Dr. Leshner: As far as I can tell, there is general agreement that it has been a terrific morning, and therefore we are putting terrific pressure on the afternoon speakers, so don’t let us down. People have been very well behaved and stayed on target, asked questions, didn’t make long speeches, so everybody so far has behaved very well, identified gaps.
Dr. Levitt: Good afternoon. My name is Pat Levitt. In this session, there is one content session and then the discussion; this is the session that has the unenviable task of putting together environment and biology.
That is the title of it.
The first speaker is Art Beaudet, who is professor and chair in the Department of Molecular and Human Genetics at the Baylor College of Medicine.
HOW MAY ENVIRONMENTAL FACTORS IMPACT POTENTIAL MOLECULAR AND
EPIGENETIC MECHANISMS?10 Dr. Arthur Beaudet
Dr. Beaudet: I am going to maybe be a bit provocative and try to argue that there is a substantial chunk of autism where we now can predict what is going on. I made a diagram here. You heard earlier this morning the mention of maybe 10 percent of autism being genetic.
I’d like to argue that this is 40 or 50 percent. This is maybe an exaggeration, maybe it won’t be quite that high, but there is quite a group that we know where we are going to end up.
These individuals have chromosomal defects and single gene defects.
You have seen some of these mentioned. You have heard someone mention how we have better and better techniques for how to
10Throughout Dr. Beaudet’s presentation, he may refer to slides that can be found online at http://www.iom.edu/?id=42981.
search for these. But we know the way we search for them today is like looking at icebergs and only looking above the water line. So we know that this group is much larger because of our inadequate ability to detect small changes.
Then I would argue there is a second group over here that is much more unknown as far as what is going on, and much more likely to be a candidate for involvement with environmental interactions, epigenetics, you name it. I think we just don’t know where we are there.
This is a review from about a year ago that is very nice, indicating how many known definitive chromosomal abnormalities are seen in autism. These are mostly so-called de novo events in children. Their parents are normal. Duplications of chromosome 15 are by far the greatest, and there are quite a lot of deletions of chromosome 22 as well.
So we know there are deletions and duplications that can involve every single chromosome that can give rise to autism.
There are a couple of papers that have appeared recently that further emphasize this, using a methodology called array comparative genomic hybridization to detect larger events across the genome with greater efficiency. One paper recently reported detecting abnormalities that are presumed causative in children, with 27 and a half of the children with syndromic autism. These are children who are dysmorphic. They are likely more cognitively impaired, probably both mentally retarded and autistic in most cases, and unusual looking. If you see them in a grocery store, you will see that they have some physical abnormalities.
The report by Jonathan Sebat has been mentioned, where he found in about 10 percent of simplex cases these kinds of abnormalities. We know that these methods being used will miss many, many kinds of genetic lesions which would give the same functional effect.
We have heard about advanced paternal age. Dr. Susser has this publication here. I will just say that we have an ability to make a very good guess what the problem is with advanced paternal age. It is probably point mutations, so it is probably causing a de novo effect on a single gene. I think the fact that we are seeing it more in females than males will make sense in a minute.
If you were to take away anything from my presentation, I would say this is the message. I would say there is a group of mutations that are identified, chromosomal, single gene, in autistic patients. We do see what their primary defect is. It is a strong genetic effect, it is a very
highly penetrant effect. This group tends to be dysmorphic and they tend to have cognitive impairment.
From what we know about how we found these, we know that our ascertainment method is pretty terrible, so I am speculating that there has got to be more of these which are likely, more of the same mutations.
That would give you then a residual, very small group of females who are the pink section up here, and this huge group of males who are less impaired, less dysmorphic, and more puzzling as to their etiology.
The reason the paternal age effect makes sense is that we know that paternal age effects will be relevant to this group of mutations up here.
For almost all of these we have equal male–female distribution. So I think there is a big chunk of autism which we maybe would have said was 10 percent or 5 percent 5 years ago, that I think is going to be closer to 40 or 50 percent of the total. That leads to the second phenomenon down here, which seems very different.
Geneticists think about things being heritable. You have heard comments about monozygous twins. I just want to make the point that de novo genetic events are highly heritable in genetic terminology. That is, if you take Down syndrome and you have identical twins, they will both have Down syndrome 100 percent of the time. So their phenotype of Down syndrome is determined by their phenotype, and we say the heritability is 100 percent. But their parents don’t have Down syndrome.
The abnormality is not inherited.
I think this is the case for all of the autism genetic defects that we know about at present. They are by and large de novo genetic events. We would expect them to be highly concordant in monozygous twins and much less frequently concordant in dizygous twins, which is what the bulk of the twin data says about autism.
The rest of it, I have to say, turns more to this leftover group that we understand less. I have worked with a couple of disorders, Prader-Willi syndrome and Angelman syndrome, that involve the phenomenon of genomic imprinting that I don’t have time to go into here.
