A systematic review of childhood maltreatment and dna methylation: candidate gene and epigenome-wide approaches

ABSTRACT Childhood maltreatment is a major risk factor for chronic and severe mental and physical health problems across the lifespan. Increasing evidence supports the hypothesis that

maltreatment is associated with epigenetic changes that may subsequently serve as mechanisms of disease. The current review uses a systematic approach to identify and summarize the

literature related to childhood maltreatment and alterations in DNA methylation in humans. A total of 100 empirical articles were identified in our systematic review of research published

prior to or during March 2020, including studies that focused on candidate genes and studies that leveraged epigenome-wide data in both children and adults. Themes arising from the

literature, including consistent and inconsistent patterns of results, are presented. Several directions for future research, including important methodological considerations for future

study design, are discussed. Taken together, the literature on childhood maltreatment and DNA methylation underscores the complexity of transactions between the environment and biology

across development. SIMILAR CONTENT BEING VIEWED BY OTHERS DNA METHYLATION SIGNATURES OF CHILDHOOD TRAUMA PREDICT PSYCHIATRIC DISORDERS AND OTHER ADVERSE OUTCOMES 17 YEARS AFTER EXPOSURE

Article 11 May 2022 DNA METHYLATION MEDIATES THE LINK BETWEEN ADVERSITY AND DEPRESSIVE SYMPTOMS Article 02 December 2024 COMBINED EFFECTS OF GENOTYPE AND CHILDHOOD ADVERSITY SHAPE

VARIABILITY OF DNA METHYLATION ACROSS AGE Article Open access 01 February 2021 INTRODUCTION Childhood maltreatment is a highly prevalent public health problem that often has devastating

effects on physical and mental health. There are now numerous studies linking childhood maltreatment and other early adversities to nearly all forms of mental illness, as well as chronic

medical conditions that cut across multiple organ systems1,2,3,4,5,6,7. Unfortunately, the negative sequelae of childhood maltreatment often begin early in life, and persist across

adulthood1,6, posing risk for premature mortality. Indeed, adults with numerous adverse experiences in childhood die nearly 20 years earlier than those with no early adversity1.

Understanding the mechanisms of biological influence is therefore critical to developing innovative targets for intervention to enhance health outcomes among highly vulnerable children with

this major adverse exposure. Over the past decade a rapidly growing body of literature has underscored the significant role of epigenetics in the sequelae of childhood maltreatment.

Epigenetic processes allow the body to respond to environmental influences by altering gene expression through chemical modifications that regulate chromatin structure and/or DNA

accessibility without inducing changes to the DNA sequence8. DNA methylation is among the most commonly studied epigenetic processes and involves the addition of a methyl group at sites in

the DNA where a cytosine nucleotide occurs next to a guanine nucleotide (CpG dinucleotides). Initial work focused on understanding associations of early life stress and DNA methylation

examined these processes in rodents and found that low levels of maternal care (licking and grooming and arched-back nursing) was associated with greater methylation of the glucocorticoid

receptor (GR) gene in offspring9,10. Furthermore, this work suggested that methylation was a mechanism of the effect of maternal care on the offspring HPA stress response. Emerging from this

groundbreaking work with animal models, some of the earliest studies in humans documenting altered DNA methylation in association with childhood maltreatment focused on the glucocorticoid

receptor (GR) gene, _NR3C1_, which modifies responsiveness of the HPA axis to stress exposure. Our group was the first to demonstrate altered leukocyte DNA methylation of _NR3C1_ in adults

with childhood maltreatment11, associations of _NR3C1_ methylation with behavior problems and symptoms in children with early adversity12, and maltreatment as a predictor of change in

_NR3C1_ methylation over time13. In 2016, we completed a literature review focused on altered methylation of glucocorticoid signaling genes in association with childhood maltreatment14.

Several other recent reviews have also focused on methylation of glucocorticoid signaling genes15,16,17, as well as methylation of genes associated with the serotonin system18,19 in relation

to adverse exposures. We expand upon these prior reviews to consider the recent explosion of research in this area that has examined additional genes as well as epigenome-wide effects.

Cecil and colleagues recently conducted a systematic review of research focused on childhood maltreatment, specifically experiences of abuse and neglect, and DNA methylation20. The current

review provides an important addition to the literature by utilizing a broader conceptualization of childhood maltreatment that includes any experience that involved potential for harm to

the child. Our systematic approach to identify and summarize the literature related to childhood maltreatment and other adversities in association with DNA methylation captures research

through March, 2020 and includes research focused on candidate genes and studies that leveraged epigenome-wide data in both children and adults. METHODS In accordance with the Preferred

Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines21, we conducted a systematic review of human studies investigating the relationship between childhood

maltreatment and other adversities and DNA methylation. For the purposes of this review, childhood was defined as events occurring between birth and age 18. Maltreatment was defined as any

experience that involved potential for harm to the child, including emotional, sexual, and physical abuse, and emotional and physical neglect. Other adversities included exposure to intimate

partner violence or other violence, early parental death or separation, institutional deprivation, and indentured labor. Empirical articles investigating DNA methylation in association with

other forms of childhood stress such as low socioeconomic status or famine, without consideration of other adversities, were not included. Studies focusing on experimentally induced

stressors were also not included. In addition, because our focus was on childhood maltreatment after birth; we also did not include studies focusing on prenatal stress or substance use and

