DEPRESSION AND ANXIETY 32:307–315 (2015)
Research Article EFFECT OF THE APOE ε4 ALLELE AND COMBAT EXPOSURE ON PTSD AMONG IRAQ/AFGHANISTAN-ERA VETERANS Nathan A. Kimbrel, Ph.D.,1,2,3 ∗ Michael A. Hauser, Ph.D.,4,5 Melanie Garrett, M.S.,4,5 Allison Ashley-Koch, Ph.D.,4,5 Yutao Liu, Ph.D.,4,5 Michelle F. Dennis, B.A.,1,2,3 Rebecca C. Klein, Ph.D.,1,2,3 Veterans Affairs Mid-Atlantic Mental Illness Research, Education, and Clinical Center Workgroup,2 and Jean C. Beckham, Ph.D.1,2,3
Background: The apolipoprotein E (APOE) ε4 allele has been implicated in a range of neuropsychiatric conditions. The present research examined if the ε4 allele of the APOE gene moderated the effect of combat exposure on posttraumatic stress disorder (PTSD) among Iraq/Afghanistan-era veterans. Method: Participants included 765 non-Hispanic White (NHW) and 859 non-Hispanic Black (NHB) Iraq/Afghanistan-era veterans. A structured interview established psychiatric diagnoses. Combat exposure and PTSD symptom severity were assessed via self-report. Results: The most common lifetime diagnoses were depression (39.2%), PTSD (38.4%), and alcohol dependence (24.38%). After correcting for multiple comparisons, no significant effects were observed on any of the outcomes among the NHW sample; however, within the NHB sample, significant gene × environment (G × E) interactions were observed for lifetime PTSD (P = .0029) and PTSD symptom severity (P = .0009). In each case, the APOE ε4 allele had no effect on the outcomes when combat exposure was low; however, when combat exposure was high, an additive effect was observed such that ε4 homozygotes exposed to high levels of combat reported the highest rates of PTSD (92%) and the worst symptom severity scores on the Davidson Trauma Scale (M = 79.5). Conclusions: Although preliminary, these findings suggest that the APOE ε4 allele, in conjunction with exposure to high levels of combat exposure, may increase veterans’ risk for developing PTSD. Depression and Anxiety 32:307–315, C 2015 Wiley Periodicals, Inc. 2015. Key words: APOE; genetic; PTSD; depression; veteran; comorbidity
1 Durham
Veterans Affairs Medical Center, Durham, North Carolina 2 The VA Mid-Atlantic Mental Illness Research, Education, and Clinical Center, Durham, North Carolina 3 Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 4 Department of Medicine, Duke University Medical Center, Durham, North Carolina 5 Duke Center for Human Genetics, Durham, North Carolina Contract grant sponsor: VA Mid-Atlantic Mental Illness Research, Education, and Clinical Center; Contract grant sponsor: Research & Development and Mental Health Services of the Durham Veterans
C 2015 Wiley Periodicals, Inc.
Affairs Medical Center; Contract grant sponsor: Clinical Science Research and Development Service of the VA Office of Research and Development. ∗ Correspondence
to: Nathan A. Kimbrel, Durham Veterans Affairs Medical Center, 508 Fulton Street, Durham, NC 27705. E-mail: [email protected] Received for publication 21 January 2014; Revised 12 May 2014; Accepted 11 December 2014 DOI 10.1002/da.22348 Published online 24 February 2015 in Wiley Online Library (wileyonlinelibrary.com).
Kimbrel et al.
308
INTRODUCTION
Iraq/Afghanistan-era veterans are at increased risk for
anxiety, mood, and substance use disorders.[1–3] One obvious explanation for the high rates of psychiatric problems and psychiatric comorbidity observed among returning veterans is combat exposure, which has been associated with a variety of problems, including posttraumatic stress disorder (PTSD), depression, and substance use.[1,4–6] Although the impact of combat exposure on postdeployment psychiatric adjustment is evident, genetic factors are also likely to play a role.[7–13] Indeed, it is estimated that approximately 30% of susceptibility to PTSD among Vietnam Veterans is due to genetic factors.[7] Behavioral genetics studies further suggest that genetic factors may partially explain the cooccurrence of other disorders observed among veterans, such as the comorbidity observed between PTSD and depression.[8] Much of the psychiatric genetics research conducted with veterans to date has involved candidate gene studies aimed at identifying the genetic basis of PTSD;[9–12] however, the molecular genetic basis of PTSD has remained elusive, leading some researchers to propose the need for more gene × environment (G × E) investigations of PTSD.[13] For example, although it is clear that trauma severity—particularly combat exposure severity—is associated with the development of PTSD,[1, 4–6] there is also substantial variability in response to traumatic events,[14] which suggests the possibility of G × E interactions.[15] Moreover, a number of studies have already found evidence for G × E effects on PTSD.[13, 16–20] There is also evidence for G × E effects on other closely related disorders, such as other anxiety disorders, depression, and alcohol use disorders.[21–24] To date, however, there has been only limited research on whether the apolipoprotein E (APOE) gene might moderate the effect of trauma exposure on PTSD and other closely related disorders.[20, 25] The human APOE gene contains three polymorphic alleles, ε2, ε3, and ε4, that are estimated to have worldwide frequencies of, 8.4, 77.9, and 13.7%, respectively.[26] APOE supports injury repair in the brain by transporting cholesterol and other lipids to neurons; however, the ε4 allele of the APOE gene is associated with a wide range of neurological problems, including apoptosis, cortical atrophy, abnormalities in the hippocampus, inflammation, increased amyloid– β (Aβ) peptide aggregation, decreased lipid and glucose metabolism, and decreased neurogenesis.[26, 27] The ε4 allele is also associated with cardiovascular disease, difficulty in recovering from traumatic brain injuries (TBI), and Alzheimer disease (AD).[26–31] Most notable are the additive genetic effects observed for the APOE ε4 allele on risk for AD. For example, among non-Hispanic Whites (NHWs), the odds ratio (OR) of developing AD is 2.6 for ε2/ε4, 3.2 for ε3/ε4, and 14.9 among ε4/ε4 homozygotes relative to ε3/ε3 homozygotes.[26] Age of onset for AD Depression and Anxiety
is also affected by APOE ε4 in a dose-dependent manner.[26] These findings, in conjunction with prior research suggesting an increased risk for dementia among veterans with PTSD,[32] recently lead Lyons and colleagues to examine whether the ε4 allele of the APOE gene might moderate susceptibility to PTSD in a sample of predominantly NHW male Vietnam-era veterans (N = 172).[20] Consistent with their hypothesis, they found that ε4 carriers who scored highly on a self-report measure of combat exposure were significantly more likely to have PTSD symptoms and to meet criteria for PTSD.[20] Although promising, the conclusions that can be drawn from this study are limited by its relatively small sample size, which prevented the authors from examining the potential additive effects of ε4 on PTSD. An additional concern relates to disparate findings between this study and other studies on APOE and PTSD. The first such study examined the relationship between APOE genotype and PTSD symptoms among 54 male NHW combat veterans, all of whom met criteria for combat-related PTSD.[33] Carriers of the ε2 allele— which is associated with decreased risk for AD[26] — reported more re-experiencing symptoms and worse memory functioning than the rest of the sample; however, the findings from this study are also limited by the small sample size and the fact that all of the participants met criteria for PTSD. A more recent study examined the relationship between APOE, PTSD, and alcohol use in Korean veterans of the Vietnam War.[25] Absence of the ε2 allele was associated with decreased risk for PTSD in this study after accounting for combat exposure. In addition, a significant interaction between APOE ε2 and harmful drinking was observed, such that the ε2 noncarriers with harmful drinking were more likely to have PTSD compared with ε2 noncarriers without harmful drinking. Although novel, this study was also limited by its small sample size (N = 256) and the small number of ε4 homozygotes present (n = 3). HYPOTHESES
Based on the recent findings from Lyons et al.[20] as well as prior research demonstrating additive effects for ε4 on AD,[26] we hypothesized that the ε4 allele would moderate the effect of combat exposure on PTSD in an additive fashion, such that ε4 homozygotes exposed to high levels of combat would exhibit the highest rate of PTSD and have the worst symptom severity. In addition, given previous findings suggesting that much of the comorbidity observed between PTSD and other psychiatric disorders is due to genetic factors[8] as well as evidence for G × E effects on other closely related disorders, such as other anxiety disorders, depression, and alcohol use-disorders,[21–24] we further hypothesized that the APOE ε4 × combat interaction would be associated with psychiatric comorbidity in general, such
Research Article: APOE, Combat Exposure, and PTSD
that ε4 homozygotes exposed to high levels of combat would also exhibit the highest overall rates of psychiatric comorbidity.
