Open Access

Epigenetic alterations following early postnatal stress: a review on novel aetiological mechanisms of common psychiatric disorders

  • Magdalene C. Jawahar1,
  • Chris Murgatroyd2,
  • Emma L. Harrison1, 3 and
  • Bernhard T. Baune1Email author
Clinical EpigeneticsThe official journal of the Clinical Epigenetics Society20157:122

https://doi.org/10.1186/s13148-015-0156-3

Received: 18 September 2015

Accepted: 8 November 2015

Published: 14 November 2015

Abstract

Stressor exposure during early life has the potential to increase an individual’s susceptibility to a number of neuropsychiatric conditions such as mood and anxiety disorders and schizophrenia in adulthood. This occurs in part due to the dysfunctional stress axis that persists following early adversity impairing stress responsivity across life. The mechanisms underlying the prolonged nature of this vulnerability remain to be established. Alterations in the epigenetic signature of genes involved in stress responsivity may represent one of the neurobiological mechanisms. The overall aim of this review is to provide current evidence demonstrating changes in the epigenetic signature of candidate gene(s) in response to early environmental adversity. More specifically, this review analyses the epigenetic signatures of postnatal adversity such as childhood abuse or maltreatment and later-life psychopathology in human and animal models of early life stress. The results of this review shows that focus to date has been on genes involved in the regulation of hypothalamic-pituitary-adrenal (HPA) axis and its correlation to subsequent neurobiology, for example, the role of glucocorticoid receptor gene. However, epigenetic changes in other candidate genes such as brain-derived neurotrophic factor (BDNF) and serotonin transporter are also implicated in early life stress (ELS) and susceptibility to adult psychiatric disorders. DNA methylation is the predominantly studied epigenetic mark followed by histone modifications specifically acetylation and methylation. Further, these epigenetic changes are cell/tissue-specific in regulating expression of genes, providing potential biomarkers for understanding the trajectory of early stress-induced susceptibility to adult psychiatric disorders.

Keywords

Early life stressMaternal separationEpigeneticsDNA methylationStress-responsive genesHistone acetylationPsychopathology

Background

Early life stress (ELS) encompasses childhood abuse, neglect, poverty and parental illness, alongside a multitude of other stressors. Some forms of ELS affect 30–40 % of the Western population and have been implicated in approximately half of all childhood and a third of adulthood psychiatric disorders [1, 2]. Exposure to ELS results in enhanced susceptibility to neuropsychiatric disorders such as major depressive disorder, generalised anxiety disorder, schizophrenia and autism spectrum disorders [1, 3, 4]. Chronic health conditions such as obesity, type two diabetes mellitus, respiratory disorders and cardiovascular diseases are also increased in individuals with a history of ELS [5, 6].

The impact of early adversity on the susceptibility to psychiatric disorders in later life is influenced by a number of factors. Environmental factors include nature of stressors [7], time of exposure in development [8, 9] and severity and cumulative exposure effects [1012]. Biological factors include gender, age of assessment [13] and predisposing genetic polymorphisms in genes associated with mood regulation, stress response and inflammatory processes. These include genes such as serotonin transporter (5-HTT), brain-derived neurotrophic factor (BDNF) and FK506 binding protein (FKBP5) [1416]. A dysfunctional hypothalamic-pituitary-adrenal (HPA) stress axis and impaired immune responses such as increased cytokines have also been implicated in the increased vulnerability to ELS [17, 18]. In spite of the increasing knowledge, the molecular mechanisms underlying ELS-mediated long-term vulnerability to later-life stressors are unclear.

Gene-environment interactions, such as those occurring when exposed to ELS, often encompass epigenetic changes. Epigenetics processes occur at the level above the genome, which includes DNA methylation, posttranslational histone modifications and gene regulation by micro-RNA (miRNA). Collectively, these epigenetic changes can stably mark the genome in response to environment, potentially altering gene expression across lifespan [19, 20] and across generations [21]. As such, epigenetic alterations may represent one of the key mechanisms underlying the long-lasting nature of ELS-induced changes in neurobiology, behaviour and disease susceptibility [22, 23].

We first present a brief overview of the stress response pathways followed by a detailed review of evidence demonstrating epigenetic alterations following ELS in animal models and humans. Changes in the epigenetic signature of candidate genes and alterations in genome-wide methylation will be reported. Finally, we aim to understand how these changes mediate long-term effects such as their role in risk to developing psychiatric disorders in adulthood.

The central role of hypothalamus-pituitary-adrenal system

Adversities during early postnatal life are able to shape the experience-dependent maturation of stress-regulating pathways, such as the HPA system. This can lead to persistent alterations in stress responsivity during adulthood—a phenomenon often referred to as “early-life programming”. Tight regulation of the HPA axis is therefore core to the long-term control of systems governing stress responsivity. The HPA axis involves the release, following a stressor, of the neuropeptide corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the paraventricular nucleus (PVN) of the hypothalamus. These bind to their specific receptors (the CRHR1 and V1b receptors) in the anterior pituitary that stimulate the release of adrenocorticotrophic hormone (ACTH) which stimulates the adrenal cortex to release glucocorticoid (GC) hormones, cortisol in human and corticosterone in rodents. These GCs in turn mobilise glucose from energy stores and increase cardiovascular tone, among further widespread effects. Feedback loops, primarily mediated through glucocorticoid receptors (GRs) in the PVN and pituitary, regulate responsiveness of the HPA axis ensuring a return to a homeostatic balance when it is no longer challenged (Fig. 1).
Fig. 1

The hypothalamic-pituitary-adrenal (HPA) axis and its response to stress stimuli: the signalling events (green, solid lines) in the HPA axis in response to stress stimuli and how glucocorticoids (GCs) produced by the adrenal gland can have a negative feedback role in maintaining GC levels in the blood. The negative feedback in the hypothalamus and pituitary (red, dotted lines) are both mostly regulated by glucocorticoid receptors (GRs), and dysfunctional negative feedback system is often seen associated with chronic exposure to stress stimuli

A loss of this negative feedback control, particularly following periods of chronic stress, may influence the development of affective disorders. Indeed, dysregulated HPA activity is one of the most commonly observed neuroendocrine symptoms in major depressive disorder (for review see [24]). Childhood stress has also been shown to be a strong predictor of impaired inhibitory feedback regulation of the HPA axis with evidence linking to a role of CRH and/or AVP systems. For example, postmortem brain tissue of depressed individuals revealed elevated CRH and AVP in the hypothalamus [25, 26]. Studies in rodent models further support the concept that exposure to a chronic stressor can lead to long-term changes in HPA regulation and behaviour stemming from changes in neuropeptide regulation (for review see [27]).

The role of candidate genes outside of the HPA axis

Candidate genes such as the serotonin transporter SLC6A4 and BDNF have been highly implicated in stress response and in increased risk for psychiatric disorders [2832]. BDNF is the most prevalent growth factor in the central nervous system (CNS) and important in neuronal development and plasticity [33]. Serotonin transporter is involved in the reuptake of serotonin from the brain synapses regulating serotonin signalling and is the target for many antidepressants [34]. SLC6A4 or 5-HTT carries a genetic polymorphism in the promoter region resulting in a short “s” and a long “l” allele version of the promoter [35]. The “s” allele is associated with poor transcriptional efficiency of SLC6A4 compared to “l” allele [35]. The BDNF gene carries a Val66Met polymorphism which impacts an activity-dependent expression of BDNF and the intracellular trafficking [36]. In combination with exposure to ELS events, both SLC6A4 and BDNF polymorphisms have been attributed to increased risk for depression in later life [28, 30, 37]. Further, steroid hormone estrogen and its receptors have been shown to influence brain function and psychiatric disorders (for review see [38]). Animal models analysing maternal care in rats identified estrogen receptor α expression was altered with the type of maternal care, and this was passed across generations [39]. A detailed analysis of the role of these and other candidate genes implicated in ELS and later-life psychopathology is reviewed in the following sections.

