Multi-omics analysis identifies mitochondrial pathways associated with anxiety-related behavior.

Stressful life events are major environmental risk factors for anxiety disorders, although not all individuals exposed to stress develop clinical anxiety. The molecular mechanisms underlying the influence of environmental effects on anxiety are largely unknown. To identify biological pathways mediating stress-related anxiety and resilience to it, we used the chronic social defeat stress (CSDS) paradigm in male mice of two inbred strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), that differ in their susceptibility to stress. Using a multi-omics approach, we identified differential mRNA, miRNA and protein expression changes in the bed nucleus of the stria terminalis (BNST) and blood cells after chronic stress. Integrative gene set enrichment analysis revealed enrichment of mitochondrial-related genes in the BNST and blood of stressed mice. To translate these results to human anxiety, we investigated blood gene expression changes associated with exposure-induced panic attacks. Remarkably, we found reduced expression of mitochondrial-related genes in D2 stress-susceptible mice and in exposure-induced panic attacks in humans, but increased expression of these genes in B6 stress-susceptible mice. Moreover, stress-susceptible vs. stress-resilient B6 mice displayed more mitochondrial cross-sections in the post-synaptic compartment after CSDS. Our findings demonstrate mitochondrial-related alterations in gene expression as an evolutionarily conserved response in stress-related behaviors and validate the use of cross-species approaches in investigating the biological mechanisms underlying anxiety disorders.

Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-κB-driven inflammation and cardiovascular risk.

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

DeepWAS: Multivariate genotype-phenotype associations by directly integrating regulatory information using deep learning.

Genome-wide association studies (GWAS) identify genetic variants associated with traits or diseases. GWAS never directly link variants to regulatory mechanisms. Instead, the functional annotation of variants is typically inferred by post hoc analyses. A specific class of deep learning-based methods allows for the prediction of regulatory effects per variant on several cell type-specific chromatin features. We here describe "DeepWAS", a new approach that integrates these regulatory effect predictions of single variants into a multivariate GWAS setting. Thereby, single variants associated with a trait or disease are directly coupled to their impact on a chromatin feature in a cell type. Up to 61 regulatory SNPs, called dSNPs, were associated with multiple sclerosis (MS, 4,888 cases and 10,395 controls), major depressive disorder (MDD, 1,475 cases and 2,144 controls), and height (5,974 individuals). These variants were mainly non-coding and reached at least nominal significance in classical GWAS. The prediction accuracy was higher for DeepWAS than for classical GWAS models for 91% of the genome-wide significant, MS-specific dSNPs. DSNPs were enriched in public or cohort-matched expression and methylation quantitative trait loci and we demonstrated the potential of DeepWAS to generate testable functional hypotheses based on genotype data alone. DeepWAS is available at https://github.com/cellmapslab/DeepWAS.

Effects of stressful life-events on DNA methylation in panic disorder and major depressive disorder.

BACKGROUND: Panic disorder (PD) is characterized by recurrent panic attacks and higher affection of women as compared to men. The lifetime prevalence of PD is about 2-3% in the general population leading to tremendous distress and disability. Etiologically, genetic and environmental factors, such as stress, contribute to the onset and relapse of PD. In the present study, we investigated epigenome-wide DNA methylation (DNAm) in respond to a cumulative, stress-weighted life events score (wLE) in patients with PD and its boundary to major depressive disorder (MDD), frequently co-occurring with symptoms of PD. METHODS: DNAm was assessed by the Illumina HumanMethylation450 BeadChip. In a meta-analytic approach, epigenome-wide DNAm changes in association with wLE were first analyzed in two PD cohorts (with a total sample size of 183 PD patients and 85 healthy controls) and lastly in 102 patients with MDD to identify possible overlapping and opposing effects of wLE on DNAm. Additionally, analysis of differentially methylated regions (DMRs) was conducted to identify regional clusters of association. RESULTS: Two CpG-sites presented with p-values below 1 × 10-05 in PD: cg09738429 (p = 6.40 × 10-06, located in an intergenic shore region in next proximity of PYROXD1) and cg03341655 (p = 8.14 × 10-06, located in the exonic region of GFOD2). The association of DNAm at cg03341655 and wLE could be replicated in the independent MDD case sample indicating a diagnosis independent effect. Genes mapping to the top hits were significantly upregulated in brain and top hits have been implicated in the metabolic system. Additionally, two significant DMRs were identified for PD only on chromosome 10 and 18, including CpG-sites which have been reported to be associated with anxiety and other psychiatric phenotypes. CONCLUSION: This first DNAm analysis in PD reveals first evidence of small but significant DNAm changes in PD in association with cumulative stress-weighted life events. Most of the top associated CpG-sites are located in genes implicated in metabolic processes supporting the hypothesis that environmental stress contributes to health damaging changes by affecting a broad spectrum of systems in the body.

