A pilot investigation on DNA methylation modifications associated with complex posttraumatic symptoms in elderly traumatized in childhood
Marinova et al. BMC Res Notes
A pilot investigation on DNA methylation modifications associated with complex posttraumatic symptoms in elderly traumatized in childhood
Zoya Marinova 3
Andreas Maercker 0 2
Edna Grünblatt 3
Tomasz K. Wojdacz 1
Susanne Walitza 3
0 Department of Psychology, Division of Psychopathology and Clinical Intervention, University of Zurich , Binzmühlerstrasse 14/17, Raum BIN 3 E 14, 8050 Zurich , Switzerland
1 Aarhus Institute of Advanced Studies, University of Aarhus , Aarhus , Denmark
2 Department of Psychology, Division of Psychopathology and Clinical Intervention, University of Zurich , Binzmühlerstrasse 14/17, Raum BIN 3 E 14, 8050 Zurich , Switzerland
3 Department of Child and Adolescent Psychiatry and Psychotherapy, Psychiatric Hospital, University of Zurich , Zurich , Switzerland
Objective: Complex posttraumatic stress disorder (CPTSD) is a newly proposed diagnosis in the International Classification of Diseases-version 11, which is currently intensively investigated. Childhood trauma is regarded as main source of CPTSD symptoms, even in later life. Induction of DNA methylation changes by childhood trauma may contribute to its long-lasting adverse health consequences. The current study analyzed the correlation of genome-wide DNA methylation profiles with complex posttraumatic sequelae in buccal epithelial cells from 31 elderly former indentured child laborers (Verdingkinder) using the Infinium Illumina 450k Human DNA methylation chip. Results: DNA methylation modifications indicated experiment-wide significant associations with the following complex posttraumatic symptom domains: dissociation, tension reduction behavior and dysfunctional sexual behavior. Differentially methylated CpG sites were mapped to the genes huntington associated protein 1 (HAP1), RAN binding protein 2 (RANBP2) and proteasome subunit alpha 4 (PSMA4), respectively. In addition, the methylation of cg07225277 located in carnosine synthase 1 (CARNS1) correlated with trauma symptom complexity. Our pilot data suggest correlation of DNA methylation modifications with complex posttraumatic symptoms in elderly individuals subjected to prolonged and complex childhood trauma. More comprehensive and elaborated studies should be carried out to analyze epigenetic modifications associated with CPTSD.
Childhood trauma; Complex posttraumatic stress disorder; DNA methylation; Epigenetic modifications
Exposure to childhood adversities has been associated
with increased risk for mental illness in later life,
including posttraumatic stress disorder (PTSD) and complex
posttraumatic stress disorder (CPTSD) [
Symptoms characteristic of PTSD include re-experiencing
the traumatic event, avoiding stimuli associated with
it and hyperarousal. CPTSD according to the
International Classification of Diseases-version 11 (ICD-11)
incorporates in addition to the PTSD symptoms also
disturbances in other mental and behavioral symptom
]. A high number of different childhood
traumatic events has been correlated to increased complexity
of posttraumatic symptoms in adulthood [
DNA methylation is a mechanism of epigenetic
regulation, which affects gene transcription. Various
environmental factors including stress can alter the DNA
methylation pattern of cells [
]. Childhood adversities
have been associated with differential DNA methylation
of genes involved in neuronal development, cell
signaling, inflammation and disease biomarkers by several
whole-genome studies [
]. Genome-wide DNA
methylation studies in PTSD patients have implicated among
others genes involved in immune function [
the link between DNA methylation changes and complex
posttraumatic symptoms has not been specifically
Swiss former indentured child laborers
(Verdingkinder) were removed as children from their families by
the authorities due to different reasons (poverty, being
born out of wedlock) and were placed to live and work
on farms. This was a practice applied until the 1950s and
many of the Verdingkinder were subjected to childhood
trauma and neglect during the indentured labor [
have previously identified differentially methylated genes
in elderly former child laborers compared to a control
group, which included genes involved in neuronal
projections and neuronal development [
In the current investigation, we assessed the correlation
of genome-wide DNA methylation profiles with complex
posttraumatic symptoms according to ICD-11 CPTSD in
a group of elderly Swiss former indentured child laborers.
