DNA methylation at specific CpG sites is often used for chronological age estimation. Most methodologies require bisulfite conversion to preserve the methylation signature during PCR amplification; however, bisulfite conversion involves chemical modification of the DNA and harsh pH and temperature conditions, which causes substantial DNA fragmentation and loss. High DNA amounts (>50 ng) are required for a successful conversion, making the current methodologies incompatible with many forensic trace samples, where DNA is often limited.

In this study, a novel methodology, the EpiDirect®-Clock (PentaBase), was developed and tested. The EpiDirect®-Clock is a bisulfite-free, real-time PCR based assay that enables direct methylation estimation without any pretreatment of the sample. The assay relies on intercalating nucleic acid modified primers, that present different affinity to the DNA sequence depending on the methylation status. The EpiDirect®-Clock targets seven CpGs sites in the ELOVL2 gene and quantifies their overall methylation level.

DNA purified from a total of 150 blood samples was diluted to 1 ng/µL (DNA input 5 ng) and analysed in duplicates using the EpiDirect®-Clock assay on a QuantStudio5 (Thermo Fisher Scientific). Ct values were used to develop and validate an age prediction model. Several models were tested using repeated 5-fold cross-validation on the training dataset (90 samples). The best performance was achieved with a third-degree polynomial regression model. The selected model was validated on 60 samples. The performance of the model on the training and test datasets was measured as mean absolute error (MAE) of 4.47 years (root mean squared error (RMSE) of 5.95 years, R2 = 0.83) and MAE of 4.73 years (RMSE of 6.27, R2 = 0.81), respectively. These results are comparable with or even outperform other age prediction models currently in use. Most importantly, a preliminary sensitivity analysis showed that the DNA input could be lowered to 500 pg without greatly compromising model efficiency. This represents a 100-fold reduction in DNA input compared to bisulfite-based methods (>50 ng) which makes the EpiDirect®-Clock compatible with trace forensic samples.

The EpiDirect®-Clock provides a rapid, low input, cost-efficient, and bisulfite-free method for age estimation, with significant advantages for forensic casework. The limited biological material needed combined with a simple protocol for instrumentation already present in most forensic genetic laboratories make this approach highly appealing and may replace more expensive and sample-consuming methods such as sequencing or microarray technology.

Authors

• Monica Giuffrida (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark)
• Maria Mindegaard (R&D, PentaBase A/S, Denmark)
• Rasmus Koefoed Petersen (R&D, PentaBase A/S, Denmark)
• Erik Sørensen (Copenhagen Hospital Biobank Unit, Department of Clinical Immunology, Copenhagen University Hospital Rigshospitalet, Denmark)
• Marie-Louise Kampmann (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark)
• Claus Børsting (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark)
• Vania Pereira (Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark)