Transcriptome analysis reveals novel genes and immune networks dysregulated in veterans with PTSD.

BACKGROUND: Posttraumatic stress disorder (PTSD) is a serious condition that emerges following trauma exposure and involves long-lasting psychological suffering and health-issues. Uncovering critical genes and molecular networks is essential to understanding the biology of the disorder. We performed a genome-wide scan to identify transcriptome signatures of PTSD. METHODS: Genome-wide peripheral blood transcriptomic data from 380 service personnel were investigated. This included a discovery sample of 96 Australian Vietnam War veterans and two independent pre and post-deployment replication samples of U.S. Marines (N = 188 and N = 96). RESULTS: A total of 60 transcripts were differentially expressed between veterans with and without PTSD, surviving Bonferroni multiple testing correction. Genes within the cytokine-cytokine receptor interaction, Jak-STAT signaling and Toll-like receptor signaling pathways were enriched. For 49% of the genes, gene expression changes were also accompanied by DNA methylation changes. Using replication data from two U.S. Marine cohorts, we observed that of the differentially expressed genes, 71% genes also showed significant gene expression changes between pre and post-deployment. Weighted gene co-expression networks revealed two modules of genes associated with PTSD. The first module (67 genes, p-value = 6e-4) was enriched for genes within the 11p13 locus including BDNF. The second module (266 genes, p-value = 0.01) was enriched for genes in 17q11 including SLC6A4, STAT5A and STAT5B. CONCLUSIONS: We identified novel transcriptomic loci and biological pathways for PTSD in service personnel. Network analysis revealed enrichment of loci harboring key candidate genes in PTSD. These findings highlight the role of transcriptional biomarkers in the molecular etiology of PTSD.

Clinically proven drug targets differentially expressed in the prefrontal cortex of schizophrenia patients.

BACKGROUND: Due to the heterogeneous nature of schizophrenia, understanding the genetic risk for the disease is a complex task. Gene expression studies have proven to be more reliable than association studies as they are consistently replicated in a tissue specific manner. METHODS: Using RNA-Seq we analysed gene expression in the frontal cortex of 24 individuals with schizophrenia and 25 unaffected controls. RESULTS: We identified 1146 genes that were differentially expressed in schizophrenia, approximately 60% of which were up-regulated and 366 of 1146 (32%) also have aberrant DNA methylation (p=2.46×10-39). The differentially expressed genes were significantly overrepresented in several pathways including inflammatory (p=8.7×10-3) and nitric oxide pathways (p=9.2×10-4). Moreover, these genes were significantly enriched for those with a druggable genome (p=0.04). We identified a number of genes that are significantly up-regulated in schizophrenia as confirmed in other gene expression studies using different brain tissues. Of the 349 genes associated with schizophrenia from the Psychiatric Genomics Consortium we identified 16 genes that are significant from our list of differentially expressed genes. CONCLUSIONS: Our results identified biological functional genes that are differentially expressed in schizophrenia. A subset of these genes are clinically proven drug targets. We also found a strong pattern of differentially expressed immune response genes that may reflect an underlying defect in schizophrenia.

Genetic and serum biomarker evidence for a relationship between TNFα and PTSD in Vietnam war combat veterans.

BACKGROUND: Posttraumatic stress disorder (PTSD) is associated with increased inflammation and comorbid medical conditions. However, study findings for individual inflammatory marker levels have been inconsistent. Some research suggests that resilience may play a role in decreased inflammation. A polymorphism in the promoter region of the tumor necrosis factor α gene (TNFα), TNFA -308 (rs1800629) is associated with psychiatric illness but its role in PTSD is yet to be elucidated. OBJECTIVE: This study investigates a key inflammatory marker, TNFα, for its role in PTSD severity. METHOD: In a cohort of trauma-exposed Vietnam War veterans (n=299; 159 cases, 140 controls) TNF α serum levels and TNFα polymorphism rs1800629 were correlated with PTSD severity and resilience scores. RESULTS: The polymorphism was associated with PTSD severity (p=0.045). There were significant group differences between cases and controls with regards to serum TNFα levels (p=0.036). Significant correlations were found between PTSD severity and elevated TNFα levels (r=0.153; p=0.009), and between resilience and decreased TNFα levels at a trend level (p=0.08) across the entire cohort. These relationships were non-significant after controlling for covariates. In the PTSD diagnostic group, a correlation of TNFα and PTSD severity was observed on a trend level (p=0.06), the relationship between TNFα and resilience remained non-significant. CONCLUSIONS: To our knowledge, this is the first time rs1800629 has been investigated in PTSD contributing to a growing body of literature that identifies the GG as a risk genotype for psychiatric disorders in Caucasian cohorts. However, more research is needed to replicate our results in larger, equally well-characterized cohorts. The relationship between serum TNFα levels and PTSD severity and resilience requires further investigation.