On chromosome 15, if you have a deletion of a particular region, you have Prader-Willi syndrome, and if you have a deletion in the same region on the maternal chromosome you have Angelman syndrome. If you inherit two copies of chromosome 15 from your mother you have Prader-Willi syndrome, and two copies of chromosome 15 from your father you have Angelman syndrome.
The deletions are genetic. If you sequence the genome, you will find 5 or 6 million base pairs of DNA have been lost. These are epigenetic. If you sequence the gene in the epigenetic cases, the sequence is perfectly normal, but the fact that these genes behave differently whether they are of maternal origin or paternal origin explains the problem.
Both of these events, which are the bulk of events that cause this kind of abnormality, are de novo. That is, the parents don’t have the deletions and they don’t have the two copies of a chromosome from a single parent.
This is emphasized for us that a diagnosis could be quite hard to figure out. If you have some cases in the mix being epigenetic, some being genetic, the epigenetic or genetic events could be de novo or inherited. It creates quite a complicated model, and so we have tried to explore this mostly as it relates to the patients who we understand less.
This de novo component fits very well with what I have been talking about, the genetic group. We don’t really have definitive evidence as to whether the epigenetic component is going to be an important one or not.
In this, I am very interested, particularly from the epigenetic status, about the environmental interaction, particularly folic acid. The genotype has to have a certain epigenetic state in order to give rise to the pheno- type, so I am very supportive of the idea that there will be environmental genetic interactions going on.
This graph is widely talked about and looked at. I just want to make the point that some people think there is a substantially increased incidence of autism. I think it is clear this is partly artifactual by how children are diagnosed and ascertainment and so on. But if there is any component of this that is real, it is very, very important to detect for the reasons that have already been stated this morning in terms of under- standing the causation and trying to develop treatment.
If there is something going on, what could be going on? I just want to mention two issues. Prenatal ultrasound, in the event that it might not get mentioned otherwise. Paternal age we have already talked about. I want to talk about folic acid a bit more.
This is a paper from last year in PNAS looking at ultrasound exposure of mice and the effect on the neuronal migration in these developing mice. I think this is a good example of the kind of area that we need to be thinking about as far as any kind of environmental factor.
These people made some recommendations that we shouldn’t be doing
prenatal ultrasound as a recreational activity, and that we need more research in this area.
This is how prenatal ultrasound has increased over the right year interval. You have seen this figure before about folic acid, and I will use it now to transition to folic acid, just to make a few points.
When I have tried to express some concern that folic acid could be a problem that could be increasing the incidence of autism, people have said it was that the fortification came too late. But I think if you look at the data, that is not correct. In the NHANES studies in the 1970s, we had 23 percent of people reporting they took a daily vitamin. In the later 1970s and 1980s, 35 percent, and this went up with time. The FDA (Food and Drug Administration) prohibited putting much folic acid in vitamins until the mid-70s, and most vitamins had none, but a few had a tiny amount. But in 1973 they raised the limit to 0.4 milligrams of folic acid. So one-a-day vitamins went from none to 0.4 milligrams in 1976 and Vidaylin went from none to 0.4 milligrams in 1977. This is before the neural tube defect perspective.
There are data from the Framingham study that people who reported that they took a daily vitamin or ate ready-to-eat cereal had a folic acid level roughly two to three times higher than people who reported they did not. This was in the 1990s before fortification. We had two groups in the population, those who were taking a folate supplement and those who were not.
So I think these changes are reasonably compatible with the possibility that timewise, folic acid is a potential factor.
Why have we been very interested in it? My laboratory has been interested in epigenetics, and it is known that using folic acid intake in mice and in humans, you can alter gene expression because of the way it contributes to DNA methylation and histone methylation.
This is a publication from some time back, where coat color in these mice is under a particular genetic element which is responsive to DNA methylation. You can change the coat color of the mice by feeding the mother differing amounts of folic acid and other methylation-related compounds during the pregnancy.
This is a study from humans. I won’t try to take you through the technology, but just to say it demonstrates that folic acid can change gene expression in humans as well. This is a gene which should have only one of the two bands here present in a normal situation. These are patients with renal failure in high homocysteines, and those with the
highest homocysteines are expressing both the maternal and paternal copy of the gene, so they have two bands; that is abnormal. But when you put them on folate supplementation they go back to expressing just one band, which is the normal state.
So again, folic acid can influence gene expression in mice and in humans in certain situations, and often this involves this phenomenon of genomic imprinting, where the maternal copy of a gene and the paternal copy can differ.
So folic acid definitely changes the action of some genes, probably especially imprinted genes. The laboratory acid intake of the population at large, and particularly reproductive-age women, has dramatically increased over the last three decades. Your folate level and maybe imprinted gene expression are different today than they were 15 years ago, and we need to know more about whether folic acid intake is increasing or decreasing the intake of any diseases.
