Dual Role of microRNA-146a in Experimental Inflammation in Human Pulmonary Epithelial and Immune Cells and Expression in Inflammatory Lung Diseases.

microRNA (miR)-146a emerges as a promising post-transcriptional regulator in various inflammatory diseases with different roles for the two isoforms miR-146a-5p and miR-146a-3p. The present study aimed to examine the dual role of miR-146a-5p and miR-146a 3p in the modulation of inflammation in human pulmonary epithelial and immune cells in vitro as well as their expression in patients with inflammatory lung diseases. Experimental inflammation in human A549, HL60, and THP1 via the NF-kB pathway resulted in the major upregulation of miR-146a-5p and miR-146a-3p expression, which was partly cell-specific. Modulation by transfection with miRNA mimics and inhibitors demonstrated an anti-inflammatory effect of miR-146a-5p and a pro-inflammatory effect of miR-146a-3p, respectively. A mutual interference between miR-146a-5p and miR-146a-3p was observed, with miR-146a-5p exerting a predominant influence. In vivo NGS analyses revealed an upregulation of miR-146a-3p in the blood of patients with cystic fibrosis and bronchiolitis obliterans, while miR-146a-5p levels were downregulated or unchanged compared to controls. The reverse pattern was observed in patients with SARS-CoV-2 infection. In conclusion, miR-146a-5p and miR-146a-3p are two distinct but interconnected miRNA isoforms with opposing functions in inflammation regulation. Understanding their interaction provides important insights into the progression and persistence of inflammatory lung diseases and might provide potential therapeutic options.

Effectiveness of a positive psychology and mindfulness-based app on mental health for parents of children with a neurodevelopmental disorder: study protocol of a pragmatic international randomized controlled trial.

INTRODUCTION: Parents of children with a neurodevelopmental disorder (NDD) experience more stress than parents of typically developing children. In a cocreation process with experts and parents, a low-threshold application that uses exercises based on the principles of positive psychology and mindfulness was developed. This application, called "Adappt," aims at enhancing the ability to adapt of the parents and caregivers of children with NDDs and at supporting their mental health. This protocol describes the evaluation study of the effectiveness of Adappt, its core working mechanisms and user experiences. METHOD: A pragmatic international multicenter randomized controlled trial will compare the effectiveness of Adappt with a (delayed) waitlist control condition. At least 212 parents or primary caregivers of children younger than 18 years diagnosed with or suspected of a NDD will be randomly assigned to the intervention or waitlist control condition. Participants are excluded if they have severe anxiety or depression levels or are in treatment for mental health issues. Measures will be collected online at baseline, post-intervention (1 month after baseline), and 4 and 7 months after baseline. The primary outcome is the improvement in generic sense of ability to adapt as measured with the Generic Sense of Ability to Adapt Scale (GSAAS; (Front Psychol 14:985408, 2023)) at 4-month follow-up. Secondary outcomes are mental well-being, (parental) distress, and client satisfaction with "Adappt." DISCUSSION: Results of this study will contribute to knowledge on the effectiveness of a low-threshold application for parents of children with a NDD in multiple countries. If the application is found to be effective in improving mental health, recommendations will be made for implementation in health care. TRIAL REGISTRATION: This study is registered on clinicaltrials.gov (NCT06248762) on February 8, 2024, and the Open Science Framework ( https://osf.io/5znqv ).

Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis.

Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4-/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4-/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4-/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.

MicroRNA Profiling of the Inflammatory Response after Early and Late Asthmatic Reaction.

A high proportion of house dust mite (HDM)-allergic asthmatics suffer from both an early asthmatic reaction (EAR) and a late asthmatic reaction (LAR) which follows it. In these patients, allergic inflammation is more relevant. MiRNAs have been shown to play an important role in the regulation of asthma's pathology. The aim of this study was to analyze the miRNA profile in patients with mild asthma and an HDM allergy after bronchial allergen provocation (BAP). Seventeen patients with EAR/no LAR and 17 patients with EAR plus LAR, determined by a significant fall in FEV1 after BAP, were differentially analyzed. As expected, patients with EAR plus LAR showed a more pronounced allergic inflammation and FEV1 delta drop after 24 h. NGS-miRNA analysis identified the down-regulation of miR-15a-5p, miR-15b-5p, and miR-374a-5p after BAP with the highest significance in patients with EAR plus LAR, which were negatively correlated with eNO and the maximum decrease in FEV1. These miRNAs have shared targets like CCND1, VEGFA, and GSK3B, which are known to be involved in airway remodeling, basement membrane thickening, and Extracellular Matrix deposition. NGS-profiling identified miRNAs involved in the inflammatory response after BAP with HDM extract, which might be useful to predict a LAR.