31 elderly former indentured child laborers (17 M/14 F)
were included in the current study. Mean age (± SD) was
76.4 (± 6.3) years. Mean age of indenture (± SD) was 4.7
(± 4.5) years. Mean duration of indenture (± SD) was
11.8 (± 5) years. The participants were part of a larger
investigation analyzing mental and physical sequelae
in elderly former indentured child laborers [
approval was granted by the Cantonal Ethic Commission
of Zurich (KEK-ZH-Nr. 2012-0245). Inclusion criteria
for the study were: indentured child labor, upbringing in
rural areas of Switzerland, at least 65 years of age,
voluntary participation, (Swiss) German speaking. Exclusion
criteria were: under 65 years of age, insufficient
knowledge of German.
Assessment of complex posttraumatic symptoms
Complex posttraumatic symptoms were evaluated with
the trauma symptom inventory (TSI) [
]. TSI is
a 100-item self-report instrument, which includes 10
clinical scales to assess multiple complex posttraumatic
sequelae dimensions (anxious arousal, depression, anger
and irritability, intrusive experiences, defensive
avoidance, dissociation, sexual concerns, dysfunctional sexual
behavior, impaired self reference and tension reduction
behavior), as well as three validity scales and twelve
critical items. Items are rated on scales ranging from 0 (never)
to 3 (often) depending on how often symptoms occurred
in the previous 6 months. The validation of the German
version of the TSI was carried out in a group of former
indentured child laborers and showed overall good
reliability and validity [
To assess symptom complexity, TSI scores were
dichotomized as previously described [
]. For each of the 10
clinical scales values at least 1.5 standard deviations
above the median for all former indentured child
laborers included in our project were considered as clinically
elevated. The number of clinically elevated TSI scales for
each participant were used as an indicator of symptom
DNA isolation, bisulfite conversion and Infinium Illumina
450k chip analysis
Buccal swabs collection and DNA stabilization was
carried out as previously described [
]. Buccal swabs were
processed at the “Barts and The London Genome
Centre” in the United Kingdom. DNA isolation was
performed with the Isohelix buccal DNA isolation kit (Cell
Projects Ltd) and purification with the Zymo ZR-96 DNA
clean-up kit (Zymo Research Corporation, Irvine, United
States) according to the manufacturer’s
recommendations. DNA concentration was controlled with the Qubit
2.0 Fluorometer (Life Technologies) and integrity with
agarose gel electrophoresis. DNA bisulfite conversion
was carried out with the EZ DNA Methylation kit (Zymo
Research Corporation) according to the manufacturer’s
recommendations. Bisulfite converted DNA was
hybridized to the Infinium Illumina 450k Human DNA
methylation chip, which encompasses 485,000 methylation sites
per sample [
Analysis was carried out in the R environment (http://
cran.r-project.org) using raw intensity data files [
Data import, quality control and normalization were
carried out with the minfi Bioconductor package [
Probes with detection p-values > 0.01 in any of the
samples were excluded. Background correction and
normalization were carried out with the Illumina method
implemented in minfi (bg.correct = TRUE,
normalize = “controls”). Probes on the Illumina Infinium 450k
array assaying single nucleotide polymorphisms (SNPs)
as well as non-CpG methylation were filtered out. So
were the ones containing SNPs at the investigated CpG
site or at a single base extension position. Probes located
on the sex chromosomes were also excluded. The minfi
preprocessed data were analyzed with the CpGassoc
package to assess correlation of DNA methylation
profiles with complex posttraumatic symptoms measured by
the TSI scales [
]. Age, gender, the estimated proportion
of buccal epithelial cells, array and slide were included
as covariates in the analysis. The proportion of buccal
epithelial cells was estimated according to a previously
published protocol [
]. The threshold for statistical
significance of the results was set at 5 × 10−7 according to a
previously recommended threshold to account for
multiple testing [
DNA methylation profiles of elderly former indentured
child laborers were significantly associated (p < 5 × 10−7)
with complex posttraumatic sequelae in the symptom
dimensions dissociation, tension reduction behavior and
dysfunctional sexual behavior (Table 1). No significant
(p < 5 × 10−7) association of DNA methylation patterns
were observed for the scales anxious arousal, depression,
anger and irritability, intrusive experiences, defensive
avoidance, sexual concerns and impaired self reference.
