Glatiramer acetate (GA) is a first-line disease-modifying therapy for multiple sclerosis (MS) with well-established moderate efficacy and high safety, yet its mechanisms of action remain incompletely understood. DNA methylation plays a significant role in MS development and is modulated by various environmental factors, including therapeutic drugs. In this pilot study, we report the first prospective analysis of genome-wide DNA methylation changes in peripheral blood mononuclear cells (PBMCs) from four female relapsing-remitting MS patients before GA initiation and after approximately four and eight months of therapy. We identified 365 loci that are characterized by differential methylation, distinguishing post-treatment time points from baseline, with significant enrichment in CpG islands, shores, and promoter regions. Two distinct temporal patterns emerged: (1) non-monotonic DNA methylation changes peaking at four months and associated with response to foreign antigenic stimuli, and monotonic changes progressively increasing by eight months and related to mTOR-associated pathways relevant to chronic inflammation and neurodegeneration. Integration of DNA methylation and transcriptomic data revealed significant methylation-expression correlations for eight genes, including HLA-DMA, PDE4A, and SMOX-genes with established roles in MS-associated antigen presentation, immunoregulation, and neuroinflammation. Cell composition of PBMCs remained stable throughout treatment. In general, GA therapy for MS appears to induce dynamic, locus-specific DNA methylation changes in PBMCs, with distinct temporal patterns suggesting a biphasic response of the immune system. However, given that none of the individual DMPs reached genome-wide significance, the results presented in this pilot study strongly require validation in larger independent cohorts. Nevertheless, we believe that our findings provide insights into the immunomodulatory effects of GA and lay the foundation for future hypothesis-driven studies to develop epigenetic biomarkers for therapeutic monitoring and generic GA product assessment.

Multiple sclerosis (MS) is a heterogeneous autoimmune disorder of the central nervous system of polygenic nature. Uncovering the genetic predictors of MS phenotype can help to explain the nature of the disease’s clinical heterogeneity, and contribute to the development of novel tools for precise disease prognosis. We conducted a retrospective genetic association study of 35 polymorphic variants in immune-related genes with MS severity assessed using the Multiple Sclerosis Severity Score (MSSS) in a sample of 548 Russian relapsing-onset MS patients who have not previously received immunomodulatory therapy. Variants in the CXCR5, EOMES, TNFRSF1A, IRF8, PVT1, CCR5, HLA-DRB1, IL6, TCF7, and CD40 genes were identified as MSSS-associated in at least two of the three models analyzed (MSSS > 3.5 versus ≤3.5; MSSS > 5.0 versus <2.5; MSSS as a continuous variable). Among them, variants in CCR5, HLA-DRB1 and IL6 genes were associated with MSSS only in women, while variants in the TCF7 and CD40 genes only in men. The variant in CXCR5 was MSSS-associated both in the total sample and in subgroups of female and male MS patients. Thus, we demonstrate that several GWAS-identified MS risk genes, along with other immunological loci, act as modifiers of the MS phenotype.

Background: Relapsing-remitting multiple sclerosis (RRMS) is a most common form of multiple sclerosis in which periods of neurological worsening are followed by periods of clinical remission. RRMS relapses are caused by an acute autoimmune inflammatory process, which can occur in any area of the central nervous system. Although development of exacerbation cannot yet be accurately predicted, various external factors are known to affect its risk. These factors may trigger the pathological process through epigenetic mechanisms of gene expression regulation, first of all, through changes in DNA methylation. Methods: In the present work, we for the first time analyzed genome-wide DNA methylation patterns in CD4+ T lymphocytes and CD14+ monocytes of the same RRMS patients in relapse and remission. The effects of the differential methylation on gene expression were studied using qPCR. Results: We found 743 differentially methylated CpG positions (DMPs) in CD4+ cells and only 113 DMPs in CD14+ cells. They were mostly hypermethylated in RRMS relapse in both cell populations. However, the proportion of hypermethylated DMPs (as well as DMPs located within or in close proximity to CpG islands) was significantly higher in CD4+ T lymphocytes. In CD4+ and CD14+ cells we identified 469 and 67 DMP-containing genes, respectively; 25 of them were common for two cell populations. When we conducted a search for differentially methylated genomic regions (DMRs), we found a CD4+ specific DMR hypermethylated in RRMS relapse (adj. p = 0.03) within the imprinted GNAS locus. Total level of the protein-coding GNAS transcripts in CD4+ T cells decreased significantly in the row from healthy control to RRMS remission and then to RRMS relapse (adj. p = 3.1 x 10-7 and 0.011, respectively). Conclusion: Our findings suggest that the epigenetic mechanism of DNA methylation in immune cells contributes to the development of RRMS relapse. Further studies are now required to validate these results and shed light on the molecular mechanisms underlying the observed GNAS methylation and expression changes.

