Neurology Neuroimmunology & Neuroinflammation

BackgroundMultiple Sclerosis (MS) is a complex autoimmune disease caused by a combination of genetic and environmental factors. Translation of Genome-Wide Association Study (GWAS) findings in MS into therapeutics and effective preventive strategies has been limited to date. MethodsWe used Summary Data-Based Mendelian Randomisation (SMR) to synthesise findings from public expression quantitative trait locus (eQTL; eQTLgen and CAGE), methylation quantitative trait locus (mQTL; Lothian Birth Cohort and Brisbane Systems Genetics Study), and MS GWAS datasets (International Multiple Sclerosis Genetics Consortium). By correlating the effects of methylation on MS (M-2-MS), methylation on expression (M-2-E), and expression on MS susceptibility (E-2-MS), we prioritise genetic loci with strong evidence of causally influencing MS susceptibility. We overlay these findings onto a list of druggable genes, i.e. genes which are currently, or could theoretically, be targeted by therapeutic compounds. We use GeNets and STRING to identify protein-protein interactions and druggable pathways enriched in our results. We extend these findings to a model of Epstein-Barr Virus-infected B cells, Lymphoblastoid Cell Lines (LCLs). We conducted a systematic review of prioritised genes using the Open Targets platform to identify completed and planned trials targeted prioritised genes in MS and related disease areas. ResultsExpression of 45 genes in peripheral was strongly associated with MS susceptibility (False discovery rate 0.05). Of these 45 genes, 20 encode a protein which is currently targeted by an existing therapeutic compound. These genes were enriched for Gene Ontology terms pertaining to immune system function and leukocyte signalling. We refined this prioritised gene list by restricting to loci where CpG site methylation was associated with MS susceptibility (M-2-MS), with gene expression (M-2-E), and where expression was associated with MS susceptibility (E-2-MS). This approach yielded a list of 15 prioritised druggable target genes for which there was evidence of a causal pathway linking methylation, expression, and MS. Five of these 15 genes are targeted by existing drugs (CD40, ERBB2, VEGFB, MERTK, and PARP1), and three were replicated in a smaller eQTL dataset (CD40, MERTK, and PARP1). In LCLs, SMR prioritised 7 druggable gene targets, of which only one was priortised by the multi-omic approach in peripheral blood (FCRL3). Systematic review of Open Targets revealed multiple early-phase trials targeting 13/20 prioritised genes in disorders related to MS. ConclusionsWe use public datasets and SMR to identify a list of prioritised druggable genetic targets in Multiple Sclerosis. We hope our findings could be translated into effective repurposing of existing drugs to provide novel therapies for MS and, potentially, provide a platform for developing preventive therapies.

ImportanceThe identification of a prodrome in multiple sclerosis (MS) is the key to early prevention and the targeting of new interventions. ObjectiveTo assess the associations between health conditions diagnosed in primary care and the risk of incident MS relative to other autoimmune inflammatory diseases. DesignA case-control study in the UK and France was conducted from Jan 1, 1996 to March 28, 2022 in the UK and from Jan 4, 1998 to March 28, 2022 in France. SettingData were obtained from electronic health records from the Health Improvement Network database. ParticipantsWe included all individuals with at least two years of history in the database and a recorded diagnosis of either MS, lupus or Crohns disease. Three controls matched for sex, age at index date and year at index date were randomly assigned to each individual with a diagnosis of MS. Main outcome measuresWe agnostically tested the associations between 113 different diagnoses and multiple sclerosis diagnosis during the five years before or the five years after the diagnosis of MS. Unadjusted odds ratios (ORs) and 95% CIs were estimated, and p values were corrected for multiple comparisons. We also stratified for sex, age at diagnosis, and year of diagnosis. A logistic regression analysis (adjusted for sex and age at diagnosis) was performed to compare MS patients with lupus and Crohns disease patients. ResultsThe study population consisted of patients with MS (UK: 15,808; and France: 4,366), Crohns disease (UK: 20,872; and France: 9,605) or lupus (UK: 5,296; and France: 2,041). We identified twelve health conditions as significantly positively associated with the risk of MS. After considering health conditions suggestive of demyelinating events as the first diagnosis of MS, five health conditions remained significantly associated with MS: depression (UK OR 1.22 [95%CI 1.11-1.34]), sexual dysfunction (1.47 [1.11-1.95]), constipation (1.5 [1.27-1.78]), cystitis (1.21 [1.05-1.39]), and urinary tract infections (1.38 [1.18-1.61]). However, none of these conditions was selectively associated with MS in comparisons with both lupus and Crohns disease. During the five years after MS diagnosis, all five health conditions identified here were still associated with MS. Conclusion and relevanceThe identified symptoms may be considered to be not only prodromal, but also early-stage symptoms, albeit not specific to MS. Key pointsO_ST_ABSQuestionC_ST_ABSWhat prediagnostic manifestations of multiples sclerosis (MS) occur in primary care settings and how do they differ from those of other autoimmune diseases? ResultsIn this agnostic study of 15,808 MS patients from the UK and 4,366 MS patients from France, we identified five health conditions as positively associated with the risk of MS when recorded in the five years preceding MS diagnosis. We show that most of these health conditions were also present in the early presentations of lupus and Crohns disease. MeaningOur findings suggest that the prodromal phase of MS is largely similar to the prediagnostic manifestations of other autoimmune diseases.

