- Email: [email protected]
Genetic association of FKBP5 with PTSD in US service members deployed to Iraq and Afghanistan
Journal of Psychiatric Research 122 (2020) 48–53
Contents lists available at ScienceDirect
Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires
Genetic association of FKBP5 with PTSD in US service members deployed to Iraq and Afghanistan
T
Lei Zhanga,∗,1, Xian-Zhang Hua,1, Tianzheng Yub,1, Ze Chena, Jacob Dohlb, Xiaoxia Lia, David M. Benedeka, Carol S. Fullertona, Gary Wynna, James E. Barrettc, Mian Lid, Dale W. Russella,b, Biomarker team, Robert J. Ursanoa,1 a
Center for the Study of Traumatic Stress, Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA Consortium for Health and Military Performance, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA c Department of Neurology, Drexel University College of Medicine Philadelphia, PA, 19102-1192, USA d Department of Neurology, Washington DC VA Medical Center, Washington, DC, 20422, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: PTSD FKBP5 Single-nucleotide polymorphism (SNP) Haplotype Military
Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with a prevalence of more than 7% in the US population and 12% in the military. An interaction of childhood trauma with FKBP5 (a glucocorticoidregulated immunophilin) has been reported to be associated with PTSD in the general population. However, there are few reports on the association of FKBP5 with PTSD, particularly in important high-risk population such as the military. Here, we examined the association between four single-nucleotide polymorphisms (SNPs; rs3800373, rs9296158, rs1360780, rs9470080) covering the FKBP5 gene and probable PTSD in US service members deployed to Iraq and Afghanistan, a high-risk military population (n = 3890) (Hines et al., 2014). We found that probable PTSD subjects were significantly more likely to carry the A-allele of rs3800373, G-allele of rs9296158, C-allele of rs1360780, and C-allele of rs9470080. Furthermore, the four SNPs were in one block of strong pairwise linkage disequilibrium (r = 0.91–0.96). Within the block there were two major haplotypes of CATT and AGCC (rs3800373-rs9296158-rs1360780-rs9470080) that account for 99% of haplotype diversity. The distribution of the AGCC haplotype was significantly higher in probable PTSD subjects compared to nonPTSD (p < .05). The diplotype-based analysis indicated that the AGCC carriers tended to be probable PTSD. In this study, we demonstrated the association between FKBP5 and probable PTSD in US service members deployed to Iraq and Afghanistan, indicating that FKBP5 might be a risk factor for PTSD.
1. Introduction Posttraumatic stress disorder (PTSD) is a chronic and debilitating condition with characteristic symptoms, including re-experiencing of trauma, avoidance, negative alterations in cognition and mood, and hyperarousal triggered by life-threatening traumatic events. APA, 2013 (Kessler et al., 1995). However, PTSD is only observed in a fraction of those exposed to trauma (Stein et al., 2002). A lifetime trauma incidence is 40%–90% in the general population, but the overall lifetime prevalence for PTSD ranges from 7% to 12% (Kessler et al., 1995), indicating that a complex interplay of multiple factors is decisive (Stein et al., 2002). Genetic susceptibility might also play a role in the development of PTSD (Koenen, 2007). Children whose parents have PTSD
show higher rates of PTSD than the general population (Stein et al., 2002). Twin and family studies also show a genetic component in the development of PTSD (Koenen et al., 2005; Stein et al., 2002). It is believed that genetic influences account for about one-third of the variance in risk of developing PTSD (True et al., 1993). Binder et al. (2008) identified four single nucleotide polymorphisms (SNPs) in the FKBP5 gene interacting with childhood trauma to predict an increased risk of developing adult PTSD (Binder et al., 2008). FKBP5 is a key regulator in the stress hormone system, which mediates responses to traumatic stress – the relationships between PTSD and the dysfunction of hypothalamic-pituitary-adrenal stress axis (HPA) and genetic association have been intensively investigated (Binder, 2009; Binder et al., 2008; Kang et al., 2019; Klengel et al., 2013; Scharf et al., 2011;
∗
Corresponding author. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpsychires.2019.12.014 Received 1 August 2019; Received in revised form 23 December 2019; Accepted 24 December 2019 0022-3956/ © 2019 Published by Elsevier Ltd.
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
2. Material and methods
Schmidt et al., 2015; Zannas et al., 2015). One of the key systems mediating long-term effects of stress and the development of PTSD is the glucocorticoid receptor (GR) system in the HPA. Those with PTSD may be susceptible to stress or mitigated depending on the molecular abnormalities within the GR stress-signaling pathway. As a ligand-activated transcription factor, GR translocates from the cytosol into the nucleus after ligand binding and initiates gene transcription. This process is regulated by a large molecular complex that includes FKBP5 (Binder et al., 2008; Hartmann et al., 2012; Schiene-Fischer and Yu, 2001; Schmidt et al., 2015). FKBP5 is a part of the FKBP/GR complex that regulates GR sensitivity. Glucocorticoid (GC) binds with lower affinity to GR in the FKBP5/GR complex which results in less GR nuclear translocation. FKBP5 polymorphisms are associated with GC-induced FKBP5 expression and GR sensitivity. It is known that FKBP5 plays a key role in an intracellular ultra-short negative feedback loop for GR activity (Reynolds et al., 1998, 1999). Alterations in FKBP5 expression are involved in abnormal GR-mediated signaling in neurons involved in the stress response and memory formation (Fujii et al., 2014; Szabo et al., 2014). A cortisol-induced higher level of FKBP5 is associated with PTSD (Binder, 2009), indicating the role of FKBP5 in PTSD in the GR system. FKBP5 is one of the intensively investigated genes in PTSD (Binder et al., 2008; Boscarino et al., 2011; Levy-Gigi et al., 2013; Sarapas et al., 2011; Watkins et al., 2016; Wilker et al., 2014; Xie et al., 2010; Yehuda et al., 2009, 2016). Notably, the interaction of FKBP5 SNPs with childhood trauma, but not with adult trauma, predicts greater self-reported PTSD symptoms (Binder et al., 2008; Xie et al., 2010). Lower mRNA expression of FKBP5 predicts higher severity of PTSD symptoms after combat (Skelton et al., 2012). A study in two nationally representative samples of European-American (EA) U.S. military veterans revealed that the four FKBP5 SNPs are associated with PTSD symptom severity. SNP rs9470080 in the main sample, and all four SNPs in the replication sample interact with childhood abuse to predict PTSD severity. An association of FKBP5 polymorphisms with PTSD directly and/or with childhood abuse interactively can predict severity of lifetime PTSD symptoms (Watkins et al., 2016). Another study in nationally representative samples of US military veterans shows that FKBP5, childhood abuse and an insecure attachment style are associated with greater severity of PTSD symptoms, indicating gene x environment interactions (Tamman et al., 2019). FKBP5 expression level is also associated with the size of various brain regions and therapeutic responses to PTSD. PTSD patients who have a smaller hippocampus and medial orbitofrontal cortex have lower expression levels of FKBP5 compared to non-PTSD controls (Levy-Gigi et al., 2013). Cognitive-behavioral therapy significantly increases FKBP5 expression and hippocampal volume in patients, suggesting that an improvement in PTSD symptoms may be predicted by increased FKBP5 expression and increased hippocampal volume (Levy-Gigi et al., 2013). Genotypes of FKBP5 are associated with hippocampal function (Fani et al., 2013), cingulum structure (Fani et al., 2014), and a higher risk of PTSD symptoms (Koenen, 2005). Moreover, 4 SNPs (rs9296158, rs3800373, rs1360780, and rs9470080) significantly interacted with the severity of child abuse, but not with non-child abuse trauma exposure, to effect on the level of adult PTSD symptoms (Binder et al., 2008). PTSD is one of only a few disorders in the DSM (American Psychiatric Association, 2013) that require an etiological factor, a traumatic event, for its diagnosis. In addition, approximately half of people with PTSD also suffer from Major Depressive Disorder (Breslau et al., 1997; Rytwinski et al., 2013). Based on the findings above, we investigated these four SNPs in a large sample of US Army soldiers who served in Afghanistan or Iraq between 2008 and 2016, to study the main effects of FKBP5 and effects of interaction of FKBP5 with environmental variables on PTSD. Our results may add to the well-recognized role of FKBP5 in the risk of developing PTSD.