Objective: to look for differences in the transcriptome profiles in mononuclear blood cells of a group of patients with radiologically isolated syndrome (RIS) who developed symptoms of multiple sclerosis (MS) in the following three years of observation and a group of patients with RIS who did not develop MS during this period.Material and methods. The study included 19 patients with RIS (9 men and 10 women), six of whom developed symptoms of MS during the three-year follow-up period. The transcription profiles of blood mononuclear cells were compared between the groups of patients with RIS who developed or did not develop MS symptoms during this period. The work was conducted in the format of a prospective study; the time of blood collection was taken as the reference point. Full transcriptome profiling was performed using RNA sequencing on an MGISEQ-200 platform. Differential gene expression analysis was performed using the DESeq2 package for the R programming language. Subsequent analysis involved constructing a network of interactions between the protein products of the detected differentially expressed genes based on data from the STRING database, identifying a cluster of interacting proteins, and analyzing the enrichment of this cluster by participants in pathways annotated in the KEGG database.Results. The expression of 146 genes differed significantly (p<0.05; |log2FC| >1) in the studied groups of patients with RIS: in patients with subsequent manifestation of MS symptoms, the expression of 67 genes was lower and expression of 79 genes was higher than in patients without MS symptoms. The decrease in expression of two of the 67 genes (ADGRG7 and LGALS9C) remained significant even after correction for multiple comparisons (padj=2.17⋅10-11 and padj=6.19⋅10-6, respectively). Analyzing the network of interactions between the protein products of the differentially expressed genes allowed the identification of a cluster of 12 genes: APBB2, CCL4, CCL4L2, CDH2, DAZL, FOSB, H2BC17, JUN, KLF4, KLF5, MAPK8IP1, SYCE1; it is over-represented by components of the Toll-like receptor signaling pathway.Conclusion. The transcriptome profiles of blood mononuclear cells differ in groups of patients with RIS who did or did not develop MS symptoms during the three-year follow-up period. The decrease in the expression level of ADGRG7 and LGALS9C genes detected in this study as a sign of rapid conversion of RIS to MS needs to be confirmed in independent samples.

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the сoronavirus disease 2019 (COVID-19) have become a global health threat. At the height of the pandemic, major efforts were focused on reducing COVID-19-associated morbidity and mortality. Now is the time to study the long-term effects of the pandemic, particularly cognitive impairment associated with long COVID. In recent years much attention has been paid to the possible relationship between COVID-19 and Alzheimer’s disease, which is considered a main cause of age-related cognitive impairment. Genetic predisposition was shown for both COVID-19 and Alzheimer’s disease. However, the analysis of the similarity of the genetic architecture of these diseases is usually limited to indicating a positive genetic correlation between them. In this review, we have described intrinsic linkages between COVID-19 and Alzheimer’s disease, pointed out shared susceptibility genes that were previously identified in genome-wide association studies of both COVID-19 and Alzheimer’s disease, and highlighted a panel of SNPs that includes candidate genetic risk markers of the long COVID-associated cognitive impairment.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory and neurodegenerative disease of the central nervous system, which is characterized by significant clinical heterogeneity. Primary progressive MS (PPMS) develops in 10-15% of patients. Unlike the most common relapsing-remitting form of MS, PPMS involves steady progress of neurodegeneration and, as a consequence, a persistent gradual increase in neurological symptoms. The peculiarities of epigenetic regulation of gene expression may be one of the reasons for the differences in the pathogenesis of the two MS forms. DNA methylation is one of the key epigenetic mechanisms, which remains almost unexplored in different cell populations of PPMS patients. The goal of this work was to identify differential methylation profiles of the CpG sites in the CD14+ monocyte DNA, which characterize PPMS. A genome-wide analysis of DNA methylation in PPMS patients and healthy individuals has identified 169 differentially methylated positions (DMPs), 90.5% of which were hypermethylated in PPMS patients. More than half of all DMPs are located in/near known genes and within CpG islands and their neighboring regions, which indicates their high functional significance. We have found six differentially methylated regions (DMRs) in the OR2L13, CAT, LCLAT1, HOXA5, RNF39, and CRTAC1 genes involved in inflammation and neurodegeneration, which indicates active epigenetic regulation of their expression.