DNA methylation, which have been addressed in other reviews22,23. Studies were identified by searching PubMed/Medline for empirical articles published in March 2020 or earlier using the

following search terms: (((methylation OR epigenetic) AND (child AND (maltreat OR adversity OR trauma OR early life stress OR abuse OR neglect OR ACE))), filtered by Humans. Additional

searches in PubMed/Medline for studies published during this timeframe were also conducted using the following search terms: (1) emotion regulation AND methylation; (2) abuse AND methylation

NOT substance NOT alcohol; (3) trauma AND methylation; (4) early life stress AND methylation; (5) maltreatment AND methylation; (6) adversity AND methylation. Additional articles published

during this timeframe identified through other sources (Google Scholar alerts) were also included. Our qualitative synthesis included empirical articles that: (1) examined the relationship

between childhood maltreatment/adversity and DNA methylation; (2) provided statistical indicators to examine the impact of maltreatment during childhood on DNA methylation; and (3) described

maltreatment/adversity that occurred prior to age 18. Empirical articles were excluded if they: (1) did not provide information on childhood maltreatment/adversity and DNA methylation; (2)

childhood maltreatment/adversity was not clearly distinguished from adult adversities in study measurement/hypothesis testing; (3) only quality (or quantity) of parental care or support was

included as a predictor; (4) were not written in English; (5) were only conducted with animals; and (6) did not use a quantitative approach to summarize research findings (i.e., were

qualitative, reviews, comments, or other editorials). We did not omit articles based on small sample size; this information is included in the tables so that it can be considered when

evaluating results. Figure 1 documents the methods of our systematic review to generate the final empirical articles included in our qualitative synthesis. Of note, consistent with PRISMA

guidelines, records screened includes screening of both titles and abstracts. RESULTS A total of 100 empirical articles were identified in our systematic review focused on the relationship

between childhood maltreatment and DNA methylation. This includes 69 empirical articles focused on candidate genes and 31 empirical articles that leveraged epigenome-wide data. Twenty-eight

empirical articles measured DNA methylation in childhood and 72 measured DNA methylation in adulthood. Findings are summarized in Tables 1–4, and examples are described below. Major results

of each empirical article are included in the tables, though results of specific CpG sites are not detailed given significant design and analytic variability across studies. Given the focus

of the current review, only the major study findings related to the association of childhood maltreatment and other adversities with DNA methylation are included in the tables. Empirical

articles that focused on another condition (e.g., parental substance use, psychiatric conditions, as described in Sample description in the tables) but included a result pertaining to

maltreatment are presented. Importantly, several empirical articles drew upon overlapping samples. We included information regarding the name of the study in the tables if the study name was

provided in the empirical article. However, we were unable to denote all articles that may have utilized overlapping samples as the relevant information was not consistently provided in the

literature. CHILDHOOD MALTREATMENT AND METHYLATION OF CANDIDATE GENES CHILDREN As displayed in Table 1, 16 empirical articles focused on childhood maltreatment and methylation of candidate

genes in children. Most of these studies involved saliva DNA, but one used buccal cell DNA and several examined DNA from blood. The most commonly studied candidate genes were those that

regulate glucocorticoid signaling, including _NR3C1_ which encodes the glucocorticoid receptor (GR) (six studies represented in eight empirical articles) and _FKBP5_ which modulates

sensitivity of the GR24 (two studies represented in three empirical articles). Most studies of children support the hypothesis that childhood adversity is associated with higher levels of

methylation of _NR3C1_. In our own study of preschoolers, we found that childhood maltreatment status and the number of stressful life events was associated with greater _NR3C1_

methylation25, and _NR3C1_ methylation mediated effects of maltreatment and stressful life events on child internalizing behavior problems12. Likewise, in 534 school-aged children with low

socioeconomic status, children who experienced maltreatment beginning in infancy or early childhood had greater _NR3C1_ methylation than children who had no maltreatment history26.

Interestingly, there were no differences between children who experienced maltreatment that started in the preschool period or later, and those children with no maltreatment history.

However, more chronic maltreatment and more types of maltreatment, were associated with greater _NR3C1_ methylation. Using data from the Bucharest Early Intervention Project, time in

institutional care was associated with lower levels of methylation of _FKBP5_, which modulates sensitivity of the GR, when children were 12 years old27. Lower levels of _FKBP5_ methylation

in association with childhood maltreatment were also observed in our sample of preschoolers28. Another study of Tanzanian children found evidence that maltreatment was associated with

hypermethylation of _CRH_, the gene that encodes the hypothalamic corticotropin releasing hormone (CRH) and its downstream target, _POMC_, which encodes the pituitary precursor of ACTH29.

Taken together, these findings indicate that childhood maltreatment may be associated with altered methylation of genes that regulate the child stress response, suggesting a possible

mechanism for the effect of maltreatment and other adversities on poor health outcomes. Epigenetic alterations with childhood maltreatment have been documented in additional candidate genes.

