calculation question

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please complete the spreed sheet and provide calculation equations

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Interaction of PON1 and Low-Level Nerve Agent Exposure with Gulf War Illness, Haley 2022.
Numbers and stratum-specific ORs as displayed in Table 2
PON1 genotype
QQ
QR
Heard alarms
Cases Controls
Cases Controls
No
43
130
50
120
Yes
129
104
177
96
ORe
3.75
4.43
ORg1
1.26
1.49
Numbers and ORs in “2×4 table” format, using QQ/N as reference group for each stratum
PON1 genotype Alarms?
Cases Controls OR vs QQ/N
Add (SI)
RR
Y
N
QR
Y
N
QQ
Y
N
43
PON1
QQ
QR
RR
130
1.00
Unexposed Exposed
1.00
3.75
1.26
5.57
1.47
13.10
PON1 and Exposure Interac
15.00
10.00
5.00
0.00
QQ
QR
Unexposed
RR
Cases Controls
18
37
91
21
8.91
ORg2
1.47
3.49
Mult
PON1 and Exposure Interaction
Exposed
Unexposed
QR
Unexposed
RR
Exposed
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-reported environmental exposure measures
inflated their associations with GWI.” How did the authors address each of these concerns?
2. How does demonstrating interaction of PON1 with self-reported exposure to chemical warfare
alarms contribute to our understanding of GWI? How do you explain occurrence of GWI in some
persons with only G or E exposure, but not both?
Table 2 is hard to read online, so it’s copied here from the PDF.
Measure of additive interaction (synergy index): (RRge-1) / [(RRg-1)+(RRe-1)]
Measure of multiplicative interaction:
RRge / (RRg*RRe)
Reference:
A Tutorial on Interaction. VanderWeele TJ, Knol MJ. Epidemiologic Methods. 2014.
Discusses additive and multiplicative scales of interaction, including estimation, confounding, and
interpretation. Explains synergy index (S) and “relative excess risk due to interaction” (RERI).
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.
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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
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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.
Data availability: The data that support the findings of this
study are the property of the U.S. Department of Veterans Affairs
and were analyzed by the authors under a data use agreement
with the University of Texas Southwestern Medical Center.
Role of the funding source: None of the funding agencies had
any role in the design and conduct of the study; in collection,
management, analysis, and interpretation of the data; or in the
preparation, review, or approval of the manuscript. The
corresponding author had full access to all the data (including
statistical reports and tables) in the study, takes responsibility for
the integrity of the data and the accuracy of the data analysis, and
takes final responsibility for the decision to submit for publication.
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