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Multiple sclerosis (MS) is a chronic autoimmune disease with huge heterogeneity in terms of clinical course, disease severity and treatment response. The need for a tailored treatment approach has emerged over the last few years. The present observational study aims to assess fingolimod (FTY) effectiveness in RRMS patients, stratifying them according to the disease-modifying treatments used before FTY, to identify subjects who could benefit more from this treatment.We prospectively included 554 RRMS patients who started FTY at San Raffaele Hospital between 2012 and 2018. We classified them into three categories according to previous treatments: naïve patients, subjects previously treated with first-line drugs, and patients previously treated with second-line drugs. We compared disease activity during a 2-years follow-up using No-Evidence-of-Disease-Activity (NEDA-3) and Time-To-First-Relapse (TTFR) outcomes, applying logistic and Cox proportional hazard regression respectively.The proportion of patients who maintained NEDA-3 status was higher in the naïve group despite a higher level of baseline disease activity (naïve versus first-line p = 0.025, naïve versus second-line p 0.001). In the multivariable analyses, patients switching to FTY from first- and second-line treatments showed a higher risk of disease reactivation (p = 0.041, OR = 1.86 and p = 0.002, OR = 2.92, respectively) and a shorter TTFR (p = 0.017, HR = 4.35 and p = 0.001, HR = 8.19, respectively).Naïve patients showed a better response to FTY compared to patients switching to FTY from other drugs. Our findings support the early use of FTY in patients with active MS.

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.

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.

Fingolimod (FTY), a second-line oral drug approved for relapsing remitting Multiple Sclerosis (RRMS) acts in preventing lymphocyte migration outside lymph nodes; moreover, several lines of evidence suggest that it also inhibits myeloid cell activation. In this study, we investigated the transcriptional changes induced by FTY in monocytes in order to better elucidate its mechanism of action. CD14+ monocytes were collected from 24 RRMS patients sampled at baseline and after 6 months of treatment and RNA profiles were obtained through next-generation sequencing. We conducted pathway and sub-paths analysis, followed by centrality analysis of cell-specific interactomes on differentially expressed genes (DEGs). We investigated also the predictive role of baseline monocyte transcription profile in influencing the response to FTY therapy. We observed a marked down-regulation effect (60 down-regulated vs. 0 up-regulated genes). Most of the down-regulated DEGs resulted related with monocyte activation and migration like IL7R, CCR7 and the Wnt signaling mediators LEF1 and TCF7. The involvement of Wnt signaling was also confirmed by subpaths analyses. Furthermore, pathway and network analyses showed an involvement of processes related to immune function and cell migration. Baseline transcriptional profile of the HLA class II gene HLA-DQA1 and HLA-DPA1 were associated with evidence of disease activity after 2 years of treatment. Our data support the evidence that FTY induces major transcriptional changes in monocytes, mainly regarding genes involved in cell trafficking and immune cell activation. The baseline transcriptional levels of genes associated with antigen presenting function were associated with disease activity after 2 years of FTY treatment.

BACKGROUND AND PURPOSE Multiple sclerosis (MS) susceptibility is influenced by genetics; however, little is known about genetic determinants of disease expression. We aimed at assessing genetic factors influencing quantitative neuroimaging measures in two cohorts of progressive MS (PMS) and relapsing-remitting MS (RRMS) patients. METHODS Ninety-nine PMS and 214 RRMS patients underwent a 3-T brain magnetic resonance imaging (MRI) scan, with the measurement of five MRI metrics including T2 lesion volumes and measures of white matter, grey matter, deep grey matter, and hippocampal volumes. A candidate pathway strategy was adopted; gene set analysis was carried out to estimate cumulative contribution of genes to MRI phenotypes, adjusting for relevant confounders, followed by single nucleotide polymorphism (SNP) regression analysis. RESULTS Seventeen Kyoto Encyclopedia of Genes and Genomes pathways and 42 Gene Ontology (GO) terms were tested. We additionally included in the analysis genes with enriched expression in brain cells. Gene set analysis revealed a differential pattern of association across the two cohorts, with processes related to sodium homeostasis being associated with grey matter volume in PMS (p = 0.002), whereas inflammatory-related GO terms such as adaptive immune response and regulation of inflammatory response appeared to be associated with T2 lesion volume in RRMS (p = 0.004 and p = 0.008, respectively). As for SNPs, the rs7104613T mapping to SPON1 gene was associated with reduced deep grey matter volume (β = -0.731, p = 3.2*10-7 ) in PMS, whereas we found evidence of association between white matter volume and rs740948A mapping to SEMA3A gene (β = 22.04, p = 5.5*10-6 ) in RRMS. CONCLUSIONS Our data suggest a different pattern of associations between MRI metrics and functional processes across MS disease courses, suggesting different phenomena implicated in MS.