ELS-induced epigenetic modifications in animal models

A variety of animal models are currently being used to model ELS paralleling childhood adversity in humans (Table 1). Each paradigm facilitates investigation into ELS-induced alterations in the developing animal and centres on the importance of mother for normal nervous, immune and endocrine system development [4042]. A majority of the literature on animal models discussed in this review will therefore be on variations in maternal care.
Table 1

Commonly used models of early adversity in animal studies

Paradigm

Description

Rationale

References

Handling

Daily separation of pups as a litter from mother for 15 min from PND1–14 (up to PND21)

Allows comparison of mild vs severe stress-induced effects during the early postnatal period

[94]

Early weaning

Weaning of pups between PND14 and PND21

Enables assessment of prolonged stress after the early postnatal period

[42, 95]

Maternal separation

Daily separation of pups individually or as a litter from mother for 3 h (up to 6 h) from PND1–14 (up to PND21)

Ethnologically relevant as it models repeated episodes of mild stress rather than 1 episode of severe stress during the early postnatal period

[96]

Maternal deprivation

Single episode of separation of pups as a litter from mother for 24 h, usually on PND3 or 9

Allows determination of stress-induced effects at specific developmental time windows within the early postnatal period

[41, 47, 95]

Low vs high LG-ABN

Pups raised by biological mothers who are characterised as either low or high care dams on the basis of the level of licking grooming arched-back nursing they perform

Enables comparison of the effects of variations in maternal care on development rather than stress exposure

[39, 97]

h hour, LG-ABN licking grooming arched-back nursing, PND postnatal day

ELS-induced epigenetic changes in HPA axis genes

Given the central role of the HPA axis in stress responsivity and adaptation to ELS, the genes involved in regulating this system have been of focus in ELS-induced epigenetic studies (see Table 2 for summary of studies).
Table 2

Early stress-induced epigenetic changes in stress-regulatory genes in animal studies

Candidate gene

Objective

Model/tissue type

Early stress/assessment age

Epigenetic and expression changes

Interpretation

References

GR promoter

Determine the effect of maternal care on exon 17 GR promoter methylation and histone H3K9 acetylation

Long-Evans rats

Maternal care variations: high vs low LG-ABN

Assessed: PND6, 21 and 90

↓ methylation 5′ CpG of NGFI-A response element in high vs low LG-ABN offspring (↔3′ CpG)

↑ histone H3K9 acetylation in high vs low LG-ABN offspring

↑ NGFI-A binding to exon 17 GR promoter in high vs low LG-ABN offspring

↑ GR mRNA in high vs low LG-ABN offspring

↑ CBP associated with exon 17 GR promoter in high vs low LG-ABN offspring

High maternal care was associated with ↑ exp of GR mRNA and protein and ↑ binding of NGFI-A in the hippocampus. This correlated with ↓ exon 17 GR promoter methylation and ↑Histone H3K9 acetylation

Changes were persistent from early life to adulthood

[20, 43]

Determine strain-specific epigenetic alterations of MS in mice

C57BL/6 J and DBA/2 J mice

Hippocampus

MS: PND9, 24 h separation

Assessed: 11–12 weeks

↔ Nr3c1 methylation in C57BL/6J mice

↑Nr3c1 CpG 13, 14 and 17 methylation in MS-treated DBA/2J mice

MS in DBA/2J mice ↑ methylation of CpG 13, 14 and 17 in Nr3c1 at 3 months of age

[45]

Determine the effect of MS on exon 17 GR promoter methylation

Sprague Dawley rats

Hippocampus

MS: PND2–14, 3 h/day

Assessed: PND21

↔ methylation of exon 17 GR promoter or NGFI-A binding site

↑ NGF mRNA exp

↔NT-3

No effect of MS on methylation status of exon 17 of the GR promoter or the NGFI-A binding site in hippocampus

[44]

GR gene locus (7 million base pairs)

Determine the effect maternal care on DNA methylation and H3K9 acetylation of a region of Chr 18 containing the Nr3c1 gene

Long-Evans Rats

Hippocampus

High vs low LG- ABN

Assessed: PND90

723 RDme and 471 RDac in GR gene were identified across the entire locus in high vs low LG-ABN

Clustering patterns revealed changes in exp of Pcdh family genes. 20 out of 33 had ↑ exp in high compared to low LG-ABN offspring

Identified that variations in maternal care affect a broad genomic region and epigenetic and exp changes act on a family of genes localised in that broad genomic region

Finding of the association of the Pcdh cluster of genes involved in synaptic plasticity

[46]

Crh promoter

Determine the effect of MS on Crh promoter methylation

Sprague Dawley rats

Hypothalamus

MS: PND2–13, 4 h/day

Assessed: PND61

↓ methylation of Crh promoter in hypothalamus (Met-C2)

↓ methylation of Met-C2

↑ phosphoCREB binding to CRH CRE

Crh hnRNA exp in hypothalamus (↔ amygdala)

Crh mRNA exp in hypothalamus and amygdala

MS ↓ methylation of Met-C2 and

↑ transcriptional activity of Crh in the PVN on PND61

[47]

Determine the effect of MS on Crh promoter methylation

Sprague Dawley rats

Hippocampus CA1

MS: PND1–10, 3 h/day

Assessed: 10 weeks of age

↑ H3 acetylation of the Crh promoter in MS vs no MS pups

↓methylation and ↓ binding of MeCP2 in the Crh promoter

Crh mRNA in MS vs no MS pups

Enriched environment reversed the epigenetic up-regulation of Crh

MS ↑ acetylation of the Crh promoter region thereby allowing for increased transcriptional activity of Crh which was reversed when mice were treated to enriched environment

[48]

Crfr2

Determine the effect of MS on Crfr2 methylation

C57Bl/6 mice

Genomic DNA from sperm in F1 and F2 males

Cortex in F2 females

Unpredictable MS: PND1–14, 3 h/day in F1

Assessed: 3–8 months of age

F1

↓ methylation of 5′ CpG of Crfr2 in M

F2a

↓ methylation of 5′ CpG of Crfr2 in F

Changes in gene exp were accompanied by ↓ mRNA exp

Early stress ↓ methylation of Crfr2 and mRNA exp in adult C57/BL6 mice and that is transmitted across generations

[21]

Avp enhancer

Determine the effect of MS on Avp enhancer methylation and mRNA exp

C57Bl/6 mice

PVN of hypothalamus

MS: PND1–10, 3 h/days

Assessed: PND10, 6 weeks, 3 months and 1 year

Avp enhancer methylation at 6 weeks, 3 months and 1 year

↓ methylation of CpGs largely mapped to CG13 of Avp enhancer from 6 weeks (↔ at PND10)

↓ binding of CG13 Avp enhancer at PND10 and 6 weeks

↓ methylation of CpGs w age (11 CpGs at 6 weeks, 7 at 3 months, 3 at 1 year)

↑ AVP mRNA exp from 6 weeks

MS causes ↓ methylation of the Avp enhancer from 6 weeks of age in C57/BL6 mice which is accompanied by persistent up-regulation of Avp exp in parvocellular neurons in the PVN

[23]

Determine strain-specific epigenetic alterations of MS in mice

C57BL/6J and DBA/2J mice

Hippocampus

MS: PND9, 24-h separation

Assessed: 11–12 weeks

↑ methylation of CpG1 of Avp in MS treated M of C57BL/6J and DBA/2J strain

MS ↑ methylation of CpG 1 unit in the Avp enhancer DNA sequence

[45]

↑ increased, ↓ decreased, ↔ no change, Avp arginine vasopressin, CBP CREB binding protein, Crfr2 corticotrophin-releasing hormone receptor 2, Crh corticotrophin-releasing hormone, exp expression, F female, GR glucocorticoid receptor, h hour, hnRNA heterogeneous nuclear ribonucleic acid, LG-ABN licking grooming arched-back nursing, M male, mRNA messenger ribonucleic acid, MS maternal separation, NGF nerve growth factor, NGFI-A nerve growth factor inducible protein A, NT3 neurotophin 3, PND postnatal day, PVN paraventricular nucleus, RDac regional differences in acetylation, RDme regional differences in methylation, Pcdh: Protocadherin, CA-1 Cornu Ammonis area 1, MeCP2 Methyl CpG binding protein 2

aF2: MS M and control F were bred to produce F2 offspring

Glucocorticoid receptor gene

Pioneering studies on epigenetic alterations in GR promoter in response to variations in maternal care were first shown by Weaver and colleagues [20]. They reported increased methylation of the 5′ exon17 GR promoter and decreased H3K9 acetylation both associated with reduction in GR messenger RNA (mRNA) expression in the hippocampus of pups raised by low licking grooming arched-back nursing (LG-ABN) dams [20]. Extended studies showed that increased 5′ cytosine phosphate guanine (CpG) site methylation in the low LG-ABN pups reduced binding of transcription factor nerve growth factor inducible protein A (NGFI-A) to GR exon 17 promoter and reduced recruitment of CREB binding protein (CBP), subsequently reducing the levels of GR mRNA in hippocampus [20, 43]. These changes were observed both at postnatal day (PND) 6 (early) and PND90 (adulthood) suggesting the long-lasting nature of the epigenetic mark. In contrast, Daniels and colleagues reported no differences in the methylation status of exon 17 GR promoter in maternally separated (MS) compared to control rats on PND21 [44]. The conflicting results could be due to differences in the early stress model (maternal care vs MS) and strain (Long-Evans vs Sprague Dawley) which may exert different effects on the epigenetic signature of the glucocorticoid receptor. Other studies also report epigenetic alterations in the GR promoter using different ELS models of rat/mouse strains [45, 46] (Table 2).

Crh

Chen and colleagues reported hypomethylation of the Crh promoter in the PVN of maternally deprived (MD) Sprague Dawley rats on PND61 [47]. This was associated with increased phosphoCREB binding to the Crh cAMP response element (CRE), critical in the regulation of transcription of Crh. Similarly, Wang and colleagues reported increased H3 acetylation and hypomethylation of the Crh promoter region in the hippocampal cornu ammonis 1 (CA1) region of rats with postnatal MS [48]. Franklin and colleagues reported hypomethylation of the CRH receptor 2 (Crhr2) in maternally separated male C57/BL6 mice at 3–8 months of age and demonstrated a transgenerational effect [21]. The hypomethylation was associated with decreased Crhr2 gene expression which in this case was assessed in vitro using zebularine, a DNA methylation inhibitor [21]. This is in contrast to conventional understanding that DNA methylation is repressive [49, 50]; however, results correlate with the expected ELS-induced changes in HPA axis regulation and decreased Crhr2 expression.

