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Nitric oxide pathway genes (NOS1AP and NOS1) are involved in PTSD severity, depression, anxiety, stress and resilience
Bruenig, Dagmar; Morris, Charles P; Mehta, Divya; et.al. https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12150219300002621?l#13151196550002621
Bruenig, D., Morris, C. P., Mehta, D., Harvey, W., Lawford, B., Young, R., & Voisey, J. (2017). Nitric oxide pathway genes (NOS1AP and NOS1) are involved in PTSD severity, depression, anxiety, stress and resilience. Gene, 625, 42–48. https://doi.org/10.1016/j.gene.2017.04.048 Document Type: Accepted Version
Link to Published Version: https://doi.org/10.1016/j.gene.2017.04.048
USC Research Bank: https://research.usc.edu.au [email protected] CC BY-NC-ND V4.0 Copyright © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ Downloaded On 2021/09/28 07:36:42 +1000
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Nitric oxide pathway genes (NOS1AP and NOS1) are involved in PTSD severity, depression,
anxiety, stress and resilience
Dagmar Bruenig1,2, Charles P. Morris1, Divya Mehta1, Wendy Harvey2, Bruce Lawford1,
Ross McD Young1, Joanne Voisey1*
1 Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences
60 Musk Avenue
Queensland University of Technology
Kelvin Grove, Queensland, 4059
Australia
2 Gallipoli Medical Research Institute,
Greenslopes Private Hospital,
Newdegate Street,
Greenslopes QLD 4120
*Corresponding author
Joanne Voisey
School of Biomedical Sciences
Faculty of Health
Institute of Health and Biomedical Innovation (IHBI)
60 Musk Avenue
Queensland University of Technology
Kelvin Grove, Queensland, 4059
Australia
Email: [email protected] 2
Phone: +61 7 3138 6261
Fax. +61 7 3138 6030
3
Abstract
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.
Key Words: PTSD, depression, anxiety, resilience, NOS1AP, NOS1
4
Highlights
• Association of NOS1AP and NOS1 with PTSD, comorbidities and resilience
(coping) was tested
• A well-screened cohort of combat exposed cases and controls was utilised
• Association between NOS1AP and PTSD severity, depression, anxiety, and stress
was found
• Association between NOS1 and PTSD severity and stress was found
• NOS1AP and NOS1 were both associated with resilience
5
1. Introduction
Posttraumatic stress disorder (PTSD) is a severe psychiatric disorder that some individuals
may develop following from the experience of a traumatic event (American Psychiatric
Association, 2013). Despite similar traumatic exposure only a small portion of the population
develops PTSD while the majority will show no clinically relevant symptoms (Schmidt et al.,
2013). There is a strong genetic component to PTSD with the heritability generally reported
to be about 40% (Lyons et al., 1993; Sartor et al., 2012). The molecular aetiology of PTSD is
still poorly understood. The biological stress system (hypothalamic pituitary-adrenal axis;
HPA axis) and genes of that pathway have typically been the focus of investigations into the
aetiology of the disorder due to its centrality in the stress response.
The activation of the stress cascade initiates processes within the HPA axis and in adjacent
systems, often with detrimental effect. The nitric oxide pathway is directly linked with the
HPA axis and has been hypothesised to play an integral role in development of PTSD
(Freudenberg et al., 2015). Hippocampal atrophy is often observed in individuals with PTSD
(Smith, 2005), and HPA feedback dysfunction is thought to be at the centre of the disorder
due to a dysregulation of glucocorticoids (Yehuda, 2006). The nitric oxide pathway may
contribute to these symptoms as nitric oxide impacts on gamma-aminobutyric acid (GABA)
and glutamate levels. Furthermore, recent research indicates that there might be a direct link
between nitric oxide synthase 1 (NOS1) and glucocorticoid regulation (Chen et al., 2015).