Admission rates and clinical profiles of children and youth with eating disorders treated as inpatients before and during the COVID-19 pandemic in a German university hospital.

Introduction: Children and youth at risk for mental health disorders, such as eating disorders (ED), were particularly affected by the COVID-19 pandemic, yet evidence for the most seriously affected and thus hospitalized youth in Germany is scarce. Methods: This crosssectional study investigated anonymized routine hospital data (demographic information, diagnoses, treatment modalities) of patients admitted (n = 2,849) to the Department of Child and Adolescence Psychiatry, Psychosomatics and Psychotherapy (DCAPPP) of a German University Hospital between 01/2016 and 02/2022. Absolute and relative number of inpatients with or without ED prior to (01/2016-02/2020) and during the COVID-19 pandemic (03/2020-02/2022) were compared. The effect of school closures as part of social lockdown measures for COVID-19 mitigation on inpatient admission rate was explored as it has been discussed as a potential risk factor for mental health problems in youth. Results: During the COVID-19 pandemic, ED inpatient admission rate increased from 10.5 to 16.7%, primarily driven by Anorexia Nervosa (AN). In contrast to previous reports, we found no change in somatic and mental disorder comorbidity, age or sexratio for hospitalized youth with ED. However, we did observe a shortened length of hospital stay (LOS) for hospitalized youth with and without ED. In addition, non-ED admissions presented with an increased number of mental disorder comorbidities. In contrast to our hypothesis, school closures were not related to the observed increase in ED. Discussion: In summary, the COVID-19 pandemic was associated with an increased rate of inpatient treatment for youth suffering from AN, and of youth affected by multiple mental disorders. Accordingly, we assume that inpatient admission was prioritized for individuals with a higher burden of disease during the COVID-19 pandemic. Our findings pinpoint the need for adequate inpatient mental health treatment capacities during environmental crises, and a further strengthening of child and adolescence psychiatry services in Germany.

Association between Polyunsaturated Fatty Acid Profile and Bronchial Inflammation in Bronchiolitis Obliterans.

Introduction: Bronchiolitis obliterans (BO) is a chronic lung disease, which occurs after an insult to the lower airways, in particular after airway infections or after stem cell transplantation, and which results in persistent inflammation. N-3 and n-6 polyunsaturated fatty acids (PUFA) have been shown to influence the inflammatory processes in chronic inflammatory conditions. Since BO is maintained by persistent pulmonary inflammation, a disbalanced n-6/n-3 fatty acid profile could support the inflammatory process in patients with BO and therefore, could become an approach to new therapeutic options. Methods: Twenty-five patients with BO (age: 13; 7-39) and 26 healthy controls (age: 19; 7-31) participated in the study. Lung function (forced viral capacity (FVC), forced expiratory volume 1 (FEV1), residual volume (RV)), and lung clearance index (LCI) were measured. Induced sputum was analyzed for cytology and cytokine levels (IL-1ß, IL-6, IL-8, TNF-α) using cytometric bead array (CBA). The PUFA profile was determined in the serum and induced sputum by gas chromatography. Results: Patients presented with significantly lower FVC and FEV1 as well as higher RV and LCI measurements compared to the control group. Apart from a massive airway inflammation indicated by elevated numbers of total cells and neutrophils, the CBA analysis showed significantly increased levels of IL-1ß, IL-6, and IL-8. The analysis of PUFA in sputum and serum revealed a significant difference in the ratio between the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) (AA : DHA). Furthermore, the AA : DHA ratio significantly correlated with the inflammatory cytokines in induced sputum. Conclusion: Lung function in BO is significantly impaired and associated with uncontrolled neutrophil-dominated airway inflammation. Furthermore, the imbalance in the AA/DHA ratio in favor of n-6 PUFA demonstrates a pro-inflammatory microenvironment in the cell membrane, which correlates with the inflammatory cytokines in induced sputum and might be an option for an anti-inflammatory therapy in BO.

