Archive for the ‘Intriguing’ Category
Implications for Autism Or Just Interesting? “Epigenetic and immune function profiles associated with posttraumatic stress disorder”
Posted on: June 25, 2010
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:
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).
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
Confusing and Fascinating Findings – Gestational Age at Delivery and Special Educational Need
Posted on: June 11, 2010
Hello friends –
I ran across this one on accident the other day (why wasn’t it in one of my pubmed alerts?):
Background
Previous studies have demonstrated an association between preterm delivery and increased risk of special educational need (SEN). The aim of our study was to examine the risk of SEN across the full range of gestation.
Methods and Findings
We conducted a population-based, retrospective study by linking school census data on the 407,503 eligible school-aged children resident in 19 Scottish Local Authority areas (total population 3.8 million) to their routine birth data. SEN was recorded in 17,784 (4.9%) children; 1,565 (8.4%) of those born preterm and 16,219 (4.7%) of those born at term. The risk of SEN increased across the whole range of gestation from 40 to 24 wk: 37–39 wk adjusted odds ratio (OR) 1.16, 95% confidence interval (CI) 1.12–1.20; 33–36 wk adjusted OR 1.53, 95% CI 1.43–1.63; 28–32 wk adjusted OR 2.66, 95% CI 2.38–2.97; 24–27 wk adjusted OR 6.92, 95% CI 5.58–8.58. There was no interaction between elective versus spontaneous delivery. Overall, gestation at delivery accounted for 10% of the adjusted population attributable fraction of SEN. Because of their high frequency, early term deliveries (37–39 wk) accounted for 5.5% of cases of SEN compared with preterm deliveries (<37 wk), which accounted for only 3.6% of cases.
Conclusions
Gestation at delivery had a strong, dose-dependent relationship with SEN that was apparent across the whole range of gestation. Because early term delivery is more common than preterm delivery, the former accounts for a higher percentage of SEN cases. Our findings have important implications for clinical practice in relation to the timing of elective delivery
[Full paper from link. Emphasis is mine]
Essentially the authors evaluated gestational lengths with a fine tooth comb to discern if ‘early’, though not technically ‘pre-term’ delivery was associated with a ‘special education need’ (SEN), which in this case embodies a range of developmental problems including dyslexia, autism, or even physical problems like deafness or vision problems.
What the authors found was that there were subtle, but real effects in the likelyhood of having a special education need for non full term births that was dose dependent, but even included children that would not necessarily be considered early by existing standards.
Our study demonstrated a strong trend of decreasing risk of SEN with advancing gestational age at birth. The key finding of the present analysis is that this trend continued across gestational ages classified as term. Although the risk of SEN was highest among infants who were delivered preterm (<37 wk gestation), these accounted for only 5.1% of deliveries. Therefore, only a relatively small proportion of SEN (3.5%) could be attributed to preterm delivery. By contrast, 39.6% of infants were delivered between 37 and 39 wk gestation. Therefore, whilst these early term infants had only a moderately increased risk, 5.3% of SEN cases could be attributed to early term delivery.
The authors claim that the finding of effects at early, but not pre-term gestational lengths is one that is largely missing from existing studies, which have not taken these date ranges into consideration, or the ones that did, were not studying for cognitive problems, and indeed, excluded children with these criteria. Curiously, they also report an increase in SEN in children who had extra gestational periods, i.e., > 41 weeks in some studies.
The authors make absolutely no speculation as to what might be driving increased special education needs as the result of premature or early birth.
Looking at their results, one of the most striking things is that the impact did not alter if elective (i.e. C-Section) versus non-elective births were used as a variable. But this has deep ramifications for the autism storyline, which holds that if there are environmental factors that can contribute to autism, they are prenatal, and indeed, are often thought to involve insults very early in the prenatal period. In this case, we know that a genetic or environmental force isn’t contributing to the early birth, because it didn’t matter if the birth was spontaneous or not. The only area for an effect is postnatal. That is a big, big difference in the narrative.
Is this a matter of some just in time epigenetic programming happening in the womb that doesn’t get a chance to finish up in early births? Alternatively it could be that early birth allows for environmental exposures that the infant is not quite prepared to deal with. Or it could be both, or neither, or an illusory finding, but if these findings can be replicated, it raises a lot of questions about the sacred line between prenatal and postnatal environmental influences.
Unfortunately, the raw data for this project doesn’t seem to be available online; it might be really nice to see if there were patterns to be observed had particular salience to our population of interest.
– pD
Fascinating Study – Immune transcriptome alterations in the temporal cortex of subjects with autism
Posted on: June 7, 2010
Hello friends –
I’ve been referencing this paper in some discussions online for a while; I’ve read it, and in fact, while working on another project, got the opportunity to speak with one of the authors of the paper. It’s a very cool paper with a lot of information in it, some of which, could be considered inconvenient findings. Here is the abstract:
Immune transcriptome alterations in the temporal cortex of subjects with autism
Autism is a severe disorder that involves both genetic and environmental factors. Expression profiling of the superior temporal gyrus of six autistic subjects and matched controls revealed increased transcript levels of many immune system related genes. We also noticed changes in transcripts related to cell communication, differentiation, cell cycle regulation and chaperone systems. Critical expression changes were confirmed by qPCR (BCL6, CHI3L1, CYR61, IFI16, IFITM3, MAP2K3, PTDSR, RFX4, SPP1, RELN, NOTCH2, RIT1, SFN, GADD45B, HSPA6, HSPB8and SERPINH1). Overall, these expression patterns appear to be more associated with the late recovery phase of autoimmune brain disorders, than with the innate immune response characteristic of neurodegenerative diseases. Moreover, a variance-based analysis revealed much greater transcript variability in brains from autistic subjects compared to the control group, suggesting that these genes may represent autism susceptibility genes and should be assessed in follow-up genetic studies.
(emphasis is mine) [Full paper freely available from that link]
I am particularly intrigued by the second bolded sentence regarding the “these expression patterns appear to be more associated with the later recovery phase of autoimmune brain disorders, than the innate immune response characteristic of neurodegenerative diseases”. I’ve had it put to me previously that we should not necessarily implicate neuroinflammation in autism, the argument being that even though we had evidence of chronically activated microglia, what we do not seem to have evidence for is actual damage to the brain, and ergo, the neuroinflammation may actually be a byproduct of having autism, as opposed to playing a causative role, or that in fact, the neuroinflammation might even be beneficial. There have been some other places where the claim has been made that because our profile of neuroinflammation doesn’t match more classically recognized neurodegenerative disorders (i.e., MS/Alzheimer’s/Parkinson’s), that therefore, certain environmental agents need not be fully investigated as a potential contributor to autism. This is the first time that I am aware that someone has attempted to classify the neuroinflammatory pattern observed in autism not only as distinctly different from classical neurodegenerative diseases, but to also go so far as to provide a more refined example.
From the Introduction:
In order to better understand the molecular changes associated with ASD, we assessed the transcriptome of the temporal cortex of postmortem brains from autistic subjects and compared it to matched healthy controls. This assessment was performed using oligonucleotide DNA microarrays on six autistic-control pairs. While the sample size is limited by the availability of high-quality RNA from postmortem subjects with ASD, this sample size is sufficient to uncover robust and relatively uniform changes that may be characteristic of the majority of subjects. Our study revealed a dramatic increase in the expression of immune system-related genes. Furthermore, transcripts of genes involved in cell communication, differentiation, cell cycle regulation and cell death were also profoundly affected. Many of the genes altered in the temporal cortex of autistic subjects are part of the cytokine signaling/regulatory pathway, suggesting that a dysreactive immune process is a critical driver of the observed ASD-related transcriptome profile.
I was initially very skeptical about this, with a sample set so small, wasn’t it difficult to ascertain if their findings were by chance or not? It turns out, the answer depends on the type of datapoint you are evaluating against. A powerful tool in use by the researchers is a recent addition to the genetic analysis research suite, not only the ability to scan for thousands of gene activity levels simultaneously, but the use of known gene networks to identify if among those thousands of results, related genes are being expressed differentially. This is important for some amazingly robust findings presented later in the paper, so lets sidetrack a little bit. Here is a nice overview of the process being used:
Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. A common approach involves focusing on a handful of genes at the top and bottom of L (i.e., those showing the largest difference) to discern telltale biological clues. This approach has a few major limitations.
(i) After correcting for multiple hypotheses testing, no individual gene may meet the threshold for statistical significance, because the relevant biological differences are modest relative to the noise inherent to the microarray technology.
(ii) Alternatively, one may be left with a long list of statistically significant genes without any unifying biological theme. Interpretation can be daunting and ad hoc, being dependent on a biologist’s area of expertise.
(iii) Single-gene analysis may miss important effects on pathways. Cellular processes often affect sets of genes acting in concert. An increase of 20% in all genes encoding members of a metabolic pathway may dramatically alter the flux through the pathway and may be more important than a 20-fold increase in a single gene.
(iv) When different groups study the same biological system, the list of statistically significant genes from the two studies may show distressingly little overlap (3).
So, back to Garbett, not only did the authors find a great number of genes overexpressed in the autism group (and a smaller number, underexpressed), when they threw their thousands of results of individual genes into the GSEA, what came back was that several genetic pathways were very significantly altered, many of them immune mediated. This is a big step in understanding in my opinion. I believe we have likely come full circle on our understanding of very high penetrance genes that might be driving towards a developmental trajectory of autism; i.e., Rhett, Fragile-X. But using this technique we can determine if entire biological pathways are altered by measuring the output of genes. Specifically the point made in bullet (iii) stands out to me; having a twenty fold increase in a single gene might not be too big a deal if the other participants in the proteins function cannot be altered by twenty fold as a result due to other rate limiting constraints; but if we can see related sets of genes with similar expression profiles, we can get a much better picture of the biological results of different expression.
The methods get dense pretty quickly, but are worth a shot to show how thorough the researchers were to insure that their findings were likely to be signficant. Essentially they performed three different statistical tests against their results of differentially expressed genes and broke their results down into genes that passed all three tests, two of three test, or one of three tests. Furthermore, a selected twenty genes were targeted with qPCR validation, which in all cases showed the expected directionality; i.e., if the expression was increased in the transcriptome analysis, qPCR analysis confirmed the increased expression.To provide another benchmark, they tested for other genes known to be associated with autism, REELN, and GFAP and found results consistent with other papers.
Having determined a large number of differentially expressed genes, the authors then went to try to analyze the known function of these genes.
These classifications were performed on a selected gene set that is differentially expressed between AUT and CONT subjects; based on the success of our qPCR validation, we decided to perform this analysis using transcripts that both reported an |ALR>1| and that reached p<0.05 in at least 2/3 statistical significance comparisons. Of 221 such transcripts, 186 had increased expression in AUT compared to CONT, while only 35 genes showed reduced expression in the AUT samples. We subjected these transcripts to an extensive literature search and observed that 72 out of 193 (37.3%) annotated and differentially expressed transcripts were either immune system related or cytokine responsive transcripts (Supplemental Material 2). Following this first classification, we were able to more precisely sub-classify these 72 annotated genes into three major functional subcategories, which overlap to a different degree; 1) cell communication and motility, 2) cell fate and differentiation, and 3) chaperones (Figure 3). The deregulation of these gene pathways might indicate that the profound molecular differences observed in the temporal cortex of autistic subjects possibly originate from an inability to attenuate a cytokine activation signal.
That last sentence packs a lot of punch for a couple of reasons. It would seem to be consistent with their statements regarding a “late recovery phase” of an autoimmune disorder; i.e., an immune response was initiated at some point in the past, but has yet to be completely silenced. This also isn’t the first time that the idea of problems regulating an immune response (i.e., the inability to attenuate a cytokine activation signal) has been suggested from clinical findings, for example, in Decreased transforming Growth Factor Beta1 in Autism: A Potential Link Between Immune Dysregulation and Impairment in Clinical Behavioral Outcomes, the authors found an inverse correlation between TGF-Beta1 and autism behavioral severity:
Given that a major role of TGFβ1 is to control inflammation, the negative correlations observed for TGFβ1 and behaviors may suggest that there is increased inflammation and/or ongoing inflammatory processes in subjects that exhibit higher (worse) behavioral scores.
As such, TGFβ has often been considered as one of the crucial regulators within the immune system and a key mediator in the development of autoimmune and systemic inflammation.
In summary, this study demonstrates that there is a significant reduction in TGFβ1 levels in the plasma of young children who have ASD compared with typically developing children and with non-ASD developmentally delayed controls who were frequency-matched on age. Such immune dysregulation may predispose to the development of autoimmunity and/or adverse neuroimmune interactions that could occur during critical windows in development.
[full paper from the link]
The theme of a critical window of development and enduring consequences of insults during that window is one that is getting more and more attention recently; this is an area that is going to get more and more attention as time goes by, and eventually, as the clinical data continues to pile up, meaningless taglines aren’t going to be enough to keep us from dispassionately evaluating our actions.