METHODS PARTICIPANTS
Participants were drawn from an ongoing multisite research study known as the Veterans Affairs (VA) Mid-Atlantic Mental Illness Research, Education and Clinical Center (MIRECC) Repository. To be eligible, participants had to have served in the U.S. military after September 11, 2001. Additional inclusion criteria for the current analyses included: (1) being either an NHW or non-Hispanic Black (NHB) Iraq/Afghanistan-era veteran; (2) having data available regarding level of combat exposure; (3) completion of the diagnostic interview; and (4) being genotyped for APOE. The final sample of participants (total N = 1,624) included 765 NHW and 859 NHB Iraq/Afghanistan-era veterans. Note that the current analyses were restricted to NHB and NHW veterans because they represented the two largest subgroups of participants (1,624/1,686 or 96.3%) with genetic data available for analysis from the Repository at the time of the study. The final sample was predominantly male (79.5%), although females were also well represented (20.5%). The majority of participants had served in Iraq (82.5%). A substantial proportion had also served in Afghanistan (49.8%). Approximately 41.6% of the sample had served in both Iraq and Afghanistan. PROCEDURES
Participants were recruited through fliers, advertisements, VA clinic referrals, and invitation letters from four VA Medical Centers located in the mid-Atlantic region of the United States. Approval was obtained from each site’s Institutional Review Board (IRB) prior to data collection. After a complete description of the study was provided to participants, written informed consent was obtained. Following informed consent procedures, a structured diagnostic interview was administered to participants along with a battery of self-report measures. A peripheral blood sample was obtained through venipuncture.
309
(n = 35). Genotype frequencies for the NHW sample (n = 765) were as follows: ε2/ε2 (n = 8), ε2/ε3 (n = 89), ε3/ε3 (n = 459), ε4/ε2 (n = 16), ε4/ε3 (n = 175), and ε4/ε4 (n = 18). Thus, there were approximately twice as many ε4 homozygotes among the NHB sample as in the NHW sample. Neither distribution varied from Hardy– Weinberg equilibrium (P’s > .05). MEASURES
Trained clinical interviewers administered the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I)[35] to determine psychiatric diagnoses. Reliability among clinical interviewers (N = 22) scoring seven SCID-I training videos was excellent for both current and lifetime diagnoses (Fleiss’ κ = 0.94). For the purposes of the current analyses, lifetime mood (depression, bipolar I, bipolar II), anxiety (PTSD, panic disorder, social anxiety disorder, obsessive-compulsive disorder, specific phobia), and substance use disorders (alcohol, sedatives, cannabis, stimulants, opioid, cocaine, hallucinogens) were summed to create a lifetime psychiatric comorbidity variable. Psychotic (schizophrenia lifetime prevalence = 0.2%) and eating disorders (anorexia lifetime prevalence = 0.3%; bulimia lifetime prevalence = 0.3%) were excluded from the analyses due to low base rates. On average, participants had 1.5 lifetime psychiatric disorders (SD = 1.3; range = 0–9). The most common diagnoses were major depression (39.2%), PTSD (38.4%), and alcohol dependence (24.4%). The Combat Exposure Scale (CES)[6] is a brief selfreport measure used to assess combat exposure. The scoring procedures outlined by Keane et al.[6] were followed in the current study. The mean score on the CES was 11.4 (SD = 10.5; range = 0–40). To maximize interpretability of the findings, a median split was used to classify level of combat exposure as either high or low, such that scores from 0 to 5 were recoded as 0 (low) and scores from 6 to 40 were recoded as 1 (high). Internal consistency for the CES was good (α = .86). The Davidson Trauma Scale (DTS)[36] was used to assess PTSD symptom severity. It contains 34 items that allow participants to rate each of the 17 DSM-IV-TR PTSD symptoms on frequency and severity. The full range of possible DTS scores was observed (range = 0– 136; M = 39.4; SD = 39.1). Internal consistency was excellent (α = .98).
GENOTYPING
Genomic DNA was extracted from the blood samples using standard salting techniques (Gentra, Minneapolis, MN). APOE genotypes for rs429358 and rs7412 were determined with TaqMan-based allelic discrimination assays by use of Assays-On-Demand products. Products were run on the ViiA7 Realtime PCR system with 384well block according to the standard protocols from the manufacturer (Applied Biosystems, Foster City, CA).[34] Genotype frequencies for the NHB sample (n = 859) were as follows: ε2/ε2 (n = 6), ε2/ε3 (n = 117), ε3/ε3 (n = 414), ε4/ε2 (n = 33), ε4/ε3 (n = 254), and ε4/ε4
DATA ANALYSIS PLAN
The NHW and NHB samples were examined separately to help protect against potential population stratification effects. Binary logistic regression was used to examine the effects of the APOE ε4 allele and combat exposure on PTSD diagnosis. Due to small cell counts for higher numbers of psychiatric disorders, the number of psychiatric disorders was truncated at 3. Ordinal logistic regression was used to examine the relationship between the APOE ε4 allele and combat exposure on number of psychiatric disorders (0, 1, 2, or ࣙ3), whereas linear Depression and Anxiety
Kimbrel et al.
310
regression was utilized to study the relationship between the APOE ε4 allele, combat exposure, and PTSD symptom severity, as determined by the DTS. For each model, the main effects of APOE and combat exposure were entered into the model along with the interaction term. An additive genetic model was employed and therefore APOE was considered continuously in all of the analyses (zero, one, or two copies of the ε4 allele). Contrasts were specified within each model to inspect the relationship between APOE ε4 and the outcome at each level of combat exposure; CES < 6 was considered to be low combat exposure and CES ࣙ 6 was considered to be high combat exposure. Because three hypotheses were tested in two different samples (six total tests), a Bonferroni correction was used and α was set at .0083. Statistical tests were performed using SAS version 9.2 (SAS Systems, Cary, NC).
RESULTS As shown in Table 1, no significant G × E effects were observed within the NHW sample. In contrast, significant G × E effects were observed for all three of the main outcomes examined in the NHB sample (all P’s < .0083). A significant interaction between APOE and combat exposure was observed for lifetime PTSD diagnosis (P = .0029). For each additional ε4 allele, the odds of having a lifetime PTSD diagnosis increased by
1.61, but only among those with high combat exposure (P = .0077). The rates of lifetime PTSD among the high combat exposure group were 49.3% for participants with no ε4 alleles, 56.4% for those with one ε4 allele, and 92.3% for those with two ε4 alleles (Fig. 1). APOE and combat exposure also interacted to predict current PTSD symptom severity (P = .0009). Again, as the number of ε4 alleles increased, the mean severity score on the DTS increased by 10.25 points, but only among those with high combat exposure (P = .0011, Fig. 2). Finally, APOE and combat exposure were found to also interact to predict lifetime psychiatric comorbidity (P = .0065) among NHB veterans, such that the odds of having a greater number of lifetime psychiatric disorders increased by 1.55 for each additional ε4 allele, but only among those with high combat exposure (P = .0053, Fig. 3). To determine if the latter finding was being driven by the presence or absence of a PTSD diagnosis, a post hoc analysis was conducted in which PTSD diagnosis was removed from the calculation of the lifetime psychiatric comorbidity score. The interaction term continued to be statistically significant in this follow-up analysis (P = .0202). In addition, contrasts indicated that the odds of having a greater number of lifetime psychiatric disorders other than PTSD increased by 1.50 (1.10, 2.03) with each additional ε4 allele, but only among those with high combat exposure (P = .0098). When this analysis was repeated with lifetime PTSD diagnosis included as a covariate, the interaction term
