Archive for April 2010
Super Cool Study: Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain
Posted April 25, 2010on:
So this is a really cool paper by some folks that have a series of interesting stuff: Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain. Here is the abstract:
Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.
[As always, any emphasis is my own.]
This group has published a couple of papers that utilized similar study groups, methodologies, and means to display their findings, all of which I would recommend to anyone interested in learning; specifically, Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes [full paper available!], Gene expression profiling differentiates autism case-controls and phenotypic variants of autism spectrum disorders: evidence for circadian rhythm dysfunction in severe autism [full version available!], and Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: altered pathways in neuronal development and steroid biosynthesis [full paper available!].
There are a couple of things I really like about their methodology and presentation style.
1) Several studies, including the most recent, included twins with discordant autism severity as study participants as a way to gain insight into the impact of genetic expression, as opposed to genetic structure on autistic behaviors. The highly cited heritability of autism in twins is used as evidence that the condition is predominantly mediated through genetics, and while no doubt genetic structure is important, by using genetic clones with different manifestations of autism severity, the authors are able to ascertain information about which genes are being affected in twins.
2) The two stage nature of the study design allows for both large scale analysis of a great number of genes being expressed differentially by genome wide scan, the results of which can be used for highly targeted confirmation by tissue analysis. Further, the use of cells available in the periphery, lymphobastoid cell lines (LLCs) as measurement points for genetic expression, allows for well thought out investigations of a very rare resource, post morten brain tissue from autistics. In this instance, different methylation profiles identified from LLCs from blood samples gave the researchers a starting point for what to look for in the brain tissue.
3) This paper ties together both genetic expression and epigenetics; i.e., not only that genes are being used differently, but it forwards our understandings of the means by which this is happening. Earlier studies by this group have found differences in genetic expression previously, but hadn’t elucidated on the specific mechanisms of action, in this case, over methylation, and consequent silencing of genetic protein production.
4) This is the first group of papers I’ve seen that have been using a bioinformatics approach to understanding the pathways affected by their findings; there may be other papers out there in the autism realm, (and almost certainly in others), that have been performing this type of analysis, but I haven’t run into them. Several of their papers, including the circadian rhythm paper, provide illustrations of associations to biological conditions and pathologies associated with affected networks. Here is an example from the latest paper. (Sarcastic apologies for those running at 800 / 600)
This type of illustration is the death knell for the argument that autism is a condition to be handled by psychologists; there are a couple of similar ones in the paper.
Considering those points, here are some juicy parts from the paper itself. From the introduction:
In this study, we use global methylation profiling of discordantly diagnosed monozygotic twins and their nonautistic siblings on CpG island arrays to test the hypothesis that differential gene expression in idiopathic autism is, at least in part, the result of aberrant methylation. Our study reveals distinct methylation differences in multiple genes between the discordant MZ twins as well as common epigenetic differences distinguishing the twins (the undiagnosed twin exhibiting milder autistic traits that are below the threshold for diagnosis) from nonautistic sibling controls.
There are essentially three groups, twins with different autism severity, and non autistic siblings. One thing that I’m not cerrtain of here is whether or not there were methylation differences found between the twins and their non autistic siblings or not; the text above is a little unclear; i.e., as there are different mechanisms by which genetic expression can be modified besides methylation, this may mean that while there were expression differences found between autism and controls, those differences were not found to be attributed to differential methylation levels. (?)
From the results:
Network analysis was then performed to examine the relationship between this set of genes and biological processes. As shown in Fig. 1B, many of the associated processes within the network, including synaptic regulation, fetal development, morphogenesis, apoptosis, inflammation, digestion, steroid biosynthesis, and mental deficiency, have been associated with autism. Two genes from this network, BCL-2 and RORA, were selected for further study because of their respective roles in apoptosis and morphogenesis/inflammation. Interestingly, BCL-2 protein has been previously demonstrated to be reduced in the cerebellum and frontal cortex of autistic subjects relative to control subjects (31, 32), but RORA, a nuclear steroid hormone receptor and transcriptional activator that is involved in Purkinje cell differentiation (33) and cerebellar development (34), has never before been implicated in autism. In addition, RORA, a regulator of circadian rhythm (35), is also neuroprotective against inflammation and oxidative stress (36), both of which are increased in autism (37, 38).