Avp

Murgatroyd and colleagues reported hypomethylation of the AVP enhancer sequence in the parvocellular neurons of the PVN [23]. Hypomethylation of the AVP enhancer was associated with increased Avp expression following ELS (maternal separation) from 6 weeks of age and was still evident at 1 year suggesting the long-lasting nature of this epigenetic mark. Mechanistic analysis using mouse hypothalamic-like cells revealed that the hypomethylated CpG sites bound MeCP2 during postnatal life that in turn recruited DNA methyl transferase (DNMTs) and histone deacetylase (HDACs) to regulate expression of Avp [23] and that MeCP2 occupancy further depended on polycomb binding earlier in hypothalamic development [51]. Contrasting results were reported in a more recent study showing hypermethylation of the Avp enhancer CpG site in the hippocampus of maternally deprived C57BL/6 and DBA/2 males [45]. This could be due to the type of tissue analysed and the fact that the CpG sites analysed by these two studies did not overlap.

ELS-induced epigenetic changes in genes outside of the HPA axis

Brain-derived neurotrophic factor

ELS has been shown to decrease Bdnf expression whilst enhancing anxiety and depression-like behaviours [41, 52]. Roth and colleagues exposed Long-Evans rat pups to an abusive mother during the first week of life [53]. They reported hypermethylation of the Bdnf exons IV and IX in the prefrontal cortex on PND8 with associated reduction of Bdnf mRNA which persisted into adulthood. Bai and colleagues reported significantly decreased Bdnf mRNA and protein and increased miR-16 expression in the hippocampus of MD rats compared to those exposed to chronic unpredictable stress in adulthood (CUPS) and control rats [54] (see Table 3). The results suggested significant association of depression induced by MD to Bdnf and miR-16 levels but not the late-life stressors such as the CUPS thus emphasising the role of ELS-induced epigenetic alterations.
Table 3

Early stress-induced epigenetic modifications in other candidate genes

Candidate gene

Objective

Model/tissue type

Early stress/assessment age

Epigenetic and expression changes

Interpretation

References

Er-α

Determine the effect of maternal care on Er-α mRNA exp and Erα1b promoter methylation

Long-Evans hooded rats

MPOA of Amygdala

Maternal care variations: high vs low LG-ABN

Assessed: PND6

Erα1b promoter methylation in high vs low LG-ABN offspring

Stat5 binding to Erα1b promoter in high vs low LG-ABN offspring

Er-α mRNA exp in high vs low LG-ABN offspring

Higher levels of maternal care cause ↓Erα1b promoter methylation, ↑Er-α mRNA exp and ↑ Stat5 binding to Erα1b promoter in the MPOA in Long-Evans hooded rats on PND6

[59]

5-HTT

Determine the relationship between early stress and 5-HTT gene methylation

Rhesus macaques

Peripheral blood DNA

Rearing variations: MR vs NR

Assessed: 90–120 days (infants)

↑ average 5-HTT gene methylation in carrier of the low-expressing rh5-HTTLPR

5-HTT mRNA in PBMCs

Variations in maternal care were associated with average differences in 5-HTT gene methylation and this was dependent on the 5-HTT polymorphism in Rhesus macaques

[57]

Determine the effect of early stress on 5-HTT gene and whole genome DNA methylation

F bonnet macaques

Peripheral blood DNA

VFD from 3–8 months age

Assessed: mean = 8.4 years

5-HTT and whole genome methylation

5-HTT and whole genome methylation ↑ stress reactivity in VFD

5-HTT gene and whole genome methylation confer an ↑ stress reactivity following early stress in adolescent F bonnet macaques

[58]

Gad1

Determine the effect of maternal care on the Gad1 promoter in the hippocampus

Long-Evans hooded rats

Hippocampus

Maternal care variations: high vs low LG-ABN

Assessed: 3–4 months of age except for NGFI-A association to Gad1 promoter at PND4

↓ methylation of Gad1 promoter in high vs low LG-ABN offspring

↑ histone H3K9 acetylation of Gad1 promoter in high vs low LG-ABN offspring

↑ NGFI-A association w Gad1 promoter in high vs low LG-ABN offspring

Gad1 mRNA exp in high vs low LG-ABN offspring

High levels of maternal care ↓ Gad1 promoter methylation, ↑ Gad1 promoter histone acetylation and ↑ Gad1 mRNA in the hippocampus of Long-Evans hooded rats

[66]

Bdnf

Determine the effect of early stress on DNA methylation

Long-Evans hooded rats

Hippocampus and PFC

Exposure to abusive or caring mother from PND1–7 for 30 min

Assessed: PND8, 30 and 90

↑ methylation of Bdnf exon IV and IX in PFC

Bdnf mRNA exp in PFC

Bdnf mRNA exp in hippocampus

Early abuse ↑ methylation of Bdnf exon IV and IX in the PFC that persists into adulthood in Long-Evans hooded rats

[53]

Determine the effect of early stress on Bdnf mRNA, protein and miR-16 exp

Sprague Dawley rats

Hippocampus

MS: PND1–13

CUPS: 10–13 weeks

C: no stress

Assessed: 13–14 weeks

Only Bdnf transcribed from 5ʹ exon VI and 3ʹ common exon analysed

Bdnf mRNA exp in MS vs CUPS and C

Bdnf protein in MS vs CUPS and C

↑ miR-16 exp in MS vs CUPS and C

Bdnf mRNA exp in MS correlated significantly with ↑ miR-16 exp

MS induced significantly different BDNF and miR-16 expression in rats.

[54]

BDNF and miR-16 levels were inversely related in the presence of early stress

Reelin (Reln)

Determine the effect of MS on Reln expression

Wistar rats

Hippocampus

MS: PND2–15 for 3 h

Assessed: PND22

Reln gene methylation

Reln mRNA exp

MS ↑ Reln gene methylation and ↓ Reln mRNA exp in Wistar rats on PND22

[67]

↑ increased, ↓ decreased, ↔ no change, 5-HTT serotonin transporter, PND postnatal day, Bdnf brain-derived neurotrophic factor, DNA deoxyribonucleic acid, Er-α estrogen receptor alpha, exp expression, F female, Gad1 glutamic acid decarboxylase 1, LG-ABN licking grooming arched-back nursing, MPOA medial preoptic area, MR mother reared, NR nursery reared, mRNA messenger ribonucleic acid, PBMC peripheral blood mononuclear cells, VFD variable foraging demand, MS maternal separation, NGFI-A nerve growth factor inducible protein A, PFC prefrontal cortex, PND postnatal day, Stat5 signal transducer and activator of transcription 5, mi-R micro-RNA, CUPS chronic unpredictable stress, C control

Serotonin transporter (5-HTT or Slc6a4) gene

Reduction in 5-HTT expression has been observed in response to ELS in non-human primate and rodent models [55, 56]. Kinnaly and colleagues investigated the role of ELS-induced epigenetic modifications in the 5-HTT gene using non-human primate models [57, 58] (see Table 3). These studies assessed the relationship between 5-HTT expression and 5-HTT gene methylation following ELS in infant rhesus macaques [57] and adolescent bonnet macaques [58]. No significant effect of rearing was observed on the 5-HTT methylation status in either study. However, carriers of the short allele of the 5-HTT polymorphism had higher mean 5-HTT CpG methylation, and this was significantly associated to the levels of peripheral 5-HTT expression in the infant rhesus macaques [57]. In adolescent and adult Bonnet macaques, genome-wide methylation levels were associated with 5-HTT expression in those exposed to early stress as infants compared to controls [58]. The above studies suggest that 5-HTT gene methylation is not susceptible to variations in maternal care; however, polymorphisms in the 5-HTT gene and methylation at other cis or Trans regulating sites as may be conferring increased stress reactivity.

Estrogen receptor-α gene

Increased expression of estrogen receptor-α (ERα) in response to variations in maternal care was first reported by Champagne and colleagues using the LG-ABN rat model [39]. The study reported elevated ERα mRNA expression in the medial preoptic area of high LG-ABN dams compared to low LG-ABN, and this effect was transmitted to the female offspring of the high LG-ABN dams [39, 59]. Champagne and colleagues then demonstrated decreased ERα1b promoter methylation in high vs low LG-ABN offspring on PND6 [59] (Table 3). It was characterised that this hypomethylation of the promoter region enhanced binding of the STAT5 transcription factor to the ERα1b promoter and a corresponding increase in ERα mRNA expression in response to increased maternal care.

