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Background: Vitamin D (VitD) affects the risk of multiple sclerosis (MS), but the impact on disease activity is controversial. We assessed whether VitD is associated with the No-Evidence of Disease Activity-3 (NEDA-3) status at 2 years from disease-modifying treatment (DMT) start, and whether this association is causal or the result of confounding factors. Furthermore, we explored if a genetic predisposition to higher VitD levels affects the risk of disease activity., Methods: 230 untreated relapsing-remitting MS patients underwent serum 25-OH-vitamin-D measurement, and the association between seasonally adjusted VitD and disease activity was tested. Modelling a Polygenic Risk Score from a Genome-Wide Association Study on ~400 000 individuals, we studied the impact of genetic predisposition to higher VitD on the NEDA-3 status in 1408 independent MS patients. Two-sample Mendelian randomisation (MR) was used to assess causality., Results: Lower baseline VitD was associated with decreased probability of NEDA-3 at 2 years (p=0.019). Particularly, VitD levels <20 ng/mL conferred an over twofold risk of disease activity (OR 2.36, 95% CI 1.30 to 3.88, p=0.0037). Genetic predisposition to higher VitD levels was associated with delayed age at MS onset (p=0.018) and with a higher probability of NEDA-3 status (p=0.034). MR confirmed causality between VitD and the risk of disease activity (p=0.041)., Conclusions: VitD levels before DMT start affect the risk of disease activity in MS. Genetic predisposition to higher VitD levels confers a lower risk of disease activity and is associated with delayed MS onset. Our work prompts future prospective studies regarding VitD supplementation and lifestyle interventions to hamper disease activity in MS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Multiple sclerosis (MS) is a complex disorder characterized by high heterogeneity in terms of phenotypic expression, prognosis and treatment response. In the present study, we aimed to explore the genetic contribution to MS disease activity at different levels: genes, pathways and tissue-specific networks. Two cohorts of relapsing-remitting MS patients who started a first-line treatment (n = 1294) were enrolled to evaluate the genetic association with disease activity after 4 years of follow-up. The analyses were performed at whole-genome SNP and gene level, followed by the construction of gene-gene interaction networks specific for brain and lymphocytes. The resulting gene modules were evaluated to highlight key players from a topological and functional perspective. We identified 23 variants and 223 genes with suggestive association to 4-years disease activity, highlighting genes like PON2 involved in oxidative stress and in mitochondria functions and other genes, like ILRUN, involved in the modulation of the immune system. Network analyses led to the identification of a brain module composed of 228 genes and a lymphocytes module composed of 287 genes. The network analysis allowed us to prioritize genes relevant for their topological properties; among them, there are MPHOSPH9 (connector hub in both brain and lymphocyte module) and OPA1 (in brain module), two genes already implicated in MS. Modules showed the enrichment of both shared and tissue-specific pathways, mainly implicated in inflammation. In conclusion, our results suggest that the processes underlying disease activity act on shared mechanisms across brain and lymphocyte tissues., Competing Interests: Declarations. Ethics Approval: The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Ospedale San Raffaele (study protocol: GR2016-01). Consent to Participate: Informed consent was obtained from all subjects involved in the study. Consent for Publication: Not applicable. All presented data refer to summary statistics obtained combining genetic data from a large group of patients. No individual level data are presented. Competing Interests: E. Ma., V.N., L.F., M.S., F.C., A.C., S.S., A.G., K.M., M.C., L.Mi. and E.Mo. declare no conflict of interest. L.Mo.: received compensation for speaking activities, and/or consulting services from Merck, Biogen, Novartis, Roche, Sanofi, and TEVA; V.M.: received compensation for speaking and/or for consultancy and support for travel expenses and participation in Congresses from Biogen, Merck-Serono, Novartis, Genzyme and Teva Pharmaceutical Industries; M.F.: Editor-in-Chief of the Journal of Neurology, Associate Editor of Human