Several of the tables are pretty cumbersome to paste in, but do provide more detailed functional level impacts of some of the functions of the differentially methylated genes identified. Even with the text above, however, we can see a lot of sweet spots being touched on, including several that were identified in previous studies by this group of researchers. It also illustrates some of the very powerful techniques in use; a broad array of genes were scanned for differential expression, some with different expression and significant roles in processes known to be abnormal in the autism population are identified, and used for further, more pinpointed analysis.
As noted, Fatemi found reduced BCL-2 in post mortem brain samples in two studies; one of the roles played by BCL-2 is apoptosis, or programmed cell death. By way of example, here is a study that shows that knockout (or in this case, knockup) mice that overexpress BCL-2 have more Purkinje cells than their non modified counterparts, which states, in part:
Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.
Considering that reductions in Purkinje cells is among the most commonly found brain difference in autism, a reduction in BCL-2 seems appropriate. The fact that it in this case it was methylation levels leading to a reduction in BCL-2 might also be of interest in regards to the Fairytale Of The Static Rate of Autism; here we have evidence that mechanisms other than genetic structure are leading to decreases in a protein known to protect Purkinje cells from apoptosis.
I don’t know anything about RORA, but its list of functions make a lot of sense when we consider other findings; a relative dearth of a protein known to protect against neuroinflammation and oxidative stress and a regulatory role in the sleep cycle.
The authors also noticed a dose dependent relationship between expression levels, which in this case represented a silencing of genes and autism severity.
Quantitative RT-PCR was used to confirm decreased expression of BCL-2 and RORA in autistic samples and to evaluate the effect of a global methylation inhibitor, 5-Aza-2-deoxycytidine, on gene expression. For both BCL-2 and RORA, gene expression was significantly higher (P_0.05) in the unaffected control than autistic co-twins (Fig. 4A). Generally, the diagnosed autistic co-twin (_A) had the lowest level of expression of BCL-2 and RORA, while the milder undiagnosed co-twin (_M) exhibited transcript levels between that observed for unaffected sibling controls and autistic co-twins. This suggests a quantitative relationship between phenotype and gene expression of these 2 genes, although additional studies are required to confirm this observation
Again, this makes plenty of sense if we believe that things like a neuroinflammation, oxidative stress have parts to play in the behavioral manifestation of autism; in this case, get more methylation, and hence, less RORA and BCL-2, which, in turns, makes you more susceptible to neuroinflammation, oxidative stress, and Purkinje cell development abnormalities.
If we take the predisposition towards problems with inflammation for a closer look, we can find that several other papers, including Grigorenko, Enzo, and Ashwood have all found that a propensity for inflammation, or a propensity towards abnormal regulation of inflammation have correlations with autism severity. Though potentially inconvenient, this would seem to lend additional evidence for a causal role of immune based pathology in autism, as opposed to autism causing immune abnormalities.
The discussions section has a lot of good text that is largely a touch up on what we already have here. Here are some good quotes:
In particular, functional and pathway analyses of the differentially methylated/expressed genes showed enrichment of genes involved in inflammation and apoptosis, cellulardifferentiation, brain morphogenesis, growth rate, cytokine production, myelination, synaptic regulation, learning, and steroid biosynthesis, all of which have been shown to be altered in ASDs. The candidate genes were prioritized for further analyses by identifying the overlap between the differentially methylated genes and those that had been shown to be differentially expressed in the same set of samples in previous gene expression analyses (18). Pathway analyses of this filtered set of genes thus focused our attention on 2 genes, BCL-2 and RORA, as potential candidate genes for ASDs whose expression may be dysregulated byaberrant methylation. As shown in Figs. 3 and 4, respectively, RORA was confirmed to be inversely differentially methylated and expressed in LCLs from autistic vs. nonautistic siblings,with expression dependent on methylation, as demonstrated by the absence of methylation in the presence of 5-Aza-2-deoxycytidine. Notably, we also show by immunohistochemical staining of cerebellar and frontal cortex regions of autistic vs. normal brain (Figs. 5, 8), that RORA protein is noticeably reduced in the majority ofthe autistic samples relative to age- and sex-matched controls. This reduction is also specifically demonstrated in Purkinje cells, which are dependent on RORA for both survival and differentiation (Fig. 7). These findings thus link molecular changes identified in a peripheral cell model of ASDs to actual pathological changes in the autistic brain, suggesting that LCLs is an appropriate surrogate for studies on autism.
