Research

Research A Section 508–conformant HTML version of this article is available at https://doi.org/10.1289/EHP9009. Evaluation of a Gene–Environment Interaction of PON1 and Low-Level Nerve Agent Exposure with Gulf War Illness: A Prevalence Case–Control Study Drawn from the U.S. Military Health Survey’s National Population Sample Robert W. Haley,1 1 2 Gerald Kramer,1 Junhui Xiao,1 Jill A. Dever,2 and John F. Teiber1 Division of Epidemiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA RTI International, Washington, District of Columbia, USA BACKGROUND: Consensus on the etiology of 1991 Gulf War illness (GWI) has been limited by lack of objective individual-level environmental exposure information and assumed recall bias. OBJECTIVES: We investigated a prestated hypothesis of the association of GWI with a gene–environment (GxE) interaction of the paraoxonase-1 (PON1) Q192R polymorphism and low-level nerve agent exposure. METHODS: A prevalence sample of 508 GWI cases and 508 nonpaired controls was drawn from the 8,020 participants in the U.S. Military Health Survey, a representative sample survey of military veterans who served during the Gulf War. The PON1 Q192R genotype was measured by real-time polymerase chain reaction (RT-PCR), and the serum Q and R isoenzyme activity levels were measured with PON1-specific substrates. Low-level nerve agent exposure was estimated by survey questions on having heard nerve agent alarms during deployment. RESULTS: The GxE interaction of the Q192R genotype and hearing alarms was strongly associated with GWI on both the multiplicative [prevalence odds ratio (POR) of the interaction = 3:41; 95% confidence interval (CI): 1.20, 9.72] and additive (synergy index = 4:71; 95% CI: 1.82, 12.19) scales, adjusted for measured confounders. The Q192R genotype and the alarms variable were independent (adjusted POR in the controls = 1:18; 95% CI: 0.81, 1.73; p = 0:35), and the associations of GWI with the number of R alleles and quartiles of Q isoenzyme were monotonic. The adjusted relative excess risk due to interaction (aRERI) was 7.69 (95% CI: 2.71, 19.13). Substituting Q isoenzyme activity for the genotype in the analyses corroborated the findings. Sensitivity analyses suggested that recall bias had forced the estimate of the GxE interaction toward the null and that unmeasured confounding is unlikely to account for the findings. We found a GxE interaction involving the Q-correlated PON1 diazoxonase activity and a weak possible GxE involving the Khamisiyah plume model, but none involving the PON1 R isoenzyme activity, arylesterase activity, paraoxonase activity, butyrylcholinesterase genotypes or enzyme activity, or pyridostigmine. DISCUSSION: Given gene–environment independence and monotonicity, the unconfounded aRERI >0 supports a mechanistic interaction. Together with the direct evidence of exposure to fallout from bombing of chemical weapon storage facilities and the extensive toxicologic evidence of biochemical protection from organophosphates by the Q isoenzyme, the findings provide strong evidence for an etiologic role of low-level nerve agent in GWI. https://doi.org/10.1289/EHP9009 Introduction In the 1991 Persian Gulf War, approximately 700,000 U.S. military personnel and 300,000 people from 41 Coalition countries were deployed to the Kuwaiti Theater of Operations (KTO) for a 5-wk air war punctuated by a 5-d ground war.1 For months after the short deployment, tens of thousands of previously fit personnel developed an often-disabling set of symptoms, termed Gulf War illness (GWI), including fatigue, memory and concentration impairment, difficulty finding words, insomnia, diarrhea or constipation, cutaneous tingling and numbness, balance disturbance and vertigo attacks, body temperature dysregulation, and often severe somatic pain,2–4 which have persisted.5 Rates of these symptoms were higher in the KTO-deployed than in the nondeployed U.S. force.6,7 Among the deployed, both combat and support personnel were affected,8–10 and psychological explanations do not fully explain the illness.11 Clinical case–control studies employing neuroimaging, electroencephalography, and autonomic testing have identified Address correspondence to Robert W. Haley, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390 USA. Email: [email protected] Supplemental Material is available online (https://doi.org/10.1289/EHP9009). The authors declare they have no actual or potential competing financial interests. Received 21 January 2021; Revised 23 February 2022; Accepted 21 March 2022; Published 11 May 2022. Note to readers with disabilities: EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days. Environmental Health Perspectives abnormalities of brain and peripheral nerve function or metabolism underlying the symptoms.12–20 In the first published epidemiological study of environmental risk factors completed 4 y after the war (n = 249), our group found the strongest associations of GWI with self-reported lowlevel organophosphate nerve agent exposure and having