2.1. Subjects Samples (n = 3890) were collected from US Army service members who served during combat operations in Afghanistan and/or Iraq between 2008 and 2016 (Hines et al., 2014). Individuals who did not provide biological samples (e.g. saliva or blood) were excluded. The Institutional Review Board at the Uniformed Service University (USUHS) approved all study procedures and all participants were given written informed consent. 2.2. Survey measures PTSD symptoms were assessed using the PTSD Checklist (PCL), a 17item, DSM-based, self-report measure with well-established validity and reliability (Gelaye et al., 2017; Karstoft et al., 2017). Cronbach's alpha coefficients indicate high internal consistency for the total scale (0.91) and for the theoretical dimensions of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) (0.83, 0.81, and 0.80). Three symptom clusters account for 48.9% of the variance, namely, intrusions, avoidance, and numbing-hyperarousal (Lima Ede et al., 2012). Probable PTSD was diagnosed based on the endorsement of DSM-IV criteria and a PCL total score ≥50. PTSD symptom severity was assessed using the PCL total score. To determine conditions of medication, subjects were asked if any medication (including vitamins, those prescribed by a doctor or one bought at the pharmacy/store, and/ or substance) had been taken in the past 24 h. Subjects with substance abuse, or any medication use were excluded. Demographic data, such as age, gender, and race, were collected. Stressful life events including combat exposure were determined using the Life Events Checklist (LEC, 1995 version) (Gray et al., 2004). Non-PTSD controls might also experience lifetime traumatic events, such as combat or exposure to a war-zone. Subjects’ lifetime traumatic events, including events prior to deployment, were determined. The participants (probable PTSD and non-PTSD controls) in this study were military active duty volunteers. Depression was determined by using the PHQ-9 (Chagas et al., 2013). The PHQ-9 is an adequate instrument for the screening depression. The internal consistency of the scale is 0.83 (Chagas et al., 2013). Sixty-six (1.5%) veterans screened positive for depression. Probable PTSD subjects with comorbidity of depression were enrolled in the study group. Probable PTSD comorbid with other anxiety disorders or somatic disorders were not evaluated in this study. 2.3. Saliva sample collection and DNA extraction Saliva samples were collected using Oragene saliva DNA tube kit (Oragene). Genomic DNA was extracted from a 200 μl saliva sample using the QIAmp protocol (Qiagen, Chatsworth, CA). The quantity and quality of the genomic DNA isolate were determined by 260/280 ultraviolet spectrophotometry (Nanodrop SD-1000 spectrophotometer). 2.4. Genotyping Four SNPs with frequencies of > 5% covering FKBP5 were selected from dbSNP and a published paper indicating two potentially functional SNPs (rs1360780, and rs3800373) and two other SNPs (rs9296158 and rs9470080) that have strong interactions with childhood abuse on adult depression symptoms and PTSD. The TaqMan allelic discrimination assay was used for genotyping on the ABI 7900HT instrument (Applied Biosystems, Foster City, California). Predesigned and validated TaqMan assay reagent kits contain one pair of polymerase chain reaction primers and one pair of fluorescently labeled probes (Applied Biosystems). Polymerase chain reactions (PCR) were performed in 5 μL reaction volumes, in a 384-well plate, and contained 5 ng of DNA. Amplification 49
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
glucocorticoid receptor response element. Fig. 1c shows these four SNPs that span FKBP5 were in one block of strong pairwise linkage disequilibrium (r = 0.91–0.96, Fig. 1c). Within the block there are two haplotypes of CATT and AGCC (rs3800373-rs9296158-rs1360780rs9470080) that account for 99% of haplotype diversity. The distribution of AGCC haplotype was significantly higher in the probable PTSD group compared to the non-PTSD group (p < .05, Table 3a). The diplotype-based analysis indicated that the AGCC carriers tended to fall within the probable PTSD group (Table 3b). The Nagelkerke R Square was 0.265. No correlation between probable PTSD and the interactions of genotypes and lifetime traumatic events (either combat or noncombat related) was found (Table 4). In addition, there was no association between the SNPs of FKBP5 and lifetime traumatic events including child abuse. Moreover, these SNPs did not moderate the association between child abuse and probable PTSD.
conditions were 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles at 96 °C for 15 s and at 60 °C for 60 s. The software of SDS version 2.3 (Applied Biosystems) was used for allelic discrimination. For quality control, 20% of the samples, randomly selected, were genotyped in duplicate. The error rate was < 0.006, and the completion rate was > 0.95. 2.5. Haplotype analysis We used Haploview 4.0 to determine the linkage disequilibrium (LD) structure of the four SNPs covered the FKBP5 gene. LD was indicated by r and visualized using Haploview (Barrett et al., 2005). We used the ‘confidence intervals’ approach to explore the putative block structure. Haplotypes and diplotypes were used to test for association in addition to individual SNPs because they are known to often provide more information.
4. Discussion 2.6. Statistical analyses Population-based association studies (case-control studies) are a widely used design to determine the impact of genetic variants on the risk of developing a particular complex disease (Cardon and Bell, 2001). Many association studies have been performed in order to elucidate the genetic contribution to complex diseases. Through these studies, several low penetrance genes have been found to behave as PTSD risk modifiers, contributing to the understanding of traumatic stress in many types of stress-related disorders and leading to advances in diagnosis and therapy. In this study, we presented the first evidence for an association of FKBP5 variation with lifetime probable PTSD in US Army service members. We found that four SNPs (rs3800373, rs9296158, rs1360780, rs9470080) covering the FKBP5 gene were associated with lifetime probable PTSD in these soldiers. It is consistent with other studies, showing that SNP markers rs16969968, rs9470080, and rs4680 were individually associated with PTSD and that a cumulative allele model using these SNPs was associated with a higher PTSD risk (Boscarino et al., 2011). However, the interaction of the SNPs with LEC showed no effects on PTSD, suggesting that the SNPs might not be moderators for the onset of PTSD. There was no effect of interactions of genotypes and life time traumatic events (either combat or non-combat related) on PTSD, although previous studies reported that two FKBP5 minor allele carriers, childhood abuse and insecure attachment style were associated with greater severity of PTSD symptoms in veterans (Tamman et al., 2019). Those inconsistent results might be related to differences in sampling or characteristics of life time traumatic events. Nevertheless, our results, particularly the data of combat exposure and trauma history, suggested that gene–environment interactions may play the role in PTSD development in the U.S. military population. Service
All analyses were performed using SPSS version 18. Chi-square tests were performed to test the differences in distributions of sex, race, educational levels, and marital status among the groups as well as the association between FKBP5 and lifetime probable PTSD. The parameter data are presented as mean ± S.D. The statistically significant differences in age among the subjects were analyzed using Student's t-test. We utilized regression analysis to examine the effects of the interactions of stressful life events with FKBP5 on probable PTSD and the differences in the magnitudes of observed groups. Covariates included age, sex, and ethnicity. The Bonferroni correction was used for our multiple tests. Our tests included four highly correlated SNPs, the main effects of the SNPs, the effects of LEC with each SNPs on probable PTSD. 3. Results Table 1 shows that participant ages ranged from 18 to 62 years old, with the mean being 29.4 years old. The distribution of ethnicities was not significantly different between the probable PTSD and non-PTSD groups. The participants with probable PTSD were more likely to report combat or exposure to a war-zone than non-PTSD controls (p < .05). The four loci were in Hardy–Weinberg equilibrium (HWE) in our population (p > 0.05). Table 2 shows the frequencies of genotype/allele and haplotype/diplotype in probable PTSD and non-PTSD controls. We found that probable PTSD subjects were significantly more likely to carry the A-allele of rs3800373, G-allele of rs9296158, C-allele of rs1360780, and C-allele of rs9470080. Fig. 1b illustrates the organization of the FKBP5 gene, the location of the four SNPs (Fig. 1a) and the Table 1 Demographic information of the army service member. Total
Race White African American Hispanic Asian or Pacific Islander Others Gender Male Female Combat or exposure to a war-zone Age (mean ± sd; range) Male Female Total
PTSD
Control
N
%
N
%
N
%
1876 274 874 647 219
48.2 7.0 22.5 16.6 5.6
164 19 22.5 39 39
47.7 5.5 24.1 11.3 11.3
1712 255 791 608 180
48.3 7.2 22.3 17.1 5.1
3379 476 1021
87.6 12.4 26.2
295 46 136
86.5 16.5 39.5
3084 430 885
87.8 12.2 24.3**
29.7 ± 8.5; 18-62 27.4 ± 7.2; 18-55 29.4 ± 8.4; 18-62
30.3 ± 8.4; 18-56 28.1 ± 8.0; 18-52 30.1 ± 8.3; 18-56
29.6 ± 8.5; 18–62* 27.3 ± 7.1; 18-55 29.3 ± 8.4; 18-62
sd: standard deviation. * Student t-test, P < 0.05 (males vs females of controls).**χ2 = 23.9, P < 0.0001 (OR = 1.8, 95% CV: 1.4–2.3). 50
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Table 2 Genotype and allele distributions of FKBP5 SNPs in PTSDs and controls. Genotypes
rs1360780 (1: T, 2: C) rs3800373 (1: C, 2: A) rs9296158 (1: G, 2: A) rs9470080 (1: T, 2: C)
Control PTSD Control PTSD Control PTSD Control PTSD
Alleles
11
12
22
P value
1
2
OR, 95% CI
P value
388 (0.10) 15 (0.04) 404 (0.11) 20 (0.06) 1449 (0.40) 163 (0.47) 430 (0.12) 22 (0.06)
1618 (0.42) 155 (0.45) 1541 (0.44) 150 (0.44) 1648 (0.47) 157 (0.46) 1672 (0.48) 163 (0.47)
1840 (0.48) 171 (0.50) 1566 (0.45) 174 (0.50) 444 (0.13) 24 (0.07) 1349 (0.39) 159 (0.46)
0.002
2394 (0.34) 191 (0.28) 2349 (0.33) 190 (0.28) 4556 (0.64) 483 (0.70) 2532 (0.37) 207 (0.30)
4698 (0.66) 497 (0.72) 4673 (0.67) 498 (0.72) 2546 (0.36) 205 (0.30) 4370 (0.63) 481 (0.70)
1.3, 1.1-1.6
0.001
13, 1.1-1.6
0.002
0.2, 0.2-0.3
0.002
2.9, 2.4–3.4
0.001
0.004 0.003 0.001
Fig. 1. The description of FKBP5 gene location, SNPs, glucocorticoid receptor response element (GRE), and haplotypes. (a) Schematic diagram of FKBP5 location on chromosome 6. FKBP5 gene localizes to chromosome 6p21.31 and spans over 154 kbp from 35541362 to 35696360 on the reverse strand. The region surrounding the FKBP5 gene is enlarged. (b) Schematic diagram of the locations of the SNPs around the FKBP5 gene and GRE sites. (c) The relative positions of FKBP5 SNPs and their haplotype block structure. The numbers in the squares refer to pair-wise linkage disequilibrium (LD, r > 0.90).
Table 3a Haplotype distribution in participants with probable PTSD and controls.
Table 3b Diplotype distribution in PTSDs and controls.
Haplotype distribution
PTSD Control
Diplotype distribution
CATT
AGCC
Chi square
P value
185 2141
455 4001
9.112
0.0025
PTSD Control
RR = 1.09; OR = 1.32.
CATT/CATT
AGCC carrier
Chi square
P value
18 344
302 2727
9.45
0.002
RR = 1.06; OR = 2.12.
51
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Table 4 Logistic regression analyses of the effects of the interactions of lifetime traumatic event with SNPs of FKBP5 on PTSD. Variables in the Equation
rs1360780 rs3800373 rs9296158 rs9470080
x x x x
lifetime lifetime lifetime lifetime
traumatic traumatic traumatic traumatic
event event event event
B .023 .024 -.019 .001
S.E. .055 .044 .020 .044
Wald .178 .292 .857 .001
df 1 1 1 1
Sig. .673 .589 .355 .982
Exp(B) 1.023 1.024 .982 1.001
95% C.I. .919 .939 .944 .918
1.140 1.116 1.021 1.091
personnel with PTSD. Our study excluded the subjects who reported medication use within the last month. Those concerns will be considered in future studies. Accumulative association studies show that FKBP5 is indirectly associated with PTSD (Binder et al., 2008; Xie et al., 2010). However, the GWAS meta-analyses show no strong FKBP5 association with PTSD (Zannas et al., 2016). We have also noticed that there is an inconsistency about the findings of the directionality of the risk alleles in publications (Binder et al., 2008). These discrepancies between ours and other's might be due to the sampling. For example, the subjects in this study are active duty military service members who are exposed to unique war-related traumatic events while their subjects are civilians who may have experience with non-war-related traumatic events. In addition, the differences of evaluation procedures might be associated with the discrepancies. In addition, the difference of disease stages between the studies may also be a reason for this inconsistency. Moreover, there are statistical power issues in candidate gene studies. The limitation of the studies are relatively small sample size. Therefore, well-designed conformation studies are needed. In summary, we demonstrated that four SNPs covering the FKBP5 gene were associated with PTSD in US service members deployed to Iraq and Afghanistan. These findings suggest that FKBP5 might be involved in the pathophysiology of PTSD and a potential biomarker for PTSD.
members with different FKBP5 genotypes are affected differently by exposure to the same environmental factors, and thus gene–environment interactions can result in different phenotypes (Grant, 2006; Ridley, 2003). The AGCC haplotype carriers have the higher risks of PTSD. These interactions are of particular interest for predicting PTSD and useful for developing the methods of prevention with respect to public health as well. Since a statistical analysis based on haplotypes is more efficient than separate analyses of the individual markers (Fallin et al., 2001), we also conducted a linkage disequilibrium (LD) analysis revealing strong LD among SNPs in close proximity only. It is known that rs1360780 C/T leads to an allelic-specific RNA expression (Binder et al., 2004). Thus, we considered CATT and AGCC to be yin-yang haplotypes. The AGCC was a high risk haplotype for PTSD. Our data, as well as others’, suggested that psychological alterations are associated not only with inborn genetic bases, but also environmental stressors (Binder et al., 2008). The participants with probable PTSD were more likely to report combat or exposure to a war-zone, indicating that environment (traumatic stress) plays an important role in PTSD development, while gene variants of FKBP5 may promote PTSD symptoms. Life events associated with symptom severity of PTSD, suggesting that symptoms are induced by the trauma. The inconsistency of these results might be due to the differences in sample sizes of the two studies (small vs large) and the characteristics of the samples (civilian vs military). However, these assumptions need to be examined. It is known that several parts of the brain are involved in the pathology of stress response and PTSD, including the amygdala, prefrontal cortex, hippocampus and hypothalamic pituitary adrenal axis (HPAaxis) (Akiki et al., 2017; Neumeister et al., 2017; Reznikov et al., 2017). Functionally, FKBP5, and GR are in a central position to mediate acute and chronic response to stress in the HPA-axis (Binder et al., 2008). In order to respond to stress, the HPA is activated by GR and regulates the on/off production of hormones. FKBP5 inhibits GR translocation to the nucleus. Therefore, it is crucial in an ultra-short feedback loop (Binder et al., 2008; Cioffi et al., 2011), in which FKBP5 regulates GR sensitivity and bioavailability (Binder et al., 2008). In the absence of GC, the GR/ FKBP5 complex is in an inactive stable state. When GC is present, GC is bound to GR, which dissociates from FKBP5/GR complex and is translocated into the nucleus (Binder, 2009; Binder et al., 2004, 2008; Galigniana et al., 2001). In the nucleus, the GR binds to the glucocorticoid response element (GRE) of the FKBP5 gene, leading to FKBP5 mRNA transcription (Binder, 2009; Binder et al., 2004, 2008; Galigniana et al., 2001). Subsequently, FKBP5 inhibits the GR nuclear translocation that is called an ultrashort, feedback loop (inhibition of GR activity) (Binder, 2009). A major limitation of this study is the use of the candidate gene approach. This method may be inappropriate when the genes are already selected. In addition, it chooses a gene either upstream of the points of action or in the downstream signaling pathways. Also, the SNPs selected may provide incomplete coverage of all variants. It sometimes uses small case and control samples with less statistical power, which may be misleading. Moreover, it often relies on prior hypotheses about disease mechanisms which have been reported previously. Therefore, a confirmation study is required. Also, high levels of depressive symptoms, sleep disturbances, and frequent use of medications for insomnia and depression have been reported in military
Funding Center for Traumatic Stress, Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. CRediT authorship contribution statement Lei Zhang: Data curation, Supervision. Xian-Zhang Hu: Data curation. Tianzheng Yu: Data curation. Ze Chen: Data curation. Jacob Dohl: Writing - review & editing. Xiaoxia Li: Data curation. David M. Benedek: Data curation. Carol S. Fullerton: Data curation. Gary Wynn: Data curation. James E. Barrett: Writing - review & editing. Mian Li: Writing - review & editing. Dale W. Russell: Data curation. Robert J. Ursano: Project administration, Writing - original draft. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We thank Supriya Prabhakar, Berwin Yuan, Alexis Shahidi, and Nora Wang for editing the manuscript. The manuscript was improved following comments from Dr. Li Zheng of NIMH, NIH. References Akiki, T.J., Averill, C.L., Wrocklage, K.M., Schweinsburg, B., Scott, J.C., Martini, B., Averill, L.A., Southwick, S.M., Krystal, J.H., Abdallah, C.G., 2017. The association of PTSD symptom severity with localized Hippocampus and amygdala abnormalities. Chronic Stress (Thousand Oaks) 1. APA, 2013. Diagnostic and Statistical Manual of Mental Disorders, 5th. American Psychiatric Association, Arlington, VA, USA.
52
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Koenen, K.C., Hitsman, B., Lyons, M.J., Niaura, R., McCaffery, J., Goldberg, J., Eisen, S.A., True, W., Tsuang, M., 2005. A twin registry study of the relationship between posttraumatic stress disorder and nicotine dependence in men. Arch. Gen. Psychiatr. 62 (11), 1258–1265. Levy-Gigi, E., Szabo, C., Kelemen, O., Keri, S., 2013. Association among clinical response, hippocampal volume, and FKBP5 gene expression in individuals with posttraumatic stress disorder receiving cognitive behavioral therapy. Biol. Psychiatry 74 (11), 793–800. Lima Ede, P., Barreto, S.M., Assuncao, A.A., 2012. Factor structure, internal consistency and reliability of the Posttraumatic Stress Disorder Checklist (PCL): an exploratory study. Trends Psychiatr. Psychother. 34 (4), 215–222. Neumeister, P., Feldker, K., Heitmann, C.Y., Buff, C., Brinkmann, L., Bruchmann, M., Straube, T., 2017. Specific amygdala response to masked fearful faces in post-traumatic stress relative to other anxiety disorders. Psychol. Med. 1–11. Reynolds, P.D., Roveda, K.P., Tucker, J.A., Moore, C.M., Valentine, D.L., Scammell, J.G., 1998. Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis). Lab. Anim. Sci. 48 (4), 364–370. Reynolds, P.D., Ruan, Y., Smith, D.F., Scammell, J.G., 1999. Glucocorticoid resistance in the squirrel monkey is associated with overexpression of the immunophilin FKBP51. J. Clin. Endocrinol. Metab. 84 (2), 663–669. Reznikov, R., Bambico, F.R., Diwan, M., Raymond, R.J., Nashed, M.G., Nobrega, J.N., Hamani, C., 2017. Prefrontal Cortex Deep Brain Stimulation Improves Fear and Anxiety-like Behavior and Reduces Basolateral Amygdala Activity in a Preclinical Model of Posttraumatic Stress Disorder. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology. Ridley, M., 2003. What makes you who you are. Time 161 (22), 54–60 63. Rytwinski, N.K., Scur, M.D., Feeny, N.C., Youngstrom, E.A., 2013. The co-occurrence of major depressive disorder among individuals with posttraumatic stress disorder: a meta-analysis. J. Trauma. Stress 26 (3), 299–309. Sarapas, C., Cai, G., Bierer, L.M., Golier, J.A., Galea, S., Ising, M., Rein, T., Schmeidler, J., Muller-Myhsok, B., Uhr, M., Holsboer, F., Buxbaum, J.D., Yehuda, R., 2011. Genetic markers for PTSD risk and resilience among survivors of the World Trade Center attacks. Dis. Markers 30 (2–3), 101–110. Scharf, S.H., Liebl, C., Binder, E.B., Schmidt, M.V., Muller, M.B., 2011. Expression and regulation of the Fkbp5 gene in the adult mouse brain. PLoS One 6 (2), e16883. Schiene-Fischer, C., Yu, C., 2001. Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett. 495 (1–2), 1–6. Schmidt, U., Buell, D.R., Ionescu, I.A., Gassen, N.C., Holsboer, F., Cox, M.B., Novak, B., Huber, C., Hartmann, J., Schmidt, M.V., Touma, C., Rein, T., Herrmann, L., 2015. A role for synapsin in FKBP51 modulation of stress responsiveness: convergent evidence from animal and human studies. Psychoneuroendocrinology 52, 43–58. Skelton, K., Ressler, K.J., Norrholm, S.D., Jovanovic, T., Bradley-Davino, B., 2012. PTSD and gene variants: new pathways and new thinking. Neuropharmacology 62 (2), 628–637. Stein, M.B., Jang, K.L., Taylor, S., Vernon, P.A., Livesley, W.J., 2002. Genetic and environmental influences on trauma exposure and posttraumatic stress disorder symptoms: a twin study. Am. J. Psychiatry 159 (10), 1675–1681. Szabo, C., Kelemen, O., Keri, S., 2014. Changes in FKBP5 expression and memory functions during cognitive-behavioral therapy in posttraumatic stress disorder: a preliminary study. Neurosci. Lett. 569, 116–120. Tamman, A.J.F., Sippel, L.M., Han, S., Neria, Y., Krystal, J.H., Southwick, S.M., Gelernter, J., Pietrzak, R.H., 2019. Attachment style moderates effects of FKBP5 polymorphisms and childhood abuse on post-traumatic stress symptoms: results from the National Health and Resilience in Veterans Study. World J. Biol. Psychiatry : Off. J. World Feder. Soc. Biol. Psychiatr. 20 (4), 289–300. True, W.R., Rice, J., Eisen, S.A., Heath, A.C., Goldberg, J., Lyons, M.J., Nowak, J., 1993. A twin study of genetic and environmental contributions to liability for posttraumatic stress symptoms. Arch. Gen. Psychiatr. 50 (4), 257–264. Watkins, L.E., Han, S., Harpaz-Rotem, I., Mota, N.P., Southwick, S.M., Krystal, J.H., Gelernter, J., Pietrzak, R.H., 2016. FKBP5 polymorphisms, childhood abuse, and PTSD symptoms: results from the national health and resilience in veterans study. Psychoneuroendocrinology 69, 98–105. Wilker, S., Pfeiffer, A., Kolassa, S., Elbert, T., Lingenfelder, B., Ovuga, E., Papassotiropoulos, A., de Quervain, D., Kolassa, I.T., 2014. The role of FKBP5 genotype in moderating long-term effectiveness of exposure-based psychotherapy for posttraumatic stress disorder. Transl. Psychiatry 4, e403. Xie, P., Kranzler, H.R., Poling, J., Stein, M.B., Anton, R.F., Farrer, L.A., Gelernter, J., 2010. Interaction of FKBP5 with childhood adversity on risk for post-traumatic stress disorder. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 35 (8), 1684–1692. Yehuda, R., Cai, G., Golier, J.A., Sarapas, C., Galea, S., Ising, M., Rein, T., Schmeidler, J., Muller-Myhsok, B., Holsboer, F., Buxbaum, J.D., 2009. Gene expression patterns associated with posttraumatic stress disorder following exposure to the World Trade Center attacks. Biol. Psychiatry 66 (7), 708–711. Yehuda, R., Daskalakis, N.P., Bierer, L.M., Bader, H.N., Klengel, T., Holsboer, F., Binder, E.B., 2016. Holocaust exposure induced intergenerational effects on FKBP5 methylation. Biol. Psychiatry 80 (5), 372–380. Zannas, A.S., Provencal, N., Binder, E.B., 2015. Epigenetics of posttraumatic stress disorder: current evidence, challenges, and future directions. Biol. Psychiatry 78 (5), 327–335. Zannas, A.S., Wiechmann, T., Gassen, N.C., Binder, E.B., 2016. Gene-stress-epigenetic regulation of FKBP5: clinical and translational implications. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 41 (1), 261–274.
Barrett, J.C., Fry, B., Maller, J., Daly, M.J., 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21 (2), 263–265. Binder, E.B., 2009. The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders. Psychoneuroendocrinology 34 (Suppl. 1), S186–S195. Binder, E.B., Bradley, R.G., Liu, W., Epstein, M.P., Deveau, T.C., Mercer, K.B., Tang, Y., Gillespie, C.F., Heim, C.M., Nemeroff, C.B., Schwartz, A.C., Cubells, J.F., Ressler, K.J., 2008. Association of FKBP5 polymorphisms and childhood abuse with risk of posttraumatic stress disorder symptoms in adults. J. Am. Med. Assoc. : J. Am. Med. Assoc. 299 (11), 1291–1305. Binder, E.B., Salyakina, D., Lichtner, P., Wochnik, G.M., Ising, M., Putz, B., Papiol, S., Seaman, S., Lucae, S., Kohli, M.A., Nickel, T., Kunzel, H.E., Fuchs, B., Majer, M., Pfennig, A., Kern, N., Brunner, J., Modell, S., Baghai, T., Deiml, T., Zill, P., Bondy, B., Rupprecht, R., Messer, T., Kohnlein, O., Dabitz, H., Bruckl, T., Muller, N., Pfister, H., Lieb, R., Mueller, J.C., Lohmussaar, E., Strom, T.M., Bettecken, T., Meitinger, T., Uhr, M., Rein, T., Holsboer, F., Muller-Myhsok, B., 2004. Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nat. Genet. 36 (12), 1319–1325. Boscarino, J.A., Erlich, P.M., Hoffman, S.N., Rukstalis, M., Stewart, W.F., 2011. Association of FKBP5, COMT and CHRNA5 polymorphisms with PTSD among outpatients at risk for PTSD. Psychiatry Res. 188 (1), 173–174. Breslau, N., Davis, G.C., Peterson, E.L., Schultz, L., 1997. Psychiatric sequelae of posttraumatic stress disorder in women. Arch. Gen. Psychiatr. 54 (1), 81–87. Cardon, L.R., Bell, J.I., 2001. Association study designs for complex diseases. Nat. Rev. Genet. 2 (2), 91–99. Chagas, M.H.N., Tumas, V., Rodrigues, G.R., Machado-de-Sousa, J.P., Filho, A.S., Hallak, J.E.C., Crippa, J.A.S., 2013. Validation and internal consistency of Patient Health Questionnaire-9 for major depression in Parkinson's disease. Age Ageing 42 (5), 645–649. Cioffi, D.L., Hubler, T.R., Scammell, J.G., 2011. Organization and function of the FKBP52 and FKBP51 genes. Curr. Opin. Pharmacol. 11 (4), 308–313. Fallin, D., Cohen, A., Essioux, L., Chumakov, I., Blumenfeld, M., Cohen, D., Schork, N.J., 2001. Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease. Genome Res. 11 (1), 143–151. Fani, N., Gutman, D., Tone, E.B., Almli, L., Mercer, K.B., Davis, J., Glover, E., Jovanovic, T., Bradley, B., Dinov, I.D., Zamanyan, A., Toga, A.W., Binder, E.B., Ressler, K.J., 2013. FKBP5 and attention bias for threat: associations with hippocampal function and shape. JAMA Psychiatr. 70 (4), 392–400. Fani, N., King, T.Z., Reiser, E., Binder, E.B., Jovanovic, T., Bradley, B., Ressler, K.J., 2014. FKBP5 genotype and structural integrity of the posterior cingulum. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 39 (5), 1206–1213. Fujii, T., Ota, M., Hori, H., Hattori, K., Teraishi, T., Matsuo, J., Kinoshita, Y., Ishida, I., Nagashima, A., Kunugi, H., 2014. The common functional FKBP5 variant rs1360780 is associated with altered cognitive function in aged individuals. Sci. Rep. 4, 6696. Galigniana, M.D., Radanyi, C., Renoir, J.M., Housley, P.R., Pratt, W.B., 2001. Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus. J. Biol. Chem. 276 (18), 14884–14889. Gelaye, B., Zheng, Y., Medina-Mora, M.E., Rondon, M.B., Sanchez, S.E., Williams, M.A., 2017. Validity of the posttraumatic stress disorders (PTSD) checklist in pregnant women. BMC Psychiatry 17 (1), 179. Grant, S.G.N., 2006. Review of genes and behavior: nature-nurture interplay explained. Genes Brain Behav. 5 (3) 303-303. Gray, M.J., Litz, B.T., Hsu, J.L., Lombardo, T.W., 2004. Psychometric properties of the life events checklist. Assessment 11 (4), 330–341. Hartmann, J., Wagner, K.V., Liebl, C., Scharf, S.H., Wang, X.D., Wolf, M., Hausch, F., Rein, T., Schmidt, U., Touma, C., Cheung-Flynn, J., Cox, M.B., Smith, D.F., Holsboer, F., Muller, M.B., Schmidt, M.V., 2012. The involvement of FK506-binding protein 51 (FKBP5) in the behavioral and neuroendocrine effects of chronic social defeat stress. Neuropharmacology 62 (1), 332–339. Hines, L.A., Sundin, J., Rona, R.J., Wessely, S., Fear, N.T., 2014. Posttraumatic stress disorder post Iraq and Afghanistan: prevalence among military subgroups. Canadian journal of psychiatry. Rev. Canad. Psychiatr. 59 (9), 468–479. Kang, J.I., Kim, T.Y., Choi, J.H., So, H.S., Kim, S.J., 2019. Allele-specific DNA methylation level of FKBP5 is associated with post-traumatic stress disorder. Psychoneuroendocrinology 103, 1–7. Karstoft, K.I., Andersen, S.B., Nielsen, A.B.S., 2017. Assessing PTSD in the military: validation of a scale distributed to Danish soldiers after deployment since 1998. Scand. J. Psychol. 58 (3), 260–268. Kessler, R.C., Sonnega, A., Bromet, E., Hughes, M., Nelson, C.B., 1995. Posttraumatic stress disorder in the national comorbidity survey. Arch. Gen. Psychiatr. 52 (12), 1048–1060. Klengel, T., Mehta, D., Anacker, C., Rex-Haffner, M., Pruessner, J.C., Pariante, C.M., Pace, T.W., Mercer, K.B., Mayberg, H.S., Bradley, B., Nemeroff, C.B., Holsboer, F., Heim, C.M., Ressler, K.J., Rein, T., Binder, E.B., 2013. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions. Nat. Neurosci. 16 (1), 33–41. Koenen, K.C., 2005. Nature-nurture interplay: genetically informative designs contribute to understanding the effects of trauma and interpersonal violence. J. Interpers Violence 20 (4), 507–512. Koenen, K.C., 2007. Genetics of posttraumatic stress disorder: Review and recommendations for future studies. J. Trauma. Stress 20 (5), 737–750.
53
Contents lists available at ScienceDirect
Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires
Genetic association of FKBP5 with PTSD in US service members deployed to Iraq and Afghanistan
T
Lei Zhanga,∗,1, Xian-Zhang Hua,1, Tianzheng Yub,1, Ze Chena, Jacob Dohlb, Xiaoxia Lia, David M. Benedeka, Carol S. Fullertona, Gary Wynna, James E. Barrettc, Mian Lid, Dale W. Russella,b, Biomarker team, Robert J. Ursanoa,1 a
Center for the Study of Traumatic Stress, Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA Consortium for Health and Military Performance, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA c Department of Neurology, Drexel University College of Medicine Philadelphia, PA, 19102-1192, USA d Department of Neurology, Washington DC VA Medical Center, Washington, DC, 20422, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: PTSD FKBP5 Single-nucleotide polymorphism (SNP) Haplotype Military
Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with a prevalence of more than 7% in the US population and 12% in the military. An interaction of childhood trauma with FKBP5 (a glucocorticoidregulated immunophilin) has been reported to be associated with PTSD in the general population. However, there are few reports on the association of FKBP5 with PTSD, particularly in important high-risk population such as the military. Here, we examined the association between four single-nucleotide polymorphisms (SNPs; rs3800373, rs9296158, rs1360780, rs9470080) covering the FKBP5 gene and probable PTSD in US service members deployed to Iraq and Afghanistan, a high-risk military population (n = 3890) (Hines et al., 2014). We found that probable PTSD subjects were significantly more likely to carry the A-allele of rs3800373, G-allele of rs9296158, C-allele of rs1360780, and C-allele of rs9470080. Furthermore, the four SNPs were in one block of strong pairwise linkage disequilibrium (r = 0.91–0.96). Within the block there were two major haplotypes of CATT and AGCC (rs3800373-rs9296158-rs1360780-rs9470080) that account for 99% of haplotype diversity. The distribution of the AGCC haplotype was significantly higher in probable PTSD subjects compared to nonPTSD (p < .05). The diplotype-based analysis indicated that the AGCC carriers tended to be probable PTSD. In this study, we demonstrated the association between FKBP5 and probable PTSD in US service members deployed to Iraq and Afghanistan, indicating that FKBP5 might be a risk factor for PTSD.
1. Introduction Posttraumatic stress disorder (PTSD) is a chronic and debilitating condition with characteristic symptoms, including re-experiencing of trauma, avoidance, negative alterations in cognition and mood, and hyperarousal triggered by life-threatening traumatic events. APA, 2013 (Kessler et al., 1995). However, PTSD is only observed in a fraction of those exposed to trauma (Stein et al., 2002). A lifetime trauma incidence is 40%–90% in the general population, but the overall lifetime prevalence for PTSD ranges from 7% to 12% (Kessler et al., 1995), indicating that a complex interplay of multiple factors is decisive (Stein et al., 2002). Genetic susceptibility might also play a role in the development of PTSD (Koenen, 2007). Children whose parents have PTSD
show higher rates of PTSD than the general population (Stein et al., 2002). Twin and family studies also show a genetic component in the development of PTSD (Koenen et al., 2005; Stein et al., 2002). It is believed that genetic influences account for about one-third of the variance in risk of developing PTSD (True et al., 1993). Binder et al. (2008) identified four single nucleotide polymorphisms (SNPs) in the FKBP5 gene interacting with childhood trauma to predict an increased risk of developing adult PTSD (Binder et al., 2008). FKBP5 is a key regulator in the stress hormone system, which mediates responses to traumatic stress – the relationships between PTSD and the dysfunction of hypothalamic-pituitary-adrenal stress axis (HPA) and genetic association have been intensively investigated (Binder, 2009; Binder et al., 2008; Kang et al., 2019; Klengel et al., 2013; Scharf et al., 2011;
∗
Corresponding author. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpsychires.2019.12.014 Received 1 August 2019; Received in revised form 23 December 2019; Accepted 24 December 2019 0022-3956/ © 2019 Published by Elsevier Ltd.
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
2. Material and methods
Schmidt et al., 2015; Zannas et al., 2015). One of the key systems mediating long-term effects of stress and the development of PTSD is the glucocorticoid receptor (GR) system in the HPA. Those with PTSD may be susceptible to stress or mitigated depending on the molecular abnormalities within the GR stress-signaling pathway. As a ligand-activated transcription factor, GR translocates from the cytosol into the nucleus after ligand binding and initiates gene transcription. This process is regulated by a large molecular complex that includes FKBP5 (Binder et al., 2008; Hartmann et al., 2012; Schiene-Fischer and Yu, 2001; Schmidt et al., 2015). FKBP5 is a part of the FKBP/GR complex that regulates GR sensitivity. Glucocorticoid (GC) binds with lower affinity to GR in the FKBP5/GR complex which results in less GR nuclear translocation. FKBP5 polymorphisms are associated with GC-induced FKBP5 expression and GR sensitivity. It is known that FKBP5 plays a key role in an intracellular ultra-short negative feedback loop for GR activity (Reynolds et al., 1998, 1999). Alterations in FKBP5 expression are involved in abnormal GR-mediated signaling in neurons involved in the stress response and memory formation (Fujii et al., 2014; Szabo et al., 2014). A cortisol-induced higher level of FKBP5 is associated with PTSD (Binder, 2009), indicating the role of FKBP5 in PTSD in the GR system. FKBP5 is one of the intensively investigated genes in PTSD (Binder et al., 2008; Boscarino et al., 2011; Levy-Gigi et al., 2013; Sarapas et al., 2011; Watkins et al., 2016; Wilker et al., 2014; Xie et al., 2010; Yehuda et al., 2009, 2016). Notably, the interaction of FKBP5 SNPs with childhood trauma, but not with adult trauma, predicts greater self-reported PTSD symptoms (Binder et al., 2008; Xie et al., 2010). Lower mRNA expression of FKBP5 predicts higher severity of PTSD symptoms after combat (Skelton et al., 2012). A study in two nationally representative samples of European-American (EA) U.S. military veterans revealed that the four FKBP5 SNPs are associated with PTSD symptom severity. SNP rs9470080 in the main sample, and all four SNPs in the replication sample interact with childhood abuse to predict PTSD severity. An association of FKBP5 polymorphisms with PTSD directly and/or with childhood abuse interactively can predict severity of lifetime PTSD symptoms (Watkins et al., 2016). Another study in nationally representative samples of US military veterans shows that FKBP5, childhood abuse and an insecure attachment style are associated with greater severity of PTSD symptoms, indicating gene x environment interactions (Tamman et al., 2019). FKBP5 expression level is also associated with the size of various brain regions and therapeutic responses to PTSD. PTSD patients who have a smaller hippocampus and medial orbitofrontal cortex have lower expression levels of FKBP5 compared to non-PTSD controls (Levy-Gigi et al., 2013). Cognitive-behavioral therapy significantly increases FKBP5 expression and hippocampal volume in patients, suggesting that an improvement in PTSD symptoms may be predicted by increased FKBP5 expression and increased hippocampal volume (Levy-Gigi et al., 2013). Genotypes of FKBP5 are associated with hippocampal function (Fani et al., 2013), cingulum structure (Fani et al., 2014), and a higher risk of PTSD symptoms (Koenen, 2005). Moreover, 4 SNPs (rs9296158, rs3800373, rs1360780, and rs9470080) significantly interacted with the severity of child abuse, but not with non-child abuse trauma exposure, to effect on the level of adult PTSD symptoms (Binder et al., 2008). PTSD is one of only a few disorders in the DSM (American Psychiatric Association, 2013) that require an etiological factor, a traumatic event, for its diagnosis. In addition, approximately half of people with PTSD also suffer from Major Depressive Disorder (Breslau et al., 1997; Rytwinski et al., 2013). Based on the findings above, we investigated these four SNPs in a large sample of US Army soldiers who served in Afghanistan or Iraq between 2008 and 2016, to study the main effects of FKBP5 and effects of interaction of FKBP5 with environmental variables on PTSD. Our results may add to the well-recognized role of FKBP5 in the risk of developing PTSD.