Background and ObjectivesThe 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.MethodsWe 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.ResultsWe 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.DiscussionOur 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.

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.To analyze a large cohort of Italian individuals to identify markers associated with MS with potential functional impact in the disease.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.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.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.

Known multiple sclerosis (MS) susceptibility variants can only explain half of the disease’s estimated heritability, whereas low-frequency and rare variants may partly account for the missing heritability. Thus, here we sought to determine the occurrence of rare functional variants in a large Italian MS multiplex family with five affected members. For this purpose, we combined linkage analysis and next-generation sequencing (NGS)-based whole exome and whole genome sequencing (WES and WGS, respectively). The genetic burden attributable to known common MS variants was also assessed by weighted genetic risk score (wGRS). We found a significantly higher burden of common variants in the affected family members compared to that observed among sporadic MS patients and healthy controls (HCs). We also identified 34 genes containing at least one low-frequency functional variant shared among all affected family members, showing a significant enrichment in genes involved in specific biological processes—particularly mRNA transport—or neurodegenerative diseases. Altogether, our findings point to a possible pathogenic role of different low-frequency functional MS variants belonging to shared pathways. We propose that these rare variants, together with other known common MS variants, may account for the high number of affected family members within this MS multiplex family.

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.

Multiple Sclerosis (MS) is caused by a still unknown interplay between genetic and environmental factors. Epigenetics, including DNA methylation, represents a model for environmental factors to influence MS risk. Twenty-six affected and 26 unaffected relatives from 8 MS multiplex families were analysed in a multicentric Italian study using MeDIP-Seq, followed by technical validation and biological replication in two additional families of differentially methylated regions (DMRs) using SeqCap Epi Choice Enrichment kit (Roche®). Associations from MeDIP-Seq across families were combined with aggregation statistics, yielding 162 DMRs at FDR ≤ 0.1. Technical validation and biological replication led to 2 hypo-methylated regions, which point to NTM and BAI3 genes, and to 2 hyper-methylated regions in PIK3R1 and CAPN13. These 4 novel regions contain genes of potential interest that need to be tested in larger cohorts of patients.

Background: Fingolimod (FTY) is an effective second-line drug for relapsing-remitting multiple sclerosis, with ~50% patients showing no evidence of disease activity (NEDA) after 2 years. Nonetheless, the early identification of non-responders is extremely important, to promptly address them to more aggressive drugs. Objectives: This cohort study evaluates FTY medium-term effectiveness, searching for early markers of treatment failure. Patients and methods: Three hundred eighty patients starting FTY were enrolled and classified according to NEDA and time to first relapse criteria at 4-year follow-up. Logistic and Cox regression analyses were applied to identify early predictors of non-response. Results: At 4 years, 65.6% of patients were free from relapses and 35.4% had NEDA. Female gender was associated with a higher risk of non-response. Moreover, evidence of clinical and/or magnetic resonance imaging (MRI) activity during the first year of treatment was highly predictive of disease activity in the follow-up: the positive predictive value for non-response was 0.74 for the presence of ⩾1 relapse, 0.73 for the presence of ⩾1 active MRI lesion, and 0.83 for the presence of both clinical and MRI activity. Conclusions: FTY effectiveness persists at medium-term follow-up; a close monitoring during the first year of treatment is warranted to early identify non-responders requiring treatment optimization.

