A Ge.F.I. Collaborative Study: Evaluating Reproducibility and Accuracy of a DNA‐Methylation‐Based Age‐Predictive Assay for Routine Implementation in Forensic Casework

ABSTRACT

The increasing interest in DNA methylation (DNAm) analysis within the forensic scientific community prompted a collaborative project by Ge.F.I. (Genetisti Forensi Italiani). The study evaluated a standardized bisulfite conversion–based Single Base Extension (SBE) protocol for the analysis of the methylation levels at five age‐predictive loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59). The study encompassed three phases: (1) setting up and validating the protocol to ensure consistency and reproducibility; (2) comparing fresh peripheral blood with blood spots; and (3) evaluating sources of intra‐ and inter‐laboratory variability. Samples from 22 Italian volunteers were analyzed by 6 laboratories in replicates for a total of 528 records. From phase I emerged that the choice of genetic sequencer significantly contributed to inter‐laboratory data variation, resulting in separate regression analyses performed for each laboratory. In phase II, blood spots were found to be a reliable source for DNAm analysis, despite exhibiting increased experimental variation compared to fresh peripheral blood. In phase III, a strong correlation between the individual's predicted and true ages was observed across different laboratories. Analysis of variance (ANOVA) of the residuals indicated that one‐third of the total variance could be attributed to laboratory‐specific factors, whereas two‐thirds could be attributed to inter‐individual biological differences. The leave‐one‐out cross‐validation (LOO‐CV) method yielded an overall mean absolute deviation (MAD) value of 4.41 years, with an average 95% confidence interval of 5.24 years. Stepwise regression analysis proved that a restricted model (ELOVL2, C1orf132/MIR29B2C, and TRIM59) produced results virtually indistinguishable from the five‐loci model. Additionally, the analysis of samples in replicates greatly improved the fit of the regression model, balancing the slight effects of intra‐laboratory variability. In conclusion, the bisulfite conversion–based SBE protocol, combined with replicate analysis and in‐lab calibration of a regression‐prediction model, proves to be a reliable and easily implementable method for age prediction in forensic laboratories.