FHL2 DNA methylation in tooth root surfaces as a tool for age estimation

Age-at-death estimation is critical in forensic investigations, particularly for skeletal and dental remains. Accurate age estimation is essential for constructing biological profiles in forensic investigations. DNA methylation, particularly in age-related genes such as FHL2, has emerged as a promising epigenetic marker for this purpose. While previous studies have applied methylation assays to the entire tooth, these methods often result in the destruction of the dental structures. In this study, we assessed whether FHL2 methylation levels measured using methylation-sensitive high-resolution melting (MS-HRM) in root surface samples could serve as a reliable marker for age estimation in Japanese individuals. Samples were collected from the root surfaces of extracted third molars using one to three strokes with a dental scaler, primarily obtaining cementum or cementum with minor dentin content. MS-HRM analysis was performed using primers previously validated for saliva and blood. The FHL2 methylation scores were significantly correlated with chronological age, despite the use of non-fresh samples stored at room temperature. Notably, the methylation levels were unaffected by self-reported coffee consumption and smoking habits. Additionally, while alveolar bone resorption status was associated with both age and methylation scores, it did not alter the strength of this correlation. Although a regression formula derived from FHL2 alone yielded low accuracy, these results suggest that root surface methylation data may be valuable for age estimation, particularly when combined with additional genetic markers. This minimally invasive method preserves tooth morphology and may be applicable even in decomposed remains, offering practical advantages in forensic contexts.