Identify Child Maltreatment Biomarkers for Early Detection

Child maltreatment (CM), including abuse and neglect, profoundly impacts children’s physical and mental health, increasing risks for psychiatric disorders, cardiovascular diseases, and suicide [1]. Epigenetic mechanisms, particularly DNA methylation, have been proposed as mediators linking early-life adversity to long-term health outcomes. Such epigenetic alterations may leave specific molecular signatures in children exposed to CM [2]. Consequently, numerous epigenetic studies have aimed not only to elucidate underlying biological mechanisms but also to identify biomarkers for early detection and prevention of CM.

However, most previous epigenetic studies have employed candidate-gene approaches based on specific hypotheses, such as stress-related [3] or sociability-related genes [4, 5]. These studies were not designed to discover novel or unexpected epigenetic markers beyond existing knowledge. Therefore, epigenome-wide association studies (EWAS) are essential for comprehensively identifying DNA methylation alterations associated with CM. Although promising, many prior EWAS have been conducted in adults retrospectively reporting childhood maltreatment, rather than in children themselves [6]. Such retrospective studies have limited utility for developing preventive biomarkers due to the substantial time gap between maltreatment exposure and methylation assessment. Additionally, methylation changes identified in adults may not reflect dynamic responses directly attributable to CM, as intervening environmental factors could confound results.

Conducting EWAS in children presents unique ethical and practical challenges. Obtaining informed consent from minors is complicated, particularly given sensitive family backgrounds and legal considerations. Consequently, only a limited number of epigenetic studies have directly investigated children exposed to maltreatment. To date, fewer than ten EWAS have examined DNA methylation in children close to the time of maltreatment exposure [7,8,9,10,11,12,13]. However, these studies have limitations, such as lack of functional interpretation [13], reliance on candidate genes [7], qualitative analyses [11], or small sample sizes [8]. A recent prospective study identified methylation sites dynamically altered by childhood adversity and highlighted sensitive developmental periods [9]. However, reliance on maternal self-reports may underestimate CM prevalence. Thus, further studies using cohorts with objectively verified CM exposure are necessary to confirm and extend these findings.

To address these gaps, we conducted EWAS in three independent cohorts comprising judicially or socially certified CM cases and matched controls. Our primary aim was to identify robust epigenetic signatures associated with CM. We conducted EWAS separately in each cohort, followed by meta-analysis to identify common methylation markers. We further examined associations between these markers and CM-related biological indicators, and evaluated their utility in classifying CM exposure. To our knowledge, this is the first EWAS of CM employing multiple independent cohorts analyzed in parallel using a unified analytical pipeline, enabling subsequent meta-analysis and synthesis of findings. Our study thus provides a critical step toward identifying reliable epigenetic biomarkers for early detection and prevention of CM, overcoming limitations of previous research.

Participants and cohorts

This study included three Japanese cohorts comprising a total of 226 children for genome-wide DNA methylation analyses (Table 1). The cohorts consisted of judicial autopsy cases, children sheltered in residential childcare facilities, and typically developing (TD) children raised by biological families recruited from the local community as controls. Children in residential childcare facilities had been legally removed from their biological parents by Child Protection Services or equivalent authorities, and most had documented histories of physical, emotional, or sexual abuse, or neglect prior to placement. Participants with documented maltreatment histories were classified as the CM group (ICD-10-CM Code T74). Psychosocial difficulties and depressive symptoms were assessed using the Strengths and Difficulties Questionnaire (SDQ) [14] and the Depression Self-Rating Scale for Children (DSRS-C) [15], respectively.

Judicial autopsy cases

Twenty-six children whose deaths were judicially authenticated by a forensic pathologist (M.N.) between 2000 and 2021 were included. Of these, 15 cases (CM) had causes of death attributed to child abuse or neglect, and the remaining 11 cases (TD) were due to fatal accidents or illnesses (Supplementary Table S1). Thymus weight records were available for 24 cases (CM:15, TD:9). Whole blood samples from 18 cases (CM:11, TD:7) had been stored at −20 °C. Formalin-fixed paraffin-embedded (FFPE) brain tissues or formalin-immersed brain blocks were preserved for 24 cases (CM:13, TD:11). Prefrontal cortex tissues were selected for methylation analysis, as this region was consistently preserved across all cases. The study protocol was approved by the Ethics Committee of the University of Fukui (approval no. 20200030) and the Research Ethics Review Board of Hiroshima University (approval no. E–2032), and was conducted in accordance with the Declaration of Helsinki.