Methylation of serotonin signaling genes, including _SLC6A4_, which encodes the serotonin receptor, and _HTR2A_, the gene that encodes the serotonin receptor subtype, 5-HT2A, have been

investigated in association with childhood maltreatment and other interpersonal adversities27,30,31,32. Childhood adversity was associated with greater _SLC6A4_ methylation in two

studies31,32, but lower levels of methylation in association with institutional care27. Using a subsample (_n_ = 785) of the Avon Longitudinal Study of Parents and Children (ALSPAC), Barker

et al.33 generated an inflammation-related epigenetic polygenic risk score (i-ePGS). Adversity between birth and 7 years of age was related to higher i-ePGS, which, in turn, was an indirect

pathway by which adversity was associated with internalizing behavior problems in later childhood. The receptor gene for the pituitary hormone oxytocin (_OXTR_), best known for its role in

social behavior and attachment, has also been studied in relation to early adversity in children. Fujisawa et al.34 demonstrated greater methylation of _OXTR_ in Japanese children and

adolescents with a maltreatment history (_n_ = 44) compared with controls (_n_ = 41), and found that _OXTR_ methylation was negatively associated with gray matter volume in the left

orbitofrontal cortex. ADULTS As displayed in Table 2, 53 empirical articles focused on childhood maltreatment and methylation of candidate genes in adults. Most studies involved analysis of

DNA from blood, with a handful of studies examining DNA from saliva or buccal cells, and a few studies of DNA from brain tissue. The most commonly studied candidate genes included _NR3C1_

(20 empirical articles), _FKBP5_ (eight empirical articles), _SLC6A4_ (six empirical articles), and _OXTR_ (six empirical articles). Taken together, the majority of studies focused on

_NR3C1_ methylation observed significant associations of childhood maltreatment and methylation of this gene, but there is considerable variability in the findings. Several studies found

increased _NR3C1_ methylation with childhood maltreatment, but others found no effect, or reduced methylation with maltreatment. Melas and colleagues35 found that childhood adversity was

associated with saliva DNA _NR3C1_ hypermethylation, with childhood parental loss linked to higher methylation near a transcription factor binding site. Two reports analyzed DNA from the

Quebec Suicide Brain Bank in relation to childhood maltreatment retrospectively assessed via psychological autopsy, including a group with suicide and childhood maltreatment, a group with

suicide but no maltreatment, and a control group. The suicide with maltreatment group had greater hippocampal _NR3C1_ methylation and reduced _NR3C1_ expression across exon 1F36, reduced

_NR3C1_ gene expression across exons 1B, 1C, and 1H, and reduced _NR3C1_ methylation at exon 1H37, in comparison with the other two groups. In contrast, both suicide groups differed from

controls in regions of exon 1B, with hypermethylation at two CpG sites and hypomethylation at one CpG site37. In a diverse sample of adults, childhood maltreatment was associated with

greater methylation of leukocyte _NR3C1_, and maltreatment was associated with lower _NR3C1_ gene expression38. Likewise, in two samples with psychiatric disorders, childhood maltreatment

was associated with greater _NR3C1_ methylation39,40. In contrast, in a recent study, we found that early adversity, and the number of adverse exposures, was associated with lower _NR3C1_

methylation in healthy adults41. Other studies found no association of maltreatment and _NR3C1_ methylation42,43,44,45,46, but one of these found that _NR3C1_ methylation moderated effects

of maltreatment on cortisol response to the Trier Social Stress Test. The literature focused on _FKPB5_ methylation in adults also demonstrates inconsistent effects. Klengel et al.47 found

that childhood maltreatment was associated with lower methylation of _FKBP5_ in adults with the rs1360780 T risk allele, and this effect was observed in both a subsample of participants from

the Grady Trauma Project and a replication sample. This effect was more recently replicated by Tozzi and colleagues48 who also reported that childhood maltreatment was associated with lower

_FKBP5_ methylation among those with the T risk allele of the rs1360780 SNP. In contrast, Harms et al.49 found significant _positive_ associations between stress in childhood and

methylation of _FKPB5_ in young adulthood using a prospective longitudinal design, and a number of studies did not find significant associations among childhood maltreatment and _FKBP5_

methylation50,51,52,53, or moderation by FKBP5 risk allele50,51. Turning to the serotonin system, several studies have examined whether childhood maltreatment is associated with methylation

of _SLC6A4_, the gene for the serotonin transporter. Most studies have found a positive association of childhood maltreatment with methylation of regions of this gene54,55,56,57,58 although

some studies show equivocal or trend-level effects59,60 or no effect61 of maltreatment on _SLC6A4_ methylation. Based on the hypothesis that oxytocin may play a protective role in the

biological response to stress and trauma62, five studies investigated methylation of the oxytocin-receptor-gene (_OXTR)_ in adulthood; all five reported no overall association of childhood

maltreatment with _OXTR_ methylation. A number of these studies did report indirect or moderation effects. In a sample of 309 African American men, childhood adversity had a significant

indirect effect on _OXTR_ methylation through socioeconomic instability38. No significant direct effect of childhood adversity on _OXTR_ methylation was observed. Likewise, while Smearman

and colleagues63 did not observe simple associations of child abuse history and _OXTR_ methylation when accounting for multiple comparisons in their sample of 393 African American adults,

_OXTR_ methylation moderated the association of childhood abuse and psychiatric symptoms. Several other candidate genes have been examined in individual studies. For example, childhood

trauma was associated with lower methylation of the proinflammatory _IL-6_ promoter, and lower methylation in turn was associated with greater salivary IL-6 in response to the Trier Social