Among multiple sclerosis (MS) susceptibility genes, the strongest non-human leukocyte antigen (HLA) signal in the Italian population maps to the TNFSF14 gene encoding LIGHT, a glycoprotein involved in dendritic cell (DC) maturation. Through fine-mapping in a large Italian dataset (4,198 patients with MS and 3,903 controls), we show that the TNFSF14 intronic SNP rs1077667 is the primarily MS-associated variant in the region. Expression quantitative trait locus (eQTL) analysis indicates that the MS risk allele is significantly associated with reduced TNFSF14 messenger RNA levels in blood cells, which is consistent with the allelic imbalance in RNA-Seq reads (P < 0.0001). The MS risk allele is associated with reduced levels of TNFSF14 gene expression (P < 0.01) in blood cells from 84 Italian patients with MS and 80 healthy controls (HCs). Interestingly, patients with MS are lower expressors of TNFSF14 compared to HC (P < 0.007). Individuals homozygous for the MS risk allele display an increased percentage of LIGHT-positive peripheral blood myeloid DCs (CD11c+, P = 0.035) in 37 HCs, as well as in in vitro monocyte-derived DCs from 22 HCs (P = 0.04). Our findings suggest that the intronic variant rs1077667 alters the expression of TNFSF14 in immune cells, which may play a role in MS pathogenesis.

[Background] Multiple sclerosis (MS) is a disease in which biomarker identification is fundamental to predict response to treatments and to deliver the optimal drug to patients. We previously found an association between rs7298096, a polymorphism upstream to the NINJ2 gene, and the 4-year response to interferon-β (IFNβ) treatment in MS patients., [Objectives] To analyse the association between rs7298096 and time to first relapse (TTFR) during IFNβ therapy in MS patients and to better investigate its functional role. [Methods] Survival analysis was applied in three MS cohorts from different countries (n = 1004). We also studied the role of the polymorphism on gene expression using GTEx portal and a luciferase assay. We interrogated GEO datasets to explore the relationship between NINJ2 expression, IFNβ and TTFR. [Results] Rs7298096AA patients show a shorter TTFR than rs7298096G-carriers (Pmeta-analysis = 3 × 10−4, hazard ratio = 1.41). Moreover, rs7298096AA is associated with a higher NINJ2 expression in blood (p = 7.0 × 10−6), which was confirmed in vitro (p = 0.009). Finally, NINJ2 expression is downregulated by IFNβ treatment and related to TTFR. [Conclusions] Rs7298096 could influence MS disease activity during IFNβ treatment by modulating NINJ2 expression in blood. The gene encodes for an adhesion molecule involved in inflammation and endothelial cells activation, supporting its role in MS.

Background The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data. Results The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis. Conclusions The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples. Electronic supplementary material The online version of this article (10.1186/s12867-018-0108-5) contains supplementary material, which is available to authorized users.

Background: Several studies have shown an association between 25-hydroxyvitamin D (25[OH]D) levels and multiple sclerosis (MS) susceptibility and/or level of disease activity in patients treated with first line drugs. Aims: To investigate whether baseline 25[OH]D values could influence disease activity also during treatment with the second-line drug fingolimod (FTY). Patients and methods: We enrolled 176 MS patients who started FTY at the San Raffaele Hospital (OSR) MS center with available 25[OH]D measurement at the time of treatment start. We then prospectively followed them for 2 years with periodic clinical examinations and MRI scans. Results: We found no linear correlation between baseline 25[OH]D levels and annualized relapse rate (ARR) or time to first relapse. However, we observed that patients with serum 25[OH]D ≥ 100 nmol/l showed a lower number of Gd+ and combined unique activity (CUA) lesions at baseline compared to patients with the lowest 25[OH]D levels (less than 50 nmol/l, p value < 0.05). Moreover, they showed fewer CUA lesions at 2-year follow-up also when accounting for baseline level of disease activity (p value < 0.05). Conclusions: In patients treated with FTY, those with the highest baseline 25(OH)D levels had a significantly lower number of active lesions at baseline; the same effect, even if weaker, was observed also at 2-year follow-up when adjusting for baseline disease activity. Given Vitamin D supplementation safety profile, also if a causal effect has not yet been shown, most of MS patients could probably benefit from 25[OH]D levels above those currently considered to be sufficient.

