Epigenetic biotypes of post-traumatic stress disorder in war-zone exposed veteran and active duty males

Abstract

Post-traumatic stress disorder (PTSD) is a heterogeneous condition evidenced by the absence of objective physiological measurements applicable to all who meet the criteria for the disorder as well as divergent responses to treatments. This study capitalized on biological diversity observed within the PTSD group observed following epigenome-wide analysis of a well-characterized Discovery cohort (N = 166) consisting of 83 male combat exposed veterans with PTSD, and 83 combat veterans without PTSD in order to identify patterns that might distinguish subtypes. Computational analysis of DNA methylation (DNAm) profiles identified two PTSD biotypes within the PTSD+ group, G1 and G2, associated with 34 clinical features that are associated with PTSD and PTSD comorbidities. The G2 biotype was associated with an increased PTSD risk and had higher polygenic risk scores and a greater methylation compared to the G1 biotype and healthy controls. The findings were validated at a 3-year follow-up (N = 59) of the same individuals as well as in two independent, veteran cohorts (N = 54 and N = 38), and an active duty cohort (N = 133). In some cases, for example Dopamine-PKA-CREB and GABA-PKC-CREB signaling pathways, the biotypes were oppositely dysregulated, suggesting that the biotypes were not simply a function of a dimensional relationship with symptom severity, but may represent distinct biological risk profiles underpinning PTSD. The identification of two novel distinct epigenetic biotypes for PTSD may have future utility in understanding biological and clinical heterogeneity in PTSD and potential applications in risk assessment for active duty military personnel under non-clinician-administered settings, and improvement of PTSD diagnostic markers.

Fig. 1. Two epigenetic biotypes G1 and G2 were identified based on 100 clinical-meaningful DNAm genes.

a Thirty-four well-characterized symptomatic assessments (Table S1) were grouped into five clusters based on Pearson’s correlations values; we assigned clinical-related names to these groups (labels on the left). The values were translated to a color gradient schema in which, red indicates a value of 1 (i.e., positive correlation) and blue indicates −1 (i.e., negative correlation). b Heatmap illustrating the z-scores of the components of two CCA latent clinical factors. The first factor is focused on PTSD core symptoms, named ‘Psychological’, and the second factor contains physical and dissociative, named ‘Physical and dissociative’. Colors span dark blue to dark red where dark blue denotes a z-score of −2, and dark red indicates a z-score of 2. Black boxes were added to highlight the components highly ranked based on absolute z-scores. c The scatter plot shows the DNAm Psychological and Physical score paired with their associated Psychological and Physical clinical features. The highly correlated pairs were identified by PCA + CCA approach based on Discovery cohort (N = 157, r = 0.49, p = 3e−8, and r = 0.41, p = 2.6e−4, respectively). The gray dots are controls, and PTSD individuals are presented in colored dots. d LDA classifier was trained on DNAm ‘Psychological’ and ‘Physical and dissociative’ to assign any PTSD individuals to G1 or G2 biotypes. The gray dots are controls, the blue and red dots are PTSD G1 and G2 biotype.

Fig. 2. Individuals of the G2 biotype have significantly higher PTSD severity than G1.

a We compared the difference in CAPS scores between the PTSD individuals in our Discovery (DISC) (G1, N = 39; G2, N = 41) and Replication (REP) (G1, N = 11; G2, N = 15) cohorts. The statistical significance was defined by a two-tailed t-test and double asterisks indicate p < 0.005, asterisk indicates p < 0.05. b PTSD severity comparison in the veteran Bronx VA cohort. Comparison of current and lifetime CAPS scores between the PTSD individuals in veteran Bronx VA cohort (G1, N = 14; G2, N = 14). c Correlation between biotype score and CAPS score for different cohorts.

Fig. 3. Validation in the 3-year Follow-up cohort.

a The CAPS scores between G1 and G2 in the Follow-up cohort (G1, N = 11; G2, N = 12) were compared. b Plot illustrates the Current CAPS total scores at Discovery and the 3-year Follow-up time points for the 19 (out of 83) PTSD-positive individuals and 29 PTSD negative individuals (at Discovery) with data for both time point.

Fig. 4. Validation in the active duty Fort Campbell cohort.

Comparison of the PCL-5 score of PTSD-positive G1 and G2 individuals from the active duty Fort Campbell cohort pre-deployment (G1, N = 7 and G2, N = 3), 3-days returning from duty (G1, N = 11 and G2, N = 10) and 3–6 months post-deployment (G1, N =23 and G2, N = 22).

Fig. 5. The biotypes differentiate in anxiety and depressive symptoms.

The ternary plot shows the relative similarity of each clinical feature to three PTSD core subcategories. The statistical significance of the difference between the two biotypes is colored from red to black (red is for false discovery rate (FDR) < 0.01, black for FDR = 1, with other colors in between as denoted in the figure). The clinical features with significant biotype difference (FDR < 0.05) and no difference (FDR = 1) were labeled.

Fig. 6. The biotypes oppositely regulated in dopaminergic and serotonergic pathways.

a Comparison of the differentially methylated genes (DMG) of G1 versus control, and G2 versus control, and the combined PTSD+ group (G1 and G2) versus control, from the combined Discovery and Replication cohorts. The orange and blue bars indicate the number of hyper-/hypo-methylated genes, respectively. b The differentially methylated pathways, identified from the analysis of the DMGs, showed overlap between the subtypes in the Dopamine-cAMP-PKA-CREB and GABA-PKC-CREB signaling pathway. *Only the probe for DRD5 is located in the promoter region. For all others, an upward arrow indicates hypermethylation corresponding to activation, and a downward arrow to hypomethylation, corresponding to suppression.