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

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. 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. 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.

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.

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.

Background Sex-related differences affect multiple sclerosis (MS), but the impact of sex hormones on disease progression remains unclear. We investigated whether sex hormone-related factors influence progression independent of relapse activity (PIRA) in women with MS over a long-term follow-up. Methods The study analysed 1210 female MS patients from the San Raffaele MS Center using data from an environmental survey (2019–2023). PIRA was defined as 12-week confirmed disability progression independent of recent relapses (<30 days). Cox proportional-hazard models (adjusted for confounding factors) were used to assess the effect of hormone-related factors on PIRA risk. Results Patients who used oral contraceptives before MS diagnosis had a 26% lower risk of PIRA and a delayed median time to the first PIRA event (9.94 vs 7.5 years; HR=0.74; 95% CI 0.61 to 0.89; p=0.0018). Conversely, menopause at diagnosis (HR=1.82; 95% CI 1.24 to 2.67; p=0.0022) and pregnancy before diagnosis (HR=1.22; 95% CI 1.006 to 1.47; p=0.043) were associated with a shorter time to PIRA. No significant differences were found with abortion, menstrual irregularity or fertility therapy. Conclusions This study suggests that early oral contraceptives may delay future disability progression, supporting the importance of sex hormones in MS and prompting further prospective investigations on oral contraceptives to slow disease progression.

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.