Impact of early environment on children's mental health : lessons from DNA methylation studies with monozygotic twins

Over the past decade, epigenetic analyses have made important contributions to our understanding of healthy development and a wide variety of adverse conditions such as cancer and psychopathology. There is increasing evidence that DNA methylation is a mechanism by which environmental factors influence gene transcription and, ultimately, phenotype. However, differentiating the effects of the environment from those of genetics on DNA methylation profiles remains a significant challenge. Monozygotic (MZ) twin study designs are unique in their ability to control for genetic differences because each pair of MZ twins shares essentially the same genetic sequence with the exception of a small number of de novo mutations and copy number variations. Thus, differences within twin pairs in gene expression and phenotype, including behavior, can be attributed in the majority of cases to environmental effects rather than genetic influence. In this article, we review the literature showing how MZ twin designs can be used to study basic epigenetic principles, contributing to understanding the role of early in utero and postnatal environmental factors on the development of psychopathology. We also highlight the importance of initiating longitudinal and experimental studies with MZ twins during pregnancy. This approach is especially important to identify: (1) critical time periods during which the early environment can impact brain and mental health development, and (2) the specific mechanisms through which early environmental effects may be mediated. These studies may inform the optimum timing and design for early preventive interventions aimed at reducing risk for psychopathology

Peripheral SLC6A4 DNA Methylation Is Associated with In Vivo Measures of Human Brain Serotonin Synthesis and Childhood Physical Aggression

The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET) measures of brain serotonin (5-HT) synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA) had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC). Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25) who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20) where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function

Combined effects of genotype and childhood adversity shape variability of DNA methylation across age

Lasting effects of adversity, such as exposure to childhood adversity (CA) on disease risk, may be embedded via epigenetic mechanisms but findings from human studies investigating the main effects of such exposure on epigenetic measures, including DNA methylation (DNAm), are inconsistent. Studies in perinatal tissues indicate that variability of DNAm at birth is best explained by the joint effects of genotype and prenatal environment. Here, we extend these analyses to postnatal stressors. We investigated the contribution of CA, cis genotype (G), and their additive (G+CA) and interactive (GxCA) effects to DNAm variability in blood or saliva from five independent cohorts with a total sample size of 1074 ranging in age from childhood to late adulthood. Of these, 541 were exposed to CA, which was assessed retrospectively using self-reports or verified through social services and registries. For the majority of sites (over 50%) in the adult cohorts, variability in DNAm was best explained by G+CA or GxCA but almost never by CA alone. Across ages and tissues, 1672 DNAm sites showed consistency of the best model in all five cohorts, with GxCA interactions explaining most variance. The consistent GxCA sites mapped to genes enriched in brain-specific transcripts and Gene Ontology terms related to development and synaptic function. Interaction of CA with genotypes showed the strongest contribution to DNAm variability, with stable effects across cohorts in functionally relevant genes. This underscores the importance of including genotype in studies investigating the impact of environmental factors on epigenetic marks

The developmental origins of chronic physical aggression: biological pathways triggered by early life adversity

Longitudinal epidemiological studies with birth cohorts have shown that physical aggression in humans does not appear suddenly in adolescence as commonly thought. In fact, physically aggressive behaviour is observed as early as 12 months after birth, its frequency peaks around 2-4 years of age and decreases in frequency until early adulthood. However, a minority of children (3-7%) maintain a high frequency of physical aggression from childhood to adolescence and develop serious social adjustment problems during adulthood. Genetic factors and early social experiences, as well as their interaction, have been shown to play an important role in the development of chronic aggressive behaviour. However, the biological mechanisms underlying these associations are just beginning to be uncovered. Recent evidence suggests that epigenetic mechanisms are responsive to adverse environments and could be involved in the development of chronic aggression. Using both gene candidate and genomic approaches, recent studies have identified epigenetic marks, such as DNA methylation alterations in genes involved in the stress response and the serotonin and immune systems to be partly responsible for the long-lasting effects of early adversity. Further longitudinal studies with biological, environmental and behavioural assessments from birth onwards are needed to elucidate the sequence of events that leads to these long-lasting epigenetic marks associated with early adversity and aggression

Association between child maltreatment and depressive symptoms in emerging adulthood: The mediating and moderating roles of DNA methylation.