Glutamate decarboxylase-1 and Reelin genes

Glutamate decarboxylase 1 (GAD1) is a key enzyme in the synthesis of gamma amino-butyric acid (GABA), and Reelin (Reln) is important in migration of new neurons in the central nervous system (CNS). Postmortem studies of schizophrenic patient brains have shown decreased forebrain expression of GAD1 [60, 61] and increased methylation of the GAD1 promoter [62, 63] and decreased RELN expression [64, 65]. Zhang and colleagues assessed the methylation status of the Gad1 promoter in the offspring of the high vs low LG-ABN dams [66] (See Table 3). Hypomethylation, increased histone H3K9 acetylation and increased NGFI-A association with the Gad1 promoter were observed in those raised by high LG-ABN dams leading to increased Gad1 mRNA expression [66]. Qin and colleagues reported hypermethylation of the Reln gene and subsequent down-regulation of Reln mRNA in the hippocampus of Wistar rats exposed to MS from PND2–15 compared to controls [67].

Early stress-induced epigenetic modifications in humans

Epigenetic alterations in candidate genes

In human studies, most focus to date has been on the role of GR due to its negative feedback control in stress responsivity and serotonin transporter (5-HTT) due to its polymorphisms and role in mediating ELS and later-life stress effects on adult depression status [14, 68] (see Table 4 for summary of studies).
Table 4

Early stress-induced epigenetic modifications of candidate genes in humans

Candidate gene

Objective

Model/tissue type

Early stress/assessment age

Epigenetic and expression changes

Interpretation

References

rRNA promoter

Determine the effect of childhood abuse on methylation status of the rRNA promoter

Retrospective

Hippocampus

CA + suicide vs no CA + no suicide

Assessed

Mean age 35 yrs.

N = 30

↑ methylation rRNA promoter and 5′ regulatory region in CA

↑ methylation of 21 of 26 CpG sites in CA

↓ rRNA expression in hippocampus

Childhood abuse ↑ methylation of rRNA promoter in the hippocampus of suicide completers in adulthood

[98]

GR promoter

Determine the effect of childhood abuse on the methylation status of the GR promoter and GR mRNA exp

Retrospective

Hippocampus

Child abuse

Assessed

Mean age 34.6 yrs.

N = 36 M

↑ methylation of CpGs in GR promoter

↓ GR mRNA exp

↓ GR 1F mRNA exp

Childhood abuse ↑ methylation of individual CpGs within the GR promoter and GR1F exon resulting in ↓ GR and GR 1F mRNA expression in adult hippocampus

[19]

Determine the effect of early stress on methylation status of the GR promoter

Retrospective

Peripheral blood WBCs

Early stressa

Assessed: mean age 27.4 yrs.

N = 99

↑ CpG1 methylation w ↓ parental care or loss

↑ CpG3 methylation w maltreatment or parental loss

↔ CpG2, 4, 5–13 methylation w childhood adversity

Early stress ↑ methylation of individual CpGs of the GR promoter in adulthood

[71]

Determine the effect of childhood maltreatment and severity on NR3C1 promoter methylation

Retrospective

Peripheral blood WBCs

Childhood maltreatmentb

Assessed: mean ages and sample size

BPD 30.76 yrs. N = 101

MDD 41.63 yrs. N = 99

MDD + PTSD 37.33 yrs.

N = 15

↑ CpG 2–8 methylation of the NR3C1 gene promoter in maltreated participants

↑ severity of maltreatment ↑ methylation status of NR3C1 except for CpG1 site

Childhood maltreatment was associated with ↑ methylation of the NR3C1 gene promoter in peripheral blood in adulthood.

Number and severity of maltreatment correlated positively with methylation status

[70]

NR3C1 locus (6.5 million base pairs)

Determine the effect of CA on a 6.5 Mbp loci centred on NR3C1

Retrospective

Hippocampus

CA + suicide vs no CA + no suicide

Assessed: adulthood

N = 24

281 DMRs were identified

↑ methylation in 126 DMRs in controls

↑methylation in 155 DMRs in abused

3 clusters of DMRs mapped within α-, β- and γ- protocadherin (PCDH) gene family

DMRs enriched more in α-PCDH in the abused brains

Early stress has a broader epigenomics imprint expanding to promoters of genes both upstream and downstream to the NR3C1 gene and includes the PCDH gene family implicated in synaptic plasticity

[69]

5HTT or SLC6A4

Determine the effect of CA on SLC6A4 promoter methylation and mRNA expression

Retrospective

Peripheral blood DNA

Childhood adversitya

Assessed: adulthood

N = 102 MDD with or without CA

↑average methylation in promoter of SLC6A4 with CpG7 showing higher methylation in CA to no CA

↑ methylation in CpG2 of SLC6A4 associated with physical abuse

Childhood adversities were significantly associated with higher SLC6A4 promoter methylation in people with current MDD

[76]

Determine the effect of CA on the methylation status of SLC6A4 promoter

Retrospective

Lymphoblast cell lines

Childhood maltreatment

Assessed: mean age: M 49 yrs.

F 47 yrs.

N = 192

↑ overall SLC6A4 promoter methylation in abused male and female

↑ methylation of CpG1 and CpG 3 of SLC6A4 in abused females compared to non-abused

↔ in individual CpG sites in males

Childhood abuse increased methylation of CpGs in the promoter of SLC6A4 in adults

[74, 75]

↑ increased, ↓ decreased, ↔ no change, yrs. years, CA child abuse, DNA deoxyribonucleic acid, mRNA messenger ribonucleic acid, exp expression, rRNA ribosomal ribonucleic acid, GR glucocorticoid receptor, M male, F Female, WBC white blood cell, BPD borderline personality disorder, PTSD post-traumatic stress disorder, MDD major depressive disorder, DMRs differentially methylated regions, PCDH protocadherin, NR3C1 nuclear receptor subfamily 3, group C, member 1, SLC6A4 solute carrier family 6, member 4 (neurotransmitter transporter)

aEarly stress included: low levels of parental care, parental loss and childhood maltreatment

bSexual, physical and emotional abuse; BPD had higher childhood maltreatment; MDD had lower maltreatment

GR gene

McGowan and colleagues were the first to demonstrate hypermethylation of the GR promoter exon 1F and decreased GR mRNA in the hippocampus of adults who were exposed to childhood abuse [19]. When compared to suicide victims or controls with no childhood abuse the GR promoter of suicide victims with a history of childhood abuse showed a significant increase (p < 0.05; d = 1.07) in methylation. This study was thus able to translate the results previously described in LG-ABN rat model [43]. McGowan and colleagues extended research by analysing a wider locus containing the GR gene on chromosome 5 and reported differential methylation in promoters of the protocadherin (PCDH) gene family which are implicated in synaptic plasticity [69]. Whilst McGowan and colleagues analysed the epigenetic changes in hippocampal GR gene, other human studies have reported similar hypermethylation GR gene promoter in peripheral blood leukocytes of adults exposed to early childhood stress [70, 71]. Murgatroyd and colleagues have recently demonstrated maternal stroking to modify CpG methylation within this GR region further translating the rat LG results [72]. These studies have shown that hypermethylation of specific CpG sites were associated to type of early stress or the severity of early maltreatment (see Table 4).

5-HTT gene

Philibert and colleagues were the first to report hypermethylation of the 5-HTT promoter and subsequent reduction in 5-HTT mRNA; however, this study did not assess the role of early maltreatment [73]. Further work from Beach and colleagues reported association of increased methylation of the 5-HTT promoter to childhood abuse. In their 2010 study, Beach and colleagues report significant association of overall methylation of the 5-HTT promoter region (CpGs analysed = 71, CpGs used in analyses = 26) to childhood abuse (p < 0.0004, d = 0.73) [74]. In particular, increased methylation of the sites CpG1 and CpG3 in the 5-HTT promoter was significantly associated in abused females. In their 2011 study, the same group showed significant association of childhood abuse to 5-HTT promoter methylation, 5-HTT mRNA levels and a correlation to 5-HTT genotype and adult anti-social personality disorder [75]. The study reported that 9 % of the variation in the personality disorder was attributed to increased 5-HTT promoter methylation observed only in those that were sexually abused (see Table 4 for details). Kang and colleagues also reported similar hypermethylation of the 5-HTT promoter (total of seven CpG sites) in abused compared to non-abused people with current MDD [76]. The average methylation percentage of the seven CpG sites for any adversity was significantly higher in those who were abused in childhood (p < 0.001, d = 1.1).

Genome-wide methylation

A number of studies have assessed the effect of early stress on genome-wide methylation patterns (See Table 5 for summary of studies). Naumova and colleagues demonstrated hypermethylation of 28 genes involved in brain development and function including those regulating the arginine vasopressin 1A receptor (AVPR1A), GABA A receptor (GABRA5), glutamate receptor (GRM5), among others, in 7- to 10-year-old children following institutionalisation [77]. Hypermethylation of candidate genes has also been shown in adulthood, with Labonte and colleagues reporting hypermethylation of 248 gene promoters and associated decreases in mRNA expression of these promoters in the hippocampi of adult men who completed suicide [78]. Genes responsible for cellular and neuronal plasticity were the most differentially methylated, including the alsin (ALS2) gene promoter. Many other studies have also shown significant global methylation differences between those exposed to early adversity [7981]. These studies suggest that alterations in the early environment have the ability to cause changes in the methylation status of numerous genes across the genome, including those involved in control of nervous and immune system development and function.
Table 5

Effects of early stress on genome-wide methylation in humans

Objective

Model/tissue type

Early stress/assessment age

Epigenetic and expression changes

Interpretation

References

Determine the effect of childhood SES on genome-wide methylation in adulthood

Retrospective

Peripheral blood DNA

High vs low childhood SES

Assessed: 45 yrs.

