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Early adversity can influence gene expression via epigenetic mechanisms, including DNA methylation. Peripheral tissues are essential in psychiatric epigenetics, as methylation generally cannot be assessed in the living human brain. Several magnetic resonance imaging (MRI) studies show associations of peripheral serotonin transporter gene (SLC6A4) methylation with function and/or structure of frontal-limbic circuits and brain's resting-state. Commonly used samples are derived from blood, saliva or buccal cells. However, little is known regarding which peripheral tissue is most strongly associated with human brain processes. The aim of the current study was to compare the extent of the association between peripheral SLC6A4 promoter methylation and frontal-limbic function, structure and resting-state in healthy individuals across peripheral tissues. Forty healthy prospectively-followed adults underwent anatomical, resting-state and functional MRI. Saliva-, blood- and buccal-derived DNA methylation was assessed by pyrosequencing. Blood-derived SLC6A4 methylation was positively associated with superior frontal gray matter (GM) volume and with right lateral parietal area (RLP)-frontal pole regional resting-state functional connectivity (rsFC). Saliva-derived SLC6A4 methylation was positively associated with superior frontal GM volume. Buccal-derived SLC6A4 methylation was positively associated with superior and inferior frontal and anterior cingulate cortical (ACC) GM volumes, and with RLP-ACC, frontal pole and medial prefrontal regional rsFC. Current results confirmed the relevance of peripheral methylation for frontal-limbic processes in humans. Buccal cells may be the most sensitive cell type when studying SLC6A4 promoter methylation and its associated risk for neural vulnerability and resilience for psychopathologies in which serotonin is implicated. These data should be further validated in clinical populations., (Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.)

Highlights • Daily life stress is associated with HPA-axis related gene methylation. • DNA methylation in HPA-axis genes is stable over time in healthy volunteers. • DNA methylation-brain associations are gene and tissue specific. • Measuring methylation in different tissues in the same persons can be recommended. Abstract Environmental factors can influence gene expression via epigenetic modifications such as DNA methylation. DNA methylation levels of regulatory regions in Hypothalamo-Pituitary-Adrenal (HPA) axis-related genes assessed from brain tissues as well as from surrogate, peripheral tissues have been associated with vulnerability to stress-related psychopathologies. Commonly used peripheral samples to assess DNA methylation in living humans are derived from blood, saliva or buccal cells. Although psychiatric epigenetic studies are increasingly relying on peripheral measures of DNA methylation, it is still unknown to what extent methylation patterns across peripheral tissues are associated with each other and with measures of brain processes and behavioural stress. In the present study, with a sample of 51 healthy adults, we assessed cross-tissue correlations of DNA methylation patterns in the glucocorticoid receptor (NR3C1) 1 F promoter and the FK506 Binding Protein 5 (FKBP5) gene intron 7 region using saliva and buccal cell samples, and assessed two-year stability in both tissues in a male subsample (N = 14). We also investigated associations between peripherally-derived DNA methylation and measures of neural function and perceived daily stress, and compared the extent of these associations across tissue samples. DNA methylation cross-tissue correlations were highly significant for FKBP5 , but not significant for NR3C1. DNA methylation in both genes remained stable for two years. Tissue- and gene-specific associations were found for brain resting state connectivity and neural responses to sadness, thereby suggesting that saliva- and buccal cell-derived DNA methylation levels of NR3C1 -1 F and FKBP5 gene regions might differently capture different measures of putatively related brain processes. It was also found that greater buccal cell- (but not saliva-) derived NR3C1 -1 F methylation was associated with lower perceived daily life demands. Results of the present study may inform the design of future epigenetic studies on FKBP5 -intron-7 and NR3C1 -1 F-promoter methylation in relation to neuro-imaging and behavioural measures, and provide insight for the development of peripheral DNA methylation correlates of stress sensitivity and resilience. [ABSTRACT FROM AUTHOR]

Numerous studies have been using peripheral tissues (e.g., blood or saliva) for DNA methylation analyses in psychiatric epigenetics. The glucocorticoid receptor gene (NR3C1) was among the first ones to show DNA methylation changes due to adverse social environment. Importantly, the NR3C1 gene has several alternative promoters, out of which the 1-F showed increased DNA methylation level at a transcription factor binding site. Afterwards, its co-chaperone molecule, FK506-binding protein 5 (FKBP5) gained interest, showing allele-specific demetylation in its glucocorticoid response element of the 7th intron. This decreased methylation at FKBP5 can lead to NR3C1 resistance, because of an impaired intracellular negative feedback loop. While it is now standard for epigenome-wide DNA methylation analyses to control for cell heterogeneity using cell count estimates derived from the DNA methylation profiles, this important technical variable of cell composition is rarely included in candidate gene analyses. Therefore, we aimed to (1) develop a panel of pyrosequencing assays for cell-specific CG-sites, and (2) evaluate the effect of cell ratio on DNA methylation levels of candidate gene regions in frequently used peripheral tissues, such as Whole Blood (WB), Peripheral Blood Mononuclear Cell (PBMC), as well as non-invasively collected buccal, mouthwash, and saliva samples in two cohorts. Samples were collected from 51 adults participating in a Montreal-based longitudinal cohort, out of which 16 men had been assessed twice. In addition, 38 female patients with borderline personality disorder and 21 control women were recruited at a psychiatric clinic in Budapest. Candidate gene regions of the NR3C1-1F and FKBP5-intron7, as well as tissue-specific CG-sites were assessed by pyrosequencing at both sites. The DNA methylation level of the CG-island in the NR3C1-1F promoter region showed little variance in all of the tissue types we investigated. However, the low CG-density region of FKBP5-intron7 was highly variable across samples, especially in saliva. Notably, methylation level of the epithelial cell marker PTPN7 cg18384097 varied widely among the mouth-related samples in both cohorts (ranging from 9%-95%), even in buccal swab samples, where there was ~ 80% buccal cell ratio on average with 10–12% SD. The effect of epithelial cell composition was substantial on FKBP5-intron7 methylation levels in saliva and mouthwash samples (r=0.5–0.7), but it was less pronounced in buccal swab samples (r=0.2–0.4). Higher ratio of epithelial cells resulted in lower methylation level at the studied FKBP5 CG-sites. Importantly, the stability of the DNA methylation level of this gene region was significant both in saliva and buccal swab samples (r=0.6–0.8, p<0.01). In addition, methylation level of the T lymphocyte marker CD3E cg06164961 also showed correlation with FKBP5-intron7 methylation in blood samples (WB: r=0.5–0.7, PBMC: r=0.3–0.5). Our preliminary results show that cell composition should be taken into account at candidate gene DNA methylation analyses, especially when measuring low CG-density regions in heterogeneous peripheral tissues, such as whole blood or saliva. Using more homogeneous biological samples, e.g., PBMC or purified leukocytes would result in less technical variability. The non-invasively obtained buccal swabs can also yield a relatively homogeneous surrogate sample in psychiatric epigenetics. [ABSTRACT FROM AUTHOR]