Brain Mapping, Associate Editor of Radiology, and Associate Editor of Neurological Sciences, received compensation for consulting ser-vices from Alexion, Almirall, Biogen, Merck, Novartis, Roche, Sanofi, speaking activities from Bayer, Biogen, Celgene, Chiesi Italia SpA, Eli Lilly, Genzyme, Janssen, Merck-Serono, Neopharmed Gentili, Novartis, Novo Nordisk, Roche, Sanofi, Takeda, and TEVA, participation in Advisory Boards for Alexion, Biogen, Bristol-Myers Squibb, Merck, Novartis, Roche, Sanofi, Sanofi-Aventis, Sanofi-Genzyme, Takeda, scientific direction of educational events for Biogen, Merck, Roche, Celgene, Bristol-Myers Squibb, Lilly, Novartis, Sanofi-Genzyme, he receives research support from Biogen Idec, Merck-Serono, Novartis, Roche, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and ARiSLA (Fondazione Italiana di Ricerca per la SLA); F.E.: received consulting and speaking fees from Novartis, Sanofi Genzyme., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Inter-individual differences in treatment response are marked in multiple sclerosis (MS). This is true for Natalizumab (NTZ), to which a subset of patients displays sub-optimal treatment response. We conducted a multi-centric genome-wide association study (GWAS), with additional pathway and network analysis to identify genetic predictors of response to NTZ., Methods: MS patients from three different centers were included. Response to NTZ was dichotomized, nominating responders (R) relapse-free patients and non-responders (NR) all the others, over a follow-up of 4 years. Association analysis on ~ 4.7 M imputed autosomal common single-nucleotide polymorphisms (SNPs) was performed fitting logistic regression models, adjusted for baseline covariates, followed by meta-analysis at SNP and gene level. Finally, these signals were projected onto STRING interactome, to elicit modules and hub genes linked to response., Results: Overall, 1834 patients were included: 119 from Italy (R = 94, NR = 25), 81 from Germany (R = 61, NR = 20), and 1634 from Sweden (R = 1349, NR = 285). The top-associated variant was rs11132400 T (p = 1.33 × 10 -6 , OR = 0.58), affecting expression of several genes in the locus, like KLKB1. The interactome analysis implicated a module of 135 genes, with over-representation of terms like canonical WNT signaling pathway (p adjust  = 7.08 × 10 -6 ). Response-associated genes like GRB2 and LRP6, already implicated in MS pathogenesis, were topologically prioritized within the module., Conclusion: This GWAS, the largest pharmacogenomic study of response to NTZ, suggested MS-implicated genes and Wnt/β-catenin signaling pathway, an essential component for blood-brain barrier formation and maintenance, to be related to treatment response., Competing Interests: Declarations Competing interests F. Clarelli, A. Corona, K. Pääkkönen, M. Sorosina, A. Zollo, F. Piehl, T. Olsson, P. Stridh, M. Jagodic, B. Hemmer, C. Gasperi, A. Harroud, K. Shchetynsky, A. Mingione, E. Mascia, K. Misra, A. Giordano, M.L. Terzi Mazzieri, A. Priori, J. Saarela, I. Kockum, M. Filippi, F. Esposito, and F. Martinelli Boneschi have no competing interests to declare that are relevant to the topic of the present study. T. Olsson has received honoraria from Biogen, Merck, Novartis, and Sanofi for lectures/advisory Boards, and unrestricted MS research grants from the same companies. B. Hemmer has served on scientific advisory boards for Novartis; he has served as DMSC member for AllergyCare, Sandoz, Polpharma, Biocon, and TG therapeutics; his institution received research grants from Roche for multiple sclerosis research. He has received honoraria for counseling (Gerson Lehrmann Group). He holds part of two patents; one for the detection of antibodies against KIR4.1 in a subpopulation of patients with multiple sclerosis and one for genetic determinants of neutralizing antibodies to interferon. He is associated with DIFUTURE (Data Integration for Future Medicine) [BMBF 01ZZ1804[A-I]]. C. Gasperi received funding from the German Federal Ministry of Education and Research (BMBF—161L0216), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—GA 2913/3–1, project number 513308106), and the Hertie Foundation (P1200018). A. Harroud has received consultancy fees from Biogen and Pfizer, and received compensation for serving on a scientific advisor board for Amgen. A. Priori is the founder and the chair of the scientific advisory board of Newronika SpA, Milan Italy. M. Filippi is Editor-in-Chief of the Journal of Neurology, Associate