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Additional file 2: Table S1. Top 20 CpGs differentially associated with changes in glycemic measures, RYGB vs. IMI. Table S2. Top 20 CpGs differentially associated with changes in glycemic measures, BAND vs. IMI. Table S3. Top 20 CpGs differentially associated with changes in glycemic measures, RYGB vs. BAND. Table S4. CpGs differentially associated with changes in glycemic measures, after Bacon correction. Table S5. Top 20 CpGs differentially associated with changes in glycemic measures, RYGB vs. IMI.

Additional file 1: Fig. S1. Heatmap showing the correlations between surrogate variableand cell type compositions. Fig. S2. Manhattan plots depicting CpGs differentially associated with glycemic outcomes in response to BAND vs. IMI and RYGB vs. BAND. Fig. S3. Q–Q plots of p-values and λ for the epigenome-wide differential associations between DNA methylation and glycemic outcomes, Bacon-correctedvs. conventional approach.

Background. This study investigates the impact of childhood maltreatment (CM) on hypo- thalamic-pituitary-adrenal (HPA)-axis functioning and on anxiety perception. Moreover, the influence of CM severity and frequency was also explored. Methods. In total, 187 participants aged 7-17 were assessed for CM history using validated questionnaires and ad hoc interviews to be classified according to the criteria of the Tool for Assessing the Severity of Situations in which Children are Vulnerable (TASSCV). Psychopathology was ascertained using the K-SADS-PL5. To assess HPA-axis functioning, salivary cortisol samples were collected throughout a normal day and during an acute psycho- social stressor, the Trier Social Stress Test for children (TSST-C). Subjective anxiety was evaluated using STAI/-C. Results. Youth with a CM history had higher overall diurnal cortisol levels (p=0.001), blunted cortisol response to acute psychosocial stress ( p = 0.002) and greater perceived anx- iety ( p = 0.003), than those without CM. Specifically, participants exposed to moderate/severe or often/frequent CM showed the greater diurnal cortisol output ( pseverity = 0.002; pfrequency = 0.003), and blunted cortisol response during the TSST-C ( pseverity = 0.006; pfrequency = 0.008). Meanwhile, youth with low CM severity/frequency exhibited a similar cortisol response to those without CM. However, perceived anxiety was higher in those exposed to CM ( p < 0.001), regardless of its severity/frequency. Conclusions. Disturbances in HPA-axis functioning are already evident early after CM expos- ure, while psychological and physiological responses to an acute stressor are dissociated in youth exposed to CM. The dose-response relationship described in this paper highlights the need to comprehensively evaluate CM so that vulnerable children can be identified and assigned to proper interventions. Supported by the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III through the University of Barcelona multicenter project (PI15/00097)-PI L. Fañanas, Hospital Universitario Araba (PI15/00793)-PI I. Zorrilla, Hospital Gregorio Marañón (PI15/00723)-PI M. Rapado and Hospital Clinic (PI15/00685)-PI S. Romero, co-financed by the European Regional Development Fund project, ‘A way of making Europe’. We thank the Network Centre for Biomedical Research in Mental Health (CIBERSAM). This study was facilitated by a pre-doctoral research grant from the Catalonian authorities to Laia Marques-Feixa (AGAUR-FI_B100023-2018). Supported by a NARSAD Distinguished Investigator Grant awarded to Professor Lourdes Fañanas (26887) and by the Comissionat per a Universitats i Recerca del DIUE, of the Generalitat de Catalunya regional authorities (2017SGR1577). Dr Rapado-Castro was supported by a Ramon y Cajal Research Fellowship (RYC-2017-23144), Spanish Ministry of Science, Innovation and Universities; an NARSAD independent investigator grant (24628) from the Brain & Behaviour Research Foundation; Fundación Familia Alonso and Fundación Alicia Koplowitz.

Aging is the single most important risk factor for diseases that are currently the leading causes of morbidity and mortality. However, there is considerable inter-individual variability in risk for aging-related disease, and studies suggest that biological age can be influenced by multiple factors, including exposure to psychosocial stress. Among markers of biological age that can be affected by stress, the present article focuses on the so-called measures of epigenetic aging: DNA methylation-based age predictors that are measured in a range of tissues, including the brain, and can predict lifespan and healthspan. We review evidence linking exposure to diverse types of psychosocial stress, including early-life stress, cumulative stressful experiences, and low socioeconomic status, with accelerated epigenetic aging as a putative mediator of the effects of psychosocial environment on health and disease. The chapter also discusses methodological

