We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice ( Cardiotoxicity due to anthracyclines (CDA) is a frequent problem in cancer patients that limits the efficacy of chemotherapy . Long-teThe diversity of the results observed when attempting to identify CDA genetic markers may result from the CDA being a complex polygenic disease or complex trait influenced by multiple genes contributing at systemic, tissue, cellular, and molecular levels. It is estimated that CDA has a proportion of phenotype variation due to genetics . HoweverIt was previously proposed that the missing heritability of complex traits could be due to genes that exert their influence at the level of intermediate phenotypes, such as arterial hypertension. However, they would not be powerful enough and contribute with sufficient effect to be detected at the level of the primary complex phenotype, such as acute myocardial infarction, in which they participate in its pathogenesis ,17. ThisLoci (eQTLs) may control blood gene expression [It is predicted that common small-effect genes affecting a complex trait might be located throughout much of the genome. For example, between 71% and 100% of 1 Mb windows could contribute to the heritability of schizophrenia , and thopression . Additiopression . These opression ,25. Thuspression . It is dpression . Howeverpression ,27.In this work, we propose that genetic determinants linked to intermediate molecular phenotypes of CDA could help quantify susceptibility to this chemotherapy complication. We illustrate the rationale of our study in FVB/N-Tg(MMTVneu)202Mul/J transgenic mice (hereafter FVB), carrying the Avian erythroblastosis oncogene B2/Neuroblastoma-derived (ErbB2/cNeu) protooncogene, expressed under the mouse mammary tumor virus (MMTV) promoter (MMTV-Erbb2/Neu transgene) and allowed to develop breast cancer as shown by Castellanos et al. [We generated a genetically heterogeneous mouse cohort by backcrossing two inbred strains. We crossed a breast cancer-resistant mouse strain, C57BL/6 (hereafter C57), with a susceptible strain, FVB/J, to generate F1 mice. To generate the backcross mice , the non-transgenic F1 mice were crossed with s et al. ,28.2 when the tumors were bigger than 15 mm or showed signs of suffering, such as weight loss, dyspnea, and skin wounds. FVB transgenic mice were obtained from the Jackson Laboratories, and wild-type FVB/N and C57BL/6 mice were purchased from Charles River.Each mouse from the backcross cohort carried a unique combination of alleles from the two strains (FVB and C57) in variable proportions. In this combination, the genetic component from the FVB strain was predominant since it was used to generate the backcross with the F1 mice. FVB alleles can be homozygous or heterozygous, while the C57 component is reduced and heterozygous when present. Because each mouse is genetically unique, we can explore the phenotype variation (heart damage) among individuals throughout the cohort ,30,31. Tn = 87, from them 70 were F1BX mice) was treated with doxorubicin every ten days with a dose of 5 mg/kg, and another group  received the combined therapy of doxorubicin (Pfizer) (5 mg/kg) plus docetaxel (Sanofi Aventis) (25 mg/kg), administered intraperitoneally every ten days. The age of the FVB and F1 mice was also heterogeneous because they were treated when the animals developed breast cancer, thus varying between 51 and 114 weeks of age. Without therapy, FVB control mice were used to evaluate subendocardial (n = 5) and subepicardial zones (n = 5). Litters from the same cage and age were randomly ascribed to each treatment group. The drug doses used mimicked the clinically relevant drug concentration [All mice were housed in ventilated filter cages in the Animal Research Facility of our Institution under specific-pathogen-free (SPF) conditions, environmental enrichment, and fed and watered ad libitum. We evaluated cardiotoxicity in 164 mice: 130 F1BX, 18 FVB, and 16 F1 (the latter having been generated after crossing FVB transgenic mice with C57). One group  for 24 h and then processed in an automatic system . The subsequent samples were sectioned, embedded in paraffin, and stained with hematoxylin-eosin with a standard protocol or the Masson-Goldner Trichrome kit  to evaluate the cardiac fibrosis and cardiomyocyte area. We robotically quantified heart fibrosis and the average area of myocardial fibers as pathophenotypes of CDA using the Ariol slide scanner to avoid intra- and inter-observer deviations. Histopathological damage was measured in the subendocardium and subepicardium from five randomly chosen regions of each sample. Regarding protocols for quantifying intermediate molecular phenotypes, see the ® technology  and conducted in accordance with the manufacturer’s guidelines on a Bioplex 200 device . Protein quantification adhered to specifications set forth by the manufacturer , with each sample containing 18.5 µg of total protein. The fluorescence readings from the heart samples fell within the linear dynamic range. Using a 7 Plex DNA Damage/Genotoxicity Magnetic Bead Kit , we quantified levels of ATR , CHK1(pS345), CHK2(pT68), H2AX(pS139), P53(pS15), MDM2 , and P21 . Buffer-compatible MAPmates assays were included to measure levels of activated caspase-3 and β-tubulin (#46-713MAG). A separate test employed a 9 Plex Multi-Pathway Magnetic Bead Kit (Milliplex Map Kit #48-680MAG) to quantify ERK1/2(pT185/pY187), P38(pT180/pY182), NFκB(pS536), JNK(pT183/pY185), AKT(pS473), P70S6K(pT412), CREB(pS133), STAT3(pS727), and STAT5(pY694/699). Lastly, the Milliplex MAP TGFβ Magnetic-Bead 3 Plex Kit  was utilized to quantify TGFβ-1, TGFβ-2, and TGFβ-3 levels.The quantification of signaling proteins within mouse heart protein lysates was achieved using multiplex assays. These were based on Millipore Luminex xMAP−ΔΔCt method as proposed by Livak and Schmittgen in 2001 [The miRNAs assess comprised miR-21a-5p, miR-29b-3p, miR-34a-5p, and the let-7 and miR families. Heart tissue miRNAs were analyzed using the 96:96 Fluidigm BioMark platform. A FAM-labeled, assay-specific TaqMan fluorescence probe Mix  was employed, with each sample measured in triplicate. The QPCR protocol was as follows: 6.7 µL of the sample and 0.49 µL of the master mix were subjected to an incubation cycle at 50 °C for 2 min, followed by 70 °C for 30 min, and 25 °C for 10 min. This was succeeded by 50 °C for 2 min and 96.5 °C for 10 min, for 40 cycles in total. The final step consisted of 15 s at 96 °C and 1 min at 60 °C. Data analysis and cycle threshold (CT) value determination were facilitated by BioMark (Singapore) real-time PCR analysis software . Quantification followed the 2 in 2001 .p < 0.05), determined via a “drop-one-QTL-at-a-time” analysis, which evaluates the impact of single QTLs or interactions.Regarding mouse genotyping, see the The association of genetic variants with CDA was evaluated in three patient cohorts previously published. In the first two cohorts, comprising 71 anthracycline-treated pediatric cancer patients  (PaediatCardiac magnetic resonance (CMR) examinations were conducted with a Philips 1.5-Testa Achieva whole-body scanner  equipped with a 16-element phased-array cardiac coil . The ima2R < 0.7 were removed from the analysis before proceeding further. Data were analyzed in R v3.6.0 (1). The SNPassoc package v2 was used to explore associations between mutations. Employing the “association” function, we performed case/control analyses of the possible genetic models  to examine the associations between phenotypes and input mutations. SNVs from allelic forms of the genes encoding intermediate molecular phenotypes associated with myocardium damage in mice were used to generate prognosis risk scores in humans. A penalized restrictive analysis was carried out with the Least Absolute Shrinkage and Selection Operator (LASSO) machine-learning multivariate regression to reduce the risk of identification of false positives.The existence of associations of CDA, measured using echocardiography or CMR, with SNVs, was evaluated in the three patient cohorts. We looked for alleles encoding proteins with levels in the myocardium that were associated with CDA in mice. SNVs were genotyped using the Infinium™ Global Screening Array-24 v2.0 BeadChip. Data were imputed using the Michigan Imputation Server with Minimac4 . After rt-test, or the Mann–Whitney U test, was used to compare pairs of groups based on the