Generation of four human induced pluripotent stem cells derived from ADHD patients carrying different genotypes for the risk SNP rs1397547 in the ADHD-associated gene ADGRL3.

Single nucleotide polymorphisms (SNPs) in the ADGRL3 gene have been significantly associated with the development of ADHD, the aetiology of which remains poorly understood. The rs1397547 SNP has additionally been associated with significantly altered ADGRL3 transcription. We therefore generated iPSCs from two wild type ADHD patients, and two ADHD patients heterozygous for the risk SNP. With this resource we aim to facilitate further investigation into the complex and heterogenous pathology of ADHD. Furthermore, we demonstrate the feasibility of using magnetic activated cell sorting to allow the unbiased selection of fully reprogrammed iPSCs.

Association of polygenic risk scores and hair cortisol with mental health trajectories during COVID lockdown.

The COVID-19 pandemic is a global stressor with inter-individually differing influences on mental health trajectories. Polygenic Risk Scores (PRSs) for psychiatric phenotypes are associated with individual mental health predispositions. Elevated hair cortisol concentrations (HCC) and high PRSs are related to negative mental health outcomes. We analyzed whether PRSs and HCC are related to different mental health trajectories during the first COVID lockdown in Germany. Among 523 participants selected from the longitudinal resilience assessment study (LORA), we previously reported three subgroups (acute dysfunction, delayed dysfunction, resilient) based on weekly mental health (GHQ-28) assessment during COVID lockdown. DNA from blood was collected at the baseline of the original LORA study (n = 364) and used to calculate the PRSs of 12 different psychopathological phenotypes. An explorative bifactor model with Schmid-Leiman transformation was calculated to extract a general genetic factor for psychiatric disorders. Hair samples were collected quarterly prior to the pandemic for determining HCC (n = 192). Bivariate logistic regressions were performed to test the associations of HCC and the PRS factors with the reported trajectories. The bifactor model revealed 1 general factor and 4 sub-factors. Results indicate a significant association between increased values on the general risk factor and the allocation to the acute dysfunction class. The same was found for elevated HCC and the exploratorily tested sub-factor "childhood-onset neurodevelopmental disorders". Genetic risk and long-term cortisol secretion as a potential indicator of stress, indicated by PRSs and HCC, respectively, predicted different mental health trajectories. Results indicate a potential for future studies on risk prediction.

ADHD-associated PARK2 copy number variants: A pilot study on gene expression and effects of supplementary deprivation in patient-derived cell lines.

Recent studies show an association of Parkin RBR E3 ubiquitin protein ligase (PARK2) copy number variations (CNVs) with attention deficit hyperactivity disorder (ADHD). The aim of our pilot study to investigate gene expression associated with PARK2 CNVs in human-derived cellular models. We investigated gene expression in fibroblasts, hiPSC and dopaminergic neurons (DNs) of ADHD PARK2 deletion and duplication carriers by qRT PCR compared with healthy and ADHD cell lines without PARK2 CNVs. The selected 10 genes of interest were associated with oxidative stress response (TP53, NQO1, and NFE2L2), ubiquitin pathway (UBE3A, UBB, UBC, and ATXN3) and with a function in mitochondrial quality control (PINK1, MFN2, and ATG5). Additionally, an exploratory RNA bulk sequencing analysis in DNs was conducted. Nutrient deprivation as a supplementary deprivation stress paradigm was used to enhance potential genotype effects. At baseline, in fibroblasts, hiPSC, and DNs, there was no significant difference in gene expression after correction for multiple testing. After nutrient deprivation in fibroblasts NAD(P)H-quinone-dehydrogenase 1 (NQO1) expression was significantly increased in PARK2 CNV carriers. In a multivariate analysis, ubiquitin C (UBC) was significantly upregulated in fibroblasts of PARK2 CNV carriers. RNA sequencing analysis of DNs showed the strongest significant differential regulation in Neurontin (NNAT) at baseline and after nutrient deprivation. Our preliminary results suggest differential gene expression in pathways associated with oxidative stress, ubiquitine-proteasome, immunity, inflammation, cell growth, and differentiation, excitation/inhibition modulation, and energy metabolism in PARK2 CNV carriers compared to wildtype healthy controls and ADHD patients.

Effects of Hypoxia on RNA Cargo in Extracellular Vesicles from Human Adipose-Derived Stromal/Stem Cells.