Background and ObjectivesMultiple sclerosis (MS) is a chronic autoimmune disease with limited treatment options. Thus, drug discovery and repurposing are essential to enhance treatment efficacy and safety. MethodsWe obtained summary statistics for protein quantitative trait loci (pQTL) of 2,004 plasma proteins and 1,443 brain proteins, a genome-wide association study (GWAS) of MS susceptibility with 14,802 cases and 26,703 controls, and expression quantitative trait loci (eQTL) for 8,000 genes in peripheral blood and 16,704 genes in brain tissue. Our integrative analysis included a proteome-wide association study to identify MS-associated proteins, followed by summary-data-based Mendelian randomization (SMR) to determine causal associations. We used the HEIDI test and Bayesian colocalization analysis to distinguish pleiotropy from linkage. Proteins passing SMR, HEIDI, and colocalization analyses were considered potential drug targets. We further conducted pathway annotations, protein-protein interaction (PPI) network analysis, and examined mRNA levels of these targets. ResultsWe identified hundreds of MS-associated proteins in plasma and brain, confirming the causal roles of 18 proteins (nine in plasma and nine in brain). Among these, we found 78 annotated pathways and 16 existing non-MS drugs targeting six proteins. We also discovered intricate PPIs among seven potential drug targets and 19 existing MS drug targets, as well as PPIs of four targets across plasma and brain. Combining expression data, we identified two targets adhering to the central dogma of molecular biology. DiscussionWe prioritized 18 potential drug targets in plasma and brain, elucidating the underlying pathology and providing evidence for drug discovery and repurposing in MS.

ImportanceThe risk of multiple sclerosis (MS) is significantly influenced by polygenic inheritance. Polygenic risk scores (PRS) for MS can help identify high-risk individuals and stratify populations for clinical trials. However, most genome-wide association studies (GWAS) have been conducted in populations of European ancestry, raising questions about the accuracy of these PRS in other ancestries. ObjectiveTo determine whether a PRS for MS can effectively stratify individuals of non-European ancestries. Design, setting and participantsThis cross-sectional study utilized prospectively collected data from the All of Us Research Program (2018-2023). It included participants who had both whole genome sequencing and electronic health record (EHR) data. Exposure(s)A PRS comprising 282 independent single nucleotide variants for MS, divided into quintiles. Main Outcome(s) and Measure(s)Prevalence of multiple sclerosis ascertained through ICD-10 or SNOMED codes. ResultsIn this study population, MS cases comprised 1.0% (327 cases) of the European population, 0.56% (183 cases) of the African population, and 0.46% (150 cases) of the Latino/admixed American population. In analyses adjusting for age, sex, and genetic principal components, the PRS associates with MS risk in the European population, with a 141% increase in the risk of MS for individuals in the highest compared to the lowest PRS quintile (OR: 2.41 [1.69-3.50], test-for-trend p<0.001). Similarly, the PRS appropriately partitions the Latino/admixed population into increasing MS risk groups (OR: 2.56 [1.45-4.78], test-for-trend p-value <0.001). However, it did not significantly stratify the African population into distinct MS risk categories (OR: 1.45 [0.95-2.25], test-for-trend p=0.10). Conclusions and RelevanceA PRS for MS effectively stratified individuals of European and Latino/admixed ancestries but not African ancestry. This highlights the need for ancestry-specific PRS development to ensure accurate risk prediction across diverse populations, emphasizing the importance of including non-European groups in MS genetic research.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system with a complex etiology. Recent genomic studies highlight the contribution of expression quantitative trait loci (eQTLs) in modulating gene expression and disease susceptibility. Given the emerging role of circular RNAs (circRNAs) in MS, we hypothesized that genetic variants may regulate circRNA expression through circRNA-specific eQTLs (circ-eQTLs). We performed a cis-circ-eQTL analysis integrating circRNA expression and whole-genome genotyping data from 30 MS patients and 18 healthy controls using a linear regression model adjusted for disease status and sex. Candidate circ-eQTLs were prioritized based on MS-associated regions and known splicing QTLs (sQTLs) from GTEx and validated in an independent cohort (67 MS, 64 controls). Association analysis in a larger cohort (2831 MS, 3191 controls) evaluated two candidate variants for MS risk. We identified 42,077 significant cis-circ-eQTLs and validated three. Two SNPs, rs7214410 and rs11079784, modulated hsa_circ_0106983 expression, and rs7214410 also acted as an sQTL affecting EFCAB13 splicing. rs7214410 showed stronger association with MS than rs11079784. Our findings reveal extensive genetic regulation of circRNA expression and highlight rs7214410 as a dual-function variant refining the MS susceptibility locus on chromosome 17.

