Accurate age estimation from blood samples of Han Chinese individuals using eight high-performance age-related CpG sites.

Age-related CpG sites (AR-CpGs) are currently the most promising biomarkers for forensic age estimation. In our previous studies, we first validated the age correlation of seven reported AR-CpGs in blood samples of Chinese Han population. Subsequently, we screened some good age predictors from blood samples of Chinese Han population, and built pyrosequencing-based age prediction models. However, it is still important to select a set of high-performance AR-CpGs in a specific racial group and establish a simple and efficient method for accurate age estimation for forensic purpose. In this study, eight AR-CpGs, namely chr6: 11,044,628 (ELOVL2), cg06639320 (FHL2), chr1: 207,823,723 (C1orf132), cg19283806 (CCDC102B), cg14361627 (KLF14), cg17740900 (SYNE2), cg07553761 (TRIM59), and cg26947034, were selected based on our previous studies, and a multiplex methylation SNaPshot assay was developed to investigate DNA methylation levels at these AR-CpGs in 529 blood samples (aged 2-82 years) from Han Chinese population. All selected CpG sites showed strong age correlation with the correlation coefficient (r) from 0.8363 to 0.9251. Multiple linear regression (MLR) and support vector regression (SVR) age prediction models were simultaneously established to fit change characteristics of DNA methylation levels of eight AR-CpGs with the age in 374 donors' blood samples. The MLR model enabled age prediction with R2 = 0.923, mean absolute error (MAE) = 3.52, while the SVR model enabled age prediction with R2 = 0.935, MAE = 2.88. One hundred fifty-five independent samples were used as a validation set to test the two models' performance, and the prediction MAE for the validation set was 3.71 and 3.34 for the MLR and SVR models, respectively. For the MLR and SVR models, the correct prediction rate at ± 5 years reached a high level of 79.35% and 83.23%, respectively. In general, these statistical parameters indicated that the SVR model outperformed the MLR model in age prediction of the Han Chinese population. In addition, our method provides sufficient sensitivity in forensic applications and allows for 100% efficiency when examining bloodstains kept in room conditions for up to 43 days. These results indicate that our multiplex methylation SNaPshot assay is a reliable, effective, and accurate method for age prediction in blood samples from the Chinese Han population.