Changes in DNMT1 expression as a marker of epigenetic regulation disturbanses in multiple sclerosis patients

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Abstract

BACKGROUND: Multiple sclerosis is a chronic neurodegenerative autoimmune disease characterized by the presence of foci of inflammation and demyelination in the central nervous system. The initiation of pathological processes in multiple sclerosis is caused by a complex interaction of genetic factors, unfavorable environmental factors and epigenetic influences. Progressive neurological symptoms caused by axonal conduction disorders, axonal death and neurodestruction lead to a significant decreased patients’ quality of life and disability. The search for a new markers to improve diagnostic and therapeutic methods, including taking into account the genetic background and epigenetic interactions, is an urgent task.

AIM: The work was aimed to study the changes in DNMT1 mRNA expression in multiple sclerosis patients with different disease duration, to analyze methylation of DNMT1 promoter, and compare the changes in the level of DNMT1 expression with the homocysteine content in the blood, and the presence of polymorphic variants in genes coding the key folate cycle enzymes.

MATERIALS AND METHODS: The level of DNMT1 mRNA expression in peripheral mononuclear blood cells was assessed by reversed transcription followed by polymerase chain reaction. Fluorescent polymerase chain reaction followed by methyl-sensitive analysis of high-resolution melting curves was used to analyze methylation of the DNMT1 promoter. The content of homocysteine in the blood was determined by chemiluminescence immunoassay. The real-time polymerase chain reaction was used for genotyping by polymorphism of folate cycle genes; the fluorescent probes with the LNA modifications were used to discriminate alleles.

RESULTS: It has been shown that in multiple sclerosis patients, including those at the onset of the disease, the level of DNMT1 mRNA expression is significantly lower than in the control group. No relationship was found between the decrease in DNMT1 expression and the level of promoter methylation. Strong positive relationship between the level of DNMT1 mRNA expression and homocysteine content in patients with multiple sclerosis and the combined effects of the genotypes of MTR A2756G and MTHFR C677T polymorphism on the expression of DNMT1 have been shown. These findings suggest that genetically determined features of folate metabolism may contribute to the disruption of epigenetic regulation in multiple sclerosis.

CONCLUSIONS: The obtained results indicate the promise of research aimed to identifying the factors causing epigenetic changes in multiple sclerosis. Studying the mechanisms of the folate cycle genes polymorphic variants contribution to the pathogenesis of multiple sclerosis could be one of the possible ways to improve diagnostic and therapeutic approaches.

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About the authors

Evgenia A. Tsymbalova

Institute of Experimental Medicine

Email: evgesha.tsymbalova@mail.ru

research assistant of I.P. Pavlov Department of Physiology

Russian Federation, Saint Petersburg

Ekaterina А. Chernyavskaya

Institute of Experimental Medicine

Email: kate-chernjavskaja@yandex.ru
ORCID iD: 0009-0003-0421-9819

research assistant of I.P. Pavlov Department of Physiology

Russian Federation, Saint Petersburg

Darja Е. Ryzhkova

Institute of Experimental Medicine

Email: dashstepanova@gmail.com
ORCID iD: 0009-0004-3745-3203

specialist of I.P. Pavlov Department of Physiology

Russian Federation, Saint Petersburg

Gennady N. Bisaga

Almazov National Medical Research Center

Email: bisaga@yandex.ru
ORCID iD: 0000-0002-1848-8775
SPIN-code: 9121-7071

MD, Dr. Sci. (Med.), Professor of the Department of Neurology with the Clinic

Russian Federation, Saint Petersburg

Irina N. Abdurasulova

Institute of Experimental Medicine

Email: i_abdurasulova@mail.ru
ORCID iD: 0000-0003-1010-6768
SPIN-code: 5019-3940

Cand. Sci. (Biol.), Head of I.P. Pavlov Department of Physiology

Russian Federation, Saint Petersburg

Viktoria I. Lioudyno

Institute of Experimental Medicine

Author for correspondence.
Email: vlioudyno@mail.ru
ORCID iD: 0000-0002-1449-7754
SPIN-code: 8980-8497

Cand. Sci. (Biol.), Senior Research Associate of I.P. Pavlov Department of Physiology

Russian Federation, Saint Petersburg

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2. Fig. 1. Schematic representation of the DNMT1 promoter region and the positions of the primers used to amplify the promoter region. Cross lines indicate the position of CpG dinucleotides (double lines correspond to CGCG regions). The numbers indicate positions on the chromosome for the sequence NC_000019.10 located in the NCBI

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3. Fig. 2. The changes in the DNMT1 mRNA expression level in peripheral blood mononuclear cells in patients with multiple sclerosis (MS). Data are presented as means and the error of the mean; * significant differences between groups according to the results of post-hoc analysis, p < 0.05 (Tukey’s test for unequal samples)

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4. Fig. 3. Normalized melting curves for amplified DNMT1 promoter fragments. Blue lines correspond to melting curves for calibration samples with known percentage of methylation; green line — melting curve for a sample obtained from the HeLa tumor cell line (calculated percentage of methylation 5.5%); orange line — sample obtained from peripheral blood mononuclear cells of a patient with multiple sclerosis (calculated percentage of methylation 1.15%)

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5. Fig. 4. The combined effect of genotypes for polymorphisms C677T of the MTHFR gene and A2756G of the MTR gene on the level of DNMT1 expression in control group subjects and patients with multiple sclerosis

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