Mesenchymal stromal/stem cells and their derivates are the most promising cell source for cell therapies in regenerative medicine. The application of extracellular vesicles (EVs) as cell-free therapeuticals requires particles with a maximum regenerative capability to enhance tissue and organ regeneration. The cargo of mRNA and microRNA (miR) in EVs after hypoxic preconditioning has not been extensively investigated. Therefore, the aim of our study was the characterization of mRNA and the miR loading of EVs. We further investigated the effects of the isolated EVs on renal tubular epithelial cells in vitro. We found 3131 transcripts to be significantly regulated upon hypoxia. Only 15 of these were downregulated, but 3116 were up-regulated. In addition, we found 190 small RNAs, 169 of these were miRs and 21 were piwi-interacting RNAs (piR). However, only 18 of the small RNAs were significantly altered, seven were miRs and 11 were piRs. Interestingly, all seven miRs were down-regulated after hypoxic pretreatment, whereas all 11 piRs were up-regulated. Gene ontology term enrichment and miR-target enrichment analysis of the mRNAs and miR were also performed in order to study the biological background. Finally, the therapeutic effect of EVs on human renal tubular epithelial cells was shown by the increased expression of three anti-inflammatory molecules after incubation with EVs from hypoxic pretreatment. In summary, our study demonstrates the altered mRNA and miR load in EVs after hypoxic preconditioning, and their anti-inflammatory effect on epithelial cells.

The methylome in females with adolescent Conduct Disorder: Neural pathomechanisms and environmental risk factors.

Conduct Disorder (CD) is an impairing psychiatric disorder of childhood and adolescence characterized by aggressive and dissocial behavior. Environmental factors such as maternal smoking during pregnancy, socio-economic status, trauma, or early life stress are associated with CD. Although the number of females with CD is rising in Western societies, CD is under-researched in female cohorts. We aimed at exploring the epigenetic signature of females with CD and its relation to psychosocial and environmental risk factors. We performed HpaII sensitive genome-wide methylation sequencing of 49 CD girls and 50 matched typically developing controls and linear regression models to identify differentially methylated CpG loci (tags) and regions. Significant tags and regions were mapped to the respective genes and tested for enrichment in pathways and brain developmental processes. Finally, epigenetic signatures were tested as mediators for CD-associated risk factors. We identified a 12% increased methylation 5' of the neurite modulator SLITRK5 (FDR = 0.0046) in cases within a glucocorticoid receptor binding site. Functionally, methylation positively correlated with gene expression in lymphoblastoid cell lines. At systems-level, genes (uncorr. P < 0.01) were associated with development of neurons, neurite outgrowth or neuronal developmental processes. At gene expression level, the associated gene-networks are activated perinatally and during early childhood in neocortical regions, thalamus and striatum, and expressed in amygdala and hippocampus. Specifically, the epigenetic signatures of the gene network activated in the thalamus during early childhood correlated with the effect of parental education on CD status possibly mediating its protective effect. The differential methylation patterns identified in females with CD are likely to affect genes that are expressed in brain regions previously indicated in CD. We provide suggestive evidence that protective effects are likely mediated by epigenetic mechanisms impairing specific brain developmental networks and therefore exerting a long-term effect on neural functions in CD. Our results are exploratory and thus, further replication is needed.

Neuroendocrine Stress Response in Female and Male Youths With Conduct Disorder and Associations With Early Adversity.

OBJECTIVE: Conduct disorder (CD) involves aggressive and antisocial behavior and is associated with blunted cortisol stress response in male youths. Far less is known about cortisol stress responsivity in female youths with CD or other neuroendocrine responses in both sexes. Although CD is linked to early adversity, the possibility that neuroendocrine alterations may mediate the relationship between early adversity and CD has not been systematically investigated. METHOD: Within the European FemNAT-CD multi-site study, salivary cortisol, testosterone, the testosterone/cortisol ratio, oxytocin, and psychological stress response to a standardized psychosocial stress test (the Trier Social Stress Test [TSST]), together with common pre- and postnatal environmental risk factors, were investigated in 130 pubertal youths with CD (63% female, 9-18 years of age) and 160 sex-, age-, and puberty-matched healthy controls (HCs). RESULTS: The TSST induced psychological stress in both CD and HCs. In contrast, female and male youths with CD showed blunted cortisol, testosterone, oxytocin, and testosterone/cortisol stress responses compared to HCs. These blunted stress responses partly mediated the relationship between environmental risk factors and CD. CONCLUSION: Findings from this unique sample, including many female youths with CD, provide evidence for a widespread attenuated stress responsivity of not only stress hormones, but also sex hormones and neuropeptides in CD and its subgroups (eg, with limited prosocial emotions). Results are the first to demonstrate blunted neuroendocrine stress responses in both female and male youths with CD. Early adversity may alter neuroendocrine stress responsivity. Biological mechanisms should be investigated further to pave the way for personalized intervention, thereby improving treatments for CD.