Editor of Human Brain Mapping, Associate Editor of Radiology, and Associate Editor of Neurological Sciences. He received compensation for consulting services from Alexion, Almirall, Biogen, Merck, Novartis, Roche, and Sanofi; speaking activities from Bayer, Biogen, Celgene, Chiesi Italia SpA, Eli Lilly, Genzyme, Janssen, Merck-Serono, Neopharmed Gentili, Novartis, Novo Nordisk, Roche, Sanofi, Takeda, and TEVA; participation in Advisory Boards for Alexion, Biogen, Bristol-Myers Squibb, Merck, Novartis, Roche, Sanofi, Sanofi-Aventis, Sanofi-Genzyme, and Takeda; scientific direction of educational events for Biogen, Merck, Roche, Celgene, Bristol-Myers Squibb, Lilly, Novartis, and Sanofi-Genzyme, and he receives research support from Biogen Idec, Merck-Serono, Novartis, Roche, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and ARiSLA (Fondazione Italiana di Ricerca per la SLA). F. Esposito received consulting and speaking fees from Novartis and Sanofi Genzyme. F. Martinelli Boneschi has received compensation for consulting services and/or speaking activities from Teva Pharmaceutical Industries, Sanofi Genzyme, Merck-Serono, Biogen Idec, Roche, Medday, and Excemed, and received research support from Merck, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and Fondazione Cariplo., (© 2024. The Author(s).)

Objective: Multiple sclerosis (MS) has a complex pathobiology, with genetic and environmental factors being crucial players. Understanding the mechanisms underlying heterogeneity in disease activity is crucial for tailored treatment. We explored the impact of DNA methylation, a key mechanism in the genetics-environment interplay, on disease activity in MS., Methods: Peripheral immune methylome profiling using Illumina Infinium MethylationEPIC BeadChips was conducted on 249 untreated relapsing-remitting MS patients, sampled at the start of disease-modifying treatment (DMT). A differential methylation analysis compared patients with evidence of disease activity (EDA) to those with no evidence of disease activity (NEDA) over 2 years from DMT start. Utilizing causal inference testing (CIT) and Mendelian randomization (MR), we sought to elucidate the relationships between DNA methylation, gene expression, genetic variation, and disease activity., Results: Four differentially methylated regions (DMRs) were identified between EDA and NEDA. Examining the influence of single nucleotide polymorphisms (SNPs), 923 variants were found to account for the observed differences in the 4 DMRs. Importantly, 3 out of the 923 SNPs, affecting DNA methylation in a DMR linked to the anti-Mullerian hormone (AMH) gene, were associated with disease activity risk in an independent cohort of 1,408 MS patients. CIT and MR demonstrated that DNA methylation in AMH acts as a mediator for the genetic risk of disease activity., Interpretation: This study uncovered a novel molecular pathway implicating the interaction between DNA methylation and genetic variation in the risk of disease activity in MS, emphasizing the role of sex hormones, particularly the AMH, in MS pathobiology. ANN NEUROL 2024;96:289-301., (© 2024 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Small-Fiber Neuropathy (SFN) is a disorder of the peripheral nervous system, characterised by neuropathic pain; approximately 11% of cases are linked to variants in Voltage-Gated Sodium Channels (VGSCs). This study aims to broaden the genetic knowledge on painful SFN by applying Whole-Exome Sequencing (WES) in Early-Onset (EO) cases. A total of 88 patients from Italy (n = 52) and the Netherlands (n = 36), with a disease onset at age ≤ 45 years old and a Pain Numerical Rating Score ≥ 4, were recruited. After variant filtering and classification, WES analysis identified 142 potentially causative variants in 93 genes; 8 are Pathogenic, 15 are Likely Pathogenic, and 119 are Variants of Uncertain Significance. Notably, an enrichment of variants in transient receptor potential genes was observed, suggesting their role in pain modulation alongside VGSCs. A pathway analysis performed by comparing EO cases with 40 Italian healthy controls found enriched mutated genes in the "Nicotinic acetylcholine receptor signaling pathway". Targeting this pathway with non-opioid drugs could offer novel therapeutic avenues for painful SFN. Additionally, with this study we demonstrated that employing a gene panel of reported mutated genes could serve as an initial screening tool for SFN in genetic studies, enhancing clinical diagnostics.