DNA methylation values for CpG probes used to discard the presence of maternal contamination. (DOCX 29.6 kb)

[eng] Psychiatric disorders are highly prevalent and one of the leading causes of disease burden worldwide. Nevertheless, there are no validated biomarkers to be used for either diagnostic or prognostic purposes and their etiology remains largely unknown. Thanks to heritability studies, it has been recognized that both nature (genetics) and nurture (environment) contribute to the onset of mental disorders. Accordingly, exposure to both prenatal stress and childhood maltreatment has been reliably identified as a risk factor for a wide range of psychiatric disorders. However, not all individuals facing this kind of early environmental insults ends up developing psychopathological conditions. Thus, gene—environment (GxE) interactions have been proposed to be involved in the physiopathology of mental disorders; epigenetic mechanisms such as DNA methylation have been hypothesized to mediate these interactions. In this framework, the present dissertation aimed to disentangle the role of DNA methylation both in increasing anxious-depressive liability, and embedding the experience of severe prenatal stress, thanks to two independent samples of monozygotic twins. NR3C1 gene, encoding the glucocorticoid receptor, was selected as a candidate gene of interest due to its crucial role in the regulation of the stress response. A systematic review of the available scientific literature revealed a great interest in NR3C1 methylation, and specifically in its exon 1F, in the context of both exposure to stress and stress-related disorders, highlighting the association between exposure to early life stress and NR3C1 hypermethylation. Additionally, this revision also pointed out the lack of studies regarding other promoter regions of this gene. Thus, we explored NR3C1 exon 1D methylation near a glucocorticoid responsive element (GRE) describing its association with both (i) the familial component of anxious-depressive liability, and (ii) hippocampal connectivity within the brain emotional network. Our second candidate gene of interest was SLC6A4, encoding the serotonin transporter, which is the primary pharmacological target of the most widely prescribed antidepressants to date (SSRIs). Again, a systematic review of the literature revealed the association between SLC6A4 methylation and exposure to early life stress, further suggesting its mediator role in previously discussed GxE interactions involving a long polymorphic region of this gene (5-HTTLPR). Complementarily, we assessed SLC6A4 methylation in a CpG island shore region characterized by a sex-differential methylation pattern; specifically, women exhibit higher methylation at this region when compared to men. Remarkably, we found an association between methylation at this region and the presence of somatization symptoms, suggesting the involvement of SLC6A4 methylation in known sexual differences regarding internalizing psychopathology globally. Since epigenetic signatures can arise in a stochastic fashion, monozygotic twins were also analyzed to identify methylation outliers which could potentially contribute to anxious-depressive psychopathology. In this regard, we reported the presence of methylation outliers in genes previously involved in neuropsychiatric disorders. Finally, the role of prenatal stress in fetal DNA methylation signatures was also explored. First, a meta-analysis on maternal psychosocial stress experienced during pregnancy and newborn NR3C1 methylation exhibited the association between both variables in line with non-genetic intergenerational transmission of stress hypotheses. On the other hand, a sample of monochorionic monozygotic newborn twins exposed to several prenatal complications was epigenomically assessed with regard to an obstetric routine measure predictive of perinatal morbid risk. The latter study revealed newborns exposed to prenatal stress during the third trimester of pregnancy exhibit developmental immaturity in terms of epigenetic (DNA methylation based) age; furthermore, differential CpG-specific methylation was found in EP300 gene, a previously identified candidate gene conferring risk for schizophrenia and involved in hypoxia response. Together, these findings support the notion that DNA methylation is involved both in the embedding of early life stress exposure and the pathophysiology of several psychiatric disorders., [spa] El objetivo de la presente tesis doctoral fue explorar la metilación del ADN tanto en genes candidatos de relevancia para la regulación del estrés y el riesgo para desarrollar depresión (NR3C1, SLC6A4) como realizar aproximaciones masivas centradas en la identificación de valores de metilación atípicos (outliers) y patrones que muestren plasticidad ante la exposición a estrés temprano. Estas aproximaciones se desarrollaron gracias al estudio de dos muestras independientes de gemelos monocigóticos; la primera, tomada de población general adulta mientras que la segunda se constituye por recién nacidos expuestos a distintos tipos de complicaciones obstétricas. Así, describimos la asociación entre la metilación en una nueva región del gen NR3C1, el exón 1D, tanto con el riesgo de padecer psicopatología ansioso-depresiva como de mostrar una menor conectividad hipocampal. De la misma forma, la metilación sexo-específica del gen SLC6A4 se asoció con una mayor presencia de sintomatología psicosomática. También se identificaron outliers de metilación en genes previamente implicados en trastornos neuropsiquiátricos. Finalmente, la exposición a estrés prenatal severo se asoció con una mayor inmadurez en términos epigenéticos y con una menor metilación en el gen EP300, previamente identificado en estudios de asociación para esquizofrenia, funcionalmente implicado en la respuesta a hipoxia. Todos estos hallazgos sugieren el rol de mecanismos epigenéticos y, más específicamente, de la metilación del ADN, en la etiopatogenia de los trastornos psiquiátricos. Además, la metilación del ADN puede ser utilizada como biomarcador de la exposición a estrés. Sin embargo, se necesitan estudios longitudinales para poder determinar tanto la causalidad como la estabilidad temporal de dichas marcas.