Prospective studies suggest that child maltreatment substantially increases the risk for depression in adulthood. However, the mechanisms underlying this association require further elucidation. In recent years, DNA methylation has emerged as a potential mechanism by which maltreatment experiences (a) could partly explain the emergence or aggravation of depressive symptoms (i.e., mediation) and/or (b) could increase (or decrease) the risk for depressive symptoms (i.e., moderation). The present study tested whether the methylation levels of nine candidate genes mediated and/or moderated the association between maltreatment experiences in childhood and depressive symptoms in emerging adulthood. The sample comprised 156 men aged between 18 and 35 years. Maltreatment experiences and depressive symptoms were assessed retrospectively using self-reported questionnaires. Methylation levels of nine candidate genes (COMT, FKBP5, IL6, IL10, MAOA, NR3C1, OXTR, SLC6A3 and SLC6A4), previously reported to be sensitive to early-life stress, were quantified from saliva samples. Maltreatment experiences in childhood were significantly associated with depressive symptoms in emerging adulthood. Both maltreatment experiences and depressive symptoms were associated with the methylation levels of two genomic sites, which cumulatively, but not individually, explained 16% of the association between maltreatment experiences in childhood and depressive symptoms in emerging adulthood. Moreover, maltreatment experiences in childhood interacted with the methylation levels of fourteen genomic sites, which cumulatively, but not individually, modulated the level of depressive symptoms in young male adults who were maltreated as children. However, none of these effects survived multiple testing correction. These findings bring attention to the cumulative effects of DNA methylation measured in several candidate genes on the risk of reporting depressive symptoms following maltreatment experiences in childhood. Nonetheless, future studies need to clarify the robustness of these putative cumulative effects in larger samples and longitudinal cohorts

Association between child maltreatment and depressive symptoms in emerging adulthood: The mediating and moderating roles of DNA methylation

Prospective studies suggest that child maltreatment substantially increases the risk for depression in adulthood. However, the mechanisms underlying this association require further elucidation. In recent years, DNA methylation has emerged as a potential mechanism by which maltreatment experiences (a) could partly explain the emergence or aggravation of depressive symptoms (i.e., mediation) and/or (b) could increase (or decrease) the risk for depressive symptoms (i.e., moderation). The present study tested whether the methylation levels of nine candidate genes mediated and/or moderated the association between maltreatment experiences in childhood and depressive symptoms in emerging adulthood. The sample comprised 156 men aged between 18 and 35 years. Maltreatment experiences and depressive symptoms were assessed retrospectively using self-reported questionnaires. Methylation levels of nine candidate genes (COMT, FKBP5, IL6, IL10, MAOA, NR3C1, OXTR, SLC6A3 and SLC6A4), previously reported to be sensitive to early-life stress, were quantified from saliva samples. Maltreatment experiences in childhood were significantly associated with depressive symptoms in emerging adulthood. Both maltreatment experiences and depressive symptoms were associated with the methylation levels of two genomic sites, which cumulatively, but not individually, explained 16% of the association between maltreatment experiences in childhood and depressive symptoms in emerging adulthood. Moreover, maltreatment experiences in childhood interacted with the methylation levels of fourteen genomic sites, which cumulatively, but not individually, modulated the level of depressive symptoms in young male adults who were maltreated as children. However, none of these effects survived multiple testing correction. These findings bring attention to the cumulative effects of DNA methylation measured in several candidate genes on the risk of reporting depressive symptoms following maltreatment experiences in childhood. Nonetheless, future studies need to clarify the robustness of these putative cumulative effects in larger samples and longitudinal cohorts