Stress Test64. Although this study drew upon a relatively small sample, the findings are consistent with other work demonstrating effects of childhood trauma and maltreatment on inflammatory

processes. CHILDHOOD MALTREATMENT AND EPIGENOME-WIDE ASSOCIATION STUDIES Arising in part out of replication inconsistencies as well as interest in identifying novel variation, the past

decade has included an increase in the number of Genome Wide Association Studies (GWAS), which are agnostic by design and interrogate genetic variation across millions of common genetic

variants the entire genome. However, a limitation of GWAS is the extremely large samples needed to detect effects after adjusting for multiple testing. Similar to GWAS approaches,

epigenome-wide association studies interrogate markers across the entire genome. Consideration of the biology of DNA methylation as well as optimization of power has led to the development

of new frameworks for analyzing multiple markers, including examination of CpG sites that are in close proximity to one another (differentially methylated regions, DMRs), examination of DNA

methylation in biological pathways known to be involved in the condition, and application of analytic tools that use a ranking approach rather than relying on p values. Researchers have also

developed quality control standards and approaches for addressing both concerns with Type I error as well as population stratification that may arise in diverse samples65. Given that

biological processes function as part of a larger interrelated system, research has increasingly focused on how either systems or patterns of alterations in methylation may be important in

efforts to understand epigenetic modifications. Studies have begun to explore DNA methylation as a measure of molecular aging, with evidence that this epigenetic “clock” is associated with

age-related disorders and mortality66,67,68,69,70 as well as with age-related development such as menopause71,72. Several analytical methods have been developed to measure epigenetic age as

indicated by epigenome-wide methylation profiles73,74,75,76,77. This emerging area of research has the potential to provide exciting evidence for understanding the impact of early adversity

on molecular age. CHILDREN As displayed in Table 3, 12 empirical articles focused on epigenome-wide effects of maltreatment and other adversities in children. These studies suggest that

maltreatment is associated with variation in methylation across the genome, and several differentially methylated regions and genes have been identified. In a racially and ethnically diverse

sample of 548 children, Cicchetti et al.78 found differential whole-genome methylation in children with a maltreatment history relative to children with no maltreatment. Maltreated children

had higher levels of methylation at sites where methylation was generally low, and lower methylation at sites where methylation was generally high, compared to non-maltreated children.

Using data from a subsample (_n_ = 774) of the ALSPAC, Dunn et al.79 examined associations among childhood adversity and epigenome-wide methylation in children at 7 years of age.

Thirty-eight differentially methylated CpG sites were identified in association with early adversity, and the developmental timing of adversity was the most salient predictor of methylation.

Neither the simple presen-ce/absence of adversity, recency of adversity, nor accumulation of adverse experiences was associated with altered methylation. Studies of early adversity and

epigenetic age are just emerging in samples of children. Very recently in a sample of 247 children who were 8–16 years of age, threat-related early adversity (including abuse and violence

exposure), but not deprivation (including neglect), was associated with accelerated DNA methylation age80. Furthermore, threat-related early adversity exerted a significant indirect effect

on depressive symptoms through accelerated DNA methylation age, suggesting that this measure of early adversity is clinically relevant to psychiatric outcomes. This is consistent with

Jovanovic et al.81 that demonstrated that violence exposure was associated with greater DNA methylation age acceleration in African American children who were 6–13 years of age. ADULTS As

displayed in Table 4, 19 empirical articles focused on epigenome-wide effects of childhood maltreatment in adults. Collectively, these studies suggest that childhood maltreatment exerts

epigenetic effects across the genome, yet these studies differed in their overall focus and methodology. In the ALSPAC and the MRC National Survey of Health and Development, Houtepen et

al.82 identified nine differentially methylated regions (DMRs) in the genome that replicated across the two cohorts and were associated with childhood adversity. No individual CpG sites in

their epigenome-wide analysis replicated across the cohorts. O’Donnell et al.83 used a different methodological approach to describe variation in DNA methylation, and found that childhood

maltreatment was associated with variation in methylation at 27 years of age utilizing principal components scores to describe variation in methylation. Lutz et al.84 examined genome-wide

DNA methylation and gene expression in postmortem brain samples of adults with a history of depression who died by suicide. This study found that child abuse history was associated with

differential methylation specifically in oligodendrocytes in the cingulate cortex, as well as expression of myelin-related genes. Investigations using epigenome-wide data to explore effects

of early adversity on accelerated epigenetic aging have shown mixed outcomes. Lawn et al.85 used the ALSPAC and the MRC National Survey of Health and Development to examine associations of

childhood psychosocial adversity, including abuse and neglect, and DNA methylation age acceleration. Childhood sexual abuse was associated with methylation age acceleration in ALSPAC, and

this effect remained significant when controlling for socioeconomic position. Data regarding sexual abuse was not available in the MRC survey, however Tamman et al.86 also found that

childhood sexual abuse was associated with increased DNA methylation age. Neither individual adversity types nor a cumulative measure of adversity were associated with methylation age

acceleration in ALSPAC and the MRC National Survey of Health and Development85. Han et al.87 also reported a positive association between a history of childhood trauma and epigenetic aging