distribution type. p < 0.05 was considered significant. Multiple regression was applied to determine which intermediate molecular phenotypes were associated with the definition of global heart fibrosis and the global cardiomyocyte area in mice. To construct the multivariate models, those variables (intermediate molecular phenotype levels in the myocardium associated with CDA (heart fibrosis or cardiomyocyte area) with p < 0.05) were chosen.Pearson or Spearman correlation coefficients, depending on whether a Kolmogorov–Smirnov test indicated that the data were normally or non-normally distributed. The Principal component analyses (PCAs) were performed on the data with the function “prcomp” of “stats” R package. The results of the PCA analysis were plotted with “fviz_pca_biplot” function (factoextra” Rpackage), showing biplots of individuals and variables. The specific test conducted in each study is indicated in the legend of each figure or table. General statistical analyses were conducted using GraphPad Prism5, JMP 12, and R v3.6.0 . The SNPCDA is a complex trait, and as such, identifying the genetic component in humans that influences CDA susceptibility is a challenging task . HoweverPrevious studies showed that, after anthracycline chemotherapy, a subclinical injury could be detected at the histopathological level in the myocardium even before functional damage had occurred ,47. Anthp = 0.0149) and subendocardial (p = 0.0043) fibrosis than did FVB mice (p = 0.0066), subendocardial (p = 0.0083), and subepicardial fibrosis (p = 0.0546) than when doxorubicin was administered alone  but not significantly after therapy solely with doxorubicin in global heart and subepicardial and subendocardial zones (p = 0.0052) and the combined treatment (p = 0.021) but not in young mice  to determine whether they could account for more of the CDA phenotype variation than that explained solely via cda-QTLs. Accordingly, we looked for ipQTLs and cdaQTLs in the F1BX genetically heterogeneous mice that could subsequently be used in the genetic models . Thus, fFollowing on, we searched for cdaQTLs associated with heart fibrosis and the cardiomyocyte area based on the type of chemotherapy  in different conditions: heart zone  and age (young or old mice) A. QTLs lEighty cda-QTLs were identified, but some were in the same chromosome (Chr.) and were genetic regions simultaneously linked to several CDA conditions. In the end, we identified 27 cda-QTLs in full . For exaOur next step was integrating the ipQTLs with cda-QTLs into the genetic models  to evaluWe then assessed whether ipQTLs linked to myocardium molecules increased the amount of global heart fibrosis explained by cdaQTL6 in old mice and cdaQTL13 in young mice. The intermediate molecular phenotypes that were correlated with global fibrosis under these conditions and their ipQTLs are shown in Concerning global heart fibrosis in young mice, we observed that the phenotypic variation due to cda-QTL13 increased after including some of the ipQTLs associated with P70S6K in genetic models B. The ipRPS6KB1 levels with siRNA in hiPSC-CMs increases their sensitivity to doxorubicin, confirming the role of P70S6K as an intermediate molecular phenotype of CDA , considering an LVEF reduction of 5% or more during the first six months or throughout the complete follow-up. We only evaluated SNVs from those genes that encoded molecules associated with CDA in mice, testing the most probably genetic model . SeveralAKT1, STAT3, TP53, MAPK8, MAPK11, and RelA/P65; the pediatric model consisted of SNVs of the same first five genes.CDA susceptibility is partly different in adults and in pediatric patients ,38,69. SOur results highlight the value of genetic mouse models as tools for identifying the intermediate phenotypes that contribute to the variation of CDA and the use of the genes encoding them as potential susceptibility markers.Chronic CDA is a common side effect that can be very severe and affect the continuity of chemotherapy treatment . CDA susThe use of intermediate phenotypes to identify a part of the genetic component of complex traits has been proposed in psychiatric disorders  and exteThe network of intermediate phenotypes at the systemic, tissue, cellular, and molecular levels that determines the pathogenesis of a complex trait is regulated by a multitude of genes acting at all those levels. This scenario coincides with the omnigenic model, involving a series of gene networks with core genes and many peripheral genes that has recently been proposed to explain missing heritability ,25. It iFrom a practical point of view, the challenge is to find a way to determine the genetic markers of susceptibility to a complex trait. The focus on the genetic determinants associated with the variation of intermediate phenotypes  makes it possible to identify essential genes that can be susceptibility markers of diseases of complex genesis. In this sense, the approach proposed in this study could be adopted as a general strategy for better identifying genetic markers of high-prevalence complex diseases, e.g., type II diabetes, autoimmune diseases, thrombosis, cardiovascular diseases, sporadic cancers, and CDA .It is difficult to determine the polygenic component of complex human population traits because of their genetic complexity and sophisticated environmental interactions . IdentifThis work has identified some genetic components linked to intermediate molecular phenotypes associated with CDA in a mouse backcross model, which helped identify some genetic elements related to the CDA susceptibility itself. Indeed, variable susceptibility to doxorubicin-induced cardiotoxicity observed in humans can be modeled in a panel of the collaborative cross strains . Since acis. So, if genetic variants of these genes determine the protein intermediate phenotype levels, they will also contribute to the heritability of the complex trait. For this reason, we looked for variants of these genes to evaluate in the human population.Although ipQTLs were not directly linked to CDA, we used genetic models to demonstrate that these ipQTLs account for a part of the heritability of the CDA. These ipQTLs help control in trans the levels of intermediate protein phenotypes in the myocardium. Protein levels depend on cis factors, the most important being the DNA regulatory sequences in the gene encoding the molecule and genetic elements in “trans.” We have used ipQTLs in –“trans” to demonstrate that genetic determinants linked to the levels of the molecule contribute to the phenotypic variation of CDA. Although we do not know which genes drive the effects of ipQTLs, we know the gene encoding the intermediate protein phenotype in An enormous number of intermediate phenotypes affect the pathogenesis and variation in a complex trait like CDA, so, unsurprisingly, many of the contributing genetic determinants cannot be detected among those of the central phenotype, for which reason they are responsible for much of the missing heritability . FurtherIn summary, a genetically diverse group of mice can be leveraged to uncover the genetic influences on protein levels in the myocardium associated with histopathological damage following chemotherapy. This process will shed light on elusive genetic factors related to CDA in both mice and humans. By pinpointing the genetic and molecular factors contributing to elevated CDA risk, we can enhance our capabilities in predicting and mitigating CDA. Our findings suggest that the proposed approach may broadly facilitate the identification of susceptibility markers for complex diseases. Furthermore, the genetic markers discovered could assist in identifying patients at heightened risk of developing CDA. This would enable more personalized patient care and optimized chemotherapy regimens, potentially reducing the risk of severe adverse drug reactions.In terms of study limitations, further epidemiological investigations are required to ascertain the extent to which genetic determinants linked to intermediate phenotypes of a complex trait can aid in identifying useful genetic markers for risk prediction. Although these genetic determinants might not be potent enough to be identified as associated with the primary complex phenotype, they should exert a sufficient impact to contribute to risk explanation within a polygenic model context. Such genetic influences could help elucidate the likelihood of disease manifestation.


The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (μ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies.In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, β1, μ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for μ1A, the polarized epithelial cells specific to μ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the  AP1S2 is encoded on the X-chromosome in mice and man and its deficiency causes severe X-linked intellectual disability and impaired motor coordination [Proteins and lipids are transported between organelles by vesicles. Several secretory transport routes starting at the trans-Golgi network and endocytic routes are mediated by heterotetrameric adaptor protein (AP) complexes. Eukaryotic cells have five homologous AP-complexes named: AP1, AP2, AP3, AP4 and AP5. Each AP complex has a characteristic intracellular distribution, protein sorting motif-signal recognition, specificity and function. Only AP1 and AP2 complexes form clathrin-coated vesicles (CCVs). Tissue-specific isoforms exist for AP1, AP2 and AP3 in vertebrates [dination .In the past, the generation of knockout and knockdown animal models highlighted the important roles of AP1 subunits in vesicular traffic and their indispensable functions in the development of vertebrates and their various tissues and the immune system ,8.It also demonstrated that μ1B can partially substitute μ1A and that also σ1B and σ1C can only partially substitute σ1A . However, we do not know all isoform-specific functions of the adaptins and the AP1 complexes they are part of.AP1G1 hypomorphic mutation may be the result of mislocalized membrane proteins in polarized cells of the affected tissues [AP1G1 heterozygous mice, which indicates that this shortened γ1 mutant may have dominant negative effects only in specific tissues and cell types [Ap1g1 (γ1) knockout zebrafish model using the CRISPR/CAS9 technique. Our previous studies on AP1 adaptin functions in zebrafish demonstrated the conservation of vertebrate adaptin functions [−/− fish cease development during the transition between the blastula and gastrula stage, in line with the γ1−/− mouse phenotype, which ceases development prior to the blastocyte hatching out of the zona pellucida. Interestingly, adult heterozygous fish showed impaired cell adhesion in epithelial tissues, such as cadherin protein sorting and adherent junction (AJ) formation. Female and male heterozygous animals have reduced fertility due to impaired oocyte and sperm maturation. This analysis revealed novel essential functions of the vertebrate AP1G1 complexes, which cannot be fulfilled by its highly homologous AP1G2 complex. These data demonstrate the usefulness of the zebrafish model systems for future analyses of human adaptinopathies.Recent research led to the identification of adaptin mutations that reduce adaptin levels and impair functions. These are novel adaptinopathies whose molecular mechanisms are not known. Mice with a spontaneous deletion of two γ1 amino acids are viable, but exhibit multiple phenotypes in the inner ear, retina, thyroid and testes. They also showed circling behaviour and were unable to swim. The multiple pathologies associated with the  tissues . Comparall types . The expll types ,11. The ll types ,13,14. Oll types . Vertebrll types ,17. Addill types ,19. Bialll types ,21. Recell types . Faint ell types . The prell types . Patientll types . Alteratll types ,27. Recell types  All thesunctions ,28,29. Tap1g1 transcript is expressed from early to late stages of development, particularly in the developing central nervous system (CNS) and somites [ap1g1 gene using a CRISPR/Cas9 approach [https://chopchop.cbu.uib.no, accessed on 1 March 2015). The sgRNA, injected together with Cas9 mRNA, caused mutation at the expected target site in the ap1g1 gene in F0 embryos and adults. Out of 200 injected embryos, 136 reached reproductive age and 52 out of 136 were genotyped at 6 weeks by DNA sequence analysis to verify the presence of the mutation (F0). As confirmed by sequencing of PCR fragments, five individuals had a mutation in the proximity of the sgRNA target sequence . We decided to carry out our further analysis on individuals with the mutation of two nucleotide insertion lines whose genotypes will hereinafter be referred to as +/+ap1g1; +/−ap1g1 and ap1g1−/−. At 4.5 h post-fertilization (hpf), we analyzed using qPCR the expression profile of ap1g1 mRNA and we observed a significant reduction (>90%) in −/−ap1g1 mutant embryos compared to ap1g1+/+ and ap1g1+/− individuals, suggesting the activation of the nonsense-mediated mRNA decay process (The zebrafish Ap1g1 protein consists of 819 amino acids and shows a high amino acid identity (95%) with the human and mouse counterparts. Zebrafish genome encodes only for one ortholog protein, which has 91% identity with humans and mice; the  somites . To geneapproach . A suitasequence . Of the  process D.ap1g1−/− animals and we suggested that the lack of ap1g1 subunit is lethal. In the F2 progeny at 3.5 hpf, it was possible to differentiate two main phenotypes: embryos with normal morphology and embryos with dramatic morphological abnormalities in dividing cells of the yolk syncytial layer. Genotype analysis revealed that embryos with normal morphology were either +/+ or +/− while the aberrant ones were −/−. Therefore, we genotyped fertilized eggs (n = 376) of the F2 generation at 3.5 hpf and we found a genotype distribution as follows: 27% ap1g1+/+ (n = 104); 47% of ap1g1+/− (n = 177) and 26% ap1g1−/− (n = 95) (desmoplakin b (dspb) expression by whole mount in situ hybridization (WISH). For epidermal morphogenesis, dspb is required, and its transcripts are maternally provided at the blastula stage, detectable before zygotic transcription activation, and widely distributed at the gastrula stage [dspb with the same distribution, indicating the proper embryonic development of ap1g1+/+, ap1g1+/− and impaired ap1g1−/− individuals until that developmental stage  as shown in ap1g1−/− embryos. This is in line with our previous studies performed in zebrafish with ap1g1 antisense morpholino and the data from Ap1g1 mouse knockout embryos, which ceased development at the blastocyst stage [We analyzed the genotypes of F2 progeny at different developmental stages: 3.5 hpf and subsequently at 3-, 28- and 90-days post fertilization (dpf) A. At 3 d(n = 95) B. We folla stage . At the al stage A. At 4 hal stage . Althougap1g1+/− fish reached at least 18 mpf (months post-fertilization) as the ap1g1+/+ counterpart, but we observed a 50% reduction in their fertility rate in heterozygous mating of 9 months old females compared to ap1g1+/+. At the same age (9 months), in ten different matings, we observed a reduced number of laid eggs. In a representative experiment only, 52 (SD +/− 0.6) of ap1g1+/− eggs were deposed compared to 131 (SD +/− 0.8) of ap1g1+/+. We also analyzed the percentage of fertilized eggs and observed that 91% of ap1g1+/+ eggs were fertilized compared to 52% of ap1g1+/−  compared to ap1g1+/+  at 3, 6, 9, 12 and 18 months. Concerning the body length, we did not observe any differences, but the body mass analysis enhanced differences for ap1g1+/+ compared to ap1g1+/− animals, as shown in ap1g1+/− animals displayed a sloughing epithelium at the fold tips sigh of mucosa detachment and disorganization of lamina propria.  and the body mass of propria. B. Previoelopment . The mouneurod1) gene promoter Tg(neurod1:EGFP), a transcription factor known to play important roles in post-mitotic neurons during neurodevelopment of different areas as telencephalon, diencephalon, hindbrain, midbrain, midbrain–hindbrain boundary, trigeminal ganglia, olfactory placode, retinal neuroepithelial and caudally in spinal cord neurons [+/−ap1g1 (carrying the insertion of two nucleotides) with the Tg(neurod1:EGFP). Individuals of the F1 progeny were selected based on the contemporary presence of ap1g1 mutation and EGFP expression. The larvae were raised to adulthood to establish the stable transgenic/knock-out line, hereinafter named Tg(neurod1:EGFP-ap1g1). As shown in neurod1-driven fluorescence was evident in the anterior part of CNS corresponding to telencephalon (tel), midbrain (mb), hindbrain (hb), midbrain–hindbrain boundary (mhb), spinal cord neurons (scn) where cells positive for EGFP expression were detected in Tg(neurod1:EGFP ap1g1 +/+). In general, Tg embryos had a similar pattern of EGFP expression compared to Tg (neurod1:EGFPap1g1 +/+) counterpart, but specific brain regions or cells were not decorated: fluorescence was clearly missing in the midbrain and in particular at the level of the midbrain–hindbrain boundary and also in anterior part as telencephalon. At 48 hpf in the trunk, spinal cord neurons (scn) were expressing EGFP, but we observed a marked decrease in the number of snc in heterozygous embryos compared to control embryos (+/+)  compared to ap1g1+/+ fish (11.89 mm ± 0.022 mean ± s.e.m.)  compared to ap1g1+/+ fish  at 6 dpf  in the total brain volume of ap1g1+/− compared to ap1g1+/+. The reconstruction of the 3D distribution of EGFP fluorescence in 48 hpf +/+ and +/− embryos clearly evidenced the lack of EGFP signal in the anterior part of the CNS, in the hindbrain region and in particular in the midbrain–hindbrain boundary (neurod1:EGFPap1g1+/−) line suggests that normal expression of ap1g1 at early stages of neurodevelopment is required for determination and/or differentiation of specific neuronal populations located in different brain areas and spinal cord neurons.Neurons are highly polarized cells with morphologically and functionally distinct neurites, dendrites and axons which contain different proteins for specialized functions. AP-1 complex has been implicated in the polarized sorting of a subset of transmembrane receptors and transporters to the somatodendritic domain ,32. The  neurons ,34,35. Tos (+/+) E. The obat 6 dpf A. Althou s.e.m.) A,B, therat 6 dpf B, recapiboundary C–G. Altoap1g1−/− zebrafish blastula, the inability of Ap1g1−/− mouse blastocysts to hatch out of the zona pellucida and the inability of their ES-cells to adhere to feeder cells and to grow in cell culture strongly suggested AJ dysfunction. Morula of E-cadherin KO mice undergo compaction, but the cell–cell contacts are not stable and single cells can be lost, but the development can proceed to the blastocyst stage. However, these blastocysts are unable to hatch out of the zona pellucida, just like the Ap1g1−/− mouse blastocyst [ap1g1+/− embryos at 48 hpf compared to controls. This indicates that the reduced fertility of zebrafish ap1g1+/− females is not only due to the reduced production of eggs, but also due to the production of eggs with much-reduced mRNA and protein contents, which may not suffice to perform the earliest developmental stages and formation of epithelial polarized cells. E-cadherin +/− KO mice do not show a phenotype in blastocyst development and thus the lower E-cadherin level in ap1g1+/− zebrafish should suffice to mediate blastula development. Thus, failure of E-cadherin sorting to and maintenance of AJ, impaired by the lack of ap1g1 mediated E-cadherin sorting, appears to be responsible for the failure of blastocyst development. The resulting E-cadherin miss-sorting may lead to its trapping in a TGN-endosomal compartment, contributing to or causing the increase in protein over mRNA level. The BMP signalling cascade plays a central role in dorso-ventral patterning of zebrafish embryos. We know that Bone Morphogenetic Proteins (BMPs) are extracellular proteins which belong to Tgfβ superfamily. During zebrafish gastrulation, different bmp genes are expressed: bmp2a, bmp4 and bmp7 that are essential for embryonic development, organogenesis, tissue specification and regeneration. BMP4, in particular, has a variety of biological functions and plays a prominent role in the regulation of epithelial cell differentiation [ap1g1+/− mutants compared to wild-type larvae.Critical steps in early vertebrate development are the morula to blastula transition and the development of the blastocyst are critically dependent on the adherens junction (AJ) protein E-cadherin. Gastrulating cells may use E-cadherin-mediated cell–cell contact as a migration substrate and real movements that drive convergence and extension. In addition, cadherin molecules are necessary to maintain the integrity and survival of precordal plate derivatives and cellular adhesion between epithelial cells; failure to maintain these structures may have resulted in axis pattern defects due to reduced organized activity of different pathways. The morphological alterations of the astocyst ,36. Realntiation . As showtrans-Golgi network and on endosomes. Due to their regulation of intracellular protein sorting, they are also indirectly involved in the regulation of protein levels in the plasma membrane [fgt) to reflect the atypical movement pattern (circling behaviour) of mutant mice (fgt/fgt). These mutant mice are viable and exhibit multiple phenotypic abnormalities, such as hearing loss and eyes, thyroid and testes defects. In humans, impaired functions of different subunits of AP complexes have been associated with severe inherited disorders. Individuals of families with variants in the AP1G1 gene show clinical phenotypes common to neurodevelopmental disorders (NDD), including global developmental delay (GDD) and intellectual disability (ID), which varies from mild to severe. In particular, they show clinical features such as epilepsy, developmental delay, speech delay, hyperactivity and aggressive behaviour. Additionally, recessive loss-of-function variants in AP1B1 cause a multi-organ disorder with clinical manifestations such as enteropathy, hearing impairment, peripheral neuropathy, keratodermia encephalopathy and intellectual disability [AP1S1 mutations cause MEDNIK  syndrome [−/−ap1g1 embryos ceased their development at 3.5 hpf during the transition period of the blastula-gastrula stage. On the contrary, the heterozygous fish reached the adult stage, but they showed a reduced growth rate and reductions in their fertility rate. Males and females have defects in spermatogenesis and oocyte maturation, respectively. One-year-old males showed a reduction in sperm concentration and motility, while females showed impairment in follicle maturation. The zebrafish ap1g1 knock-out model revealed the indispensable function of the family of AP1 complexes for vertebrate development and that heterozygosity causes disease phenotypes. This result confirms the suitability of the zebrafish model system because these data are in line with the phenotypes observed in the Ap1g1 knock-out mouse model. The −/−Ap1g1 mice ceased development prior to hatching out of the zona pellucida and when these blastocysts were seeded onto feeder cells, they did not adhere to the feeder cells and did not proliferate. The molecular basis of this phenotype was not analyzed at the time. The Ap1g1+/− inner cell mass cells grew just like isogenic wild-type cells [Ap1g1+/− mice was not quantified, but they reproduced less efficiently than the isogenic wild-type mice. However, the Ap1g1+/− mice grew slower, specifically during nursing than the wt control animals. In addition, they had a defect in the formation of CD4 single-positive T-cells, while CD8 single positive T-cell numbers were normal. These data point to impaired precursor cell differentiation processes. In zebrafish, we observed a strong reduction of E-Cadherin and BMP factors. Morula of E-cadherin knockout mice do undergo compaction, but the cell–cell contacts are not stable and single cells are lost. The development does proceed to the blastocyst stage, but these blastocysts are unable to hatch out of the zona pellucida [−/−Ap1g1 KO mouse blastocysts, also has this phenotype. E-cadherin has functions in numerous tissues. Tissue-specific functions have been observed in almost every epithelium. E-cadherin sorting plays a role in the establishment and maintenance of adherens junction (AJ) complexes. In E-cadherin heterozygous animals, only very few tissues show morphological alterations. These are the gut, ovaries and brain. In Drosophila melanogaster, cell-type specific AP1 γ1 knockdowns, E-cadherin is transported to cell–cell contact sites, but E-cadherin is not able to support cell–cell contacts upon increasing plasma membrane tension [ap1g1+/− fish may also be caused by defects in the organization of AJ due to inefficient cadherin sorting [ap1g1+/− heterozygous embryos till adult age, indicates that ap1g1 expression levels suffice to permit cadherin-mediated AJ functions. However, cadherins have functions in signal transduction pathways, which are independent of their functions in AJ and one can speculate that those activities may not suffice for proper tissue development [ap1g1 heterozygous animals and is associated with markedly reduced growth during nursing and reduced fertility. In conclusion, our data demonstrate that zebrafish is a suitable model system to study the molecular mechanisms causing human AP1-mediated adaptinopathies. The study highlighted the important role of the subunit in different organs and tissues and its function in polarized cells as neurons or epithelial tissues. The findings might help to understand why the adaptinopathies have a common clinical phenotype. However, it also might suggest deeper studies in patients in order to clarify potential defects in polarized cells. The study of the biological basis of these kinds of diseases could provide relevant topics to better characterize the pathogenesis and treat adaptinopaties.Cellular trafficking via transport vesicles and tubules is essential to maintain critical biological functions and thus is mediated by tightly regulated machinery. AP family complexes are components of membrane protein coats that mediate cargo selection and coated vesicle formation . Vertebrmembrane . The twomembrane . Later, membrane  describesability . AP1S1 msyndrome ; AP1S2 msyndrome ; and AP1syndrome ,39. Thessyndrome . We havepe cells . The ferellucida . The zeb tension . This in tension ,43,44. T sorting ,46. The elopment . AJ are elopment ,48. Firselopment ,48. In telopment ,49. Howeelopment ,51. In oDanio rerio (zebrafish) were maintained in 3 L of water at a temperature-controlled (28.5 °C) with 14 h light-and 10 h dark cycle and fed as described by Kimmel and colleagues (Zebrafish: Housing and husbandry recommendations [neurod1-EGFP) [All experiments were performed in accordance with the Italian and European Legislations (Directive 2010/63/EU)9 and with permission for animal experimentation from the Local Committee for Animal Health  of the University of Brescia and the Italian Ministry of Health (Authorization Number 287/2018-PR). ndations ,53. Matid1-EGFP) .ap1g1 mutant lines following the protocol of Gagnon et al., 2014. Single guide RNA (sgRNA) was designed using the CHOPCHOP algorithm , to specifically target an optimal CRISPR sequence on exon 1 of γ1ap1 gene (NM_199682.1). The chosen sgRNA  was used for the identification and sequence characterization of heterozygous mutants generated by the CRISPR/Cas 9 technique.We used the CRISPR/Cas9-mediated genome editing technique to generate the gRNA see  was trangRNA see  were des+/−ap1g1 line was crossed with the Tg(neurod1-EGFP) transgenic line [The nic line . The res+/−ap1g1 individuals and genotyped (as described above) at different developmental stages: 3.5 hpf, 3 dpf, 28 dpf and 90 dpf.At 3 months, we crossed +/+ap1g1, +/−ap1g1 and −/−ap1g1 zebrafish embryos were collected at 3 hpf and fixed with 4% (v/v) paraformaldehyde (PFA) , dehydrated in 100% (v/v) methanol, and stored at −20 °C. The WISH was performed as previously described in [dspb) DIG-labelled. Embryos were washed with ascending scale of Hybe Wash/PBS and SSC/PBS and then incubated with anti-DIG antibody conjugated with alkaline phosphatase overnight at 4 °C. The staining was performed with NBT/BCIP  alkaline phosphatase substrates. Images were taken with Zeiss Axio Zoom V16 equipped with Zeiss Axiocam 506 colour digital camera and processed using Zen 3.5 (Blue Version) software from Zeiss  The experiments were done two times with n = 50 for each genotyped condition.ribed in  and sele+/−ap1g1 mutants were fixed for 24 h in Bouin’s solution  at room temperature. Samples were dehydrated through graded series of ethanol, infiltrated with xylene and embedded in Paraplast plus . The samples were serially cut into 7–8-μm sections on an LKB microtome. After rehydration, the sections were stained with hematoxylin and eosin for microscopic analysis. The images were acquired using an optical microscope .Wild type and rpl13a as a reference gene.Total mRNA was extracted from 30 larvae at different developmental stages using TRI-Reagent  according to the manufacturer’s protocol. RNA was quantified using My Spect spectrophotometer  and controlled by electrophoretic separation on a 1% TAE-agarose gel. 1.5 μg of total RNA was retrotranscribed to cDNA using Im-Prom Reverse Transcriptase  and oligo (dT) primers. Primers were designed by the real-time PCR tool from IDT and the sequences are reported in 2, 20 mM HEPES, pH = 8.5) 21. A total number of 8 males, 4 with an +/+ap1g1 genotype and 4 to +/−ap1g1 were stripped.Fish were isolated for 2 days before stripping, to guarantee the replenishment of sperm reserve. Males were anaesthetized in Tricaine 1X (0.16 mg/mL), gently dried on a paper towel and placed in a dampened sponge ventral side up, with their genital papilla exposed. The ventral surface of the fish was further dried to remove any excess water that could prematurely activate the sperm. The fish abdomen was gently pushed with soft plastic tweezers and the ejaculate was collected from the genital papilla by means of a Drummond micro dispenser equipped with a 5 μL microcapillary tube. The whole ejaculate was diluted and preserved until analyses (within 1 h) in 20 μL of zebrafish sperm immobilizing solution , only VAP was considered in the following analyses. The measurement was repeated twice per male, yielding a repeatability of 0.68 ± 0.14, and mean values were used for statistical analyses.Sperm were activated by gently mixing with a Gilson micropipette 1 μL of ejaculate preserved in ZSI with 10 μL of aged tap water. Three microliters of samples were then quickly placed in separate wells on a 12-well multi-test slide and covered with an 18 × 18 mm coverslip. Both slide and coverslip were previously coated with 1% polyvinyl alcohol (Sigma–Aldrich), to avoid sperm sticking to the glass slide Sperm velocity was measured using a CEROS Sperm Tracker  in the first 10 s after activation. The measurement was repeated on three subsamples per male, leading to a repeatability of 0.68 ± 0.1422, and mean values were used for statistical analyses. Mean velocity measurements were based on 280 ± 169 (mean ± s.d.) sperm tracks per male. Since sperm velocity parameters  were highly correlated  were captured using a Zeiss Axio Zoom V16 equipped with Zeiss Axiocam 506 colour digital camera and processed using Zen 3.5 (Blue Version) software from Zeiss  Confocal Images were acquired using a Plan-Neofluar 10X/0.3NA objective. A Z-stack was performed to acquire the whole central nervous system and Maximum Intensity Projection was obtained using Zen Black software. For Light Sheet analysis embryos were first anaesthetized using tricaine (0.02% in fish water) and subsequently included using in a low melting agarose matrix  at 0.5% in fish water. Images were acquired using Zeiss LightSheet microscope V1 supported by ZenPro software using a 488–30 nm laser and 505–545 nm filter. Images from the same experiment were taken with the same laser intensity and exposure time to generate comparable images. After the acquisition, 3D images were generated and manipulated using Arivis Vision 4D  3D reconstructions of EGFP-positive cells were manipulated to obtain pictures comparable to each other in terms of fluorescence intensity. 