Abstract

Introduction

Childhood trauma is common and imposes a substantial psychological burden on youth. Approximately two-thirds of youth are exposed to psychological trauma before they reach adulthood, and up to 16% of all youth develop post-traumatic stress disorder (PTSD) by the age of 18 (1, 2). Youth suffering with PTSD often show lower academic achievement than their non-affected counterparts; this decrease in achievement is coupled with increased rates of depression, suicide attempts, and substance abuse into adulthood (3, 4). These statistics highlight the need for additional research into the neurodevelopmental underpinnings of pediatric PTSD, with the goals of improving detection, prognosis, and treatment.

The developmental origins of health and disease hypothesis posits that early life exposures, including exposure to childhood trauma, will have prolonged effects on child health, including neurodevelopmental outcomes. Epigenetic alterations provoked by exposure to childhood trauma are likely to play a central role in the molecular mechanisms underlying the emergence of PTSD in youth. Specifically, trauma-related alterations in DNA methylation (DNAm) at cytosine-guanine junctions (CpGs) may influence the developmental programming of neural circuitry underlying stress responses, including the hippocampus, amygdala, and medial prefrontal cortex (mPFC) (5).

Important strides have been made in identifying epigenetic substrates of adult PTSD using peripheral DNAm markers (6-8). Other studies have also reported associations between DNAm and brain phenotypes in predominantly healthy cohorts (9-12). However, despite these promising results, it remains unclear whether similar DNAm markers are present in a pediatric PTSD, which is of critical importance given ongoing physiological and neurodevelopment in youth. It is also unclear whether these DNAm markers are related to neural abnormalities previously identified in pediatric PTSD, which is important to identify potential molecular/cellular pathways associated with altered neurodevelopment contributing to pediatric PTSD (13). In addition, previous studies examining childhood trauma are mostly based on a priori searches of candidate genes(14). However, as is common with a priori searches, this approach increases the risk of confirmation biases, thereby increasing the risk of false negatives for useful epigenetic biomarkers of developmental psychopathology after adversity.

To address these knowledge gaps, we performed a Methylome-wide association study (MWAS) using a discovery cohort of youth with PTSD, compared with two control groups. The first group (Dutch sample) consisted of trauma-exposed youth without PTSD (a resilient group), and a second group of healthy non traumatized youth. We then attempted to replicate these DNAm findings in an independent (American) cohort of youth with PTSD and a non-traumatized comparison group.

In addition to our MWAS, we investigated whether altered methylation was further related to brain structure in regions involved in emotion regulation. This combined approach has two major advantages. First, the use of independent cohorts for discovery and replication of DNAm findings should reduce the likelihood of false positive findings and enhance generalizability of results. Second, identifying which peripherally-derived methylation differences are linked to structural brain differences in trauma-exposed and PTSD youth may point to genes with the most biological relevance for pediatric trauma and PTSD through their link to central nervous system abnormalities. Given prior work implicating altered methylation in glucocorticoid pathway genes in childhood trauma and adult PTSD, we hypothesized that youth with PTSD would show altered methylation in genes annotated to the HPA axis, which would then be further associated with prefrontal and hippocampal gray matter volume abnormalities previously identified in pediatric PTSD (15-19). We aimed to minimize DNAm findings that were either likely to occur by chance (by requiring replication) and that may not have an impact on neural systems (by requiring association with gray matter volume). Complementing these specific hypotheses, we aimed to identify novel methylation abnormalities and methylation-brain relationships in pediatric PTSD through our MWAS.

METHODS

Clinical and Behavioral Assessments

Assessments in these cohorts have been previously described. Briefly, participants and their caregivers completed structured psychiatric and trauma screening including the CAPS-CA. Additional self- and parent-report of youth symptoms of depression and anxiety were obtained using standardized questionnaires. Please see supplementary methods and materials for additional detail. To facilitate comparisons between study samples, Table 1 provides the rates of children that score above clinical cut-off of internalizing and externalizing symptoms based on these questionnaires.

Table 1:

Demographic information, trauma history and clinical characteristics in the Dutch cohort

	PTSD youth (n=74)	Traumatized Controls (n=75)	Healthy non-traumatized
Sex
Boys	31 (41.9 %)	37 (49.3 %)	36 (48.0 %)
Girls	43 (58.1 %)	38 (50.7 %)	39 (52.0 %)
Age	12.13 (3.44)	11.95 (2.28)	10.77 (2.15)a**
Ethnicity
Caucasian	44 (59.5 %)	61 (81.3 %) c**
Other	30 (40.5 %)	14 (18.7 %)	64 (85.3 %)c**
Index trauma
Interpersonal violence	38 (54.4%)	21 (35.6%)	X
Sexual abuse	11 (14.9%)	0 (0%)	X
Accidents/medical	9 (12.2%)	37 (49.3%)	X
Comorbid diagnosis
Internalizing problems	23 (31.1 %)	2 (2.7 %)c**	0 (0 %)c**
Externalizing problems	18 (24.3 %)	3 (4.0 %)c**	0 (0 %)c**
CAPS-CA Severity Score	52.91 (26.07)	14.45 (12.88)b**	X
CRIES-13 Severity Score	37.44 (15.35)	11.04 (15.07)b**	X

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Continuous variables presented as mean (standard deviation); categorical variables presented as frequency (percentage).