The following are suggestions for potential research areas. I think genomewide studies at the exxon level and single-gene level and single- nucleotide level will expand this group, which I propose will turn out to be genetic, but we don’t have very good ability to detect them right now.
This will separate out this strong mutation group from the other puzzling group that is left. I think that epigenetic approaches are very worthwhile for the idiopathic portion that has not got specific genetic lesions.
Dr. Levitt: We have time for one or two clarifying questions.
Dr. Pessah: When I started out in looking at autism many years ago, only about 10 years ago actually, the emphatic view was 90 to 95 percent heritable genetics. What has changed over the last 10 years to make it 50- 50?
Dr. Beaudet: Well, I don’t know. It is different opinions about what the heritability is. I think one question had been, why is it so concordant in monozygote twins and nonconcordant in dizygote twins? That is totally explained by de novo events. Whether it is advanced paternal age causing a point mutation or whether it is trisomy 21, these de novo copy number variants, they all will give you 100 percent concordance in monozygote twins and a much lower concordance in dizygote twins.
I think also, this whole issue that these people have genetic conditions, their genotype determines their phenotype, but it is not inherited. So if you try to compare their genotype to their parents’
genotype, you don’t find the expected implications.
Dr. Herbert: You said that the known genetic mutations had a 100 percent concordance monozygotic and 5 percent dizygotic. Where are those data from?
Dr. Beaudet: I would say on general principles, if you take any new mutation event, whether it is trisomy 21 or whether it is achondroplasia, Rett syndrome, any new mutation event happens prior to fertilization or prior to twinning. The monozygote twinning takes place later on, and the twins have the identical genotype, including the genotypic error that they have.
Dr. Herbert: You are saying they are concordant for autism. We don’t know whether the gene causes it or it is just a risk factor. If it is just a risk factor, then you can’t assume that it is going to be 100 percent concordance.
So if it is 100 percent concordance in these genetic errors, are there data that support what you said?
Dr. Beaudet: The question is, when you find these kinds of errors, how convinced are you that they are the cause of that child’s abnormality?
Dr. Herbert: Close to a risk factor, a high risk factor.
Dr. Beaudet: Right, or totally irrelevant. I think that there is some of that. If you look for these de novo events in the control population, you do see some de novo events in the normal population. But statistically, most of these new events are almost certainly the cause of the child’s disability.
They all have major effects, for the most part. There may be weaker effects that we haven’t discovered yet, but the ones that we are looking at here, they mostly have physical abnormalities associated with them in terms of dysmorphic features and birth defects, they are mostly mentally retarded, and they meet the criteria for autism.
Dr. Levitt: That’s it. We have a lot of time for discussion this afternoon, and the godfather is looking at me. Thanks very much, Art.
Mark Nobel is our next speaker. He is going to talk about environ- mental factors impacting cell function. Mark is a professor of genetics at the University of Rochester Medical Center.
HOW MAY ENVIRONMENTAL FACTORS IMPACT POTENTIAL CELL-BASED MECHANISMS?11
Dr. Mark Noble
Dr. Noble: Thank you so much for this opportunity to come and learn from you all. It is very exciting to me to have the opportunity to take part in this discussion.
I am going to approach this talk from the perspective of our efforts to develop a comprehensive approach to the field of stem cell medicine.
This work began with our initial isolation of CNS progenitor cells almost 25 years ago, and now extends to cover many components of stem cell medicine that are separate from the use of cell transplantation to repair damaged tissue.
In order to discuss our work, I have to introduce you to some of the cellular players in the CNS. The only point that I want to make with this slide is that when people talk about development, they mostly talk about stem cells and they talk about differentiated cell types, neurons and myelin-forming oligodendrocytes and astrocytes. From our attempts to understand the cellular basis of developmental maladies, however, it seems the most interesting cells are the progenitor cells that lie in the middle. These lineage-restricted progenitor cells are the workhorses of building tissues. They are the ones that are responding to environmental signals. They are the ones that are building your whole nervous system during development, and these are the ones where we focus our attention.
There are several such progenitors that we study.
Our greatest interest, however, has been studying myelination because of damage to myelin being the largest category of neurological disorder, showing up in all traumatic injuries, most chronic degenerative conditions, and in respect to this meeting, with some very interesting findings in respect to autism.
Through our studies of all these progenitors and what happens to them during development, we have come to realize that many develop- mental maladies are diseases of precursor cells. You have abnormalities in the generation of specific cells, with specific cell types being generated too early in some conditions, and not at all in others. Or you don’t make enough of certain cell types. We have been trying to
11Throughout Dr. Noble’s presentation, he may refer to slides that can be found online at http://www.iom.edu/?id=42465.