Background and ObjectivesOral cladribine tablet (CladT) therapy is efficacious for relapsing multiple sclerosis (MS). However, the mechanisms by which cladribine exerts benefit in MS remain unclear, particularly regarding its effects on compartmentalized inflammation within the cerebrospinal fluid (CSF). MethodsTranscriptional profiles along with T and B lymphocyte receptor repertoires from CSF and blood were obtained by single cell sequencing methods from a single site participating in a phase IV clinical trial investigating the impact of cladribine treatment for MS. All subjects provided blood and CSF samples immediately before starting CladT therapy and were randomized to also provide samples at either five weeks, ten weeks, one year, or two years post-CladT therapy. Thirty-four samples from 13 individuals with relapsing MS before and after treatment were available to test the hypothesis that CladT alters the composition and phenotype of lymphocytes in the CSF. ResultsWe found that treatment with CladT profoundly altered cellular composition, but not the transcriptional phenotype, of immune cells in the CSF. In particular, we identified a reduction of switched memory B cells but recovery of naive B cells in the CSF, similar to our findings in blood. Additionally, populations of CD4 Treg cells emerged early after CladT therapy and remained elevated one year later in the CSF, but not in the blood. Antigen receptor sequencing revealed a moderate decrease in numbers of large clonally expanded CD8 T cell clones (>10 cells/clone) primarily in the CSF, but also in the blood post-CladT treatment. DiscussionOur results identified unique cellular dynamics and changes in T and B cell clonality in both tissues which can potentially explain long-term beneficial effects of CladT therapy in MS, including preservation of immune function and relatively low number of side effects. Altogether, this study demonstrates that CladT treatment had a substantial impact not only on blood, but also on the CSF compartment highlighting the importance of cross-tissue analysis for better understanding of effect and the mechanism of action of DMTs.

BackgroundMultiple sclerosis is a neurodegenerative autoimmune disorder of the central nervous system (CNS) in which autoreactive immune cells migrate through a damaged blood brain barrier, resulting in focal demyelinating lesions of both the white and grey matter. Of increasing interest is the repeated observation that beyond focal lesions, there are also diffuse, surface-in gradients of pathology in MS, wherein damage is most severe directly adjacent to cerebrospinal fluid (CSF)-contacting surfaces, such as the subpial and periventricular areas. This observation suggests that toxic factors within MS CSF may be contributing to the emergence and/or evolution of surface-in gradients. Directly separating the CSF from the periventricular parenchyma are ependymal cells - a glial epithelium that are equipped with tufts of motile cilia which are critical for circulating CSF solutes and regulating local fluid flow. While damage to ependymal cilia has the potential to drastically modify CSF homeostasis and thus contribute to the damage of CSF exposed regions, these motile cellular structures have yet to be investigated in the context of MS. MethodsWe first conducted single cell RNA sequencing of fresh human periventricular brain tissue containing ependymal cells from MS patients and non-MS disease controls. We subsequently collected CSF from MS patients and exposed cultured rodent ependymal cells to this CSF in order to evaluate impact on ependymal ciliary function. To complement our direct evaluation of cilia in the context of MS, we also confirmed whether cilia were altered in a classic animal model of MS, experimental autoimmune encephalomyelitis (EAE), and also designed a novel transgenic animal model to evaluate the cellular and behavioural effect(s) of adult ependymal ciliary disruption. ResultsSingle cell RNA sequencing analysis of human ependymal cells in MS demonstrated largescale dysregulation of ciliary genes and in situ stains of MS brain tissue confirmed a loss of ependymal cilia. Exposure of ependymal cells to MS CSF led to transcriptional modification of ciliary gene and protein expression and reduced ciliary beating frequency. Likewise, analysis of ependymal cells in EAE also demonstrated altered cilia gene and protein expression. Conditional knockout in adult mice, of the critical cilia-associated gene Ccdc39 in ependymal cells led to transient ventricular enlargement, increased periventricular microglial density, and alterations in nesting behaviour. ConclusionThese data suggest that motile cilia in ependymal cells are dysregulated in CNS autoimmunity. More importantly, however, they provide evidence to suggest that ependymal cilia disruption could play an active role in the development of periventricular pathology in MS and can lead to behavioural deficits that may underlie aspects non-motor MS symptomatology.

