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A recent proof-of-concept pilot study proposed using microRNA (miRNA) markers for time of death determination. The markers - miRNA-142-5p and miRNA-541, were reported to show considerable expression differences in vitreous humor between individuals who died during the day or night. Here, we investigated whether these miRNA markers show the same diurnal expression pattern in blood, which would make them useful for estimating bloodstain deposition time to allow molecular alibi testing for forensic casework. We analyzed venous blood samples collected from 12 healthy individuals every 4h during the 24hday/night period under controlled sleep-laboratory conditions. MiRNA-142-5p normalized against miRNA-222 showed no statistically significant expression differences between blood samples collected during daytime and nighttime (one-way ANOVA p=0.81), and also no statistically significant rhythmicity during the 24hday/night period (cosine fit for all individuals p>0.05, averaged data p=0.932). MicroRNA-541 amplification in blood was above the 34-cycle threshold applied in the study, indicating too low quantities for obtaining reliable data. Overall, we conclude that the two miRNA markers previously suggested for time of death determination in vitreous humor are not suitable for estimating the deposition time of forensic bloodstains. Future studies may find out if miRNA markers with significant diurnal expression patterns can be identified and how useful they would be for forensic trace deposition timing., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Recently, the field of predicting phenotypes of externally visible characteristics (EVCs) from DNA genotypes with the final aim of concentrating police investigations to find persons completely unknown to investigating authorities, also referred to as Forensic DNA Phenotyping (FDP), has started to become established in forensic biology. We previously developed and forensically validated the IrisPlex system for accurate prediction of blue and brown eye colour from DNA, and recently showed that all major hair colour categories are predictable from carefully selected DNA markers. Here, we introduce the newly developed HIrisPlex system, which is capable of simultaneously predicting both hair and eye colour from DNA. HIrisPlex consists of a single multiplex assay targeting 24 eye and hair colour predictive DNA variants including all 6 IrisPlex SNPs, as well as two prediction models, a newly developed model for hair colour categories and shade, and the previously developed IrisPlex model for eye colour. The HIrisPlex assay was designed to cope with low amounts of template DNA, as well as degraded DNA, and preliminary sensitivity testing revealed full DNA profiles down to 63pg input DNA. The power of the HIrisPlex system to predict hair colour was assessed in 1551 individuals from three different parts of Europe showing different hair colour frequencies. Using a 20% subset of individuals, while 80% were used for model building, the individual-based prediction accuracies employing a prediction-guided approach were 69.5% for blond, 78.5% for brown, 80% for red and 87.5% for black hair colour on average. Results from HIrisPlex analysis on worldwide DNA samples imply that HIrisPlex hair colour prediction is reliable independent of bio-geographic ancestry (similar to previous IrisPlex findings for eye colour). We furthermore demonstrate that it is possible to infer with a prediction accuracy of >86% if a brown-eyed, black-haired individual is of non-European (excluding regions nearby Europe) versus European (including nearby regions) bio-geographic origin solely from the strength of HIrisPlex eye and hair colour probabilities, which can provide extra intelligence for future forensic applications. The HIrisPlex system introduced here, including a single multiplex test assay, an interactive tool and prediction guide, and recommendations for reporting final outcomes, represents the first tool for simultaneously establishing categorical eye and hair colour of a person from DNA. The practical forensic application of the HIrisPlex system is expected to benefit cases where other avenues of investigation, including STR profiling, provide no leads on who the unknown crime scene sample donor or the unknown missing person might be., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The ability to predict Externally Visible Characteristics (EVCs) from DNA, also referred to as Forensic DNA Phenotyping (FDP), is an exciting new chapter in forensic genetics holding great promise for tracing unknown individuals who are unidentifiable via standard forensic short tandem repeat (STR) profiling. For the purpose of DNA-based eye colour prediction, we previously developed the IrisPlex system consisting of a multiplex genotyping assay and a prediction model based on genotype and phenotype data from 3804 Dutch Europeans. Recently, we performed a forensic developmental validation study of the highly sensitive IrisPlex assay, which currently represents the only validated tool available for DNA-based prediction of eye colour in forensic applications. In the present study, we validate the IrisPlex prediction model by extending our initially described model towards genotype and phenotype data from multiple European populations. We performed IrisPlex analysis on 3840 individuals from seven sites across Europe as part of the European Eye (EUREYE) study for which DNA and high-resolution eye