595 - Sex-Specific DNA Methylation of Neurodevelopmental, Stress-Response and Reward Pathway Genes Associated with Need for Pharmacological Treatment in Opioid-Exposed Newborns

Publication Number: 4583.595

Angelica Aragon Vasquez, Brown University, Providence, RI, United States; Marie Camerota, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Elisabeth Conradt, Duke University School of Medicine, Durham, NC, United States; James Padbury, UCSF Benioff Children's Hospital San Francisco, San Francisco, CA, United States; barry lester, The Warren Alpert Medical School of Brown University, east greenwich, RI, United States

Background: Neonatal Opioid Withdrawal Syndrome (NOWS) has increased alongside rising opioid use during pregnancy. NOWS involves physiological instability and neurobehavioral dysregulation with possible long-term effects. Not all opioid-exposed infants develop NOWS, and those who do differ in symptom onset and severity, suggesting biological heterogeneity. Male infants often show greater NOWS severity, yet the mechanisms behind these sex differences remain unclear. Because sex shapes stress regulation and epigenetic programming, examining sex-specific patterns of DNA methylation (DNAm) may reveal pathways contributing to variability in NOWS.

Objective: To investigate sex-specific associations between DNAm of neurodevelopmental, stress, and reward pathway genes and need for pharmacological treatment in opioid-exposed infants.

Design/Methods: Participants in the Child and Family Study were recruited from sites in Rhode Island and Utah (2019-2024). Buccal swabs were collected at birth and assayed for DNAm at CpG sites within neurodevelopmental (BDNF), stress (AVP, NR3C1), and reward pathway genes (DRD2, DRD4). Receipt of pharmacological treatment was obtained via medical record. Differences in DNAm by treatment status and sex were examined using robust linear models with a Sex × Treatment interaction term, adjusting for study site. Estimated marginal means were derived to quantify adjusted DNAm differences between treated and untreated male and female infants.

Results: Analyses included 181 opioid-exposed newborns (Table 1). CpG sites in the promoter regions of BDNF, AVP, NR3C1, DRD2, and DRD4 showed significant Sex × Treatment interactions (all p ≤ .05; Table 2). Methylation was generally higher in males who received pharmacological treatment for NOWS. The largest effects occurred in DRD4 (-362 from transcription start site [TSS]: β = 3.65, Fig. 1A; -358 from TSS: β = 0.54). Similar patterns were observed in DRD2 (285 from TSS; β = 0.14, Fig. 1B), BDNF (-2021 from TSS: β = 0.29; -2010 from TSS: β = 0.39), and AVP (-6014 from TSS; β = 2.98). In NR3C1, one CpG (220 from TSS: β = -0.17) showed lower methylation in treated males, whereas another CpG (267 from TSS; β = 0.13) showed higher methylation.

Conclusion(s): DNAm of neurodevelopmental, stress, and reward pathway genes showed sex-specific associations with NOWS treatment among opioid-exposed infants. These differences may reflect epigenetic mechanisms underlying variability in NOWS severity. Consideration of sex-specific risk factors could guide more personalized care for opioid-exposed newborns.

Table 1: Maternal and Infant Characteristics by Infant Pharmacological Treatment Group and Sex


Table 2: Summary of Model Results from CpG Sites with Significant Sex × Treatment Interaction Effects
Note: *indicates p < 0.05, 1indicates main effect of treatment on DNA methylation in model without interaction term; 2indicates main effects of treatment by sex and sex X treatment interaction in model with interaction term.

Figure 1: Representative CpG Sites Showing Significant Sex-Specific Associations between DNA Methylation and Need for Pharmacological Treatment for NOWS