We aimed to investigate whether NLR family, pyrin domain containing 3 (NLRP3) polymorphisms are associated with the response to interferon-beta (IFNβ) in multiple sclerosis (MS) patients. A total of 14 NLRP3 polymorphisms were genotyped in a cohort of 665 relapsing-remitting MS patients recruited across 5 centers and classified into responders and non-responders according to clinical-radiological criteria after 1 year of IFNβ treatment. A meta-analysis failed to demonstrate significant associations between the response to IFNβ and NLRP3 polymorphisms. These findings do not support a role of polymorphisms located in the NLRP3 gene and the response to IFNβ in MS patients.

Sequencing large number of individuals, which is often needed for population genetics studies, is still economically challenging despite falling costs of Next Generation Sequencing (NGS). Pool-seq is an alternative cost- and time-effective option in which DNA from several individuals is pooled for sequencing. However, pooling of DNA creates new problems and challenges for accurate variant call and allele frequency (AF) estimation. In particular, sequencing errors confound with the alleles present at low frequency in the pools possibly giving rise to false positive variants. We sequenced 996 individuals in 83 pools (12 individuals/pool) in a targeted re-sequencing experiment. We show that Pool-seq AFs are robust and reliable by comparing them with public variant databases and in-house SNP-genotyping data of individual subjects of pools. Furthermore, we propose a simple filtering guideline for the removal of spurious variants based on the Kolmogorov-Smirnov statistical test. We experimentally validated our filters by comparing Pool-seq to individual sequencing data showing that the filters remove most of the false variants while retaining majority of true variants. The proposed guideline is fairly generic in nature and could be easily applied in other Pool-seq experiments.

Multiple sclerosis (MS) is a disease in which biomarker identification is fundamental to predict response to treatments and to deliver the optimal drug to patients. We previously found an association between rs7298096, a polymorphism upstream to theTo analyse the association between rs7298096 and time to first relapse (TTFR) during IFNβ therapy in MS patients and to better investigate its functional role.Survival analysis was applied in three MS cohorts from different countries (Rs7298096Rs7298096 could influence MS disease activity during IFNβ treatment by modulating

Up to 50% of multiple sclerosis (MS) patients do not respond to interferon-beta (IFN-β) treatment and determination of response requires lengthy clinical follow-up of up to 2 years. Response predictive genetic markers would significantly improve disease management. We aimed to identify IFN-β treatment response genetic marker(s) by performing a two-stage genome-wide association study (GWAS). The GWAS was carried out using data from 151 Australian MS patients from the ANZgene/WTCCC2 MS susceptibility GWAS (responder (R)=51, intermediate responders=24 and non-responders (NR)=76). Of the single-nucleotide polymorphisms (SNP) that were validated in an independent group of 479 IFN-β-treated MS patients from Australia, Spain and Italy (R=273 and NR=206), eight showed evidence of association with treatment response. Among the replicated associations, the strongest was observed for FHIT (Fragile Histidine Triad; combined P-value 6.74 × 10-6) and followed by variants in GAPVD1 (GTPase activating protein and VPS9 domains 1; combined P-value 5.83 × 10-5) and near ZNF697 (combined P-value 8.15 × 10-5). Refereed/Peer-reviewed