The Discussion section is particularly nice, I’ll try not to just quote the entire thing. Here are the really juicy parts.
The results of our study suggest that 1) in autism, transcript induction events greatly outnumbers transcript repression processes; 2) the neocortical transcriptome of autistic individuals is characterized by a strong immune response; 3) the transcription of genes related to cell communication, differentiation and cell cycle regulation is altered, putatively in an immune system-dependent manner, and 4) transcriptome variability is increased among autistic subjects, as compared to matched controls. Furthermore, our study also provides additional support for previously reported involvement of MET, GAD1, GFAP, RELN and other genes in the pathophysiology of autism. While the findings were obtained on a limited sample size, the statistical power, together with the previously reported postmortem data by other investigators suggest that the observed gene expression changes are likely to be critically related to the pathophysiology seen in the brain of the majority of ASD patients.
There is some description of studies using gene expression testing in the autism realm where the authors ultimately conclude that technical and methodological differences between the studies make them difficult to tie together coherently. There is another small section re-iterating the findings that were similar to single gene studies; i.e., REELN, MET, and GAD genes.
The most prominent expression changes in our dataset are clearly related to neuroimmune disturbances in the cortical tissue of autistic subjects. The idea of brain inflammatory changes in autism is not novel; epidemiological, (DeLong et al., 1981; Yamashita et al., 2003; Libbey et al., 2005) serological studies (Vargas et al., 2005; Ashwood et al., 2006) and postmortem studies (Pardo et al., 2005; Vargas et al., 2005; Korkmaz et al., 2006) over the last 10 years have provided compelling evidence that immune system response is an essential contributor to the pathophysiology of this disorder (Ashwood et al. 2006). Finally, converging post-mortem assessments and measurements of cytokines in the CSF of autistic children (Vargas et al., 2005), may indicating an ongoing immunological process involving multiple brain regions
Nothing really new here to anyone that is paying attention, but good information for the extremely common, gross oversimplification that ‘immune abnormalities’ have been found in autism, but we don’t have any good reason to think they may be part of the problem. Of course, this is an argument you’ll see a lot of the time regarding everyone’s favorite environmental agent.
Altered immune system genes are often observed across various brain disorders, albeit there are notable differences between the observed transcriptome patterns. The majority of neuroimmune genes found activated in the autistic brains overlap with mouse genes that are activated during the late recovery or “repair” phase in experimental autoimmune encephalomyelitis (Baranzini et al., 2005). This suggests a presence of an innate immune response in autism. However, the altered IL2RB, TH1TH2, and FAS pathways suggest a simultaneously occurring, T cell-mediated acquired immune response. Based on these combined findings we propose that the expression pattern in the autistic brains resembles a late stage autoimmune event rather than an acute autoimmune response or a non-specific immune activation seen in neurodegenerative diseases. Furthermore, the presence of an acquired immune component could conceivably point toward a potential viral trigger for an early-onset chronic autoimmune process leading to altered neurodevelopment and to persistent immune activation in the brain. Interestingly, recently obtained gene expression signatures of subjects with schizophrenia (Arion et al., 2007) show a partial, but important overlap with the altered neuroimmune genes found here in autism. These commonly observed immune changes may represent a long-lasting consequence of a shared, early life immune challenge, perhaps occurring at different developmental stages and thus affecting different brain regions, or yielding distinct clinical phenotypes due to different underlying premorbid genetic backgrounds.
The last sentence, regarding ‘long-lasting’ consequences of early life immune challenges is one that has a large, and growing body of evidence in the literature that report physiological and behavioral similarities to autism. We also have recent evidence that hospitilization for viral or bacterial infection during childhood is associated with an autism diagnosis. There is, of course, a liberal sprinkling of ‘mays’, ‘propose’, and ‘conceivably’ caveats in place here.
Earlier I mentioned that the authors studied gene networks in addition to single gene expressions., and that some of those findings were very significant. The results of this are found in Table 2. In one discussion, I had it pointed out to me by ScienceMom that it appeared that some of the networks were not found to be statistically significant (and ergo we should not necessarily assume that immune dysfunction was a participant in autism). [If you look at Table 2, some networks like a p value of 0000]. I decided to use the data in this paper for another project that isn’t ready yet, but in that process I was able to speak directly with one of the authors of this paper. I asked him about this, and he told me that this was a function of space limitations; all of the gene networks described were found to be statistically signficant, but in some instances there wasn’t enough space to typeset the p value. In fact, some networks were found to be differentially expressed with a p-value of .000000000000001. (!!!!!!!!) That isn’t a value that you see very often.
