Investigating the Epigenetic Discrimination of Identical Twins Using Buccal Swabs, Saliva, and Cigarette Butts in the Forensic Setting

Abstract

Monozygotic (MZ) twins are typically indistinguishable via forensic DNA profiling. Recently, we demonstrated that epigenetic differentiation of MZ twins is feasible; however, proportions of twin differentially methylated CpG sites (tDMSs) identified in reference-type blood DNA were not replicated in trace-type blood DNA. Here we investigated buccal swabs as typical forensic reference material, and saliva and cigarette butts as commonly encountered forensic trace materials. As an analog to a forensic case, we analyzed one MZ twin pair. Epigenome-wide microarray analysis in reference-type buccal DNA revealed 25 candidate tDMSs with >0.5 twin-to-twin differences. MethyLight quantitative PCR (qPCR) of 22 selected tDMSs in trace-type DNA revealed in saliva DNA that six tDMSs (27.3%) had >0.1 twin-to-twin differences, seven (31.8%) had smaller (<0.1) but robustly detected differences, whereas for nine (40.9%) the differences were in the opposite direction relative to the microarray data; for cigarette butt DNA, results were 50%, 22.7%, and 27.3%, respectively. The discrepancies between reference-type and trace-type DNA outcomes can be explained by cell composition differences, method-to-method variation, and other technical reasons including bisulfite conversion inefficiency. Our study highlights the importance of the DNA source and that careful characterization of biological and technical effects is needed before epigenetic MZ twin differentiation is applicable in forensic casework.

Applications of massively parallel sequencing in forensic genetics

“…The first study to report that this epigenetic modification differs between MZ twins was published in 2005 ( Fraga et al, 2005 ), and, since then, many other attempts to discriminate them by methylation analysis have been made. So far, the methodology employed in such studies includes array chips ( Park et al, 2017 ), array followed by qPCR ( Vidaki et al, 2017b ; Vidaki et al , 2018 ), PCR-high resolution melting ( Marqueta-Gracia et al, 2018 ), and other techniques ( Xu et al, 2015 ; Du et al, 2015 ). In all the studies, at least a fraction of MZ twin pairs were able to be discerned.…”

Developments in forensic DNA analysis

“…The addition of a methyl group (-CH 3 ) to the 5 0 position of cytosine residues in the human genome, primarily those found in cytosine-guanine dinucleotides (known as CpG sites), is one of the most widely studied epigenetic modifications, and observations of differential methylation patterns with age and across tissue types has led to interest in forensic applications of DNA methylation analysis [101]. Research in this area has focused on estimation of the age of the donor of a DNA sample and the identification of tissue-type for body fluids and other forensically relevant biological samples [102][103][104][105], although DNA methylation analysis has a range of other forensic applications including the discrimination of monozygotic twins [105] and the determination of smoking status [106].…”

Interpol review of forensic biology and forensic DNA typing 2016-2019

“…DNA methylation studies have explored the outcome of DNA methylation analysis using simulated low amounts of DNA [ 161 ], evaluated MPS methods for forensic methylation profiling [ 162 ], and investigated epigenetic discrimination of identical twins using buccal swabs, saliva, and cigarette butts [ 163 ].…”

“…A special issue on forensic genomics was organized in Genes [ 217 ] by guest editors Manfred Kayser and Walther Parson with 11 articles published between November 2017 and December 2018 (see https://www.mdpi.com/journal/genes/special_issues/Forensic_Genomics ). Topics for these open access articles include performing molecular analysis of the RNA transcriptome for human organ tissue identification to assist in investigations of traumatic injury [ 218 ], investigating the epigenetic discrimination of identical twins using reference sample buccal swabs and saliva and cigarette butts common to forensic evidence [ 163 ], recovering fragmented nuclear DNA from human hair shafts [ 219 ], using high-throughput sequencing to recover nuclear DNA from a 4000-year-old Egyptian mummy head [ 220 ], examining mitochondrial DNA heteroplasmy with MPS to help distinguish maternal relatives [ 221 ], dating juvenile blow flies to assist forensic entomology with postmortem interval estimation [ 222 ], predicting the postmortem interval using microbiome data [ 223 ], applying NGS probe capture enrichment techniques to examine the whole mitochondrial genome and 426 nuclear SNPs on individual telogen hairs [ 224 ], and demonstrating that flanking region variation can impact rates of stutter product formation in STR markers [ 225 ].…”