Background: Treatment choice in multiple sclerosis (MS) is crucial for optimizing risk–benefit profile. Objective: To assess fingolimod (FTY) effectiveness and identify baseline features associated to disease activity in a large Italian cohort of Relapsing–Remitting (RR) MS patients. Methods: Three-hundred sixty-seven RRMS patients starting FTY treatment at San Raffaele Hospital (Milan-Italy) underwent clinical and MRI evaluations for 2 years. Treatment response was assessed considering the proportion of patients with no evidence of disease activity (NEDA) and recording the time to first relapse. Primary analyses were performed stratifying for Natalizumab (NTZ) treatment in the year before (NO_NTZ vs NTZ group), to account for post-NTZ reactivation. Results: Almost half of patients were NEDA after 2 years, 53.4% in the NO_NTZ group and 36.2% in the NTZ group. Despite an opposite trend during the first 6–12 months, at 2-year follow-up the two groups were comparable for relapses and number of new/enlarging T2 and Gd-enhancing lesions. Baseline parameters of higher disease activity (ARR, Gd enhancing lesions and age at onset) were associated with increased likelihood of failing NEDA criteria or with shorter time to relapse (p < 0.05). Conclusions: Our data strengthen FTY effectiveness in everyday clinical practice, even in patients switching from NTZ treatment. Baseline parameters of inflammatory activity are the most important prognostic factors for mid-term disease reactivation also during second-line treatment with FTY, providing hints on how to select therapies towards a more personalized management.

We assembled and analyzed genetic data of 47,351 multiple sclerosis (MS) subjects and 68,284 control subjects and establish a reference map of the genetic architecture of MS that includes 200 autosomal susceptibility variants outside the major histocompatibility complex (MHC), one chromosome X variant, and 32 independent associations within the extended MHC. We used an ensemble of methods to prioritize up to 551 potentially associated MS susceptibility genes, that implicate multiple innate and adaptive pathways distributed across the cellular components of the immune system. Using expression profiles from purified human microglia, we do find enrichment for MS genes in these brain - resident immune cells. Thus, while MS is most likely initially triggered by perturbation of peripheral immune responses the functional responses of microglia and other brain cells are also altered and may have a role in targeting an autoimmune process to the central nervous system.One Sentence Summary:We report a detailed genetic and genomic map of multiple sclerosis, and describe the role of putatively affected genes in the peripheral immune system and brain resident microglia.

The aim of the present study is to investigate the molecular pathways underlying amyotrophic lateral sclerosis (ALS) pathogenesis within the peripheral nervous system. We analyzed gene expression changes in human motor nerve diagnostic biopsies obtained from eight ALS patients and seven patients affected by motor neuropathy as controls. An integrated transcriptomics and system biology approach was employed. We identified alterations in the expression of 815 genes, with 529 up-regulated and 286 down-regulated in ALS patients. Up-regulated genes clustered around biological process involving RNA processing and protein metabolisms. We observed a significant enrichment of up-regulated small nucleolar RNA transcripts (p = 2.68*10-11) and genes related to endoplasmic reticulum unfolded protein response and chaperone activity. We found a significant down-regulation in ALS of genes related to the glutamate metabolism. Interestingly, a network analysis highlighted HDAC2, belonging to the histone deacetylase family, as the most interacting node. While so far gene expression studies in human ALS have been performed in postmortem tissues, here specimens were obtained from biopsy at an early phase of the disease, making these results new in the field of ALS research and therefore appealing for gene discovery studies.

Previous studies suggest that genetic variants in CHRNA7, which encodes for the major subunit of the acetylcholine receptor (α7-nAChR), are associated with the clinical response to cholinesterase inhibitors (ChEI) in Alzheimer's disease (AD) patients. We sought to replicate the association of two SNPs in the CHRNA7 gene, rs6494223 and rs8024987, with response to ChEI treatment in an Italian cohort of 169 AD patients, further extending the study to gene-level analysis. None of the tested variants was associated with clinical response. However, rs6494223 showed a consistent effect direction (OR = 1.4; p = 0.17), which after meta-analysis with previous study yielded a significant result (OR = 1.57, p = 0.02, I2 = 0%).

