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

Abstract

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 $\text{interaction}=3.41$; 95% confidence interval (CI): 1.20, 9.72] and additive (synergy $\text{index}=4.71$; 95% CI: 1.82, 12.19) scales, adjusted for measured confounders. The Q192R genotype and the alarms variable were independent (adjusted POR in the $\text{controls}=1.18$; 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 $\text{aRERI}>0$ 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.

Figure 1.

Selection of the stage 1 USMHS population sample and the stage 2 prevalence case–control subsample. (A) Includes all U.S. military personnel on active duty or in the Reserves or National Guard on 2 August 1990. (B) Those not deployed to the Kuwaiti Theater of Operations (KTO) included medically nondeployable personnel. “Special Studies” included twins, members of the 24th Reserve Naval Mobile Construction Battalion (Seabees), and parents of children with Goldenhar Complex. Counts for subgroups are suppressed to maintain confidentiality according to terms of the Certificate of Confidentiality. (C) The sampling frame was stratified by age, sex, race, service branch, military rank, active duty/reserve status, special studies strata, and KTO location on 20 January 1991. (D) The denominator of the survey-weighted contact rate includes the number of known survey-eligible persons and the estimated number of eligible persons among those with an undetermined survey eligibility status. (E) The eligibility rate was calculated with survey weights applied among sample members with known survey eligibility. (F) The survey-weighted response rate is the American Association for Public Opinion Research’s Response Rate 4 (RR4), calculated as the number of confirmed and estimated eligible cases among those initially selected for the CATI phase of the study. (G) The universe for the current prevalence case–control study includes all subjects in (F). (H) In selecting the prevalence case–control sample, the CATI algorithm selected all GWI cases by the Research and Kansas case definitions and a 12.5% random sample of the rest of the USMHS participants. The slight deviations from these selection percentages resulted from late adjustments in the CATI algorithm. (L) Veterans who met any GWI case definition specifically met the GWI Research, CDC, or Kansas (without exclusions) case definitions. The 165 developmental subjects excluded after (L) were those from the Seabees battalion who participated in the initial study to develop the GWI Research case definition. (M) To minimize misclassification, the 508 who met the GWI Research case definition—a close subset of the CDC and Modified Kansas definitions—were separated from those meeting only the CDC or Modified Kansas definitions and were used as cases in the prevalence case-control study. Unaffected subjects, those meeting none of the three GWI case definitions, constituted the controls in the prevalence case–control study. The left-hand column (A–F) of the figure was adapted from the original USMHS sampling flowchart published in Iannacchione et al. Note: CATI, computer-assisted telephone interview; CDC, U.S. Centers for Disease Control and Prevention; GWI, Gulf War Illness; USMHS, U.S. Military Health Survey.

Figure 2.

(A) Map of the Kuwaiti Theater of Operations (KTO) showing the locations of major chemical weapons storage facilities bombed on the night of 18–19 January and location of U.S. military units and sites of sarin and other chemical weapon detections on 19–21 January. (B) Weather satellite image of the large debris cloud (light tan in color and demarcated by white arrows) containing dispersed chemical weapon vapor. Sequential images from every-2-h passes showed the debris rising from the bombed chemical weapons storage facilities at Muthanna and Fallujah and drifting southward to encompass U.S. troop positions. This image was taken at approximately 14:30 h local time on 19 January showing the debris reaching Hafir Al Batin, the day 10,000 nerve agent alarms began sounding and chemical weapons experts using sophisticated equipment detected ambient sarin and other agents at multiple sites across U.S. positions.^, The light green cloud bank extending from northeast to southwest indicated a stationary weather front that held the sarin-containing debris cloud over U.S. troop positions for a week. (C) Numbers of reports of alarms, warnings, etc., logged within the Nuclear, Biological, and Chemical cells of the Central Command, Army Central Command, and VII Army Corps during the Conflict Period of the Gulf War; the red vertical arrow marks the night of 18–19 January just before the satellite image in (B) was taken. Figures (A) and (B) reproduced from Neuroepidemiology by permission of S. Karger AG, Basel, and (C) from the June 1994 report of the Defense Science Board Task Force on Persian Gulf War Health Effects.

Figure 3.

Classification of the 1,016 Gulf War-era veterans of the prevalence case-control sample into the three PON1 Q192R phenotypes (QQ, QR, and RR) by their serum hydrolytic activity for substrates diazoxon (“diazoxonase activity”) and paraoxon (“paraoxonase activity”). QQ subjects have only the Q isoenzyme (green circles); RR subjects have only the R isoenzyme (blue squares); and the QR subjects have some of each (red triangles). The relative amounts of the Q and R isoenzymes in each QR subject is estimated by an interpolation equation.^,

Figure 4.

The association of measures of low-level nerve agent exposure and genetic predisposition with Gulf War Illness. The measures are (A) the number of times veterans were possibly exposed to low-level nerve agent indicated by nerve agent alarms sounding where they were present; (B) the number of days veterans were in an area exposed to a possible plume of low-level nerve agent from postwar demolition of artillery shells containing sarin and cyclosarin in the Khamisiyah ammunition dump according to U.S. government computer modeling; (C) the PON1 Q192R genotype; (D) serum PON1 192Q isoenzyme activity; (E) serum PON1 192R isoenzyme activity; (F) the BChE genotype; (G) serum BChE enzyme activity; and (H) dibucaine number. Statistics are unadjusted. Note: BChE, serum butyrylcholinesterase activity; CI, confidence interval; GWI, Gulf War Illness; POR, prevalence odds ratio; ${\tau }_{\mathrm{c}}$, Stuart and Kendall’s tau-c nonparametric correlation coefficient and its asymptotic standard error testing monotonicity; P, two-tailed significance test of ${\tau }_{\mathrm{c}}=0$. The numerical values for the graphs are given in Table S5.