OBJECTIVE:To study the whole-genome DNA methylation profiles of peripheral blood mononuclear blood cells (PBMCs) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and relapse in order to assess the contribution of this epigenetic mechanism of gene expression regulation to the activity of the pathological process.MATERIAL AND METHODS:Eight patients with RRMS in remission and 6 patients in relapse were included in the study. Methylation levels of DNA CpG sites in PBMCs were analyzed using Infinium HumanMethylation450 BeadChip DNA microarrays.RESULTS:Seven differentially methylated positions (DMPs) were identified, of which 3 were hypermethylated (cg02981003, cg18486102, cg19533582) and 4 were hypomethylated (cg16814680, cg1964802, cg18584440, cg08291996) during RRMS relapse. Five DMPs are located in protein-coding genes (GPR123, FAIM2, BTNL2, ZNF8, ASAP2), one in microRNA gene (MIR548N), and one in an intergenic region. For all identified DMPs, we observed a change in DNA methylation levels of more than 20% (range 20.2-57.5%). Hierarchical clustering of DNA samples on the heatmap shows their clear aggregation into separate clusters corresponding to RRMS patients in the stages of relapse and remission.CONCLUSION:For the first time it was shown that during relapse and remission of RRMS there are differences in the DNA methylation profile that allow discrimination between these clinical stages. These data indicate the involvement of the epigenetic mechanism of DNA methylation in the activation of the pathological process in RRMS.

Multiple sclerosis (MS) is an autoimmune inflammatory neurodegenerative disease of the central nervous system. The disease is characterized by a heterogeneous clinical course, which is reflected in the fact that there are various types, such as relapsing-remitting MS (RRMS), primary and secondary progressive MS (PPMS and SPMS, respectively). Currently, there is an active search for MS biomarkers capable of predicting and assessing disease progression with high sensitivity and specificity, which would be of great benefit in determining treatment tactics and evaluating their efficacy. MicroRNAs (miRNAs) are short (21–25 nucleotides) non-coding RNA molecules that are primarily involved in post-transcriptional regulation of gene expression. miRNAs play an essential role in tissue development, homeostasis, immune system regulation, and immune cell maturation; they are also involved in the pathophysiology of MS. In addition, high hopes are pinned on miRNAs as disease biomarkers, mainly due to their stability and ability to be released from cells into the extracellular space and circulate there for a long time. The review considers published data on miRNAs in different types of MS. In the future, changes in their levels may be used to create a panel of prognostic markers for disease progression. Studies of miRNAs levels in both circulating fluids (plasma, serum, cerebrospinal fluid) and brain tissue of MS patients were reviewed. Based on the aggregated data from the studies reviewed, it can be confirmed that the accumulated data are quite sufficient to recognize that regulatory miRNAs molecules are involved in the pathophysiological mechanisms of MS progression. However, there is still a long way to go to establish a panel of circulating miRNAs that predict the rate of progression of MS.