BackgroundLong COVID (LC) is a novel condition that is characterized by persistent symptoms that last from months to years following a SARS-CoV-2 infection. While LC symptoms vary widely, neuropathy is one of the most prevalent symptoms and drastically affects patients quality of life. However, the underlying pathophysiology of LC neuropathy remains poorly understood. Here, we investigated the prevalence and potential mechanisms of LC neuropathy in the largest LC neuropathy cohort to date. MethodsWe conducted an observational study of 977 adults with LC at Dell Medical School. Participants underwent clinical assessments, skin punch biopsy, and comprehensive metabolic, endocrine and immunological profiling. A subset of patients received treatment with intravenous immunoglobulin (IVIG). FindingsNeuropathic symptoms were reported by 55% (534/977) participants, with skin biopsy confirming small fiber neuropathy in 56.5% (48/85) cases, affecting both epidermal and autonomic nerve fibers. Common risk factors for neuropathy, including metabolic and endocrine disorders, did not fully explain neuropathic symptoms. While general immunological markers (lymphocyte, T cell, and B cell count and C reactive protein were unremarkable, unexpectedly, we detected anti-ganglioside antibodies (AGAs) in 25% of patients with LC neuropathy, a comparable rate to other AGA-associated neuropathies. Longitudinal testing revealed persistent AGA positivity, and multiple elevated AGAs in a subset of patients. In a pilot treatment cohort of eight patients, IVIG treatment resulted in improvement of patient reported neuropathic symptoms. InterpretationOur findings reveal a high prevalence of small fiber neuropathy in LC, with evidence suggesting an autoimmune mechanism involving AGAs in one in four LC neuropathy patients. The therapeutic response to IVIG further supports an autoimmune pathophysiology, suggesting potential benefits of immunomodulation in LC neuropathy patients.

Multiple sclerosis (MS) is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 (FLOT-1/2) complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 (ANO2) or neurofascin-155 (NF155) have been previously described in MS patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against NF155, ANO2 and the FLOT-1/2 complex in MS. Serum (n=252) and CSF (n=50) samples from 282 MS patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-FLOT1/2, anti-ANO2 and anti-NF155 antibodies were tested by cell-based assays using transfected-HEK293 cells. We identified 6 MS patients with antibodies against the FLOT-1/2 complex (2.1%) and 1 MS patient with antibodies against ANO2 (0.35%). All MS patients were negative for anti-NF155 antibodies. Three of the anti-FLOT1/2 positive patients showed anti-FLOT-1/2 positivity in other serum samples extracted at different moments of their disease. IgG subclasses of anti-FLOT-1/2 antibodies were predominantly IgG1 and IgG3. We confirm that antibodies targeting the Flotillin-1/2 complex are present in a subgroup of patients with MS. Further studies are needed to understand the clinical and pathological relevance of anti-FLOT-1/2 autoantibodies in MS.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system. The cause of the disease is unknown but both genetic and environmental factors are strongly implicated in its pathogenesis. We derived cerebral and spinal cord organoids from induced pluripotent stem cells (iPSC) from healthy controls as well as from primary progressive MS (PPMS), secondary progressive MS (SPMS) and relapsing-remitting MS (RRMS) patients to investigate and compare oligodendrocyte differentiation and myelination capacity in healthy subjects and MS subtypes. In MS organoids, particularly in PPMS, we observed a decrease in p21 expression associated with a dysregulation of PAK1 and E2F1 expression. In parallel, a decrease in oligodendrocyte maturation was detected in long-term cultured cerebral and spinal cord organoids, especially in PPMS, leading to a reduced myelination capacity. Disruption of astrocyte and neuronal populations was also observed. Our findings demonstrate that in MS, inherent deficits in the p21 pathway may alter glial and neuronal cell populations and may contribute to the disease pathogenesis by reducing the capacity for myelin repair. Summary StatementUsing cerebral and spinal cord organoids derived from multiple sclerosis patients, we found an innate disruption of oligodendrocyte differentiation and myelination capacity as well as excitotoxicity, associated with PAK1 and E2F1-induced p21 dysregulation.

