LINE-1 Methylation Status in Multiple Sclerosis Patients Is Associated with Changes in Folate Metabolism

Abstract

The disruption of epigenetic regulation and the development of abnormal DNA methylation patterns are crucial steps in the pathogenesis of neurodegenerative diseases. Methylation alterations in multiple sclerosis (MS) patients may contribute to the dysregulation of gene expression linked to the regulation of inflammation, myelin production, and the preservation of the integrity of the myelin sheath. The possibility that epigenetic alterations could be reversed provides a rationale for studying their mechanisms. In this study, we evaluated the methylation status of LINE-1 retrotransposons in the peripheral blood cells of patients with MS and healthy controls. In healthy individuals, LINE-1 methylation levels were observed to decrease with advancing age. MS patients exhibited a positive correlation between LINE-1 methylation and MS duration. The study indicates that the level of LINE-1 methylation is notably higher in progressive MS compared to the remitting type. LINE-1 methylation variations in MS patients were observed to be associated with the serum levels of homocysteine and vitamin B9, and dependent on the genotype for the C677T polymorphism of the MTHFR gene as well. The data obtained point to the contribution of the C677T polymorphism to the appearance of epigenetic disorders in MS development and suggest that hypermethylation may be mediated by disruptions in the folate metabolism that accompany MS.

Keywords: C677T polymorphism; LINE-1; folate metabolism; homocysteine; methylation; multiple sclerosis.

Fig. 1

Assessment of LINE-1 methylation levels via methyl-sensitive high-resolution melting curve analysis (MS-HRM). (A) – raw melting curves and melting peaks of fully methylated (100%) and fully unmethylated (0%) standard samples; (B) – melting curves for the standard sample (0%) and the three tested samples with high (56%), low (12%), and medium (26%) methylation levels, converted to difference plots

Fig. 2

LINE-1 methylation levels in peripheral blood mononuclear cells. (A) – comparison of indices in the control group and in the group of MS patients; (B) – comparison of indices in patients with RR MS, PP MS, and SP MS; (C) – degree of neurologic deficit in patients with remitting and progressive MS. * – statistically significant differences between groups; p < 0.05; ns – no significant differences

Fig. 3

Correlation analysis data to detect changes in LINE-1 methylation levels. (A) – data from both MS patients and those in the control group; (B) – the blue line shows the correlation between the LINE-1 methylation level and disease duration for the whole group of patients, and the green line shows the results of the analysis of patients with a remitting course (RR MS) and duration of over 1 year

Fig. 4

Homocysteine serum levels in the control subjects and MS patients. The concentration ranges correspond to the quartiles established for the control group. The data for each range are expressed as percentages of the entire cohort within their respective groups

Fig. 5

Alterations in the ratio of serum homocysteine and folic acid concentrations (Hcy/B9) in individuals with MS (A), and the correlation between alterations in homocysteine levels (B) and Hcy/B9 ratios (C) with LINE-1 methylation levels in peripheral blood mononuclear cells. ** – statistically significant differences between the groups, p < 0.05 (Kruskal– Wallis test with subsequent pairwise comparisons)

Fig. 6

Concentration of vitamin B9 (A) and homocysteine (B), and the Hcy/B9 ratio (C) in the individuals of the control group and the MS patients depending on the genotype for the C677T polymorphism of the MTHFR gene. ♦ – values deviating from the median by more than 1.5 interquartile range. * – statistically significant differences between the groups, p < 0.05; ** – statistically significant differences between the groups, p < 0.01