Background and Objectives: The major histocompatibility complex (MHC) locus has a predominant role in the genetic predisposition to multiple sclerosis (MS), with 32 associations found to be involved. We aimed to investigate the impact of MHC MS-risk alleles on T-cell repertoire in patients with MS., Methods: We studied 161 untreated patients with relapsing-remitting MS for whom Class I and II human leukocyte antigen (HLA) alleles were inferred from whole-genome genotyping data, and T-cell receptor (TCR) CDR3 sequences were obtained through next-generation sequencing. T-cell repertoire features including diversity, public clones, and architecture were evaluated., Results: We identified 5 MS-risk loci associated with TCR diversity: HLA-DRB1*15:01 (7.65 × 10 -3 ), rs9271366 (1.96 × 10 -3 ), rs766848979 A (1.89 × 10 -2 ), rs9277626 (2.95 × 10 -2 ), and rs11751659 (1.92 × 10 -2 ), with evidence of expanded clonotypes in carriers of risk alleles. Moreover, HLA-DRB1*15:01 (4.99 × 10 -3 ), rs9271366 (6.54 × 10 -3 ), rs1049079 C (4.37 × 10 -2 ), AA DQΒ1 position -5 L (1.05 × 10 -3 ), and AA DQΒ1 position 221 Q (9.39 × 10 -4 ) showed an association with the CDR3 aminoacidic sequence architecture, suggesting an impact on the antigen recognition breadth as well. Evaluating the sharing of clones across MS-risk allele carrier individuals revealed the presence of highly shared clonotypes predicted to target viral antigens, including Epstein-Barr virus., Discussion: Our study supports the association between MHC-risk alleles and macrofeatures of the T-cell repertoire in the context of MS. Further studies are needed to understand the underlying molecular mechanisms., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

A personalized approach is strongly advocated for treatment selection in Multiple Sclerosis patients due to the high number of available drugs. Machine learning methods proved to be valuable tools in the context of precision medicine. In the present work, we applied machine learning methods to identify a combined clinical and genetic signature of response to fingolimod that could support the prediction of drug response. Two cohorts of fingolimod-treated patients from Italy and France were enrolled and divided into training, validation, and test set. Random forest training and robust feature selection were performed in the first two sets respectively, and the independent test set was used to evaluate model performance. A genetic-only model and a combined clinical-genetic model were obtained. Overall, 381 patients were classified according to the NEDA-3 criterion at 2 years; we identified a genetic model, including 123 SNPs, that was able to predict fingolimod response with an AUROC= 0.65 in the independent test set. When combining clinical data, the model accuracy increased to an AUROC= 0.71. Integrating clinical and genetic data by means of machine learning methods can help in the prediction of response to fingolimod, even though further studies are required to definitely extend this approach to clinical applications.