Personalized medicine for patients with COPD: where are we?

Chronic airflow limitation is the common denominator of patients with chronic obstructive pulmonary disease (COPD). However, it is not possible to predict morbidity and mortality of individual patients based on the degree of lung function impairment, nor does the degree of airflow limitation allow guidance regarding therapies. Over the last decades, understanding of the factors contributing to the heterogeneity of disease trajectories, clinical presentation, and response to existing therapies has greatly advanced. Indeed, diagnostic assessment and treatment algorithms for COPD have become more personalized. In addition to the pulmonary abnormalities and inhaler therapies, extra-pulmonary features and comorbidities have been studied and are considered essential components of comprehensive disease management, including lifestyle interventions. Despite these advances, predicting and/or modifying the course of the disease remains currently impossible, and selection of patients with a beneficial response to specific interventions is unsatisfactory. Consequently, non-response to pharmacologic and non-pharmacologic treatments is common, and many patients have refractory symptoms. Thus, there is an ongoing urgency for a more targeted and holistic management of the disease, incorporating the basic principles of P4 medicine (predictive, preventive, personalized, and participatory). This review describes the current status and unmet needs regarding personalized medicine for patients with COPD. Also, it proposes a systems medicine approach, integrating genetic, environmental, (micro)biological, and clinical factors in experimental and computational models in order to decipher the multilevel complexity of COPD. Ultimately, the acquired insights will enable the development of clinical decision support systems and advance personalized medicine for patients with COPD.

Polymorphism in Tmem132d regulates expression and anxiety-related behavior through binding of RNA polymerase II complex.

TMEM132D is a candidate gene, where risk genotypes have been associated with anxiety severity along with higher mRNA expression in the frontal cortex of panic disorder patients. Concurrently, in a high (HAB) and low (LAB) trait anxiety mouse model, Tmem132d was found to show increased expression in the anterior cingulate cortex (aCC) of HAB as compared to LAB mice. To understand the molecular underpinnings underlying the differential expression, we sequenced the gene and found two single-nucleotide polymorphisms (SNPs) in the promoter differing between both lines which could explain the observed mRNA expression profiles using gene reporter assays. In addition, there was no difference in basal DNA methylation in the CpG Island that encompasses the HAB vs. LAB Tmem132d promoter region. Furthermore, we found significantly higher binding of RNA polymerase II (POLR2A) to the proximal HAB-specific SNP (rs233264624) than the corresponding LAB locus in an oligonucleotide pull-down assay, suggesting increased transcription. Virus mediated overexpression of Tmem132d in the aCC of C57BL/6 J mice could confirm its role in mediating an anxiogenic phenotype. To model gene-environmental interactions, HAB mice exposed to enriched environment (HAB-EE) responded with decreased anxiety levels but, had enhanced Tmem132d mRNA expression as compared to standard-housed HAB (HAB-SH) mice. While LAB mice subjected to unpredictable chronic mild stress (LAB-UCMS) exhibited higher anxiety levels and had lower mRNA expression compared to standard-housed LAB (LAB-SH) mice. Chromatin immunoprecipitation revealed significantly higher binding of POLR2A to rs233264624 in HAB-EE, while LAB-UCMS had lower POLR2A binding at this locus, thus explaining the enhanced or attenuated expression of Tmem132d compared to their respective SH controls. To further investigate gene-environment interactions, DNA methylation was assessed using Illumina 450 K BeadChip in 74 panic disorder patients. Significant methylation differences were observed in two CpGs (cg26322591 and cg03283235) located in TMEM132D depending on the number of positive life events supporting the results of an influence of positive environmental cues on regulation of Tmem132d expression in mice.

Correction to: Lifetime stress accelerates epigenetic aging in an urban, African American cohort: relevance of glucocorticoid signaling.

Upon publication of the original article [1] it was highlighted by the authors that a transposition error affected Additional file 1, causing the misplacement of several columns and rendering the table difficult to read. This transposition does not influence any of the results nor analyses presented in the paper and has since been formally noted in this correction article; the corrected file is available here as an Additional File. The publisher apologizes for this error.

Anxiety Associated Increased CpG Methylation in the Promoter of Asb1: A Translational Approach Evidenced by Epidemiological and Clinical Studies and a Murine Model.