Two key genes in the nitric oxide pathway are of interest in mental health research. The nitric
oxide synthase 1 adaptor protein gene (NOS1AP; previously known as carboxyl-terminal
PDZ ligand of neuronal nitric oxide synthase protein gene: CAPON) produces an adaptor
protein that binds to the enzyme NOS1 (encoded by the nitric oxide synthase 1 gene, NOS1),
and influences n-methyl-d-aspartate (NMDA) receptor activity. Changes in receptor activity 6
have been associated with modulation of nitric oxide production. Increased nitric oxide causes a decrease in overall GABA level leading to HPA axis feedback dysfunction and an increase in glutamate causing excito-toxicity in the hippocampus (for a review, see (Chen et al., 2015) and (Wang et al., 2016)). A simplified schematic of the nitric oxide pathway including two of the key genes in connection with the HPA axis is shown in Figure 1.
7
Figure 1: A simplified schematic of the relationship between the HPA-Axis and the nitric oxide pathway including two key genes (NOS1AP and NOS1)
Figure Legend: Triggering of the HPA axis initiates nitric oxide synthase 1 (NOS1)
production. NOS1 is encoded by the NOS1 gene. Nitric oxide synthase 1 adaptor protein
encoded by the NOS1AP gene binds with NOS1 and mediates N-methyl-D-aspartate
(NMDA) receptor availability. Increased NOS1 activity down-regulates NMDA receptors and increases nitric oxide. Nitric oxide is known to lower GABA levels causing increased glutamate levels. Low GABA levels are associated with HPA axis feedback dysfunction and adrenal stress. Increased glutamate levels can cause cell burnout (excito-toxicity) leading to hippocampal atrophy as observed in PTSD patients (Chen et al., 2015; Freudenberg et al.,
2015; Wang et al., 2016).
8
NOS1AP has been studied in a range of psychiatric disorders, including schizophrenia
(Brzustowicz et al., 2004), autism spectrum disorder and obsessive-compulsive disorder
(Delorme et al., 2010). Associations of NOS1AP variants with depression have previously been found in a cohort with schizophrenia (Cheah et al., 2015) and also with PTSD severity in a cohort of untreated combat veterans (Lawford et al., 2013). For NOS1, associations have been found with anxiety and depression in healthy cohorts (Harro et al., 2010; Kurrikoff et al., 2012) and a large general population sample (Sarginson et al., 2014). A genome-wide association study of personality traits and psychological distress established an effect of
NOS1 on psychological distress (Luciano et al., 2012).
The systematic investigation of the genetics of resilience and coping is still in the early phases (Dunn and Conley, 2015). A few genes have been identified to potentially play a role in resilience and coping, such as the sodium-dependent serotonin transporter and solute carrier family 6 member 4 gene (SLC64A) and the dopamine receptor D2 gene (DRD2),
(Wilhelm et al., 2007; van der Zwaluw et al., 2011), the beta-2 adrenergic receptor gene
(ADRB2) (Poole et al., 2006; Liberzon et al., 2014), and the brain-derived neurotrophic factor gene (BDNF) (Caldwell et al., 2013). Some of these studies identified genetic association with maladaptive coping strategies, indicating a risk rather than a protective factor (van der
Zwaluw et al., 2011). The finding of associations between NOS1 and neuroticism, which is commonly associated with maladaptive coping behaviour (Luciano et al., 2012), further support the role of NOS1 in poor coping outcomes. Research into another PTSD candidate
gene BDNF indicates that the Val allele of the rs6265 (Val66Met) single nucleotide
polymorphism (SNP) may have protective properties whereas the Met allele may be the risk
factor for the disorder (Jiang et al., 2005; Bruenig et al., 2016). 9
Due to an increased body of research suggesting involvement of the nitric oxide pathway in mental health, it would be beneficial to understand any protective factors that may derive from genes of the nitric oxide pathway. Such genetic factors may contribute to enhanced
homeostasis from the biological stress reaction and allow for more effective recovery from
extreme stress. Understanding the role of genes in resilience and coping may lead to
improved prevention methods for stress-related maladaptive behaviours (Dunn and Conley,
2015).
