Dr. Falk: Thank you very much, Alan.
There was a lot of discussion yesterday about issues we will be talking about this morning, so I think there is a lot of interest in these sessions. We will start with Irva Hertz-Picciotto, who has a Ph.D. in epidemiology from the University of California–Berkeley. She was on the University of North Carolina–Chapel Hill faculty for 12 years, and is now at UC–Davis Department of Public Health Sciences. Her research interests are in environmental exposures, pregnancy outcomes, and epidemiological methods. She is on the editorial boards of the American Journal of Epidemiology, Environmental Health Perspectives, and Epidemiology, and was on the scientific advisory board for the U.S.
EPA. Thank you very much, Irva.
ENVIRONMENTAL EPIDEMIOLOGY STUDIES:
NEW TECHNIQUES AND TECHNOLOGIES TO USE EPIDEMIOLOGY TO FIND ENVIRONMENTAL TRIGGERS15
Dr. Irva Hertz-Picciotto
Dr. Hertz-Picciotto: Thank you. I am going to provide an overview of environmental epidemiology and epidemiology generally. I’ll talk about different study designs and what we have learned from them, and then make some recommendations.
The first couple of study designs I am going to go through somewhat quickly, because I think most of the meat is really at the end, in terms of the future for the field.
Starting with focused clinical studies, these are generally self- selected populations or a group of patients in a clinic. These studies are descriptive. They usually have small numbers of subjects in them.
Sometimes the hypotheses are generated a posteriori. These are the
15Throughout Dr. Hertz-Picciotto’s presentation, she may refer to slides that can be found online at http://www.iom.edu/?id=42469.
studies that have taught us about sibling recurrence, about twin concordance, male–female ratio, the comorbidities, and the genetic syndromes that seem to also go sometimes with autism, as well as seizure disorders and gastrointestinal symptoms. They have taught us about the heterogeneity of onset, including the regression phenomenon that seems to happen in a lot of cases, and the data on anthropometrics, such as head size, have come from these studies as well.
The second kind of study, described in the next slide, is based on administrative databases. These are large databases that are collected for administrative purposes. The diagnosis of autism is frequently done by whoever the clinician is who happens to see that child. We have learned about perinatal factors and about time trends from these studies. There are two ways in which these studies assess exposure. When exposures are not assessed in the individual and the outcomes are summarized at the group level, for instance, by area, it is called an ecologic study; in the other design, both exposure and outcomes are assessed at the individual level.
This is an example of a time trend study conducted in Denmark using an administrative database of diagnoses. It was a study looking at the removal of thimerosal from vaccines and the rates of autism before and after. What you can see from this time line, which was not necessar- ily obvious from the original paper, was that before thimerosal was removed, there was a period of time when only inpatients were in the database, and during part of the “after-removal” period, which covered all the way out to 2000 in this study, there was an interval when both outpatients and inpatients were included. This study, therefore, is not a rigorous design, because as you can see, you can’t really compare the before and after periods because of artifacts in how the database was constructed, and specifically, in how that changed over time.
The next slide shows another administrative database study, quite a good one done from the Swedish Birth Registry, which was linked to their inpatient register. It gave us some information about some of the aspects of the perinatal period that seemed to be associated with higher risk for autism.
From these administrative database studies, we have learned about patterns: We have learned about the age effect of the parents, obstetric complications as risk factors, and aspects of the time trends in autism.
Moving along to the genetic studies, these again, like the clinical studies, are volunteer samples. The largest such study right now is the
Autism Genetic Resource Exchange (AGRE) database. It has over 1,000 families right now. They are all multiplex families with at least two members who are affected, and they are focusing on genetics. We have learned from these studies how highly concordant monozygotic twins are, but not entirely. We have learned that this is not a condition that follows simple Mendelian inheritance. We have also seen the slide show yesterday by Isaac Pessah and others depicting the large number of chromosomes that may be involved, indicating multiple genes. But at this point those studies have focused on genes in isolation, although Clara Lajonchere has been working with me to figure out how to collect more environmental data on the AGRE families.
Just a few words about studying environmental factors in autism. I think there are some misconceptions, maybe not in this room, but certainly out in the field of science and the community at large. We know from the genetic studies that about 60 to 90 percent of cases have some genetic component.
What can we conclude about environment? Let’s look at these two pies. This is the sufficient causes model from epidemiology. Each pie represents a set of sufficient causes that will cause autism in at least one individual out there. It might be that A is a genetic factor, that B is another gene, that C is an environmental factor in the prenatal period, and D might be something happening at birth or postnatally, just hypothetically.
Looking at the lower pie, this is another set of sufficient causes, where A and C can be substituted with some other set of events. Each set, that is, each causal pie is sufficient. So what that means is, if you take away B from either one of those pies you don’t get autism.
This is gene–environment interaction. Because of gene–environment interaction, environment plays a role in 10 to 40 percent at a minimum.
But notice that in this individual, say someone corresponding to the upper pie, it takes both. Let’s suppose that this particular set of sufficient causes produces 30 percent of the autism cases. That means 30 percent require genes and 30 percent require environment. Suppose the other pie corresponds to the remaining 70 percent of autism cases: Under this scenario, 100 percent of the cases require genes and 100 percent require the environment. In other words, the contribution from genes plus the contribution from environment do not have to sum to 100 percent, and they will not sum to 100 if there is any gene–environment interaction.
There are also several environmental factors that have been associated with autism with very high relative risk. The first is congenital rubella. In the mid-1960s, the United States experienced an epidemic of rubella. Mothers who had rubella during a pregnancy and passed it transplacentally gave birth to children at a much higher risk for autism, about 10-fold higher, and that figure is based on counting only the cases that did not seem to resolve over time.
Thalidomide: also, a very high relative risk. Just looking at a few other factors such as maternal age or male sex, that are not necessarily causal, but might be proxies for some causal factors, there are also some large relative risks.
The fourth type of study design I will talk about is the new genera- tion of case-control studies. These are population-based studies where the diagnosis is confirmed in all of the individuals. They cover a broad range of factors. Generally speaking the exposure has been assessed retrospectively, but that is not 100 percent true. If you go back to get medical records from these individuals, you are collecting essen-tially prospective data, data that were originally written down in a prospective manner. These studies also have been collecting specimens with linkage to laboratory scientists.
I’ll give an example. There aren’t very many of these studies, but you have heard yesterday a little bit about the Norwegian study, not a case-control study, but they are doing nested case-control studies within it.
This is a case-control study we are conducting in California.
CHARGE stands for Childhood Autism Risk from Genetics and the Environment. We currently have about 800 participating families. They include children with autism, children with developmental delay but not autism, and children from the general population. Each child with a potential diagnosis for autism is assessed with the ADOS and the ADI and other assessments are done on all children, including cognitive and adaptive development, a physical exam, medical history, and a structured interview that takes about an hour and 40 minutes, covering 12 domains.
Six of them are shown here, including information about the index pregnancy, household product use, metal exposures, and so forth.
Then there are some self-administered forms about comorbidities and also about treatments and services that the child receives. We collect urine, blood, hair, and we ask the mother to bring in the baby hair lock if she saved it, and many of them have. They don’t have to give all the hair;
we can do with a few strands. We also collect specimens from siblings and parents, and then we go back to get medical records. We have them sign medical record release forms. It’s a very labor-intensive process, and we try to obtain as many of the types of medical records shown on the slide as possible.
In addition, in California there is a banking of newborn blood spots on every newborn. Currently we have about 480 dried blood spots and we are applying to get more.
Overall, this is the scheme that we are working with. On the left side is a panel of broad classes of exposures that we drafted as priority exposures to take a look at, and on the right side are the methods for collecting data about those exposures. Just a few examples: From blood, we can measure pesticides, and we can ask about what pesticides were used in the home. We can also link the residential information the mother provides with some databases that are also available in California, which have a record of every commercial application of a pesticide anywhere in the state. The database has geographic locations of applications, which we then can link to the residence of the mother at the time of birth, or at any other time because we collect those residential data.
Measurement of metals can be conducted in blood, hair, the baby lock, the newborn blood spot. We ask about fish consumption and other household product use for metals. Another example, data on infections can be abstracted from the medical records and is collected by interview from the medical history. You get the picture.
The CHARGE study is in progress. With regard to other case-control studies of this type, there is also an autism phenome project, which Sue Swedo talked about yesterday; it involves NIMH and the M.I.N.D.
Institute. The CADDRE study we will be hearing about later in the session from Diana Schendel.
Just briefly, some of the things that we are starting to see: In CHARGE, we are finding very different immunologic profiles in the children with autism as compared to the control groups. A wide range of immune markers appear different in the children with autism.
We have also examined gene expression, and have observed a set of genes that seem to be differentially expressed in the children with autism, especially in cells from the immune system called the natural killer cells.
There are also some hints now that there are perinatal factors, potentially avoidable ones, that might be linked to autism. We have looked at the metals and last year reported at the IMFAR meeting those
results. This year I will be reporting on PBDEs; that work was funded by Cure Autism Now, which is now in the process of merging with Autism Speaks.
The CHARGE study, by the way, is funded by the National Institute for Environmental Health Sciences as part of one of the children’s centers. It began in 2001, and we are now in our second 5-year period.
