DNA Methylation in the Anti-Mullerian Hormone Gene and the Risk of Disease Activity in Multiple Sclerosis.

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.

Vitamin D affects the risk of disease activity in multiple sclerosis.

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.

Early evidence of disease activity during fingolimod predicts medium-term inefficacy in relapsing-remitting multiple sclerosis.

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.

Assessment of the genetic contribution to brain magnetic resonance imaging lesion load and atrophy measures in multiple sclerosis patients.

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 (ß = -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 (ß = 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.

A pharmacogenetic study implicates NINJ2 in the response to Interferon-ß in multiple sclerosis.

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-ß (IFNß) treatment in MS patients. OBJECTIVES: To analyse the association between rs7298096 and time to first relapse (TTFR) during IFNß 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, IFNß 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 IFNß treatment and related to TTFR. CONCLUSIONS: Rs7298096 could influence MS disease activity during IFNß 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.

Laser capture microdissection for transcriptomic profiles in human skin biopsies.

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.

NLRP3 polymorphisms and response to interferon-beta in multiple sclerosis patients.

We aimed to investigate whether NLR family, pyrin domain containing 3 (NLRP3) polymorphisms are associated with the response to interferon-beta (IFNß) 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 IFNß treatment. A meta-analysis failed to demonstrate significant associations between the response to IFNß and NLRP3 polymorphisms. These findings do not support a role of polymorphisms located in the NLRP3 gene and the response to IFNß in MS patients.

Inverse correlation of genetic risk score with age at onset in bout-onset and progressive-onset multiple sclerosis.

We correlated the weighted genetic risk score measured using 107 established susceptibility variants for multiple sclerosis (MS) with the age at onset in bout-onset (BOMS, n=906) and progressive-onset MS Italian patients (PrMS) (n=544). We observed an opposite relationship in the two disease courses: a higher weighted genetic risk score was associated with an earlier age at onset in BOMS (rho= -0.1; p=5 × 10(-3)) and a later age at onset in PrMS cases (rho=0.07; p=0.15) (p of difference of regression=1.4 × 10(-2)). These findings suggest that established MS risk variants anticipate the onset of the inflammatory phase, while they have no impact on, or even delay, the onset of the progressive phase.

The burden of multiple sclerosis variants in continental Italians and Sardinians.

BACKGROUND: Recent studies identified > 100 non-HLA (human leukocyte antigen) multiple sclerosis (MS) susceptibility variants in Northern European populations, but their role in Southern Europeans is largely unexplored. OBJECTIVE: We aimed to investigate the cumulative impact of those variants in two Mediterranean populations: Continental Italians and Sardinians. METHODS: We calculated four weighted Genetic Risk Scores (wGRS), using up to 102 non-HLA MS risk variants and 5 HLA MS susceptibility markers in 1691 patients and 2194 controls from continental Italy; and 2861 patients and 3034 controls from Sardinia. We then assessed the differences between populations using Nagelkerke's R(2) and the area under the Receiver Operating Characteristic (ROC) curves. RESULTS: As expected, the genetic burden (mean wGRS value) was significantly higher in MS patients than in controls, in both populations. Of note, the burden was significantly higher in Sardinians. Conversely, the proportion of variability explained and the predictive power were significantly higher in continental Italians. Notably, within the Sardinian patients, we also observed a significantly higher burden of non-HLA variants in individuals who do not carry HLA risk alleles. CONCLUSIONS: The observed differences in MS genetic burden between the two Mediterranean populations highlight the need for more genetic studies in South Europeans, to further expand the knowledge of MS genetics.

Genetic burden of common variants in progressive and bout-onset multiple sclerosis.

BACKGROUND: The contribution of genetic variants underlying the susceptibility to different clinical courses of multiple sclerosis (MS) is still unclear. OBJECTIVE: The aim of the study is to evaluate and compare the proportion of liability explained by common SNPs and the genetic burden of MS-associated SNPs in progressive onset (PrMS) and bout-onset (BOMS) cases. METHODS: We estimated the proportion of variance in disease liability explained by 296,391 autosomal SNPs in cohorts of Italian PrMS and BOMS patients using the genome-wide complex trait analysis (GCTA) tool, and we calculated a weighted genetic risk score (wGRS) based on the known MS-associated loci. RESULTS: Our results identified that common SNPs explain a greater proportion of phenotypic variance in BOMS (36.5%±10.1%) than PrMS (20.8%±6.0%) cases, and a trend of decrease was observed when testing primary progressive (PPMS) without brain MRI inflammatory activity (p = 7.9 × 10(-3)). Similarly, the wGRS and the variance explained by MS-associated SNPs were higher in BOMS than PPMS in males (wGRS: 6.63 vs 6.51, p = 0.04; explained variance: 4.8%±1.5% vs 1.7%±0.6%; p = 0.05). CONCLUSIONS: Our results suggest that the liability of disease is better captured by common genetic variants in BOMS than PrMS cases. The absence of inflammatory activity and male gender further raise the difference between clinical courses.