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

Ablation of CNTN2+Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation

Corticothalamic axons express Contactin-2 (CNTN2/TAG-1), a neuronal recognition molecule of the immunoglobulin superfamily involved in neurogenesis, neurite outgrowth, and fasciculation. TAG-1, which is expressed transiently by cortical pyramidal neurons during embryonic development, has been shown to be fundamental for axonal recognition, cellular migration, and neuronal proliferation in the developing cortex. Although Tag-1(-/-) mice do not exhibit any obvious defects in the corticofugal system, the role of TAG-1+ neurons during the development of the cortex remains elusive. We have generated a mouse model expressing EGFP under the Tag-1 promoter and encompassing the coding sequence of Diptheria Toxin subunit A (DTA) under quiescence with no effect on the expression of endogenous Tag-1. We show that while the line recapitulates the expression pattern of the molecule, it highlights an extended expression in the forebrain, including multiple axonal tracts and neuronal populations, both spatially and temporally. Crossing these mice to the Emx1-Cre strain, we ablated the vast majority of TAG-1+ cortical neurons. Among the observed defects were a significantly smaller cortex, a reduction of corticothalamic axons as well as callosal and commissural defects. Such defects are common in neurodevelopmental disorders, thus this mouse could serve as a useful model to study physiological and pathophysiological cortical development

Validated inference of smoking habits from blood with a finite DNA methylation marker set

Inferring a person’s smoking habit and history from blood is relevant for complementing or replacing self-reports in epidemiological and public health research, and for forensic applications. However, a finite DNA methylation marker set and a validated statistical model based on a large dataset are not yet available. Employing 14 epigenome-wide association studies for marker discovery, and using data from six population-based cohorts (N = 3764) for model building, we identified 13 CpGs most suitable for inferring smoking versus non-smoking status from blood with a cumulative Area Under the Curve (AUC) of 0.901. Internal fivefold cross-validation yielded an average AUC of 0.897 ± 0.137, while external model validation in an independent population-based cohort (

Differentially methylated embryonal Fyn-associated substrate (EFS) gene as a blood-specific epigenetic marker and its potential application in forensic casework

DNA methylation patterns have the ability to reveal the activities of genes within a certain tissue at a particular time point. Tissue-specific DNA methylation patterns have been previously investigated for their applicability in the identification of forensically relevant body fluids, however there is still a lack in robust markers. While following a genome-wide scale investigation has a great potential to reveal useful tissue-specific changes, a gene-targeted approach can also lead to significant outcomes, especially in genomic locations not included in the genome-wide experiments. In this study, the potential of the candidate embryonal Fyn-associated substrate (EFS) gene for the positive identification of whole blood was investigated. For this purpose, the methylation profile of a selected genomic region containing a total of 10 CpG sites was analysed in 124 individuals via bisulfite pyrosequencing. Volunteers donated various forensically relevant tissues, including whole blood, saliva, seminal fluid, vaginal fluid and menstrual secretion. Whole blood showed the highest levels of DNA methylation (mean = 0.67), while semen samples were found to be very low methylated (mean = 0.06). The remaining tissues demonstrated partial mean methylation levels; more specifically, saliva − 0.43, vaginal fluid − 0.22 and menstrual blood − 0.22. One out of the 10 analysed CpG sites, CpG4, showed to be more robust, resulting in not only the highest methylation difference between blood and the rest of the tissues, but also the lowest inter-individual methylation difference. The proposed pyrosequencing assay was found to be accurate, linear and reproducible. Lastly, the method's applicability to forensic casework was assessed via the analysis of very old bloodstains stored up to 18 years, blood DNA samples stored long-term up to 9 years, mixed stains as well as other ‘forensic-like’ samples. In the majority of cases the expected methylation ratios were obtained indicating a stable DNA methylation pattern, however caution is necessary when analysing low quantity and/or quality samples due to potential stochastic effects. Future validation experiments can shed more light into the usefulness of EFS locus as a promising blood-specific epigenetic marker

Tag1 deficiency results in olfactory dysfunction through impaired migration of mitral cells

The olfactory system provides mammals with the abilities to investigate, communicate and interact with their environment. These functions are achieved through a finely organized circuit starting from the nasal cavity, passing through the olfactory bulb and ending in various cortical areas. We show that the absence of transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice results in a significant and selective defect in the number of the main projection neurons in the olfactory bulb, namely the mitral cells. A subpopulation of these projection neurons is reduced in Tag1- deficient mice as a result of impaired migration. We demonstrate that the detected alterations in the number of mitral cells are well correlated with diminished odor discrimination ability and social long-term memory formation. Reduced neuronal activation in the olfactory bulb and the corresponding olfactory cortex suggest that Tag1 is crucial for the olfactory circuit formation in mice. Our results underpin the significance of a numerical defect in the mitral cell layer in the processing and integration of odorant information and subsequently in animal behavior. © 2015. Published by The Company of Biologists Ltd

Revisiting genetic artifacts on DNA methylation microarrays exposes novel biological implications

Background: Illumina DNA methylation microarrays enable epigenome-wide analysis vastly used for the discovery of novel DNA methylation variation in health and disease. However, the microarrays’ probe design cannot fully consider the vast human genetic diversity, leading to genetic artifacts. Distinguishing genuine from artifactual genetic influence is of particular relevance in the study of DNA methylation heritability and methylation quantitative trait loci. But despite its importance, current strategies to account for genetic artifacts are lagging due to a limited mechanistic understanding on how such artifacts operate. Results: To address this, we develop and benchmark UMtools, an R-package containing novel methods for the quantification and qualification of genetic artifacts based on fluorescence intensity signals. With our approach, we model and validate known SNPs/indels on a genetically controlled dataset of monozygotic twins, and we estimate minor allele frequency from DNA methylation data and empirically detect variants not included in dbSNP. Moreover, we identify examples where genetic artifacts interact with each other or with imprinting, X

Estimating the Time Since Deposition of Saliva Stains With a Targeted Bacterial DNA Approach

Information on the time when a stain was deposited at a crime scene can be valuable in forensic investigations. It can link a DNA-identified stain donor with a crime or provide a post-mortem interval estimation in cases with cadavers. The available methods for estimating stain deposition time have limitations of different types and magnitudes. In this proof-of-principle study we investigated for the first time the use of microbial DNA for this purpose in human saliva stains. First, we identified the most abundant and frequent bacterial species in saliva using publicly available 16S rRNA gene next generation sequencing (NGS) data from 1,848 samples. Next, we assessed time-dependent changes in 15 identified species using de-novo 16S rRNA gene NGS in the saliva stains of two individuals exposed to indoor conditions for up to 1 year. We selected four bacterial species, i.e., Fusobacterium periodonticum, Haemophilus parainfluenzae, Veillonella dispar, and Veillonella parvula showing significant time-dependent changes and developed

Differentiating between monozygotic twins through DNA methylation specific high resolution melt curve analysis

Although STR profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterised by bi-sulfite treatment followed by pyrosequencing. However, this can be time consuming and expensive, thus unlikely to be widely used by investigators. If the sequences are different, then, in theory, the melting temperature should be different. Thus the aim of this study is to assess whether high resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs which underwent extraction, quantification, bi-sulfite treatment, PCR amplification and high resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins, targeting Alu-E2F3, and in four out of five MZ twins, targeting Alu-SP (p<0.05). Thus it has been demonstrated that bi-sulfite treatment followed by high resolution melting curve analysis could be used to differentiate between MZ twins