Examination of Genetic and Epigenetic Characteristics of Patients with Hyperhomocysteinemia Following High-Dose Folic Acid Consumption.

Purpose: Homocysteine (HCY) metabolism is regulated by the methionine cycle, which is essential for DNA methylation and is associated with the folate cycle. This study examines the alterations in DNA methylation signature including epigenetic age changes, measure cell-free DNA (cfDNA), and HCY concentrations, and identifies genetic markers that may influence homocysteine response following folic acid (FA) supplementation in individuals with hyperhomocysteinemia (HHC). Methods: Blood samples were obtained from 43 HHC patients undergoing FA supplementation. We quantified FA and HCY levels, separated plasma and white blood cell fractions, and evaluated global DNA methylation using LINE-1 bisulfite pyrosequencing. Biological age was determined using Illumina BeadArray technology, and whole-exome sequencing was performed to investigate the patients' genetic backgrounds. Results: Following FA supplementation, cfDNA levels significantly decreased and correlated positively with HCY (r = 0.2375). Elevated average LINE-1 methylation of cfDNA and PBMC-origin DNA was observed, with mean relative changes of 1.9% for both sample types. Regarding HCY levels, we categorized patients based on their response to FA supplementation. FA responders showed decreased HCY from 15.7 ± 5.5 to 11 ± 2.9 µmol/L, while in FA non-responders, an opposite trend was detected. The average biological age was reduced by 2.6 years, with a notable reduction observed in 80% of non-responders and 48% of responders. Sequencing identified mutations in several genes related to the one-carbon cycle, including MTRR, CHAT, and MTHFD1, with strong correlations to the non-responder phenotypes found in genes like PRMT3, TYMS, DNMT3A, and HIF3A. Conclusions: FA supplementation influences the HCY level, as well as affects the cfDNA amount and the DNA methylation pattern. However, genetic factors may play a crucial role in mediating individual responses to folate intake, emphasizing the need for personalized approaches in managing hyperhomocysteinemia.