Athina Vidaki

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

Forensic DNA methylation profiling - Potential opportunities and challenges

Investigating the DNA sequence is the most powerful tool that can be employed in forensic genetics for the identification of an individual, or to determine specific ethnic and phenotypic characteristics. However, there are also other heritable changes in gene function or cellular phenotype which are caused by mechanisms other than differences in the DNA sequence itself. Over the last decade it has become evident that epigenetic markers can be of substantial forensic significance. The determination of possible alterations in DNA methylation patterns could aid various forensic investigations, such as differentiating monozygotic twins, identifying the tissue source or determining the age of tissue donors. This review aims to give a brief overview of the possible advantages of forensic DNA methylation profiling and sheds light on the limitations of this approach.

DNA methylation-based forensic age prediction using artificial neural networks and next generation sequencing

Highlights • Blood DNA methylation profiles of 1156 individuals were assessed for age correlation.• Stepwise regression identified 23 age-associated CpG sites in DNA from blood.• A machine learning model based on 16 markers predicted age with a mean error of 3.8 years.• The model predicted age successfully for twins and ‘diseased’ individuals.• A new NGS-based method was combined with machine learning for age prediction.

RNA/DNA co-analysis from human skin and contact traces – results of a sixth collaborative EDNAP exercise

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

The helix-loop-helix protein Id2 is expressed differentially and induced by myc in T-cell lymphomas

Inhibitor of DNA binding 2 (Id2), a helix‐loop‐helix protein of the inhibitor of differentiation (ID) family, is involved in hematopoiesis, mainly through interaction with the E‐family of transcription factors. Recent studies have shown that Id2 is overexpressed in some types of B‐cell lymphoma, including classical Hodgkin lymphoma. The authors of the current study hypothesized that Id2 also is overexpressed in T‐cell lymphomas.

Discovery of potential DNA methylation markers for forensic tissue identification using bisulphite pyrosequencing

The presence of specific body fluids at crime scenes could be linked with particular types of crime, therefore attributing a DNA profile to a specific tissue could increase the evidential significance of a match with a suspect. Current methodologies such as tissue‐specific mRNA profiling are useful but drawbacks include low tissue specificity and applicability to degraded samples. In this study, the potential of 11 tissue‐specific differentially methylated regions, initially identified following large‐scale methylation analysis of whole blood, buccal cells and sperm, was explored in order to identify markers for blood, saliva and semen. Bisulphite pyrosequencing analysis supported previous findings, but tissue‐specific differentially methylated regions for blood and buccal cells did not show enough specificity to be proposed as markers for blood and saliva, respectively. For some CpGs, a large inter‐individual variation in methylation levels was also observed. Two of the semen markers (cg04382920 and cg11768416) were used for further validation on a large set of stains. These two semen‐specific assays showed high sensitivity (as low as 50 pg) and stability. Future experiments will shed light on the usefulness of these markers in forensic casework.

Epigenetic discrimination of identical twins from blood under the forensic scenario

Monozygotic (MZ) twins share the same STR profile, demonstrating a practical problem in forensic casework. DNA methylation has provided a suitable resource for MZ twin differentiation; however, studies addressing the forensic feasibility are lacking. Here, we investigated epigenetic MZ twin differentiation from blood under the forensic scenario comprising i) the discovery of candidate markers in reference-type blood DNA via genome-wide analysis, ii) the technical validation of candidate markers in reference-type blood DNA using a suitable targeted method, and iii) the analysis of the validated markers in trace-type DNA. Genome-wide methylation analysis in blood DNA from 10 MZ twin pairs resulted in 19-111 twin-differentially methylated sites (tDMSs) per pair with >0.3 twin-to-twin differences. Considering all top three candidate tDMSs across all pairs in the technical validation based on methylation-specific qPCR, 67.85% generated >0.1 twin-to-twin differences. Of the validated tDMSs, 68.4% showed >0.1 twin-to-twin differences with qPCR in trace-type DNA across 8 pairs. Using an updated marker selection strategy, 8 additional candidate tDMSs were obtained for an example MZ pair, of which 7 showed >0.1 twin-to-twin differences in both reference- and trace-type DNA. Lastly, we introduce a high-resolution melting curve analysis of the entire fragment that can complement the proposed approach. Overall, our study demonstrates the general feasibility of epigenetic twin differentiation in the forensic context and highlights that the number of informative tDMSs in the final trace DNA analysis is crucial, as some candidate markers identified in reference DNA were shown not informative in the trace DNA due to various, including technical, reasons. Future studies will need to address the optimal number of epigenetic markers required for reliable identification of MZ twin individuals including statistical considerations.

From forensic epigenetics to forensic epigenomics: broadening DNA investigative intelligence

Human genetic variation is a major resource in forensics, but does not allow all forensically relevant questions to be answered. Some questions may instead be addressable via epigenomics, as the epigenome acts as an interphase between the fixed genome and the dynamic environment. We envision future forensic applications of DNA methylation analysis that will broaden DNA-based forensic intelligence. Together with genetic prediction of appearance and biogeographic ancestry, epigenomic lifestyle prediction is expected to increase the ability of police to find unknown perpetrators of crime who are not identifiable using current forensic DNA profiling.

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

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.

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.