Finally, this paper generated a lot of press, in part (I think), because somewhere, someone (the authors?), apparently made note of the fact that this type of feature, hypermethylation, is potentially treatable, raising the possibility of palliative avenues. (Or was this just a function of the fact that it was a finding that wasn’t truly genetic, and thus, ‘fixable’?) While technically true, I am of the opinion that this is a long ways off; the authors found large numbers of differentially methylated genes; some were also hypomethylated. The drugs that we know are capable of epigenomic modifications right now, some are used in advanced cancer patients, for example, are not discriminatory in their actions. What we really would need would be targeted unmethylators that we could use to attach to RORA and BCL-2 genes and specifically free them up to produce more protein. The same week that this paper came out, another paper was published, entitled Epigenetic approaches to psychiatric disorders which speaks towards this complexity.
Hello friends –
A while ago I saw a completely fascinating Nova called Ghost In Your Genes concerning the nascent field of epigenetics, the study of the relative expression of genes, which is a bit different than the presence or absence of genetic differences. I’d recommend this program to anyone interested in learning.
In a general sense, our genes are simply blueprints for the production of proteins; the traditional model of genetic research involves structural changes in the genetic blueprints, so that we might understand that a person with a particular mutation might produce more, or less, of a particular protein than someone without that mutation. As protein gradients are altered, physiological effects accumulate, and we can begin to associate genetic differences with identifiable classifications.
But. It turns out, structural differences in the DNA aren’t the only way to affect the production of genes. Genes can also be regulated by a variety of factors, and these changes in regulation, in turn, are measured as expression of genes, essentially a measure of which genes are active, or inactive, and to what extent.
In biology, epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επί– over, above) –genetics. These changes may remain through cell divisions for the remainder of the cell’s life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism’s genes to behave (or “express themselves”) differently.
For another interesting write up, see this one by PZ Meyers; it gets technical quickly, but is a nice read. Specific mechanisms aside, it is sufficient for our purposes that epigenetics is the study of how a variety of non structural changes can affect how our genes operate.
An analogy might of a series of car engines, sitting at idle. All of them power a car, but at a structural level there are differences, most models are roughly generating the same amount of force at idle, but, for example, the Prius engine is generating much lower force than Porsche engine. At a very general level, we might consider physical mutations of our genome and protein generation capacities to be equivalent to the difference between the Porshce and the Prius at idle. But there are other means to affect the energy being put out by the engine, the accelerator, tweaking the cylinders, or a variety of other means. This is a big shift and weights heavily on the ‘genetic and environment interaction’ theme that gets a lot play in the autism realm. Despite a lot of studies, and spectrums of dollars there have been very few findings involving autism genes that do anything but confer a very limited risk of a diagnosis. Furthermore, a lot of the studies are finding that seemingly very common mutations are implicated, but with very delicate effects. An example of this might be the MET genes, that have several neat papers (here, here, here), but the specific MET-C allele associated with autism is still very common, found in nearly fifty percent of everyone. None the less, it is just a little more prevalent in the autism cohort, but the impact is very subtle, and likely dependent on the presence of a variety of other genes (or expression patterns), or other factors. Excepting known genetic conditions that confer great risk, but can be responsible for only a fraction of our autism, mutations such as Fragile-X or Rhett Syndrome, the vast majority of genetic findings impart small increases in risk.
But once we start looking at the wide array of different genetic expression in autism, it becomes clear that which genes you use, and to what extent, might be as important as which genes you are born with. By way of example, a very cool paper out recently, “Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain ” that is available in full online and I would recommend to anyone interested in how analyzing epigenetic changes and the accompanying differential gene expression can teach us more about autism. A lot of the press around this study involves the hope that eventually this kind of finding might lead a treatment opportunities, something I personally consider to be a long term goal that still faces significant technical hurdles; but it does gives us insight into the nature of autism, and the usefulness of the half truth ‘differently wired’ argument concerning autism treatments.
Note: Updated link to PZ.
Posted April 14, 2010on:
I hate to write another vaccination related post, but I keep on running into the same, tired argument, and thought it might be nice to have a single place to list and link the reasons that one of the most commonly used defenses of why we don’t need to study the vaccination schedule can be dismantled. The scary part, the really fucking scary part, is how easy it is to deconstruct the metrics being provided by experts as to why questioning the process of vaccination need not be thoroughly evaluated, and how people that ought to know better keep regurgitating the antigen gambit despite its obvious shortcomings when held to the most primitive logical tests.
