Pilot study on CpG methylation of the NRF2 promoter across different ages and sexes in healthy and lung cancer prediagnostic individuals

This pilot study introduces the concept of a "redox clock," an NRF2-based epigenetic clock that reflects age-related changes in oxidative stress regulation. We examined CpG methylation of the NRF2 promoter across two independent populations: 101 healthy participants (56 males, 45 females) from Gyeongsang National University Hospital (GNUH) and 150 healthy participants (111 males, 39 females) from the National Cancer Center (NCC). Methylation-sensitive HpaII restriction enzyme assays targeted three promoter regions (A, B, and C), while Illumina MethylationEPIC microarray analysis identified two specific CpG sites (cg03988329 and cg15484591) that correlated significantly with age (p < 0.01). Males, in particular, showed heightened CpG methylation in regions A and C with advancing age, alongside higher Ct values for NRF2 and HO-1 transcripts, indicating reduced gene expression. Using these methylation patterns, we developed the "redox clock" to model accelerated aging (AA). Notably, this redox clock discriminated prediagnostic lung cancer cases from healthy controls by revealing significantly greater AA in the cancer cohort, whereas established epigenetic clocks (Horvath, Hannum, and PhenoAge) did not detect this difference. These findings support CpG methylation of the NRF2 promoter, as captured by the redox clock, as a promising biomarker for biological aging and potential risk assessment for age-related diseases such as lung cancer.