N = 40 M

666 gene promoters ↑ and 586 promoters ↓ methylation in high vs low childhood SES

The genes involved fall into extra and intracellular signalling, DNA signalling and metabolic signalling categories.

Variations in childhood SES cause changes in genome-wide methylation in adulthood with genes in extra and intra cellular signalling and metabolic functioning

[79]

Determine the effect of early environment on genome-wide methylation levels

Retrospective

Peripheral blood DNA

Institutional care vs raised by biological parents

Assessed: mean 8.25 yrs. N = 28

Differential methylation of 914 of 26,214 CpG sites from 838 gene promoters across groupsa

↑ methylation of 744 promoters in institutionalised

↓ methylation of 94 promoters in institutionalised

Promoters mainly involved in control of cellular signalling and the immune response

Early environmental alterations cause changes in methylation of a number of genes important for control of cellular signalling and the immune response in childhood

[77]

Determine the effect of childhood abuse on genome-wide DNA methylation

Retrospective

Hippocampus

CA + suicide vs no CA + suicide

Assessed: adulthood

N = 41 M

Differentially methylated promoters in CA vs no CA were spread across the genome

248 (68.5 %) promoters ↑ methylation in CA

114 (31.5 %) promoters ↓ methylation in CA

↑ methylation associated w ↓ mRNA exp

Childhood abuse causes alteration in the methylation of gene promoters and mRNA exp in adulthood specifically genes involved in neuronal plasticity

[93]

Retrospective

Peripheral blood DNA

PTSD + CA vs PTSD + no CA

Assessed: mean age CA = 39.6 yrs.

No CA = 43.69

N = 61

Differential methylation in promoters of abused vs non-abused PTSD patients

↑ methylation in transcripts of PTSD + CA group (11.78 %) vs PTSD + no CA (0.8 %)

14 transcripts differentially methylated in CA vs no CA

Childhood abuse causes alteration to the methylation of CpG sites in both promoter regions and gene body and include specifically genes involved in CNS development in the abused PTSD group

[80]

Determine the effect of childhood abuse in methylation status of immune system and cytokine regulation

Retrospective

Peripheral blood DNA

PTSD + CA, PTSD + no CA, C + CA, C + no CA

Assessed: adulthood

N = 110

↑ global methylation in PTSD vs C

↔ methylation due to CA in PTSD or C

Gene specific associations with found in BDNF, HSF1, TLR8 for PTSD and CA

↑plasma TNFα in CA vs no CA

Childhood abuse early in life can alter global and gene specific DNA methylation patterns specifically involved in immune dysregulation

[81]

↑ increased, ↓ decreased, ↔ no change, yrs years, SES socioeconomic status, M Male, DNA deoxyribonucleic acid, mRNA messenger ribonucleic acid, exp expression, CA childhood abuse, PTSD post-traumatic stress disorder, C controls, BDNF brain-derived neurotrophic factor, HSF1 heat shock transcription factor 1, TLR8 toll-like receptor 8, TNFα tumour necrosis factor alpha, CNS central nervous system

aGenes modified by rearing environment include those involved in control of the dopaminergic system (TERF2IP), serotonin biosynthesis and serotonin receptor activity (TPH, HTR1D, HTR1F), glucocorticoid and steroid biosynthesis and their receptor activity (NRIP1, PPARGC1B, UGT), genes encoding the arginine vasopressin receptor, glutamate, cadherin and cholinergic receptors, and others which are collectively responsible for neural communication, memory formation and learning and retention

Discussion

As evidenced in the studies reviewed above, changes in DNA methylation in response to the early environment remain the best characterised to date. This could be due to DNA methylation being a robust epigenetic mark and available to study across lifespan and generations [21, 82]. More recent studies however have also analysed the role of microRNAs [54], histone modifications [83] and DNA hydroxymethylation [84] in mediating early stress effects.

Results from the animal models reviewed suggest that ELS exerts significant methylation changes in the stress-regulatory genes. Enhanced Crh and Avp expression in the hypothalamus may lead to a hypersensitive HPA axis [23, 47], whilst reduced Crhr2 and GR expression (in the hypothalamus and hippocampus respectively) may affect negative feedback of the HPA response to stress [20, 21]. Epigenetic signatures in the GR gene promoter in humans exposed to a variety of early adversity translate the results observed in animal models and confirm the central role of GR methylation in the dysfunctional negative feedback of HPA axis [19, 70, 71]. Analysis of a broader GR locus (~7 Mpb centred with Nr3c1) in both rats and humans exposed to early adversity revealed conserved methylated sequences in the GR locus including the GR promoter methylations thus emphasising an evolutionary role of the GR locus in stress responsivity [46, 69] (See Tables 2 and 4 for details).

Studies of animal models have centred on other candidate genes implicated in the pathogenesis of neuropsychiatric disorders. Altered 5-HTT expression early in life may moderate the impact of early environment on emotion development [28] as also shown by pharmacological blocking studies [85, 29]. The findings on 5-HTT promoter methylation therefore support the role of reduced 5-HTT activity in the onset of adult depression-like behaviours [57, 58]. Reduced BDNF expression has been implicated in anxiety and depression-like behaviours [41, 52]; therefore, hypermethylation of the Bdnf gene alongside reduced Bdnf mRNA expression in the prefrontal cortex (PFC) of rats exposed to abuse (67) suggests early stress may play a role in the development of anxiety and depression disorders. In humans exposed to early adversity, 5-HTT hypermethylation has been associated to adult depression status depending on the polymorphic status of the 5-HTT gene, emphasising the role of genetic polymorphisms on epigenetic effects [14, 76]. Interestingly, studies to date have only analysed the effect of prenatal stress on the methylation status of the BDNF gene in humans [86, 87] and not variations in postnatal stress. BDNF is known to regulate neuronal development, serotonergic functions and signalling [88]. For example, BDNF promotes the development and function of serotonergic neurons where high affinity receptors for BDNF, Trkb, are also found [89]. Therefore, ELS-induced reduced BDNF could also potentially lead to decreased function of the serotonergic system thus leading to mood and affective behavioural dysfunctions as reported in the above studies. In addition, BDNF has been shown to be regulated by estrogen via ERα within the hippocampus of rat brains [90]. In this context, the increased ERα expression in the brains of the high LG-ABN rats [59] suggests that estrogen is important in regulating normal brain development and possibly involves maturation of neural systems via BDNF regulation. The down-regulation of Reelin post ELS [67] may negatively impact hippocampal function, potentially altering stress axis regulation and predisposing to anxiety and depression across the lifespan. A neurodevelopmental origin for schizophrenia is shown from the study suggesting increased methylation and reduced Gad1 expression in rats [66] which fits with the observations in postmortem brains of schizophrenic patients [62, 63].

One of the major questions in psychiatric epigenetics is whether epigenetic changes observed in the periphery reflect the changes in the brain. For example, hypermethylation of GR gene promoter and subsequent increase in GR mRNA was reported in both peripheral leukocytes [70, 71] and in the neurons of the hippocampus [19]. Analysis of methylome of cells in the PFC and T cells in mother vs surrogate-reared rhesus macaques revealed similarities in the methylation of cells in PFC and T cells, specifically the GR receptor promoter region alongside genes in the immune response, transcription and response to stimulus [91]. These findings suggest that peripheral GR methylation or cell specific (example T cells) methylation changes could be a potential biomarker for assessing early stress-induced HPA dysfunction. Genome-wide methylation studies show that childhood maltreatment leaves a systemic, genome-wide mark by altering the methylation status of key genes in regulatory pathways such as intra and extra cellular signalling [77, 79, 80, 92, 93]. Although most work to date has focused on the stress response, it may be interesting to consider whether prolonged immune dysfunction that often persists after early stress is secondary to epigenetic changes in immunoregulatory genes as reported by Smith and colleagues in PTSD [81] or if it results indirectly from epigenetic changes in stress-regulatory genes.

Conclusions

Research to date highlights the remarkable susceptibility of the genome, and particularly of genes involved in stress and emotion regulation, to environmental alterations early in the lifespan. Many of these changes persisted into adulthood, and epigenetic mechanisms have been shown to contribute to the long-lasting nature of ELS and its contribution to an individual’s disease risk and susceptibility to neuropsychiatric conditions across the lifespan.