Based on the reported role of NOS1AP and NOS1 in psychiatric disorders, this study investigated the association between these genes and PTSD and its common comorbidities in a cohort of Australian Vietnam war veterans with comparable trauma exposure. Based on previous findings, it was hypothesised that SNPs of NOS1AP and NOS1 will be associated with PTSD severity as well as depression, anxiety and stress within our cohort. Extending from the idea that an adequately working biological stress response allows for recovery from severe stress, it was further hypothesised that SNPs may also be associated with resilience measures.
2. Materials and methods
2.1 Participants
A total of 299 gender- and age-matched Vietnam veterans were recruited through
Greenslopes Private Hospital and the Returned and Services League of Australia by the
Gallipoli Medical Research Foundation. For inclusion in the study, participants had to be
deployed to Vietnam during the war. The mean age of the all-male cohort was 68.82 years
(SD = 4.2). Diagnostic status was achieved through structured interviews by psychiatrists
specialised in the assessment and differential diagnosis of PTSD at the hospital. A total of 10
159 participants received a PTSD diagnosis, and 140 participants did not meet diagnostic
criteria despite comparable trauma-exposure. Psychologists used validated psychological
measures for further assessment. Table 1 shows an overview of the characteristics of the
cohort by diagnostic status.
2.2 Ethics
Each participant gave written informed consent before commencement of data collection. Ethics
approval for the project was obtained from the Human Research Ethics Committee of the Queensland
University of Technology and Greenslopes Private Hospital. This study was carried out in accordance
with The Code of Ethics of the World Medical Association (Declaration of Helsinki).
2.2 Genotyping
Blood samples were sent to the Australian Genome Research Facility (AGRF) for DNA
extraction and genotyping. Upon collection, samples were stored at -20°C. Using
MACHERY-NAGEL NucleoSpin L (MACHEREY-NAGEL GmbH & Co. KG, Dueren,
NRW, Germany) genomic DNA was extracted from a 8 ml blood sample. Quality of the
DNA was assessed through resolution on a 0.8% agarose gel at 130 V for 60 minutes.
Samples were normalised to 200 ng of DNA in 4 μL.
The Illumina PsychArray-24 BeadChip scanned with the Illumina iScan systems was used for
genotyping. Data was exported and quality checks were performed using GenomeStudio
v2011.1 (Illumina, San Diego, CA, USA) with Genotyping Module 1.9.4 software using the
InfiniumPsychArray-24v1-1_A1 manifest and cluster files to call the genotypes. The
genotype call rate was 99.59% across the remaining variants after quality control.
Eleven samples were performed in duplicate for quality assurance with 99.9% reproducibility
rate across all SNPs from NOS1AP and NOS1. Of the SNPs within NOS1 and NOS1AP we 11
filtered for > 5% minor allele frequency (MAF), genotyping call rate of > 90% and Hardy-
Weinberg Equilibrium (HWE, p > 0.05), allowing 16 of 44 SNPs in NOS1 and 49 of 54 SNPs
in NOS1AP for subsequent analyses. An overview of all SNPs included in the analyses and
their position can be found in the supplementary material (Table S1).
2.3 Scales
Clinician-Administered PTSD Scale (CAPS): Trained psychologists assessed severity of
PTSD with the Clinician Administered PTSD Scale for DSM 5 (CAPS; Weathers et al.
(2014)). This instrument is regarded as the gold standard for the assessment of current PTSD
symptoms. Higher scores reflect increased PTSD severity.
The MINI (Mini International Neuropsychiatric Interview DSM IV) is an instrument
designed to assess major axis 1 disorders and was used in this study to assess common
psychiatric comorbidities. It has high validity and reliability (Sheehan et al., 1998).
The Depression Anxiety Stress Scale 21 (DASS) is a self-report scale. Three subscales measure three different constructs of psychological states: stress, depression and anxiety
(Lovibond and Lovibond, 1995a). The scales have shown only moderate correlation despite a common factor, and a three-scale model had been confirmed by second-order factor analysis
(Lovibond and Lovibond, 1995b). Higher scores reflect increased symptoms of depression, anxiety and stress, respectively. Cronbach’s Alpha was high: α = 0.95.