experienced adverse effects of antinerve gas tablets containing the carbamate pyridostigmine bromide.21 To try to explain why only a fraction of those exposed to these agents developed GWI, we followed up with a prevalence case–control study (n = 40) drawn from the first cohort, in which we found GWI inversely associated with serum activity of the Q isoenzyme of the paraoxonase-1 (PON1) gene, a known genetic determinant of susceptibility to organophosphate cholinesterase-inhibiting chemicals including nerve agents.22 The PON1 enzyme hydrolyzes several important substrate molecules such as paraoxon (the active metabolite of the pesticide parathion) and diazoxon (the active metabolite of the pesticide diazinon) as well as nerve agents like sarin and soman. A given subject’s PON1 enzyme hydrolyzes these substrates at very different levels of catalytic efficiency. For example, one’s PON1 enzyme can have very high catalytic activity against sarin (high sarinase activity) but very low activity against paraoxon (low paraoxonase activity). The enzyme was named “paraoxonase” after the first substrate it was found to hydrolyze. The PON1 gene contains a common polymorphism in codon 192 that directs the production of either the 192 glutamine (Q) isoenzyme or the 192 arginine (R) isoenzyme, the only catalytic enzymes in humans that hydrolyze, and thus inactivate, organophosphates. QQ homozygous individuals produce only the Q isoenzyme, which efficiently hydrolyzes nerve agents like sarin; RR homozygotes produce only the R isoenzyme, which is relatively ineffective against nerve agents; and QR heterozygotes produce 057001-1 130(5) May 2022 variable proportions of both23,24; moreover, within genotype the level of Q isoenzyme activity varies >10-fold.23,25 As hypothesized, we found GWI was significantly elevated in veterans with an R allele (RR or QR genotypes) and in those with lower serum activity of the Q isoenzyme—a pattern compatible with an increased susceptibility to nerve agents.22 The sample size was too small to test for a gene–environment (GxE) interaction. The butyrylcholinesterase (BChE) enzyme (serum cholinesterase, pseudocholinesterase) normally contributes to protection from organophosphates and pyridostigmine by covalently binding and sequestering them. An early case report suggested that variants of the BChE gene with lower organophosphate binding activity may have contributed to GWI.26 Our study22 and a later one27 found no association of GWI with genetic variants or the serum activity level of BChE, but the Steele et al. study27 suggested that the uncommon BChE gene variants K/K, U/AK, U/A, A/F, and AK/F may have modified the association of GWI with having taken pyridostigmine antinerve agent tablets. Despite subsequent evidence further linking GWI with widespread exposure to cholinesterase-inhibiting chemicals, reviewed exhaustively by Michalovicz et al.,28 no consensus on the role of these environmental exposures has developed because of common study design flaws, including small, unrepresentative samples of veterans; self-selection of volunteer participants; and post hoc exploratory analyses of multiple risk factors.29 The criticism most often cited has been the assumption that recall bias from self-reported environmental exposure measures inflated their associations with GWI.30 We thus undertook the present study to test the prestated hypothesis that, if low-level organophosphate nerve agent exposure caused GWI, nerve agent–exposed veterans homozygous for the minor RR genotype (having no Q isoenzyme) and those QQ or QR individuals with lower levels of Q isoenzyme activity would have higher rates of GWI. To overcome the challenges, we performed a large population-representative random sample survey, measured PON1 and BChE genotypes and enzyme activity levels in a large prevalence case–control subsample drawn from the survey participants, tested the primary prestated hypothesis of a GxE interaction between the PON1 Q192R genotype and lowlevel nerve agent exposure on both the additive and multiplicative scales, controlled selection bias by random sample selection and confounding by multivariable analysis, and addressed recall bias and unmeasured confounding by sensitivity testing. Methods National Survey of Gulf War-Era Veterans To obtain information from a representative sample of veterans, from 2007 to 2010 we conducted a national prevalence survey known as the U.S. Military Health Survey (USMHS) via a computer-assisted telephone interview (CATI) with 8,020 veterans.7 The original sample of 14,812 veterans was randomly selected from the target population of 3,492,407 on active duty, National Guard, and Reserves in the personnel file of the Gulf War-era military population covering 2 August 1990 to 1 July 1991 maintained by the Defense Manpower Data Center, Seaside, California. We stratified the personnel file by the following design parameters prior to sample selection: a flag indicating deployment to the KTO; age (0 criterion, these findings establish a mechanistic interaction. We