*3.2. PTSD and Epigenetic E*ff*ects*

The *NR3C1*, *FKBP5,* and *CRHR1* genes methylation have all been shown to be associated with childhood adversity [8,14,108,154]. These modifications could constitute risk factors for PTSD development upon later exposure to a TE. Epigenetic modifications could also result from TE proper exposure and influence the pathophysiology of PTSD development. Methylation levels of *NR3C1* and *FKBP5* have been repeatedly associated with the status of PTSD diagnosis [155].

Despite the different methodologies across studies, a recent systematic review found strong evidence of an association between *NR3C1* increased methylation levels and decreased gene expression, which suggests a role for this gene's methylation in stress-related psychopathology [156]. Methylation levels of the *NR3C1* gene have been studied in the promoter regions of exons 1B, 1C, and 1F, which are rich CpG sites. Hypermethylation of 1F is associated with a decreased expression of the gene and accordingly with GR resistance [108]. An association between the demethylation of the GR gene exon 1F promoter and both PTSD and plasma cortisol decline on the dexamethasone suppression test was found [157]. In this study, war-related PTSD male subjects showed greater cortisol suppression after the administration of dexamethasone, and higher levels of peripheral blood mononuclear cell lysozyme inhibition in the lysozyme suppression test. Accordingly, another study of survivors of the Rwanda genocide found that the sex-dependent salivary GR gene exon 1F promotor DNA methylation was associated with PTSD [158]. Male but not female survivors with increased GR gene exon 1F promotor methylation, which was associated with lower *NR3C1* expression, showed less intrusive memory of the traumatic event and reduced PTSD risk. In these studies, it was not possible to distinguish the effects of trauma from the effects of PTSD. Another study focussing on female victims of the same Rwanda Tutsi genocide, found higher peripheral blood leukocytes *NR3C1* exon 1F promotor methylation levels than those of Tutsi non-victims [159]. However, the association between PTSD and *NR3C1* gene exon 1F promotor methylation levels was not addressed, and in this case, hypermethylation could be attributed to trauma exposure. *NR3C1* exon 1F promotor methylation levels negatively correlated both with cortisol levels and *NR3C1* mRNA expression levels. In this same study, those exposed to trauma showed higher methylation of CpGs located within the *NR3C2* coding sequence than non-exposed subjects. On the other hand, peripheral T lymphocytes *NR3C1* 1B and 1C promoters' methylation levels were found to be lower in subjects with lifetime PTSD related to different types of trauma, when compared with non-traumatised controls. Cortisol levels were inversely correlated with *NR3C1* 1B mRNA expression. Furthermore, overall and CpG site-specific methylation levels were inversely correlated with total *NR3C1* and 1B mRNA expression [160]. However, in this study, the *NR3C1* exon 1F promotor methylation did not associate with PTSD risk. Another study of female interpersonal violence victims found PTSD severity to be negatively correlated with the mean percentage of *NR3C1* exon 1F methylation [161].

*FKBP5* methylation has also been extensively studied. Klengel et al. [14] found that being a carrier of the *FKBP5* SNP rs1360780 risk allele T and also being exposed to early life trauma lead to *FKBP5* intron 7 demethylation. *FKBP5* intron 7 is situated in a GC response element zone, which is subject to the action of GCs as part of the ultra-short feedback loop between FKBP5 and the GR, which leads to GR resistance, as demethylation increases levels of this co-chaperone. However, [162] no main effects of PTSD diagnosis on *FKBP5* intron 7 methylation were found in a sample of Holocaust survivors, nor in their offspring and comparison subjects. A recent study did find significantly higher intron 7 methylation levels among veterans with PTSD carrying the rs1360780 risk allele when they were compared to the non-PTSD group [163]. On the other hand, *FKBP5* exon 1 promoter methylation was associated with lower plasma cortisol levels in subjects with combat-related PTSD [33], which supports previous findings of lower *FKBP5* expression in PTSD [146].

In the case of GILZ—which is a transcription factor that is up-regulated by GC action [46]—mRNA levels were associated with PTSD in males, and were negatively associated with the methylation of the respective gene. Furthermore, the number of TEs correlated negatively with *GILZ* mRNA levels, and positively with the percentage methylation of *GILZ* just in the case of males [164].

Other approaches to the epigenomic study of PTSD pathophysiology are still being carried out. A longitudinal study of combat-related PTSD found decreases in DNA methylation in three novel genomic regions *(ZFP57*, *RNF39* and *HIST1H2APS2*) across the period of exposure which constituted marks of susceptibility to PTSD [165]. Noncoding RNAs have also been implicated in the relationship between stress and GCs and these could prove to be useful biomarkers to facilitate the prescription of personalised medicines for trauma-related disorders, as the majority of PTSD blood based microRNA studies report reduced expression in PTSD [110].

Differences in gene expression can reflect epigenetic modifications and/or interactions with SNPs. Changes in gene expression have been found to occur after combat exposure [166]. However, no longitudinal studies have examined the combination of DNA methylation and gene expression—which limits our understanding of associated TE exposure DNA methylation on gene expression [166].

The epigenetic regulation of gene expression is very much cell-type specific [112]. The previous studies researched methylation in non-CNS cells, and therefore we are unable to conclude on the neuronal methylation status, although this could well reflect exposure to environmental influences [100], as has been shown for psychosocial adversity [167] and combat [165], which accordingly could represent biomarkers of the brain related phenotype. In addition, substantial correlations between blood and brain samples have been observed in post-mortem studies [168]. On the other hand, the epigenetic modifications could be the result of PTSD effects on the HPA axis and immune system, as DNA methylation changes in response to environmental exposure can be induced by altered GC signalling [169].

There are also reports of intergenerational transmission of these epigenetic modifications [105,162], which could constitute one of the many mechanisms for intergenerational transmission of PTSD [28].

In brief, PTSD has been consistently associated with lower levels of cortisol and GR hypersensitivity. Furthermore, a meta-analysis found associations between variability in the *NR3C1* and *FKBP5* genes and PTSD. Other SNPs in genes involved in HPA axis regulation have also been found to be associated with PTSD, such as the *CRHR1.* Epigenome studies have found evidence of associations between levels of methylation in several genes with a regulatory role in HPA axis functioning—mainly *NR3C1* and *FKBP5*—which can depend on the gene's SNPs and on the gene's location of methylation. These findings are consistent with previous evidence of lower cortisol levels and GR hypersensitivity in PTSD. All this genetic makeup variability has been found to interact with environmental influences, consubstantiating a new paradigm of gene × trauma × epigenetic interactions [155].

### **4. PTSD Treatments Which Influence the HPA Axis**