For some background, lets start with basic immunology and the hows and whys of how vaccines actually work. But even before that, lets be clear: Vaccines work. I have absolutely no doubt that the purpose of vaccines, providing protection against microbial invaders is successful, and saves millions of lives every year. What I’m not so sure of, is whether or not this is the only thing our increasingly aggressive vaccination schedule has been accomplishing.
The functional success of vaccination is that we have crafted a technique that allows us to train our immune system to recognize some very nasty, dangerous, and deadly bacterial and viral pathogens. How is this done? Well, it turns out that at a very detailed molecular level, many bacteria and viruses have very specific patterns on their exterior, for our purposes, an immunological fingerprint that identifies, for example, the tetanus bacteria from the diphtheria bacteria. These fingerprints are known as antigens, and our immune systems use them to store a memory of particular pathogens we have been exposed to, so the next time such a pattern is encountered, a robust immune response can be mounted rapidly, before the pathogen gets a chance to reproduce and get us sick. The memorization of these molecular patterns, the fingerprints of specific bacteria and viruses, is the foundational premise of vaccination; by presenting these antigens to our immune system in a hopefully(?) harmless way, we train our immune system to respond to these invaders without actually having to endure the virulence of the actual bacteria or virus. Making things a bit more complicated, some pathogens have more than one molecular face to present, and as such, more than one fingerprint is necessary for our immune system to recognize. Some others, such as flu, regularly shift their molecular fingerprint; this is why there are seasonal flu shots, each year scientists must make educated guesses as to which particular influenza fingerprints will be most prevalent; when they guess correctly, the vaccine mostly works, because we have trained our immune system to see that particular antigen pattern. Other pathogens, like HIV, undergo such rapid transformation of their outward facing molecular structure that tailoring a molecular portrait of them has proven exceedingly difficult.
So, again at a very high level, vaccines work because they present antigens, immune fingerprints, from viruses or bacteria to our bodies, without the associated virulence of the organisms. The hows of creating the antigens without the problems of actual infection aren’t necessary for this discussion; lets just assume that for our purposes, you can have bacterial or viral fingerprints introduced in a vaccine without having to worry about the traditional ramifications of the actual bacteria or virus they came from. Great!
Given that, lets imagine you are a skeptic and are a bit bothered by the fact that our existing vaccine and autism research seems to be wholly comprised of studies involving either thimerosal, or the MMR. It seems a bit confusing that these two types of studies are sufficient for us to have certainty that the act of vaccination itself, or other vaccines administered at very different ages might be contributing to our apparent observations of increases in autism (or other behavioral or autoimmune disorders). If you raise a question involving this glaring blind spot in our research, a lot of the time you’ll see a response like some of these:
The only thing that makes biological sense in the discussion really is antigens and excipients and if you look at that, today’s kids get FAR fewer than say, my generation.
What is relevant is the number of antigens, and not the number of vaccines, that matters. Antigens are the active part of the vaccine which stimulates the immune response.
Another point directed to those who think that multiple vaccines overload the immune system. In actual fact, even though we are vaccinating against more diseases than in the past, we are actually using fewer antigens (the part of the vaccine which stimulates the immune response) in these vaccines than was previously the case.
You get the picture; the only measurement of interest is the number of antigens in vaccines. To be completely fair to some people that use the antigen gambit, it is in response to its equally simplistic counterpart, the ‘Vaccines Overload The Immune System’ gambit. That’s no excuse, at the end of the day, the people using crank arguments are supposed to be the cranks. What worries me is the people using the antigen gambit, are in many cases, the experts, and in the rest of the cases, folks that have listened to the experts, and parrot something that sounds sciency. It is a frightening day when you realize that if infectious disease experts had a reason, a real reason, we shouldn’t study the entire vaccination schedule, they’d provide one better than the antigen gambit.
The tour de force take down of the Vaccines Overload the Immune System gambit is “Addressing Parents’ Concerns: Do Multiple Vaccines Overwhelm or Weaken the Infant’s Immune System?“, by Paul Offit and others. It’s my guess that this document, published in the highly read Pediatrics journal, plays a big part in people believing that the only important thing about the vaccine schedule is the number of antigens involved. Here is the abstract:
Recent surveys found that an increasing number of parents are concerned that infants receive too many vaccines. Implicit in this concern is that the infant’s immune system is inadequately developed to handle vaccines safely or that multiple vaccines may overwhelm the immune system. In this review, we will examine the following: 1) the ontogeny of the active immune response and the ability of neonates and young infants to respond to vaccines; 2) the theoretic capacity of an infant’s immune system; 3) data that demonstrate that mild or moderate illness does not interfere with an infant’s ability to generate protective immune responses to vaccines; 4) how infants respond to vaccines given in combination compared with the same vaccines given separately; 5) data showing that vaccinated children are not more likely to develop infections with other pathogens than unvaccinated children; and 6) the fact that infants actually encounter fewer antigens in vaccines today than they did 40 or 100 years ago.
The biggest problem here is that the acknowledged, ‘implicit’ concern is that multiple vaccines may overwhelm the immune system. The concern we should be more concerned with is, can vaccines modify the immune system in ways that we cannot predict? This is a question that is not addressed here, but if your premise starts with the wrong question, or in this case, a bad question your conclusions shouldn’t be worth much.
All of the bullet points provided suffer from one or more maladies, including a foundational structure of gross over simplifications, insulting the intelligence of the reader, or in one case, wildly optimistic claims of a study conclusions; the same kind of thing what would get you a special article by the Chicago Tribune if you recommended children with autism try not to eat wheat for a few weeks and see what happens.
For this post, we’ll just focus on the last bullet point, and the text that supports it:
6) the fact that infants actually encounter fewer antigens in vaccines today than they did 40 or 100 years ago
This is the lead in for this question:
Parents who are worried about the increasing number of recommended vaccines may take comfort in knowing that children are exposed to fewer antigens (proteins and polysaccharides) in vaccines today than in the past.
To prove this comforting point, the authors provide this fancy table:
(Bigger view on the link to full paper – they don’t have this table exploded as its own supplement link). The good news is in green here, as noted in the text, the only reduction count in the vaccine schedule after 1960 was the change from DTP to DTAP.
The bad news is that, if counting antigens were a meaningful metric, of well, anything, the chicken pox vaccine, Varicella, now contains more antigens than the rest of the shot schedule combined.
This puts us in somewhat of a conundrum. If the ‘number of antigens’ in vaccines is what is relevant, does this mean that the Varicella vaccine puts nine times more stress on the immune system than the Pneumococcus vaccine? Does the Varicella vaccine initiate an immune response sixty nine times more strenuous than the diphtheria component of the DTAP vaccine? [Good luck finding a study to measure the innate immune response to any of those vaccines in a pediatric population.]
The DTAP was licensed in the 1980s, but Varicella didn’t get licensed until 1990; so this means that children who got DTAP, but didn’t get Varicella, got far fewer antigens, half as many, than children born just a few years later. Is this meaningful?
Here is an interesting way to view the question. Imagine the CDC was addressing a set of parents whose children was born in 1985 who were concerned about those vaccinations overloading the immune system of their children, and this was the response:
Parents who are worried about the increasing number of recommended vaccines may take comfort in knowing that your children were exposed to fewer antigens (proteins and polysaccharides) than in vaccines today.
Does this sound like a good argument?
We might also take a look at how frequently children experience mild side effects from vaccination, according to the CDC web site. Fever is an indicator of innate immune activation, though you will occasionally see arguments made that it is insufficiently characterized to draw conclusions from, but if we are trying to understand if addition of antigens is a useful measurement or not, it would seem the rates of side effects are valid goalposts. Here are some quotes; there isn’t a fancy table of this information yet.
- Varicella: Fever (1 person out of 10, or less) [69 antigens]
- Pneumococcal: Up to about 1 out of 3 had a fever of over 100.4 degrees Fahrenheit, and up to about 1 in 50 had a higher fever (over 102.2 degrees Fahrenheit). [8 antigens]
- MMR Fever (up to 1 person out of 6) [24 antigens]
- DTAP: Fever (up to about 1 child in 4) [4 – 7 antigens]
Now that is curious. According to the CDC, the vaccine with the most antigens causes fever far less frequently than vaccines with many times fewer antigens in them. If we can use addition to gain comfort from the fact that the current vaccine schedule includes fewer antigens than it used to, how do we incorporate in this information?
But if we can’t use addition for our purposes? What if, in fact, the system we are interacting with is much, much too complicated to be usefully outlined with simple addition? What if antigens aren’t the only relevant measuring point in evaluating vaccine impact on the immune system? In this case, why use the reduction in antigens in vaccines as an argument to ‘address parents concerns’? Why has such a gross over simplification achieved ubiquity in the blogosphere and indeed, why was it promulgated by the most frequently interviewed physician when the subject is autism and vaccination?
Ponder the above at your own risk.