Tobacco Smoking Influences ELOVL2 Methylation in Buccal Cells without Affecting Age Estimation Accuracy

抄録

CpG methylation in the upstream region of elongation of very long chain fatty acids-like 2 (ELOVL2) is a robust epigenetic marker associated with chronological age. However, the influence of tobacco smoking on ELOVL2 methylation remains unclear. Therefore, this study aimed to assess the effect of smoking on ELOVL2 methylation and its impact on age estimation accuracy. DNA was extracted from the buccal mucosa of 111 individuals, and ELOVL2 methylation levels were quantified using real-time methylation-specific PCR. Smoking status was determined using a self-administered questionnaire, and individuals were classified into three groups: non-smokers, current smokers, and former smokers. To assess how age, smoking status, and their interaction affect ELOVL2 methylation, a regression model was fitted. ELOVL2 methylation-related differences among the three groups were evaluated using analysis of covariance (ANCOVA), followed by Tukey–Kramer multiple comparisons of age-adjusted ELOVL2 methylation levels. An age estimation model based on ELOVL2 methylation was constructed using data from 51 non-smokers and applied to all three groups to estimate chronological age, followed by a comparison of estimation accuracy among the groups. Regression analysis revealed no significant interaction between chronological age and smoking status, suggesting that age affected ELOVL2 methylation consistently across all groups. ANCOVA revealed that smoking status significantly affected ELOVL2 methylation. Tukey–Kramer multiple comparisons showed that current smokers exhibited significantly higher methylation levels than both non-smokers and former smokers. In contrast, no significant difference was observed between former smokers and non-smokers, suggesting that smoking cessation abolished the effects of smoking on ELOVL2 methylation. Age estimation accuracy did not differ among the three groups. In conclusion, ELOVL2 methylation is influenced by both chronological age and current smoking status; however, smoking status does not impair the accuracy of ELOVL2 methylation-based age estimation. Thus, CpG sites within ELOVL2 remain applicable as predictors for age estimation in forensic medicine.