While the role of common genetic variants in multiple sclerosis (MS) has been elucidated in large genome-wide association studies, the contribution of rare variants to the disease remains unclear. Herein, a whole-genome sequencing study in four affected and four healthy relatives of a consanguineous Italian family identified a novel missense c.1801T > C (p.S601P) variant in the GRAMD1B gene that is shared within MS cases and resides under a linkage peak (LOD: 2.194). Sequencing GRAMD1B in 91 familial MS cases revealed two additional rare missense and two splice-site variants, two of which (rs755488531 and rs769527838) were not found in 1000 Italian healthy controls. Functional studies demonstrated that GRAMD1B , a gene with unknown function in the central nervous system (CNS), is expressed by several cell types, including astrocytes, microglia and neurons as well as by peripheral monocytes and macrophages. Notably, GRAMD1B was downregulated in vessel-associated astrocytes of active MS lesions in autopsied brains and by inflammatory stimuli in peripheral monocytes, suggesting a possible role in the modulation of inflammatory response and disease pathophysiology.

Multiple sclerosis (MS) is a complex disease of the central nervous system for which human leukocyte antigen (HLA) alleles are major contributors to susceptibility. Several investigations have focused on the relationship between HLA and clinical parameters, while few studies have evaluated its correlation with brain magnetic resonance imaging (MRI) measures. We investigated the association between the HLA genetic burden (HLAGB), originating from the most updated HLA alleles associated with MS, and neuroimaging endophenotypes, with a specific focus on brain atrophy metrics. A monocentric Italian cohort of 334 MS patients with imputed HLA alleles and cross-sectional volumetric measures of white matter (WM), gray matter (GM), hippocampus, thalamus and T2-hyperintense lesions was investigated. Linear regression models with covariate adjustment were fitted for each metric. We detected no effect of HLAGB on WM and GM volumes. Interestingly, we found a marginal correlation between higher HLAGB and lower hippocampal volume (β = -0.142, p = 0.063) and a nominal association between higher HLAGB and lower thalamic volume (β = -0.299, p = 0.047). No association was found with T2 lesion volumes. The putative impact of higher HLAGB on hippocampus and thalamus suggests, if replicated in independent cohorts, a possible cumulative contribution of HLA risk loci on brain volumetric traits linked to clinical deficits in MS.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disorder of the central nervous system (CNS). The migration of immune cells into the CNS is essential for its development, and plasma membrane molecules play an important role in triggering and maintaining the inflammation. We previously identified ninjurin2, a plasma membrane protein encoded by NINJ2 gene, as involved in the occurrence of relapse under Interferon-β treatment in MS patients. The aim of the present study was to investigate the involvement of NINJ2 in inflammatory conditions and in the migration of monocytes through the blood-brain barrier (BBB). We observed that NINJ2 is downregulated in monocytes and in THP-1 cells after stimulation with the pro-inflammatory cytokine LPS, while in hCMEC/D3 cells, which represent a surrogate of the BBB, LPS stimulation increases its expression. We set up a transmigration assay using an hCMEC/D3 transwell-based model, finding a higher transmigration rate of monocytes from MS subjects compared to healthy controls (HCs) in the case of an activated hCMEC/D3 monolayer. Moreover, a positive correlation between NINJ2 expression in monocytes and monocyte migration rate was observed. Overall, our results suggest that ninjurin2 could be involved in the transmigration of immune cells into the CNS in pro-inflammatory conditions. Further experiments are needed to elucidate the exact molecular mechanisms., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Background: Over 200 genetic loci have been associated with multiple sclerosis (MS) explaining ~ 50% of its heritability, suggesting that additional mechanisms may account for the "missing heritability" phenomenon., Objective: To analyze a large cohort of Italian individuals to identify markers associated with MS with potential functional impact in the disease., Methods: We studied 2571 MS and 3234 healthy controls (HC) of continental Italian origin. Discovery phase included a genome wide association study (1727 MS, 2258 HC), with SNPs selected according to their association in the Italian cohort only or in a meta-analysis of signals with a cohort of European ancestry (4088 MS, 7144 HC). Top associated loci were then tested in two Italian cohorts through array-based genotyping (903 MS, 884 HC) and pool-based target sequencing (588 MS, 408 HC). Finally, functional prioritization through conditional eQTL and mQTL has been performed., Results: Top associated signals overlap with already known MS loci on chromosomes 3 and 17. Three SNPs (rs4267364, rs8070463, rs67919208), all involved in the regulation of TBKBP1, were prioritized to be functionally relevant., Conclusions: No evidence of novel signal of association with MS specific for the Italian continental population has been found; nevertheless, two MS loci seems to play a relevant role, raising the interest to further investigations for TBKBP1 gene., (© 2022. The Author(s).)