Multiple sclerosis (MS) is a chronic autoimmune and degenerative disease of the central nervous system, which develops in genetically predisposed individuals upon exposure to environmental influences. Environmental triggers of MS, such as viral infections or smoking, were demonstrated to affect DNA methylation, and thus to involve this important epigenetic mechanism in the development of pathological process. To identify MS-associated DNA methylation hallmarks, we performed genome-wide DNA methylation profiling of two cell populations (CD4+ T-lymphocytes and CD14+ monocytes), collected from the same treatment-naive relapsingremitting MS patients and healthy subjects, using Illumina 450 K methylation arrays. We revealed significant changes in DNA methylation for both cell populations in MS. In CD4+ cells of MS patients the majority of differentially methylated positions (DMPs) were shown to be hypomethylated, while in CD14+ cells - hypermethylated. Differential methylation of HLA-DRB/ gene in CD4+ and CD14+ cells was associated with carriage of DRB1 *15 allele independently from the disease status. Besides, about 20% of identified DMPs were shared between two cell populations and had the same direction of methylation changes; they may be involved in basic epigenetic processes occuring in MS. These findings suggest that the epigenetic mechanism of DNA methylation in immune cells contributes to MS; further studies are now required to validate these results and understand their functional significance.

Vasovagal syncope (VVS) is the most common cause of sudden loss of consciousness. VVS results from cerebral hypoperfusion, due to abnormal autonomic control of blood circulation, leading to arterial hypotension. It is a complex disease, and its development is largely associated with genetic susceptibility. Since abnormal neurohumoral regulation plays an important role in VVS development, we analyzed the association of VVS with polymorphic variants of ADRA1A, ADRB1, HTR1A, ADORA2A, COMT, and NOS3 genes, the products of which are involved in neurohumoral signaling, in patients with a confirmed VVS diagnosis (157 subjects) and individuals without a history of syncope (161 subjects). We were able to identify the associations between VVS and alleles/genotypes ADRA1A rs1048101, ADRB1 rs1801253, ADORA2A rs5751876, and COMT rs4680, as well as NOS3 rs2070744 in biallelic combination with COMT rs4680. Thus, we are the first to observe, within a single study, the role of the genes that encode α- and β-adrenergic receptors, catechol-O-methyltransferase, adenosine receptors and nitric oxide synthase in VVS development. These findings demonstrate that the genes involved in neurohumoral signaling pathways contribute to the formation of a genetic susceptibility to VVS.

Radiologically isolated syndrome (RIS) is a nosological form in which magnetic resonance imaging (MRI) reveals lesions of the white matter of the brain and/or spinal cord characteristic of multiple sclerosis (MS) in individuals in the absence of clinical symptoms of the disease. Among the studies devoted to RIS, the number of works aimed at studying the molecular mechanisms underlying its formation is very small. Previously, using next generation sequencing (NGS), we for the first time revealed significant differences in the expression profiles of several genes in peripheral blood mononuclear cells (MNCs) of individuals with RIS and healthy controls.Objective: to conduct a validation analysis of changes in the expression of the CCR2, CX3CR1, and TNF genes that were observed during NGS in the MNCs of individuals with RIS compared with healthy individuals.Patients and methods. Analysis of the expression of the CCR2, CX3CR1, and TNF genes was performed on independent validation cohorts (in MNCs of 14 subjects with RIS and 14 without RIS) by reverse transcription followed by real-time PCR. Results and discussion. In MNCs of subjects with RIS, the TNF gene expression was significantly reduced compared to healthy controls (p=0.035; FC=0.78). No significant differences in expression levels were found for other genes.Conclusion. The obtained data shows that disturbances of TNF gene expression preceding the clinical manifestations of MS, at least in individuals with RIS, which can lead to further dysregulation of several processes.

The pathogenesis of multiple sclerosis (MS), a chronic disease of the CNS, includes autoimmune and neurodegenerative components. In most cases, patients develop relapsing-remitting MS (RRMS), while 10–15% of patients develop primary progressive MS (PPMS), which differs from RRMS in the mechanisms of the pathological process, some demographic, and some clinical characteristics. These differences may be explained by the epigenetic regulation of gene expression in PPMS including DNA methylation as one of the key epigenetic processes. The features of DNA methylation in various cell populations in PPMS patients remain understudied. The goal of this study is to identify differentially methylated CpG sites (DMSs) of the genome of CD4+ T lymphocytes, which characterize PPMS. The study included eight treatment-naïve PPMS patients and eight healthy controls. Genome-wide analysis of DNA methylation of CD4+ T lymphocytes was performed using high-density DNA microarrays. We have identified 108 DMSs, which distinguish PPMS patients from healthy controls. In PPMS patients 81% of the DMSs are hypermethylated . More than a half of the identified DMSs are located in known genes in CpG islands and adjacent regions, which indicates a high functional significance of these DMSs in PPMS development. Analysis of the overrepresentation of DMS-containing genes in the main biological processes demonstrates their involvement in the regulation of cell adhesion to the extracellular matrix and the development of the immune response, i.e., antigen processing and presentation, and development of the immune system. Genome-wide analysis of DNA methylation in CD4+ T lymphocytes of PPMS patients indicates the involvement of this epigenetic process in the immunopathogenesis of the disease. These results may help better understand the pathogenesis of this severe form of MS.