images were available. The accuracy rate of correctly predicting an individual's eye colour as being blue or brown, above the empirically established probability threshold of 0.7, was on average 94% across all seven European populations, ranging from 91% to 98%, despite the large variation in eye colour frequencies between the populations. The overall prediction accuracies expressed by the area under the receiver characteristic operating curves (AUC) were 0.96 for blue and 0.96 for brown eyes, which is considerably higher than those established before. The IrisPlex prediction model parameters generated from this multi-population European dataset, and thus its prediction capabilities, were highly comparable to those previously established. Therefore, the increased information regarding eye colour phenotype and genotype distributions across Europe, and the system's ability to provide eye colour predictions across Europe accurately, both highlight additional evidence for the utility of the IrisPlex system in forensic casework., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The panels of 9-17 Y-chromosomal short tandem repeats (Y-STRs) currently used in forensic genetics have adequate resolution of different paternal lineages in many human populations, but have lower abilities to separate paternal lineages in populations expressing low Y-chromosome diversity. Moreover, current Y-STR sets usually fail to differentiate between related males who belong to the same paternal lineage and, as a consequence, conclusions cannot be drawn on the individual level as is desirable for forensic interpretations. Recently, we identified a new panel of rapidly mutating (RM) Y-STRs, composed of 13 markers with mutation rates above 1 × 10(-2), whereas most Y-STRs, including all currently used in forensics, have mutation rates in the order of 1 × 10(-3) or lower. In the present study, we demonstrate in 604 unrelated males sampled from 51 worldwide populations (HGDP-CEPH) that the RM Y-STRs provide substantially higher haplotype diversity and haplotype discrimination capacity (with only 3 haplotypes shared between 8 of the 604 worldwide males), than obtained with the largest set of 17 currently used Y-STRs (Yfiler) in the same samples (33 haplotypes shared between 85 males). Hence, RM Y-STRs yield high-resolution paternal lineage differentiation and provide a considerable improvement compared to Yfiler. We also find in this worldwide dataset substantially less genetic population substructure within and between geographic regions with RM Y-STRs than with Yfiler Y-STRs. Furthermore, with the present study we provide enhanced data evidence that the RM Y-STR panel is extremely successful in differentiating between closely and distantly related males. Among 305 male relatives, paternally connected by 1-20 meiotic transfers in 127 independent pedigrees, we show that 66% were separated by mutation events with the RM Y-STR panel whereas only 15% were with Yfiler; hence, RM Y-STRs provide a statistically significant 4.4-fold increase of average male relative differentiation relative to Yfiler. The RM Y-STR panel is powerful enough to separate closely related males; nearly 50% of the father and sons, and 60% of brothers could be distinguished with RM Y-STRs, whereas only 7.7% and 8%, respectively, with Yfiler. Thus, by introducing RM Y-STRs to the forensic genetic community we provide important solutions to several of the current limitations of Y chromosome analysis in forensic genetics., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We analyzed 67 short tandem repeat polymorphisms from the non-recombining part of the Y-chromosome (Y-STRs), including 49 rarely studied simple single-copy (ss)Y-STRs and 18 widely used Y-STRs, in 590 males from 51 populations belonging to 8 worldwide regions (HGDP-CEPH panel). Although autosomal DNA profiling provided no evidence for close relationship, we found 18 Y-STR haplotypes (defined by 67 Y-STRs) that were shared by two to five men in 13 worldwide populations, revealing high and widespread levels of cryptic male relatedness. Maximal (95.9%) haplotype resolution was achieved with the best 25 out of 67 Y-STRs in the global dataset, and with the best 3-16 markers in regional datasets (89.6-100% resolution). From the 49 rarely studied ssY-STRs, the 25 most informative markers were sufficient to reach the highest possible male lineage differentiation in the global (92.2% resolution), and 3-15 markers in the regional datasets (85.4-100%). Considerably lower haplotype resolutions were obtained with the three commonly used Y-STR sets (Minimal Haplotype, PowerPlex Y, and AmpFlSTR Yfiler. Six ssY-STRs (DYS481, DYS533, DYS549, DYS570, DYS576 and DYS643) were most informative to supplement the existing Y-STR kits for increasing haplotype resolution, or - together with additional ssY-STRs - as a new set for maximizing male lineage differentiation. Mutation rates of the 49 ssY-STRs were estimated from 403 meiotic transfers in deep-rooted pedigrees, and ranged from approximately 4.8 x 10(-4) for 31 ssY-STRs with no mutations observed to 1.3 x 10(-2) and 1.5 x 10(-2) for DYS570 and DYS576, respectively, the latter representing the highest mutation rates reported for human Y-STRs so far. Our findings thus demonstrate that ssY-STRs are useful for maximizing global and regional resolution of male lineages, either as a new set, or when added to commonly used Y-STR sets, and support their application to forensic, genealogical and anthropological studies.