The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, we provide an update of the publically available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for 3 eye, 4 hair, and 5 skin colour categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin colour prediction, and reflects the first forensically validated tool for simultaneous eye, hair and skin colour prediction from DNA

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a+ NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a+ CXCR6+ NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance

NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity

T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)-interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5-IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5-IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a; +; NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5-IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ-inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a; +; CXCR6; +; NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance

Methods for studying the zebrafish brain: Past, present and future

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience

Functional properties of habenular neurons are determined by developmental stage and sequential neurogenesis.

The developing brain undergoes drastic alterations. Here, we investigated developmental changes in the habenula, a brain region that mediates behavioral flexibility during learning, social interactions, and aversive experiences. We showed that developing habenular circuits exhibit multiple alterations that lead to an increase in the structural and functional diversity of cell types, inputs, and functional modules. As the habenula develops, it sequentially transforms into a multisensory brain region that can process visual, olfactory, mechanosensory, and aversive stimuli. Moreover, we observed that the habenular neurons display spatiotemporally structured spontaneous activity that shows prominent alterations and refinement with age. These alterations in habenular activity are accompanied by sequential neurogenesis and the integration of distinct neural clusters across development. Last, we revealed that habenular neurons with distinct functional properties are born sequentially at distinct developmental time windows. Our results highlight a strong link between the functional properties of habenular neurons and their precise birthdate

Towards broadening Forensic DNA Phenotyping beyond pigmentation: Improving the prediction of head hair shape from DNA

Human head hair shape, commonly classified as straight, wavy, curly or frizzy, is an attractive target for Forensic DNA Phenotyping and other applications of human appearance prediction from DNA such as in paleogenetics. The genetic knowledge underlying head hair shape variation was recently improved by the outcome of a series of genome-wide association and replication studies in a total of 26,964 subjects, highlighting 12 loci of which 8 were novel and introducing a prediction model for Europeans based on 14 SNPs. In the present study, we evaluated the capacity of DNA-based head hair shape prediction by investigating an extended set of candidate SNP predictors and by using an independent set of samples for model validation. Prediction model building was carried out in 9674 subjects (6068 from Europe, 2899 from Asia and 707 of admixed European and Asian ancestries), used previously, by considering a novel list of 90 candidate SNPs. For model validation, genotype and phenotype data were newly collected in 2415 independent subjects (2138 Europeans and 277 non-Europeans) by applying two targeted massively parallel sequencing platforms, Ion Torrent PGM and MiSeq, or the MassARRAY platform. A binomial model was developed to predict straight vs. non-straight hair based on 32 SNPs from 26 genetic loci we identified as significantly contributing to the model. This model achieved prediction accuracies, expressed as AUC, of 0.664 in Europeans and 0.789 in non-Europeans; the statistically significant difference was explained mostly by the effect of one EDAR SNP in non-Europeans. Considering sex and age, in addition to the SNPs, slightly and insignificantly increased the prediction accuracies (AUC of 0.680 and 0.800, respectively). Based on the sample size and candidate DNA markers investigated, this study provides the most robust, validated, and accurate statistical prediction models and SNP predictor marker sets currently available for predicting head hair shape from DNA, providing the next step towards broadening Forensic DNA Phenotyping beyond pigmentation traits

Further evidence for population specific differences in the effect of DNA markers and gender on eye colour prediction in forensics

The genetics of eye colour has been extensively studied over the past few years, and the identified polymorphisms have been applied with marked success in the field of Forensic DNA Phenotyping. A picture that arises from evaluation of the currently available eye colour prediction markers shows that only the analysis of HERC2-OCA2 complex has similar effectiveness in different populations, while the predictive potential of other loci may vary significantly. Moreover, the role of gender in the explanation of human eye colour variation should not be neglected in some populations. In the present study, we re-investigated the data for 1020 Polish individuals and using neural networks and logistic regression methods explored predictive capacity of IrisPlex SNPs and gender in this population sample. In general, neural networks provided higher prediction accuracy comparing to logistic regression (AUC increase by 0.02–0.06). Four out of six IrisPlex SNPs were associated with eye colour in the studied population. HERC2 rs12913832, OCA2 rs1800407 and SLC24A4 rs12896399 were found to be the most important eye colour predictors (p < 0.007) while the effect of rs16891982 in SLC45A2 was less significant. Gender was found to be significantly associated with eye colour with males having ~1.5 higher odds for blue eye colour comparing to females (p = 0.002) and was ranked as the third most important factor in blue/non-blue eye colour determination. However, the implementation of gender into the developed prediction models had marginal and ambiguous impact on the overall accuracy of prediction confirming that the effect of gender on eye colour in this population is small. Our study indicated the advantage of neural networks in prediction modeling in forensics and provided additional evidence for population specific differences in the predictive importance of the IrisPlex SNPs and gender