passionless Droning about autism

Posts Tagged ‘Epigenome

Hello friends –

One of my tangential pubmed alerts notified me of this study the other day:  Epigenetic and immune function profiles associated with posttraumatic stress disorder

The biologic underpinnings of posttraumatic stress disorder (PTSD) have not been fully elucidated. Previous work suggests that alterations in the immune system are characteristic of the disorder. Identifying the biologic mechanisms by which such alterations occur could provide fundamental insights into the etiology and treatment of PTSD. Here we identify specific epigenetic profiles underlying immune system changes associated with PTSD. Using blood samples (n = 100) obtained from an ongoing, prospective epidemiologic study in Detroit, the Detroit Neighborhood Health Study, we applied methylation microarrays to assay CpG sites from more than 14,000 genes among 23 PTSD-affected and 77 PTSD-unaffected individuals. We show that immune system functions are significantly overrepresented among the annotations associated with genes uniquely unmethylated among those with PTSD. We further demonstrate that genes whose methylation levels are significantly and negatively correlated with traumatic burden show a similar strong signal of immune function among the PTSD affected. The observed epigenetic variability in immune function by PTSD is corroborated using an independent biologic marker of immune response to infection, CMV—a typically latent herpesvirus whose activity was significantly higher among those with PTSD. This report of peripheral epigenomic and CMV profiles associated with mental illness suggests a biologic model of PTSD etiology in which an externally experienced traumatic event induces downstream alterations in immune function by reducing methylation levels of immune-related genes.

Essentially the authors took a bunch of people that are more likely to experience stressful situations and PTSD, urban Detroit residents, who amazingly report PTSD symptoms at twice the level that previous studies have found in analysis of larger areas.  [Apparently, getting physically attacked is more common there, which gives rise to PTSD even more than ‘other traumatic event types’, and was reported by 50% of the participants from a larger study which formed the population pool of this study. (!!)]  With this population base, blood was drawn and methylation profiles were analyzed between participants who reported PTSD symptoms (n=23) and those who ‘only’ had ‘potentially traumatic events’ (PTE).  PTSD and ‘controls’ where matched by race, age, sex, and blood profiles.

Once methylation levels were identified, a functional annotation clustering analysis was performed, which I believe is similar a pathway analysis; essentially a bioinformatic tool to gain insight into which biological functions were being manipulated as a result of differential methylation of the genome. This is a powerful new tool in discerning what is happening in autism and elsewhere, and I expect it will provide some surprising answers in the future.   Here is their text on what they found:

Consistent with previous findings from gene expression (4, 5) and psychoeneuroimmunologic studies (3), each of the top three FACs determined from uniquely unmethylated  genes among PTSD-affected individuals shows a strong  signature of immune system involvement. This signature includes  genes from the innate immune system (e.g.,TLR1 andTLR3), as well  as from genes that regulate innate and adaptive immune system  processes (e.g., IL8, LTA, and KLRG-1). In contrast, pathways and  processes relevant to organismal development in general—and  neurogenesis in particular—figure prominently among the genes  uniquely unmethylated in the PTSD-unaffected group (e.g., CNTN2  and TUBB2B; Fig. S2). Notably, similar clusters were obtained using  an alternative approach based on genes differentially methylated  between the two groups at P < 0.01, with annotations in the top five  FACs that include signal, cell proliferation, developmental process,  neurologic system process, and inflammatory response

Keeping in mind that reduced methylation results in increased gene expression, if we take a look at Table 1, some of the parallels to autism jump out a little more robustly:

Table 1

In the ‘Uniquely Unmethylated’ (i.e., higher expression), area, we find that participants affected by PTSD had showed greater enrichment in genes related to the immune response, and specifically the inflammatory response and innate immune response.  Our evidence for similar immunological profiles in the autism realm is deep, and includes multiple observations of an active immune response in the CNS, highly significant over expression of genes related to immune function in the CNS, several observations of known upregulators of the innate immune response that are associated with inflammatory conditions, and multiple studies finding an exaggerated innate immune response in vitro when compared to controls.   The correlations with developmental process and neuron creation are pretty straightforward.

In the ‘Uniquely Methylated’ area (i.e., lower expression), the sensory perception differences hit close to home, and xenobiotic metabolism has been implicated by several studies.

Going further, the researchers attempted to evaluate for correlations between the number of potentially traumatic experiences and the methylation profile, and somewhat unsurprisingly found that as the number of experiences increased, the methylation differentials showed wider variation.

