Methylomic Changes in MTHFR Promoter Region, along with the Heterozygous C677T Polymorphism, Contribute to the Risk of Thrombotic Stroke

Abstract

Stroke is the second leading cause of death globally and a major contributor to disability. Developing countries report the highest rates of stroke, with ischemic stroke being the most prevalent type. This study aimed to explore the potential association between specific single nucleotide polymorphisms (SNPs) and thrombotic strokes in Egyptian patients, as well as the role of DNA methylation in the promoter regions of genes associated with these SNPs. The study involved 100 adult patients who were consecutively admitted to the International Medical Center. These patients, diagnosed with acute ischemic stroke, were compared to age-matched control subjects (± 3 years). Molecular analysis was conducted on six thrombosis-related SNPs: FV (R506Q, H1299R, Y1702C), FII (G20210A), and MTHFR (C677T, A1298C) using blood samples from both stroke patients and healthy controls. DNA methylation in the promoter regions of the FV, FII, and MTHFR genes was assessed through a sodium bisulfite conversion protocol and genomic DNA digestion with the methylation-dependent restriction enzyme MspJI, using specific primers for the promoter regions of FV, FII, and MTHFR in all derived samples. The biochemical analysis of the derived samples revealed elevated levels of homocysteine, ESR, and LDL in stroke patients, alongside reduced levels of both vitamin B12 and serum folate. The SNP analysis of samples from healthy controls and stroke patients, conducted using the TaqMan™ SNP genotyping assay, identified the homozygous SNPs in the FV, FII, and MTHFR genes. The results clearly show that the MTHFR C677T heterozygous mutation is present in nearly all stroke patient samples, with a very low likelihood of this mutation co-occurring with SNP mutations in the other indicated genes. Analysis of methylation activities in the promoter regions of the indicated genes showed hypermethylation in the MTHFR promoter region, while methylation levels in the FV and FII promoter regions were normal. The analysis showed increased methylation of cytosine nucleotide in the MTHFR promoter region, potentially inhibiting MTHFR expression and contributing to the development of thrombotic strokes in patients. Overall, the data support an association between the MTHFR C677T mutation, hypermethylation in its promoter region, and stroke development in the study participants.

Keywords: FII; FV; MTHFR; Methylation activates; SNPs; Thrombotic strokes.

Fig. 1

Identification of homozygous and heterozygous variants of six SNPs linked to thrombotic stroke patients. The identification of six wild-type SNPs (R506Q, H1299R, Y1702C), (G20210A), and (C677T, A1298C), along with heterozygous SNPs (R506R, H1299H, Y1702Y), (G20210G), and (C677C, A1298A) in the FV, FII, and MTHFR genes was performed using blood samples from stroke patients and healthy controls. The genotyping was conducted using the TaqMan™ SNP genotyping assay, which included specific melting curves for each SNP. The study involved 100 blood samples from stroke patients, with 100 samples from healthy individuals used as controls

Fig. 2

Statistical diagram illustrating the distribution of heterozygous and homozygous SNPs in stroke patient samples. (A and B) The number of wild-type and heterozygous SNPs detected in the FV, FII, and MTHFR genes from 100 blood samples of healthy individuals (Control group), with a separate, detailed chart highlighting the MTHFR gene analysis. (C and D) The number of wild-type and heterozygous SNPs identified in the FV, FII, and MTHFR genes from 100 blood samples collected from stroke patients, accompanied by a focused, standalone chart specifically illustrating the MTHFR gene analysis

Fig. 3

Methylation activities within the promoter regions of FV, FII, and MTHFR. (A) Agarose gel electrophoresis shows the MspJI-cleaved and amplified fragments of genomic DNA, which was purified from three healthy control samples and three stroke patient samples. The DNA was amplified via conventional PCR using primers specific to the promoter regions of the FV, FII, and MTHFR genes. (B) The relative methylation levels in sodium bisulfite-converted DNA were measured using qRT-PCR with a specific primer for the FV gene promoter region. The results are presented as fold-change in methylation activity for the three stroke patient samples compared to a sample from a healthy control. (C) The relative methylation levels for the FII gene promoter region were similarly assessed in sodium bisulfite-converted DNA using qRT-PCR, with the data indicating fold-change in stroke patients versus healthy controls. (D) The relative methylation levels for the MTHFR gene promoter region were also measured using qRT-PCR in sodium bisulfite-converted DNA, with the results shown as fold-change in stroke patients compared to healthy controls. Error bars represent the standard deviation from three replicates. A two-tailed t-test was used to assess data differences, with * indicating P < 0.05 for statistical significance and ** indicating P < 0.01 for high statistical significance. The data reflect 100 patient samples and 100 healthy control samples