The EpiTect Methyl qPCR Assay as novel age estimation method in forensic biology

Human aging is associated with epigenetic modification of the genome. DNA methylation at cytosines appears currently as the best characterised modification that occurs during the mammalian lifetime. Such methylation changes at regulatory region can provide insights to track contributor age for criminal investigation. The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands in the promoter regions of a number of age related genes, of which four successfully showed changes across the lifespan (NPTX2, KCNQ1DN, GRIA2 and TRIM58). This technique is based on the detection of remaining input genome after digestion with a methylation-sensitive restriction enzyme. This study examined DNA specimens from 80 female subjects of various ages (18-91 years) obtained from blood, using primers designed to flank the studied gene loci. The data obtained from DNA methylation quantification showed successful discrimination among volunteered ages. Overall, the difference between predicted and real age was about 11 years and absolute mean differences (AMD) was only 7.2 years error. We suggest the EpiTect system can be used as fast and simple innovative tool in future forensic age estimation

Quantification of global mitochondrial DNA methylation levels and inverse correlation with age at two CpG sites

Mammalian ageing features biological attrition evident at cellular, genetic and epigenetic levels. Mutation of mitochondrial DNA, and nuclear DNA methylation changes are well established correlates of ageing. The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. Here we describe the bisulphite sequencing of mtDNA from 82 individuals aged 18-91 years. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse correlation with subject age suggesting their utility as epigenetic markers of ageing

Epigenetic markers in forensics and ageing

This PhD project describes the development of two novel forensic approaches the use of trace DNA isolation and profiling from fired gun cartridges, and the development and application of epigenetic analyses of the autosomal and mitochondrial genomes in order to estimate age.In the first part of the work two novel swabs (forensiX) and a standard cotton swab (EUROTUBE/Deltalab) were compared for the collection of forensic samples. Additionally, the effect of long-term storage of these samples was assessed. The forensiX swabs generally produced a higher (two- to four-fold) DNA yield compared to standard swab (around 750 pg and 250 pg, respectively, from 0.5 μL of saliva). These findings demonstrate the importance of 'active drying' performance in the preservation of DNA and swab selection. Using the forensiX swabs, we subsequently produced data that indicated that DNA samples deposited on cartridges during loading can survive firing from eight different types of gun.It has been shown that the methylation status of certain human DNA loci correlates with ageing. Blood sample DNA from a cohort of 82 women aged 18 to 91 years was obtained. We used Illumina MiSeq next generation sequencing platform to investigate the promoter regions of 23 genes suggested to have age-correlated methylation levels. Methylation levels at three CpGs located in the ASPA, ITGA2B and PDE4C genes showed an epigenetic signature of ageing with only a 6 year error range from chronological age.The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. We describe the bisulphite sequencing of mtDNA from the same cohort of individuals. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse relationship with age.We wished to create a fast and simple forensic tool (compared to next generation sequencing) for practical age estimation. The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands of the promoter regions of 4 age related genes (NPTX2, KCNQ1DN, GRIA2 and TRIM58). The data obtained from DNA methylation quantification showed successful estimation of subject age (11 year accuracy).This thesis describes practical steps to obtain forensic DNA samples while also applying novel techniques to explore the use of epigenetic profiling as a means to age prediction. The data generated suggest that the analysis of methylation patterns of both specific autosomal and mitochondrial gene sequences from biological evidence left at crime scene may help build up an accurate picture of an offender's age and may help investigators obtain better descriptive information about a contributor from DNA, regardless of database inclusion.This PhD project describes the development of two novel forensic approaches the use of trace DNA isolation and profiling from fired gun cartridges, and the development and application of epigenetic analyses of the autosomal and mitochondrial genomes in order to estimate age.In the first part of the work two novel swabs (forensiX) and a standard cotton swab (EUROTUBE/Deltalab) were compared for the collection of forensic samples. Additionally, the effect of long-term storage of these samples was assessed. The forensiX swabs generally produced a higher (two- to four-fold) DNA yield compared to standard swab (around 750 pg and 250 pg, respectively, from 0.5 μL of saliva). These findings demonstrate the importance of 'active drying' performance in the preservation of DNA and swab selection. Using the forensiX swabs, we subsequently produced data that indicated that DNA samples deposited on cartridges during loading can survive firing from eight different types of gun.It has been shown that the methylation status of certain human DNA loci correlates with ageing. Blood sample DNA from a cohort of 82 women aged 18 to 91 years was obtained. We used Illumina MiSeq next generation sequencing platform to investigate the promoter regions of 23 genes suggested to have age-correlated methylation levels. Methylation levels at three CpGs located in the ASPA, ITGA2B and PDE4C genes showed an epigenetic signature of ageing with only a 6 year error range from chronological age.The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. We describe the bisulphite sequencing of mtDNA from the same cohort of individuals. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse relationship with age.We wished to create a fast and simple forensic tool (compared to next generation sequencing) for practical age estimation. The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands of the promoter regions of 4 age related genes (NPTX2, KCNQ1DN, GRIA2 and TRIM58). The data obtained from DNA methylation quantification showed successful estimation of subject age (11 year accuracy).This thesis describes practical steps to obtain forensic DNA samples while also applying novel techniques to explore the use of epigenetic profiling as a means to age prediction. The data generated suggest that the analysis of methylation patterns of both specific autosomal and mitochondrial gene sequences from biological evidence left at crime scene may help build up an accurate picture of an offender's age and may help investigators obtain better descriptive information about a contributor from DNA, regardless of database inclusion