Forensic Application Assessment Of A DNA Methylation Panel For Discriminating Monozygotic Twins

Objective: The identification of monozygotic twins(MZT)remains an unsolved problem in Forensic science.In our previous study,we developed a set of DNA methylation detection panel containing 9 sequences of 37 CpG sites.120 pairs of MZT were studied using this panel,94.20% of them show methylation difference greater than 5% and 55.33% of them with difference larger than 10%.In order to further evaluate the practical application value of the panel in Forensic science,this study carried out research on sensitivity,stability,tissue consistency,and simulation of forensic actual samples.Methods: We measured 26 pairs of MZT(0-80 years old)peripheral blood to verify the efficacy of the panel.Twenty healthy adult peripheral blood samples were collected four times at different time with 3-months interval to assess the longitudinal stability.Four kinds of tissues(heart,liver,kidney,brain)from 3 autopsy individuals were detected to evaluate tissue consistency.We also analyzed the DNA recovery rate after bisulfite conversion using two kits(EZ DNA Methylation-Gold Kit and EpiTect Fast DNA Bisulfite Kit),and the sensitivity of pyrosequencing at different concentrations of bisulfite-converted DNA.Blood stains,buccal swabs and 2 hairs with hair follicles were examined to validate the application values for common samples in forensic cases.The whole genome DNA of blood samples was extracted using E.Z.N.A Blood DNA Midi kit(OMEGA).The whole genome DNA of hair,buccal swabs,bloodstains,and tissue were extracted by QIAamp DNA Investigator Kit(Qiagen).Genomic DNA samples were treated with bisulfite conversion using EZ DNA Methylation-Gold Kit(ZYMO Research).The bisulfite-converted DNA was used as a template to amplify nine targetsequences of LINE-1,GS1,GS3,IGS1,IGS2,IGS3,IGS4,IGS5 and IGS6.The amplified products were sequenced using Pyromark Q48 Autoprep pyrosequencer(Qiagen)and the methylation values were calculated automatically by the Pyromark Q48 software.Statistical analysis was carried out using Excel and SPSS 24.0 software.Results:The mean methylation values of 132 tested blood samples were 61.68%,91.23%,91.45%,76.26%,75.45%,84.05%,80.86%,74.21%,and 82.52% for LINE-1,GS1,GS3,IGS1,IGS2,IGS3,IGS4,IGS5,IGS6 sequences respectively.In this study,the results of 26 pairs of MZT testing set showed that under the conditions of methylation difference thresholds of 5% and 10%,the number of MZT discriminated by the nine sequences was 22 pairs(84.62%)and 10 pairs(38.46%),respectively.There was no statistical difference with results of the training set in our previous study.The results of longitudinal stability indicated that no significant methylation differences were found among the four blood samples with a time span of 9 months,confirming the stability of 9 sequences methylation in a relatively short period of time.However,comparing the methylation degree of a pair of MZT in 2011 and2018,three sequences located in the genic region(LINE-1,GS1 and GS3)are stable without methylation variations,while other six sequences at intergenic regions(IGS1,IGS2,IGS3,IGS4,IGS5,and IGS6)showed a significant methylation decrease in 2018.The results of tissue difference testing demonstrated that the LINE-1,IGS1,IGS5 and IGS6 did not have DNA methylation differences in the four tissue samples of heart,liver,kidney and brain,but GS1,GS3,IGS2,IGS3 and IGS4 have significant tissue differences.The DNA methylation degree of GS1 in liver is higher than the other three tissues.The DNA methylation degree of IGS3 in brain tissue is lower than the other three tissues,and the DNA methylation difference is larger than 40%.Sensitivity study found that the average conversion recovery of EZ DNA Methylation-Gold Kit and EpiTect Fast DNA Bisulfite Kit was 74.24% and64.76%,respectively.For sequences LINE-1,GS1,GS3,IGS1 and IGS2,we were able to obtain successful pyrosequencing results when using 5 ng of bisulfate modified DNA.When the samples were amplified for the IGS3,IGS4,IGS5 and IGS6 sequences,good results were obtained using 10 ng of bisulfate modified DNA.For three kinds of forensic simulated samples,blood stains,buccal swabs and hair,the panel performed good and got results from all of them.However,significant methylation differences were observed among these three types of samples in GS1,IGS2,IGS4,and IGS5.Conclusions:The DNA methylation panel with nine sequences constructed by Pyrosequencing technology was evaluated for forensic application.The panel is capable of discriminating MZT to some degree.The methylation degree of 9sequences in the panel is relative stable within one year,but that of the intergenic sequences is changed after 7 years.The methylation of the GS1,GS3,IGS2,IGS3 and IGS4 show tissue differences.The panel can be used for forensic samples such as blood stains,buccal swabs and hair.The lowest concentration consistently detected varied from 5 to 10 ng,depending on the sequences,and the sensitivity of the panel can meet the requirements of regular amounts of forensic samples,which confirming the value for forensic application.However,the efficacy and sensitivity of the panel needs to be further improved.