Abbreviations

5-HTT: 

5-hyrodxytryptamine transporter (serotonin transporter)

ACTH: 

adrenocorticotrophic hormone

AVP: 

arginine vasopressin

BDNF: 

brain-derived neurotrophic factor

CA1: 

cornu ammonis 1

CpG: 

cytosine phosphate guanine

CRH: 

corticotrophin-releasing hormone

DNMT: 

DNA methyl transferase

ELS: 

early life stress

GAD1: 

glutamate decarboxylase 1

GR: 

glucocorticoid receptor

HDAC: 

histone deacetylase

HPA: 

hypothalamic-pituitary-adrenal axis

MD: 

maternal deprivation

MS: 

maternal separation

PCDH: 

protocadherin

PFC: 

prefrontal cortex

PND: 

postnatal day

PVN: 

para ventricular nucleus

Declarations

Acknowledgements

The presented work is partly supported by the National Health and Medical Research Council Australia (APP1003788 to BTB). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Discipline of Psychiatry, School of Medicine, University of Adelaide
(2)
School of HealthCare Science, Manchester Metropolitan University
(3)
School of Medicine and Dentistry, James Cook University

References

  1. Green JG, McLaughlin KA, Berglund PA, Gruber MJ, Sampson NA, Zaslavsky AM, et al. Childhood adversities and adult psychiatric disorders in the national comorbidity survey replication I: associations with first onset of DSM-IV disorders. Arch Gen Psychiatry. 2010;67(2):113–23.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Kessler RC, McLaughlin KA, Green JG, Gruber MJ, Sampson NA, Zaslavsky AM, et al. Childhood adversities and adult psychopathology in the WHO World Mental Health Surveys. Br J Psychiatry. 2010;197(5):378–85.PubMed CentralView ArticlePubMedGoogle Scholar
  3. Bernet CZ, Stein MB. Relationship of childhood maltreatment to the onset and course of major depression in adulthood. Depress Anxiety. 1999;9(4):169–74.View ArticlePubMedGoogle Scholar
  4. Varese F, Smeets F, Drukker M, Lieverse R, Lataster T, Viechtbauer W, et al. Childhood adversities increase the risk of psychosis: a meta-analysis of patient-control, prospective- and cross-sectional cohort studies. Schizophr Bull. 2012;38(4):661–71.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Scott KM, Von Korff M, Angermeyer MC, Benjet C, Bruffaerts R, de Girolamo G, et al. Association of childhood adversities and early-onset mental disorders with adult-onset chronic physical conditions. Arch Gen Psychiatry. 2011;68(8):838–44.PubMed CentralView ArticlePubMedGoogle Scholar
  6. Scott KM, Von Korff M, Alonso J, Angermeyer MC, Benjet C, Bruffaerts R, et al. Childhood adversity, early-onset depressive/anxiety disorders, and adult-onset asthma. Psychosom Med. 2008;70(9):1035–43.View ArticlePubMedGoogle Scholar
  7. Gershon A, Sudheimer K, Tirouvanziam R, Williams LM, O’Hara R. The long-term impact of early adversity on late-life psychiatric disorders. Curr Psychiatry Rep. 2013;15(4):013–0352.View ArticleGoogle Scholar
  8. Kaplow JB, Widom CS. Age of onset of child maltreatment predicts long-term mental health outcomes. J Abnorm Psychol. 2007;116(1):176–87.View ArticlePubMedGoogle Scholar
  9. Schoedl AF, Costa MC, Mari JJ, Mello MF, Tyrka AR, Carpenter LL, et al. The clinical correlates of reported childhood sexual abuse: an association between age at trauma onset and severity of depression and PTSD in adults. J Child Sex Abus. 2010;19(2):156–70.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Evans GW, Schamberg MA. Childhood poverty, chronic stress, and adult working memory. Proc Natl Acad Sci. 2009;30:2009.Google Scholar
  11. Felitti VJ, Anda RF, Nordenberg D, Williamson DF, Spitz AM, Edwards V, et al. Relationship of childhood abuse and household dysfunction to many of the leading causes of death in adults. The Adverse Childhood Experiences (ACE) Study. Am J Prev Med. 1998;14(4):245–58.View ArticlePubMedGoogle Scholar
  12. Wise LA, Zierler S, Krieger N, Harlow BL. Adult onset of major depressive disorder in relation to early life violent victimisation: a case-control study. Lancet. 2001;358(9285):881–7.View ArticlePubMedGoogle Scholar
  13. Gershon A, Minor K, Hayward C. Gender, victimization, and psychiatric outcomes. Psychol Med. 2008;38(10):1377–91.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Caspi A, Sugden K, Moffitt TE, Taylor A, Craig IW, Harrington H, et al. Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science. 2003;301(5631):386–9.View ArticlePubMedGoogle Scholar
  15. Buchmann AF, Hellweg R, Rietschel M, Treutlein J, Witt SH, Zimmermann US, et al. BDNF Val 66 Met and 5-HTTLPR genotype moderate the impact of early psychosocial adversity on plasma brain-derived neurotrophic factor and depressive symptoms: a prospective study. Eur Neuropsychopharmacol. 2013;23(8):902–9.View ArticlePubMedGoogle Scholar
  16. Binder EB, Bradley RG, Liu W, Epstein MP, Deveau TC, Mercer KB, et al. Association of FKBP5 polymorphisms and childhood abuse with risk of posttraumatic stress disorder symptoms in adults. JAMA. 2008;299(11):1291–305.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Heim C, Newport DJ, Mletzko T, Miller AH, Nemeroff CB. The link between childhood trauma and depression: insights from HPA axis studies in humans. Psychoneuroendocrinology. 2008;33(6):693–710.View ArticlePubMedGoogle Scholar
  18. Danese A, Pariante CM, Caspi A, Taylor A, Poulton R. Childhood maltreatment predicts adult inflammation in a life-course study. Proc Natl Acad Sci U S A. 2007;104(4):1319–24.PubMed CentralView ArticlePubMedGoogle Scholar
  19. McGowan P, Sasaki A, D’Alessio AC, Dymov S, Labonte B, Szyf M, et al. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse. Nat Neurosci. 2009;12(3):342–8.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Weaver IC, Cervoni N, Champagne FA, D’Alessio AC, Sharma S, Seckl JR, et al. Epigenetic programming by maternal behavior. Nat Neurosci. 2004;7(8):847–54.View ArticlePubMedGoogle Scholar
  21. Franklin TB, Russig H, Weiss IC, Graff J, Linder N, Michalon A, et al. Epigenetic transmission of the impact of early stress across generations. Biol Psychiatry. 2010;68(5):408–15.View ArticlePubMedGoogle Scholar
  22. McGowan PO, Szyf M. The epigenetics of social adversity in early life: implications for mental health outcomes. Neurobiol Dis. 2010;39(1):66–72.View ArticlePubMedGoogle Scholar
  23. Murgatroyd C, Patchev A, Wu Y, Micale V, Bockmuhl Y, Fischer D, et al. Dynamic DNA methylation programs persistent adverse effects of early-life stress. Nat Neurosci. 2009;12(12):1559–66. doi:10.038/nn.2436. Epub 009 Nov 8.Google Scholar
  24. Holsboer F. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology. 2000;23(5):477–501.View ArticlePubMedGoogle Scholar
  25. Meynen G, Unmehopa UA, van Heerikhuize JJ, Hofman MA, Swaab DF, Hoogendijk WJ. Increased arginine vasopressin mRNA expression in the human hypothalamus in depression: A preliminary report. Biol Psychiatry. 2006;60(8):892–5.View ArticlePubMedGoogle Scholar
  26. Raadsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swaab DF. Increased numbers of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients. Neuroendocrinology. 1994;60(4):436–44.View ArticlePubMedGoogle Scholar
  27. Murgatroyd C, Spengler D. Epigenetics of early child development. Front Psychiatry. 2011;2:16.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Bennett AJ, Lesch KP, Heils A, Long JC, Lorenz JG, Shoaf SE, et al. Early experience and serotonin transporter gene variation interact to influence primate CNS function. Mol Psychiatry. 2002;7(1):118–22.View ArticlePubMedGoogle Scholar
  29. Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA. Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science. 2004;306(5697):879–81.View ArticlePubMedGoogle Scholar
  30. Gatt JM, Nemeroff CB, Dobson-Stone C, Paul RH, Bryant RA, Schofield PR, et al. Interactions between BDNF Val66Met polymorphism and early life stress predict brain and arousal pathways to syndromal depression and anxiety. Mol Psychiatry. 2009;14(7):681–95.View ArticlePubMedGoogle Scholar
  31. Cirulli F, Francia N, Berry A, Aloe L, Alleva E, Suomi SJ. Early life stress as a risk factor for mental health: role of neurotrophins from rodents to non-human primates. Neurosci Biobehav Rev. 2009;33(4):573–85.PubMed CentralView ArticlePubMedGoogle Scholar