BackgroundDisorders affecting the spinal cord (myelopathies) can cause severe disability. Despite diagnostic advances, approximately 12-18% of myelopathy cases continue to elude an etiological diagnosis, hampering effective treatment. MethodsThis retrospective, multicenter, tertiary care cohort study conducted from 2014 to 2025 evaluated archived biofluids from patients with IM, known autoimmune myelitis, or other neurological diseases (ONDs). Proteome-wide phage display was used to discover novel autoantibodies. Targeted immunoassays were used to screen for a candidate autoantibody. Downstream metabolites were measured in the cerebrospinal fluid (CSF). ResultsAutoantibodies targeting the transcobalamin receptor (CD320) responsible for cellular transport of vitamin B12 were identified in 18 out of 32 IM patients (56%) in a discovery cohort. Bioactive B12 concentration was decreased in the CSF of anti-CD320 positive patients compared to OND controls (P = 0.0273), indicative of autoimmune B12 central deficiency (ABCD). Compared to anti-CD320 negative IM cases, anti-CD320 positive IM cases demonstrated a higher frequency of subacute time course (56% vs 7%, P = 0.008), normal CSF profile (83% vs 50%, P = 0.044), and dorsolateral spinal cord abnormalities on magnetic resonance imaging (MRI) (61% vs 7%, P = 0.003). In two independent validation cohorts comprising 94 and 25 patients with IM, anti-CD320 was detected in 43 (46%) and 12 (48%) patients, respectively. Comorbid anti-CD320 was detected in a smaller proportion of patients with other known autoimmune etiologies of myelopathy. Five anti-CD320 positive IM patients received B12 supplementation with or without concurrent immunosuppression, and four out of five clinically improved. ConclusionsABCD is associated with a substantial proportion of IM. Screening for anti-CD320 followed by metabolic confirmation of a CNS-restricted B12 deficiency may be considered in the diagnostic evaluation of myelopathy.

BackgroundMultiple sclerosis (MS) is an inflammatory disease characterized by demyelination and neuro-axonal degeneration in the central nervous system. Except for neurofilament light protein, identification of biomarkers has been difficult to assess in the blood, presumably due partly to sensitivity. To detect traces of disease activities in the periphery and identify low-abundance protein biomarkers, this study conducts an exploratory examination of the plasma proteome of MS using proximity extension technology, a high-sensitivity multiplex PCR-based immunoassay. MethodsA case-control cohort consisting of 52 MS cases (relapsing-remitting=30, progressive=22) and 17 healthy controls were enrolled at the Karolinska University Hospital. EDTA plasma was analyzed for 1157 unique protein targets across thirteen proximity extension assays. Protein associations to disease outcomes and related clinical measures were assessed using a multivariable linear regression model corrected for sex and age at sampling. ResultsAHCY and CHR levels were higher among MS cases than controls, while FABP2 was lower among those with relapsing-remitting disease than controls (Pdiscovery<0.05, Preplication<0.05), although not significant after multiple test corrections. Furthermore, PTN and CYR61 levels were higher in progressive MS than in relapsing-remitting disease (P<0.0002, PFDR<0.05), and CRNN and CXCL13 were associated with more severe disability at sampling (P<0.0001, PFDR<0.05), independent of disease course. CTSF was positively correlated with disease duration (P=4.1x10-5, PFDR=0.044), while RRM2B level correlated with intrathecal immunoglobulin production (IgG Index) in relapsing-remitting MS (P=1.7x10-5, PFDR=0.018). ConclusionWe provide several candidates for characterizing MS, particularly progressive disease, which may help monitor disease progression and treatment response in a clinical setting.

ObjectiveAutoimmune nodopathies (AINs) are a group of rare, acquired autoimmune neuropathies with distinct clinical features and the presence of circulating autoantibodies - often of the immunoglobulin G4 (IgG4) subclass - targeting proteins at the node of Ranvier. Defects in B cell tolerance checkpoints have been implicated in several autoimmune diseases. Prior work identified defective B cell tolerance--reflected by a high frequency of self-reactive naive B cells--in patients with MuSK-positive myasthenia gravis (MG), mediated by IgG4 autoantibodies. Here, we investigated whether tolerance defects exist in neurofascin-155-mediated AIN (NF155-AIN), similar to MuSK+ MG. Additionally, we analyzed B and T cell transcriptomics and interactions at the single-cell level to explore the underlying pathomechanism. MethodsUsing a well-established assay, we assessed B cell tolerance fidelity by generating recombinant antibodies from new emigrant (NE) and mature naive (MN) B cell populations-- directly downstream of key tolerance checkpoints--from three NF155-AIN patients, and testing these antibodies for polyreactivity and autoreactivity, thereby determining the frequency of polyreactive and autoreactive B cells. The transcriptome of peripheral blood mononuclear cells (PBMC) was studied, with a special focus on naive B cells and CD4+ T cells at the single-cell level, along with characterization of cell-cell interactions. ResultsNF155-AIN patients have an elevated frequency of polyreactive B cells in the NE (37.4% compared to 9.7% in healthy controls (HCs), p = 0.03) and MN (31.5% compared to 10.5% in HCs, p = 0.03) compartments with increased B cell clones expressing autoreactive antibodies, consistent with a breach in early tolerance checkpoints. We observed abnormal B cell receptor (BCR) signaling characterized by low CD79B, CSK, BLNK, and BTK expression, which may contribute to a breach in B cell tolerance. We also observed evidence of impaired follicular helper T cells (Tfh) and regulatory T cells (Treg), which may limit the normal development and suppression of autoreactive B cells. Moreover, comparative gene expression analysis of B cells and CD4+ T cells from three patients with chronic inflammatory demyelinating polyneuropathy (CIDP) --a related autoimmune neuropathy-- confirmed that these differences are largely specific to NF155-AIN, supporting a distinct pathophysiology in this subset. ConclusionThese findings demonstrated a breach in early B cell tolerance checkpoints, defective BCR signaling, and disrupted T cell-B cell interactions in NF155-AIN, all of which may contribute to the development of pathogenic autoreactivity. These immunologic abnormalities appear distinct from those seen in CIDP, supporting NF155-AIN as a unique immunopathologic entity.