Resilience was measured through the Connor-Davidson Resilience Scale (CD-RISC). The scale is comprised of items that reflect strategies that have been shown to be successful mediators in dealing with stress (Connor and Davidson, 2003). The scale has good psychometric properties (Bezdjian et al., 2016). Higher scores indicate higher resilience.
Cronbach’s Alpha was high: α = 0.92. 12
2.4 Statistical Analysis
Hardy-Weinberg Equilibrium was calculated using Utility Programs for Analysis of Genetic
Linkage (Ott, 1988). To correct for multiple testing, the false detection rate (FDR) methodology was used with a 10% threshold using R (https://www.r-project.org/). All other statistics were performed using SPSS (2015).
Analyses of variance (ANOVA) were used to perform genetic analyses on genotype level unless there was a small representation of the minor homozygotes in which case dominant models were pursued (independent t-test). If a genotype-based analysis suggested potential for a dominant model the dominant model was further pursued for that particular SNP
(independent t-test).
3 Results
3.1 Demographics
An all-male cohort of 299 participants was used in this study. Table 1 shows an overview of the characteristics of the cohort by diagnostic status. The PTSD group had significantly more common psychiatric co-morbidities than the control group as would be expected. Higher numbers of participants in the PTSD group than the control group were taking psychotropic medications, such as anti-depressants and anti-anxiety medications.
Mean scores for all scales (CAPS, DASS, CD-RISC) were significantly different between the groups, with higher mean scores for all scales in the PTSD group than the controls with the resilience scale showing reverse results as would be expected (Table 3). 13
After screening for ethnicity and relatedness, 294 participants remained for analyses (156
cases, 138 controls). Any additional exclusions were based on failed genotype calls per SNP only.
14
Table 1: Demographics and clinical summary of the cohort by diagnostic status
PTSD No PTSD p-value
(159) (140)
Age M (SD) 68.47 (4.16) 69.23 (4.13) 0.113
Marital Status (current) 0.494
Married (current) 116 108
Divorced/Separated (current) 9 8
Psychotropic Medication Yes: 94 Yes: 15 5.4979E-19
No: 51 No: 112
Education level 0.005
Less than year 10 26 8
Year 10 29 23
Vocational 32 20
Year 11 or 12 34 33
University 37 56
Comorbidities1
Major depression 21 2 0.000419
Suicide risk 31 2 0.000002
Agoraphobia 33 6 0.000080
Social phobia 8 0 0.017
Alcohol dependence 22 6 0.019
Alcohol abuse 4 1 0.029
Generalised anxiety disorder 12 3 0.092 15
Note. M = mean; SD = standard deviation; 1all comorbidity counts as per Mini International
Neuropsychiatric Interview (MINI) for DSM IV (Sheehan et al., 1998). Only a subset of all comorbidities is shown. Rare comorbidities with no current information or both groups = 0 were excluded from the table. 16
3.2 Scales
Correlations between scales met expectations with moderate to high positive correlations between all DASS scales and moderate positive correlations between all DASS scales and
PTSD severity. Resilience was moderately negatively correlated with all other scales. All scale correlations can be found in Table 2.
Table 2: Correlations (Spearman’s rho) of scales across the entire cohort
CAPS Severity DASS Depression DASS Anxiety DASS Stress CD RISC
CAPS 1 0.610; 0.680 0.677 -0.473 Severity p = 8.7143E-31 p = 1.834E-40 p = 6.0803E-40 p = 1.8605E-17
DASS 0.610 1 0.683 0.702 -0.555 Depression p = 8.7143E-31 p = 7.7049E-41 p = 3.3321E-43 p = 1.2335E-24
DASS 0.680 0.683 1 0.767 -0.494 Anxiety p = 1.834E-40 p = 7.7049E-41 p = 8.175E-59 p = 3.7887E-19
DASS Stress 0.677 0.702 0.767 1 -0.558 p = .0803E-40 p = 3.3321E-43 p = 8.175E-59 p = 5.8173E-25
CD RISC -0.473 -0.555 -0.494 -0.558 1 p = 1.8605E-17 p = 1.2335E-24 p = 3.7887E-19 p = 5.8173E-25
Scale distributions in the case and control groups were accepted as normal, respectively, with the exception of CAPS severity and DASS depression and DASS anxiety. Even though the chosen analyses are relatively stable against violations of normality the analyses were performed both parametrically and non-parametrically on those three scales, and findings were only accepted as significant if they were significant in both models.