Interaction of multiple gene variants and their effects on schizophrenia phenotypes.

BACKGROUND: Schizophrenia is a clinically heterogeneous disorder and may be explained by its complex genetic architecture. Many schizophrenia susceptibility genes were identified but the picture remains unclear due to inconsistent or contradictory genetic association studies. This confusion may, in part, be because symptoms result from the combined interaction of many genes and these interacting genes are associated with specific sub-phenotypes of schizophrenia rather than schizophrenia as a whole. This study investigates the relationship between schizophrenia susceptibility genes and schizophrenia sub-phenotypes by identifying multiple gene variant interactions. MATERIALS AND METHODS: Fifty SNPs from 21 genes were genotyped in 235 Australian participants with schizophrenia screened for various phenotypes. Schizophrenia participants were grouped into relevant phenotype clusters using cluster analysis and normalized phenotype cluster scores were calculated for each patient. The relationship between genotypes and normalized phenotype cluster scores were analyzed by linear regression analysis. RESULTS: Three phenotype clusters were identified. There was some overlap in symptoms between phenotype clusters, particularly for depression. However, cluster 1 appears to be characterized by speech disorder and affective behavior symptoms, cluster 2 has predominantly hallucination symptoms and cluster 3 has mainly delusion symptoms. Interaction of five SNPs was found to have an effect on cluster 1 symptoms; ten SNPs on cluster 2 symptoms; and eight SNPs on cluster 3 symptoms. CONCLUSION: The interaction of specific susceptibility genes is likely to lead to specific clinical sub-phenotypes of schizophrenia. Larger patient cohorts with more extensive clinical data will improve the detection of gene interactions and the resultant schizophrenia clinical phenotypes.

A Case-Control Study and Meta-Analysis Reveal BDNF Val66Met Is a Possible Risk Factor for PTSD.

Posttraumatic stress disorder (PTSD) is a debilitating condition that develops in some people after exposure to a traumatic event. Brain-derived neurotrophic factor (BDNF) is highly expressed in the mammalian brain and is thought to be involved in learning and memory processes. A nonsynonymous polymorphism in the BDNF gene, rs6265 (Val66Met), has been hypothesised to be associated with PTSD. Association studies examining the Val66Met polymorphism and PTSD have been inconclusive, likely due to the variability in type of trauma exposure analysed. Vietnam veterans (n = 257) screened for PTSD and controlled for trauma exposure were genotyped for BDNF Val66Met. The association was not significant so we incorporated our data into a meta-analysis to obtain greater statistical power. A comprehensive search of more than 1237 articles revealed eight additional studies suitable for meta-analysis (n = 3625). A random-effects meta-analysis observed a potential protective factor of the Val/Val genotype. After removing two studies with violation of Hardy-Weinberg equilibrium, findings for the Val/Val genotype reached significance. Subgroup analyses confirmed a trend for this finding. Limitations of some studies that inform this meta-analysis include poorly screened controls and a lack of examination of population stratification. Effectively designed studies should inform this line of research in the future.

Investigation of C-reactive protein and AIM2 methylation as a marker for PTSD in Australian Vietnam veterans.

Chronic inflammation is a key factor in symptomology and comorbidities of post-traumatic stress disorder (PTSD). Levels of a proinflammatory marker, C-reactive protein (CRP) are increased in individuals with PTSD but it is not clear if this is due to trauma exposure or PTSD. Our study aimed to assess the relationship between serum CRP levels, CRP SNPs, methylation, mRNA expression and PTSD in a homogenous trauma exposed Australian Vietnam veteran cohort. We hypothesized that decreased DNA methylation would be associated with increased gene expression and increased peripheral CRP levels in PTSD patients and that this would be independent of trauma. Participants were 299 Vietnam veterans who had all been exposed to trauma and approximately half were diagnosed with PTSD. We observed higher levels of serum CRP in the PTSD group compared to the non-PTSD group but after controlling for BMI and triglycerides the association did not remain significant. No association was found between CRP SNPs and PTSD or CRP levels. Absent in Melanoma 2 (AIM2) which is a mediator of inflammatory response and a determinant of CRP levels was analysed for DNA methylation and mRNA expression. We observed a trend level association between PTSD and AIM2 methylation after controlling for age, smoking, triglycerides, BMI and cell types. There was no significant interaction between PTSD and CRP levels on AIM2 methylation after controlling for covariates. We observed that as AIM2 methylation levels decreased, AIM2 mRNA expression increased. Elevated CRP levels were associated with AIM2 mRNA in the trauma exposed cohort but there was no significant interaction effect with PTSD. Our results could not confirm that CRP is a marker of PTSD independent of trauma in this group of older veterans. CRP may be a broad marker of disease risk, or a marker of PTSD in younger cohorts than those in this study.

Differential BDNF methylation in combat exposed veterans and the association with exercise.

Brain-derived neurotrophic factor (BDNF) gene is associated with increased risk of posttraumatic stress disorder (PTSD) and plays a role in neuroplasticity, cognition and memory. BDNF has strong potential as a therapeutic target as studies have shown that antidepressants, electroconvulsive treatment and exercise modulate BDNF expression and methylation. In this study we examined the role of BDNF methylation and expression in PTSD and the implications of exercise in mediating these effects. BDNF DNA methylation and gene expression analysis was performed in a sample of 96 male Vietnam veterans. Cases were combat-exposed veterans with current PTSD (n = 48) and controls were combat exposed veterans with no past or current PTSD diagnosis (n = 48). No association between BDNF mRNA and PTSD was identified. PTSD was associated with decreased methylation at three BDNF CpG sites (cg01546433 P = 0.004835; cg24650785 P = 0.000259 and cg002298481 P = 0.000672). Differential BDNF methylation was associated with exercise, with active exercise associated with lower methylation levels at three CpG sites (cg04481212 P = 0.005; cg01546433 P = 0.025 and cg00298481 P = 0.035). Given that exercise mediates BDNF action on cognitive plasticity, exercise may be a non-invasive, drug free option in the treatment of PTSD.

DNA methylation from germline cells in veterans with PTSD.

In this study we investigated genome-wide sperm DNA methylation patterns in trauma-exposed Vietnam veterans. At the genome-wide level, we identified 3 CpG sites associated with PTSD in sperm including two intergenic and one CpG within the CCDC88C gene. Of those associated with PTSD in sperm at a nominal level, 1868 CpGs were also associated with PTSD in peripheral blood (5.6% overlap) including the RORA, CRHR1 and DOCK2 genes that have been previously implicated in PTSD. A total of 10 CpG sites were significantly associated with a reported history of a diagnosed mental health condition in children and reached genome-wide significance. CpGs associated with a history of a reported mental health condition in children were also enriched (90% of tested genes) for genes previously reported to be resistant to demethylation, making them strong candidates for transgenerational inheritance. In conclusion, our findings identify a unique sperm-specific DNA methylation pattern that is associated with PTSD.

Correlation between interferon γ and interleukin 6 with PTSD and resilience.

Posttraumatic Stress Disorder (PTSD) is a debilitating psychiatric disorder with decreased general health prognosis and increased mortality. Inflammation has been hypothesised to be a link between PTSD and the most common co-morbid medical disorders. However, the relationship between inflammation and PTSD is not clear. Individual inflammatory markers have shown variable associations with PTSD. This study investigates the correlations between serum cytokines, PTSD and resilience in a cohort of Caucasian Vietnam combat veterans (n = 299). After correction for multiple testing, PTSD severity was correlated with small but significant decreases in interleukin 6 and interferon γ (p = 0.004, p = 0.013, respectively) whereas resilience was correlated with increased levels of interleukin 6 and interferon γ (p = 0.023; p = 0.007, respectively). Analyses of sub-symptoms of PTSD revealed that mood and arousal symptoms showed the most significant effect on interleukin 6 and interferon γ. More research is needed to further elucidate the mechanisms underlying the relationship between cytokine levels, PTSD sub-symptoms and trauma outcomes to improve the knowledge base of differences in trauma response and the biological system.

Accelerated DNA methylation aging and increased resilience in veterans: The biological cost for soldiering on.

Accelerated epigenetic aging, the difference between the DNA methylation-predicted age (DNAm age) and the chronological age, is associated with a myriad of diseases. This study investigates the relationship between epigenetic aging and risk and protective factors of PTSD. Genome-wide DNA methylation analysis was performed in 211 individuals including combat-exposed Australian veterans (discovery cohort, n = 96 males) and trauma-exposed civilian males from the Grady Trauma Project (replication cohort, n = 115 males). Primary measures included the Clinician Administered PTSD Scale for DSM-5 and the Connor-Davidson Resilience Scale (CD-RISC). DNAm age prediction was performed using the validated epigenetic clock calculator. Veterans with PTSD had increased PTSD symptom severity (P-value = 3.75 × 10-34) and lower CD-RISC scores (P-value = 7.5 × 10-8) than veterans without PTSD. DNAm age was significantly correlated with the chronological age (P-value = 3.3 × 10-6), but DNAm age acceleration was not different between the PTSD and non-PTSD groups (P-value = 0.24). Evaluating potential protective factors, we found that DNAm age acceleration was significantly associated with CD-RISC resilience scores in veterans with PTSD, these results remained significant after multiple testing correction (P-value = 0.023; r = 0.32). This finding was also replicated in an independent trauma-exposed civilian cohort (P-value = 0.02; r = 0.23). Post-hoc factor analyses revealed that this association was likely driven by "self-efficacy" items within the CD-RISC (P-value = 0.015; r = 0.35). These results suggest that among individuals already suffering from PTSD, some aspects of increased resilience might come at a biological cost.

Nitric oxide pathway genes (NOS1AP and NOS1) are involved in PTSD severity, depression, anxiety, stress and resilience.

The nitric oxide pathway in the hippocampus is involved in the biological stress response with detrimental consequences to cells and HPA axis feedback. Hippocampal atrophy and HPA axis feedback dysfunction are associated with posttraumatic stress disorder (PTSD). This study systematically investigates two genes of the nitric oxide pathway NOS1AP and NOS1 for a potential involvement in PTSD, comorbidities and resilience. A cohort of age and gender matched Vietnam veterans including trauma-exposed cases and controls was recruited and comprehensively assessed (n=299). A total of 49 NOS1AP and 16 NOS1 polymorphisms were analysed and genotypes correlated with gold standard clinical measures to assess PTSD severity and related phenotypes (depression, anxiety, stress, resilience) based on diagnostic status. Multiple NOS1AP polymorphisms were associated across all measures, and NOS1 polymorphisms were associated with PTSD severity, stress and resilience. The GG genotype of NOS1 polymorphism rs10744891 was associated with PTSD severity (surviving multiple correction) while the combined TT-TG genotypes were associated with resilience (p=0.005; p=0.033, respectively). This study indicates that NOS1AP and NOS1 from the nitric oxide pathway are likely to play a key role in PTSD, its comorbidities and resilience. Given the essential role of NOS1AP and NOS1 in stress response they may be reliable targets for screening and intervention strategies.

mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain.

Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

Expression and methylation of BDNF in the human brain in schizophrenia.

OBJECTIVES: To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. METHODS: DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. RESULTS: There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. CONCLUSIONS: Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

Progress towards understanding the genetics of posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is a complex syndrome that occurs following exposure to a potentially life threatening traumatic event. This review summarises the literature on the genetics of PTSD including gene-environment interactions (GxE), epigenetics and genetics of treatment response. Numerous genes have been shown to be associated with PTSD using candidate gene approaches. Genome-wide association studies have been limited due to the large sample size required to reach statistical power. Studies have shown that GxE interactions are important for PTSD susceptibility. Epigenetics plays an important role in PTSD susceptibility and some of the most promising studies show stress and child abuse trigger epigenetic changes. Much of the molecular genetics of PTSD remains to be elucidated. However, it is clear that identifying genetic markers and environmental triggers has the potential to advance early PTSD diagnosis and therapeutic interventions and ultimately ease the personal and financial burden of this debilitating disorder.

NOS1AP is associated with increased severity of PTSD and depression in untreated combat veterans.