in adults with MDD. DNA methylation age was also examined in the sample of 27 year olds described above, with analyses revealing no differences in epigenetic age as a function of childhood

adversity83. Although there have been more studies focused on childhood adversity and accelerated epigenetic age in adults, few studies overall have been completed. DISCUSSION This

systematic review examined associations of childhood maltreatment and DNA methylation in children and adults. One hundred empirical articles focused on humans were identified. These studies

included both candidate gene and epigenome-wide approaches. Strengths of the literature included: (1) rigorous approaches to measure childhood maltreatment in studies focused on DNA

methylation in children, including record review methods; (2) several racially and ethnically diverse samples in the child studies; (3) diverse and innovative approaches to measuring

epigenome-wide effects of maltreatment, including exploration of how early adversity may lead to epigenetic age acceleration; and (4) several replication studies focused on childhood

maltreatment and methylation of glucocorticoid signaling genes in children and adults. Collectively, these studies provide evidence that childhood maltreatment and other adversities are

associated with DNA methylation. Genes and pathways observed to have altered methylation in relation to childhood maltreatment, and the expected epigenetic pathways from maltreatment to

health and mental health outcomes, are displayed in Fig. 2. Studies of childhood adversity and DNA methylation often focused on methylation of candidate genes that regulate glucocorticoid

signaling including _NR3C1_ and _FKBP5_. Most studies in children demonstrated increased _NR3C1_ methylation with maltreatment. In adults, several studies documented greater _NR3C1_

methylation in those exposed to childhood maltreatment, whereas others demonstrated no associations of maltreatment and _NR3C1_ methylation. Furthermore, in our own recent work we found

lower _NR3C1_ methylation in association with childhood adversity41, and other studies have identified CpG sites in _NR3C1_ that are hypomethylated in association with maltreatment or

suicide37 and post-traumatic stress disorder (PTSD)88,89. For _FKBP5_, Klengel and colleagues first published findings of an interaction of childhood maltreatment and _FKBP5_ genotype such

that maltreatment was associated with lower _FKBP5_ methylation in adults with the T risk allele47. Lower _FKBP5_ methylation with maltreatment—although not interacting with genotype—has

been seen in the two studies with published data in children. The maltreatment by genotype interaction predicting lower _FKBP5_ methylation in adults was recently replicated48, but other

studies have not observed an effect of maltreatment, or an interaction of maltreatment and genotype, and one study of young adults found that stressful life events in childhood were

associated with greater _FKBP5_ methylation, which in turn mediated effects of early stress on prefrontal brain activity49. Many studies in adults utilized samples with chronic disease and

mental health diagnoses, and did not exclude participants with consistent medication use, which may have influenced the findings. Overall levels of methylation of the _NR3C1_ gene promoter

are generally very low, and this may also limit the ability to reliably detect group differences. However, there is evidence that _NR3C1_ methylation at exons 1F, 1B, and 1C, is inversely

associated with GR gene expression36,37. Since GR mediates negative feedback to the HPA axis, higher methylation—and lower GR gene expression—is expected in conditions with elevated cortisol

and there is some evidence for this90,91. But variable findings for methylation of _NR3C1_ and _FKBP5_ are perhaps not surprising given that maltreatment, trauma, and stress-related

psychiatric disorders have been linked to both exaggerated and diminished basal and provoked cortisol concentrations, which may in part depend on measured or unmeasured individual and trauma

characteristics92,93. Moreover, many genes and gene products regulate HPA axis function, and complex interactions among participant characteristics (including age and genetic ancestry),

genotype (with allele frequencies that may differ based on genetic ancestry), and stress exposure may best characterize these effects. Interaction effects may also characterize methylation

of oxytocin system genes, given findings of indirect or moderation effects in the absence of simple effects63,94. Although the potential risk for statistical artifacts provide important

cautions to investigations exploring interaction effects, interactions and conditional effects are known to occur in biological systems and may be appropriate to examine in large samples

and/or using stratification or model invariance strategies. Methylation of serotonin signaling genes was also examined in multiple studies. Most studies of adults and some work in children

found elevated methylation of _SLC6A4_ with maltreatment, but there are also reports of lower _SLC6A4_ methylation at some CpG sites with adversity, or no difference between adversity

groups. Differences in epigenetic findings in _SLC6A4_ as well as other candidate genes may also be related to inconsistencies in the ways that the number of differentially methylated

regions are assessed. For example, _SLC6A4_ has 81 CpG sites with studies adopting a range of approaches to assessing this large region, including selection of target sites based on the

literature, multiple testing of sites, and strategies for binning methylation at multiple sites95. Given evidence for ancestry-related differences in methylation, differences across studies

related to ancestry may also potentially contribute to apparent inconsistencies96. Studies using agnostic approaches to exploring multiple markers across the epigenome also suggested that

maltreatment is associated with variation in methylation, though these epigenome-wide studies have generally failed to identify commonly studied candidate genes, leading to questions about

candidate approaches. Several reasons for these inconsistencies have been advanced, including large effect sizes needed to detect an effect after multiple testing corrections, biological

considerations such as the potential combinatory impact of genotype and DNA methylation (see ref. 83 for an example of integration of genotype and epigenetic influences), interactions among

systems of genes, and differences in aspects of the phenotyping and heterogeneity of different studies. Due to traditional approaches to corrections for multiple testing, studies that

analyze methylation of multiple markers across the genome tend to require large samples which are often heterogeneous and may have less intensive phenotyping and measurement of maltreatment

and other relevant exposures. Promising markers identified with epigenome-wide approaches show some consistency in terms of their roles in neural cell development (_BDNF, KITLG_, and