^a= One-way ANOVA;

^b= Kruskal-Wallis Test;

^c= Chi-Square Test.

^**significant group differences (p > .05). PTSD=post-traumatic stress disorder; CAPS-CA = Clinician Administered PTSD Scale for Children and Adolescents; CRIES-13 = Children’s revised Impact of Event Scale, Revised. Comorbid internalizing and externalizing symptoms were based on the Revised Children's Anxiety and Depression Scale (RCADS; T- scores ≥70), and the Child Behavioral Checklist (CBCL) and Youth Self Report (YSR; T score ≥63).

RESULTS

Demographic and mental health measures of participating youth are shown in Table 1 and ​and22 and further described in supplement.

Table 2:

Demographic Information, Trauma History and Clinical Characteristics in the USA cohort

	PTSD Youth (n=22)	Healthy non-traumatized
Sex
Boys	8	7
Girls	14	13
Age	14.22 (2.70)	14.21 (2.80)
Ethnicity
Caucasian	18	17
Other	4	3
IQ	103.2 (13.0)	110.5 (12.5)
Left Handed	0	0
Index Trauma
Interpersonal Violence	3 (13.6%)	-
Sexual Abuse	11 (50%)	-
Severe	3 (13.6%)	-
Accident/medical trauma Other (Traumatic News, Natural Disaster)	5 (22.7%)	-
Comorbid Diagnoses
Depression	19 (86.4%)	-
Anxiety	14 (63.6%)	-
ADHD	6 (27.3%)	-
Previous Psychotropic Medication-Use
Depression	8 (36.4%)	-
Anxiety	3 (13.6%)	-
ADHD	7 (31.8%)	-
CAPS-CA Severity Score	77.11 (16.75)	-
PTSD-RI Severity Score	55.36 (11.14)	-

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Continuous variables presented as mean (standard deviation); categorical variables presented as frequency (percentage). There were no significant group differences (PTSD Youth vs. Healthy Youth; p > 0.05) in sex, ethnicity, or handedness distribution, age, or IQ (based on χ² tests of independence and independent samples t-tests respectively). PTSD = Post-Traumatic Stress Disorder; ADHD = Attention Deficit, Hyperactivity Disorder; CAPS-CA = Clinician-Administered PTSD Scale for Children and Adolescents; PTSD-RI = UCLA PTSD Reaction Index. Comorbid diagnosis were based on the Kiddie Schedule for Affective Disorders and Schizophrenia (KSADS), Mood and Feelings Questionnaire (MFQ), and Screen for Child Anxiety Related Emotional Disorders (SCARED).

DISCUSSION

To our knowledge, this is the first study in which biological pathways of altered gene expression and brain structures implicated in pediatric PTSD have been investigated. Using two independent, international cohorts, the results of this study confirm for the first time the hypothesis that pediatric PTSD is associated with epigenetic modifications which, in turn, are associated with structural neurophenotypes implicated in pediatric PTSD. Most notably, altered DNA methylation on genes annotated to PM20D1, TNXB and OLFM3 genes were replicated across cohorts and exhibited a relationship to neural structure in at least one of the cohorts. Because this is the first genome wide methylation study in youth with PTSD to date, we cannot compare these results with other cohorts (15). It is notable, however, that our results do not overlap with recent findings in adult PTSD (Smith, Ratanatharathorn (6). This is perhaps not surprising, given that the current study focused on a developmental sample, in which trauma load, comorbidities, substance use, and many other factors markedly differ from adult PTSD samples. However, our findings do thematically overlap with studies of childhood trauma and adult PTSD, suggesting abnormal methylation in genes annotated to the inflammation and stress response systems (15).

First, changes observed on OLFM3 gene were related to hippocampal volume in both cohorts. This may represent an interaction between methylation and neuronal change related to early exposure to traumatic events and the development of PTSD at an early stage in life. OLFM3 is a neuronal protein, found throughout the brain and related to the development of microglia (27, 28). Microglia serve as key immune cells of the brain, and are activated in response to signals they receive or detect in their microenvironment. For example, they respond to injuries and infections and are able to modify the structure and function of a cell. They can for example lead to neuronal degeneration, impaired microglia activation has been linked to the development of brain disorders, such as neurodegenerative diseases (29). Epigenetic mechanisms have emerged as important regulators in this process (30). In addition to their essential role in the development of the central nervous system (CNS), microglial dysfunction is suggested to be involved in stress vulnerability and depression recurrence (31). These findings are in line with our results that suggest that hyper methylation of OLFM3 may be a molecular mechanism by which hippocampal volume is decreased in youth with PTSD, potentially via enhanced CNS inflammation in affected youth.