Background Nabiximols (Sativex®) is a cannabinoid-based compound used for the treatment of moderate to severe spasticity in multiple sclerosis (MS). The aim of the study is to investigate the effect of the administration of Nabiximols on blood transcriptome profile of MS patients and to interpret it in the context of pathways and networks. Methods Whole-genome expression profiling was performed in whole blood of 33 MS subjects at baseline (B) and after 4 weeks (4W) of drug treatment. Patients were classified in responders (n=19) and non-responders (n=14). Pathway and network analyses on genes modulated by the drug were performed, followed by in vitro stimulations of peripheral blood mononuclear cells (PBMCs) with pro-inflammatory agents, to support the immunomodulatory properties of the drug. Results Individual effect size was modest, however, we observed a downregulation of several immune-related pathways after 4 weeks of treatment which was more pronounced when restricting analyses to responders. Interesting hub molecules functionally related to the immune system emerged from network analysis, including NFKB1, FYN, MAP14 and TP53. The immunomodulatory properties of the drug were confirmed through in vitro assays in PBMCs collected from MS patients. Conclusions Our findings support the immunomodulatory activity of cannabinoids in MS patients. Further studies in more specific cell types are needed to refine these results. This article is protected by copyright. All rights reserved.

The aim of the study is the identification of genetic factors that influence the long-term response to interferon-β (IFNβ) (4-year follow-up). We performed a genome-wide association study in 337 IFNβ-treated Italian multiple sclerosis patients at the extreme of treatment response, and we meta-analyzed association effects, integrating results with pathway analysis, gene-expression profiling of IFNβ-stimulated peripheral blood mononuclear cells from 20 healthy controls (HC) and expression quantitative locus (eQTL) analyses. From meta-analysis, 43 markers were associated at P

Background: The role of genetic factors in influencing the clinical expression of multiple sclerosis (MS) is unclear. Objective: The objective of this paper is to identify genes, pathways and networks implicated in age at onset (AAO) and severity, measured using the Multiple Sclerosis Severity Score (MSSS), of primary-progressive MS (PPMS). Methods: We conducted a genome-wide association study (GWAS) of 470 PPMS patients of Italian origin:. Allelic association of 296,589 SNPs with AAO and MSSS was calculated. Pathway and network analyses were also conducted using different tools. Results: No single association signal exceeded genome-wide significance in AAO and MSSS analyses. Nominally associated genes to AAO and MSSS were enriched in both traits for 10 pathways, including: “oxidative phosphorylation” (FDRAAO=9*10−4; FDRMSSS=3.0*10−2), “citrate (TCA) cycle” (FDRAAO=1.6*10−2; FDRMSSS=3.2*10−3), and “B cell receptor signaling” (FDRAAO=3.1*10−2; FDRMSSS=2.2*10−3). In addition, an enrichment of “chemokine signaling pathway” (FDR=9*10−4) for AAO and of “leukocyte transendothelial migration” (FDR=2.4*10−3) for MSSS trait was observed, among others. Network analysis revealed that p53 and CREB1 were central hubs for AAO and MSSS traits, respectively. Conclusions: Despite the fact that no major effect signals emerged in the present GWAS, our data suggest that genetic variants acting in the context of oxidative stress and immune dysfunction could modulate the onset and severity of PPMS.

Objectives: To identify clinical predictors of effectiveness of a motor rehabilitation treatment in a cohort of multiple sclerosis (MS) patients. Materials and methods: We analysed 212 consecutive patients who underwent a short-term (3–7 weeks) intensive (two hours per day, five days per week), individualised, goal-oriented inpatient rehabilitation program. Activity limitation and impairment were measured on admission and discharge of the rehabilitation trial using the motor sub-items of the Functional Independence Measure (mFIM) and the Expanded Disability Status Scale (EDSS) score. Multivariate logistic regression models have been tested to evaluate the role of clinical baseline features on rehabilitation effectiveness. Results: According to pre-defined outcome measures, 75.1% of MS patients improved in either activity limitation (≥5 points delta mFIM) or impairment (≥1.0 delta EDSS score if baseline EDSS was ≤5.5, or ≥0.5 if baseline EDSS was >5.5), and 35.4% of MS patients improved in both outcomes. A relapsing-remitting course of disease, a more severe baseline impairment and activity limitation level, a shorter disease duration and a less severe balance dysfunction were predictive of the effectiveness of rehabilitation. Discussion: These data confirm that an intensive inpatient rehabilitation program is able to produce a short-term relevant improvement on clinical and functional outcome measures and suggest some clinical features which can be considered as potential predictors of the outcome of rehabilitative intervention.

