Demelza Smeeth, PhD; Simone Ecker, PhD; Olga Chervova, PhD; Fiona McEwen, PhD; Elie Karam, MO; Stephan Beck. PhD; Michael Pluess, PhD

IMPORTANCE Exposure to war is associated with poor mental health outcomes. Adverse and traumatic experiences can lead to long-lasting DNA methylation changes, potentially mediating the link between adversity and mental health. To date, limited studies have investigated the impact of war on DNA methylation in children or adolescents, hampering our understanding of the biological impact of war exposure.
OBJECTIVE To identify salivary DNA methylation differences associated with war exposure in refugee children and adolescents.
DESIGN, SETTING, AND PARTICIPANTS This cohort study included Syrian refugee children and adolescents, and their primary caregiver were recruited from tented settlements in Lebanon. Data collection was carried out in 2 waves, 1 year apart, from October 2017 to January 2018 and October 2018 to January 2019. Children and their caregiver were interviewed, and children provided saliva samples for DNA extraction. Data analysis was conducted in 2022, 2023, and 2024.
EXPOSURE War exposure assessed by interviewing children and their caregiver using the War Events Questionnaire.
MAIN OUTCOMES AND MEASURES Salivary DNA methylation levels were assayed with the lnfinium MethylationEPIC BeadChip (lllumina). Epigenetic aging acceleration was estimated using a set of preexisting epigenetic aging clocks. A literature search was conducted to identify previously reported DNA methylation correlates of childhood trauma.
RESULTS The study population included 1507 children and adolescents (mean [SD] age, 11.3
[2.4] years; age range, 6-19 years; 793 female [52.6%]). A total of1449 children provided saliva samples for DNA extraction in year 1, and 872 children provided samples in year 2. Children who reported war events had a number of differentially methylated sites and regions. Enrichment analyses indicated an enrichment of gene sets associated with transmembrane transport, neurotransmission, and intracellular movement in genes that exhibited differential methylation. Sex-stratified analyses found a number of sex-specific DNA methylation differences associated with war exposure. Only 2 of 258 (0.8%) previously reported trauma-associated DNA methylation sites were associated with war exposure
(B = -0.004; 95% Cl, -0.005 to -0.003; Bonferroni P = .04 and B = -0.005; 95% Cl,
-0.006 to -0.004; Bonferroni P = .03). Any war exposure or bombardment was nominally associated with decreased epigenetic age using the Horvath multitissue clock (B = -0.39; 95% Cl, -0.63 to -0.14; P = .007 and B = -0.42; 95% Cl, -0.73 to -0.11; P = .002).
CONCLUSIONS AND RELEVANCE In this cohort of Syrian refugee children and adolescents, war exposure was associated with a small number of distinct differences in salivary DNA methylation.

War Exposure and DNA Methylation in Syrian Refugee Children and Adolescents