Table S1. Datasets included in our study. Table S2. Age, gender and pmi distributions between cases and control samples at each step of the preprocessing procedure at the asd defferentail gene expression meta-analysis. Table S3. Final samples included in the asd meta-analysis. Table S4. Jointly same direction derregulated genes in asd ans sddcs and jointly oposite direction derregulated genes in asd and oddcs. Table S5. Genes jointly deregulated in asd and in sddcs and oddcs. Figure S1. Comaprison of differential gene expression analysis using limma with rlog transformed data and two state of the art rnaseq differential expression metdos. Figure S2. Patient overlap in the three asd studies. Figure S3. Metaqc and mean intersample correlation results. Figure S4. Partition around medoids (pam) clustering of the differential gene expression profiles of asd and the 22 included cancer types. Figure S5. Top upregulated and downregulated pathways in cancer (halmmarks molecular signature). Figure S6. Top upregulated and downretualted pathways in cancer (c2 canonical pathways molecular signature). Figure S7. Intersection analysis of the two control diseases (ulcerative colitis and malaria). Figure S8. Percentaje of pathways deregulated in the same or opposite directions between asd and cancer (DOCX 3835 kb)

BACKGROUND AND AIMS: The hypothalamic-pituitary-adrenal (HPA) axis is the main regulator of the stress response and its malfunction may underlie the etiology of mental disorders. The role of HPA axis in psychopathology has been scarcely studied in child and adolescent populations1. The main aim of this study is to describe the activity and regulation of the HPA axis in a representative sample of children and adolescents (affected and non-affected by psychiatric disorders) from the Spanish multicentric EPI_Young_Stress_Project. Additionally, a secondary aim was to explore the influence of sex and pubertal stage in HPA axis functioning. METHODS: A preliminary sample of 127 children from 7 to 17 years old (mean 13.6) was analyzed: consisting of 77 psychiatric patients (assessed by K-SADS-PL) and 50 healthy stratified controls. Pubertal stage was analyzed by Tanner scale: children (Tanner stages I and II) and adolescents (Tanner stages III, IV and V). Reactivity of HPA axis was measured during Trier Social Stress Test for Children (TSST-C)2. TSST-C is an experimental psychosocial stress procedure where subjects have to perform an arithmetic task and a public speaking task in front of a tribunal and a camera. This stress paradigm has been reliably shown to activate the HPA axis allowing the measurement of salivary cortisol before, during, and after social stress (four saliva samples collected during the time points displayed in the figure). TSSTs were scheduled at 4:30PM to control the circadian nature of cortisol levels. Cortisol reactivity was operationalized as the maximum difference between two time points.To analyze basal function of HPA axis two saliva samples were collected in another weekday at awakening and half an hour later in order to explore Cortisol Awaking Response (CAR). RESULTS AND DISCUSSION: In our sample HPA axis reactivity increased according to Tanner stage (p=0.02). With regard to sex, pre-pubertal subjects displayed comparable levels of HPA axis reactivity during TSST; however, adolescent females exhibited higher reactivity of HPA axis when compared to males (p=0.04). Subjects affected by psychiatric disorders exhibited lower HPA axis reactivity during TSST when compared to controls (p=0.03). There were no significant differences in CAR as a function of either psychiatric status or pubertal stage. Both, the pubertal stage and sex, influence the variability of HPA axis reactivity and have to be taken into account to understand the stress response. According to previous studies focused on adult population HPA axis reactivity is influenced by the presence of psychiatric disorders in children and adolescents3,4. However, further studies are needed to disentangle the putative role of pharmacological treatment and childhood maltreatment in HPA axis dysregulation5,6.