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A Ge.F.I. Collaborative Study: Evaluating Reproducibility and Accuracy of a DNA-Methylation-Based Age-Predictive
Martina Onofri1, Federica Alessandrini2, Serena Aneli3
1Section of Legal Medicine, Department of Medicine and Surgery, University of Perugia, Terni, Italy.
Electrophoresis
|January 7, 2025
Summary
Forensic DNA methylation analysis using a bisulfite conversion-based Single Base Extension (SBE) protocol reliably predicts age. This method is reproducible across labs and effective with blood spots, offering a valuable tool for age estimation.
Area of Science:
- Forensic Science
- Genetics
- Epigenetics
Background:
- Growing interest in DNA methylation (DNAm) for forensic age prediction.
- Need for standardized and validated protocols for DNAm analysis in forensic settings.
- Previous studies highlighted variability in DNAm age prediction methods.
Purpose of the Study:
- To evaluate a standardized bisulfite conversion-based Single Base Extension (SBE) protocol for DNA methylation analysis.
- To assess the protocol's reliability using fresh blood versus blood spots.
- To identify and quantify sources of intra- and inter-laboratory variability in age prediction.
Main Methods:
- A collaborative study involving 6 Italian laboratories analyzed DNA methylation levels at five age-predictive loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, TRIM59).
- Protocol validation, comparison of fresh blood and blood spots, and variability assessment were conducted in three phases.
- Leave-one-out cross-validation (LOO-CV) and Analysis of Variance (ANOVA) were used to evaluate prediction accuracy and variance sources.
Main Results:
- Blood spots are a reliable source for DNA methylation analysis, though with increased experimental variation compared to fresh blood.
- A strong correlation between predicted and true ages was observed across laboratories, with a mean absolute deviation (MAD) of 4.41 years.
- A restricted three-loci model (ELOVL2, C1orf132/MIR29B2C, TRIM59) yielded results comparable to the five-loci model.
- Laboratory-specific factors accounted for one-third of the total variance, while inter-individual differences accounted for two-thirds.
Conclusions:
- The bisulfite conversion-based SBE protocol is a reliable and implementable method for forensic age prediction.
- Replicate analysis and in-laboratory calibration of regression models are crucial for improving accuracy and managing variability.
- The protocol offers a robust approach for estimating chronological age in forensic casework.