are then left with the question whether this mechanistic GxE interaction actually represents a biological, or functional, 057001-12 130(5) May 2022 interaction, i.e., where low-level nerve agent exposure and the genotype of the PON1 Q192R polymorphism interacted biochemically to produce GWI. Although the presence of a mechanistic interaction suggests this, it must be further supported by a convincing mosaic of basic research establishing a compatible biological mechanism.51 Extensive biochemical and clinical research has elucidated both the adverse brain effects of low-level sarin at doses comparable to levels to which soldiers would have been exposed during the Gulf War (Table S19) and the ability of the PON1 gene through its type Q isoenzyme to efficiently hydrolyze sarin at the low physiological concentrations expected from subsymptomatic exposure and prevent its neurotoxic effects23 (Table S20). Confounding by Measured Characteristics In the design of the USMHS we measured all the characteristics considered possible confounders and incorporated them in the random sampling design as stratification variables to ensure adequate power to control for them in the multivariable analyses.7 Controlling for these seven confounders only strengthened the GxE interaction effects, indicating negative confounding. Confounding by Unmeasured Characteristics Our finding a significant GxE interaction under a strong assumption of GxE independence means that either the GxE interaction truly exists or it is due to an interaction between the genotype and an unmeasured confounder of the environment effect.59 In our case, however, the fact that the PON1 Q192R genotype was randomly assigned at birth and was unknown to participants during and after the war makes associations between the genotype and unmeasured confounders unlikely.59 If there is no genotype– confounder association, then the GxE interaction on either the additive or multiplicative scales cannot be biased by confounding even if the unmeasured confounder is not controlled for.51 In the unlikely event that, notwithstanding these considerations, unmeasured confounding was present, our sensitivity analysis found that our measures of GxE interaction on the additive and multiplicative scales were robust to all plausible patterns and ranges of unmeasured confounding. In addition, the finding of negative confounding by the measured confounders limits the possibility of confounding by unmeasured characteristics associated with those we measured. Three possible confounders not analyzed in this study are pesticide exposure,85 rocket or jet fuel or vehicle exhaust exposure,86 and severe fright from hearing nerve agent alarms.8 Both organophosphate pesticides and fuel or exhaust fumes are capable of causing falsely positive nerve agent alarms from the M8A1 nerve agent detectors,86 but exposures to both were ubiquitous long before the approximately 10,000 alarms began sounding at the start of the air campaign when Coalition bombing of Iraqi chemical weapon facilities released the fallout cloud that reached U.S. troop concentrations just as sarin was detected at multiple sites37,87,88,39,38 (Figure 2). Whereas heavy repetitive exposure to organophosphate pesticides might cause chronic cognitive problems, the PON1 R isoenzyme is the more efficient detoxifier of most pesticides.23 Jet exhaust is not known to have neurotoxic effects,89 and PON1 does not metabolize petroleum-derived hydrocarbons. Thus, if the wide array of pesticides used in the Gulf War85 or jet exhaust were responsible for most of the alarms, the GxE interaction involving the Q isoenzyme would not have occurred. Although severe fright can produce posttraumatic stress disorder (PTSD), psychological explanations including PTSD do not explain GWI fully,11 and the studies suggesting that GWI was PTSD were shown to be falsely positive misinterpretations of psychological screening tests.90 Environmental Health Perspectives Limitations of the Study A limitation of this study is that it focused on environmental exposures to low-level sarin nerve agent to the exclusion of other risk factors implicated in epidemiological studies, such as exposures to pesticides, pyridostigmine bromide, antibiotics, immunizations, insect repellants, and psychological effects of deployment.91 This focus was due to the unique opportunity afforded by the PON1 Q192R polymorphism to develop objective genetic and biochemical support for their etiologic role in GWI by studying GxE interactions. Another limitation was that we were unable to apply Mendelian randomization to strengthen the evidence for a causal relationship between low-level nerve agent exposure and GWI, because the PON1 Q192R polymorphism is acting as an effect modifier rather than an instrumental variable, which is required to apply that approach.92 The USMHS survey participation rate of 60% leaves the possibility of selection bias; however, the application of survey weights that control for such bias had little effect on parameter estimates.7 Our