Stella Iurato

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

BackgroundGestational 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.ResultsWe 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.ConclusionsDNA 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.

An epigenome-wide association study meta-analysis of educational attainment

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications—in our case, CpG methylation—and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

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 (β-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 (β=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.

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.

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.

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

Importance Tumor necrosis factor &agr; (TNF-&agr;) is a proinflammatory cytokine with manifold consequences for mammalian pathophysiology, including cardiovascular disease. A deeper understanding of TNF-&agr; biology may enhance treatment precision. Objective To conduct an epigenome-wide analysis of blood-derived DNA methylation and TNF-&agr; levels and to assess the clinical relevance of findings. Design, Setting, and Participants This meta-analysis assessed epigenome-wide associations in circulating TNF-&agr; 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-&agr; 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-&agr; levels were associated with methylation of 7 cytosine-phosphate-guanine (CpG) sites, 3 of which were located in or near DTX3L-PARP9 at cg00959259 (&bgr; [SE] = −0.01 [0.003]; P = 7.36 × 10−8), cg08122652 (&bgr; [SE] = −0.008 [0.002]; P = 2.24 × 10−7), and cg22930808(&bgr; [SE] = −0.01 [0.002]; P = 6.92 × 10−8); NLRC5 at cg16411857 (&bgr; [SE] = −0.01 [0.002]; P = 2.14 × 10−13) and cg07839457 (&bgr; [SE] = −0.02 [0.003]; P = 6.31 × 10−10); or ABO, at cg13683939 (&bgr; [SE] = 0.04 [0.008]; P = 1.42 × 10−7) and cg24267699 (&bgr; [SE] = −0.009 [0.002]; P = 1.67 × 10−7), after accounting for multiple testing. Of these, negative associations between TNF-&agr; concentration and methylation of 2 loci in NLRC5 and 1 in DTX3L-14 PARP9 were replicated. Replicated TNF-&agr;–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-&agr; concentration in blood samples and linked these loci to coronary heart disease risk, opening opportunities for validation and therapeutic applications.

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.

An epigenome-wide association study of educational attainment (n = 10,767)

The epigenome has been shown to be influenced by biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption, and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here, we provide evidence on the associations between epigenetic modifications—in our case, CpG methylation—and educational attainment (EA), a biologically distal environmental factor that is arguably among of the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10,767 individuals. While we find that 9 CpG probes are significantly associated with EA, only two remain associated when we restrict the sample to never-smokers. These two are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, their effect sizes on EA are far smaller than the known associations between CpG probes and biologically proximal environmental factors. Two analyses that combine the effects of many probes—polygenic methylation score and epigenetic-clock analyses—both suggest small associations with EA. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

SEX-SPECIFIC DNA METHYLATION SIGNATURES IN PANIC DISORDER

Background Panic disorder (PD) is characterized by sudden episodes of acute anxiety occurring without any apparent reason. PD is the most disabling anxiety disorder and it affects twice as many women as men. The heritability of PD is estimated to be up to 48% and epidemiological studies show that both cumulative and specific life events are risk factors for the development of PD. Therefore, the investigation of genetic factors and gene-environment interaction is of high importance for understanding the pathophysiology of PD. As such, examining epigenetic differences in PD patients is of great interest given that environmental factors, in combination with genetic variation, can influence DNA methylation. Methods We conducted an Epigenome-Wide Association Study (EWAS) comparing non-medicated PD patients (49 females, 40 males) with healthy controls (48 females, 28 males). Replication was sought in an independent sample (131 cases, 190 controls) and further confirmed with a meta-analysis across both samples (220 cases, 266 controls). DNA methylation levels were assessed in whole blood using the Infinium HumanMethylation450 BeadChip. Failed probes were excluded based on a detection p-value larger than 0.01 in >50% of the samples. X chromosome, Y chromosome and non-specific binding probes were removed. The data were normalized with functional normalization in Minfi and batch-corrected using ComBat. Regression analyses accounting for cellular heterogeneity, sex and age were performed to test for associations between PD status and DNA methylation. To further assess functionality of the significant CpG, gene expression profiles (Illumina HumanHT-12 v3.0 array) in peripheral blood before and after exposure to the glucocorticoid receptor agonist dexamethasone were tested for association with DNA methylation in another independent female sample (N=71). Results No genome-wide associations were observed in PD patients compared to controls in the whole sample. Interestingly when stratified by sex, only the comparison of female PD patients with controls yielded one genome-wide association surviving FDR of 5% (P= 1.094 x 10-7 , P-adj=0.046). Specifically, cg07308824, located in the promoter of the HECA gene, was hypermethylated in female PD patients (N=49) compared to controls (N=48). The same CpG was also hypermethylated in female PD patients in the replication sample (P=0.035) and the genome-wide significant association was confirmed in the meta-analysis (P-adj=0.007). Methylation at this CpG site was associated with mRNA expression of HECA both at baseline (P= 0.046) and after induction by dexamethasone (P= 0.029). Pathway analyses using Web Gestalt investigating the top 50 associated CpG sites showed enrichment in female-specific pathways, e.g. female infertility pathways. Discussion Our study is the first to examine epigenome wide differences in peripheral blood for PD patients. Interestingly, our results point to possible sex-specific and functional methylation changes in PD. Further studies are needed to examine a possible contribution of the HECA gene methylation to female specific factors in PD.

Aging- and stress-related epigenetic disinhibition of FKBP5 contributes to NF-κB-driven inflammation and cardiovascular risk

Abstracts of the WASAD Conference 2017, 14–16 September, Würzburg, Germanys of the WASAD Conference 2017, 14–16 September, Würzburg, Germany