To our knowledge, this study represents the first analysis that utilised a genome-wide approach to explore whether DNAm mediates the relationship between childhood adversity, and for the subtypes abuse and neglect in people with a FEP. We used the DACT approach, specifically developed to address mediation effects in EWAS data15, that allowed us to interrogate mediating effects of DNAm in 614719 CpG sites across the entire genome.
Although none of the analyses survived to Bonferroni correction, we report nominally significant (P < 5e-5) DMPs, located in multiple genes of interest, mediating the association between composite measure of adversity, abuse, and neglect with psychosis. Some of these have been previously associated with various phenotypes, including SCZ, as shown in Table 5. These included PANK1, SPEG, TTC7B, ZHX2, HDAC5, NEK6, PKNOX2, TSNARE1; TMEM114, SORT1, PPP2R2D, VARS, NMB and LRRC27. One of the replicated genes, PANK1, is the top ranked gene mediating both the composite measure of adversity and neglect with psychosis, which has previously been associated to SCZ in an EWAS study34. It codes a protein belonging to the pantothenate kinase family, which is a key regulatory enzyme in the biosynthesis of coenzyme A (CoA) in mammalian cells, important for oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle49. This enzyme has a crucial role in mitochondrial functioning, which has recently appeared as key biological process involved in psychosis aetiopathogenesis50. SPEG was the second top ranked gene for both analyses with composite adversity and neglect. Two previous EWAS studies have found that increased methylation at this level was associated with SCZ26, 34, and another found an association with Alzheimer Disease35. This gene encodes a protein with similarity to members of the myosin light chain kinase family, essential for myocyte cytoskeletal development. The top ranked DMP (cg08457495) for analyses of abuse lies within a CpG island at the level of the TSS1500 within the gene TBKBP1, which is an important gene regulating immunity. TBKBP and its homologue IKKε are serine–threonine kinases that mediate the induction of type I Interferon in antiviral innate immunity51 ; although its molecular role in psychiatric conditions remain unknown, a recent GWAS study in frontotemporal dementia patients found that a genetic variant within that loci was associated with the condition52. Abundant evidence supports the role of neuroinflammation and altered immune processes in the aetiopathogeneses of psychosis53, 54. Various EWAS studies having found DMPs located in genes involved in the immune system in those with SCZ 55–58, as well as related to CA in different conditions such as borderline personality disorder and post traumatic stress disorder59–61. Exploring the specific implications of DNAm changes in TBK1 in immune processing involved with the disorder is a necessary target for future research.
Another important gene that appeared among the top ranked DMPs mediating composite adversity and psychosis is TSNARE1. As shown in SM Table 8, it has also been associated to SCZ 26, and with child abuse in EWAS studies37. Importantly, a GWAS study involving SCZ patients of Caucasian ancestry identified that two SNPs within TSNARE1 were associated with SCZ62, which was later replicated in another whole exome sequency study63. A follow-up study in a Chinese population with SCZ confirmed that the minor allele of the SNPs (rs10098073) within TSNARE1 was associated with a reduced risk of SCZ. Furthermore, gene expression data also points to a dysregulation of TSNARE1 in SCZ, and another study in pluripotent stem cells showed synergistic effects between TSNARE1 with other common variants associated to SCZ in altering pre and post synaptic neuronal deficits64. TSNARE1 plays key roles in docking, priming, and fusion of synaptic vesicles with the presynaptic membrane in neurons, thus synchronizing neurotransmitter release into the synaptic cleft65, and experimental preclinical studies suggest that it is a negative regulator of endosomal trafficking in cortical neurons66. Altogether, strong genetic, epigenetic, gene expression and stem cells in vitro evidence point at a possible involvement of TSNARE1 in SCZ, with epigenetic evidence suggesting a concurrent involvement of CA in DNAm changes in this gene, which provides a possible pathway in the CA-SCZ association. Other genes involved in the neurodevelopment of the central nervous system that require further exploration are the ITGAX, involved in the modulation of neural differentiation through its action on microglia67; ADGRG1, involved in myelinisation processes68, 69 and extracellular matrix70, 71; and MFF, involved in oligodendrocytes formation72. All these processes are important for SCZ aetiopathogenesis73–75, and therefore are of interest for future research.
None of our gene ontology enrichment analyses passed the FDR threshold, therefore drawing conclusions on the potential biological pathways that stem from our enrichment analyses is not possible. Taking this limitation into consideration, a preliminary exploration of the top 10 biological pathways that survived a more conservative p-value of < 0.05 could still provide some insights, as previously done by others33, 76. The top 1 and top 5 pathways for composite measure of adversity and abuse respectively was the histamine-induced gastric acid secretion, led by HRH2, (coding for the histamine receptor H2), which was the top 4 gene for composite adversity and abuse. Action on the histaminergic system alongside serotoninergic (5-HT), norepinephrinergic modulation putatively underlies the beneficial effect of atypical antipsychotics in mood and anxiety disorders 77. The H2R is a G-protein coupled receptor located post-synaptically that has high expression in the central nervous system (CNS), as well as the heart and stomach78, 79. Within the CNS, H2R has a high density of expression in the cortex, basal ganglia, amygdala, and hippocampus80. H2R is commonly co-expressed with H1R which may account for their similar excitatory function, such as memory consolidation, inflammation, and motor function81–84. Clinically, H2R antagonists are widely used to inhibit gastric acid secretion85. However, following a case report detailing the resolution of acute psychotic symptoms of a patient treated with famotidine, 7 RCTs have explored treatment of SCZ with H2R antagonists. These studies have had mixed findings on improvement of positive and negative symptoms. Nevertheless, the impact on cognition was not included in their analyses, which should be considered for future research given preclinical findings suggesting the role of H2R on cognitive processes86–92. Genetic variations of H2R in SCZ have been studied and subsequently identified H2R 64949G allele, the presence of which was 80% more frequent in patients with SCZ compared to controls, while homozygous manifestations of the allele were found to be 180% more frequent in patients93, 94. Given the above, future studies examining the implications of HR2 DNAm on clinical outcomes, and its modulating effect in cognitive processes could be an important target for future research.