Epigenetic regulation in anxiety is suggested, but evidence from large studies is needed. We conducted an epigenome-wide association study (EWAS) on anxiety in a population-based cohort and validated our finding in a clinical cohort as well as a murine model. In the KORA cohort, participants (n=1522, age 32-72 years) were administered the Generalized Anxiety Disorder (GAD-7) instrument, whole blood DNA methylation was measured (Illumina 450K BeadChip), and circulating levels of hs-CRP and IL-18 were assessed in the association between anxiety and methylation. DNA methylation was measured using the same instrument in a study of patients with anxiety disorders recruited at the Max Planck Institute of Psychiatry (MPIP, 131 non-medicated cases and 169 controls). To expand our mechanistic understanding, these findings were reverse translated in a mouse model of acute social defeat stress. In the KORA study, participants were classified according to mild, moderate, or severe levels of anxiety (29.4%/6.0%/1.5%, respectively). Severe anxiety was associated with 48.5% increased methylation at a single CpG site (cg12701571) located in the promoter of the gene encoding Asb1 (ß-coefficient=0.56 standard error (SE)=0.10, p (Bonferroni)=0.005), a protein hypothetically involved in regulation of cytokine signaling. An interaction between IL-18 and severe anxiety with methylation of this CpG cite showed a tendency towards significance in the total population (p=0.083) and a significant interaction among women (p=0.014). Methylation of the same CpG was positively associated with Panic and Agoraphobia scale (PAS) scores (ß=0.005, SE=0.002, p=0.021, n=131) among cases in the MPIP study. In a murine model of acute social defeat stress, Asb1 gene expression was significantly upregulated in a tissue-specific manner (p=0.006), which correlated with upregulation of the neuroimmunomodulating cytokine interleukin 1 beta. Our findings suggest epigenetic regulation of the stress-responsive Asb1 gene in anxiety-related phenotypes. Further studies are necessary to elucidate the causal direction of this association and the potential role of Asb1-mediated immune dysregulation in anxiety disorders.

Association of Methylation Signals With Incident Coronary Heart Disease in an Epigenome-Wide Assessment of Circulating Tumor Necrosis Factor α.

Importance: Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine with manifold consequences for mammalian pathophysiology, including cardiovascular disease. A deeper understanding of TNF-α biology may enhance treatment precision. Objective: To conduct an epigenome-wide analysis of blood-derived DNA methylation and TNF-α levels and to assess the clinical relevance of findings. Design, Setting, and Participants: This meta-analysis assessed epigenome-wide associations in circulating TNF-α concentrations from 5 cohort studies and 1 interventional trial, with replication in 3 additional cohort studies. Follow-up analyses investigated associations of identified methylation loci with gene expression and incident coronary heart disease; this meta-analysis included 11 461 participants who experienced 1895 coronary events. Exposures: Circulating TNF-α concentration. Main Outcomes and Measures: DNA methylation at approximately 450 000 loci, neighboring DNA sequence variation, gene expression, and incident coronary heart disease. Results: The discovery cohort included 4794 participants, and the replication study included 816 participants (overall mean [SD] age, 60.7 [8.5] years). In the discovery stage, circulating TNF-α levels were associated with methylation of 7 cytosine-phosphate-guanine (CpG) sites, 3 of which were located in or near DTX3L-PARP9 at cg00959259 (ß [SE] = -0.01 [0.003]; P = 7.36 × 10-8), cg08122652 (ß [SE] = -0.008 [0.002]; P = 2.24 × 10-7), and cg22930808(ß [SE] = -0.01 [0.002]; P = 6.92 × 10-8); NLRC5 at cg16411857 (ß [SE] = -0.01 [0.002]; P = 2.14 × 10-13) and cg07839457 (ß [SE] = -0.02 [0.003]; P = 6.31 × 10-10); or ABO, at cg13683939 (ß [SE] = 0.04 [0.008]; P = 1.42 × 10-7) and cg24267699 (ß [SE] = -0.009 [0.002]; P = 1.67 × 10-7), after accounting for multiple testing. Of these, negative associations between TNF-α concentration and methylation of 2 loci in NLRC5 and 1 in DTX3L-14 PARP9 were replicated. Replicated TNF-α-linked CpG sites were associated with 9% to 19% decreased risk of incident coronary heart disease per 10% higher methylation per CpG site (cg16411857: hazard ratio [HR], 0.86; 95% CI, 0.78-1.95; P = .003; cg07839457: HR, 0.89; 95% CI, 0.80-0.94; P = 3.1 × 10-5; cg00959259: HR, 0.91; 95% CI, 0.84-0.97; P = .002; cg08122652: HR, 0.81; 95% CI, 0.74-0.89; P = 2.0 × 10-5). Conclusions and Relevance: We identified and replicated novel epigenetic correlates of circulating TNF-α concentration in blood samples and linked these loci to coronary heart disease risk, opening opportunities for validation and therapeutic applications.