Here again we see a distinct signature of immune-related methylation profiles among the PTSD-affected group only. More specifically, we see methylation profiles that are suggestive of immune activation among persons with more PTE exposure in the genes that are significantly negatively correlated with increasing number of PTEs—a pattern reflective of that observed for the uniquely unmethylated genes in this same group (Table 1).
Lastly, the participants were scanned for antibodies to CMV, a persistent herpesvirus found in almost all humans, and can be used as a biomarker to indicate compromised immune function.  Significant differences in antibodies were observed between the two groups.

From the discussion section:

Among the many analyses performed in this work, the immune related  functions identified in the PTSD-affected group were consistently identified only among gene sets with relatively lower levels of methylation (Tables 1 and 2). Demethylation has previously been shown to correlate with increased expression in several immune system–related genes (reviewed in ref. 22), including some identified here [e.g., IL8 (23)]. In contrast, methylation profiles among the PTSD-unaffected are distinguished by neurogenesis-related functional annotations. Neural progenitor cells have previously been identified in the adult human hippocampus (24); however, stress can inhibit cell proliferation and neurogenesis in this brain region (reviewed in ref. 25), and recent work suggests that adult neurogenesis may be regulated by components of the immune system (reviewed in ref. 26). Thus, immune dysfunction among persons with PTSD may be influenced by epigenetic profiles that are suggestive of immune activation or enhancement and also by an absence of epigenetic profiles that would be consistent with the development of normal neural-immune interactions (27).

Among the genes uniquely methylated in the PTSD-affected group, it is striking that the second most enriched cluster—sensory perception of sound—directly reflects one of the three major symptom clusters that define the disorder (Fig. 3B). Genes in this FAC thatmay be particularly salient to this symptom domain include otospiralin (OTOS),which shows decreased expression in guinea pigs after acoustic stress (28) and otoferlin (OTOF), mutations in which have been linked to nonsyndromic hearing loss in humans (29). Exaggerated acoustic startle responses, often measured via heart rate or skin conductance after exposure to a sudden, loud tone, have been well documented among the PTSD affected (30) and are indicative of a hyperarousal state that characterizes this symptom domain. Notably, prospective studies have demonstrated that an elevated startle response is a consequence of having PTSD, because the response was not present immediately after exposure to trauma but developed with time among trauma survivors who developed the disorder (30, 31).

My son had some very severe auditory related problems earlier in his life, and still occasionally struggles with either sudden loud noises, or some very specific noises, such as some dog barks, or the sound of an infant crying.  Previously the only physiology based attempt at an explanation I’d heard of for this type of response involved fine grained brain architecture and consequent filtering and/or overexcitation problems.  The idea that sound sensitivities in particular can be obtained environmentally is of particular interest to the autism community.

From the common sense angle, I find this completely fascinating; we’ve known for a long time that living with consistent stress is bad for you with a variety of nasty endpoints, but this type of finding narrows down the means by which this happens.  In the far off future, perhaps targeted methyl affecting drugs could be considered for people who experience extremely stressful events, as sort of a ‘PTSD vaccine’ [hehe] could be developed.

From an ASD perspective, increased feeling of anxiety, or just generally being ‘stressed out’ is a consistent finding both in research and from what I’ve read of readings from people with autism on the Internet.  I’ve seen several explanations, with sensory based problems being mentioned several times.  From a biological standpoint we seem to have a growing body of evidence of an abnormally regulated stress response in the autism cohort.  An internet friend of mine, Loftmatt, has written extensively on his thoughts concerning the increase in stress in modern society and the mechanisms by which this could be contributing to our apparent observations of an increase in autism.   This study would seem to provide insight towards a possible mechanism by which a frequent state of stress could lead to some of our immunological findings in the autism realm; a possibility I hadn’t considered previously when trying to detangle a means by which our observations of immune activation were not participating in autistic behavior.    The thought of a feedback loop also strikes me looking at this, something causes a feeling of extreme stress, which leads to abnormal methylation levels and genetic expression, which leads to increased physiological (and behavioral?) alterations, and even more stress.

I may try to poke through the supplementary materials to see if any specific genes or pathways found to be differentially regulated have parallels in some of the other studies we’ve seen recently such as Garbett or Hu, although this may be somewhat of a crapshoot unless I could figure out how to actually submit gene lists to GSEA and read the responses.

And we may need to consider the possibility that these types of effects can be trans-generational.  One of the most fascinating studies I’ve seen on epigentics involved exactly that, a multi-generational effect of famine in Holland, wherein the grandchildren of women who were pregnant during a time of famine bore striking differences in a variety of endpoints compared to children whose grandmothers were not pregnant during that time.

The more we learn, the more complicated the world becomes.

-pD

Hello friends –

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. 

–      pD

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.

From Wikipedia:

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;[1] instead, non-genetic factors cause the organism’s genes to behave (or “express themselves”) differently.[2]

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.

– pD

Note: Updated link to PZ.


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