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

Analysis of MCPIP1 protein nuclear localization in HepG2 cell line

ZC3H12A jest genem kodującym białko MCPIP1 (Monocyte chemoattractant protein-induced protein 1), które odgrywa istotną rolę w rozwoju stanu zapalnego. Został opisany po raz pierwszy w 2006 roku, jako nieznany dotychczas gen, którego ekspresja ulega indukcji po stymulacji makrofagów chemokiną MCP-1 [1, 2]. Prócz MCP-1, również cytokiny prozapalne takie jak IL-1b, TNFα, a także LPS aktywują ekspresję ZC3H12A [3, 4].Pierwotnie MCPIP1 opisane zostało jako białko jądrowe o funkcji czynnika transkrypcyjnego [1], jednak dane te nie zostały później potwierdzone. Ponadto, MCPIP1 został opisany również jako białko związane z cytoszkieletem komórki (aktyną) [4], oraz jako białko, które po nadekspresji formuje granule, pokrywające się przestrzennie z ciałkami GW/P, biorącymi udział w trawieniu transkryptów [5]. Różne doniesienia nie pozwalają jednoznacznie stwierdzić, czy białko MCPIP1 może lokować się w jądrze, a jeśli tak, to w jakich warunkach oraz jaką rolę może tam pełnić. Zbadanie hipotezy o jądrowej lokalizacji białka MCPIP1 było celem niniejszej pracy.W prezentowanej pracy przedstawiono wyniki dotyczące lokalizacji białka MCPIP1 w komórkach ludzkiego wątrobiaka HepG2. Przeprowadzone barwienia immunocytochemiczne pokazały, że endogenna forma białka jest obecna w jądrze. Następnie przy pomocy programu bioinformatycznego ELM [6, 7] wyszukano, a następnie zmutowano sekwencje, które mogłyby odpowiadać za jądrową lokalizację MCPIP1. Komórki stransfekowano stworzonymi konstruktami pozbawionymi sygnałów lokalizacji jądrowej (MCPIP1-NLS1+2) i eksportu jądrowego (MCPIP1-NES1+2), a także dziką formą białka. Barwienie immunocytochemiczne nie wykazało obecności nadekspresjonowanego MCPIP1 w jądrze, mimo że w analizie western blot odnotowano obecność dzikiej, nadekspresjonowanej formy tego białka we frakcji jądrowej. Uzyskany wynik barwienia nie pozwolił na ocenę funkcjonalności wyszukanych sekwencji sygnałowych. Sprawdzono także, czy wzmożona indukcja endogennej formy wpływa na jego lokalizację w komórkach. Stwierdzono, że po stymulacji komórek inhibitorami proteasomu: MG-132 [8, 9] oraz epoksomycyną [10], wzrost ekspresji endogennej formy MCPIP1 obserwuje się zarówno w jądrze jak i cytoplazmie.Podsumowując, w pracy wykazano jądrową i cytoplazmatyczną lokalizację białka MCPIP1, która zmienia się pod wpływem transfekcji.ZC3H12A is a gene encoding MCPIP1 protein (Monocyte chemoattractant protein-induced protein 1), which plays an important role during inflammation. It was described for the first time in 2006, as a previously unknown gene which expression was significantly induced after macrophage stimulation with MCP-1 chemokine [1, 2]. In addition to MCP-1, other inflammatory cytokines such as IL-1b, TNFα and LPS [3, 4] also activated expression of ZC3H12A gene.MCPIP1 was originally described as a transcription factor [1], however this data have not been confirmed. Moreover, MCPIP1 was described as a protein associated with cytoskeleton (actin) [4] and was also detected in GW/P bodies that are involved in transcripts degradation [5]. Importantly, it is still unclear whether MCPIP1 is present in the nucleus, and if so, under which conditions. Additionally, there are no data demonstrating MCPIP1 function in the nucleus. The aim of this thesis was to evaluate a cellular localization of MCPIP1 in human hepatoma cell line (HepG2).Immunocytochemistry stainings showed that endogenous form of MCPIP1 protein is present both in the nucleus and cytoplasm of HepG2 cells. Next, based on the in silico study a potential signaling sequences in the MCPIP1 protein were localized and mutated [6, 7]. We generated two constructs: first one encoding a protein devoid of nuclear localization signals (MCPIP1-NLS1+2) and second one: a protein without nuclear export signals (MCPIP1-NES1+2). Transient overexpression studies followed by immunocytochemistry staining did not show overexpression of MCPIP1 in the nucleus, although western blot analysis revealed the presence of overexpressed wild type MCPIP1 in the nuclear fraction. Lack of detectable amount of MCPIP1 in the nucleus did not allow to evaluate if mutated sequences play a role in subcellular protein localization. It was also examined whether induction of endogenous MCPIP1 affects its localization in the cell. It was found that HepG2 stimulation with proteasome inhibitor MG-132 [8, 9] and epoxomicin [10], increased expression of endogenous MCPIP1 in the nucleus and cytoplasm. In conclusion, study proved nuclear and cytoplasmic localization of endogenous MCPIP1 in HepG2 cells, which changed after transient MCPIP1 overexpression