The genetic architecture of Multiple Sclerosis (MS) susceptibility has been extensively assessed in populations of European ancestry. Greater ancestral diversity in genetic analyses of MS susceptibility is needed to improve the utility of Multiple Sclerosis genetic risk scores, fine map causal variants underlying established associations, and thereby enhance the identification of drug targets. Here we report findings from a genetic study of Multiple Sclerosis susceptibility in an ancestrally-diverse United Kingdom-based cohort. Participants with Multiple Sclerosis were recruited via clinical sites, an online platform, and through the United Kingdom Multiple Sclerosis Register. Phenotype data were gathered using a standardised questionnaire. DNA was extracted from saliva samples obtained remotely or in person, and participants were genotyped using a commercial genotyping array. Following imputation, cases were combined with controls from the United Kingdom Biobank and subjected to stringent quality control and genetic ancestry inference. We defined two broad ancestral groups of South Asian and African ancestry. We performed within-ancestry case-control genome-wide association studies of Multiple Sclerosis susceptibility using logistic models accounting for population structure and sex. We examined both single nucleotide variants and imputed classical Human Leukocyte Antigen alleles. We curated two ancestrally-matched case-control genetic datasets (South Asian ancestry: NCase=175, NControl=6744; African ancestry: NCase=113, NControl=5177). In both ancestries, we found genetic variants within the Major Histocompatibility Complex associated with Multiple Sclerosis susceptibility (South Asian ancestry: lead variant chr6:32600515:G:A on hg38 co-ordinates, Odds Ratio=1.84, nearest gene HLA-DRB1, P=4.6x10-6; African ancestry: lead variant chr6:29919337:A:G, Odds Ratio=2.24, nearest gene HLA-A P=4.3x10-5). European-ancestry susceptibility alleles were over-represented in cases from both ancestries, with the degree of concordance stronger for the South Asian ({rho}=0.31, P=8.1x10-6) than African ({rho}=0.1, P=0.3) ancestry cohort. European-derived genetic risk scores performed better than chance but less well than in European ancestry cohorts, explaining 1.6% (South Asian, P=1.0x10-4) and 0.5% (African, P=0.08) of the liability to MS. The genetic architecture of MS susceptibility shows strong concordance across ancestral groups suggesting shared disease mechanisms. Larger studies in diverse populations are likely to enhance our understanding of how genetic variation contributes to MS susceptibility in people of all ancestral backgrounds.

Background/ObjectivesSerum proteomic analysis of deeply-phenotyped samples, biological pathway modeling and network analysis were performed to elucidate the inflammatory and neurodegenerative processes of multiple sclerosis (MS) and identify sensitive biomarkers of MS disease activity (DA). MethodsOver 1100 serum proteins were evaluated in >600 samples from three MS cohorts to identify biomarkers of clinical and radiographic (gadolinium-enhancing lesions) new MS DA. Protein levels were analyzed and associated with presence of gadolinium-enhancing lesions, clinical relapse status (CRS), and annualized relapse rate (ARR) to create a custom assay panel. ResultsTwenty proteins were associated with increased clinical and radiographic MS DA. Serum neurofilament light chain (NfL) showed the strongest univariate correlation with radiographic and clinical DA measures. Multivariate modeling significantly outperformed univariate NfL to predict gadolinium lesion activity, CRS and ARR. DiscussionThese findings provide insight regarding correlations between inflammatory and neurodegenerative biomarkers and clinical and radiographic MS DA. FundingOctave Bioscience, Inc (Menlo Park, CA).