Background: Rehabilitation is fundamental for progressive multiple sclerosis (MS), but predictive biomarkers of motor recovery are lacking, making patient selection difficult. Motor recovery depends on synaptic plasticity, in which the Brain-Derived Neurotrophic Factor (BDNF) is a key player, through its binding to the Neurotrophic-Tyrosine Kinase-2 (NTRK2) receptor. Therefore, genetic polymorphisms in the BDNF pathway may impact motor recovery. The most well-known polymorphism in BDNF gene (rs6265) causes valine to methionine substitution (Val66Met) and it influences memory and motor learning in healthy individuals and neurodegenerative diseases. To date, no studies have explored whether polymorphisms in BDNF or NTRK2 genes may impact motor recovery in MS., Objectives: To assess whether genetic variants in BDNF and NTRK2 genes affect motor recovery after rehabilitation in progressive MS., Methods: The association between motor recovery after intensive neurorehabilitation and polymorphisms in BDNF (rs6265) and NTKR2 receptor (rs2289656 and rs1212171) was assessed using Six-Minutes-Walking-Test (6MWT), 10-Metres-Test (10MT) and Nine-Hole-Peg-Test (9HPT) in 100 progressive MS patients., Results: We observed greater improvement at 6MWT after rehabilitation in carriers of the BDNF Val66Met substitution, compared to BDNF Val homozygotes ( p = 0.024). No significant association was found for 10MT and 9HPT. NTRK2 polymorphisms did not affect the results of motor function tests., Conclusion: BDNF Val66Met was associated with walking function improvement after rehabilitation in progressive MS patients. This result is in line with previous evidence showing a protective effect of Val66Met substitution on brain atrophy in MS. Larger studies are needed to explore its potential as a predictive biomarker of rehabilitation outcome., Competing Interests: LL reports personal fees for speaking or consulting activities from Roche, Merck, Bristol Myers Squibb, Med-ex learning, outside the submitted work. FE received compensation for consulting services and/or speaking activities from Novartis, Sanofi Genzyme, Almirall, Teva, and Merck-Serono. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giordano, Clarelli, Cannizzaro, Mascia, Santoro, Sorosina, Ferrè, Leocani and Esposito.)

Aim: To investigate the transcriptional changes induced by Fingolimod (FTY) in T cells of relapsing remitting multiple sclerosis patients. Patients & methods: Transcriptomic changes after 6 months of FTY therapy were evaluated on T cells from 24 relapsing remitting multiple sclerosis patients through RNA-sequencing, followed by technical validation and pathway analysis. Results: Among differentially expressed genes, CX3CR1 and CCR7 resulted strongly up- and downregulated, respectively. Two relevant genes were validated with quantitative PCR and we largely confirmed findings from two previous microarray-based studies with similar design. Pathway analysis pointed to an involvement of processes related to immune function and cell migration. Conclusion: Our data support the evidence that FTY induces major transcriptional changes in genes involved in immune response and cell trafficking in T lymphocytes.

