

Background: Parkinsons disease (PD) has a prolonged prodromal phase, but minimally invasive molecular biomarkers distinguishing manifest PD from prodromal synucleinopathy remain insufficiently characterized. Isolated REM sleep behavior disorder (iRBD) represents a high-risk prodromal condition and provides an opportunity to investigate early blood-based transcriptional alterations. Objective: To identify peripheral blood transcriptomic signatures distinguishing healthy controls (HC), individuals with iRBD, and patients with PD, and to explore whether longitudinal iRBD samples exhibit movement toward a PD-like transcriptional state. Methods: Peripheral blood RNA-seq data were analyzed using harmonized metadata, DESeq2 differential-expression analysis, internally validated machine-learning models, PD-like projection, and integrated biomarker-panel prioritization. Independent baseline samples were used for cross-sectional differential-expression and machine-learning analyses. iRBD follow-up and post-conversion observations were excluded from baseline model development and reserved for exploratory longitudinal analyses. Results: Baseline analyses included 71 independent samples: 20 HC, 31 iRBD, and 20 PD. An additional 19 iRBD follow-up observations, including three post-conversion observations, were available for exploratory analyses. Differential-expression analysis identified 170 FDR-significant genes in PD versus HC and 85 in PD versus iRBD, compared with one FDR-significant gene in iRBD versus HC. Internal machine-learning validation showed stronger discrimination of manifest PD, with a best ROC-AUC of 0.883 for HC versus PD and 0.889 for iRBD versus PD. Discrimination between HC and iRBD was weak, with a best ROC-AUC of 0.584. PD-like projection scores were lowest in HC, highest in PD, and heterogeneous among baseline iRBD samples. Follow-up iRBD samples showed an exploratory upward shift in the mean PD-like projection score. Integrated prioritization produced a 24-gene PD candidate panel and a 24-gene exploratory iRBD panel, with genes in each panel supported by machine-learning feature-stability evidence and differential expression analysis. Conclusions: Manifest PD was associated with a distinct peripheral blood transcriptional signature, whereas iRBD-associated alterations were substantially weaker and more heterogeneous. The prioritized panels represent candidates for independent technical and external validation and should not yet be interpreted as clinically validated diagnostic or prognostic tests.

Background: Lysosomal dysfunction plays a crucial role in the pathogenesis of Parkinson's disease (PD), particularly among GBA1 mutation carriers. Beyond GBA1, genes such as TMEM175, SCARB2, and CTSB identified in genome-wide association studies (GWAS) are also implicated in lysosomal pathways contributing to PD risk, although their functional effects in patients remain unclear. Proteins encoded by these lysosome-related genes have been explored as potential therapeutic targets in experimental models. Biomarker profiles, including clinical measures, alpha-synuclein seeding activity, lysosomal proteins, and sphingolipids, may facilitate patient stratification and support therapeutic monitoring in future clinical trials. Aim: The aim of this study is to investigate the impact of genetic variants of three lysosomal-related genes (TMEM175, SCARB2, and CTSB) on biomarker profiles in PD with and without GBA1 variants. Cross-sectional data from two German cohorts: the Tuebingen Parkinson Cohort (TUEPAC) and the DESCRIBE PD cohort of the German Center for Neurodegenerative Diseases were used as explorative cohorts, and data from Accelerating Medicines Partnership Parkinson's Disease (AMP-PD) were used as a validation cohort. The ultimate goal is to provide new data for patient stratification based on genetics, which might serve as a readout for target engagement and treatment efficiency assessment. Methods: Three cohorts were analyzed: TUEPAC, DESCRIBE PD, and AMP-PD. TUEPAC and DESCRIBE PD were combined into a single German discovery cohort (TUEPAC-DESCRIBE-PD), while AMP-PD served as an independent validation cohort. Within each cohort, for subgroup analyses, PD patients were classified as the overall PD cohort (PDall), and further stratified by GBA1 mutation status into PD patients without GBA1 mutations (PDGBA1_wildtype), and PD patients carrying GBA1 mutations (PDGBA1). We evaluated cognitive and motor function, as well as depression using the Montreal Cognitive Assessment (MoCA), Unified Parkinson Disease Rating Scale-part III (UPDRS III), and Beck Depression Inventory-II(BDI-II) scales. Analyzed biomarkers included CSF -syn seeding activity using seed amplification assay (SAA), CSF lysosomal protein levels of lysosomal integral membrane protein 2 (LIMP2), also known as SCARB2, cathepsin B (CTSB) and lysosome-associated membrane protein 2 (LAMP2), blood-based enzyme activity of the lysosomal glucocerebrosidase (GCase), and CSF sphingolipid profiles. PD patients carrying risk alleles in TMEM175, SCARB2, and CTSB were compared to non-carriers. Results: Genotype-phenotype correlation analysis in TUEPAC-DESCRIBE-PD and AMP-PD revealed: (1) In PDall, the TMEM175 p.M393T risk variant was nominally associated with decreased cognitive function when adjusted for GBA1 mutation status in TUEPAC-DESCRIBE-PD; this association could not be replicated, although a similar trend was observed in the slightly smaller, but multicentric AMP-PD cohort; TMEM175 p.M393T was not significantly associated with BDI-II or UPDRS-III scores in either cohort. (2) In PDGBA1_wildtype, GCase activity was significantly lower in PD patients with SCARB2 rs6812193 risk allele in TUEPAC-DESCRIBE-PD, while a similar but non-significant trend was observed in AMP-PD; (3) In PDall, CSF levels of CTSB were nominally lower in carriers of CTSB rs1293298 risk allele compared to carriers of CTSB rs1293298 protective allele in TUEPAC-DESCRIBE-PD; in PDGBA1_wildtype, LAMP2 was significantly lower in carriers of CTSB rs1293298 risk allele compared to carriers of CTSB rs1293298 protective allele in TUEPAC-DESCRIBE-PD; (4) In PDall, TMEM175 p.M393T risk allele was nominally associated with altered sphingolipid profiles across both TUEPAC-DESCRIBE-PD and AMP-PD cohorts. Conclusion: These findings demonstrate that genetic variants in lysosomal-related genes (TMEM175, SCARB2, and CTSB) have a functional impact on biomarker profiles in PD patients. Integrating genetic characterization with biochemical profiling provides a framework for patient stratification and may serve as a translational strategy to monitor target engagement and evaluate treatment efficacy in future clinical trials.