2.1. Subjects Samples (n = 3890) were collected from US Army service members who served during combat operations in Afghanistan and/or Iraq between 2008 and 2016 (Hines et al., 2014). Individuals who did not provide biological samples (e.g. saliva or blood) were excluded. The Institutional Review Board at the Uniformed Service University (USUHS) approved all study procedures and all participants were given written informed consent. 2.2. Survey measures PTSD symptoms were assessed using the PTSD Checklist (PCL), a 17item, DSM-based, self-report measure with well-established validity and reliability (Gelaye et al., 2017; Karstoft et al., 2017). Cronbach's alpha coefficients indicate high internal consistency for the total scale (0.91) and for the theoretical dimensions of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) (0.83, 0.81, and 0.80). Three symptom clusters account for 48.9% of the variance, namely, intrusions, avoidance, and numbing-hyperarousal (Lima Ede et al., 2012). Probable PTSD was diagnosed based on the endorsement of DSM-IV criteria and a PCL total score ≥50. PTSD symptom severity was assessed using the PCL total score. To determine conditions of medication, subjects were asked if any medication (including vitamins, those prescribed by a doctor or one bought at the pharmacy/store, and/ or substance) had been taken in the past 24 h. Subjects with substance abuse, or any medication use were excluded. Demographic data, such as age, gender, and race, were collected. Stressful life events including combat exposure were determined using the Life Events Checklist (LEC, 1995 version) (Gray et al., 2004). Non-PTSD controls might also experience lifetime traumatic events, such as combat or exposure to a war-zone. Subjects’ lifetime traumatic events, including events prior to deployment, were determined. The participants (probable PTSD and non-PTSD controls) in this study were military active duty volunteers. Depression was determined by using the PHQ-9 (Chagas et al., 2013). The PHQ-9 is an adequate instrument for the screening depression. The internal consistency of the scale is 0.83 (Chagas et al., 2013). Sixty-six (1.5%) veterans screened positive for depression. Probable PTSD subjects with comorbidity of depression were enrolled in the study group. Probable PTSD comorbid with other anxiety disorders or somatic disorders were not evaluated in this study. 2.3. Saliva sample collection and DNA extraction Saliva samples were collected using Oragene saliva DNA tube kit (Oragene). Genomic DNA was extracted from a 200 μl saliva sample using the QIAmp protocol (Qiagen, Chatsworth, CA). The quantity and quality of the genomic DNA isolate were determined by 260/280 ultraviolet spectrophotometry (Nanodrop SD-1000 spectrophotometer). 2.4. Genotyping Four SNPs with frequencies of > 5% covering FKBP5 were selected from dbSNP and a published paper indicating two potentially functional SNPs (rs1360780, and rs3800373) and two other SNPs (rs9296158 and rs9470080) that have strong interactions with childhood abuse on adult depression symptoms and PTSD. The TaqMan allelic discrimination assay was used for genotyping on the ABI 7900HT instrument (Applied Biosystems, Foster City, California). Predesigned and validated TaqMan assay reagent kits contain one pair of polymerase chain reaction primers and one pair of fluorescently labeled probes (Applied Biosystems). Polymerase chain reactions (PCR) were performed in 5 μL reaction volumes, in a 384-well plate, and contained 5 ng of DNA. Amplification 49
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
glucocorticoid receptor response element. Fig. 1c shows these four SNPs that span FKBP5 were in one block of strong pairwise linkage disequilibrium (r = 0.91–0.96, Fig. 1c). Within the block there are two haplotypes of CATT and AGCC (rs3800373-rs9296158-rs1360780rs9470080) that account for 99% of haplotype diversity. The distribution of AGCC haplotype was significantly higher in the probable PTSD group compared to the non-PTSD group (p < .05, Table 3a). The diplotype-based analysis indicated that the AGCC carriers tended to fall within the probable PTSD group (Table 3b). The Nagelkerke R Square was 0.265. No correlation between probable PTSD and the interactions of genotypes and lifetime traumatic events (either combat or noncombat related) was found (Table 4). In addition, there was no association between the SNPs of FKBP5 and lifetime traumatic events including child abuse. Moreover, these SNPs did not moderate the association between child abuse and probable PTSD.
conditions were 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles at 96 °C for 15 s and at 60 °C for 60 s. The software of SDS version 2.3 (Applied Biosystems) was used for allelic discrimination. For quality control, 20% of the samples, randomly selected, were genotyped in duplicate. The error rate was < 0.006, and the completion rate was > 0.95. 2.5. Haplotype analysis We used Haploview 4.0 to determine the linkage disequilibrium (LD) structure of the four SNPs covered the FKBP5 gene. LD was indicated by r and visualized using Haploview (Barrett et al., 2005). We used the ‘confidence intervals’ approach to explore the putative block structure. Haplotypes and diplotypes were used to test for association in addition to individual SNPs because they are known to often provide more information.
4. Discussion 2.6. Statistical analyses Population-based association studies (case-control studies) are a widely used design to determine the impact of genetic variants on the risk of developing a particular complex disease (Cardon and Bell, 2001). Many association studies have been performed in order to elucidate the genetic contribution to complex diseases. Through these studies, several low penetrance genes have been found to behave as PTSD risk modifiers, contributing to the understanding of traumatic stress in many types of stress-related disorders and leading to advances in diagnosis and therapy. In this study, we presented the first evidence for an association of FKBP5 variation with lifetime probable PTSD in US Army service members. We found that four SNPs (rs3800373, rs9296158, rs1360780, rs9470080) covering the FKBP5 gene were associated with lifetime probable PTSD in these soldiers. It is consistent with other studies, showing that SNP markers rs16969968, rs9470080, and rs4680 were individually associated with PTSD and that a cumulative allele model using these SNPs was associated with a higher PTSD risk (Boscarino et al., 2011). However, the interaction of the SNPs with LEC showed no effects on PTSD, suggesting that the SNPs might not be moderators for the onset of PTSD. There was no effect of interactions of genotypes and life time traumatic events (either combat or non-combat related) on PTSD, although previous studies reported that two FKBP5 minor allele carriers, childhood abuse and insecure attachment style were associated with greater severity of PTSD symptoms in veterans (Tamman et al., 2019). Those inconsistent results might be related to differences in sampling or characteristics of life time traumatic events. Nevertheless, our results, particularly the data of combat exposure and trauma history, suggested that gene–environment interactions may play the role in PTSD development in the U.S. military population. Service
All analyses were performed using SPSS version 18. Chi-square tests were performed to test the differences in distributions of sex, race, educational levels, and marital status among the groups as well as the association between FKBP5 and lifetime probable PTSD. The parameter data are presented as mean ± S.D. The statistically significant differences in age among the subjects were analyzed using Student's t-test. We utilized regression analysis to examine the effects of the interactions of stressful life events with FKBP5 on probable PTSD and the differences in the magnitudes of observed groups. Covariates included age, sex, and ethnicity. The Bonferroni correction was used for our multiple tests. Our tests included four highly correlated SNPs, the main effects of the SNPs, the effects of LEC with each SNPs on probable PTSD. 3. Results Table 1 shows that participant ages ranged from 18 to 62 years old, with the mean being 29.4 years old. The distribution of ethnicities was not significantly different between the probable PTSD and non-PTSD groups. The participants with probable PTSD were more likely to report combat or exposure to a war-zone than non-PTSD controls (p < .05). The four loci were in Hardy–Weinberg equilibrium (HWE) in our population (p > 0.05). Table 2 shows the frequencies of genotype/allele and haplotype/diplotype in probable PTSD and non-PTSD controls. We found that probable PTSD subjects were significantly more likely to carry the A-allele of rs3800373, G-allele of rs9296158, C-allele of rs1360780, and C-allele of rs9470080. Fig. 1b illustrates the organization of the FKBP5 gene, the location of the four SNPs (Fig. 1a) and the Table 1 Demographic information of the army service member. Total
Race White African American Hispanic Asian or Pacific Islander Others Gender Male Female Combat or exposure to a war-zone Age (mean ± sd; range) Male Female Total
PTSD
Control
N
%
N
%
N
%
1876 274 874 647 219
48.2 7.0 22.5 16.6 5.6
164 19 22.5 39 39
47.7 5.5 24.1 11.3 11.3
1712 255 791 608 180
48.3 7.2 22.3 17.1 5.1
3379 476 1021
87.6 12.4 26.2
295 46 136
86.5 16.5 39.5
3084 430 885
87.8 12.2 24.3**
29.7 ± 8.5; 18-62 27.4 ± 7.2; 18-55 29.4 ± 8.4; 18-62
30.3 ± 8.4; 18-56 28.1 ± 8.0; 18-52 30.1 ± 8.3; 18-56
29.6 ± 8.5; 18–62* 27.3 ± 7.1; 18-55 29.3 ± 8.4; 18-62
sd: standard deviation. * Student t-test, P < 0.05 (males vs females of controls).**χ2 = 23.9, P < 0.0001 (OR = 1.8, 95% CV: 1.4–2.3). 50
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Table 2 Genotype and allele distributions of FKBP5 SNPs in PTSDs and controls. Genotypes
rs1360780 (1: T, 2: C) rs3800373 (1: C, 2: A) rs9296158 (1: G, 2: A) rs9470080 (1: T, 2: C)
Control PTSD Control PTSD Control PTSD Control PTSD
Alleles
11
12
22
P value
1
2
OR, 95% CI
P value
388 (0.10) 15 (0.04) 404 (0.11) 20 (0.06) 1449 (0.40) 163 (0.47) 430 (0.12) 22 (0.06)
1618 (0.42) 155 (0.45) 1541 (0.44) 150 (0.44) 1648 (0.47) 157 (0.46) 1672 (0.48) 163 (0.47)
1840 (0.48) 171 (0.50) 1566 (0.45) 174 (0.50) 444 (0.13) 24 (0.07) 1349 (0.39) 159 (0.46)
0.002
2394 (0.34) 191 (0.28) 2349 (0.33) 190 (0.28) 4556 (0.64) 483 (0.70) 2532 (0.37) 207 (0.30)
4698 (0.66) 497 (0.72) 4673 (0.67) 498 (0.72) 2546 (0.36) 205 (0.30) 4370 (0.63) 481 (0.70)
1.3, 1.1-1.6
0.001
13, 1.1-1.6
0.002
0.2, 0.2-0.3
0.002
2.9, 2.4–3.4
0.001
0.004 0.003 0.001
Fig. 1. The description of FKBP5 gene location, SNPs, glucocorticoid receptor response element (GRE), and haplotypes. (a) Schematic diagram of FKBP5 location on chromosome 6. FKBP5 gene localizes to chromosome 6p21.31 and spans over 154 kbp from 35541362 to 35696360 on the reverse strand. The region surrounding the FKBP5 gene is enlarged. (b) Schematic diagram of the locations of the SNPs around the FKBP5 gene and GRE sites. (c) The relative positions of FKBP5 SNPs and their haplotype block structure. The numbers in the squares refer to pair-wise linkage disequilibrium (LD, r > 0.90).
Table 3a Haplotype distribution in participants with probable PTSD and controls.
Table 3b Diplotype distribution in PTSDs and controls.
Haplotype distribution
PTSD Control
Diplotype distribution
CATT
AGCC
Chi square
P value
185 2141
455 4001
9.112
0.0025
PTSD Control
RR = 1.09; OR = 1.32.
CATT/CATT
AGCC carrier
Chi square
P value
18 344
302 2727
9.45
0.002
RR = 1.06; OR = 2.12.