The epigenetic mechanisms of gene expression regulation are a group of the key cellular and molecular pathways that lead to inherited alterations in genes' activity without changing their coding sequence. DNA methylation at the C5 position of cytosine in CpG dinucleotides is amongst the central epigenetic mechanisms. Currently, the number of studies that are devoted to the identification of methylation patterns specific to multiple sclerosis (MS), a severe chronic autoimmune disease of the central nervous system, is on a rapid rise. However, the issue of the contribution of DNA methylation to the development of the different clinical phenotypes of this highly heterogeneous disease has only begun to attract the attention of researchers. This review summarizes the data on the molecular mechanisms underlying DNA methylation and the MS risk factors that can affect the DNA methylation profile and, thereby, modulate the expression of the genes involved in the disease's pathogenesis. The focus of our attention is centered on the analysis of the published data on the differential methylation of DNA from various biological samples of MS patients obtained using both the candidate gene approach and high-throughput methods.

The presence of brain/spinal white matter lesions typical for multiple sclerosis (MS) in asymptomatic individuals is known as 'radiologically isolated syndrome' (RIS). Taking into account that RIS patients are at high risk of MS development, the understanding of mechanisms underlying its pathogenesis is of great importance. In order to investigate RIS-specific transcription signature we performed high-throughput RNA-sequencing in peripheral blood mononuclear cells (PBMCs) of 8 RIS patients and 8 age- and sex-matched healthy controls. We identified 57 differentially expressed genes (DEGs), which levels differed by more than 2 times when comparing RIS patients to healthy controls (FDR p value < 0.05). Gene ontology enrichment analysis in the "biological process" category revealed 16 signaling pathways significantly overrepresented by identified DEGs. The most significant changes in gene expression in PBMCs of RIS patients occur in pathways involved in regulation of the immune response, cytokine and chemokine signaling, cytokine production, and leukocyte migration. In general, analyzing the global transcriptome we demonstrated the dysregulation of immune processes in PBMCs of RIS patients, confirming the current assumption that RIS represents the preclinical stage and/or subclinical form of MS.

There is increasing evidence that the interaction of the mitochondrial and nuclear genomes substantially affects the risk of neurodegenerative diseases. The role of mitonuclear interactions in the development of multiple sclerosis, a severe chronic neurodegenerative disease of a polygenic nature, is poorly understood. In this work, we analyzed the association of multiple sclerosis with two-component mitonuclear combinations that include each of seven polymorphic variants of the nuclear genome localized in the region of the UCP2, and KIF1B genes and in the PVT1 locus (MYC, PVT1, and MIR1208 genes) and each of ten polymorphisms of the mitochondrial genome, as well as individual genetic variants that make up these combinations. Association of the individual components of these combinations with multiple sclerosis was also evaluated. 507 patients with multiple sclerosis and 321 healthy individuals were enrolled in the study, all participants were ethnic Russians. Two mitonuclear combinations associated with multiple sclerosis were identified: the UCP2 (rs660339) *A + MT-ATP6 (rs193303045) *G combination was characterized by p-value = 0.015 and OR = 1.39 [95% CI 1.05-1.87], and the PVT1 (rs2114358) *G + MT-ND1 (rs1599988) *C combination – by p-value = 0.012 and OR = 1.77 [95% CI 1.10-2.84]. Only one of the individual components of these combinations, allele rs660339 *A of the nuclear gene UCP2 encoding uncoupling protein 2 of the mitochondrial anion carrier family, was independently associated with multiple sclerosis (p = 0.028; OR = 1.36 [95% CI 1.01–1.84]). This study expands the current understanding of the role of mitonuclear interactions and variance of nuclear genes, whose products function in mitochondria, and in risk of MS.

