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.