In this study, we investigated the role of the dipeptidyl-peptidase-6 (DPP6) gene in the etiopathogenesis of progressive forms of multiple sclerosis (PrMS). This gene emerged as a candidate gene in a genome-wide association study (GWAS) performed in an Italian sample of PrMS and controls in which two SNPs located in the gene (rs6956703 and rs11767658) showed evidence of association (nominal p-value10(-4)) (Martinelli-Boneschi et al.) [18]. Moreover, the gene is highly expressed in the central nervous system, and it has been found to be associated with sporadic cases of amyotrophic lateral sclerosis which shares some feature with PrMS.We genotyped 19 SNPs selected using a direct and tagging approach in 244 Italian PrMS and 225 controls, and we measured the expression levels of the gene in 13 PrMS cases and 25 controls.Five out of 19 SNPs were found to be associated with the disease (adjusted p0.05), and they have been tested in an independent sample of 179 primary progressive MS and 198 controls from Northern Europe. None of the SNPs was replicated, but combined analysis confirmed the presence of association for rs2046748 (p=2.5×10(-3),OR=1.82, 95%CI=1.24-2.69).These results, inflated by the limited sample size determined by the rarity of this condition, suggest a possible role of this gene in the susceptibility to PrMS, at least in Southern Europeans. Moreover, DPP6 was over-expressed in PrMS patients compared to controls.

Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3.5) pipeline to process Affymetrix 3′ IVT GeneChips. The availability of newer technologies that demand open-source tools for microarray data analysis has impelled us to develop an updated multi-platform version, AMDA 2.13. It includes additional quality control metrics, annotation-driven (annotation grade of Affymetrix NetAffx) and signal-driven (Inter-Quartile Range) gene filtering, and approaches to experimental design. To enhance understanding of biological data, differentially expressed genes have been mapped into KEGG pathways. Finally, a more stable and user-friendly interface was designed to integrate the requirements for different platforms. AMDA 2.13 allows the analysis of Affymetrix (cartridges and plates) and whole transcript probe design (Gene 1.0/1.1 ST and Exon 1.0 ST GeneChips), Illumina Bead Arrays, and one-channel Agilent 4×44 arrays. Relative to early versions, it supports various experimental designs and delivers more insightful biological understanding and up-to-date annotations.

Background: The genetic component of multiple sclerosis (MS) is now set to 200 autosomal common variants. However, it is unclear how genetic knowledge be clinically used in the differential diagnosis between MS and other inflammatory conditions like adult-onset postinfectious neurological syndromes (PINS). The aim of this study was to investigate whether PINS and MS have a shared genetic background using an updated polygenic risk scores. Methods: Eighty-eight PINS patients have been consecutively recruited between 1996 and 2016 at Mondino Foundation of Pavia, diagnosed according to clinical, MRI and CSF findings and followed-up for several years. Patients were typed using Illumina array, and genotypes imputed using the 1000 Genomes Project reference panel. A weighted genetic risk score (wGRS) has been calculated based on autosomal MS risk loci derived from large-scale studies, and an HLA genetic burden (HLAGB) was also calculated on loci associated to MS. Results: PINS occurred as an episode of myelitis in 44% of patients, encephalomyelitis in 44%, and encephalitis in remaining cases, with an involvement of peripheral nervous system in 41% of patients. Mean age of onset was 50.1 years, and female:male ratio was 1.4. Patients were followed-up for a mean of 7.2 years, and at last visit 55% had a low disability grade (mRS 0–1). Disease was monophasic in 67% of patients, relapsing in 18% and chronic-progressive in 15%. The wGRS of PINS cases was comparable to 370 healthy controls, while significantly lower compared to 907 bout-onset MS (BOMS) cases (wGRS= 20.9 vs 21.2; p