_POU3F1)_, signaling and apoptosis (_LINGO3_ and _2NPFF2)_, neural influences on movement (_ALS2_), neuroinflammation (_ITGB1)_, and some tentative evidence for immune markers (_CXCL1)_. A

recent area of epigenome-wide research involves DNA methylation as an indicator of molecular age. A number of analytic strategies have been used to examine how early life trauma (and/or

symptoms associated with trauma such as depression or post-traumatic stress disorder) may accelerate aging. A number of adult and child studies find age acceleration among individuals

exposed to maltreatment (meaning that DNA methylation age is older than the chronological age). Childhood maltreatment and other adverse exposures alter epigenome-wide profiles, thereby

likely contributing to chronic disease and physical aging97,98,99,100,101. Interestingly, the particular markers observed to be relevant in methylation age acceleration research may point

toward biological systems implicated in the link between maltreatment and disease. For example, the method described by Horvath includes CpG sites enriched for glucocorticoid response

elements99. New methods for capturing DNA methylation age continue to be developed, and very recently Belsky and colleagues102 reported a new algorithm that was derived from longitudinal

data of 18 biomarkers of organ-system integrity to capture the rate of aging up to the time of measurement. This new algorithm, but not other previously established measures of DNA

methylation age, was associated with childhood maltreatment in sample of 1658 young adults who were longitudinally followed since childhood. As new methods to capture age acceleration

continue to develop, systematic reviews and meta-analytic approaches will continue to be important to synthesize associations of child maltreatment and the range of measures of methylation

age. Studies of maltreatment and methylation in children were often characterized by careful measurement of adverse exposures using structured record review techniques and in-depth interview

methods, and they often utilized prospective and longitudinal designs. In children, DNA methylation was most frequently measured in saliva or buccal epithelial cells, with fewer studies

measuring methylation in blood. Many of the samples were racially and ethnically diverse, and these studies covered the full developmental spectrum, from early childhood through adolescence.

In contrast, nearly all the adult studies drew upon retrospective reports of childhood maltreatment. Utilization of prospective longitudinal designs and record review techniques would

address a significant gap in the literature. Furthermore, in contrast to studies of maltreatment and methylation of candidate genes in children, most adult studies measured methylation in

leukocyte DNA. Moving beyond simple effects of maltreatment on DNA methylation, several studies highlighted the importance of the developmental timing of exposure to adversity26,27,78,79.

More research is needed to understand how early adversity at each developmental epoch may be associated with differences in epigenetic marks. Moreover, longitudinal research is needed to

explore whether epigenetic changes secondary to early adversity may “reset” during later developmental periods or perhaps may be impacted by later experiences, which either attenuate or

compound early adversity. Retrospective research with adults who report childhood adversity has numerous benefits in terms of cost- and time-effectiveness; however, reliance on retrospective

reports of childhood adversity present important methodological challenges related to recalling the timing of childhood events, ability to recall very early life events, and systematic

recall biases, introducing measurement limitations that are difficult to overcome103. Many studies use the Childhood Trauma Questionnaire104 or other self-report measures. Some self-report

measures, such as the Traumatic Life Events Questionnaire105 and the Maltreatment and Abuse Chronology of Exposure106, include trauma characteristics such as the age or frequency of

occurrence, which may be important determinants of epigenetic and phenotypic outcomes. Future research may also benefit from interview approaches that can help with retrospective recall such

as participant-tailored anchoring. Although many adult studies reviewed here relied on recall, much can be learned from the few studies that capitalized on longitudinal cohorts. For

example, Harms et al.49 assessed stress when children were 9–13 years old and then methylation approximately 10 years later. O’Donnell et al.83 assessed childhood maltreatment in the first

15 years of life and methylation at age 27. Future research should capitalize on existing studies of children that utilized rigorous measures of the environment as they develop over time to

expand the literature in adults. Likewise, very few studies drew upon repeated assessments of DNA methylation over time. In our own research, we demonstrated that childhood maltreatment and

other adversities are associated with change in methylation of glucocorticoid signaling genes over time in early childhood13,107. Future research should further examine maltreatment as a

predictor of change in methylation across development. Importantly, the majority of studies focused on exposure to childhood maltreatment without consideration of the prenatal environment.

