DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay

• Published in: Forensic science international 2020-06, Vol.311, p.110267-110267, Article 110267
• Main Authors: Dias, Helena Correia, Cordeiro, Cristina, Pereira, Janet, Pinto, Catarina, Real, Francisco Corte, Cunha, Eugénia, Manco, Licínio
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Age; Age determination; Age prediction; Aged; Aged, 80 and over; Aging - genetics; Assaying; Blood; Blood samples; Child; Child, Preschool; Chronology; CpG Islands; Datasets; Deoxyribonucleic acid; DNA; DNA Methylation; Fatty Acid Elongases - blood; Female; Females; FHL2 protein; Forensic Genetics - methods; Forensic science; Forensic sciences; Genes; Genetic Markers; Genotyping Techniques - instrumentation; Humans; Infant; Intracellular Signaling Peptides and Proteins - blood; Kruppel-Like Transcription Factors - blood; LIM-Homeodomain Proteins - blood; Linear Models; Living and deceased individuals; Loci; Male; Males; Methods; Methylation SNaPshot; Middle Aged; Multiplexing; Muscle Proteins - blood; Portugal; Prediction models; Regression analysis; Regression models; Replication study; Saliva; Software; Transcription Factors - blood; Tripartite Motif Proteins - blood; Variation; Young Adult
• ISSN: 0379-0738; 1872-6283
• DOI: 10.1016/j.forsciint.2020.110267

•A multiplex methylation SNaPshot assay was replicated in blood samples.•Postmortem changes can alter the methylation status among specific loci in blood.•Differences in the methylation levels occur between different populations.•The multiplex methylation SNaPshot is a useful method for age estimation. Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1–94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28–86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the m