Translating the Role of mTOR- and RAS-Associated Signalopathies in Autism Spectrum Disorder: Models, Mechanisms and Treatment.

Mutations affecting mTOR or RAS signaling underlie defined syndromes (the so-called mTORopathies and RASopathies) with high risk for Autism Spectrum Disorder (ASD). These syndromes show a broad variety of somatic phenotypes including cancers, skin abnormalities, heart disease and facial dysmorphisms. Less well studied are the neuropsychiatric symptoms such as ASD. Here, we assess the relevance of these signalopathies in ASD reviewing genetic, human cell model, rodent studies and clinical trials. We conclude that signalopathies have an increased liability for ASD and that, in particular, ASD individuals with dysmorphic features and intellectual disability (ID) have a higher chance for disruptive mutations in RAS- and mTOR-related genes. Studies on rodent and human cell models confirm aberrant neuronal development as the underlying pathology. Human studies further suggest that multiple hits are necessary to induce the respective phenotypes. Recent clinical trials do only report improvements for comorbid conditions such as epilepsy or cancer but not for behavioral aspects. Animal models show that treatment during early development can rescue behavioral phenotypes. Taken together, we suggest investigating the differential roles of mTOR and RAS signaling in both human and rodent models, and to test drug treatment both during and after neuronal development in the available model systems.

The MiR-320 Family Is Strongly Downregulated in Patients with COVID-19 Induced Severe Respiratory Failure.

A high incidence of thromboembolic events associated with high mortality has been reported in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infections with respiratory failure. The present study characterized post-transcriptional gene regulation by global microRNA (miRNA) expression in relation to activated coagulation and inflammation in 21 critically ill SARS-CoV-2 patients. The cohort consisted of patients with moderate respiratory failure (n = 11) and severe respiratory failure (n = 10) at an acute stage (day 0-3) and in the later course of the disease (>7 days). All patients needed supplemental oxygen and severe patients were defined by the requirement of positive pressure ventilation (intubation). Levels of D-dimers, activated partial thromboplastin time (aPTT), C-reactive protein (CRP), and interleukin (IL)-6 were significantly higher in patients with severe compared with moderate respiratory failure. Concurrently, next generation sequencing (NGS) analysis demonstrated increased dysregulation of miRNA expression with progression of disease severity connected to extreme downregulation of miR-320a, miR-320b and miR-320c. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed involvement in the Hippo signaling pathway, the transforming growth factor (TGF)-ß signaling pathway and in the regulation of adherens junctions. The expression of all miR-320 family members was significantly correlated with CRP, IL-6, and D-dimer levels. In conclusion, our analysis underlines the importance of thromboembolic processes in patients with respiratory failure and emphasizes miRNA-320s as potential biomarkers for severe progressive SARS-CoV-2 infection.

SLC25A24 gene methylation and gray matter volume in females with and without conduct disorder: an exploratory epigenetic neuroimaging study.

Conduct disorder (CD), a psychiatric disorder characterized by a repetitive pattern of antisocial behaviors, results from a complex interplay between genetic and environmental factors. The clinical presentation of CD varies both according to the individual's sex and level of callous-unemotional (CU) traits, but it remains unclear how genetic and environmental factors interact at the molecular level to produce these differences. Emerging evidence in males implicates methylation of genes associated with socio-affective processes. Here, we combined an epigenome-wide association study with structural neuroimaging in 51 females with CD and 59 typically developing (TD) females to examine DNA methylation in relation to CD, CU traits, and gray matter volume (GMV). We demonstrate an inverse pattern of correlation between CU traits and methylation of a chromosome 1 region in CD females (positive) as compared to TD females (negative). The identified region spans exon 1 of the SLC25A24 gene, central to energy metabolism due to its role in mitochondrial function. Increased SLC25A24 methylation was also related to lower GMV in multiple brain regions in the overall cohort. These included the superior frontal gyrus, prefrontal cortex, and supramarginal gyrus, secondary visual cortex and ventral posterior cingulate cortex, which are regions that have previously been implicated in CD and CU traits. While our findings are preliminary and need to be replicated in larger samples, they provide novel evidence that CU traits in females are associated with methylation levels in a fundamentally different way in CD and TD, which in turn may relate to observable variations in GMV across the brain.