Prenatal exposures exert epigenetic effects on several biological systems, particularly the development of the child stress response. For example, intimate partner violence in pregnancy is

associated with increased _NR3C1_ in late childhood and adolescence108. Smoking and depression in pregnancy have also been associated with altered methylation of placental stress-related

genes, such as _NR3C1_ and _HSD11B2_, which encodes the enzyme that inactivates cortisol109,110,111,112. Associations of childhood maltreatment and DNA methylation may be partially accounted

for by prenatal environmental factors. Conversely, maltreatment may exert a unique and independent effect on DNA methylation above and beyond prenatal exposures. Barker et al.33 found that

child adversity between birth and 7 years of age was associated with an inflammation-related epigenetic polygenic risk score (i-ePGS) at age 7, but there was no association of prenatal

adversity and the i-ePGS. Future research should disentangle prenatal exposures and adversities experienced in childhood to better understand effects of maltreatment on DNA methylation in

both childhood and adulthood. Very few studies examined sex differences in the effects of childhood maltreatment on DNA methylation. Studies focused on in utero stress exposure often find

sex differences in epigenetic pathways and observed outcomes in both preclinical and human models113,114,115. For example, Braithwaite et al.116 found that maternal depressive symptoms in

pregnancy were associated with greater methylation of _NR3C1_ in male, but not female, infants. Stroud et al.109 found that the moderating effects of placental _HSD11B2_ methylation on links

between prenatal major depressive disorder and infant cortisol response emerged most strongly for newborn daughters, whereas direct and moderating effects of _SLC6A4_ gene expression were

evident only for sons109. In one of the few studies identified in this review that examined sex differences, Cicchetti et al.78 found that boys and girls showed different directions of the

effect of maltreatment on methylation of _ALDH2_, a gene that encodes a key enzyme in the metabolism of alcohol. Sex differences were also observed in the effect of the developmental timing

of adversity. As we have shown in a meta-analysis with _ADCYAP1R1_, the adenylate cyclase activating receptor gene associated with PTSD, depending on the functional outcomes of the gene(s),

sex and developmental differences are reasonable to expect117. Future work on maltreatment and DNA methylation should consider the role of child sex to ensure that important moderation

effects are not being overlooked. An additional future direction is to move from association to causal inference. Maltreatment is often confounded with additional measures of adversity,

including poverty and other sociodemographic factors, as well as personality factors, and potentially genetic factors. Many studies reviewed utilized statistical control for potential

confounding factors; however, true random assignment designs are not ethical in humans. Thus, synthesizing across preclinical studies (where random assignment is possible) and human

association studies will be critical. Additionally, innovative designs in human studies, including random assignment to interventions to reduce maltreatment, and control groups that are

demographically and psychosocially matched to maltreatment groups, may allow the field to move closer to causal inference. Research considering intervention effects on change in methylation

over time may also address this gap in knowledge. Indeed, we found that service utilization was associated with increases in _FKBP5_ methylation over time in preschoolers with early

adversity107. More recently, Vinkers et al.118 observed changes in methylation among soldiers successfully treated for PTSD such that changes in methylation were observed among soldiers who

had reductions in PTSD symptoms. Taken together, this work provides initial evidence that psychosocial interventions exert influence on the epigenome. DNA methylation has been measured in

several tissue types, including blood, saliva, and buccal cells. Yet, the majority of studies focused on DNA methylation in children utilized saliva and buccal cells, and fewer examined

methylation in blood. Although some researchers have questioned the value of peripheral markers in research aimed at understanding psychobehavioral outcomes believed to be related to central

brain processes, with some researchers suggesting that psychiatric epigenetic research should be limited to brain tissue, a number of studies point to the value of peripheral indicators.

Research has shown reasonable concordance between gene-expression signals in blood and brain119,120,121 and primate research has identified correspondence in DNA methylation profiles in the

brain and blood122. Similar gene expression was found in the cerebellum and peripheral blood mononuclear cells (PBMCs) across 4000 unique genes123. Correspondence between DNA methylation in

PBMCs and postmortem brain tissue was identified with respect to a marker of reward and stress-induced responses124. Recent research showed good correspondence of DNA methylation in blood

and saliva125. Studies of methylation age have also shown consistency across tissue types75. Interestingly, there is some evidence126 that saliva samples may be more strongly associated with

brain methylation than blood samples, although correlations of methylation in the brain with saliva, blood, and buccal cell DNA were all observed to be high and the strength of these

associations appear to depend on the genomic region of interest127. These studies have provided helpful signals regarding the utility of blood, brain, saliva, and buccal cells, and

Epigenomic Roadmap datasets now provide some insights into the ways that peripheral methylation profiles may map on to those in brain tissue128,129. Nonetheless, cell type heterogeneity

remains a significant challenge for epigenetic research that has been addressed using a number of strategies such as cell counts, cell separation and examination of single cells, and

analytic strategies that account for cell type130,131,132,133. Another concern involves the technology and analytic strategies used to interrogate genome methylation. Power to detect true

differences and false positives are both major concerns, so large samples sizes and replication samples are required, but very large studies may not be able to provide rich data on

maltreatment. Researchers have also described the importance of considering reliability of BeadChip technology, the most frequently applied technology for interrogating the genome, as a

function of: (a) sample type, with lower reliability and replicability in dried blood spots, (b) tissue type, with some probes demonstrating greater cross-tissue concordance, (c) platform