3D reconstructions were exported as a single snap with the same compression setting. Voxel-wise comparisons were performed following the protocol of  only on t-test. The p-value is indicated with asterisks * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Differences were considered significant at p-values of less than 0.05.All the experiments described in the manuscript were performed at least two or three times using GraphPad Prism V8  to perform statistical analysis. The comparison and significance between different groups were determined by one-way ANOVA, corrected for multiple comparisons or by two-tailed unpaired Student’s


With direct-to-consumer (DTC) genetic testing enabling self-responsible access to novel information on ancestry, traits, or health, consumers often turn to social media for assistance and discussion. YouTube, the largest social media platform for videos, offers an abundance of DTC genetic testing–related videos. Nevertheless, user discourse in the comments sections of these videos is largely unexplored.This study aims to address the lack of knowledge concerning user discourse in the comments sections of DTC genetic testing–related videos on YouTube by exploring topics discussed and users' attitudes toward these videos.We employed a 3-step research approach. First, we collected metadata and comments of the 248 most viewed DTC genetic testing–related videos on YouTube. Second, we conducted topic modeling using word frequency analysis, bigram analysis, and structural topic modeling to identify topics discussed in the comments sections of those videos. Finally, we employed Bing (binary), National Research Council Canada (NRC) emotion, and 9-level sentiment analysis to identify users' attitudes toward these DTC genetic testing–related videos, as expressed in their comments.We collected 84,082 comments from the 248 most viewed DTC genetic testing–related YouTube videos. With topic modeling, we identified 6 prevailing topics on (1) general genetic testing, (2) ancestry testing, (3) relationship testing, (4) health and trait testing, (5) ethical concerns, and (6) YouTube video reaction. Further, our sentiment analysis indicates strong positive emotions  and a neutral-to-positive attitude toward DTC genetic testing–related videos.With this study, we demonstrate how to identify users' attitudes on DTC genetic testing by examining topics and opinions based on YouTube video comments. Shedding light on user discourse on social media, our findings suggest that users are highly interested in DTC genetic testing and related social media content. Nonetheless, with this novel market constantly evolving, service providers, content providers, or regulatory authorities may still need to adapt their services to users' interests and desires. Since the completion of the human genome project in 2003, dwindling genome sequencing costs and a rising interest in genomics among the general public have paved the way for direct-to-consumer (DTC) genetic testing . Today, The uprise of DTC genetic testing and self-responsible genetics has also sparked countless ethical, social, technical, and legal issues . For exaWith the increasing spread and availability of DTC genetic testing  and a geUnderstanding the topics, opinions, and attitudes discussed by the users can prove crucial for many stakeholders, as comments are the main form of user reaction and feedback on social media . ServiceExtant research regarding DTC genetic testing on social media confirms this lack of understanding. Prior research focuses on microblogging services such as Twitter ,26, ReddRQ1: What topics do YouTube users discuss in the comments sections of DTC genetic testing–related videos?RQ2: What are users' attitudes toward DTC genetic testing–related videos, as expressed in their comments on YouTube?To answer our RQs, we analyzed the 248 most viewed videos dealing with DTC genetics in a 3-step exploratory approach. First, we analyzed the selected videos regarding media type, genetic test purpose, and related health information. Second, we employed topic modeling to investigate user discourse in the comments sections of those videos. Third, we conducted a sentiment analysis unveiling users' attitudes toward the discussed topics and DTC genetic testing videos in general.Through our study, we contribute to research and practice in several ways. As for research, we add to the literature on user attitudes toward DTC genetic testing by delineating topics and opinions discussed about these genetic tests. Further, we contribute to the research stream regarding health information on social media by showing that YouTube comments provide valuable insights on user discourse on social media and demonstrate that DTC genetic testing and health information topics may generally vary from platform to platform. As for practice, our research may help providers of DTC genetic testing services and regulatory authorities gain further insights into user attitudes and consequently adapt or improve genetic testing services and regulations. As most videos are user-generated, our analysis of user discourse can provide valuable insights on the topics discussed in the comments sections of these videos, providing content creators with valuable information for improving their future DTC genetic testing–themed videos.During the past decade, social media platforms have become increasingly attractive in the digital health sector as a means of communicating medical information . In addiConsequently, information dissemination platforms  to create a list of the most relevant DTC genetic testing–related videos on YouTube. With the region set to the United States , we queried the 300 most viewed video results for each of 6 different DTC genetic testing–related search terms . Thereafter, we combined the 1800 results from the 6 queries, removed duplicates, and sorted them by video views in descending order. We further excluded all videos with less than 50,000 views because they had very few comments per video (average of 61.2), with many having no comments (n=336).Next, the remaining 468 videos were reviewed for relevance through iterative manual inspection by 2 researchers, with a third researcher breaking ties in case of differences. For this, our predefined exclusion criteria were as follows: (1) videos not focusing on DTC genetic testing, (2) videos focusing on genetic testing of animals, (3) videos focusing on clinical prenatal genetic testing, (4) videos not in English, (5) live stream videos, (6) duplicate videos , (7) videos commenting/reacting on videos , or (8) videos with disabled ratings and comments sections . As fWith the final coded set of 250 videos in place, we again used the YouTube API to download each video's 500 most recent comments. This number was chosen due to the YouTube API download limitations while still allowing meaningful analysis. Among these, 80 videos had less than 500 comments, and 2 videos were no longer available, leaving us with 84,082 comments from 248 videos, which is a sufficient number for topic modeling and sentiment analysis .To answer our first RQ, we employed topic modeling to identify common topics discussed by users in the comments sections of DTC genetic testing–related YouTube videos. Topic modeling is frequently used in medical informatics and related disciplines for text mining large data sets (such as comments or tweets) and deducing meaningful topics ,38,40,41Before conducting any topic modeling, we first separated the comments into 1-word tokens  and performed 2 essential data cleaning tasks. First, we used the SnowballC package to perform word stemming. This step was necessary to ensure that words with identical meanings  were grouped together to allow for meaningful topic modeling. For each word stem, the most frequent word was used to represent its stem . Second, we removed common stop words with the stop word list included in the tidytext package. This list comprises 1149 common stop words such as the, of, or to. As these do not hold any topical information, removing stop words reduces the data set size and benefits topic accuracy .With the cleansed word list in place, we first conducted a word frequency analysis by grouping, counting, and listing the words in descending order. This provides an overview of the most used words and can give a first insight into topics discussed most prominently .Second, we created word bigrams. We created a frequency list of 2-word tokens, which are found by pairing every 2 consecutive words in each comment . In contrast to the single word list, bigrams can be used to span a network with the number of occurrences indicating the weight of each bigram edge . To alloFinally, we conducted structural topic modeling with the help of the stm package . StructuAlthough there is no definite answer for the correct number of topics , after aWith the 50-topic model chosen, we sorted topics according to prevalence and within each topic, the words contributing to it in descending order. We then manually inspected the 50 most prevalent topics and their 10 most contributing words to deduce meaningful topics and categorized them according to their content. For this, we relied on our prior knowledge of DTC genetic testing as well as knowledge on the content of the videos that we gained during the video coding phase of the data collection step. All topic assignments were discussed among 3 researchers.Because topic modeling can only help us identify topics discussed in the comments but not users' attitudes toward the videos, we next conducted word- and comment-level sentiment analyses to answer our second RQ. Sentiment analysis is a common tool to elicit people's opinions, sentiments, emotions, and