Epigenetic age prediction in semen – marker selection and model development

Abstract

DNA methylation analysis is becoming increasingly useful in biomedical research and forensic practice. The discovery of differentially methylated sites (DMSs) that continuously change over an individual's lifetime has led to breakthroughs in molecular age estimation. Although semen samples are often used in forensic DNA analysis, previous epigenetic age prediction studies mainly focused on somatic cell types. Here, Infinium MethylationEPIC BeadChip arrays were applied to semen-derived DNA samples, which identified numerous novel DMSs moderately correlated with age. Validation of the ten most age-correlated novel DMSs and three previously known sites in an independent set of semen-derived DNA samples using targeted bisulfite massively parallel sequencing, confirmed age-correlation for nine new and three previously known markers. Prediction modelling revealed the best model for semen, based on 6 CpGs from newly identified genes SH2B2, EXOC3, IFITM2, and GALR2 as well as the previously known FOLH1B gene, which predict age with a mean absolute error of 5.1 years in an independent test set. Further increases in the accuracy of age prediction from semen DNA will require technological progress to allow sensitive, simultaneous analysis of a much larger number of age correlated DMSs from the compromised DNA typical of forensic semen stains.

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

“…In fact, a previous study has identified a potential cross hybridization for probes targeting the NOX4/FOLH1B CpG site (cg06979108) on the Illumina Infinium MethylationEPIC BeadChip [31]. However, the very same primer pair has been used in a previous study on age prediction [16] and measurement of the combined DNA methylation level from the NOX4/FOLH1B candidate CpG site and the CpG site of the co-amplified pseudogene (chr11:49,251,356) showed statistically significant age correlation in the study by [19]. Therefore, the candidate CpG site was retained in the marker set and is hereafter referred to as NOX4/FOLH1B.…”

“…Selection of age markers for the VISAGE enhanced tools for age estimation from semen was described previously [19] and includes ten newly identified candidate CpG sites and three CpG sites from Lee et al (2015) [16]. The combined marker set, termed ET-13, was designed to target the 13 candidate CpG sites listed in Table 1.…”

“…Development of an age prediction model based on data generated with the ET-13 allowed for the simultaneous analysis of CpG sites adjacent to the selected candidate CpG sites (in total 36 CpG sites). Marker selection, which was conducted using multivariable stepwise linear regression, showed that only six CpG sites located in five of the 13 candidate markers were optimal for accurate age prediction (Table 2) [19]. These CpG sites were included in the final prediction model and used to develop a second assay (ET-5).…”

“…The VISAGE enhanced tool for age estimation from semen (ET-13; Enhanced Tool-13) combines ten novel markers (regions) identified by [19] with the three markers described by Lee et al (2015) [16]. The ET-13 was validated by five VISAGE consortium laboratories in a collaborative exercise to assess the assay's reproducibility and sensitivity.…”

“…The ET-13 was validated by five VISAGE consortium laboratories in a collaborative exercise to assess the assay's reproducibility and sensitivity. Subsequent to the completed validation phase, the assay was used for data generation to establish the VISAGE age model for semen, which is presented in a separate publication that also provides details regarding marker selection [19]. As MPS allows for the analysis of CpG sites adjacent to the initially selected 13 candidate CpG sites (Table 1), 23 additional CpG sites located within the 13 marker regions were considered by .…”