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Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease of the central nervous system with an unpredictable course toward progressive disability. Understanding and treating progressive MS remains extremely challenging due to the limited knowledge of the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. We identified and replicated hypermethylation of an intergenic region within the chromosome 1q21.1 locus in the blood of PPMS patients compared to other MS patients and healthy individuals. We next revealed that methylation is under genetic control both in the blood and brain. Genetic analysis in the largest to date PPMS dataset yielded evidence of association of genetic variations in the 1q21.1 locus with PPMS risk. Several variants affected both 1q21.1 methylation and the expression of proximal genes (CHD1L, PRKAB2, FMO5) in the brain, suggesting a genetic-epigenetic-transcriptional interplay in PPMS pathogenesis. We addressed the causal link between methylation and expression using reporter systems and dCas9-TET1-induced CpG demethylation in the 1q21.1 region, which resulted in upregulation ofCHD1LandPRKAB2genes in SH-SY5Y neuron-like cells. Independent exploration using unbiased correlation network analysis confirmed the putative implication ofCHD1LandPRKAB2in brain processes in PPMS patients. Thus, several lines of evidence suggest that distinct molecular changes in 1q21.1 locus, known to be important for brain development and disorders, associate with genetic predisposition to high methylation in PPMS patients that regulates the expression of proximal genes.Significance StatementMultiple sclerosis (MS) is a long-lasting neurological disease affecting young individuals that occurs when the body’s natural guard (immune system) attacks the brain cells. There are currently no efficient treatments for the progressive form of MS disease, probably because the mechanisms behind MS progression are still largely unknown. Thus, treatment of progressive MS remains the greatest challenge in managing patients. We aim to tackle this issue using the emerging field called “epigenetics” which has the potential to explain the impact of genetic and environmental risk factors in MS. In this project, by using unique clinical material and novel epigenetic tools, we identified new mechanisms involved in MS progression and putative candidates for targeted epigenetic therapy of progressive MS patients.

DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole-genome methylation quantification of low input samples.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system. MS likely results from a complex interplay between predisposing causal gene variants (the strongest influence coming from HLA class II locus) and environmental risk factors such as smoking, infectious mononucleosis, and lack of sun exposure/vitamin D. However, little is known about the mechanisms underlying MS development and progression. Moreover, the clinical heterogeneity and variable response to treatment represent additional challenges to a comprehensive understanding and efficient treatment of disease. Epigenetic processes, such as DNA methylation and histone posttranslational modifications, integrate influences from the genes and the environment to regulate gene expression accordingly. Studying epigenetic modifications, which are stable and reversible, may provide an alternative approach to better understand and manage disease. We here aim to review findings from epigenetic studies in MS and further discuss the challenges and clinical opportunities arising from epigenetic research, many of which apply to other diseases with similar complex etiology. A growing body of evidence supports a role of epigenetic processes in the mechanisms underlying immune pathogenesis and nervous system dysfunction in MS. However, disparities between studies shed light on the need to consider possible confounders and methodological limitations for a better interpretation of the data. Nevertheless, translational use of epigenetics might offer new opportunities in epigenetic-based diagnostics and therapeutic tools for a personalized care of MS patients.

Background and purpose Multiple sclerosis (MS) is a neurological disease of the central nervous system (CNS) that causes physical and cognitive impairments. IL-7Ra is a key non-MHC gene associated with MS. IL-7Ra is a likely functional candidate for this complex disease because it is involved in the development, maturation, and homeostasis of T and B cells. Our aim was to evaluate the expression level and controlling role of lnc-IL-7R in the expression of two variants of IL-7Ra in MS patients versus healthy controls and their correlation with certain clinical features. Methods Using the real-time PCR method, we analyzed the expression levels of membrane-bound ( IL-7RB ) and soluble ( IL-7RS ) isoforms of IL-7R gene and lnc-IL-7R in 36 MS patients versus 30 healthy controls. Results Our results revealed no significant difference between the expression levels of IL-7RB and IL-7RS isoforms of IL-7R gene and lnc-IL-7R in MS patients versus healthy controls ( p = 0.7, p = 0.6 and p = 0.8, respectively). Moreover, we found a significant correlation between the expression levels of IL-7RB with lnc-IL-7R , IL-7RS with lnc-IL-7R and IL-7RB with IL-7RS in both patient and control groups. Conclusions We have probably uncovered new evidence for the controlling role of long non-coding RNAs in the expression level of genes and their roles in MS.

The hallmark of multiple sclerosis (MS) pathogenesis is the breakdown of peripheral tolerance in the immune system. However, its molecular mechanism is not completely understood. Since long non-coding RNAs (lncRNAs) has played important roles in regulation of immunological pathways, here, we evaluated the expression of a novel lncRNA, TOB1-AS1, and its putative associated coding genes in the mechanism of maintaining immune tolerance in peripheral blood of MS patients to assess their possible roles in MS pathogenesis. In this study, 39 MS patients and 32 healthy matched controls were recruited. Real-time PCR standard curve method was used to quantify transcript levels of TOB1-AS1, TOB1, SKP2, and TSG. In addition, the potential sex hormone receptor binding sites on target genes promoter were analyzed using JASPR software. This work demonstrates a negative correlation between TOB1-AS1 expression and EDSS of patients. Also, a robust dysregulation of co-expression of TOB1-AS1 lncRNA and the coding genes in MS patients compared to controls was observed. Such dysregulation in this pathway may be related to MS pathogenesis and response to interferon treatment.

Background we recently showed that a hypothesized anti-aging and anti-inflammatory protein, namely Klotho, may contribute to the etiology and/or pathogenesis of multiple sclerosis (MS). In addition, Klotho function and its gene expression are dependent on inflammatory pathways. Accordingly, the aim of this study was to investigate the Klotho gene expression within peripheral blood mononuclear cells (PBMCs) of patients with MS. Methods Altogether, 30 patients with relapsing-remitting MS (RRMS) along with 30 age and sex-matched healthy individuals were enrolled in this study. Blood samples were obtained from all participants and then PBMCs were isolated. The quantitative Real-Time PCR was carried out for Klotho mRNA derived from PBMCs. Results The results showed that klotho gene expression in the PBMCs of patients with RRMS is nearly 2.5-fold less than healthy individuals (P = 0.0006). Conclusion This is the first study demonstrating a possible role of Klotho in the PBMCs of MS patients.

Aims Coronary artery disease (CAD) can be classified as an inflammatory disease, which affected by type 2 diabetes mellitus (T2DM). Elevated levels of many inflammatory molecules were found in the serum of patients with CAD. STAT3 molecule as a transcription factor plays an important role in the cytokines expression. Here, we examined the expression levels of STAT3 and its important regulatory genes lnc-DC and SOCS1, in patients with CAD and T2DM. Methods Blood samples were obtained from 37 CAD + and 36 CAD- patients. These patients were enrolled in this study based on angiography findings and categorized based on T2DM status. The expression levels of STAT3, lnc-DC and SOCS1 genes were examined with Real time PCR method. Results A significant increase was observed in expression of STAT3 and lnc-DC genes but not SOCS1 in CAD + versus CAD- patients. These results replicated partially in some groups categorized based on T2DM and CAD status. However, severity of CAD had no effect on expressions of these genes. Moreover, we found some significant correlations between expressions of lnc-DC with SOCS1 and STAT3, which confirmed by in silico analysis. Conclusion Our results shed further light to the inflammatory aspects of CAD and T2DM with emphasis to JAK/STAT pathway and the regulatory role of long non-coding RNAs in the physiopathology of these diseases.

Background and purpose The aim of this study was to investigate the expression levels of vitamin D receptor (VDR) and NF-κB mRNAs in vitamin D (VD) supplemented multiple sclerosis (MS) patients. Methods RRMS patients received 50,000 IU vitamin D3/week as an intra-muscular injection for 2 months. Blood samples were obtained from 30 MS patients before and after VD supplementation and 32 healthy individuals, and then VDR and NF-κB mRNA levels were measured by real time PCR method and analyzed with independent and paired t-tests. Moreover, some correlations were performed between the expression levels of selected genes and some clinical features of MS and control groups. Results Surprisingly, the expression level of VDR mRNA significantly decreased after 2 months supplementation with VD in our selected patients and in contrast, the level of serum 25(OH) D increased after supplementation. Although, we didn’t find any significant difference in the expression level of NF-κB gene before and after treatment with VD, its expression significantly decreased in untreated MS cases compared with healthy controls. Conclusion In conclusion, we found some new evidences from the molecular mechanism of vitamin D effectiveness in MS treatment. Also, we need more functional studies to find the effect of VD on the expression level of VDR mRNA.

Multiple sclerosis (MS) is a chronic devastating disease of the central nervous system (CNS), which is reported to be the most common neuroinflammatory disorder of the young adults. Although the underlying etiology of MS is not completely elucidated, the number of patients and burden to societies increases worldwide. There are several CNS disorders that might have overlapping phenotypic manifestations with MS; therefore, reliable and accessible diagnostic test is highly desirable for differential diagnosis. MicroRNAs (miRNAs) are small regulatory RNAs that are proven to be master regulators of gene expression. Recently, several studies have shown potential roles for miRNAs in development, hemostasis and maturation of immune system, suggesting possible involvement of these regulatory elements in autoimmune diseases, such as MS. We reviewed recent literature to explore miRNAs that are reported to be involved in pathogenesis of MS and found some miRNAs that might have potentials as being diagnostic biomarkers.

Vitamin D regulates gene expression and affects target cell functions. IL-6 and IL-17A are pro-inflammatory cytokines associated with MS pathogenesis. The aim of this study was to investigate the vitamin D effects on the expression level of IL-6 and IL-17A in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients. Also, we performed a correlation analysis between the gene expression and some clinical features such as serum level of vitamin D and the expanded disability status scale (EDSS). Significant up-regulation of IL-6 and IL-17A gene expression was shown under vitamin D treatment. Also, some gender specific correlations between the gene expression with vitamin D levels were detected in female RR-MS patients.

IL-1B is released by monocytes, astrocytes and brain endothelial cells and seems to be involved in inflammatory reactions of the central nervous system (CNS) in multiple sclerosis (MS). This study aims to evaluate the expression level of IL-1B mRNA in peripheral blood mononuclear cells (PBMCs), genotype the rs16944 SNP and find out the role of this SNP on the expression level of IL-1B in MS patients. We found that the expression level of IL-1B in MS patients increased 3.336 times more than controls in PBMCs but the rs16944 SNP in the promoter region of IL-1B did not affect the expression level of this gene and there was not association of this SNP with MS in the examined population. Also, our data did not reveal any correlation between normalized expressions of IL-1B gene with age of participants, age of onset, and disease duration.