attitudes from written language . AlthougTherefore, we decided to conduct 2 word-level sentiment analyses and 1 comment-level sentiment analysis to deduce users' attitudes. For the word-level sentiment, we again used the tidytext package, which entails typical word-level approaches that are well suited for a first exploratory overview . We thenConsequently, we first conducted a positive and negative sentiment analysis using the Bing lexicon, which consists of approximately 6800 words that are predefined and classified as either positive or negative . SubsequWe also used the National Research Council Canada (NRC) emotion lexicon to get a more detailed overview of users' sentiments toward DTC genetic testing . This leFor the comment-level sentiment analysis, we used SentiStrength , a Java-Ethics approval was not necessary for this study, as it did not directly involve human participants. All data used in this study  were publicly available on YouTube and accessible through the YouTube API at the time of retrieval. All results are only published in aggregated form, and single references are presented anonymously and without context to protect the privacy of the comments’ authors.We examined a total of 248 videos related to DTC genetic testing, collected on September 14, 2020, with a total of 30 videos from official company accounts . Based on the media type, these included 27 advertising-related videos, 14 documentaries, 16 interviews, 12 news, 174 user-generated videos, and 5 with other media types . Among the 248 videos, 194 videos address ancestry as a test purpose, 15 address trait testing, 9 address genetic predispositions, 19 address relationship testing, and 11 address other purposes (such as how to use a test kit or comparison/presentation of multiple genetic test purposes). In total, the videos had 724,574 comments on the day of video data aggregation. We collected the comments of the videos on January 3, 2021, focusing on the 500 most recent comments of each video . An overview of the video metadata, content, and comments is provided in Word frequency analysis using the comments on DTC genetic testing–related videos provides valuable insights into the topics discussed by users. DNA , test , and people (n=9259) are by far the most frequent terms, thus indicating that users indeed primarily discuss DTC genetic testing in their comments. Additionally, we identified many words referring to ancestry testing such as ancestry (n=5015), african (n=6268), or american (n=6139). Moreover, words such as family (n=5252), dad (n=2932), or parents (n=2228) can be attributed to relationship tests. Overall, the 100 most frequent words resemble the test purposes identified from the videos themselves as well as a general excitement for DTC genetic testing videos . The bigram network of the comments provides a more fine-grained picture of the words used together often. Unlike the single word cloud, it allows us to see how multiple words are connected. Additionally, the arrows indicate in which order the words appear, whereas the shade of the edge represents the frequency of the word pair. Therefore, we can deduce possible topics discussed by users from the network.As shown in Finally, we trained structural topic models, of which we selected the 50-topic model. This topic group indicates a general interest in DTC genetic testing , entailing company names such as MyHeritage, AncestryDNA, or Ancestry.com and words of interest . Moreover, topic 16 touches on the home collection  and financial (money) aspects of DTC genetic testing.In line with our previous findings, most topics are about the results of genetic ancestry testing. Topic 8 shows a general interest in ancestry testing by users. Topics 17, 26, 37, and 47 describe findings on heritage from a specific region, whereas topic 41 is about paternal and maternal ancestry. Additionally, topic 19 might indicate that users hope to find lost relatives through ancestry testing.We also identified 3 topics about genetic relationship testing. Topics 34 and 48 deal with relationships between children such as identical twins, whereas topic 36 entails the aspects of adoption and genealogy .Although less prevalent, health genetic testing and trait testing are also covered in the top 20 topics. Topic 44 focuses on health information and data, whereas topic 28 entails words on traits such as hair or eye color.The 50-topic model also reveals some topics not contained in our previous findings. Topic 32 touches on instances of racism signified through words such as black, racist, or mad. Given the ongoing and complex debate toward instances of racism in the United States and the majority of DTC genetic testing revolving around ancestry and heritage, this could explain why this topic was found in the comments of these videos. Moreover, topic 22 deals with users' concerns regarding genetic testing and the government, with words such as lie, ad, or crime.In contrast to the previous findings, topics 18, 27, and 43 do not directly relate to genetic testing but rather entail reactions to the videos on YouTube . Further, users seem interested in personal stories .Although the bigram network and structural topic modeling use different approaches, the majority of the identified topics are present in both methods. Both approaches show strong indications of ancestry testing, relationship testing, trait testing, and health testing topics. Moreover, both methods led to the deduction of a YouTube or YouTube video–related topic. Even though topic modeling can help unveil what users discuss in the comments sections, it does not provide insights into users' attitudes toward these topics. Therefore, conducting a Bing sentiment analysis can provide a first overview of the sentiment regarding words used in the comments sections. , the single most used word (n=4751), is associated with the emotion of joy, and the most frequent emotion is trust . In contrast, disgust  has the least word occurrences.Another type of sentiment analysis is the identification of emotions with the NRC lexicon. Our results show that the most frequent words representing positive emotions, namely anticipation, joy, surprise, and trust, have higher occurrences than the words expressing negative emotions, namely anger, fear, disgust, and sadness see . This fiThe comment-level sentiment analysis provides insights into user attitudes as well as attitudes toward DTC genetic testing videos and their respective content . Although the SentiStrength sentiment can vary on a scale of –4 to 4, the average sentiment score of all comments is 0.32, meaning slightly positive. This is also reflected by almost half of all the comments  having a neutral sentiment . Grouping comment sentiment by video shows that the lowest sentiment score per video comments section is –0.62, whereas the highest is 1.33. Overall, only 30 of the 248 inspected videos have a negative sentiment, indicating an overall positive attitude toward DTC genetic testing videos.When comparing comment sentiment regarding the test purpose of the videos, our results show that from the comments with a sentiment score of 4, 91.6% (230/251) are in the comments sections of videos about ancestry testing (most frequent test purpose), whereas for comments with a sentiment score of –4, ancestry testing videos only account for 67.9% (76/112). In contrast, only 1.6% (4/251) of the comments with a sentiment score of 4 are in the responses to a video dealing with relationship testing. However, this increases to 17% (19/112) for comments with a sentiment score of –4. As shown in The analysis of comment sentiment regarding media type unveils that user-generated videos account for the most significant number of positive comments with 91.6% (230/251) for a sentiment score of 4. On the contrary, for a sentiment score of –4, user-generated videos only account for 60.7% (68/112) of the comments. Consequently, as shown in Our analysis of user comments on DTC genetic testing–related YouTube videos yields several valuable findings. The test purposes found in the videos largely resemble the most common genetic test purposes, with most videos talking about ancestry or relationship testing and fewer about trait and health testing. This finding is in line with previous research on YouTube videos related to DTC genetic testing ,31 and sMoreover, most topics found with the bigram network and structural topic modeling can be attributed to common DTC genetic testing purposes. This indicates that user discourse revolves around the contents of the videos and DTC genetic testing. In line with previous research, we also identified topics dealing with general genetic testing and users' interest in and excitement for DTC genetic testing ,51.Besides, research has also shown instances of racism regarding ancestry testing on Twitter , which wSentiment analysis revealed that users have more negative attitudes toward the content of advertisements, news, or documentary videos compared to user-generated videos on DTC genetic testing. Although this finding could be explained through some media types being more thought-provoking , another explanation might be that user-generated videos are often produced by single creators often trying to engage more with their YouTube community  than, for example, a news broadcaster or DTC genetic testing service provider. Hence, this may result in a more positive user attitude. This assumption is further supported by our findings on YouTube-related and YouTube video reaction topics. On the one hand, these findings once again indicate that users discuss and respond to the content discussed in the respective videos, and on the other hand, they suggest a more complex discussion between content creators and their community . It should be noted that the revealed user attitudes on DTC genetic testing videos do not necessarily reflect user attitudes toward DTC genetic testing in general. However, as our topic modeling results suggest that user comments largely revolve around DTC genetic testing, it is likely that users’ attitudes toward DTC genetic testing videos also reflect their attitudes toward DTC genetic testing to some degree. This notion is further supported by the finding that videos discussing the disadvantages and risks of DTC genetic testing tend to have more negative user attitudes. Comparable results on user attitudes toward DTC genetic testing were also found for Twitter and related textual platforms ,25,51, tSimilar to DTC genetic testing–related Reddit posts , we founThis study conveys several implications for research and practice. As for research, we contribute to the literature on user attitudes toward DTC genetic testing by investigating topics and opinions discussed about these genetic tests. We examined the 248 most viewed DTC genetic testing videos on YouTube in terms of their content  and analyzed users' attitudes in the form of their comments. Further, we contribute to research regarding health information on social media by showing that YouTube comments provide valuable insights into user discourse on social media. This study suggests that video content and user comments are co-dependent and should therefore be investigated together. To this end, we