Methylation changes at cg12320221 mapped to the
first exon of the gene huntington associated protein 1
(HAP1) correlated significantly and negatively with
dissociation symptoms severity (T-statistic = − 11.45; p
value = 4.54 × 10−7). Positive correlation of DNA
methylation with the scale tension reduction behavior was
detected for cg12328023, which is located in the body
of the gene RAN binding protein 2 (RANBP2;
T-statistic = 11.73; p-value = 3.61 × 10−7). For the scale
dysfunctional sexual behavior positive correlation of DNA
methylation was observed for cg20739864 in the 3′-UTR
of the gene proteasome subunit alpha 4 (PSMA4;
T-statistic = 12.05; p-value = 2.8 × 10−7).
Finally, association of DNA methylation with the
number of clinically elevated TSI scales as an indicator of
trauma symptom complexity was investigated. Positive
correlation for DNA methylation at cg07225277 located
in the body of carnosine synthase 1 (CARNS1) was found
(T-statistic = 13.41; p-value = 1.02 × 10−7).
Our pilot investigation as part of a comprehensive
psychobiologial research program [
] identified DNA
methylation alterations associated with the complex
posttraumatic symptom domains dissociation, tension
reduction behavior and dysfunctional sexual behavior in
elderly former indentured child laborers.
Experimentwide significant association of DNA methylation
modifications with trauma symptom complexity was also
For the dissociation scale differential methylation in
HAP1 was identified. HAP1 has been shown to be
critically involved in a number of neuronal functions,
including neuronal survival, inhibitory synaptic transmission
and neurogenesis [
]. Disruption of HAP1 in the
early postnatal period in mice has been associated with
depressive-like behavior in later life .
RANBP2 (with DNA methylation correlated with
tension reduction behavior) is involved in SUMOylation, a
posttranslational modification with important role in a
range of neuronal functions, including neuronal survival
and synaptic transmission [
]. PSMA4, methylation in
which was associated with dysfunctional sexual behavior,
is also involved in posttranslational modifications, since
it encodes a subunit of the 20S proteasome. The
ubiquitin–proteasome system plays an important role in the
regulation of memory formation [
Correlation of DNA methylation in carnosine
synthase 1 (CARNS1, known also as ATPGD1) with trauma
complexity was also observed. CARNS1 catalyzes the
formation of carnosine and homocarnosine, which are
compounds with anti-oxidant properties [
supplementation with carnosine has shown positive
effects on cognition on veterans suffering from Gulf War
Overall, we detected stronger association of DNA
methylation profiles with complex posttraumatic
symptoms rather than “classical” PTSD symptoms. DNA
methylation alterations were found in genes involved in
neuronal function and posttranslational modifications.
Proving the relevance of these findings would require
their replication in larger study cohorts and after
different trauma types.
Our pilot findings imply a correlation of DNA
methylation modifications with complex posttraumatic sequelae
in elderly individuals exposed to prolonged and complex
Limitations of our investigation include its
cross-sectional design, the relatively small sample size, the use
of peripheral tissue (buccal epithelial cells), the lack of
data on gene expression patterns for the genes showing
alterations in DNA methylation and the use of a CPTSD
assessment that is not fully equivalent to the ICD-11
CPTSD model [see 29]. Although certain tissue
specificity in epigenetic patterns exists, evidence that DNA
methylation alterations after early life adversity is a
system-wide phenomenon has been accumulating,
supporting the utility of surrogate peripheral tissues for analysis
PTSD: posttraumatic stress disorder; CPTSD: complex posttraumatic stress
disorder; HAP1: huntington associated protein 1; RANBP2: RAN binding
protein 2; PSMA4: proteasome subunit alpha 4; CARNS1: carnosine synthase 1;
ICD-11: International Classification of Diseases-version 11; M: male; F: female;
SD: standard deviation; TSI: trauma symptom inventory; SNP: single nucleotide
AM, SW and ZM conceived and designed the study with input from EG and
TW. ZM, AM, TW, EG and SW were involved in data analysis and interpretation.