TABLE 1. Summary of the APOE × combat exposure interaction effects observed among Iraq/Afghanistan-era veterans Outcome variable Lifetime PTSD
Number of lifetime psychiatric disorders
PTSD symptom severity
Outcome variable Lifetime PTSD
Number of lifetime psychiatric disorders
PTSD symptom severity
a Meets b Odds
Non-Hispanic White (NHW) Iraq/Afghanistan-era veterans (N = 765) APOE × CES P-value CES level Odds ratio/β b 95% Confidence interval .4532
Research Article EFFECT OF THE APOE ε4 ALLELE AND COMBAT EXPOSURE ON PTSD AMONG IRAQ/AFGHANISTAN-ERA VETERANS Nathan A. Kimbrel, Ph.D.,1,2,3 ∗ Michael A. Hauser, Ph.D.,4,5 Melanie Garrett, M.S.,4,5 Allison Ashley-Koch, Ph.D.,4,5 Yutao Liu, Ph.D.,4,5 Michelle F. Dennis, B.A.,1,2,3 Rebecca C. Klein, Ph.D.,1,2,3 Veterans Affairs Mid-Atlantic Mental Illness Research, Education, and Clinical Center Workgroup,2 and Jean C. Beckham, Ph.D.1,2,3
Background: The apolipoprotein E (APOE) ε4 allele has been implicated in a range of neuropsychiatric conditions. The present research examined if the ε4 allele of the APOE gene moderated the effect of combat exposure on posttraumatic stress disorder (PTSD) among Iraq/Afghanistan-era veterans. Method: Participants included 765 non-Hispanic White (NHW) and 859 non-Hispanic Black (NHB) Iraq/Afghanistan-era veterans. A structured interview established psychiatric diagnoses. Combat exposure and PTSD symptom severity were assessed via self-report. Results: The most common lifetime diagnoses were depression (39.2%), PTSD (38.4%), and alcohol dependence (24.38%). After correcting for multiple comparisons, no significant effects were observed on any of the outcomes among the NHW sample; however, within the NHB sample, significant gene × environment (G × E) interactions were observed for lifetime PTSD (P = .0029) and PTSD symptom severity (P = .0009). In each case, the APOE ε4 allele had no effect on the outcomes when combat exposure was low; however, when combat exposure was high, an additive effect was observed such that ε4 homozygotes exposed to high levels of combat reported the highest rates of PTSD (92%) and the worst symptom severity scores on the Davidson Trauma Scale (M = 79.5). Conclusions: Although preliminary, these findings suggest that the APOE ε4 allele, in conjunction with exposure to high levels of combat exposure, may increase veterans’ risk for developing PTSD. Depression and Anxiety 32:307–315, C 2015 Wiley Periodicals, Inc. 2015. Key words: APOE; genetic; PTSD; depression; veteran; comorbidity
1 Durham
Veterans Affairs Medical Center, Durham, North Carolina 2 The VA Mid-Atlantic Mental Illness Research, Education, and Clinical Center, Durham, North Carolina 3 Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina 4 Department of Medicine, Duke University Medical Center, Durham, North Carolina 5 Duke Center for Human Genetics, Durham, North Carolina Contract grant sponsor: VA Mid-Atlantic Mental Illness Research, Education, and Clinical Center; Contract grant sponsor: Research & Development and Mental Health Services of the Durham Veterans
C 2015 Wiley Periodicals, Inc.
Affairs Medical Center; Contract grant sponsor: Clinical Science Research and Development Service of the VA Office of Research and Development. ∗ Correspondence
to: Nathan A. Kimbrel, Durham Veterans Affairs Medical Center, 508 Fulton Street, Durham, NC 27705. E-mail: [email protected] Received for publication 21 January 2014; Revised 12 May 2014; Accepted 11 December 2014 DOI 10.1002/da.22348 Published online 24 February 2015 in Wiley Online Library (wileyonlinelibrary.com).
Kimbrel et al.
308
INTRODUCTION
Iraq/Afghanistan-era veterans are at increased risk for
anxiety, mood, and substance use disorders.[1–3] One obvious explanation for the high rates of psychiatric problems and psychiatric comorbidity observed among returning veterans is combat exposure, which has been associated with a variety of problems, including posttraumatic stress disorder (PTSD), depression, and substance use.[1,4–6] Although the impact of combat exposure on postdeployment psychiatric adjustment is evident, genetic factors are also likely to play a role.[7–13] Indeed, it is estimated that approximately 30% of susceptibility to PTSD among Vietnam Veterans is due to genetic factors.[7] Behavioral genetics studies further suggest that genetic factors may partially explain the cooccurrence of other disorders observed among veterans, such as the comorbidity observed between PTSD and depression.[8] Much of the psychiatric genetics research conducted with veterans to date has involved candidate gene studies aimed at identifying the genetic basis of PTSD;[9–12] however, the molecular genetic basis of PTSD has remained elusive, leading some researchers to propose the need for more gene × environment (G × E) investigations of PTSD.[13] For example, although it is clear that trauma severity—particularly combat exposure severity—is associated with the development of PTSD,[1, 4–6] there is also substantial variability in response to traumatic events,[14] which suggests the possibility of G × E interactions.[15] Moreover, a number of studies have already found evidence for G × E effects on PTSD.[13, 16–20] There is also evidence for G × E effects on other closely related disorders, such as other anxiety disorders, depression, and alcohol use disorders.[21–24] To date, however, there has been only limited research on whether the apolipoprotein E (APOE) gene might moderate the effect of trauma exposure on PTSD and other closely related disorders.[20, 25] The human APOE gene contains three polymorphic alleles, ε2, ε3, and ε4, that are estimated to have worldwide frequencies of, 8.4, 77.9, and 13.7%, respectively.[26] APOE supports injury repair in the brain by transporting cholesterol and other lipids to neurons; however, the ε4 allele of the APOE gene is associated with a wide range of neurological problems, including apoptosis, cortical atrophy, abnormalities in the hippocampus, inflammation, increased amyloid– β (Aβ) peptide aggregation, decreased lipid and glucose metabolism, and decreased neurogenesis.[26, 27] The ε4 allele is also associated with cardiovascular disease, difficulty in recovering from traumatic brain injuries (TBI), and Alzheimer disease (AD).[26–31] Most notable are the additive genetic effects observed for the APOE ε4 allele on risk for AD. For example, among non-Hispanic Whites (NHWs), the odds ratio (OR) of developing AD is 2.6 for ε2/ε4, 3.2 for ε3/ε4, and 14.9 among ε4/ε4 homozygotes relative to ε3/ε3 homozygotes.[26] Age of onset for AD Depression and Anxiety
is also affected by APOE ε4 in a dose-dependent manner.[26] These findings, in conjunction with prior research suggesting an increased risk for dementia among veterans with PTSD,[32] recently lead Lyons and colleagues to examine whether the ε4 allele of the APOE gene might moderate susceptibility to PTSD in a sample of predominantly NHW male Vietnam-era veterans (N = 172).[20] Consistent with their hypothesis, they found that ε4 carriers who scored highly on a self-report measure of combat exposure were significantly more likely to have PTSD symptoms and to meet criteria for PTSD.[20] Although promising, the conclusions that can be drawn from this study are limited by its relatively small sample size, which prevented the authors from examining the potential additive effects of ε4 on PTSD. An additional concern relates to disparate findings between this study and other studies on APOE and PTSD. The first such study examined the relationship between APOE genotype and PTSD symptoms among 54 male NHW combat veterans, all of whom met criteria for combat-related PTSD.[33] Carriers of the ε2 allele— which is associated with decreased risk for AD[26] — reported more re-experiencing symptoms and worse memory functioning than the rest of the sample; however, the findings from this study are also limited by the small sample size and the fact that all of the participants met criteria for PTSD. A more recent study examined the relationship between APOE, PTSD, and alcohol use in Korean veterans of the Vietnam War.[25] Absence of the ε2 allele was associated with decreased risk for PTSD in this study after accounting for combat exposure. In addition, a significant interaction between APOE ε2 and harmful drinking was observed, such that the ε2 noncarriers with harmful drinking were more likely to have PTSD compared with ε2 noncarriers without harmful drinking. Although novel, this study was also limited by its small sample size (N = 256) and the small number of ε4 homozygotes present (n = 3). HYPOTHESES
Based on the recent findings from Lyons et al.[20] as well as prior research demonstrating additive effects for ε4 on AD,[26] we hypothesized that the ε4 allele would moderate the effect of combat exposure on PTSD in an additive fashion, such that ε4 homozygotes exposed to high levels of combat would exhibit the highest rate of PTSD and have the worst symptom severity. In addition, given previous findings suggesting that much of the comorbidity observed between PTSD and other psychiatric disorders is due to genetic factors[8] as well as evidence for G × E effects on other closely related disorders, such as other anxiety disorders, depression, and alcohol use-disorders,[21–24] we further hypothesized that the APOE ε4 × combat interaction would be associated with psychiatric comorbidity in general, such
Research Article: APOE, Combat Exposure, and PTSD
that ε4 homozygotes exposed to high levels of combat would also exhibit the highest overall rates of psychiatric comorbidity.
