Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science Renata Zbiec ´ -Piekarska a , Magdalena Spo ´ lnicka a , Tomasz Kupiec b , Z ˙ anetta Makowska a , Anna Spas a , Agnieszka Parys-Proszek b , Krzysztof Kucharczyk c , Rafał Płoski d , Wojciech Branicki b, * a Central Forensic Laboratory of the Police, Aleje Ujazdowskie 7, 00-583 Warsaw, Poland b Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland c BioVectis, Pawin ´skiego 5a/d, 02-106 Warsaw, Poland d Medical University of Warsaw, Z ˙ wirki i Wigury 61, 02-091 Warsaw, Poland 1. Introduction Human chronological age is one of the most important clues employed by the investigators when tracking an unknown individual involved in a criminal case. In forensics, accurate age estimation from biological traces may have a value similar to analysis of externally visible characteristics (EVC) or biogeograph- ical ancestry. It may inform an investigation by narrowing down the number of individuals that need to be considered when a suspect or victim has to be identified but evidence DNA profiles do not match reference or database data [1–3]. The problem of estimating human age has been explored in forensics for many years [4,5]. Although it is well known that chronological age may differ from biological age and this variation may be affected not only by genetic factors but also the medical history or lifestyle of an individual, many biochemical and DNA markers which correlate with human aging have been evaluated for their possible application in forensic science as age estimation tools. This evaluation has included several age-dependent molecular pro- cesses, such as accumulation of D-aspartic acid in proteins [6], accumulation of advanced glycation end products [7], shortening of telomeres [8], accumulation of 4977 bp deletion in mitochon- drial DNA [9] or decrease in the number of sjTREC molecules caused by thymus involution occurring in the course of human life [10,11]. Interestingly, it has been found that methylation of the human genome changes with age, indicating that epigenetics may provide novel, relevant markers for forensic age estimation Forensic Science International: Genetics 14 (2015) 161–167 A R T I C L E I N F O Article history: Received 29 May 2014 Received in revised form 12 September 2014 Accepted 1 October 2014 Keywords: DNA methylation CpG islands ELOVL2 Biomarker Age prediction Forensic science A B S T R A C T Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2–75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R 2 = 0.859, prediction error = 6.85 and mean absolute deviation MAD = 5.03. Examination of a testing set of 124 blood samples (MAD = 5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched Æ7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60–78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models. ß 2014 Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Institute of Forensic Research Westerplatte Street 9, 31-033 Krako ´ w, Poland. Tel.: +4812 618 57 00; fax: +48 12 422 38 50. E-mail address: wbranicki@ies.krakow.pl (W. Branicki). Contents lists available at ScienceDirect Forensic Science International: Genetics jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/fs ig http://dx.doi.org/10.1016/j.fsigen.2014.10.002 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.