Secondly, methylation changes in the TNXB gene were observed in both cohorts. TNXB encodes an extracellular-matrix associated glycoprotein that has been implicated in cell migration processes (ProteinAtlas), and was shown to be hypermethylated in PTSD youth relative to NTC youth. Furthermore, an additional significant effect was reported comparing PTSD with NTC youth in the Dutch cohort. This suggests that there is an effect specific to pediatric PTSD (and/or its comorbid disorders) rather than trauma-exposure generally. We also observed that in the American cohort across PTSD youth and NTC youth, TNXB methylation is negatively associated with gray matter volume in the anterior hippocampus, originally identified in a group by age interaction in Keding & Herringa (2015). This relationship was not observed in the Dutch cohort, however this could be due to the missing NTC in the MRI group. Interestingly, TNXB has been shown to have protein-protein interactions with the protein products of VEGFA, VEGFB, NEURL1, and NEURL 1B (https://string-db.org/), all four of which have been heavily implicated in cellular processes in the anterior hippocampus. More specifically, these proteins are functionally related to hippocampal-dependent synaptic plasticity, learning, and memory (https://www.genecards.org/). This suggests that methylation of TNXB may act as a molecular mechanism of altered synaptic plasticity or growth affecting hippocampal volume in pediatric PTSD, which may then have implication for multiple hippocampal functions such as context discrimination, fear and extinction learning, and emotion regulation implicated in pediatric PTSD (13).

In addition to the results of the DMR analysis, we detected DNAm abnormalities on the CRHBP gene in the Dutch cohort. This gene is known for its critical role in the regulation of corticotrophin-releasing hormone binding protein (CRH-BP), which in turn plays a modulatory role in the function of the HPA axis as well as CRH signaling within the brain. The HPA-axis is thought to play an important role in the etiology of PTSD, and the regulation of emotions and behavior (15). In addition, numerous studies in rodents have shown that levels of CRH-BP in various brain regions are highly responsive to psychological stressors (32). Furthermore, studies comparing post mortem brain tissue found that altered CRH-BP expression has been associated with adult psychopathology and suicide completion (33, 34). In the case of pediatric PTSD, it is possible that altered CRH-BP methylation, and thus altered expression, may contribute to abnormal CRH signalizing both peripherally and centrally, contributing to heightened physiological and stress responses associated with PTSD. In line with earlier studies, these findings further support the involvement of abnormal gene regulation associated with HPA axis dysregulation.

While this study highlights potentially novel genetic and molecular mechanisms contributing to pediatric PTSD, several limitations should be acknowledged. First, we used data generated on different arrays between the two cohorts (HumanMethylation450 in the Dutch cohort and EPIC bead chip array in the US cohort). Data generated with these arrays are comparable (the EPIC bead chip covers the whole 450k array), but only capture a fraction of the CpG sites in the genome. Second, this study has a limited sample size in both cohorts, creating risk for both false negative and false positive findings particularly in a genetic study. To mitigate the latter, we included youth with carefully diagnosed, phenotypes to increase the probability that important, and true, group differences would be identified. However, replication is a strength that in part helps to overcome the small sample size. Another limitation regarding our cohorts are the comorbid problems that have been reported, such as ADHD and depression which may limit specificity of findings to PTSD.

Additionally, we applied strict corrections in the Dutch (MWAS)search, with replication in the American cohort at an uncorrected level, in order to reduce the overall risk of false positive results while balancing the risk of false negatives. Third, our study examined gene methylation using only saliva samples. Though this approach likely captures part of the underlying pathophysiology of pediatric PTSD, it is unlikely to represent exact DNA methylation in brain regions most relevant for PTSD. To mitigate this possibility, we have emphasized DNAm abnormalities that also map onto known structural brain abnormalities in pediatric PTSD.

Future studies would be warranted to collect longitudinal data in youth with and without trauma-exposure and PTSD to determine the temporal course of methylation-neural structure relationships in relation to childhood trauma exposure and the emergence of PTSD. Given the implication of inflammatory and glucocorticoid pathways in this initial study, future studies would also benefit from including other peripheral or central markers such as cortisol and cytokine levels, and determine whether these represent mediating factors between altered peripheral methylation and structural brain changes leading to pediatric PTSD. Finally, future studies would benefit from collecting additional tissue sources (blood, saliva and eventually post-mortem brain tissue), as well as information about RNA and protein expression.

In conclusion, our findings provide new insights into the underlying biology and potential biomarkers for PTSD in youth. To our knowledge, this is the first investigation into differential DNA methylation in pediatric PTSD and its association to structural brain abnormalities. While youth with PTSD are an inherently difficult population to recruit, we utilized a combination of two independent, international, and well-phenotyped cohorts, a common data analytic pipeline, strict statistical corrections, and linkage to neural phenotypes, to increase the likelihood of important and reproducible findings in pediatric PTSD. These findings offer future targets for hypothesis driven studies in larger samples and statistical rigor for early identification of candidate gene pathways. If replicated in subsequent work, the epigenetic markers identified here could serve as novel therapeutic targets in the prevention and treatment of pediatric PTSD.

Supplementary Material

Acknowledgements

Footnotes

References