ObjectiveThe field of multiple sclerosis (MS) has seen a tremendous expansion of treatments in the past decade. However, treatment response in individual patients can currently be determined only by waiting for breakthrough disease activity to occur. This highlights a critical need for biomarkers that can predict treatment response and stratify the risk of impeding disease activity before damage is inflicted to the CNS. Here we show that CCR6+CD3+ T-cell surveillance in peripheral blood can be used to discriminate responders and non-responders to dimethyl-fumarate. MethodsA cohort of 101 treatment-naive, dimethyl-fumarate (DMF) treated MS patients and healthy controls was immunophenotyped and then responders and non-responders were determined retrospectively after clinical and radiographic follow up. Receiver operating characteristic (ROC) curve, linear and logistic regression, mixed effects models, and cox proportional hazards were used for the analysis. ResultsAmong various clinical and immunophenotypic metrics, the percentage of CCR6+CD3+ T-cells was the most significant predictor of impending disease activity. This immunophenotypic metric was able to discriminate responders and non-responders to DMF with an area under the ROC of 0.85 (95% CI: 0.71-0.99), which was higher than that achieved using surrogate metrics for T-helper-1-like T-helper-17 or T-cytotoxic-17 cells. DMF-treated patients with the highest percentage of CCR6+CD3+ T-cells had a significantly higher risk of impending disease activity compared to patients with a low percentage. InterpretationChanges in CCR6+CD3+ T-cells in the periphery could precede disease activity by many months and potentially serve as an early biomarker of treatment response, at least for DMF. These results have implications for novel personalized treatment strategies in MS.

BackgroundProgression independent of relapses (PIRA) is a major therapeutic challenge in multiple sclerosis (MS). Nasal anti-CD3 treats animal models of progressive MS by inducing regulatory T cells (Tregs) that suppress central nervous system (CNS) inflammation and lessen clinical disease. MethodsTen patients with non-active secondary progressive MS (naSPMS) that continued to progress on B cell therapy were treated with nasal anti-CD3 (foralumab) for a minimum of six months in an open label study. Safety monitoring included otolaryngology evaluation and neurologic assessments including Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC-4), Modified Fatigue Impact Scale (MFIS), California Verbal Learning Test (CVLT-II) and Low Contract Visual Acuity (LCVA). MRI and microglial translocator protein (TSPO)-PET imaging with [F-18]PBR06 were conducted. Serum and cerebrospinal fluid (CSF) proteomic biomarkers and single cell RNA sequencing of blood was performed to evaluate foralumab-induced immunomodulation. The endpoints of our study were safety, clinical effects, microglial signal and immune measures. ResultsAll patients stabilized on EDSS scores and three of four patients treated continuously for 12 months had improvement on EDSS. Six of 10 patients had improvement in fatigue on the MFIS scale. There were no treatment-related serious adverse events (SAEs) or severe AEs and no new T2 lesions were observed on MRI. There was a reduction in TSPO-PET signal over six months (p<0.05). Changes in peripheral blood gene expression occurred as early as three months and affected antigen presentation, interferon responses and regulatory pathways in multiple cell types including FoxP3+ Tregs, CD4+ Tcm cells, CD8+ Tem cells, CD14+ and CD16+ monocytes and B cells. TGF{beta} expression was increased across cell multiple subsets. InterpretationThese findings identify a novel, non-toxic immune based therapy for the treatment for PIRA that acts by the induction of a regulatory immune responses and dampens microglial inflammation. Double blind placebo-controlled trials are warranted to explore nasal foralumab for the treatment of naSPMS.

ObjectiveBiomarkers could inform disease worsening and severity in people with MS (pwMS). Few studies have examined blood biomarkers informative of patient-reported outcome (PRO) of disability in pwMS. In this study we examine the associations between serum protein biomarker profiles and patient-reported disability in pwMS. MethodsThis cross-sectional study included adults with a neurologist-confirmed diagnosis of MS from the University of Pittsburgh Medical Center (Pittsburgh, PA) and the Rocky Mountain MS Clinic (Salt Lake City, Utah) between 2017 and 2020. For exposure, we included 19 serum protein biomarkers potentially associated with MS inflammatory disease activity and 7 key clinical factors (age at sample collection, sex, race/ethnicity, disease subtype, disease duration, disease-modifying treatment, and time interval between sample collection and closest PRO assessment). Using 6 machine learning approaches (Least Absolute Shrinkage and Selection Operator [LASSO] regression, Random Forest [RF], XGBoost, Support-Vector Machines [SVM], stacking ensemble learning, and stacking classification algorithm), we examined model performance in predicting Patient Determined Disease Steps (PDDS) as the primary outcome. We assessed model prediction of Patient-Reported Outcomes Measurement Information System (PROMIS) physical function in a subgroup. We reported model performance using the held-out testing set. ResultsWe included 431 unique participants (mean age 49 years, 81% women, 94% non-Hispanic White). Using binary outcomes, models comprising both routine clinical factors and the 19 proteins as features consistently outperformed base models (containing clinical features alone or clinical features plus single protein) in predicting severe (PDDS[&ge;]4, PROMIS<35) versus mild/moderate (PDDS<4, PROMIS[&ge;]35) disability for all machine learning approaches, with LASSO achieving the best area under the curve (AUCPDDS=0.91, AUCPROMIS=0.90). Using ordinal/continuous outcomes, LASSO models with combined clinical factors and 19 proteins as features (R2PDDS=0.31, R2PROMIS =0.35) again outperformed base models. The four LASSO models (PDDS, PROMIS; both binary and ordinal/continuous) with combined clinical and protein features shared 2 clinical features (disease subtype, disease duration) and 4 protein biomarkers (CDCP1, IL-12B, NEFL, PRTG). ConclusionsSerum protein biomarker profiles improve the prediction of real-world MS disability status beyond clinical profile alone or clinical profile plus individual protein biomarker, reaching clinically actionable performance.