"DNA Methylation signatures in panic disorder".

Panic disorder (PD) affects about four million Europeans, with women affected twice as likely as men, causing substantial suffering and high economic costs. The etiopathogenesis of PD remains largely unknown, but both genetic and environmental factors contribute to risk. An epigenome-wide association study (EWAS) was conducted to compare medication-free PD patients (n = 89) with healthy controls (n = 76) stratified by gender. Replication was sought in an independent sample (131 cases, 169 controls) and functional analyses were conducted in a third sample (N = 71). DNA methylation was assessed in whole blood using the Infinium HumanMethylation450 BeadChip. One genome-wide association surviving FDR of 5% (cg07308824, P = 1.094 × 10-7, P-adj = 0.046) was identified in female PD patients (N = 49) compared to controls (N = 48). The same locus, located in an enhancer region of the HECA gene, was also hypermethylated in female PD patients in the replication sample (P = 0.035) and the significance of the association improved in the meta-analysis (P-adj = 0.004). Methylation at this CpG site was associated with HECA mRNA expression in another independent female sample (N = 71) both at baseline (P = 0.046) and after induction by dexamethasone (P = 0.029). Of 15 candidates, 5 previously reported as associated with PD or anxiety traits also showed differences in DNA methylation after gene-wise correction and included SGK1, FHIT, ADCYAP1, HTR1A, HTR2A. Our study examines epigenome-wide differences in peripheral blood for PD patients. Our results point to possible sex-specific methylation changes in the HECA gene for PD but overall highlight that this disorder is not associated with extensive changes in DNA methylation in peripheral blood.

An epigenetic clock for gestational age at birth based on blood methylation data.

BACKGROUND: Gestational age is often used as a proxy for developmental maturity by clinicians and researchers alike. DNA methylation has previously been shown to be associated with age and has been used to accurately estimate chronological age in children and adults. In the current study, we examine whether DNA methylation in cord blood can be used to estimate gestational age at birth. RESULTS: We find that gestational age can be accurately estimated from DNA methylation of neonatal cord blood and blood spot samples. We calculate a DNA methylation gestational age using 148 CpG sites selected through elastic net regression in six training datasets. We evaluate predictive accuracy in nine testing datasets and find that the accuracy of the DNA methylation gestational age is consistent with that of gestational age estimates based on established methods, such as ultrasound. We also find that an increased DNA methylation gestational age relative to clinical gestational age is associated with birthweight independent of gestational age, sex, and ancestry. CONCLUSIONS: DNA methylation can be used to accurately estimate gestational age at or near birth and may provide additional information relevant to developmental stage. Further studies of this predictor are warranted to determine its utility in clinical settings and for research purposes. When clinical estimates are available this measure may increase accuracy in the testing of hypotheses related to developmental age and other early life circumstances.

Lifetime stress accelerates epigenetic aging in an urban, African American cohort: relevance of glucocorticoid signaling.

BACKGROUND: Chronic psychological stress is associated with accelerated aging and increased risk for aging-related diseases, but the underlying molecular mechanisms are unclear. RESULTS: We examined the effect of lifetime stressors on a DNA methylation-based age predictor, epigenetic clock. After controlling for blood cell-type composition and lifestyle parameters, cumulative lifetime stress, but not childhood maltreatment or current stress alone, predicted accelerated epigenetic aging in an urban, African American cohort (n = 392). This effect was primarily driven by personal life stressors, was more pronounced with advancing age, and was blunted in individuals with higher childhood abuse exposure. Hypothesizing that these epigenetic effects could be mediated by glucocorticoid signaling, we found that a high number (n = 85) of epigenetic clock CpG sites were located within glucocorticoid response elements. We further examined the functional effects of glucocorticoids on epigenetic clock CpGs in an independent sample with genome-wide DNA methylation (n = 124) and gene expression data (n = 297) before and after exposure to the glucocorticoid receptor agonist dexamethasone. Dexamethasone induced dynamic changes in methylation in 31.2 % (110/353) of these CpGs and transcription in 81.7 % (139/170) of genes neighboring epigenetic clock CpGs. Disease enrichment analysis of these dexamethasone-regulated genes showed enriched association for aging-related diseases, including coronary artery disease, arteriosclerosis, and leukemias. CONCLUSIONS: Cumulative lifetime stress may accelerate epigenetic aging, an effect that could be driven by glucocorticoid-induced epigenetic changes. These findings contribute to our understanding of mechanisms linking chronic stress with accelerated aging and heightened disease risk.