Evaluation of a Gene–Environment Interaction of PON1 and Low-Level Nerve Agent Exposure with Gulf War Illness..., 2022, Haley et al

[Trish]

Full title:
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
https://ehp.niehs.nih.gov/doi/10.1289/EHP9009

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 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, 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

Full text on PMC

 

[Trish]

Discussion of this research is on this thread:

Gulf War Illness - causes

 

[Hoopoe]

Organophosphate-based pesticides have been linked to a ME-like illness. My understanding is that Sarin is chemically similar to these organophosphates pesticides but much stronger. Now Sarin has been linked to a ME-like syndrome.

Can this tell us something about what might be going on in ME? That there is some damager that is occurring that is similar to that caused by organophosphate pesticides?

To clarify, ME is not like acute organophosphate poisoning but appears to be similar to the long term sequelae.

 

[belbyr]

Merged thread

https://scitechdaily.com/after-30-y...sarin-nerve-gas-as-cause-of-gulf-war-illness/

The researchers tested the samples for variants of a gene called PON1. There are two versions of PON1: the Q variant generates a blood enzyme that efficiently breaks down sarin while the R variant helps the body break down other chemicals but is not efficient at destroying sarin. Everyone carries two copies of PON1, giving them either a QQ, RR or QR genotype.

For Gulf War veterans with the QQ genotype, hearing nerve agent alarms – a proxy for chemical exposure – raised their chance of developing GWI by 3.75 times. For those with the QR genotype, the alarms raised their chance of GWI by 4.43 times. And for those with two copies of the R gene, inefficient at breaking down sarin, the chance of GWI increased by 8.91 times. Those soldiers with both the RR genotype and low-level sarin exposure were over seven times more likely to get GWI due to the interaction per se, over and above the increase in risk from both risk factors acting alone. For genetic epidemiologists, this number leads to a high degree of confidence that sarin is a causative agent of GWI.

“Your risk is going up step by step depending on your genotype, because those genes are mediating how well your body inactivates sarin,” said Dr. Haley. “It doesn’t mean you can’t get Gulf War illness if you have the QQ genotype, because even the highest-level genetic protection can be overwhelmed by higher intensity exposure.”

 

[SNT Gatchaman]

See also miRNAs: A potentially valuable tool in pesticide toxicology assessment-current experimental and epidemiological data review (2022)

 

[forestglip]

This seems like a very compelling study. It's honestly one of the most exciting papers I've read because it provides strong support for a very specific lead.

The study found a strong gene-environment (GxE) interaction between exposure to sarin gas and the PON1 Q192R allele, which is known to affect how well sarin is metabolized. Meaning that not only is exposure to sarin associated with risk of GWI, but having the Q192R allele is associated with a greater risk from sarin. So the genetic evidence further supports that sarin is actually increasing risk, as opposed to being confounded by another variable.

There was also a monotonic (consistently increasing) dose-response relationship between GWI and the number of times an individual heard nerve agent alarms (proxy for nerve agent exposure), as well as GWI and number of Q192R alleles, which supports the main finding of the study:



Here also is a link to a perspective published alongside the article:
Invited Perspective: Causal Implications of Gene by Environment Studies Applied to Gulf War Illness
Weisskopf, Marc G.; Sullivan, Kimberly A.
Web | DOI | PMC | PDF | Environmental Health Perspectives

 

[forestglip]

It should be noted that there was a comment published which brought up a potential limitation of the study: there was no matching for ancestry between cases and controls, and thus the association between GWI and allele frequency, which varies with ancestry, may be due to ancestral confounding. Two responses to the comment were published, one from the authors, and one from another individual who authored the perspective about the study. Snippets:

Comment on “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”
Curtis, David

An important concern is that the frequency of the R allele is known to vary considerably between ancestries. For example, in subjects of European, African, and South Asian ancestries, the allele frequencies are reported to be 0.286, 0.646, and 0.379, respectively.2 This means that if cases and controls tend to have different ancestries, then one will expect to observe differences in allele frequencies and genotype frequencies even if the variant in fact has no effect at all on the outcome. The study reported an allele frequency of 0.327 in controls and 0.470 in cases, both values being intermediate between those expected for subjects having European or African ancestry.

Web | DOI | PMC | PDF | Environmental Health Perspectives

Response to “Comment on ‘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, Robert W.; Dever, Jill A.; Teiber, John F.

The sensitivity analysis for unmeasured confounders in our paper demonstrated that the estimated G×E interaction was sufficiently strong that the association between an unmeasured confounder and both the environmental exposure and the disease would have to be extremely strong to explain away the finding.3

In the initial draft of our manuscript, we included Black vs. other in our multivariable models to show that race/ethnicity had no significant effect on the G×E interaction. Given that it contributed virtually nothing to the model, after the initial journal review we removed it to avoid contributing needlessly to implicit racial bias, a growing concern in science,6 particularly among geneticists.7

Web | DOI | PMC | PDF | Environmental Health Perspectives

Response to “Comment on ‘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’”
Weisskopf, Marc G.

Such case–control differences indeed may well have happened, but importantly, as Curtis points out,1 the resulting bias would be on the main effect of the gene, not the G×E interaction. (In our invited perspective,2 we outline the same scenario for recall bias of the environmental exposure.) The other question he poses, though—whether there could also be differences in nerve agent alarm exposure by ancestry—raises the possibility of G×E dependence, a violation of a key assumption necessary for the causal inference benefits of G×E interaction studies described in our perspective.

However, rather than just speculate about this possibility, Haley et al. assessed G×E dependence directly3—that is, genotype predicting exposure—in the controls (not among the cases because they would show G×E dependence if a true G×E interaction existed4). The authors reported an odds ratio of 1.18 (95% confidence interval: 0.81, 1.73) for the G×E dependence. Thus, regardless of the distribution of ancestry or whether ancestry (only an indirect proxy of genetics) predicts nerve agent alarm exposure, in these data the genotype does not predict exposure. This suggests G×E independence and that the observed G×E interaction is, if anything, biased to the null.4 Further, even if we ignore the significance and assume there is a dependence at the level of an odds ratio of 1.18, it can be shown mathematically that this level of dependence places a maximal bound on bias of the G×E interaction. This bound is far lower than the G×E interaction found by Haley et al.,3 further arguing that a true G×E interaction exists.

Web | DOI | PMC | PDF | Environmental Health Perspectives

I can't be sure the responses to the comment adequately address the comment's claim as I'm not an expert on this. Even if they don't, the study seems intriguing enough that it seems wise to go ahead and replicate with matching ancestry to eliminate doubt about it.

 

[forestglip]

It seems to me there should be a whirlwind of research following up on this. If low level sarin exposure from a war can lead to a chronic condition like GWI, and it has taken decades to unravel that association, how many other associations are there that we don't know about yet between sarin or sarin-like exposures with GWI and GWI-like conditions?

They note an interesting other similar association of sarin and disease that might be worth looking into:

A similar body of epidemiologic and clinical evidence has demonstrated a neurocognitive syndrome and dysfunction of the central nervous system and postural instability similar to those in GWI in the Japanese survivors of sarin attacks (by the Aum Shinrikyo cult on a housing subdivision in Matsumoto, Japan, in 1994 and on the Tokyo subway in 1995) in whom the severity of the chronic illness increased with the documented dose of sublethal sarin exposure.71–73

71.Yokoyama K, Araki S, Murata K, Nishikitani M, Okumura T, Ishimatsu S, et al. 1998. Chronic neurobehavioral and central and autonomic nervous system effects of Tokyo subway sarin poisoning. J Physiol Paris92(3–4):317–323, PMID: , 10.1016/s0928-4257(98)80040-5. [DOI] [PubMed] [Google Scholar]

72.Yanagisawa N, Morita H, Nakajima T. 2006. Sarin experiences in Japan: acute toxicity and long-term effects. J Neurol Sci249(1):76–85, PMID: , 10.1016/j.jns.2006.06.007. [DOI] [PubMed] [Google Scholar]

73.Yamasue H, Abe O, Kasai K, Suga M, Iwanami A, Yamada H, et al. 2007. Human brain structural change related to acute single exposure to sarin. Ann Neurol61(1):37–46, PMID: , 10.1002/ana.21024. [DOI] [PubMed] [Google Scholar]

 

[ScoutB]

This seems like a very compelling study. It's honestly one of the most exciting papers I've read because it provides strong support for a very specific lead.

Strongly agree, this study has interested me for a while but I never got around to reading it in detail. Thanks for the cliff notes version.

I assume we don't yet know how sarin could mechanistically cause GWI? I wonder if the related question of how organophosphates might be causing parkinsons has had more research. (Relatedly: it looks like there are some papers on PON1, organophosphate pesticide exposure and parkinsons but I haven't looked closely at them either yet.)

 

[forestglip]

The comment published in response to the paper about not matching ancestry has been nagging at me. I tried to understand the arguments, but instead my conclusion is that this is a complicated topic.

The main potential issue that seems like it should be a concern is the possibility of there being an ethnicity-by-environment interaction, with ethnicity being very correlated to PON1 genotype, and thus making a PON1-by-environment interaction look significant.

The "real" interaction could, for example, look like this:

Gene-by-environment interaction studies have not properly controlled for potential confounders: The problem and the (simple) solution (2013, Biol Psychiatry)

one can imagine that individuals of a certain ethnic background are more sensitive to the environmental variable than individuals of another ethnic background. This could easily occur due to, e.g., cultural differences in reporting of environmental adversity, such that the more ‘sensitive’ ethnicity only reports environmental adversity when it is more severe and harmful.

Or a true interaction could be with a different gene highly correlated to PON1 based on ethnicity.

It seems like the responses to the comment's concerns about the thread paper aren't addressing this type of confounding where there is an actual interaction. I think they may be addressing the possibility of no actual interaction, but where a correlation between gene and exposure makes it look that way. I could be misunderstanding, though.

The author's response does assuage my concerns a little bit, because they then repeated the test for interaction on two subsets of cases based on ethnicity ("Minority" and non-Hispanic White") and found that the interaction term was still in the same direction as the main result for both. However, the Minority result wasn't significant (though that could be due to low sample size), and these two categories could potentially still each include a wide variety of ancestries.

Another concern I have is the main issue pointed out in the 2013 paper quoted above: that gene-by-environment studies are often not controlling for covariates in the correct way:

Because [candidate gene-by-environment (G×E) interaction research] is inherently non-experimental, investigators have been rightly concerned that detected interactions could be driven by confounders (e.g., ethnicity, gender, age, socioeconomic status, etc.) rather than by the specified genetic or environmental variables per se. In an attempt to eliminate such alternative explanations for detected G×E interactions, investigators routinely enter the potential confounders as covariates in general linear models. However, this practice does not control for the effects these variables might have on the G×E interaction. Rather, to properly control for confounders, researchers need to enter the covariate-by-environment and the covariate-by-gene interaction terms in the same model that tests the G×E term.

Maybe Haley et al. did use covariates as recommended in that paper. I don't see the details of the model in the paper, though.

I want the result to be true. If I had to guess, I think I'd say that sarin probably was a cause of GWI. I mean, a war well-known for leaving behind thousands with a mystery illness is also a war where thousands were showered by a cloud of extremely potent brain poison. Come on...

But I don't want my comment about me being very excited about this paper to lead to false hope based on the assumption that I adequately verified that everything was done rigorously. I'd like to see clear replication, or maybe to see an expert on this specific statistics topic weigh in.

An overview of this topic, if anyone's interested: Gene–Environment Interaction: Definitions and Study Design (1996, Preventive Medicine)

 

[V.R.T.]

It should be noted that there was a comment published which brought up a potential limitation of the study: there was no matching for ancestry between cases and controls, and thus the association between GWI and allele frequency, which varies with ancestry, may be due to ancestral confounding. Two responses to the comment were published, one from the authors, and one from another individual who authored the perspective about the study. Snippets:

Comment on “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”
Curtis, David

Web | DOI | PMC | PDF | Environmental Health Perspectives

Response to “Comment on ‘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, Robert W.; Dever, Jill A.; Teiber, John F.


Web | DOI | PMC | PDF | Environmental Health Perspectives

Response to “Comment on ‘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’”
Weisskopf, Marc G.


Web | DOI | PMC | PDF | Environmental Health Perspectives

I can't be sure the responses to the comment adequately address the comment's claim as I'm not an expert on this. Even if they don't, the study seems intriguing enough that it seems wise to go ahead and replicate with matching ancestry to eliminate doubt about it.

Is there any connection between this gene and MECFS? Any studies that mention it?

A quick search threw up these posts:

GeneCards —

PON3
BCHE
STXBP5

Not sure about PON3 but PON1 was implicated in GWI: 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 (2022, Environmental Health Perspectives).

Just looking at STXBP5, it has been associated with vWF, endothelial cells and platelets. Eg from Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion (2014, The Journal of Clinical Investigation) —

Spoiler: Increased vWF and p-selectin have been shown in ME and LC (as well as the platelet-endothelial interactions). Eg recently

Blood–brain barrier disruption and sustained systemic inflammation in individuals with long COVID-associated cognitive impairment (2024, Nature Neuroscience)

Herpesvirus Infection as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (2024, Preprint)

Neutrophil degranulation, endothelial and metabolic dysfunction in unvaccinated long COVID patients (2024, European Journal of Clinical Investigation)

The physio-affective symptoms of Long COVID are strongly predicted by the severity of the acute infectious phase, and lowered antioxidant, nitric oxide, and alanine transaminase levels

Michael Maes, Chavit Tunvirachaisakul, Laura de Oliveira Semão, Ana Paula Michelin, Andressa Matsumoto, Francis Fregonesi Brinholi, Décio Sabbatini Barbosa, Yingqian Zhang, Pimpayao Sodsai, Nattiya Hirankarn, Abbas F. Almulla

Aims
This study investigated a) whether the affective, chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), respiratory, and neurological Long COVID (LC) symptom domains are intercorrelated manifestations of an underlying construct, namely the LC phenome; and b) the predictive effects of clinical symptoms and biomarkers of the COVID-19 infectious phase on LC. Methods: This cohort study included 71 LC patients who had suffered from COVID-19 six months prior. The patients complied with the LC disease criteria set forth by the World Health Organization (WHO). We used the WHO symptom list to score the LC symptoms and assessed SARS-CoV-2 positivity using real-time reverse transcription-polymerase chain reaction (RT- PCR), white blood cell counts, anion gap, alanine transaminase (ALT) levels, paraoxonase 1 (PON1) activity and genotypes, total radical trapping potential of plasma (TRAP), and nitric oxide metabolites (NOx).

Results
All four clinical LC subdomains are significantly intercorrelated, allowing for the extraction of a single latent construct encompassing affective, CFS/ME, respiratory, and neurological symptoms, labeled the physio-affective phenome of LC. The latter was strongly predicted by dyspnea and productive sputum, RT-PCR SARS-CoV-2 cycle threshold values, lower anion gap, and increased neutrophil percentage during the infectious phase. Other predictive biomarkers were lower ALT, TRAP, and NOx levels, and the PON1 QR192 over dominant gene model (protective).

Conclusions
LC may be a clinically homogeneous disease characterized by highly intercorrelated affective, CFS, neurological, and respiratory subdomains. The physio-affective phenome and its domains are strongly predicted by biomarkers of acute infection and associated inflammation and oxidative stress.

Link | PDF (Preprint)

 

[forestglip]

A quick search threw up these posts:

GeneCards —

PON3
BCHE
STXBP5

Not sure about PON3 but PON1 was implicated in GWI: 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 (2022, Environmental Health Perspectives).

Just looking at STXBP5, it has been associated with vWF, endothelial cells and platelets. Eg from Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion (2014, The Journal of Clinical Investigation) —

PON1 is being studied in GWI because it can break down organophosphates, so if organophosphates are involved, we should be able to detect whether people with the more effective PON1 gene variant are more protected.

Just based on a quick search, PON3 doesn't seem to also break down organophosphates, so it wouldn't be interesting in the same way:

Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities (2005, J Lipid Res)

Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3.

A PON for All Seasons: Comparing Paraoxonase Enzyme Substrates, Activity and Action including the Role of PON3 in Health and Disease (2022, Antioxidants)

Except for poorly hydrolyzing paraoxon, PON3 is almost devoid of OP [organophosphate] activity [4]. PON2 also demonstrates almost no paraoxonase activity [4]. However, PON1 strongly hydrolyzes the active metabolites of several OP insecticides (parathion, chlorpyrifos, malathion, and diazinon) [25] and nerve agents (tabun, sarin, soman, and cyclosarin) [18,26].

I'm not sure what the Maes paper is showing. Might be worth a look.

As far as DecodeME, there are no very significant variants directly around PON1:

 

[ScoutB]

Is there any connection between this gene and MECFS? Any studies that mention it?

From memory, based on last time I looked into this, rather than one variant of PON1 being better the other(s) at breaking all down organophosphates, the best variant depended on the organophosphate in question -- so there could be effects in opposite directions that cancel one another out. Then, even if PON1 variant+pesticide exposure did contribute to ME/CFS, it might not show up very strongly in association studies?

I want the result to be true. If I had to guess, I think I'd say that sarin probably was a cause of GWI. [...] But I don't want my comment about me being very excited about this paper to lead to false hope based on the assumption that I adequately verified that everything was done rigorously.

To be honest, I was always amazed this paper found a significant result at all. Since the gulf war happened so many years ago and data was so ad hoc and noisy (e.g. having to rely on soldiers' recollections of gas alarms decades ago) I figured any signal would be lost. When I'm less foggy I'll try to think more though what you're saying about the statistics, but I think general organophosphate research might lend some support to the claim here too.

I've only done this a little, but I suspect we might get more solid information looking at studies of modern pesticide exposure (which admittedly are more focused on Parkinson's but often investigate general neuronal and immune system changes). Some examples I've been meaning to skim through and maybe post if they are interesting:
Low-dose sarin exposure produces long term changes in brain neurochemistry of mice (sci-hub)
Brain anomalies in children exposed prenatally to a common organophosphate pesticide

 

[forestglip]

From memory, based on last time I looked into this, rather than one variant of PON1 being better the others at breaking all down organophosphates, the best variant depended on the organophosphate in question

That's at least what another GWI paper seemed to say:

- https://www.s4me.info/threads/pon1-status-in-relation-to-gulf-war-illness-evidence-of-gene–exposure-interactions-from-a-multisite-case–control-study…-2024-steele-klimas.40014/

This understanding comes from studies that demonstrated that the PON1192 Q alloform hydrolyzes sarin more rapidly than the PON1192 R alloform in vitro [24].

The second main finding of this study is that using skin pesticides during deployment was significantly associated with GWI risk for QQ and QR status veterans but not for RR veterans. This pattern is consistent with reports that the PON1192 Q alloform is less efficient than the PON1192 R alloform in protecting against pesticide oxon forms such as chlorpyrifos oxon [61,62,63,64].

 

[forestglip]

The Supplementary File of this paper has a couple lists of prior studies that might provide good reading material on this topic. The tables are called:

• Table S18. Methods and results of prior epidemiologic and clinical studies of the association of chemical weapons with GWI

• Table S19. Prior studies identifying biochemical mechanisms by which low-level subclinical sarin exposure similar to that experienced in the 1991 Persian Gulf War causes chronic cellular pathology with behavioral changes resembling GWI.