17
Table 3: Mean scores, standard deviations, and significance levels between the PTSD and no-
PTSD group for all psychological scales
CAPS DASS DASS DASS CD-RISC
Severity Depression Anxiety Stress
Group PTSD: PTSD: PTSD: PTSD: PTSD:
M (SD) 15.64 (9.79) 6.41 (4.97) 6.14 (4.46) 10.42 (4.77) 68.28 (15.37)
Group No PTSD: No PTSD: No PTSD: No PTSD: No PTSD:
M (SD) 2.52 (3.72) 2.25 (2.66) 1.24 (1.78) 4.34 (3.41) 70.12 (11.08) p value 2.6374E-36 1.8578E-17 4.9531E-28 7.7568E-30 1.3318E-11
Note. M = mean; SD = standard deviation
18
3.3 Analysis of clinical data
Univariate analyses of variance (ANOVA) and independent sample t-tests were performed to
detect associations with CAPS severity, DASS scales and CD-RISC based on diagnostic
status. Even though we found a substantial correlation between the scales we opted to not
pursue a multivariate approach as individual SNPs were not expected to be associated across
all scales.
PTSD Severity: ANOVA for NOS1AP and PTSD severity revealed four SNPs that were
associated with PTSD severity within the PTSD group. SNPs rs10918936, rs12027674,
rs10157390, and rs2264911 all reached significance in the dominant model (p = 0.042, p =
0.022, p = 0.019, p = 0.038, respectively). ANOVA for NOS1 and PTSD severity showed
that rs10744891 was associated with PTSD severity in the PTSD group (p = 0.005).
DASS Scale: ANOVA tests of the relationship between the DASS subscales and NOS1AP
revealed that rs12116744 was associated with the DASS depression subscale in the PTSD
group (p = 0.048). NOS1AP rs111479527 was associated with anxiety ratings, also in the
PTSD group (p = 0.016). None of the NOS1 SNPs were associated with DASS depression or
anxiety. We found rs164148 from NOS1AP associated with the stress subscale in the PTSD
group (p = 0.003). One SNP from NOS1, rs7295972, was also associated with stress in the
PTSD cohort (p = 0.016).
Resilience: Test for a relationship of NOS1AP and resilience showed that two SNPs were
associated with resilience, one in the PTSD group (rs17460657; p = 0.037), the other in the
control group (rs4657178; p = 0.020). For NOS1, we found rs10744891 was associated with
resilience in the PTSD group (p = 0.033). This SNP was also associated with PTSD Severity
(Figure 2). 19
Figure 2: Means and standard errors for NOS1 rs10744891 (T-dominant model) association with resilience (left) and PTSD severity (right) in the PTSD cohort.
76 20
74 72 15 70 68 10 RISC Scores
- 66
CD 64 5 CAPS CAPS Severity Scores 62 60 0 TT+TG GG TT+TG GG rs10744891 rs10744891
Significant results for all t-tests can be found in Table 4. Means and standard errors for each significant SNP can be found in the supplementary material (Table S2). Allele frequencies
and positions of the significant SNPs can be found in the supplementary material (Table S3).
The rs10744891 SNP remained associated with PTSD severity after correction for multiple
testing using the FDR method with a 10% threshold. All significant results were found in the
respective dominant model. All SNPs are intron SNPs with the exception of rs164148 which
is in the 3’ untranslated region of NOS1AP.
