Mapping DNA Methylation Signatures to Identify Epigenetic Variation Across Subcortical Regions of the Human Posttraumatic Stress Disorder Brain

Abstract

Background: Posttraumatic stress disorder (PTSD) is a mental disorder that may occur in the aftermath of severe psychological trauma. Epigenetic changes in the brain may play a critical role in understanding the neurobiology of PTSD by linking environmental traumatic stress exposure to lasting alterations in gene expression that shape neuronal function.

Methods: We examined 1,065,750 DNA methylation (DNAm) sites from 171 donors including neurotypical controls and PTSD and major depressive disorder (MDD) cases across 6 regions implicated in the fear circuitry of the brain. We performed RNA sequencing (RNA-seq) to examine changes in gene expression and linked these changes to changes in DNAm at nearby sites in a case-control manner. We created a single cell-type atlas of DNAm using a single-nucleus RNA-seq reference panel to map epigenetic changes to specific cell types. Finally, we leveraged a human PTSD ketamine trial to associate blood DNAm biomarkers of ketamine efficacy with specific changes in DNAm in the brain.

Results: We found significant differential methylation for PTSD near 195 genes, and to further resolve the changes we observed, we constructed a cell type-specific DNAm atlas defined for changes to the PTSD methylome across 6 cell types. To identify potential therapeutic intersections for PTSD, we found significant methylation levels in the MAD1L1, ELFN1, and WNT5A genes in patients with PTSD who responded to ketamine. Finally, to better understand the unique biology of PTSD, we analyzed matching methylation data for a cohort of donors with MDD with no known history of trauma or PTSD.

Conclusions: Our results implicate DNAm as an epigenetic mechanism underlying the molecular changes associated with the subcortical fear circuitry of the PTSD brain.

Keywords: Amygdala; DNA methylation; Hippocampus; Major depressive disorder; PTSD.

Figure 1.. DNA isolated from different brain regions display widespread differences in CpG methylation.

(A) Bioinformatics analysis pipeline. (B) Individual scores from principal components analysis of all samples included in the study. PC2 maximally separate subjects by sex. (C) PC1 maximally separate subjects by brain regions. (D) heatmap of median methylation levels of the 1,200 CpG sites (top 200 most significant CpG sites for each region that distinguish each of the six brain regions from the five others); only samples with data from every brain region were included. Hierarchical clustering of these 1,200 sites reveals subregion similarities within each primary region. (E) Heatmap of z-score transformed odds ratios for genic, CpG and chromatin features showing the log odds ratio of Bonferroni-corrected significant CpG sites within the feature versus excluded. Significant subcortical CpGs are more enriched in bivalent promoter, weak and active enhancer regions (P < 0.0001) and less enriched in TSS, ZNF genes, and gaps (P < 0.0001).

Figure 2.. Network analysis.

Gene network analysis was performed using IPA for DMCs in each differential methylation pattern. The most significant gene networks (network score > 30) from the six single-regional analyses were obtained and merged by integrating the hub genes and their nearest neighbors in IPA. Pie-circles were used to indicate the differential methylation regions for genes that have more than two neighboring genes. The amygdala regions (BLA: red, CeA: orange, and MeA: yellow) are in warm colors and the three hippocampus regions (CA: purple, DG: blue, and Sub: green) are in cold colors. Genes SMARCA4, ESR1, TCF4, WNT3A, and WNT5A were hub genes with the greatest number of nearest neighbors.

Figure 3.. Examples of DNAm changes for 10 GWAS-positive loci for PTSD.

(A) Examples of group-specific DNA methylation ratios for ten PTSD risk genes. MAD1L1, CAMKV, KCNIP4, SPRK2, KANSL1, CRHR1, and TCF4 are significant risk genes (identified by GWAS) for PTSD. ELFN1 is a significant TWAS hit for PTSD, and SLC32A1 is a previously identified transcriptomic key driver in PTSD. For each boxplot, y-axis dots show methylation levels at a specific CpG site. P-value corresponds to differential methylation. For box plots, center line is the median, limits are the IQR, and whiskers are 1.5X the IQR. (B) Methylation changes at nine genes identified by the largest PTSD GWAS using data from MVP were examined. Significant changes were found for KCNIP4, HSD17B11, MAD1L1, SRPK2, KANSL1, CRHR1, and TCF4 for at least one brain subregion. Significant enrichment of DNAm changes were also found in the interneuron gene ELFN1, which was a PTSD genetically regulated genes identified by TWAS.

Figure 4.. Brain Cell-type specific DNAm atlas in PTSD.

(A) Bar plots display the number of cell type-specific PTSD-associated DMCTs, calculated using CellDMC. Hypermethylated DMCs are on the left and hypomethylated DMCs are on the right (B). An example region of cell type specific DNA methylation in oligodendrocytes of the MeA at PTSD GWAS risk loci MAD1L1 (top) and ESR1 (bottom). Yellow highlight indicates position of significant methylation change. (C) Heatmap of cell type specific DMC enrichments in genic features (blue), CpG features (green), and PTSD GWAS risk variants (salmon) across brain regions and cell types. *P value<0.05, ** P value <0.005, ***P value <0.0005.

Figure 5.. Examples of ketamine altered DNA methylation ratios for three PTSD genes.

Significant hypomethylation at five sites in the promoter of MAD1L1 in patients who responded to ketamine, three in the ELFN1 promoter and one in the WNT5A promoter. For each boxplot, y-axis dots show methylation levels at a specific CpG site. P value corresponds to differential methylation. For box plots, center line is the median, limits are the IQR, and whiskers are 1.5X the IQR.

Figure 6.. Common and divergent epigenetic signatures between PTSD and MDD.

(A) Bar plot shows number of significant CpG sites in the PTSD case-control analysis (pink) and MDD case-control analysis (gray). (B) UpSet plot shows the shared DMCs across brain regions and diagnosis groups. (C, D) Enrichment analysis for genes with DNAm levels inverse to gene expression stratified by increases in PTSD (C) and increases in MDD (D). One asterisk (*) indicates adjusted p-value < 0.1, and two asterisks sign (**) indicates adjusted p-value < 0.01 (E) Locus zoom-in plots on chromosome 7 for PTSD combined-sex analysis and (F) MDD combined-sex analysis. The significant DMCs were plotted by subregion (red-BLA, orange-CeA, yellow-MeA, purple-CA, blue-DG, and green-Sub. Nominal significant (P<0.05) DMCs are plotted in blue and non-significant DMCs are colored in gray.