METHODS PARTICIPANTS
Participants were drawn from an ongoing multisite research study known as the Veterans Affairs (VA) Mid-Atlantic Mental Illness Research, Education and Clinical Center (MIRECC) Repository. To be eligible, participants had to have served in the U.S. military after September 11, 2001. Additional inclusion criteria for the current analyses included: (1) being either an NHW or non-Hispanic Black (NHB) Iraq/Afghanistan-era veteran; (2) having data available regarding level of combat exposure; (3) completion of the diagnostic interview; and (4) being genotyped for APOE. The final sample of participants (total N = 1,624) included 765 NHW and 859 NHB Iraq/Afghanistan-era veterans. Note that the current analyses were restricted to NHB and NHW veterans because they represented the two largest subgroups of participants (1,624/1,686 or 96.3%) with genetic data available for analysis from the Repository at the time of the study. The final sample was predominantly male (79.5%), although females were also well represented (20.5%). The majority of participants had served in Iraq (82.5%). A substantial proportion had also served in Afghanistan (49.8%). Approximately 41.6% of the sample had served in both Iraq and Afghanistan. PROCEDURES
Participants were recruited through fliers, advertisements, VA clinic referrals, and invitation letters from four VA Medical Centers located in the mid-Atlantic region of the United States. Approval was obtained from each site’s Institutional Review Board (IRB) prior to data collection. After a complete description of the study was provided to participants, written informed consent was obtained. Following informed consent procedures, a structured diagnostic interview was administered to participants along with a battery of self-report measures. A peripheral blood sample was obtained through venipuncture.
309
(n = 35). Genotype frequencies for the NHW sample (n = 765) were as follows: ε2/ε2 (n = 8), ε2/ε3 (n = 89), ε3/ε3 (n = 459), ε4/ε2 (n = 16), ε4/ε3 (n = 175), and ε4/ε4 (n = 18). Thus, there were approximately twice as many ε4 homozygotes among the NHB sample as in the NHW sample. Neither distribution varied from Hardy– Weinberg equilibrium (P’s > .05). MEASURES
Trained clinical interviewers administered the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I)[35] to determine psychiatric diagnoses. Reliability among clinical interviewers (N = 22) scoring seven SCID-I training videos was excellent for both current and lifetime diagnoses (Fleiss’ κ = 0.94). For the purposes of the current analyses, lifetime mood (depression, bipolar I, bipolar II), anxiety (PTSD, panic disorder, social anxiety disorder, obsessive-compulsive disorder, specific phobia), and substance use disorders (alcohol, sedatives, cannabis, stimulants, opioid, cocaine, hallucinogens) were summed to create a lifetime psychiatric comorbidity variable. Psychotic (schizophrenia lifetime prevalence = 0.2%) and eating disorders (anorexia lifetime prevalence = 0.3%; bulimia lifetime prevalence = 0.3%) were excluded from the analyses due to low base rates. On average, participants had 1.5 lifetime psychiatric disorders (SD = 1.3; range = 0–9). The most common diagnoses were major depression (39.2%), PTSD (38.4%), and alcohol dependence (24.4%). The Combat Exposure Scale (CES)[6] is a brief selfreport measure used to assess combat exposure. The scoring procedures outlined by Keane et al.[6] were followed in the current study. The mean score on the CES was 11.4 (SD = 10.5; range = 0–40). To maximize interpretability of the findings, a median split was used to classify level of combat exposure as either high or low, such that scores from 0 to 5 were recoded as 0 (low) and scores from 6 to 40 were recoded as 1 (high). Internal consistency for the CES was good (α = .86). The Davidson Trauma Scale (DTS)[36] was used to assess PTSD symptom severity. It contains 34 items that allow participants to rate each of the 17 DSM-IV-TR PTSD symptoms on frequency and severity. The full range of possible DTS scores was observed (range = 0– 136; M = 39.4; SD = 39.1). Internal consistency was excellent (α = .98).