20
Table 4
SNPs and significant findings from the independent t-tests across CAPS severity, DASS depression, anxiety, stress scale and Connor-Davidson
Resilience Scale (CD-RISC)
CAPS Severity DASS Depression DASS Anxiety DASS Stress Connor-Davidson Resilience
(CD-RISC)
NOS1AP rs10918936 t(152) = 2.055; p = 0.042;
(p = 0.023)** rs12027674 t(152) = -2.351; p = 0.022;
(p = 0.014)** rs10157390 t(152) = -2.363; p = 0.019;
(p = 0.010)** rs12725553 t(152) = -2.088; p = 0.038;
(p = 0.028)** rs12116744 t(118.176) = 1.999; p = 0.048;
(p = 0.019)** rs111479527 t(154) = -2.438; p = 0.016;
(p = 0.020)** 21
rs164148 t(152) = -2.996; p = 0.003** rs4657178 t(135) = 2.362; p = 0.020**
(No PTSD group) rs17460657 t(154) = -2.108; p = 0.037**
(PTSD group)
NOS1 rs10744891 t(146) = 2.875; p = 0.005; t(148) = -2.147; p = 0.033**
(p = 0.007)** (PTSD group) rs7295972 t(154) = -2.435; p = 0.016**
Notes: CAPS = clinician-administered PTSD scale (Weathers et al., 2014), PTSD = posttraumatic stress disorder, DASS = depression, anxiety, stress scale (Lovibond, 1995); 22
4. Discussion
In this study, the role of NOS1AP and NOS1 in PTSD, its common co-morbidities and resilience was investigated. Significant associations of the genes with psychiatric disorder and resilience were found. These two genes are essential in the regulation of the nitric oxide pathway which is closely related to the HPA axis. HPA axis activation impacts on nitric oxide synthesis with significant effects on HPA axis feedback dysfunction and hippocampal atrophy, which are both central symptoms of PTSD (Smith, 2005; Yehuda, 2006). However, for most individuals a traumatic event does not result in a psychiatric disorder. Resilience is marked by successful psychological traits and strategies that may be a deciding factor determining the onset of PTSD. Viewing resilience as the absence of the disorder alone may not be sufficient to understand the complexity of the stress response (Feder et al., 2009;
Bruenig et al., 2014; Shakespeare-Finch and Lurie-Beck, 2014)
This study found support for the hypotheses that NOS1AP SNPs are associated with PTSD severity, depression, anxiety and stress, indicating that this gene may play a vital role in mental health problems. NOS1AP is also implicated in other severe psychiatric disorders such as schizophrenia (Cheah et al., 2015; Wang et al., 2016). There was no association between
NOS1 SNPs and depression or anxiety. Other studies have found association with anxiety and depression for this gene but they focussed on polymorphisms from the promoter region of the gene (Harro et al., 2010; Kurrikoff et al., 2012). The reason this study did not detect associations may be attributed to a lack of polymorphic SNPs from the promotor region included in the Illumina psychiatric SNP array. The other two studies were also markedly different in their design from our study as they investigated mood states in the context of personality traits in healthy cohorts (Harro et al., 2010; Kurrikoff et al., 2012). It is possible that the differences in population selection and the constructs examined also account for the lack of association in our study. However, the findings of association between NOS1 SNPs 23
and PTSD severity and stress are in line with physiological expectations. Future studies should involve further investigation of this gene in cohorts with stress-, mood- and anxiety-
related disorders.
Associations between NOS1AP and NOS1 SNPs and resilience were found, both in the PTSD
group and the control group. One NOS1AP SNP (rs4657178) was associated with resilience
in the controls indicating potentially strong protective properties. Uncovering genetic variants
that may protect individuals from PTSD may have important implications for deployment
strategies in military cohorts. The association of two SNPs from NOS1AP and NOS1 with
resilience within the PTSD group indicates that potential protective properties of the SNPs
may not suffice to prevent the development of PTSD. However, they may decrease disease
severity and hence provide promising therapeutic targets for those with more severe disease.
In this context, the finding of different genotype effects for PTSD severity and resilience for
rs10744891 within the NOS1 gene may be of interest to further elucidate potential functional
consequences of the SNP.
The cohort, while exposed to severe trauma as young adults, is a surviving cohort that has been well enough to participate in an extensive research study. This indicates a relatively high level of functioning, which may be a reflection of resilience that may co-exist with severe and chronic stress. In a large meta-analysis, Shakespeare-Finch and Lurie-Beck found that posttraumatic growth and PTSD can co-exist (Shakespeare-Finch and Lurie-Beck, 2014).