51
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Table 4 Logistic regression analyses of the effects of the interactions of lifetime traumatic event with SNPs of FKBP5 on PTSD. Variables in the Equation
rs1360780 rs3800373 rs9296158 rs9470080
x x x x
lifetime lifetime lifetime lifetime
traumatic traumatic traumatic traumatic
event event event event
B .023 .024 -.019 .001
S.E. .055 .044 .020 .044
Wald .178 .292 .857 .001
df 1 1 1 1
Sig. .673 .589 .355 .982
Exp(B) 1.023 1.024 .982 1.001
95% C.I. .919 .939 .944 .918
1.140 1.116 1.021 1.091
personnel with PTSD. Our study excluded the subjects who reported medication use within the last month. Those concerns will be considered in future studies. Accumulative association studies show that FKBP5 is indirectly associated with PTSD (Binder et al., 2008; Xie et al., 2010). However, the GWAS meta-analyses show no strong FKBP5 association with PTSD (Zannas et al., 2016). We have also noticed that there is an inconsistency about the findings of the directionality of the risk alleles in publications (Binder et al., 2008). These discrepancies between ours and other's might be due to the sampling. For example, the subjects in this study are active duty military service members who are exposed to unique war-related traumatic events while their subjects are civilians who may have experience with non-war-related traumatic events. In addition, the differences of evaluation procedures might be associated with the discrepancies. In addition, the difference of disease stages between the studies may also be a reason for this inconsistency. Moreover, there are statistical power issues in candidate gene studies. The limitation of the studies are relatively small sample size. Therefore, well-designed conformation studies are needed. In summary, we demonstrated that four SNPs covering the FKBP5 gene were associated with PTSD in US service members deployed to Iraq and Afghanistan. These findings suggest that FKBP5 might be involved in the pathophysiology of PTSD and a potential biomarker for PTSD.
members with different FKBP5 genotypes are affected differently by exposure to the same environmental factors, and thus gene–environment interactions can result in different phenotypes (Grant, 2006; Ridley, 2003). The AGCC haplotype carriers have the higher risks of PTSD. These interactions are of particular interest for predicting PTSD and useful for developing the methods of prevention with respect to public health as well. Since a statistical analysis based on haplotypes is more efficient than separate analyses of the individual markers (Fallin et al., 2001), we also conducted a linkage disequilibrium (LD) analysis revealing strong LD among SNPs in close proximity only. It is known that rs1360780 C/T leads to an allelic-specific RNA expression (Binder et al., 2004). Thus, we considered CATT and AGCC to be yin-yang haplotypes. The AGCC was a high risk haplotype for PTSD. Our data, as well as others’, suggested that psychological alterations are associated not only with inborn genetic bases, but also environmental stressors (Binder et al., 2008). The participants with probable PTSD were more likely to report combat or exposure to a war-zone, indicating that environment (traumatic stress) plays an important role in PTSD development, while gene variants of FKBP5 may promote PTSD symptoms. Life events associated with symptom severity of PTSD, suggesting that symptoms are induced by the trauma. The inconsistency of these results might be due to the differences in sample sizes of the two studies (small vs large) and the characteristics of the samples (civilian vs military). However, these assumptions need to be examined. It is known that several parts of the brain are involved in the pathology of stress response and PTSD, including the amygdala, prefrontal cortex, hippocampus and hypothalamic pituitary adrenal axis (HPAaxis) (Akiki et al., 2017; Neumeister et al., 2017; Reznikov et al., 2017). Functionally, FKBP5, and GR are in a central position to mediate acute and chronic response to stress in the HPA-axis (Binder et al., 2008). In order to respond to stress, the HPA is activated by GR and regulates the on/off production of hormones. FKBP5 inhibits GR translocation to the nucleus. Therefore, it is crucial in an ultra-short feedback loop (Binder et al., 2008; Cioffi et al., 2011), in which FKBP5 regulates GR sensitivity and bioavailability (Binder et al., 2008). In the absence of GC, the GR/ FKBP5 complex is in an inactive stable state. When GC is present, GC is bound to GR, which dissociates from FKBP5/GR complex and is translocated into the nucleus (Binder, 2009; Binder et al., 2004, 2008; Galigniana et al., 2001). In the nucleus, the GR binds to the glucocorticoid response element (GRE) of the FKBP5 gene, leading to FKBP5 mRNA transcription (Binder, 2009; Binder et al., 2004, 2008; Galigniana et al., 2001). Subsequently, FKBP5 inhibits the GR nuclear translocation that is called an ultrashort, feedback loop (inhibition of GR activity) (Binder, 2009). A major limitation of this study is the use of the candidate gene approach. This method may be inappropriate when the genes are already selected. In addition, it chooses a gene either upstream of the points of action or in the downstream signaling pathways. Also, the SNPs selected may provide incomplete coverage of all variants. It sometimes uses small case and control samples with less statistical power, which may be misleading. Moreover, it often relies on prior hypotheses about disease mechanisms which have been reported previously. Therefore, a confirmation study is required. Also, high levels of depressive symptoms, sleep disturbances, and frequent use of medications for insomnia and depression have been reported in military
Funding Center for Traumatic Stress, Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, USA. CRediT authorship contribution statement Lei Zhang: Data curation, Supervision. Xian-Zhang Hu: Data curation. Tianzheng Yu: Data curation. Ze Chen: Data curation. Jacob Dohl: Writing - review & editing. Xiaoxia Li: Data curation. David M. Benedek: Data curation. Carol S. Fullerton: Data curation. Gary Wynn: Data curation. James E. Barrett: Writing - review & editing. Mian Li: Writing - review & editing. Dale W. Russell: Data curation. Robert J. Ursano: Project administration, Writing - original draft. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We thank Supriya Prabhakar, Berwin Yuan, Alexis Shahidi, and Nora Wang for editing the manuscript. The manuscript was improved following comments from Dr. Li Zheng of NIMH, NIH. References Akiki, T.J., Averill, C.L., Wrocklage, K.M., Schweinsburg, B., Scott, J.C., Martini, B., Averill, L.A., Southwick, S.M., Krystal, J.H., Abdallah, C.G., 2017. The association of PTSD symptom severity with localized Hippocampus and amygdala abnormalities. Chronic Stress (Thousand Oaks) 1. APA, 2013. Diagnostic and Statistical Manual of Mental Disorders, 5th. American Psychiatric Association, Arlington, VA, USA.
52
Journal of Psychiatric Research 122 (2020) 48–53
L. Zhang, et al.
Koenen, K.C., Hitsman, B., Lyons, M.J., Niaura, R., McCaffery, J., Goldberg, J., Eisen, S.A., True, W., Tsuang, M., 2005. A twin registry study of the relationship between posttraumatic stress disorder and nicotine dependence in men. Arch. Gen. Psychiatr. 62 (11), 1258–1265. Levy-Gigi, E., Szabo, C., Kelemen, O., Keri, S., 2013. Association among clinical response, hippocampal volume, and FKBP5 gene expression in individuals with posttraumatic stress disorder receiving cognitive behavioral therapy. Biol. Psychiatry 74 (11), 793–800. Lima Ede, P., Barreto, S.M., Assuncao, A.A., 2012. Factor structure, internal consistency and reliability of the Posttraumatic Stress Disorder Checklist (PCL): an exploratory study. Trends Psychiatr. Psychother. 34 (4), 215–222. Neumeister, P., Feldker, K., Heitmann, C.Y., Buff, C., Brinkmann, L., Bruchmann, M., Straube, T., 2017. Specific amygdala response to masked fearful faces in post-traumatic stress relative to other anxiety disorders. Psychol. Med. 1–11. Reynolds, P.D., Roveda, K.P., Tucker, J.A., Moore, C.M., Valentine, D.L., Scammell, J.G., 1998. Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis). Lab. Anim. Sci. 48 (4), 364–370. Reynolds, P.D., Ruan, Y., Smith, D.F., Scammell, J.G., 1999. Glucocorticoid resistance in the squirrel monkey is associated with overexpression of the immunophilin FKBP51. J. Clin. Endocrinol. Metab. 84 (2), 663–669. Reznikov, R., Bambico, F.R., Diwan, M., Raymond, R.J., Nashed, M.G., Nobrega, J.N., Hamani, C., 2017. Prefrontal Cortex Deep Brain Stimulation Improves Fear and Anxiety-like Behavior and Reduces Basolateral Amygdala Activity in a Preclinical Model of Posttraumatic Stress Disorder. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology. Ridley, M., 2003. What makes you who you are. Time 161 (22), 54–60 63. Rytwinski, N.K., Scur, M.D., Feeny, N.C., Youngstrom, E.A., 2013. The co-occurrence of major depressive disorder among individuals with posttraumatic stress disorder: a meta-analysis. J. Trauma. Stress 26 (3), 299–309. Sarapas, C., Cai, G., Bierer, L.M., Golier, J.A., Galea, S., Ising, M., Rein, T., Schmeidler, J., Muller-Myhsok, B., Uhr, M., Holsboer, F., Buxbaum, J.D., Yehuda, R., 2011. Genetic markers for PTSD risk and resilience among survivors of the World Trade Center attacks. Dis. Markers 30 (2–3), 101–110. Scharf, S.H., Liebl, C., Binder, E.B., Schmidt, M.V., Muller, M.B., 2011. Expression and regulation of the Fkbp5 gene in the adult mouse brain. PLoS One 6 (2), e16883. Schiene-Fischer, C., Yu, C., 2001. Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett. 495 (1–2), 1–6. Schmidt, U., Buell, D.R., Ionescu, I.A., Gassen, N.C., Holsboer, F., Cox, M.B., Novak, B., Huber, C., Hartmann, J., Schmidt, M.V., Touma, C., Rein, T., Herrmann, L., 2015. A role for synapsin in FKBP51 modulation of stress responsiveness: convergent evidence from animal and human studies. Psychoneuroendocrinology 52, 43–58. Skelton, K., Ressler, K.J., Norrholm, S.D., Jovanovic, T., Bradley-Davino, B., 2012. PTSD and gene variants: new pathways and new thinking. Neuropharmacology 62 (2), 628–637. Stein, M.B., Jang, K.L., Taylor, S., Vernon, P.A., Livesley, W.J., 2002. Genetic and environmental influences on trauma exposure and posttraumatic stress disorder symptoms: a twin study. Am. J. Psychiatry 159 (10), 1675–1681. Szabo, C., Kelemen, O., Keri, S., 2014. Changes in FKBP5 expression and memory functions during cognitive-behavioral therapy in posttraumatic stress disorder: a preliminary study. Neurosci. Lett. 569, 116–120. Tamman, A.J.F., Sippel, L.M., Han, S., Neria, Y., Krystal, J.H., Southwick, S.M., Gelernter, J., Pietrzak, R.H., 2019. Attachment style moderates effects of FKBP5 polymorphisms and childhood abuse on post-traumatic stress symptoms: results from the National Health and Resilience in Veterans Study. World J. Biol. Psychiatry : Off. J. World Feder. Soc. Biol. Psychiatr. 20 (4), 289–300. True, W.R., Rice, J., Eisen, S.A., Heath, A.C., Goldberg, J., Lyons, M.J., Nowak, J., 1993. A twin study of genetic and environmental contributions to liability for posttraumatic stress symptoms. Arch. Gen. Psychiatr. 50 (4), 257–264. Watkins, L.E., Han, S., Harpaz-Rotem, I., Mota, N.P., Southwick, S.M., Krystal, J.H., Gelernter, J., Pietrzak, R.H., 2016. FKBP5 polymorphisms, childhood abuse, and PTSD symptoms: results from the national health and resilience in veterans study. Psychoneuroendocrinology 69, 98–105. Wilker, S., Pfeiffer, A., Kolassa, S., Elbert, T., Lingenfelder, B., Ovuga, E., Papassotiropoulos, A., de Quervain, D., Kolassa, I.T., 2014. The role of FKBP5 genotype in moderating long-term effectiveness of exposure-based psychotherapy for posttraumatic stress disorder. Transl. Psychiatry 4, e403. Xie, P., Kranzler, H.R., Poling, J., Stein, M.B., Anton, R.F., Farrer, L.A., Gelernter, J., 2010. Interaction of FKBP5 with childhood adversity on risk for post-traumatic stress disorder. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 35 (8), 1684–1692. Yehuda, R., Cai, G., Golier, J.A., Sarapas, C., Galea, S., Ising, M., Rein, T., Schmeidler, J., Muller-Myhsok, B., Holsboer, F., Buxbaum, J.D., 2009. Gene expression patterns associated with posttraumatic stress disorder following exposure to the World Trade Center attacks. Biol. Psychiatry 66 (7), 708–711. Yehuda, R., Daskalakis, N.P., Bierer, L.M., Bader, H.N., Klengel, T., Holsboer, F., Binder, E.B., 2016. Holocaust exposure induced intergenerational effects on FKBP5 methylation. Biol. Psychiatry 80 (5), 372–380. Zannas, A.S., Provencal, N., Binder, E.B., 2015. Epigenetics of posttraumatic stress disorder: current evidence, challenges, and future directions. Biol. Psychiatry 78 (5), 327–335. Zannas, A.S., Wiechmann, T., Gassen, N.C., Binder, E.B., 2016. Gene-stress-epigenetic regulation of FKBP5: clinical and translational implications. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 41 (1), 261–274.
Barrett, J.C., Fry, B., Maller, J., Daly, M.J., 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21 (2), 263–265. Binder, E.B., 2009. The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders. Psychoneuroendocrinology 34 (Suppl. 1), S186–S195. Binder, E.B., Bradley, R.G., Liu, W., Epstein, M.P., Deveau, T.C., Mercer, K.B., Tang, Y., Gillespie, C.F., Heim, C.M., Nemeroff, C.B., Schwartz, A.C., Cubells, J.F., Ressler, K.J., 2008. Association of FKBP5 polymorphisms and childhood abuse with risk of posttraumatic stress disorder symptoms in adults. J. Am. Med. Assoc. : J. Am. Med. Assoc. 299 (11), 1291–1305. Binder, E.B., Salyakina, D., Lichtner, P., Wochnik, G.M., Ising, M., Putz, B., Papiol, S., Seaman, S., Lucae, S., Kohli, M.A., Nickel, T., Kunzel, H.E., Fuchs, B., Majer, M., Pfennig, A., Kern, N., Brunner, J., Modell, S., Baghai, T., Deiml, T., Zill, P., Bondy, B., Rupprecht, R., Messer, T., Kohnlein, O., Dabitz, H., Bruckl, T., Muller, N., Pfister, H., Lieb, R., Mueller, J.C., Lohmussaar, E., Strom, T.M., Bettecken, T., Meitinger, T., Uhr, M., Rein, T., Holsboer, F., Muller-Myhsok, B., 2004. Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nat. Genet. 36 (12), 1319–1325. Boscarino, J.A., Erlich, P.M., Hoffman, S.N., Rukstalis, M., Stewart, W.F., 2011. Association of FKBP5, COMT and CHRNA5 polymorphisms with PTSD among outpatients at risk for PTSD. Psychiatry Res. 188 (1), 173–174. Breslau, N., Davis, G.C., Peterson, E.L., Schultz, L., 1997. Psychiatric sequelae of posttraumatic stress disorder in women. Arch. Gen. Psychiatr. 54 (1), 81–87. Cardon, L.R., Bell, J.I., 2001. Association study designs for complex diseases. Nat. Rev. Genet. 2 (2), 91–99. Chagas, M.H.N., Tumas, V., Rodrigues, G.R., Machado-de-Sousa, J.P., Filho, A.S., Hallak, J.E.C., Crippa, J.A.S., 2013. Validation and internal consistency of Patient Health Questionnaire-9 for major depression in Parkinson's disease. Age Ageing 42 (5), 645–649. Cioffi, D.L., Hubler, T.R., Scammell, J.G., 2011. Organization and function of the FKBP52 and FKBP51 genes. Curr. Opin. Pharmacol. 11 (4), 308–313. Fallin, D., Cohen, A., Essioux, L., Chumakov, I., Blumenfeld, M., Cohen, D., Schork, N.J., 2001. Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease. Genome Res. 11 (1), 143–151. Fani, N., Gutman, D., Tone, E.B., Almli, L., Mercer, K.B., Davis, J., Glover, E., Jovanovic, T., Bradley, B., Dinov, I.D., Zamanyan, A., Toga, A.W., Binder, E.B., Ressler, K.J., 2013. FKBP5 and attention bias for threat: associations with hippocampal function and shape. JAMA Psychiatr. 70 (4), 392–400. Fani, N., King, T.Z., Reiser, E., Binder, E.B., Jovanovic, T., Bradley, B., Ressler, K.J., 2014. FKBP5 genotype and structural integrity of the posterior cingulum. Neuropsychopharmacology : Off. Publ. Am. Coll. Neuropsychopharmacol. 39 (5), 1206–1213. Fujii, T., Ota, M., Hori, H., Hattori, K., Teraishi, T., Matsuo, J., Kinoshita, Y., Ishida, I., Nagashima, A., Kunugi, H., 2014. The common functional FKBP5 variant rs1360780 is associated with altered cognitive function in aged individuals. Sci. Rep. 4, 6696. Galigniana, M.D., Radanyi, C., Renoir, J.M., Housley, P.R., Pratt, W.B., 2001. Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus. J. Biol. Chem. 276 (18), 14884–14889. Gelaye, B., Zheng, Y., Medina-Mora, M.E., Rondon, M.B., Sanchez, S.E., Williams, M.A., 2017. Validity of the posttraumatic stress disorders (PTSD) checklist in pregnant women. BMC Psychiatry 17 (1), 179. Grant, S.G.N., 2006. Review of genes and behavior: nature-nurture interplay explained. Genes Brain Behav. 5 (3) 303-303. Gray, M.J., Litz, B.T., Hsu, J.L., Lombardo, T.W., 2004. Psychometric properties of the life events checklist. Assessment 11 (4), 330–341. Hartmann, J., Wagner, K.V., Liebl, C., Scharf, S.H., Wang, X.D., Wolf, M., Hausch, F., Rein, T., Schmidt, U., Touma, C., Cheung-Flynn, J., Cox, M.B., Smith, D.F., Holsboer, F., Muller, M.B., Schmidt, M.V., 2012. The involvement of FK506-binding protein 51 (FKBP5) in the behavioral and neuroendocrine effects of chronic social defeat stress. Neuropharmacology 62 (1), 332–339. Hines, L.A., Sundin, J., Rona, R.J., Wessely, S., Fear, N.T., 2014. Posttraumatic stress disorder post Iraq and Afghanistan: prevalence among military subgroups. Canadian journal of psychiatry. Rev. Canad. Psychiatr. 59 (9), 468–479. Kang, J.I., Kim, T.Y., Choi, J.H., So, H.S., Kim, S.J., 2019. Allele-specific DNA methylation level of FKBP5 is associated with post-traumatic stress disorder. Psychoneuroendocrinology 103, 1–7. Karstoft, K.I., Andersen, S.B., Nielsen, A.B.S., 2017. Assessing PTSD in the military: validation of a scale distributed to Danish soldiers after deployment since 1998. Scand. J. Psychol. 58 (3), 260–268. Kessler, R.C., Sonnega, A., Bromet, E., Hughes, M., Nelson, C.B., 1995. Posttraumatic stress disorder in the national comorbidity survey. Arch. Gen. Psychiatr. 52 (12), 1048–1060. Klengel, T., Mehta, D., Anacker, C., Rex-Haffner, M., Pruessner, J.C., Pariante, C.M., Pace, T.W., Mercer, K.B., Mayberg, H.S., Bradley, B., Nemeroff, C.B., Holsboer, F., Heim, C.M., Ressler, K.J., Rein, T., Binder, E.B., 2013. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions. Nat. Neurosci. 16 (1), 33–41. Koenen, K.C., 2005. Nature-nurture interplay: genetically informative designs contribute to understanding the effects of trauma and interpersonal violence. J. Interpers Violence 20 (4), 507–512. Koenen, K.C., 2007. Genetics of posttraumatic stress disorder: Review and recommendations for future studies. J. Trauma. Stress 20 (5), 737–750.
53