Genome-wide association studies identified over 200 risk loci for multiple sclerosis (MS) focusing on common variants, which account for about 50% of disease heritability. The goal of this study was to investigate whether low-frequency and rare functional variants, located in MS-established associated loci, may contribute to disease risk in a relatively homogeneous population, testing their cumulative effect (burden) with gene-wise tests. We sequenced 98 genes in 588 Italian patients with MS and 408 matched healthy controls (HCs). Variants were selected using different filtering criteria based on allelic frequency and in silico functional impacts. Genes showing a significant burden (n = 17) were sequenced in an independent cohort of 504 MS and 504 HC. The highest signal in both cohorts was observed for the disruptive variants (stop-gain, stop-loss, or splicing variants) located in EFCAB13 , a gene coding for a protein of an unknown function ( p < 10 -4 ). Among these variants, the minor allele of a stop-gain variant showed a significantly higher frequency in MS versus HC in both sequenced cohorts ( p = 0.0093 and p = 0.025), confirmed by a meta-analysis on a third independent cohort of 1298 MS and 1430 HC ( p = 0.001) assayed with an SNP array. Real-time PCR on 14 heterozygous individuals for this variant did not evidence the presence of the stop-gain allele, suggesting a transcript degradation by non-sense mediated decay, supported by the evidence that the carriers of the stop-gain variant had a lower expression of this gene ( p = 0.0184). In conclusion, we identified a novel low-frequency functional variant associated with MS susceptibility, suggesting the possible role of rare/low-frequency variants in MS as reported for other complex diseases., Competing Interests: MF is Editor-in-Chief of the Journal of Neurology, Associate Editor of Human Brain Mapping, Associate Editor of Radiology, and Associate Editor of Neurological Sciences; received compensation for consulting services and/or speaking activities from Alexion, Almirall, Bayer, Biogen, Celgene, Eli Lilly, Genzyme, Merck-Serono, Novartis, Roche, Sanofi, Takeda, and Teva Pharmaceutical Industries; and receives research support from Biogen Idec, Merck-Serono, Novartis, Roche, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, and ARiSLA (Fondazione Italiana di Ricerca per la SLA). VM received compensation for speaking and/or for consultancy and support for travel expenses and participation in Congresses from Biogen, Merck-Serono, Novartis, Roche, Genzyme and Teva Pharmaceutical Industries. FMB has received compensation for consulting services and/or speaking activities from Teva Pharmaceutical Industries, Sanofi Genzyme, Merck-Serono, Biogen Idec, Roche, Medday, Excemed, and received research support from Merck, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla and Fondazione Cariplo. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clarelli, Barizzone, Mangano, Zuccalà, Basagni, Anand, Sorosina, Mascia, Santoro, Guerini, Virgilio, Gallo, Pizzino, Comi, Martinelli, Comi, De Bellis, Leone, Filippi, Esposito, Bordoni, Martinelli Boneschi and D'Alfonso.)

Objectives: Antitumour necrosis factor (TNF) therapy has revolutionised treatment of several chronic inflammatory diseases, including spondyloarthritis (SpA). However, TNF inhibitors (TNFi) are not effective in all patients and the biological basis for treatment failure remains unknown. We have analysed induced immune responses to define the mechanism of action of TNF blockers in SpA and to identify immunological correlates of responsiveness to TNFi., Methods: Immune responses to microbial and pathway-specific stimuli were analysed in peripheral blood samples from 80 patients with axial SpA before and after TNFi treatment, using highly standardised whole-blood stimulation assays. Cytokines and chemokines were measured in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory, and gene expression was monitored using nCounter assays., Results: Anti-TNF therapy induced profound changes in patients' innate immune responses. TNFi action was selective, and had only minor effects on Th1/Th17 immunity. Modular transcriptional repertoire analysis identified prostaglandin E 2 synthesis and signalling, leucocyte recirculation, macrophage polarisation, dectin and interleukin (IL)-1 signalling, as well as the nuclear factor kappa B (NF-kB) transcription factor family as key pathways targeted by TNF blockers in vivo. Analysis of induced immune responses before treatment initiation revealed that expression of molecules associated with leucocyte adhesion and invasion, chemotaxis and IL-1 signalling are correlated with therapeutic responses to anti-TNF., Conclusions: We show that TNFi target multiple immune cell pathways that cooperate to resolve inflammation. We propose that immune response profiling provides new insight into the biology of TNF-blocker action in patients and can identify signalling pathways associated with therapeutic responses to biological therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)