There is increasing evidence that the interaction of the mitochondrial and nuclear genomes substantially affects the risk of neurodegenerative diseases. The role of mitonuclear interactions in the development of multiple sclerosis, a severe chronic neurodegenerative disease of a polygenic nature, is poorly understood. In this work, we analyzed the association of multiple sclerosis with two-component mitonuclear combinations that include each of seven polymorphic variants of the nuclear genome localized in the region of the UCP2, and KIF1B genes and in the PVT1 locus (MYC, PVT1, and MIR1208 genes) and each often polymorphisms of the mitochondrial genome, as well as individual genetic variants that make up these combinations. Association of the individual components of these combinations with multiple sclerosis was also evaluated. 507 patients with multiple sclerosis and 321 healthy individuals were enrolled in the study, all participants were ethnic Russians. Two mitonuclear combinations associated with multiple sclerosis were identified: the UCP2 (rs660339)*A + MT-ATP6 (rs193303045)*G combination was characterized by p-value = 0.015 and OR= 1.39 [95% CI 1.05-1.87], and the PVT1 (rs2114358)*G + MT-ND1 (rs1599988)*С combination - by p-value = 0.012 and OR = 1.77 [95% CI 1.10-2.84]. Only one of the individual components of these combinations, allele rs660339*A of the nuclear gene UCP2 encoding uncoupling protein 2 of the mitochondrial anion carrier family, was independently associated with multiple sclerosis (p = 0.028; OR = 1.36 [95% CI 1.01-1.84]). This study expands the current understanding of the role of mitonuclear interactions and variance of nuclear genes, whose products function in mitochondria, and in risk of MS.

Multiple sclerosis (MS) is a chronic autoimmune disease, in the pathogenesis of which the concurrence of demyelination of central nervous system (CNS) axons and neurodegeneration plays a role and which is accompanied by progressive neurological dysfunction. Long-term monitoring of patients with MS is needed to rate its severity according to existing scales; it is therefore very relevant to search for genomic markers that can predict the rate of disease progression at early stages. The impact of polymorphic variants in the PVT1 locus on MS severity has not been previously studied.Objective: to analyze the association of the polymorphic variants rs4645948 in the MYC gene and rs2114358 and rs4410871) in the PVT1 genes with MS severity according to the Multiple Sclerosis Severity Scale (MSSS) separately and as part of biallelic combinations, as well as the possible linkage disequilibrium of the studied single nucleotide polymorphisms for establishing the independence of the observed associations.Patients and methods. The investigation enrolled 468 Russian MS patients who did not take immunomodulating drugs before blood testing. The patients were divided into two groups: 1) relatively mild MS (MSSS ≤3.5) and 2) relatively severe MS (MSSS >3.5). The polymorphic variants in the PVT1 locus were genotyped by a real-time polymerase chain reaction assay.Results and discussion. In the MS study group, the carriage of the allele of PVT1 (rs2114358)*G turned out to be associated with the severe course of the disease (pf=0.042; odds ratio (OR)=1.41). The significance of the association increases in the simultaneous carriage of this allele with another variant of the same gene – PVT1 (rs4410871)*T (pf=0.024; OR=1.58). There was no linkage disequilibrium between the components of the biallelic combination.Conclusion. The polymorphic variants in the PVT1 locus are associated with the severity of MS.

Increased sensitivity and availability of magnetic resonance imaging (MRI) in neurological routine practice led to the fact that more and more experts began to encounter changes typical for multiple sclerosis (MS) according to MRI in the absence of anamnestic and clinical indications of damage to the central nervous system (CNS). This nosological form has been defined as a radiologically isolated syndrome (RIS). More and more RIS cases convert to MS (up to 30% in the first 5 years after RIS diagnosis). At the moment, there are no biological markers that allow combining RIS and MS into one pathological process and early treatment with disease-modifying drugs (DMT). Prospective studies are actively being conducted to identify demographic, clinical, neuroimaging and biochemical conversion predictors. The identification of the molecular biological RIS features, combining these changes with MS, is an urgent scientific task and will allow timely initiation of therapy of the pathological process already at the subclinical stage.