The last study design I wanted to talk about is the prospective cohort studies. These are studies where we start with a pregnant woman, and follow her and the child forward. We have already heard a little bit about the National Children’s Study. A couple of other studies are now looking at high-risk cohorts. In particular, the pregnant women are ones who already have a child with autism and are carrying another child. Because of the high sibling recurrence rate, these are high-risk pregnancies. One of these studies, which is also part of our Children’s Center, is called MARBLES; we are also collaborating with the EARLI network, which is scheduled to be funded beginning in 2008, for which Craig Newschaffer will be the PI (principal investigator). What we are focusing on is trying to find out how early we can see biological signs of autism. Several baby sib studies to date have been focused on the early behavioral, but not biological, indicators, and have started postnatally.
Our aim is to determine what are the critical time windows for environmental exposures, and what are the biomarkers that we might use to identify pregnancies and children at high risk. However, any marker is only useful if we can additionally identify what are the target issues or receptors or enzymes on which we might intervene in order to interrupt the development of autism.
In conclusion then, we have had a couple of decades in which we have learned a lot about autism from psychologists and psychiatrists. We have learned how to diagnose reliably, and even some early behavioral markers. We have learned from neuroscientists about aspects of the brain and brain growth, and now is the time for the environmental epidemiol- ogy and toxicology to work together.
This is an area that has not received a lot of attention. What you see in this room is what is out there, pretty much. We have in this room about 80 percent of the environmental epidemiologists and toxicologists, who are looking at autism from this perspective. And only half the scientists in this room are currently doing autism research. So this is an area that has been understudied. There are very few studies of environ- mental factors in the causation of autism. What I have shown you is
basically about what there is.
These large epidemiologic studies can be linked to all the mechanis- tic questions that were raised yesterday. We are doing genomics and there is a potential to do metabolomics and proteomics to figure out biologically, physiologically, molecularly what is different in these children. With the prospective studies we can take the hypotheses that we are getting from the case-control studies, such as the finding from the CHARGE study of differences in expression of certain genes, and ask:
Do those same genes differ, and how early on do they differ? Does it start at 18 months, 12 months, 6 months, or what about in the cord blood? In other words, let’s go back to the very early stages of life and brain development. That is the state of the art and that is where we should be going.
Thank you.
Dr. Falk: Thank you very much, Irva. There is time maybe for one or two very quick questions.
Dr. Fombonne: Your design includes data that looks at a range of exposures, but you do not include in your model any timing of expo- sures. A lot of data suggest that early exposure is important. Therefore, my question relates to how you can modify your study design to look for common exposures at early time points.
Dr. Hertz-Picciotto: In the case-control studies, obviously that is the deficiency in the design of the case-control study, is that you are measuring things now, and you really want to know what happened before the diagnosis, perhaps in the prenatal or perinatal period.
As I said, the medical records do provide us with early information.
We have been looking at even preconception, looking at things like in vitro fertilization, medications that the mother took during pregnancy, what kind of induction or augmentation of labor happened. So there is that component.
We looked at the metals so far in the concurrent blood samples, and now we are going back and taking the baby lots. We started measuring the metals in the baby lots, which represent exposures in usually the first year of life, and then the newborn blood spot, which will tell us something about right before the time of delivery.
So yes, it is a problem. In the questionnaire we also asked whether we can get valid information. It is subject to how well people can remember what happened, and do cases remember better than parents who have a child who is developing quite typically, if you ask them what
pesticides did you use around the home when you were pregnant or in the first year of the child’s life.
Obviously that is a problem, and that is partly why we are moving now to also doing prospective studies, where all the information will be collected prior to the diagnosis.
Dr. Fombonne: May I ask another question? There are also techniques to look at clustering around different environmental exposures. There is currently a study in California looking at increased risk of autism to exposure to different pesticides at early periods of development and gestation. They have found really interesting findings that you can look at one particular exposure and map that to autism rates.
Dr. Hertz-Picciotto: Yes, that is quite a lovely analysis. That is using administrative databases and then linking them to exposure databases. Where those databases exist, I think that is a really excellent approach.
One of my graduate students has been doing—it is not looking to exposures, but she is looking at spatial clustering in California, and breaking it down. But yes, those exposure databases are quite useful, and the pesticide use report one is a very interesting database.
Dr. Falk: Thank you very much. The second speaker is Craig Newschaffer. He will speak on environmental exposures in autism international studies. Craig is professor and chairman of the Department of Epidemiology and Biostatistics at Drexel University School of Public Health. He had founded and directed the surgical office in development, disabilities, and epidemiology at Johns Hopkins previously.
ENVIRONMENTAL EXPOSURES IN AUTISM:
INTERNATIONAL STUDIES16 Dr. Craig Newschaffer
Dr. Newschaffer: Thank you. I have been tasked to talk a little bit about the promise and potential of international studies and international epidemiologic studies that shed some light on environmental exposures and autism.
This framework of looking at frequency by person, place, and time predates the formulation of epidemiology as a discipline. It guides us still
16Throughout Dr. Newschaffer’s presentation, he may refer to slides that can be found online at http://www.iom.edu/?id=42470.