Toddler social cognition cohort

One hundred twenty-two children aged 0–9 years participated in this cohort between 2017 and 2021. Participants underwent assessments of social cognitive function using gaze pattern analysis and provided buccal mucosa samples [16,17,18]. Genome-wide methylation analysis was conducted on 85 participants (CM:36, TD:49) who passed the quality control (QC) procedures described below, had no repeated measurements, and completed cognitive assessments using either the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV), Kyoto Scale of Psychological Development (KSPD), or equivalent developmental scales (Table 1). The study protocol was approved by the Ethics Committee of the University of Fukui (approval nos. 20140142, 20150068, and 20190107) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all parents or childcare facility directors.

Adolescent brain imaging cohort

Two hundred thirty-seven children and adolescents aged 6–18 years (CM:83, TD:154) participated in this cohort between 2013 and 2022, undergoing brain MRI scans [4, 5, 19,20,21,22] (Supplementary Table S2). Group comparisons of brain gray matter (GM) structures were conducted using the full dataset. Saliva samples were collected from 141 participants. Genome-wide methylation analysis was performed on a subset of 123 participants (CM:61, TD:62) who passed the QC procedures described below, had no repeated measurements, and completed full-scale IQ (FSIQ) assessments (Table 1). The study protocol was approved by the Ethics Committee of the University of Fukui (approval nos. 20110104, 20130157, 20138031, 20150068, 20190107, 20210004, 20220034, and 20220039) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all parents or childcare facility directors.

Sample collection and DNA extraction

For the Judicial Autopsy Cases, DNA was extracted from whole blood samples using the AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Venlo, Netherlands). Brain DNA was extracted from FFPE tissues using the High Pure FFPET DNA Isolation Kit (Roche, Basel, Switzerland), starting from approximately 10 mg tissue blocks or six slices (12 µm thickness, 3 cm²) until a total yield of 250 ng DNA was obtained. A modified pre-processing protocol was employed to improve extraction efficiency, including two five-minute ethanol washes, an overnight PBS wash (50 °C, 600 rpm), and overnight lysis (56 °C, 600 rpm) [23, 24].

In the Toddler Social Cognition cohort, buccal swab samples were collected using commercially available cotton swabs, with either one swab (CM:13, TD:16) or four swabs (CM:23, TD:33) per individual [16, 18]. DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Venlo, Netherlands). The first 16 TD swabs had unintentionally been stored at room temperature for an average of 461 ± 25 (SD) days before DNA extraction; thus, storage duration was included as a covariate in subsequent analyses. In the Adolescent Brain Imaging cohort, saliva samples were collected using the Oragene Discover OGR-500 kit (DNA Genotek Inc., Ottawa, Canada), and DNA was extracted using the prepIT®·L2P reagent (DNA Genotek) [4, 5, 25]. A total of 119 individuals (CM:58, TD:61) underwent both brain MRI and saliva collection and were available for imaging epigenetics analysis. Within this group, 72 (CM:38, TD:34) saliva samples were collected on the day of brain imaging or within several days; however, the remaining 47 (CM:20, TD:27) samples showed discrepancies in the dates of collection. DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).

Quality control (QC) procedures for DNA methylation

Genome-wide DNA methylation was assessed using the Infinium HumanMethylationEPIC BeadChip Kit (Illumina). DNA samples (500 ng for peripheral and 250 ng for brain tissues) were bisulfite-converted using the EZ DNA Methylation™ Kit (Zymo Research). Standardized QC procedures for peripheral tissues (blood, buccal mucosa, and saliva) were conducted separately for each cohort, including rigorous probe and sample filtering, data normalization, and correction for batch effects (see Supplementary Methods). For brain samples, customized QC criteria were applied to accommodate variations in sample quality (see Supplementary Methods).

Autopsied thymus weight ratio calculation

Thymus weight ratio, reflecting the severity and duration of child abuse or neglect [26], was calculated relative to age-specific normal ranges established by the Medico-Legal Society of Japan [27] (see Supplementary Methods).

Assessment of social cognitive function by gaze patterns for toddler social cognition cohort