provide new insights into the discourse on genetic testing on YouTube by showing that the discourse in the comments primarily revolves around the content of the videos. Our research indicates that the discourse on YouTube may differ from that on other social media platforms, and hence, a detailed and differentiated consideration of the different platforms may be necessary. We further contribute to knowledge regarding user behavior on social media by examining users' attitudes and emotions toward DTC genetic testing videos on YouTube.As for practice, our research offers important implications for DTC genetic testing service providers, content creators, and regulatory authorities regarding user attitudes, which may help adapt or improve genetic testing services, multimedia content, or regulations. Similar to the study of Lee et al  involvinThe limitations of this study are as follows. First, we only considered a limited number of videos and comments. Even though we attempted to include an appropriate sample by saturating the videos and comments using metrics such as views and number of comments, examining all the initially identified videos n=1325) and comments could provide further insight, particularly concerning topic modeling and sentiment analysis. Second, we limited our YouTube API queries to the United States because the related DTC genetic testing market is the most evolved there. However, other regions with striving markets, such as Asia , could o325 and c, and a generally neutral-to-positive attitude toward DTC genetic testing expressed through words such as love, beautiful, pretty, and cool as well as a positive attitude toward DTC genetic testing–related videos on YouTube in general. Through this study, we show how users' attitudes toward DTC genetic testing can be determined by analyzing topics and opinions based on YouTube video comments. Our findings show that users are highly interested in DTC genetic testing and related social media content. Nonetheless, with this novel market still evolving, service providers, content providers, or regulatory authorities may need to adapt their services to users' interests and desires.This study examined 248 DTC genetic testing videos and 84,082 comments on YouTube to investigate user discourse. To this end, we employed topic modeling and identified 6 prevailing topics discussed among users, which largely revolve around the test purposes mentioned within those videos, such as ancestry or relationship testing. Further, we conducted sentiment analysis, showing that users have positive emotions, as indicated by the NRC sentiments of anticipation, joy, surprise, and trust


Serotonin influences mental health and well-being. To understand the influences of genetic variations in serotonin pathway on well-being, we examined the effects of seven serotonergic polymorphisms on subjective well-being  and psychological well-being  in a larger sample. Results indicated that the cumulative genetic score, but single genetic effects of serotonergic polymorphisms, was related to individual differences in well-being. Specifically, individuals with a greater cumulative genetic score, which is related to a low risk of depression, tended to exhibit high levels of subjective well-being and psychological well-being. These findings suggest that the overall serotoninergic genetic profile, rather than a specific genetic polymorphism, could greatly influence the individual differences in well-being. Well-being (i.e. happiness) refers to individuals’ positive psychological state and a sense of personal growth and self-determination that people attain from life experiences . AccordiThere are two influential views on the aspects of well-being , reflectThere have been many efforts to identify specific genetic factors of well-being , which wTPH2) are associated with the enzyme activity of tryptophan hydroxylase 2 (5-HT1A) and the rs6311 (−1438A/G) and rs6313 (T102C) of serotonin receptor 2A gene (5-HT2A) are associated with serotonin receptors expressions (5-HTT) are related to the expression of serotonin transporter  at a significance level of 0.05 (two-tailed α = 0.05) when expected regression coefficient is close to 0.01 , participants indicated their feelings on a 7-point Likert scale . The score was calculated by averaging the responses to eight items and the Cronbach’s α was 0.89.Psychological well-being was assessed with the Chinese version of the Flourishing Scale  did not influence the results, we conducted a factor analysis. The results indicated only 37.26% of the variance was captured by the first un-rotated factor, which suggests that common method variance was not a significant issue in this study.TPH2 are associated with serotonin biosynthesis. The rs6295 of 5-HT1A is related to the firing rate of serotonergic neurons and a feedback on serotonin releasing  upstream primer and 0.25 µl (25 pmol/µl) downstream primer. We genotyped the polymorphisms with the methods of PCR combined with restriction fragment length polymorphism or PCR with polyacrylamide gel electrophoresis . To ensure the reliability of genotyping results, we randomly selected six samples to commercially sequence and 3% samples to re-genotype. The results were agreement with the first genotyping results, suggesting a high accuracy of our genotyping. Of note, the rs6295, rs6313 and 5-HTTLPR deviated from the Hardy–Weinberg equilibrium (HWE).We genotyped seven serotonergic polymorphisms by collecting 3–5 hair follicle cells from each participant to extract genomic DNA  indicating the evidence in favor of alternative hypotheses over the null hypotheses. Given that there were three outcomes  and seven independent variables (i.e. seven polymorphisms), we set the significance level at P < 0.0025 (0.05/21) for the effect of single polymorphisms after Bonferroni-corrections. In the following analyses, the associations were accepted only when both statistical approaches produce significant estimations.The associations between single polymorphisms and well-being were examined with empirical multiple linear regression and Bayesian multiple linear regression. In empirical liner regression, genotype and standardized P < 0.016 (0.05/3) because three outcomes and one independent variable (i.e. the cumulative genetic score) were involved.We also used empirical linear regression and Bayesian linear regression to examine the relationships between cumulative genetic score and the aspects of well-being. Due to no available criteria for encoding the genotypes and weighting the genetic effects, we treated the risk alleles of depression as ‘0’ and protective alleles as ‘1’ according to the results of meta-analyses , and weighted the genetic effect with standardized regression coefficient of each polymorphism impacting on well-being. Based on these procedures, we created a cumulative genetic score by summarizing the product of standardized regression coefficients of each polymorphism on well-being with the number of protective alleles. For analysis on the effect of cumulative genetic score, the significance level was set at Ps ≥ 0.146; 10 ranging from 0.073 to 0.534. Similarly, the polymorphisms were not significantly associated with psychological well-being . For global life satisfaction, empirical multiple linear regression showed that the rs6313 of HTR2A gene and rs4570625 of TPH2 were significantly associated with this aspect of well-being . However, such statistical significances disappeared after Bonferroni-correction (corrected P = 0.420 for rs6313 and corrected P = 0.945 for rs4570625), and the Bayesian multiple linear regression analysis only provided weak evidence for the existence of the associations (BF10 = 1.120–2.164). Additionally, there was no significant interaction between the single nucleotide solymorphisms within any of the genes in the aspects of well-being after Bonferroni-correction .Collinearity diagnostics indicated that there was no significant collinearity among the polymorphisms when they were used as independent variables in predicting well-being (the variance inflation factor = 1.004–2.170). Empirical multiple linear regression indicated that all the polymorphisms were not significantly associated with affective balance . Similarly, the cumulative genetic score was significantly associated with global life satisfaction . After combining affective balance with global life satisfaction as a global index of subjective well-being, the cumulative genetic score was also significantly associated with this index . In addition, the cumulative genetic score could predict psychological well-being .Cumulative genetic score was created by summing up the products of standardized regression coefficient of each polymorphism on well-being and the number of protective alleles . Empirical liner regression indicated that the cumulative genetic score of serotonergic polymorphisms was significantly associated with affective balance , and the same was true for the relationship between the cumulative genetic score and subjective well-being when controlling for psychological well-being .To examine whether the cumulative genetic score underpinned the shared or unique variances of subjective well-being and psychological well-being, we included an aspect of well-being as an outcome and the other as a covariate in empirical liner regression. The results indicated that the significant relationship between the cumulative genetic score and psychological well-being survived when controlling for subjective well-being (Previous findings have shown that the 5-HTTLPR is associated with global life satisfaction or subjective well-being (5-HTT and the −1019C/G in 5-HT1A (This study found that individuals with a greater cumulative genetic score of serotonergic polymorphisms, indexing a predisposition of lower risk of depression, tend to exhibit higher level of affective balance. These findings demonstrate the importance of the cumulative genetic effect for affective balance. Affective balance is an indicator of emotion regulation (n 5-HT1A . MoreoveThis study shows that a greater cumulative genetic score of serotonergic polymorphisms is associated with higher global life satisfaction. Global life satisfaction is a cognitive evaluation of individuals’ overall life satisfaction . ConsideIn contrast to previous studies that focused on the relationships between candidate genes and subjective well-being (Subjective well-being and psychological well-being load separately but related prosperities . As the This study suggests that the cumulative genetic score, rather than the effect of a single polymorphism, may be a more effective predictor of well-being, which further suggests the advantage of this approach in improving statistical power and generating genetic variances when compared with examining a polymorphism in isolation (Several limitations should be mentioned. Firstly, current measures on well-being were based on Western conceptions of well-being. Due to cultural differences in well-being , the assThis study demonstrates that the cumulative genetic effect of serotonergic polymorphisms can significantly predict both subjective well-being and psychological well-being, suggesting the polygenic genetic architecture of well-being.