ZM drafted the manuscript, which was revised by all authors. All authors read
and approved the final manuscript.
We thank all participants in the current study. We thank the “Barts and The
London Genome Centre” for performing the Illumina Infinium 450k DNA
methylation arrays experiments.
The authors declare that they have no competing interests.
Availability of data and materials
The ethical permission for the study does not include a permission to make
underlying patient-specific data publicly available.
Consent for publication
Ethics approval and consent to participate
Approval for the study was obtained by the Cantonal Ethic Commission of
Zurich. All participants gave their informed consent to participate.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Briere J , Scott C . Complex trauma in adolescents and adults: effects and treatment . Psychiatr Clin North Am . 2015 ; 38 : 515 - 27 .
2. Yehuda R , Flory JD , Pratchett LC , Buxbaum J , Ising M , Holsboer F. Putative biological mechanisms for the association between early life adversity and the subsequent development of PTSD . Psychopharmacology. 2010 ; 212 : 405 - 17 .
3. Marinova Z , Maercker A . Biological correlates of complex posttraumatic stress disorder-state of research and future directions . Eur J Psychotraumatol . 2015 ; 6 : 25913 .
4. Maercker A , Brewin CR , Bryant RA , Cloitre M , Reed GM , van Ommeren M, et al. Proposals for mental disorders specifically associated with stress in the International Classification of Diseases-11 . Lancet. 2013 ; 381 : 1683 - 5 .
5. Briere J , Kaltman S , Green BL . Accumulated childhood trauma and symptom complexity . J Trauma Stress . 2008 ; 21 : 223 - 6 .
6. Cloitre M , Stolbach BC , Herman JL , van der Kolk B , Pynoos R , Wang J , et al. A developmental approach to complex PTSD: childhood and adult cumulative trauma as predictors of symptom complexity . J Trauma Stress . 2009 ; 22 : 399 - 408 .
7. Booij L , Wang D , Le´vesque ML , Tremblay RE , Szyf M. Looking beyond the DNA sequence: the relevance of DNA methylation processes for the stress-diathesis model of depression . Phil Trans R Soc B . 2013 ; 368 : 51 .
8. Lutz PE , Almeida D , Fiori LM , Turecki G . Childhood maltreatment and stress-related psychopathology: the epigenetic memory hypothesis . Curr Pharm Des . 2015 ; 21 : 1413 - 7 .
9. Uddin M , Aiello AE , Wildman DE , Koenen KC , Pawelec G , de Los Santos R, et al. Epigenetic and immune function profiles associated with posttraumatic stress disorder . Proc Natl Acad Sci USA . 2010 ; 107 : 9470 - 5 .
10. Wohlwend L , Honegger A . Gestohlene Seelen: Verdingkinder in der Schweiz. Frauenfeld: Huber; 2004 .
11. Marinova Z , Maercker A , Küffer A , Robinson MD , Wojdacz TK , Walitza S , et al. DNA profiles of elderly individuals subjected to indentured childhood labor and trauma . BMC Med Genet . 2017 ; 18 : 21 .
12. Burri A , Maercker A , Krammer S , Simmen-Janevska K . Childhood trauma and PTSD symptoms increase the risk of cognitive impairment in a sample of former indentured child laborers in old age . PLoS ONE . 2013 ; 8 : e57826 .
13. Briere J . Trauma symptom inventory: professional manual . Lutz: PAR; 1995 .
14. Krammer S , Simmen-Janevska K , Maercker A . Towards 'complex PTSD': german translation of the Trauma Symptom Inventory (TSI) for the assessment of complex trauma Sequelae . Psychother Psychosom Med Psychol . 2013 ; 63 : 318 - 26 .