The Impact of School Social Experiences on Socioemotional and Behavioral Problems:The Hypothesized Role of DNA Methylation

Peer victimization (including physical, verbal, and relational forms of aggression) has a worldwide prevalence rate of nearly 13%, and has been implicated in prior literature and theory as a contributor to children’s behavioral and emotional problems, through stress-related pathways and mechanisms. However, little is known about the underlying molecular mechanisms that may account for these effects. In this chapter, we first briefly examine how the neuroendocrine system may be affected by peer victimization. Next, we describe the extent to which epigenetic mechanisms, especially DNA methylation, may be altered by life experiences and could jeopardize later development, a concept referred to as biological embedding. We conclude by outlining key methodological, biological, and statistical limitations constraining the generalization of emerging findings investigating epigenetic mechanisms in peer relationships research.</p

Childhood chronic physical aggression associates with adult cytokine levels in plasma.

An increasing number of animal and human studies are indicating that inflammation is associated with behavioral disorders including aggression. This study investigates the association between chronic physical aggression during childhood and plasma cytokine levels in early adulthood.Two longitudinal studies were used to select males on a chronic physical aggression trajectory from childhood to adolescence (n = 7) and a control group from the same background (n = 25). Physical aggression was assessed yearly by teachers from childhood to adolescence and plasma levels of 10 inflammatory cytokines were assessed at age 26 and 28 years. Compared to the control group, males on a chronic physical aggression trajectory from childhood to adolescence had consistently lower plasma levels of five cytokines: lower pro-inflammatory interleukins IL-1α (T(28.7) = 3.48, P = 0.002) and IL-6 (T(26.9) = 3.76, P = 0.001), lower anti-inflammatory interleukin IL-4 (T(27.1) = 4.91, P = 0.00004) and IL-10 (T(29.8) = 2.84, P = 0.008) and lower chemokine IL-8 (T(26) = 3.69, P = 0.001). The plasma levels of four cytokines accurately predicted aggressive and control group membership for all subjects.Physical aggression of boys during childhood is a strong predictor of reduced plasma levels of cytokines in early adulthood. The causal and physiological relations underlying this association should be further investigated since animal data suggest that some cytokines such as IL-6 and IL-1β play a causal role in aggression

Moderation analyses.

(A) Adjusted model for FKBP5_1_CpG_3. (B) Adjusted model for FKBP5_1_CpG_4. (C) Adjusted model for IL10_2_CpG_5. (D) Adjusted model for IL10_2_CpG_6. (E) Adjusted model for MAOA_3_CpG_1. (F) Adjusted model for NR3C1_1_CpG_5. (TIF)</p

HAM-TBS: high-accuracy methylation measurements via targeted bisulfite sequencing

Abstract Background The ability to accurately and efficiently measure DNA methylation is critical to advance the understanding of this epigenetic mechanism and its contribution to common diseases. Here, we present a highly accurate method to measure methylation using bisulfite sequencing (termed HAM-TBS). This novel method is able to assess DNA methylation in multiple samples with high accuracy in a cost-effective manner. We developed this assay for the FKBP5 locus, an important gene in the regulation of the stress system and previously linked to stress-related disorders, but the method is applicable to any locus of interest. Results HAM-TBS enables multiplexed analyses of up to 96 samples and regions spanning 10 kb using the Illumina MiSeq. It incorporates a triplicate bisulfite conversion step, pooled target enrichment via PCR, PCR-free library preparation and a minimum coverage of 1000×. TBS was able to resolve DNA methylation levels with a mean accuracy of 0.72%. Using this method, we designed and validated a targeted panel to specifically assess regulatory regions within the FKBP5 locus that are not covered in commercially available DNA methylation arrays. Conclusions HAM-TBS represents a highly accurate, medium-throughput sequencing approach for robust detection of DNA methylation changes in specific target regions