[Genetic risk factors and their influence on neural development in autism spectrum disorders]. / Genetische Risikofaktoren und ihre Auswirkungen auf die neurale Entwicklung bei Autismus-Spektrum-Störungen.

Genetic risk factors and their influence on neural development in autism spectrum disorders Abstract. Abstract. Autism spectrum disorders are etiologically based on genetic and specific gene x biologically relevant environmental risk factors. They are diagnosed based on behavioral characteristics, such as impaired social communication and stereotyped, repetitive behavior and sensory as well as special interests. The genetic background is heterogeneous, i. e., it comprises diverse genetic risk factors across the disorder and high interindividual differences of specific genetic risk factors. Nevertheless, risk factors converge regarding underlying biological mechanisms and shared pathways, which likely cause the autism-specific behavioral characteristics. The current selective literature review summarizes differential genetic risk factors and focuses particularly on mechanisms and pathways currently being discussed by international research. In conclusion, clinically relevant aspects and open translational research questions are presented.

DNA methylation signatures of aggression and closely related constructs: A meta-analysis of epigenome-wide studies across the lifespan.

DNA methylation profiles of aggressive behavior may capture lifetime cumulative effects of genetic, stochastic, and environmental influences associated with aggression. Here, we report the first large meta-analysis of epigenome-wide association studies (EWAS) of aggressive behavior (N = 15,324 participants). In peripheral blood samples of 14,434 participants from 18 cohorts with mean ages ranging from 7 to 68 years, 13 methylation sites were significantly associated with aggression (alpha = 1.2 × 10-7; Bonferroni correction). In cord blood samples of 2425 children from five cohorts with aggression assessed at mean ages ranging from 4 to 7 years, 83% of these sites showed the same direction of association with childhood aggression (r = 0.74, p = 0.006) but no epigenome-wide significant sites were found. Top-sites (48 at a false discovery rate of 5% in the peripheral blood meta-analysis or in a combined meta-analysis of peripheral blood and cord blood) have been associated with chemical exposures, smoking, cognition, metabolic traits, and genetic variation (mQTLs). Three genes whose expression levels were associated with top-sites were previously linked to schizophrenia and general risk tolerance. At six CpGs, DNA methylation variation in blood mirrors variation in the brain. On average 44% (range = 3-82%) of the aggression-methylation association was explained by current and former smoking and BMI. These findings point at loci that are sensitive to chemical exposures with potential implications for neuronal functions. We hope these results to be a starting point for studies leading to applications as peripheral biomarkers and to reveal causal relationships with aggression and related traits.

Knockdown of the ADHD Candidate Gene Diras2 in Murine Hippocampal Primary Cells.

Objective: The DIRAS2 gene is associated with ADHD, but its function is largely unknown. Thus, we aimed to explore the genes and molecular pathways affected by DIRAS2. Method: Using short hairpin RNAs, we downregulated Diras2 in murine hippocampal primary cells. Gene expression was analyzed by microarray and affected pathways were identified. We used quantitative real-time polymerase chain reaction (qPCR) to confirm expression changes and analyzed enrichment of differentially expressed genes in an ADHD GWAS (genome-wide association studies) sample. Results:Diras2 knockdown altered expression of 1,612 genes, which were enriched for biological processes involved in neurodevelopment. Expression changes were confirmed for 33 out of 88 selected genes. These 33 genes showed significant enrichment in ADHD patients in a gene-set-based analysis. Conclusion: Our findings show that Diras2 affects numerous genes and thus molecular pathways that are relevant for neurodevelopmental processes. These findings may further support the hypothesis that DIRAS2 is linked to etiological processes underlying ADHD.

Energy Metabolism Disturbances in Cell Models of PARK2 CNV Carriers with ADHD.

The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics.