Standards of Evidence”
ASTHMA 9 Dr. Fernando Martinez
Dr. Martinez: I hope that the examples that I will produce today are
9Throughout Dr. Martinez’s presentation, he may refer to slides that can be found online at http://www.iom.edu/?id=42463.
kind of intermediate between what Dr. Susser has shown and Dr.
Landrigan has shown in terms of our degree of understanding. I would like to say that perhaps they have to do with two issues that have been raised, natural experiments and the potential role and interests that protective effects may have. So, what I am going to talk about is what I am interested in, which is asthma. You are going to see things here that may sound very familiar to you. Asthma is a heterogeneous set of related conditions in which recurrent, partially reversible airway obstruction is the final common pathway.
The clinical expression of asthma can start at any age, but we have now found in the last 5, 10, 20 years, that the first manifestation of the disease usually occurs during the preschool years. That may sound also familiar. In our case we have well-defined intermediate phenotypes for asthma that are strongly related with the disease burden and therefore they can be studied separately and I will show you some examples of that. For example, aeroallergy, bronchial responsiveness, or total serum IgE. One thing we know about asthma is that in the last 40 years it has clearly increased in frequency and you can figure that out both through the diagnosis of asthma and through asthma symptoms as reported by parents. So, there is a strong hint that asthma is an environmental disease and, of course, we have an advantage, I think, with respect to autism in that asthma is a very variable disease, an extremely variable disease. We have known for years what the main triggers for the disease are, and, of course, some of them have been pursued as potential inceptors of the disease. In other words this is a concept that is very important. In asthma we know very well that there is a difference between what could cause the disease at its very beginning and what triggers the disease once the disease process has developed.
Unfortunately, we have not been very able to show for any of the triggers that they are involved in inception of the disease. The one that I am involved with and that is the only reason why it doesn’t have a question mark—it should have also a question mark, but we are all biased, of course—is the lack of certain protective effects. That is the one I am going to stress more today.
It all started with a form of natural experiment. A researcher in Britain, David Strachan, working with one of the largest birth cohort studies as two of the previous speakers have talked about, the 1958 birth cohort in Britain, found a startling finding, which is that children who had older siblings at home were much less likely to have what could be
considered intermediate phenotype for asthma, which is hay fever, than those who did not.
That was truer for those who had older siblings than for those who had younger siblings at home. This observation was ignored I think for years until we, in our own longitudinal study, which was started in the 1980s, tried to reproduce it. This, as you can see, is 11 years later. What we found was very interesting. Here are “in the triangles” the children who were exposed to other children, be it because they had older siblings or because they were taken to day care.
As you can see here at the beginning of life, they tend to have, of course, more viral infections, which are strongly associated with wheezing with this age period. But very interestingly if you follow them enough by the sixth to seventh year, when the atopic form of the disease, the allergic form, starts to be more prevalent, these children are clearly and significantly protected.
A series of other studies came showing that there were other protective exposures. For example, this has now been reproduced, replicated in 10 studies and not replicated in 2. If you have a dog in the home, you are less likely to develop asthma in the first years of life than if you do not have a dog.
Now, what is common between having a dog in the home and being exposed to other children? Well, in several studies now, it has been shown that day care and homes with a lot of children have high concentrations of a marker of microbial exposure in the homes, which is endotoxin. This has been shown now repeatedly in many studies. This is true for pets, as it is true for day care and homes with heavy concentration of children.
But perhaps the most interesting solid natural experiment is the one that you see here in this slide, which is a form of living, which still exists in Central Europe in which children and adults live in single-family farms as the ones you see here.
In these single-family farms, an empirical observation was that there was really very little asthma. Researchers listened to local physicians who were telling them that there was very little asthma in this environment and went to study it. What they found only a year after we published that paper on day care and other siblings was that, lo and behold, both for subjective and for objective measures of asthma, the children who live in those farms that you saw there were between 5 and 10 times less likely to have asthma than those living in the same rural
communities, but away from those farms.
They also studied endotoxin concentrations in the homes, which is here in the x axis in relation to the likelihood of having illnesses in this environment. As you can see here, immediately after the first publication about those farming environments, what they found was that there was a striking inverse relationship between endotoxin exposure and the likelihood of having asthma, particularly allergic asthma, but not nonallergic asthma, which is an important issue because the clinical expression is identical. It is impossible to distinguish them.
Even more and I don’t show this slide to take more time, but in nonatopic asthma the relation is inverse to this one. In other words the more endotoxin, the more nonatopic asthma. We will get back to this concept in a moment. Of course, this was a very extraordinary environment. So, it was necessary to try to reproduce this in a less extraordinary environment. If you want to consider Manchester in England a less extraordinary environment, here it is.
As you can see, these researchers in Manchester clearly reproduced in an urban setting the findings that had been reported before for the extraordinary environment in rural communities in Europe, in Central Europe. Now, an explanation has been proposed for this association, which is very simply as you can see here, that endotoxin or LPS increases the expression of IL-12 and IL-18, which in turn has a downregulating effect on Th2 differentiation, T helper cell 2 differentiation, which is the central and most important determinant of having atopic diseases. So, it was proposed that if you have endotoxin exposure, you deviate your immune responsiveness away from the Th2 mediated response, which is responsible for atopic asthma. If you don’t, as you see on the right side, you upregulate the likelihood of having an atopic response.
Asthma is also an allergic disease, and here I have put the latest twin studies published by the same research group with respect to asthma and interestingly with respect to autism, just published this year. You can see there are many things in common between the genetics of asthma and autism. I think the reason why the validity of asthma appears to be greater is simply because this was done earlier in life and the twin studies of autism were done later in life or perhaps because it is true since I know very little about autism, I don’t know the answer.
Something very interesting and paradoxical, however, is that these twin studies have both shown no shared environmental influences
affecting the concordance of asthma between twins, which may be true, but may also be a complete artifact, due to the fact that the models used suppose that there are no gene–environment interactions. I will get back to that concept in a moment.
But much like in the disease of your interest, in asthma what we have had is that no single study has shown strong statistical evidence of a single gene being responsible for the disease. We have 15 chromosomal regions in which there seem to be asthma genes. Sound familiar? And only three regions have been clearly reproduced in at least two studies, if not three. Same thing as for autism.
Well, one of the reasons why people have been able to reproduce linkage with asthma in chromosome 5q is because there is, I think, a large array of potential genes that can be candidates. It is a problem of luck. One of those genes is CD14, which I showed before. CD14 was in the middle of this potential pathophysiologic explanation. Why? Because CD14 is one element that is a member of the receptor system for LPS.
CD14 is a crucial member of the receptor system for the exposure and protection in the exceptional farming environments and also in the nonexceptional environments in Manchester. We sequenced that gene in populations and we found five main closely linked, single-nucleotide polymorphisms in the five prime regulatory regions, and for one of them we showed functionality. This morning, we were told that that was important and I am just showing one slide of probably 10 I could show about the functionality. You will have to believe me that transcription rates are increased in carriers of the T allele at position −159. We now know that this is a problem of balance in those particular 5' regions between SP1 and SP3 transcription factors. It was logical to suppose that if you had more CD14, you would be more sensitive to the environment and you would have less atopy. There was more atopy in our population among children who were CC and CT, who had low expression of CD14 and those who had TT. We thought we had to put ourselves in the hall of fame of geneticists, who had found something important until, of course, we fell into the same problem that every single other person working with complex diseases has fallen into, which is that three researchers were able to reproduce this and three researchers were unable to reproduce this result.
The three researchers who reproduced us called us and congratulated us. The three researchers who were unable to reproduce us said you guys don’t know what you are talking about. Of course, we immediately
thought that the right place to study this was where the exposure to endotoxin was the highest, the farming environments, because here perhaps if we could determine if these people who live this way and heavily expose their children to brown stuff that is here—about which I won’t talk before lunch—could probably heavily expose the children to endotoxin and others could not be exposed. So, we could study gene–
environment interactions. Of course here just to show to you how these things work is the relation between the same polymorphism and atopy without considering the environment among farmers, there is nothing there with CD14. The trick was to put it in relation to the environment and when we did that, something very interesting happened, which is that the sensitivity to the exposure to endotoxin was completely different, depending on your genotype.
The CCs were heavily sensitive to the environment. The CTs and TTs were not. Now we have shown in other functional experiments that baseline unstimulated production of CD14 is higher in TTs, but a stimulated production is higher in CCs and CTs. So, what happens is that you have a very flat line for TTs and CTs and a very steep line for CCs.
That creates a very interesting paradoxical situation, which is that at lower levels of exposure, the CCs are at risk whereas at high levels of exposure the TTs are at risk and the CCs are protected. This is due to the fact that the genes don’t act alone.
If you don’t believe that this is true—and I would agree if you see only one study, which was done in the exceptional environment—this next slide shows the results of the same analysis done in Manchester and the result is exactly the same. The CCs show this very steep relation- ship between risk of being allergic and the exposure to endotoxin. TTs and CTs show much less response to the exposure to the point that CC risk is lower at high level of exposure. Among African American adults, the same thing has been shown by Williams and co-workers in Detroit.
What are my proposed conclusions? From our experience, natural experiments are very important and they may be true for both risk exposures and protective exposures. In our case it was protective exposures and they provided significant cues or clues for us to understand. I think I would have to say the hygiene hypothesis as this is called is still very controversial, but I think it has focused us into an area of exposures and has allowed us to understand the disease much better.
Not only that, it has inspired new treatment. Very recently in the New
England Journal of Medicine, a paper has been published in which ligands of TLR9 are used as adjuvants for allergic desensitization, with the idea that they wanted to reproduce a little bit what could be present in the environment in this particular condition.
I believe that we have to understand biological systems as plastic with heterogeneous responses to the environment and I think the example of CD14 that I have presented to you is characteristic of what the complex related genetics are going to be. They are going to be nonlinear.
They are going to be weakly linked and strongly context dependent.
Suggested approaches: I think that following up on replicated enhancing or protective exposures may prove extremely rewarding. I am not an expert in the field, the very controversial but very interesting fact that Mexico-born mothers have children with less autism than those who are not, maybe because I am Hispanic, too, calls very much my attention.
I do understand that this may well be due to bias because they may seek less access. They may recognize this less, but being a physician who works with a lot of Mexico-born mothers, I am quite aware of how worried they are about the health of their children.
So, I am not very convinced about their argument. I think that we have now technologies, both at the genetic and epigenetic level that allow us to assess genomewide the potential for genetic and epigenetic factors to be present. That may be related to exposure. So, I think that in studies such as the National Children’s Study, we could determine if replicated exposures could be useful to determine the type of gene–
environment interactions that, only in an example, I have provided to you in the case of asthma.
Thank you.
Dr. Schwartz: Fernando, that was great.
Let me just make a suggestion, then we will open it for group discussion and focus on the general topic.
DISCUSSION
Dr. Schwartz: Your talk, Fernando, brought up a really important point, which is that environment can be used to narrow the pathophysiologic phenotype in such a way that you can understand the genetics of and also potentially the biology that underlies a very complex disorder like asthma and consequently a complex disorder like autism.
But I wanted to ask the group in general about natural experiments and whether we can expand our concept of natural experiments to autism. What are the natural populations or the cohorts that might be available and amenable to further study? Can we follow as it relates to an autism endpoint or subclinical early condition that is along the pathogenic line or clinical line or development of autism?
Phil Landrigan, you brought up and, Ezra Susser, you brought up the issue of the natural experiments. Are there populations? Clearly, the National Children’s Study is going to be an outstanding study that will allow us to follow kids over time through development, but it is also going to take a long time. Are there populations that have been exposed that we should be looking at more carefully for autism influence?
Dr. Landrigan: David, a couple of responses to that. First of all, I think the National Children’s Study is very powerful, but it is probably not going to answer every question. When I think of natural experiments, maybe because I spent many years at CDC, I think of clusters. I think of Brick Township in New Jersey, for example; I think of the group of children now three or four decades, who were exposed in utero to thalidomide and I think there is need for highly focused studies, which look at children who suffered unique exposures. I also think it is terribly important when those kinds of studies are done that we do as Ezra Susser suggested in regard to big cohort studies. That is, that we take samples and we archive them because there is always the very high possibility that new diagnostic techniques or new genetic probes will be developed in future years that will enable the scientists that follow us to examine those specimens and ask questions that are not possible to ask today.
I think with regard to the Children’s Study, I would say that it won’t be that long. It will certainly be in our lifetime that we have data on the relationship between the environment and autism now that federal funding has been made available by the Congress and in such a way that it doesn’t destroy the budget of NICHD. We are going to be moving forward. The first recruitment will take place beginning in about 12 months. That means that we will have a large number of 3-year-old children in the study in about 5 or 6 years, something like that, 7 years at the most.
So, I would argue we will begin to make data on gene–environment determinants of autism in that particular population available by 2010 or 2011, 2012, somewhere in that range, not tomorrow, but not 25 years either.