  32. Karege F, Perret G, Bondolfi G, Schwald M, Bertschy G, Aubry JM. Decreased serum brain-derived neurotrophic factor levels in major depressed patients. Psychiatry Res. 2002;109(2):143–8.View ArticlePubMedGoogle Scholar
  33. Black IB. Trophic regulation of synaptic plasticity. J Neurobiol. 1999;41(1):108–18.View ArticlePubMedGoogle Scholar
  34. White KJ, Walline CC, Barker EL. Serotonin transporters: implications for antidepressant drug development. AAPS J. 2005;7(2):E421–33.PubMed CentralView ArticlePubMedGoogle Scholar
  35. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science. 1996;274(5292):1527–31.View ArticlePubMedGoogle Scholar
  36. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, Bertolino A, et al. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell. 2003;112(2):257–69.View ArticlePubMedGoogle Scholar
  37. van der Doelen RH, Calabrese F, Guidotti G, Geenen B, Riva MA, Kozicz T, et al. Early life stress and serotonin transporter gene variation interact to affect the transcription of the glucocorticoid and mineralocorticoid receptors, and the co-chaperone FKBP5, in the adult rat brain. Front Behav Neurosci. 2014;8:355.PubMed CentralPubMedGoogle Scholar
  38. Osterlund MK, Hurd YL. Estrogen receptors in the human forebrain and the relation to neuropsychiatric disorders. Prog Neurobiol. 2001;64(3):251–67.View ArticlePubMedGoogle Scholar
  39. Champagne FA, Weaver IC, Diorio J, Sharma S, Meaney MJ. Natural variations in maternal care are associated with estrogen receptor alpha expression and estrogen sensitivity in the medial preoptic area. Endocrinology. 2003;144(11):4720–4. Epub 2003 Jul 24.View ArticlePubMedGoogle Scholar
  40. Pinheiro M, Ferraz-de-Paula V, Ribeiro A, Sakai M, Bernardi M, Palermo-Neto J. Long-term maternal separation differentially alters serum corticosterone levels and blood neutrophil activity in A/J and C57BL/6 mouse offspring. Neuroimmunomodulation. 2011;18(3):184–90. Epub 2011 Feb 9.View ArticlePubMedGoogle Scholar
  41. Roceri M, Hendriks W, Racagni G, Ellenbroek BA, Riva MA. Early maternal deprivation reduces the expression of BDNF and NMDA receptor subunits in rat hippocampus. Mol Psychiatry. 2002;7(6):609–16.View ArticlePubMedGoogle Scholar
  42. George ED, Bordner KA, Elwafi HM, Simen AA. Maternal separation with early weaning: a novel mouse model of early life neglect. BMC Neurosci. 2010;11:123.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Weaver ICG, D’Alessio AC, Brown SE, Hellstrom IC, Dymov S, Sharma S, et al. The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: altering epigenetic marks by immediate-early genes. J Neurosci. 2007;27(7):1756–68.PubMed CentralView ArticlePubMedGoogle Scholar
  44. Daniels WMU, Fairbairn L, Tilburg G, McEvoy C, Zigmond M, Russell V, et al. Maternal separation alters nerve growth factor and corticosterone levels but not the DNA methylation status of the exon 1(7) glucocorticoid receptor promoter region. Metab Brain Dis. 2009;24(4):615–27.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Kember RL, Dempster EL, Lee TH, Schalkwyk LC, Mill J, Fernandes C. Maternal separation is associated with strain-specific responses to stress and epigenetic alterations to Nr3c1, Avp, and Nr4a1 in mouse. Brain Behav. 2012;2(4):455–67.PubMed CentralView ArticlePubMedGoogle Scholar
  46. McGowan P, Suderman M, Sasaki A, Huang T, Hallett M, Meaney MJ, et al. Broad epigenetic signature of maternal care in the brain of adult rats. PLoS One. 2011;6(2):e14739.PubMed CentralView ArticlePubMedGoogle Scholar
  47. Chen J, Evans A, Liu Y, Honda M, Saavedra J, Aguilera G. Maternal deprivation in rats is associated with corticotrophin-releasing hormone (CRH) promoter hypomethylation and enhances CRH transcriptional responses to stress in adulthood. J Neuroendocrinol. 2012;24(7):1055–64.PubMed CentralView ArticlePubMedGoogle Scholar
  48. Wang A, Nie W, Li H, Hou Y, Yu Z, Fan Q, et al. Epigenetic upregulation of corticotrophin-releasing hormone mediates postnatal maternal separation-induced memory deficiency. PLoS One. 2014;9(4):e94394.PubMed CentralView ArticlePubMedGoogle Scholar
  49. Rishi V, Bhattacharya P, Chatterjee R, Rozenberg J, Zhao J, Glass K, et al. CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes. Proc Natl Acad Sci U S A. 2010;107(47):20311–6.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Zhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, et al. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc Natl Acad Sci U S A. 2005;102(12):4459–64.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Murgatroyd C, Spengler D. Polycomb binding precedes early-life stress responsive DNA methylation at the Avp enhancer. PLoS One. 2014;9(3):e90277.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Roceri M, Cirulli F, Pressina C, Peretto P, Racagni G, Riva MA. Postnatal repeated maternal deprivation produces age-dependent changes of brain-derived neurotrophic factor expression in selected rat brain regions. Biol Psychiatry. 2004;55(7):708–14.View ArticlePubMedGoogle Scholar
  53. Roth TL, Lubin FD, Funk A, Sweatt JD. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol Psychiatry. 2009;65(9):760–9. doi:10.1016/j.biopsych.2008.11.028. Epub 9 Jan 15.PubMed CentralView ArticlePubMedGoogle Scholar
  54. Bai M, Zhu X, Zhang Y, Zhang S, Zhang L, Xue L, et al. Abnormal hippocampal BDNF and miR-16 expression is associated with depression-like behaviors induced by stress during early life. PLoS One. 2012;7(10):e46921.PubMed CentralView ArticlePubMedGoogle Scholar
  55. Kinnally EL, Tarara E, Mason W, Mendoza S, Abel K, Lyons L, et al. Serotonin transporter expression is predicted by early life stress and is associated with disinhibited behavior in infant rhesus macaques. Genes Brain Behav. 2010;9(1):45–52.PubMed CentralView ArticlePubMedGoogle Scholar
  56. Lee J-H, Kim B-T, Kim HJ, Kim JG, Ryu V, Kang D-W, et al. Depressive behaviors and decreased expression of serotonin reuptake transporter in rats that experienced neonatal maternal separation. Neurosci Res. 2007;58(1):32–9.View ArticlePubMedGoogle Scholar
  57. Kinnally EL, Capitanio JP, Leibel R, Deng L, LeDuc C, Haghighi F, et al. Epigenetic regulation of serotonin transporter expression and behavior in infant rhesus macaques. Genes Brain Behav. 2010;9(6):575–82.PubMed CentralPubMedGoogle Scholar
  58. Kinnally EL, Feinberg C, Kim D, Ferguson K, Leibel R, Coplan JD, et al. DNA methylation as a risk factor in the effects of early life stress. Brain Behav Immun. 2011;25(8):1548–53.PubMed CentralView ArticlePubMedGoogle Scholar
  59. Champagne FA, Weaver ICG, Diorio J, Dymov S, Szyf M, Meaney MJ. Maternal care associated with methylation of the estrogen receptor-alpha1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring. Endocrinology. 2006;147(6):2909.View ArticlePubMedGoogle Scholar
  60. Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. Decreased glutamic acid decarboxylase 67 messenger RNA expression in a subset of prefrontal cortical gamma-aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatry. 2000;57(3):237–45.View ArticlePubMedGoogle Scholar
  61. Heckers S, Stone D, Walsh J, Shick J, Koul P, Benes FM. Differential hippocampal expression of glutamic acid decarboxylase 65 and 67 messenger RNA in bipolar disorder and schizophrenia. Arch Gen Psychiatry. 2002;59(6):521–9.View ArticlePubMedGoogle Scholar
  62. Kundakovic M, Chen Y, Costa E, Grayson D. DNA methyltransferase inhibitors coordinately induce expression of the human reelin and glutamic acid decarboxylase 67 genes. Mol Pharmacol. 2007;71(3):644–53.View ArticlePubMedGoogle Scholar
  63. Costa E, Dong E, Grayson DR, Ruzicka WB, Simonini MV, Veldic M, et al. Epigenetic targets in GABAergic neurons to treat schizophrenia. Adv Pharmacol. 2006;54:95–117.View ArticlePubMedGoogle Scholar
  64. Fatemi SH, Earle JA, McMenomy T. Reduction in Reelin immunoreactivity in hippocampus of subjects with schizophrenia, bipolar disorder and major depression. Mol Psychiatry. 2000;5(6):654–63.View ArticlePubMedGoogle Scholar
  65. Guidotti A, Sharma R, Uzunov D, Costa E, Auta J, Davis JM, et al. Decrease in Reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder: a postmortem brain study. Arch Gen Psychiatry. 2000;57(11):1061–9.View ArticlePubMedGoogle Scholar