15. Krammer S , Grossenbacher H , Goldstein N , Kaufmann C , Schwenzel A , Soyka M. Validierung der deutschen Übersetzung des revidierten Trauma Symptom Inventory (TSI-2) zur Erfassung komplexer posttraumatischer Belastungssymptomatik . Psychother Psych Med . 2017 . https://doi.org/10. 1055/s-0042-120839.
16. Bibikova M , Barnes B , Tsan C , Ho V , Klotzle B , Le JM , et al. High density DNA methylation array with single CpG site resolution . Genomics . 2011 ; 98 : 288 - 95 .
17. R_Development_Core_ Team. R. A Language and Environment for Statistical Computing . Vienna: R Foundation for Statistical Computing; 2013 .
18. Hansen KD , Ayree M , Irizary RA , Jaffe AE , Maksimovic J , Houseman A , et al. Package “minfi”: Analyze Illumina's 450k methylation arrays . R package version 1 .10.2; 2014 .
19. Barfield RT , Kilaru V , Smith AK , Conneely KN . CpGassoc: an R function for analysis of DNA methylation microarray data . Bioinformatics . 2012 ; 28 : 1280 - 1 .
20. Eipel M , Mayer F , Arent T , Ferreira MR , Birkhofer C , Gerstenmaier U , et al. Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures . Aging (Albany NY) . 2016 ; 8 : 1034 - 48 .
21. Rakyan VK , Down TA , Balding DJ , Beck S . Epigenome-wide association studies for common human diseases . Nat Rev Genet . 2011 ; 12 : 529 - 41 .
22. Kittler JT , Thomas P , Tretter V , Bogdanov YD , Haucke V , Smart TG , et al. Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating gamma-aminobutyric acid type A receptor membrane trafficking . Proc Natl Acad Sci USA . 2004 ; 101 : 12736 - 41 .
23. Xiang J , Yang H , Zhao T , Sun M , Xu X , Zhou XF , et al. Huntingtin-associated protein 1 regulates postnatal neurogenesis and neurotrophin receptor sorting . J Clin Invest . 2014 ; 124 : 85 - 98 .
24. Xiang J , Yan S , Li SH , Li XJ . Postnatal loss of hap1 reduces hippocampal neurogenesis and causes adult depressive-like behavior in mice . PLoS Genet . 2015 ; 11 : e1005175 .
25. Henley JM , Craig TJ , Wilkinson KA . Neuronal SUMOylation: mechanisms, physiology, and roles in neuronal dysfunction . Physiol Rev . 2014 ; 94 : 1249 - 85 .
26. Jarome TJ , Helmstetter FJ . The ubiquitin-proteasome system as a critical regulator of synaptic plasticity and long-term memory formation . Neurobiol Learn Mem . 2013 ; 105 : 107 - 16 .
27. Drozak J , Veiga- da -Cunha M , Vertommen D , Stroobant V , Van Schaftingen E. Molecular identification of carnosine synthase as ATP-grasp domaincontaining protein 1 (ATPGD1) . J Biol Chem . 2010 ; 285 : 9346 - 56 .
28. Baraniuk JN , El-Amin S , Corey R , Rayhan R , Timbol C. Carnosine treatment for gulf war illness: a randomized controlled trial . Glob J Health Sci . 2013 ; 5 : 69 - 81 .
29. Karatzias T , Shevlin M , Fyvie C , Hyland P , Efthymiadou E , Wilson D , et al. Evidence of distinct profiles of Posttraumatic Stress Disorder (PTSD) and Complex Posttraumatic Stress Disorder (CPTSD) based on the new ICD-11 Trauma Questionnaire (ICD-TQ) . J Affect Dis . 2017 ; 207 : 181 - 7 .
30. Szyf M , Bick J . DNA methylation: a mechanism for embedding early life experiences in the genome . Child Dev . 2013 ; 84 : 49 - 57 .