BACKGROUND Accurate differential diagnosis of complex neurological disorders remains challenging due to overlapping clinical features and heterogeneous disease presentations. Although large language models (LLMs) show promise in clinical reasoning, prior studies benchmark performance against clinician consensus rather than biological ground truth. A neuropathologically confirmed benchmark dataset for evaluating diagnostic AI in neurology is currently lacking. METHODS We introduce NeuroBench, a curated benchmark of complex neurological cases with neuropathologically confirmed gold-standard diagnoses, and DIAGNO, a confidence-aware LLM-based system for neurological diagnosis. NeuroBench comprises 203 retrospective case summaries from the Massachusetts General Hospital Brain Cutting Conference with corresponding autopsy-confirmed diagnoses. DIAGNO generated top-3 differential diagnoses, employing retrieval-augmented generation (RAG) for lower-confidence cases. Performance was assessed by three independent blinded adjudicators who evaluated both DIAGNO and neurologists against neuropathological ground truth. RESULTS NeuroBench encompassed 79 unique neuropathological diagnoses, spanning conditions including cerebrovascular disease, brain tumors, neurological infections, and various neurodegenerative and inflammatory disorders. DIAGNO matched or outperformed neurologists in top-3 accuracy (0.67 versus 0.63) and taxonomy-level accuracy (0.74 versus 0.66). In cases of disagreement, DIAGNO was more often correct than neurologists (29 versus 19 cases). Diagnostic concordance between DIAGNO and neurologists was high (90% agreement in top-3 predictions), even when both were incorrect, suggesting strong alignment in diagnostic reasoning. On NeuroBench, DIAGNO also outperformed GPT-4o baseline and DeepSeek R1 across all top-k accuracy metrics. In a real-world evaluation on eight complex cases with differentials from Mass General Brigham, neurologists rated DIAGNO's reasoning favorably (mean 4.03/5) across multiple dimensions of clinical utility and safety. CONCLUSIONS NeuroBench establishes neuropathological confirmation as the appropriate standard for evaluating diagnostic AI in neurology, moving beyond clinician-referenced benchmarking to define the ceiling of diagnostic accuracy. Evaluated against this standard, DIAGNO achieved expert-level diagnostic performance and received favorable clinician ratings in real-world applications, supporting its potential as a clinical decision-support tool in neurology.

Monogenic epilepsies are 1.6 times more likely to be treatment-resistant compared to other epilepsies, emphasizing the need for additional therapeutic strategies. Sleep dysfunction beyond sleep-related breathing disorders is common yet insufficiently characterized and treated in monogenic epilepsies. We therefore sought to study sleep phenotypes across these epilepsies, examine associations with seizure severity, and assess the diagnostic rate of sleep disorders. From 2,519 individuals enrolled in the Epilepsy Genetics Research Project at Children's Hospital of Philadelphia, we identified the monogenic epilepsies most frequently associated with sleep-related diagnoses, yielding 252 individuals across nine genetic diagnoses (STXBP1, n = 79; SCN1A, n = 57; SCN2A, n = 34; KCNQ2, n = 21; SLC6A1, n = 14; SYNGAP1, n = 13; WDR45, n = 13; KCNT1, n = 11; PCDH19, n = 10). Monogenic epilepsies exhibited distinct sleep endophenotypes, including insomnia, parasomnia, and sleep-related movement disorders in SCN1A-related disorders; frequent epileptiform discharges in sleep with insomnia symptoms in SCN2A-related disorders; sleep dysfunction restricted to the developmental and epileptic encephalopathy subtype in KCNQ2-related disorders; and insomnia without nocturnal seizure involvement in SYNGAP1-related disorders. Formal sleep diagnoses were present in only 25% of individuals (63/252), yet 58% (145/252) reported sleep difficulties, suggesting substantial underdiagnosis. Persistent seizures were associated with higher odds of sleep disorder diagnoses (OR 2.87, 95% CrI 1.57-5.36), disrupted sleep architecture (OR 2.06, 95% CrI 1.08-4.16), nocturnal seizures (OR 4.47, 95% CrI 2.50-8.28), hypersomnolence (OR 2.38, 95% CrI 1.27-4.58) and insomnia (OR 1.80, 95% CrI 1.06-3.05). Neuropsychiatric comorbidities were independently associated with sleep burden after adjustment for seizure severity (OR 2.49, 95% CrI 1.40-4.49). We find that monogenic epilepsies exhibit distinct, gene-specific sleep endophenotypes that are underdiagnosed. Treating sleep difficulties beyond obstructive sleep apnoea may improve seizure control and developmental outcomes, highlighting the need for timely diagnosis of co-occurring sleep disorders.