  66. Zhang T-Y, Hellstrom IC, Bagot R, Wen X, Diorio J, Meaney MJ. Maternal care and DNA methylation of a glutamic acid decarboxylase 1 promoter in rat hippocampus. J Neurosci. 2010;30(39):13130–7. doi:10.1523/JNEUROSCI.1039-10.2010.View ArticlePubMedGoogle Scholar
  67. Qin L, Tu W, Sun X, Zhang J, Chen Y, Zhao H. Retardation of neurobehavioral development and reelin down-regulation regulated by further DNA methylation in the hippocampus of the rat pups are associated with maternal deprivation. Behav Brain Res. 2011;217(1):142–7.View ArticlePubMedGoogle Scholar
  68. Aas M, Djurovic S, Athanasiu L, Steen NE, Agartz I, Lorentzen S, et al. Serotonin transporter gene polymorphism, childhood trauma, and cognition in patients with psychotic disorders. Schizophr Bull. 2012;38(1):15–22.PubMed CentralView ArticlePubMedGoogle Scholar
  69. Suderman M, McGowan PO, Sasaki A, Huang TC, Hallett MT, Meaney MJ, et al. Conserved epigenetic sensitivity to early life experience in the rat and human hippocampus. Proc Natl Acad Sci U S A. 2012;109 Suppl 2:17266–72.PubMed CentralView ArticlePubMedGoogle Scholar
  70. Perroud N, Paoloni-Giacobino A, Prada P, Olie E, Salzmann A, Nicastro R, et al. Increased methylation of glucocorticoid receptor gene (NR3C1) in adults with a history of childhood maltreatment: a link with the severity and type of trauma. Transl Psychiatry. 2011;1:e59.PubMed CentralView ArticlePubMedGoogle Scholar
  71. Tyrka AR, Price LH, Marsit C, Walters OC, Carpenter LL. Childhood adversity and epigenetic modulation of the leukocyte glucocorticoid receptor: preliminary findings in healthy adults. PLoS One. 2012;7(1):e30148.PubMed CentralView ArticlePubMedGoogle Scholar
  72. Murgatroyd C, Quinn J, Sharp H, Pickles A, Hill J. Effects of prenatal and postnatal depression, and maternal stroking, at the glucocorticoid receptor gene. Transl Psychiatry. 2015. In press.Google Scholar
  73. Philibert RA, Sandhu H, Hollenbeck N, Gunter T, Adams W, Madan A. The relationship of 5HTT (SLC6A4) methylation and genotype on mRNA expression and liability to major depression and alcohol dependence in subjects from the Iowa Adoption Studies. Am J Med Genet B Neuropsychiatr Genet. 2008;147B(5):543–9.View ArticlePubMedGoogle Scholar
  74. Beach SR, Brody GH, Todorov AA, Gunter TD, Philibert RA. Methylation at SLC6A4 is linked to family history of child abuse: an examination of the Iowa adoptee sample. Am J Med Genet B Neuropsychiatr Genet. 2010;153B(2):710–3.PubMed CentralPubMedGoogle Scholar
  75. Beach SR, Brody GH, Todorov AA, Gunter TD, Philibert RA. Methylation at 5HTT mediates the impact of child sex abuse on women’s antisocial behavior: an examination of the Iowa adoptee sample. Psychosom Med. 2011;73(1):83–7.PubMed CentralView ArticlePubMedGoogle Scholar
  76. Kang HJ, Kim JM, Stewart R, Kim SY, Bae KY, Kim SW, et al. Association of SLC6A4 methylation with early adversity, characteristics and outcomes in depression. Prog Neuropsychopharmacol Biol Psychiatry. 2013;44:23–8.View ArticlePubMedGoogle Scholar
  77. Naumova OY, Lee M, Koposov R, Szyf M, Dozier M, Grigorenko EL. Differential patterns of whole-genome DNA methylation in institutionalized children and children raised by their biological parents. Dev Psychopathol. 2012;24(1):143–55.PubMed CentralView ArticlePubMedGoogle Scholar
  78. Labonte B, Yerko V, Gross J, Mechawar N, Meaney MJ, Szyf M, et al. Differential glucocorticoid receptor exon 1(B), 1(C), and 1(H) expression and methylation in suicide completers with a history of childhood abuse. Biol Psychiatry. 2012;72(1):41–8.View ArticlePubMedGoogle Scholar
  79. Borghol N, Suderman M, McArdle W, Racine A, Hallett M, Pembrey M, et al. Associations with early-life socio-economic position in adult DNA methylation. Int J Epidemiol. 2012;41(1):62–74.PubMed CentralView ArticlePubMedGoogle Scholar
  80. Mehta D, Klengel T, Conneely KN, Smith AK, Altmann A, Pace TW, et al. Childhood maltreatment is associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder. Proc Natl Acad Sci U S A. 2013;110(20):8302–7.PubMed CentralView ArticlePubMedGoogle Scholar
  81. Smith AK, Conneely KN, Kilaru V, Mercer KB, Weiss TE, Bradley B, et al. Differential immune system DNA methylation and cytokine regulation in post-traumatic stress disorder. Am J Med Genet B Neuropsychiatr Genet. 2011;156B(6):700–8.PubMed CentralView ArticlePubMedGoogle Scholar
  82. Champagne FA, Meaney MJ. Transgenerational effects of social environment on variations in maternal care and behavioral response to novelty. Behav Neurosci. 2007;121(6):1353–63.View ArticlePubMedGoogle Scholar
  83. Levine A, Worrell TR, Zimnisky R, Schmauss C. Early life stress triggers sustained changes in histone deacetylase expression and histone H4 modifications that alter responsiveness to adolescent antidepressant treatment. Neurobiol Dis. 2012;45(1):488–98.PubMed CentralView ArticlePubMedGoogle Scholar
  84. Massart R, Suderman M, Provencal N, Yi C, Bennett AJ, Suomi S, et al. Hydroxymethylation and DNA methylation profiles in the prefrontal cortex of the non-human primate rhesus macaque and the impact of maternal deprivation on hydroxymethylation. Neuroscience. 2014;268:139–48.View ArticlePubMedGoogle Scholar
  85. Maciag D, Simpson KL, Coppinger D, Lu Y, Wang Y, Lin RCS, et al. Neonatal antidepressant exposure has lasting effects on behavior and serotonin circuitry. Neuropsychopharmacology. 2006;31(1):47–57.PubMed CentralPubMedGoogle Scholar
  86. Ikegame T, Bundo M, Murata Y, Kasai K, Kato T, Iwamoto K. DNA methylation of the BDNF gene and its relevance to psychiatric disorders. J Hum Genet. 2013;58(7):434–8.View ArticlePubMedGoogle Scholar
  87. Kundakovic M, Gudsnuk K, Herbstman JB, Tang D, Perera FP, Champagne FA. DNA methylation of BDNF as a biomarker of early-life adversity. Proc Natl Acad Sci U S A. 2014;112(22):6807–13. doi:10.1073/pnas.1408355111.PubMed CentralView ArticlePubMedGoogle Scholar
  88. Martinowich K, Lu B. Interaction between BDNF and serotonin: role in mood disorders. Neuropsychopharmacology. 2008;33(1):73–83.View ArticlePubMedGoogle Scholar
  89. Madhav TR, Pei Q, Zetterstrom TS. Serotonergic cells of the rat raphe nuclei express mRNA of tyrosine kinase B (trkB), the high-affinity receptor for brain derived neurotrophic factor (BDNF). Brain Res Mol Brain Res. 2001;93(1):56–63.View ArticlePubMedGoogle Scholar
  90. Solum DT, Handa RJ. Estrogen regulates the development of brain-derived neurotrophic factor mRNA and protein in the rat hippocampus. J Neurosci. 2002;22(7):2650–9.PubMedGoogle Scholar
  91. Provencal N, Suderman MJ, Guillemin C, Massart R, Ruggiero A, Wang D, et al. The signature of maternal rearing in the methylome in rhesus macaque prefrontal cortex and T cells. J Neurosci. 2012;32(44):15626–42.PubMed CentralView ArticlePubMedGoogle Scholar
  92. Khulan B, Manning JR, Dunbar DR, Seckl JR, Raikkonen K, Eriksson JG, et al. Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state. Transl Psychiatry. 2014;4:e448.PubMed CentralView ArticlePubMedGoogle Scholar
  93. Labonte B, Suderman M, Maussion G, Navaro L, Yerko V, Mahar I, et al. Genome-wide epigenetic regulation by early-life trauma. Arch Gen Psychiatry. 2012;69(7):722–31.View ArticlePubMedGoogle Scholar
  94. Levine S. Infantile experience and resistance to physiological stress. Science. 1957;126(3270):405.View ArticlePubMedGoogle Scholar
  95. Laban O, Markovic B, Dimitruevic M, Jankovic B. Maternal deprivation and early weaning modulate experimental allergic encephalomyelitis in the rat. Brain Behav Immun. 1995;9(1):9–19.View ArticlePubMedGoogle Scholar
  96. Plotsky P, Meaney M. Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Brain Res Mol Brain Res. 1993;18(3):195–200.View ArticlePubMedGoogle Scholar
  97. Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, et al. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science. 1997;277(5332):1659.View ArticlePubMedGoogle Scholar
  98. McGowan PO, Sasaki A, Huang TC, Unterberger A, Suderman M, Ernst C, et al. Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain. PLoS One. 2008;3(5):e2085.PubMed CentralView ArticlePubMedGoogle Scholar

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© Jawahar et al. 2015