decision not to adjust significance levels for multiple statistical testing is justified by use of prespecified hypotheses, the strong biochemical basis for the findings, and the relationships among the various exposures studied. Conclusion Our study supports the prestated hypothesis of a GxE interaction between the PON1 Q192R polymorphism and low-level nerve agent exposure measured by recall of hearing nerve agent alarms during the Gulf War conflict. Contemporaneous weather satellite images38 have removed the objection that originally discounted the role of nerve agent in GWI; selection of our GWI cases and controls from a large, representative sample of U.S. Gulf War– era veterans avoided selection bias; potential confounding by both measured and unmeasured confounders was ruled out; and sensitivity testing suggested that recall bias would have obscured the GxE interaction (biased toward the null), rather than causing a false positive one. The GxE interaction met the rigorous criteria for a mechanistic interaction, constituting a higher level of evidence for a causal link than a mere statistical epidemiological association. The prior research linking low-level sarin exposure with brain pathology compatible with GWI and demonstrating the biochemically modifying effects of the PON1 Q isoenzyme on the effects of sarin satisfy the higher standard of evidence for a biological interaction. These findings constitute strong evidence for a causal role of low-level nerve agent in GWI. Acknowledgments A large research team of survey specialists at RTI International contributed importantly to the design and performed the field work for the national CATI survey of Gulf War–era veterans. Research leaders included K.A. Considine, V.G. Iannacchione, J.A. Dever, C.P. Carson, H. Best, C. Bann, D. Creel, B. Alexander, A. LewisEvans, L. Trofimovich, K. Pate, A. Kenyon, J. Morton, C. Hill, and R.E. Mason. UT Southwestern team members who contributed substantially included C. Garcia, A. Lamb, R. Thompson, E. Cordell, J. Escobar, and W. Marshall. This work was funded by the following: U.S. Army Medical Research and Materiel Command grant number DAMD17-01-10741; Department of Veterans Affairs Medical Center, Dallas, Texas, IDIQ contract VA549-P-0027; and the Office of the Assistant Secretary of Defense for Health Affairs, through the Gulf War Illness Research Program under Award No. W81XWH-16-1-0740. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not 057001-13 130(5) May 2022 necessarily endorsed by the U.S. Departments of Defense or Veterans Affairs. Human subjects protection review: The protocol was approved by the institutional review boards of UT Southwestern Medical Center, RTI International, the U.S. Army, and the Department of Veterans Affairs; all subjects gave verbal informed consent for the survey and written informed consent for phlebotomy and genetic testing. 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VanderWeele TJ, Tchetgen EJT, Cornelis M, Kraft P. 2014. Methodological challenges in Mendelian randomization. Epidemiology 25(3):427–435, PMID: 24681576, https://doi.org/10.1097/EDE.0000000000000081. 057001-16 130(5) May 2022 Evaluation of a Gene–Environment Interaction of PON1 and Low-Level Nerve Agent Exposure with Gulf War Illness: A Prevalence Case–Control Study Drawn from the U.S. Military Health Survey’s National Population Sample Haley RW, et al. Environ Health Perspect. 2022 May; 130(5): 057001. 1. Briefly, what is Gulf War Illness (GWI)? What was the rationale for the current study? What was the hypothesis? 2. What was the source population for the cases and controls in this study? How were cases identified? How was the environmental exposure defined? 3. How does PON1 Q192R genotype affect the paraoxonase phenotype? Refer to Figure 3. How does this relate to the study hypothesis? 4. The first two rows in Figure 4 (panels A-E) display the individual effects of the environmental exposure (A-B) and PON1 genotype/phenotype (C-E). Briefly, what are the findings? 5. Refer to the main findings displayed in Table 2. Simply looking at the ORs, which appears to have a larger effect, genotype or exposure? Do the ORs in this Table suggest interaction? 6. The authors used automated calculations to estimate measures of additive and multiplicative interaction, but they can also be calculated directly from the data in Table 2. Calculations are more straightforward if the data are displayed in “2×4 table” format. Complete the accompanying spreadsheet to demonstrate. Discussion questions: 1. In the Introduction, the authors noted that although many studies have examined the association of GWI with exposure to nerve agents, there is no consensus on their role “because of common study design flaws, including small, unrepresentative samples of veterans; self-selection of volunteer participants; and post hoc exploratory analyses of multiple risk factors. The criticism most often cited has been the assumption that recall bias from self-rep…

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