"When I saw the first set of data in March of last year, I immediately sat up,” says neuroscientist Denise Figlewicz, an MDA-supported ALS researcher at the University of Michigan in Ann Arbor.
Figlewicz was looking at the results from a small study comparing DNA from people with ALS and without ALS in Poland.
“This was a small sample size from a genetically isolated population, and to get such a statistically significant effect is a sign that you’re looking at something that’s real. And a gene, or family of genes, already implicated in the Gulf War syndrome is certainly the kind of candidate you pay attention to,” Figlewicz says.
The something real, published online July 5 in Neurology, is an association between particular variations in genes that carry instructions for paraoxonase (PON) enzymes, and the development of sporadic ALS, the type of ALS that isn’t clearly hereditary and affects some 90 percent of those with the disease.
The three PON genes (PON1, 2 and 3), located along a stretch of chromosome 7, carry instructions for enzymes that help metabolize and detoxify a variety of man-made nervous system poisons, such as pesticides, insect repellents, nerve gas and anti-nerve gas medications.
Figlewicz recognized what she had: the first example of how a person’s genetic background can interact with environmental factors to produce ALS.
Genes, environment long suspected in ALS
It’s been known for decades that ALS can run in families, but rarely. Only about 10 percent of ALS cases are clearly familial, usually inherited in a dominant genetic pattern, and passed from parent to child.
But by the 1990s it was also becoming clear that some sort of genetic predisposition, perhaps combined with an exposure to an environmental toxin, virus or drug, was also a likely route to ALS.
By 2003, 12 years after the Gulf War conflict, surveys were showing that veterans of that war were developing ALS at twice the expected rate.
The question then became, could exposure to a neurologic toxin, combined with genetically determined impaired ability to metabolize it, be a perfect storm?
Gulf War syndromes linked to low PON, high toxins
Figlewicz says that she, like most ALS investigators, took the early Gulf War data with a grain of salt.
“There are a lot of these studies where one person finds an exposure to this or that, and probably they’re not wrong, but nobody has been able to duplicate these,” she says. “In Gulf War veterans, there were some positive studies, although they were hard to pin down. There were environmental factors possibly causing [various] neurological syndromes, not just ALS.”
It’s been hard to get funding to do this kind of epidemiologic research into environmental exposures, Figlewicz says. “You can say you’re citing previous literature as the reason for your study, but [funding agencies] may say the previous literature is garbage.” There have been reports based on “we have a feeling there are more patients than usual,” she says, but there are only a few investigators who’ve made an effort to do serious statistical studies.
One such investigator is Robert Haley, a physician-epidemiologist at the University of Texas Southwestern Medical Center in Dallas and a specialist in Gulf War syndromes.
In the early ‘90s, soldiers returning from the Persian Gulf began displaying various combinations of symptoms — impaired thinking, lack of coordination, tingling and numbness in the extremities and constant pain. (ALS, in those veterans in whom it developed, wouldn’t become apparent until years later.)
“The Pentagon and the VA [Department of Veterans Affairs] under Bill Clinton had a presidential advisory committee,” Haley says, “and they had concluded that this was due to stress, all psychological, a neurotic problem.”
But in 1997, Haley’s group published several papers showing that exposures to organophosphate pesticides (such as that used in flea collars, which many of the veterans wore to kill sand fleas), the insect repellent DEET, nerve gas, and medication given to ward off the effects of nerve gas were all correlated with the later development of neurologic symptoms.
They described the symptoms as falling into three categories: Symptom complex 1 they called “impaired cognition”; complex 2 they called “confusion-ataxia” (incoordination); and complex 3 they described as “arthromyoneuropathy,” or joint, muscle and nerve abnormalities.
In 1999, Haley, with co-workers at the University of Michigan, published a study linking sluggish PON1 enzymes, as well as chemical exposures, to these Gulf War syndromes.
The 1999 Haley study, Figlewicz says, was one of the few Gulf War studies that looked at something specific. She considers it “probably the most solid epidemiology of the studies related to Gulf War veterans.”
When blood samples were analyzed from 26 ill veterans and 20 who described themselves as not ill, low levels of PON1 activity, and the substitution of an arginine instead of a glutamine molecule at a specific position in the enzyme, were found to be associated with later development of neurologic symptoms.
The ill veterans also reported having been exposed to pesticides or nerve gas, and having had severe adverse reactions to anti-nerve gas medication.
Studies showed breakdowns neurologic, not 'Nervous'
“A lot of our man-made insecticides and herbicides are mimics of natural organophosphates,” Figlewicz says. “The reason they work is that they get into the body [of an insect] and do the same things that the native compound would do, but when they attach to an enzyme, they don’t come off.”
That enzyme they attach to is called acetylcholinesterase, or AchE, and it’s responsible for the breakdown of acetylcholine, a neurotransmitter that carries signals from nerves to muscles in the voluntary nervous system and carries other signals in the autonomic nervous system.
Organophosphates kill insects by latching onto AchE and preventing it from breaking down acetylcholine. “The insect goes into a tetanic [prolonged muscle contraction] state that is irreversible,” Figlewicz says. “It keeps on getting stimulated with acetylcholine.”
Sarin nerve gas, to which military personnel were exposed during the Gulf War when they exploded storage depots, acts similarly. It too prolongs the effect of acetylcholine in the nervous system.
In anticipation of nerve gas exposures, military personnel were given tablets containing pyridostigmine, an “antidote” to sarin and related gases, but one with its own toxicity. In the nervous system, pyridostigmine competes with sarin for a place on the AchE enzyme. If it wins the competition, it substitutes its less severe toxicity for the more deadly one from nerve gas.
“It’s pharmacologic musical chairs,” Haley says. “If the pyridostigmine is on the cholinesterase, the sarin can’t get on it.” Needless to say, if no nerve gas exposure followed pyridostigmine ingestion, the soldier would experience only the side effects, and no benefits, from the medication.
Meanwhile, the military, Haley says, was having a hard time believing that neurologic symptoms seen after the war were anything other than stress. “They said, ‘You’ve got two guys serving right together, and one gets Gulf War syndrome, and the other doesn’t. He just broke under stress.’”
Haley disagreed. “I knew from 10 years of research at the Centers for Disease Control and a bunch of investigations that it’s always shoe-leather epidemiology. Epidemiology comes up with plausible theories, and then you’ve got to go to the lab and prove them. Here we had a very plausible epidemiological association.
“I got on [the Internet database] Medline, and in about an hour, I did a search. I think I searched on ’genetic polymorphisms, enzymes and nerve gas,’ and there were two sets of literature — a lot of enzyme studies and genetics about AchE variants, and then a big literature on this other [enzyme] called PON. And there was one name that was common to both literatures, and that was Bert La Du, at the University of Michigan. I just cold-called him and said, ‘Dr. La Du, this is Dr. Robert Haley. You don’t know me, but I’ve been doing this study of Gulf War syndromes.’”
The collaboration between their two laboratories led to the identification of PON1 activity and neurologic symptoms, published in the June 15, 1999, issue of Toxicology and Applied Pharmacology.
PON gene variants found in Polish ALS patients
Some time after the Gulf War data were published, Polish neurologist Agnieszka Slowik, who was studying the possible role of PON genes in susceptibility to strokes at Jagiellonian University in Krakow, came to Denise Figlewicz’s lab in Ann Arbor for three months of training in molecular genetics.
Together, and with knowledge of Haley’s data about Gulf War neurologic syndromes, they decided to study Polish subjects with and without ALS and examine their PON gene status. “The Polish population is much more uniform than what you would get from a medical center in the United States,” Figlewicz says. “People leave there but haven’t been coming in. You can get statistically significant results with a smaller number of people.”
Slowik, Figlewicz and others studied 185 people with sporadic ALS and 437 healthy people as a control group and found that having arginine rather than glutamine at position 192 in PON1 (the same variant Haley’s group had found in the ill veterans) and cysteine rather than serine at position 311 in PON2 were significantly associated with having ALS.
A combination of the PON1 arginine and the PON2 cysteine variants occurred 3.4 times more often in ALS-affected subjects than it did in subjects without the disease.
As excited as Slowik and Figlewicz were about their data, which they announced at the American Academy of Neurology meeting in April, there’s one more step that scientists like to see completed before they start taking their own work too seriously — confirmation of the results by others, especially in different populations.
They got that, in the form of a U.S.-based study also presented at the April AAN meeting and also published online July 5 in Neurology.
North American, Polish PON variants differ
Researchers in the laboratory of neuroscientist and neurologist Teepu Siddique, at Northwestern University in Chicago, with colleagues at Vanderbilt and Duke universities, found PON gene correlations with ALS in a North American population, using a larger sample and minimizing genetic differences with a different strategy from the Figlewicz group.
Mohammad Saeed and colleagues in Siddique’s group, which included neurologist Robert Sufit, director of the MDA clinic at Northwestern, looked for possible variations in PON genes in sporadic ALS patients versus a control sample.
Their findings differ somewhat from those of Figlewicz’s group. Rather than detecting ALS-associated variations in the chemical compositions of PON1 or PON2, or in their activity levels, they found an association between developing ALS and having a variant sequence of DNA between the PON2 and PON3 genes. (The disparity between the two groups’ findings may be related to true differences between the American and Polish ALS populations. Or, the particular laboratory and statistical techniques each group applied may have identified factors potentially relevant to both groups.)
Siddique’s team analyzed DNA from 1,891 North Americans with and without ALS. In 450 of their cases, the genes of the ALS-affected person were compared with those of his or her two unaffected parents or one unaffected sibling, to minimize interference from genetic background noise arising from ethnic differences.
“The [positive ALS association] signal is from the intergenic region between PON2 and PON3,” Siddique said. “The biology has yet to be clarified.” It isn’t, he said, from within the PON1 gene.
ALS risk, Siddique’s team says in their paper, may in fact be modulated by a PON cluster, rather than a single variant in a single gene, at least in North Americans.
They also note that, because of the known function of PON enzymes, these associations “hint towards a war-related environmental exposure,” such as organophosphate pesticides and chemical nerve agents, such as sarin, “in a genetically susceptible host” as a possible causative factor in ALS.
None of Figlewicz’s subjects and probably a small number (if any) of Siddique’s subjects served in the Gulf War, indicating that PON variations may lower the threshold for ALS development without the chemical exposures implicated so far, or that the Polish and American subjects with ALS in these studies experienced some of the identified chemical exposures, such as pesticides, outside the Persian Gulf environment.
PON gene tests may come soon
Testing for PON gene variations isn’t yet widely available, but Haley says his lab has developed a rapid test that has the potential to be used on a large scale at an affordable price.
Right now, he says, such testing is available only through research studies, costs about $2,000 and is time-consuming. He expects to develop tests that can be performed “rapidly and cheaply.”
As a treatment strategy, his group has experimented with PON1 gene therapy. They recently boosted PON activity in rats by giving them genes for the glutamine type of PON1 and found “it has a pretty phenomenal effect against acute cholinesterase [AchE] inhibition.” Haley says he realizes gene therapy probably isn’t a solution for most patients but that “it might be very useful to give to troops before a battle.”
A toxin like sarin, he says, might be stored in fat cells and released a little bit at a time, after which it would travel to the brain and nervous system. “This is all fanciful,” he says, “but if that were the case and you had high PON [activity], you might mop it up before it got to your brain.”
As for other approaches, perhaps more practical and widely applicable for people who find out they have high-risk PON gene types, Haley says, “Can’t talk about it. But we’ve got some other ideas.”