BACKGROUND: Posttraumatic stress disorder (PTSD) and depressive disorder are over represented in combat veterans. Veterans with both disorders have an increased risk of suicide. The nitric oxide synthase 1 adaptor protein (NOS1AP) gene, which modulates stress-evoked N-methyl-d-aspartate (NMDA) activity, was investigated in combat veterans. METHODS: A comprehensive genetic analysis of NOS1AP and its association with PTSD was investigated in Vietnam combat veterans with PTSD (n=121) and a group of healthy control individuals (n=237). PTSD patients were assessed for symptom severity and level of depression using the Mississippi Scale for Combat-Related PTSD and the Beck Depression Inventory-II (BDI). RESULTS: The G allele of NOS1AP SNP rs386231 was significantly associated with PTSD (p=0.002). Analysis of variance revealed significant differences in BDI-II and Mississippi scores between genotypes for rs386231 with the GG genotype associated with increased severity of depression (p=0.002 F=6.839) and higher Mississippi Scale for Combat-Related PTSD scores (p=0.033). Haplotype analysis revealed that the C/G haplotype (rs451275/rs386231) was significantly associated with PTSD (p=0.001). LIMITATIONS: The sample sizes in our study were not sufficient to detect SNP associations with very small effects. In addition the study was limited by its cross sectional design. CONCLUSIONS: This is the first study reporting that a variant of the NOS1AP gene is associated with PTSD. Our data also suggest that a genetic variant in NOS1AP may increase the susceptibility to severe depression in patients with PTSD and increased risk for suicide.

KPNA3 variation is associated with schizophrenia, major depression, opiate dependence and alcohol dependence.

KPNA3 is a gene that has been linked to schizophrenia susceptibility. In this study we investigated the possible association between KPNA3 variation and schizophrenia. To investigate a wider role of KPNA3 across psychiatric disorders we also analysed major depression, PTSD, nicotine dependent, alcohol dependent and opiate dependent cohorts. Using a haplotype block-based gene-tagging approach we genotyped six KPNA3 single nucleotide polymorphisms (SNPs) in 157 schizophrenia patients, 121 post-traumatic stress disorder patients, 120 opiate dependent patients, 231 alcohol dependent patients, 147 nicotine dependent patients and 266 major depression patients. One SNP rs2273816 was found to be significantly associated with schizophrenia, opiate dependence and alcohol dependence at the genotype and allele level. Major depression was also associated with rs2273816 but only at the allele level. Our study suggests that KPNA3 may contribute to the genetic susceptibility to schizophrenia as well as other psychiatric disorders.

Persons with age-related maculopathy risk genotypes and clinically normal eyes have reduced mesopic vision.

PURPOSE: To determine whether participants with normal visual acuity, no ophthalmoscopically signs of age-related maculopathy (ARM) in both eyes, and who are carriers of the CFH, LOC387715, and HRTA1 high-risk genotypes (gene-positive) have impaired rod- and cone-mediated mesopic visual function compared with persons who do not carry the risk genotypes (gene-negative). METHODS: Fifty-three Caucasian study participants (mean 55.8 ± 6.1) were genotyped for CFH, LOC387715/ARMS2, and HRTA1 polymorphisms. Single-nucleotide polymorphisms were genotyped in the CFH (rs380390), LOC387715/ARMS2 (rs10490924), and HTRA1 (rs11200638) genes using optimized gene-expression assays. The critical fusion frequency (CFF) mediated by cones alone (long-, middle-, and short-wavelength sensitive cones, LMS) and by the combined activities of cones and rods (LMSR) were determined. The stimuli were generated using a four-primary photostimulator that provides independent control of the photoreceptor excitation under mesopic light levels. Visual function was further assessed using standard clinical tests, flicker perimetry, and microperimetry. RESULTS: The mesopic CFF mediated by rods and cones (LMSR) was significantly reduced in gene-positive compared to gene-negative participants after correction for age (P = 0.03). Cone-mediated CFF (LMS) was not significantly different between gene-positive and -negative participants. There were no significant associations between flicker perimetry and microperimetry and genotype. CONCLUSIONS: This is the first study to relate ARM risk genotypes with mesopic visual function in clinically normal persons. These preliminary results could become of clinical importance because mesopic vision may be used as a biomarker to document subclinical retinal changes in persons with risk genotypes and to determine whether those persons progress into manifest disease.