BackgroundBlood-based biomarkers are crucial for individualized management of multiple sclerosis (MS). Blood neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) have shown promising clinical utility in MS, but they are insufficient to guide clinical management. Plasma tau proteins remain underexplored despite the growing evidence of shared pathology in Alzheimers disease and MS. We aimed to: (1) assess the utility of plasma tau biomarkers (phosphorylated tau 181 [p-tau181], p-tau217, and total tau [t-tau]) in MS diagnosis, subtyping, and prognosis; and (2) compare their performance with NfL and GFAP. MethodsFrom a clinic-based prospective cohort, we included 160 people with MS (pwMS; 117 with relapsing-remitting MS [RRMS], 43 with progressive MS [PMS]) and 20 non-MS controls, all with baseline plasma samples. We measured baseline plasma concentrations of p-tau181, p-tau217, t-tau, NfL, and GFAP using ultrasensitive immunoassays. We collected demographics, clinical information, and longitudinal multi-modal outcomes (Patient Determined Disease Steps [PDDS], normalized age-related MS severity score [ARMSS], walking speed, manual dexterity, cognitive performance, retinal nerve fiber layer [RNFL] thickness, total brain volume, and gray matter volume) over a median follow-up of 3.0 years (IQR, 3.5). Adjusting for demographic and clinical covariates, we evaluated associations between biomarkers and MS diagnosis, subtypes, and prognosis. We examined the enhanced value of tau markers, in addition to NfL and GFAP, for subtype distinction and outcome prediction. Participants were enrolled between 2017 and 2023. Analyses were conducted in December 2024. ResultsParticipants (n=180) had a median age of 51 years and were predominantly women (68%) and non-Hispanic white (91%). Compared with controls, pwMS had higher levels of p-tau217 (1.0 vs 0.7 pg/mL; p=0.04) and NfL (14.1 vs 9.0 pg/mL; p<0.01). Among pwMS, higher p-tau181 (aOR [95%CI]=2.3 [1.4,4.1]) and p-tau217 (aOR [95%CI]=3.0 [1.8,5.7]) were associated with PMS. These markers improved MS subtype classification accuracy beyond clinical features, NfL, and GFAP. Higher baseline p-tau181 and p-tau217 predicted worse disability, functional outcomes, and imaging outcomes independent of other biomarkers. ConclusionsPlasma p-tau181 and p-tau217 are promising biomarkers for MS subtype classification and disability prediction, providing complementary information to NfL and GFAP. Further studies to validate their potential clinical utility in guiding MS management are warranted.

Observational studies suggest that alterations in circulating lipid levels may be associated with multiple sclerosis (MS) susceptibility and severity. Our study employs two-sample Mendelian randomization (MR) to investigate whether these relationships are causal. Genetic instruments for 249 metabolites, predominantly lipids and lipoproteins, were obtained from a combined dataset of European-ancestry individuals from the UK and Estonian Biobanks. Outcome data were obtained from the International MS Genetics Consortium GWAS studies of MS susceptibility and severity. MR analyses considered MS susceptibility and severity as distinct outcomes and utilized the inverse variance-weighted multiplicative random-effects method in the main analysis. No metabolic exposures demonstrated statistically significant evidence of a causal relationship with MS susceptibility. For MS severity, two traits showed suggestive associations - triglycerides in very-low-density lipoproteins (VLDL) ({beta} = -0.101, SE = 0.028, p = 3.9 x 10-4) and triglycerides in chylomicrons and extremely large VLDL ({beta} = -0.104, SE = 0.032, p = 1.2 x 10-3). The MR-Egger intercept suggested that these observations may be driven by horizontal pleiotropy. Sensitivity analysis with multivariable MR using Body Mass Index as a possible confounder demonstrated substantial attenuation of instrument strength once genetic overlap with BMI was taken into account. In this study we found no convincing evidence that circulating lipids or lipid-related metabolites exert a causal influence on susceptibility to, or severity of, multiple sclerosis. The nominally statistically significant result is likely a result of horizontal pleiotropy.