Several regions in the world are currently holding discussions in regard to the regulation of new genomic techniques (NGTs) and their application in agriculture. The European Commission, for instance, is proposing the introduction of specific regulation for NGT plants. Various questions need to be answered including e.g., the extent to which NGT-induced intended and unintended genetic modifications must be subjected to a mandatory risk assessment as part of an approval procedure. This review mostly focuses on findings in regard to unintended genetic changes that can be caused by the application of NGTs. More specifically, the review deals with the application of the nuclease CRISPR/Cas, which is currently the most important tool for developing NGT plants, and its potential to introduce double strand breaks (DSBs) at a targeted DNA sequence. For this purpose, we identified the differences in comparison to non-targeted mutagenesis methods used in conventional breeding. The review concludes that unintended genetic changes caused by NGT processes are relevant to risk assessment. Due to the technical characteristics of NGTs, the sites of the unintended changes, their genomic context and their frequency (in regard to specific sites) mean that the resulting gene combinations (intended or unintended) may be unlikely to occur with conventional methods. This, in turn, implies that the biological effects (phenotypes) can also be different and may cause risks to health and the environment. Therefore, we conclude that the assessment of intended as well as unintended genetic changes should be part of a mandatory comprehensive molecular characterisation and risk assessment of NGT plants that are meant for environmental releases or for market authorisation. According to EU GMO legislation , geneticFor the purposes of this review, we use the same specific terminology as Our review examines whether current EU regulation must in future continue to include the risk assessment of unintended genetic changes in NGT plants. In order to come to a sufficiently reasoned conclusion, our review first provides an overview of published findings related to unintended genetic changes caused by NGT processes in plants. Further, we identify causes for unintended genetic changes to differentiate these changes from non-targeted mutations which occur in conventional breeding. Finally, we discuss the consequences for the risk assessment of single events , and long-term accumulated effects.In short, and as summarized also by As has been shown many times [see for example ], NGTs eIn comparison to methods of conventional breeding (including non-targeted mutagenesis), NGTs can overcome the boundaries of natural genome organization: Relevant factors include repair mechanisms, gene duplications, genetic linkages and other epigenetic mechanisms . By oveIn comparison to conventional plant breeding using non-targeted mutagenesis, the overall number of mutations is typically lower in NGT plants . HoweverFurthermore, it has to be considered that the processes of NGTs involve several technical steps that, in the case of plants, very often include transformation processes which are also used in EGTs. These non-targeted methods are used to introduce the nucleases into the cells [see for example ] and mayUnintended genetic changes resulting from NGT processes can be differentiated as those with or without the insertion of transgenes, off-target changes or on-target changes, and those which are likely to be associated with or without the production of new gene products. Furthermore, this includes the identification of smaller genetic changes versus those that involve larger parts of the genome or even complex patterns of genetic changes. While some of the “types” of genetic alteration might also be observed in conventional breeding, there may also be some differences in regard to the probability of these changes occurring at specific sites in the genome (see above). In order to differentiate between unintended genetic changes resulting from NGTs and those resulting from conventional breeding, we suggest aligning them with the following five categories.At present, NGT applications in plants are in most cases a multi-step process. For example, NGTs, such as CRISPR/Cas applications in plants, typically make use of EGT techniques, i.e. non-targeted methods, to deliver the DNA coding for the nuclease into the cells . Thus, As several publications show, DSBs caused by CRISPR/Cas interventions are associated with the insertion of transgenes, especially at the target site or elsewhere in the genome. These on-target and off-target effects often include the integration of DNA from vector DNA derived from transformation processes, where, for example, fragments of the transgenes were unexpectedly integrated .Also in animal cells, it was found that unintentionally inserted foreign DNA fragments may originate from the vector construct . In someResearch is underway to develop transgene free delivery of the CRISPR/Cas molecules into the plant cells [see for example ]. HoweveVarious unintended genetic changes resulting from CRISPR/Cas applications have been described for plants. These include off-target DNA cleavage, repetitive unit deletion, indels of various sizes, larger structural changes in the targeted genomic region (with and without the insertion of transgenes) .Although some of these “types” of genetic alteration might also be observed in conventional breeding , they diFor example, larger structural genomic changes, such as translocations, deletions, duplications, inversions and scrambling of chromosomal sequences, can occur in or near the targeted genomic region which would otherwise be unlikely to occur . It shoIt is known that the nucleases rather recognize and target specific DNA sequences of a particular length rather than functional genetic elements at specific genomic sites . TherefoSince many of these undesirable effects as described above are often caused by DSBs introduced by the nuclease, other methods are under development that are purposed to introduce genetic changes without DSBs, especially in the area of human medicine such as base editing . These mChromothripsis is a genetic phenomenon where possibly hundreds of clustered chromosomal rearrangements can happen in a single catastrophic event. In mammals (including humans), the phenomenon is associated with cancer and congenital diseases. Available publications  show thaIt has been known that CRISPR/Cas applications strongly increase the likelihood of chromothripsis occurring in mammalian cells . Just reDSBs also may occur if, for example, plant cells are exposed to high dosage of radiation (non-targeted mutagenesis) . HoweverThese findings make it plausible that DSBs and chromothripsis-like effects caused by biotechnological mutagens (nucleases) should not generally be equated with those of non-targeted physical-chemical mutagens.The use of CRISPR/Cas gene scissors can induce various changes at the target sites. The targeted site  can be altered in such a way that no more mRNAs are formed, thus preventing the formation of the corresponding protein. However, new mRNAs can also be unintentionally formed, and thus cause new proteins to emerge.For example, the changes introduced by the nucleases can lead to an effect called exon skipping. In exon skipping, mRNAs can be assembled differently than planned even if the intended changes are induced at the target site. This can lead to the formation of shortened mRNAs. The resulting proteins are then also shorter, but can still carry out functions in the cell. The effects of exon skipping were described in mammalian cells  as well As a result of exon skipping and frameshift mutations, new mRNAs and proteins, or also non-coding RNAs (ncRNA) with effects on gene regulation, can be formed and fulfill new functions in cell metabolism . For exaAgain, since these unintended genetic changes may not occur randomly across the genome, its biological effects may depend on the genomic regions that are targeted by the processes of NGT. These effects can not be generally equated to those emerging from conventional breeding.According to EU regulation as cited above , it has Based on the various findings regarding unintended genetic effects that NGTs can cause, it does not appear possible to predict or control their occurrence and associated effects for specific events. As shown, unintended genetic changes can affect large sections of chromosomes and result in the emergence of unintended gene products. Since these unintended genetic changes may not occur randomly across the genome, its biological effects may depend on the genomic regions that are targeted by the processes of NGTs and therefore are also relevant for risk assessment.It is only afterwards through applying methods, e.g. whole genome sequencing (WGS) and other methods to identify long and short DNA sequence alterations  that thAs DNA sequencing will not always allow the identification of the associated unintended biological effects, additional methods, such as transcriptomics and metabolomics, should be used to draw reliable conclusions [see ]. If no After comprehensive molecular characterisation has been concluded, further steps in risk assessment should follow, such as the analysis of plant composition, agronomic and other phenotypical characteristics, that also may include further investigations in regard to human health and the environment . Data inter alia flora and fauna, soil fertility, soil degradation of organic material, the feed/food chain, biological diversity, animal health and resistance problems in relation to antibiotics.” Furthermore, similarly to Commission Directive (EU) 2018/350 , tomato (Solanum lycopersicum) or wheat (Triticum aestivum) .Even if specific unintended effects arising from molecular changes due to NGTs cannot be identified in a specific event, the regulator still has to consider cumulative effects and potential interactions which could result from future crossings within the same species or wild relative species.The resulting unintended effects may be dependent on specific combinations of intended or unintended genetic variants, which may become obvious only after exposure to stress conditions in the receiving environment .If unintended genetic changes, potentially causing adverse effects are overlooked, these may endanger health, the environment and also agricultural production. Therefore, unintended genetic changes caused by the processes of NGTs has to be included in mandatory risk assessment before the plants are released into the environment or placed onto the market.