(such as Ilumina Infinium HumanMethylation450 BeadChip with nearly 500,000 sites vs Ilumina MethylationEPIC BeadChip with nearly 850,000 sites), and (d) observed variability at each site,

with lower reliability at sites with less variability134,135,136,137. Research is needed to carefully document reliability considerations for proper assessment of reproducibility; interested

readers are directed to a thorough discussion of reliability, replication, and reproducibility in DNA methylation measurement (see ref. 134). In addition to the importance of clear site

level documentation of findings in publications, suggestions for addressing reliability concerns in ongoing research include analytic approaches such as dropping CpG sites with low observed

variability, replicating findings with procedures such as pyrosequencing, and integrating preanalysis reliability metrics134,136,137. Focusing the analyses on regions with more variability

across participants reduces the likelihood of false positives, and also reduces the number of comparisons, thereby increasing power. It is also important to note that methylation arrays

assess only CpG sites; this yields fewer sites than whole-genome bisulfite sequencing approaches, and some research suggests some important variability occurs outside of these CpG

islands137,138. Standards in this field continue to be refined, and researchers should follow these developments closely. At a minimum, researchers should implement strategies to address

quality control (including thorough evaluations of normalization procedures and strategies for addressing batch effects), cell heterogeneity, and sample ancestry65,139,140,141. Our

systematic review examined associations of maltreatment and other interpersonal adversities during childhood with DNA methylation in children and adults. We identified 100 studies, including

studies utilizing both candidate gene approaches and epigenome-wide analyses. Several strengths of the literature were identified, as well as directions for future research. Key challenges

facing the field and associated recommendations are described in Fig. 3. We included both studies focused on childhood maltreatment and other adversities, as well as studies that were not

focused specifically on early adversity but included measurement of adversity in their examination of another topic (e.g., psychiatric or physical health diagnosis). Inclusion of these

studies represents both a strength and limitation due to selection biases inherent to these designs, as well as confounds such as the presence of medical conditions and medications that may

influence methylation. We acknowledge several other limitations to the current review. Specifically, (1) the present review focused on DNA methylation and did not include studies addressing

other epigenetic modifications (e.g., microRNA, histone modifications); (2) the review did not address the functional impact (e.g., gene expression, proteins) of DNA methylation alterations

in various studies; and (3) although systematic, the review was qualitative, and thus, does not provide information regarding effect sizes or heterogeneity of included studies. Meta-analytic

reports of the most commonly studied candidate genes and EWAS results would build upon our qualitative analysis; however, the extensive number of individual CpG sites both within each

candidate gene and across the EWAS studies poses a significant challenge. Furthermore, a critical limitation of the literature is that the location of individual CpG sites is often not

consistently identified in empirical manuscripts, further precluding meta-analytic approaches. Future research should more carefully detail the precise location of CpG sites to facilitate

future meta-analysis efforts. In less than two decades, there has been an explosion of research in childhood maltreatment and epigenetics. Mirroring trends observed in genetic research,

studies have increasingly focused on sampling methylation across the genome. As with genetic research, advantages of this approach include opportunities to identify key mechanisms that would

otherwise have been overlooked. However, this approach generally requires larger samples to account for multiple testing which in turn has been criticized for less nuanced phenotyping of

samples permitted in smaller cohorts. Some of these concerns may be somewhat addressed through collaborations such as efforts undertaken by the Psychiatric Genetics Consortium working

groups, particularly when care is taken to balance genomic inflation/deflation and to address concerns that can arise with diverse samples interrogated on differing platforms65. Researchers

have also begun to explore ways that methodological approaches may improve understanding of epigenetic factors and adversity outcomes such as repeated measurement, sampling across tissues,

twin/family approaches, and psychological/pharmacological treatments. Epigenetic approaches are also perhaps best understood in combination with thorough assessments of genotype, epigenome,

and gene expression. Many factors related to the type and timing of adversity, availability and quality of buffers to adversity, and new events and time since early adversity are likely

critically important influences on negative outcomes of adversity. Ongoing recognition of the complexity of biological and environmental factors as well as phenotypic nuances is critically

needed—apparent inconsistences do not necessarily negate findings from either of the divergent studies but rather may point to important considerations for ongoing research and theoretical

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stress disorder. _Proceedings of the National Academy of Sciences_ 110, 8302–8307 (2013). Download references ACKNOWLEDGEMENTS This work was supported by NIH grants R01 HD095837 (SHP), R01

MH101107 (ART), R01 HD086487 (ART), R01 DA044504 (LRS), R01 DA031188 (LRS), R24 ES028507 (LRS), R01 MH10537 (NRN), and R01 MH108641 (NRN). L.H. received support from T32 MH019927 and F32

HD100020. T.D. received support from R25 MH101076. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of

Health. AUTHOR INFORMATION Author notes * These authors contributed equally: Stephanie H. Parade, Lindsay Huffhines * These authors contributed equally: Nicole R. Nugent, Audrey R. Tyrka

AUTHORS AND AFFILIATIONS * Initiative on Stress, Trauma, and Resilience, Department of Psychiatry and Human Behavior, Warren Alpert Medical School, Brown University, Providence, RI, USA

Stephanie H. Parade, Lindsay Huffhines, Teresa E. Daniels, Laura R. Stroud, Nicole R. Nugent & Audrey R. Tyrka * Bradley/Hasbro Children’s Research Center, E. P. Bradley Hospital, East

Providence, RI, USA Stephanie H. Parade & Lindsay Huffhines * Laboratory for Clinical and Translational Neuroscience, Butler Hospital, Providence, RI, USA Teresa E. Daniels & Audrey

R. Tyrka * Center for Behavioral and Preventive Medicine, The Miriam Hospital, Providence, RI, USA Laura R. Stroud Authors * Stephanie H. Parade View author publications You can also search

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