GENOTYPING
Genomic DNA was extracted from the blood samples using standard salting techniques (Gentra, Minneapolis, MN). APOE genotypes for rs429358 and rs7412 were determined with TaqMan-based allelic discrimination assays by use of Assays-On-Demand products. Products were run on the ViiA7 Realtime PCR system with 384well block according to the standard protocols from the manufacturer (Applied Biosystems, Foster City, CA).[34] Genotype frequencies for the NHB sample (n = 859) were as follows: ε2/ε2 (n = 6), ε2/ε3 (n = 117), ε3/ε3 (n = 414), ε4/ε2 (n = 33), ε4/ε3 (n = 254), and ε4/ε4
DATA ANALYSIS PLAN
The NHW and NHB samples were examined separately to help protect against potential population stratification effects. Binary logistic regression was used to examine the effects of the APOE ε4 allele and combat exposure on PTSD diagnosis. Due to small cell counts for higher numbers of psychiatric disorders, the number of psychiatric disorders was truncated at 3. Ordinal logistic regression was used to examine the relationship between the APOE ε4 allele and combat exposure on number of psychiatric disorders (0, 1, 2, or ࣙ3), whereas linear Depression and Anxiety
Kimbrel et al.
310
regression was utilized to study the relationship between the APOE ε4 allele, combat exposure, and PTSD symptom severity, as determined by the DTS. For each model, the main effects of APOE and combat exposure were entered into the model along with the interaction term. An additive genetic model was employed and therefore APOE was considered continuously in all of the analyses (zero, one, or two copies of the ε4 allele). Contrasts were specified within each model to inspect the relationship between APOE ε4 and the outcome at each level of combat exposure; CES < 6 was considered to be low combat exposure and CES ࣙ 6 was considered to be high combat exposure. Because three hypotheses were tested in two different samples (six total tests), a Bonferroni correction was used and α was set at .0083. Statistical tests were performed using SAS version 9.2 (SAS Systems, Cary, NC).
RESULTS As shown in Table 1, no significant G × E effects were observed within the NHW sample. In contrast, significant G × E effects were observed for all three of the main outcomes examined in the NHB sample (all P’s < .0083). A significant interaction between APOE and combat exposure was observed for lifetime PTSD diagnosis (P = .0029). For each additional ε4 allele, the odds of having a lifetime PTSD diagnosis increased by
1.61, but only among those with high combat exposure (P = .0077). The rates of lifetime PTSD among the high combat exposure group were 49.3% for participants with no ε4 alleles, 56.4% for those with one ε4 allele, and 92.3% for those with two ε4 alleles (Fig. 1). APOE and combat exposure also interacted to predict current PTSD symptom severity (P = .0009). Again, as the number of ε4 alleles increased, the mean severity score on the DTS increased by 10.25 points, but only among those with high combat exposure (P = .0011, Fig. 2). Finally, APOE and combat exposure were found to also interact to predict lifetime psychiatric comorbidity (P = .0065) among NHB veterans, such that the odds of having a greater number of lifetime psychiatric disorders increased by 1.55 for each additional ε4 allele, but only among those with high combat exposure (P = .0053, Fig. 3). To determine if the latter finding was being driven by the presence or absence of a PTSD diagnosis, a post hoc analysis was conducted in which PTSD diagnosis was removed from the calculation of the lifetime psychiatric comorbidity score. The interaction term continued to be statistically significant in this follow-up analysis (P = .0202). In addition, contrasts indicated that the odds of having a greater number of lifetime psychiatric disorders other than PTSD increased by 1.50 (1.10, 2.03) with each additional ε4 allele, but only among those with high combat exposure (P = .0098). When this analysis was repeated with lifetime PTSD diagnosis included as a covariate, the interaction term
TABLE 1. Summary of the APOE × combat exposure interaction effects observed among Iraq/Afghanistan-era veterans Outcome variable Lifetime PTSD
Number of lifetime psychiatric disorders
PTSD symptom severity
Outcome variable Lifetime PTSD
Number of lifetime psychiatric disorders
PTSD symptom severity
a Meets b Odds
Non-Hispanic White (NHW) Iraq/Afghanistan-era veterans (N = 765) APOE × CES P-value CES level Odds ratio/β b 95% Confidence interval .4532