Changes in regulators of lipid metabolism in the brain : a study of animal models of depression and hypothyroidism

Metabolic disturbances in the brain are assumed to be early changes involved in the pathogenesis of depression, and these alterations may be intensified by a deficiency of thyroid hormones. In contrast to glucose metabolism, the link between altered brain lipids and the pathogenesis of depression is poorly understood, therefore in the present study, we determine transcription factors and enzymes regulating cholesterol and fatty acid biosynthesis in the brain structures in an animal model of depression, hypothyroidism and the coexistence of these diseases. In used model of depression, a decrease in the active form of the transcription factor SREBP-2 in the hippocampus was demonstrated, thus suggesting a reduction in cholesterol biosynthesis. In turn, in the hypothyroidism model, the reduction of cholesterol biosynthesis in the frontal cortex was demonstrated by both the reduction of mature SREBP-2 and the concentration of enzymes involved in cholesterol biosynthesis. The lower expression of LDL receptors in the frontal cortex indicates the restriction of cholesterol uptake into the cells in the model of coexistence of depression and hypothyroidism. Moreover, the identified changes in the levels of SNAP-25, GLP-1R and GLP-2R pointed to disturbances in synaptic plasticity and neuroprotection mechanisms in the examined brain structures. In conclusion, a reduction in cholesterol synthesis in the hippocampus in the model of depression may be the reason for the reduction of synaptic plasticity, whereas a lower level of LDL-R occurring in the frontal cortex in rats from the model of depression and hypothyroidism coexistence could be the reason of anxiogenic and depression-like behaviors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43440-022-00395-8

The Effects of Prenatal Dexamethasone Exposure on Brain Metabolic Homeostasis in Adulthood: Implications for Depression

Since depression produces a long-term negative impact on quality of life, understanding the pathophysiological changes implicated in this disorder is urgent. There is growing evidence that demonstrates a key role for dysfunctional energy metabolism in driving the onset of depression; thus, bioenergetic alterations should be extensively studied. Brain metabolism is known to be a glucocorticoid-sensitive process, but the long-lasting consequences in adulthood following high levels of glucocorticoids at the early stages of life are unclear. We examined a possible association between brain energetic changes induced by synthetic glucocorticoid-dexamethasone treatment in the prenatal period and depressive-like behavior. The results show a reduction in the oxidative phosphorylation process, Krebs cycle impairment, and a weakening of the connection between the Krebs cycle and glycolysis in the frontal cortex of animals receiving dexamethasone, which leads to ATP reduction. These changes appear to be mainly due to decreased expression of pyruvate dehydrogenase, impairment of lactate transport to neurons, and pyruvate to the mitochondria. Acute stress in adulthood only slightly modified the observed alterations in the frontal cortex, while in the case of the hippocampus, prenatal exposure to dexamethasone made this structure more sensitive to future adverse factors

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