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 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 low-level 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 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 low-level 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 (<49y, ≥49y); sex; race/ethnicity (non-Hispanic White vs. other to ensure adequate representation of minority groups in the sample); military rank during the war (officer, enlisted); military component (active duty, Reserve/Guard); unit location in KTO on 20 January 1991 (deployed only) and special study strata, including twin pairs; members of the 24th Reserve Naval Mobile Construction Battalion; and parents of a child with Goldenhar complex birth defect (Figure 1). With 74.9% of the randomly selected veterans located and contacted and 80.2% of these agreeing to participate, the overall response rate was 60.1%.31 Of the full USMHS sample, 6,497 were deployed to the KTO, and 1,523 were nondeployed. The survey methods, extensive pilot testing and initial findings of the USMHS were described in detail elsewhere.7
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 More:
https://ehp.niehs.nih.gov/doi/10.1289/EHP9009 