Posttraumatic growth involves the ability to achieve personal growth from the experience of severe trauma and is an indicator of positive outcomes of the experience of severe stress.
Further investigations of biological processes that may moderate the relationship between genetic predisposition and phenotypes spanning the entire spectrum of possible reactions to 24
traumatic stress may clarify our understanding of the co-existence of stress states, resilience, and positive outcomes.
Findings from this study support a role for NOS1AP and NOS1 in PTSD, its most common comorbidities, and resilience. Identifying a molecular basis for risk and resilience will be of great importance for the prevention and treatment of PTSD in at-risk groups (Liberzon et al.,
2014). Given the recently growing body of evidence from animal research indicating that the nitric oxide pathway may be of importance for the development of alternative anti-depressant and anxiolytic drugs (Wegener and Volke, 2010; Gigliucci et al., 2014; Kouti et al., 2016) further studies involving key genes of the nitric oxide pathway will be of particular interest.
The study findings should be interpreted with caution. Given the modest cohort size, it is conceivable that the Type 2 error rate is inflated. Nevertheless, strict statistical criteria were applied to the findings from the large number of analyses that were conducted. Given that we operated on a hypothesis basis and our analyses were not independent, correction for multiple testing may be viewed as too restrictive; e.g. (Rothman, 1990). A number of participants in this cohort are also taking anti-depressant, anti-anxiolytic and anti-psychotic medication which may have resulted in generally lower severity scores. Hence, there is a chance that there could have been stronger and more associations of SNPs of the genes with PTSD severity, anxiety and depression than we were able to identify. Confirmation of the findings
awaits validation and replication in follow-up studies of larger cohorts with male and female
participants. The current study had a male-only cohort and findings cannot be generalised
across gender. Previous studies investigating the association of NOS1 and NOS1AP with
maladaptive behavioural traits have shown mixed findings across gender (Harro et al., 2010;
Kurrikoff et al., 2012) and a higher prevalence of association of depression and NOS1AP in
males has been reported (Cheah et al., 2015). 25
5. Conclusion
Using a tightly-controlled and well-characterised cohort of male combat veterans, this study demonstrated that NOS1AP and NOS1 may play an integral role in PTSD and its most common comorbidities. Replication of the findings using defined measures would extend our knowledge and may elucidate phenotypic differences in PTSD. Potential protective properties of NOS1AP and NOS1 genes were identified that should be investigated for their interaction with stress and resilience and future therapeutic development.
26
Conflict of Interest
None
Author Contributions
Dagmar Bruenig and Joanne Voisey substantially contributed to the study design, statistical
analyses, writing and critical editing of the manuscript. Charles P. Morris substantially
contributed to the study design, writing and critical editing of the manuscript. Divya Mehta
substantially contributed to the statistical analyses and critical editing of the manuscript.
Bruce Lawford and Ross McD Young substantially contributed to the study design and
critical editing of the manuscript. Wendy Harvey substantially contributed to the study
design, ethics submission and data collection. All authors reviewed and approved the final
version of the manuscript for publication.
Role of Funding
The PTSD Initiative (or ‘This study’) was funded by the Queensland Branch of the Returned
& Services League of Australia (RSL QLD). Financial support was also provided by the
Institute of Health and Biomedical Innovation and the School of Biomedical Sciences,
Queensland University of Technology, Australia.
Acknowledgements
The first author would like to thank the Gallipoli Medical Research Foundation for their
generous provision of a scholarship to DB, and Miriam Dwyer and Dr Sarah McLeay for
their project management support. The authors would also like to acknowledge Dr Madeline
Romaniuk for psychological input, Dr John Gibson and the team at the Keith Payne Unit, and
the staff and investigators at Greenslopes Private Hospital for their valuable contribution to 27
the study. All authors would like to extend their gratitude to the participants of our study for their generous provision of data and time. The Gallipoli Medical Research Foundation wishes to thank the RSL QLD for their generous donation, and Sullivan Nicolaides Pathology and
Queensland X-Ray for their in-kind support.
28
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