In the current study, we conducted analyses separately for the subtypes of abuse and neglect in order to explore whether DNAm changes related to abuse overlapped or differed with those related to neglect. This question stems from recent findings showing a differential impact of both type of adversities on psychopathological outcomes in those with psychosis, with abuse being specifically related to the positive symptoms of psychosis, while neglect appears to be specifically linked to the negative dimension, suggesting different trajectories3. Our results on DNAm show a lack of overlap in the genes that passed the discovery P-value between abuse and neglect, while some overlap was present between the composite measure and these two adversity subtypes: 6/27 DMP associated genes were related to abuse and the composite measure, while this was the case for 10/20 of those from neglect analyses. Although we could not relate the DNAm changes to the psychopathological domains, as described in the limitations, our results suggest different biological signatures of DNAm according to the type of traumatic experience, in the onset of psychotic disorder. Future studies exploring the DNAm relationship between these two adversities with positive and the negative dimensions of psychosis respectively will pose an interesting research area.
This study presents various strengths and limitations. A first relevant strength is the sample of FEP with a relatively young population of cases (30.5 year old sample mean age), which limits the influence of chronicity of the psychotic disease on epigenetic changes, the impact of age itself95, as well antipsychotic medication, which is known for being an important modulator of DNAm96. Moreover, we controlled for known important confounders in epigenetic studies including smoking 97, cell type composition in blood98, as well as antipsychotic medication, thereby, disentangling their confounding effects on DNAm. Third, DNAm was quantified using the Illumina EPIC array, which to date is the most robust and highly reliable, currently the best high-throughput platform with content spanning regulatory regions associated with the majority of known annotated genes, allowing us to explore 614719 CpG sites across the entire epigenome. Fourth, we used various forms of adversity measures, not only limiting our analyses to the broader composite measure where the specific biological underpinnings are diluted hampering the study of specific effects. Fifth, we used the DACT15 novel approach specifically designed to explore mediation by DNAm epigenome wide, which takes into account the composite nature of the null hypothesis of no mediation, improving the power of more traditional methods such as the Sobel test and the joint significance test13.
Nonetheless, the results from our study should be considered with care in light of some limitations. First, we could not map our findings on DNAm related to abuse and neglect with positive and negative dimension of psychosis respectively, as the sample EU-GEI was not assessed with an instrument that could capture symptom severity. This would have been interesting, given previous findings showing a differential effect of those adversities in the respective dimensions 3, as mentioned above. Second, the direction of the mediation is difficult to interpret with the information presented in the study. Whereas a positive mediation could suggest that hypermethylation associated to CA may increase the risk of psychosis, and inversely; previous work shows that such direction (hyper or hypomethylation) is strongly dependent on the genotype, which has not been accounted for in this study. Furthermore, although increased methylation is generally associated to repression of gene expression, this is not always the case99, and without measures of gene expression, it is not possible to elucidate the impact of DNAm at a molecular level in each specific gene, therefore limiting the understanding of the downstream implication of our results. Therefore, our results remain exploratory and deserve future attention with more specific hypotheses related to specific genes, where the effect of genotype is accounted for, and the functional consequences of DNAm are explored molecularly. Fourth, despite the current study being the largest to date interrogating the epigenetic signature of DNAm in relation to CA in those with psychosis, it is still underpowered, and a larger sample may be needed in future, to replicate our results and test the same hypotheses in other sources such as saliva or post-mortem brains, given the relatively low blood-brain correlation of DNAm markers100. Fifth, different biological processes operate differently across various developmental stages, therefore considering the timing of trauma could give important insights into which processes operate at different ages. Unfortunately, CTQ does not report the age of exposure to CA, which prevents us examining this in the current work. We hope that this will be the object of future attention. Lasty, we have conducted mediation analyses in a cross-sectional study, and although traumatic events were recorded before psychosis onset, we cannot exclude that psychosis itself may lead to changes in the DNAm, and not the other way around as we assume, therefore, posing a reverse causation issue. Therefore, longitudinal studies examining the outcome after the DNAm changes are required.
In conclusion, the present study was underpowered to identify putative mediation of the impact of CA on psychosis by DNA methylation. Although none of our DMP reached statistical significance based on Bonferroni correction, we reported a number of nominally significant DMPs (p < 5e-05) that are associated with genes that have been previously implicated in the pathogenesis of SCZ in genetic and epigenetic studies, as well as a number of novel candidate genes. For example, we reported differential DNA methylation in genes associated in immune and neurodevelopmental process, as well as the dopaminergic and glutamatergic pathways, which is in line with recent literature in the field suggesting a mediating role of such pathways between CA and psychosis (Alameda et al., in press). Although none of our enrichment analyses revealed pathways surviving the FDR correction, the top ranked biological process involved the histaminergic function, which can be an important target for future research, given the direct link with psychopathology and medication therapeutic effects in patients with SCZ. Lastly, low overlap between mediating genes and pathways according to abuse and neglect suggests that biological trajectories between CA and psychosis are distinct depending on the type of adversity.