I recently got a copy of Mitochondrial dysfunction in Autism Spectrum Disorders: cause or effect, which shares an author with this paper, Persico. In that paper, they reference Immune Transcriptome Alterations In the Temporal Cortex of Subjects With Autism, invoking a potential cascade effect of prenatal immune challenge, inherited calcium transport deficiencies, and resultant mitochondrial dysfunction that could lead to autism. I’ve generally stayed away from the mitochondria stuff in the discussion realm; even though I think it is probably somewhat important to some children, and critically important to a select few children, I’ve mostly found that the discussion of mitochondrial issues is comprised of two sets of people talking past one another so as to prove something, or disprove something about everyones favorite environmental agent; but this is a neat paper that I’d like to get to eventually.
– pD
Conclusions that are either correct, wrong, or somewhat in the middle; does childhood hospitilization for infection increase the risk for autism?
Posted on: May 13, 2010
Hello friends –
The abstract for Association of hospitalization for infection in childhood with diagnosis of autism spectrum disorders: a Danish cohort study hit my inbox the other morning. Here is the abstract
OBJECTIVE: To investigate the association between hospitalization for infection in the perinatal/neonatal period or childhood and the diagnosis of autism spectrum disorders (ASDs). DESIGN: A population-based cohort study. SETTING: Denmark. PARTICIPANTS: All children born in Denmark from January 1, 1980, through December 31, 2002, comprising a total of 1 418 152 children. EXPOSURE: Infection requiring hospitalization. MAIN OUTCOME MEASURE: The adjusted hazard ratio (HR) for ASDs among children hospitalized for infection compared with other children. RESULTS: A total of 7379 children were diagnosed as having ASDs. Children admitted to the hospital for any infectious disease displayed an increased rate of ASD diagnoses (HR, 1.38 [95% confidence interval, 1.31-1.45]). This association was found to be similar for infectious diseases of bacterial and viral origin. Furthermore, children admitted to the hospital for noninfectious disease also displayed an increased rate of ASD diagnoses (HR, 1.76 [95% confidence interval, 1.68-1.86]), and admissions for infection increased the rate of mental retardation (2.18 [2.06-2.31]). CONCLUSIONS: The association between hospitalization for infection and ASDs observed in this study does not suggest causality because a general association is observed across different infection groups. Also, the association is not specific for infection or for ASDs. We discuss a number of noncausal explanatory models
[Emphasis is mine.]
Considering my interest in early life immune activation, and the often difficult to predict, persistent outcomes from a variety of animal models, this study immediately struck me as an interesting one. The authors graciously sent my real world inbox a copy of this paper, as well as a similar one involving maternal infection during pregnancy, which I have yet to read.
Anyways, what strikes me very clearly here is that the authors and I have reached exactly the opposite conclusions towards the potential of a casual link between autism and hospitalization for infection in the perinatal / infancy periods. They apparently feel that the fact that an association is observed across different infectious agents (i.e., bacterial or viral), that this argues against a causal mechanism. But, as I have detailed in A Brief History of Early Life Immune Challenges and Why They (Might) Matter, we have an increasing number of animal studies that indicate that spikes in innate immune system cytokines during critical developmental timeframes can have, perverse and often baffling effects that we are only beginning to understand. Most of this research is brand new, within the past three years, and solely in the realm of animal models. However, the critical component of these studies that the Denmark study fails to take into consideration is that the innate immune response will be initiated regardless if the stimulant is viral or bacterial in nature. That is to say, the evidence from these studies tells us that the fact that we are observing differences across bacterial or viral pathogens is not necessarily an indication of lack of effect, but rather, could instead point towards a global effect, one that happens in both instances; surges in pro-inflammatory cytokines from the innate immune response.
For an example of some of these animal models, we could look to Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats, which observed a tnf-alpha driven, time dependent mechanism that ‘increases seizure susceptibility in adult rats’.
There are critical postnatal periods during which even subtle interventions can have long-lasting effects on adult physiology. We asked whether an immune challenge during early postnatal development can alter neuronal excitability and seizure susceptibility in adults. Postnatal day 14 (P14) male Sprague Dawley rats were injected with the bacterial endotoxin lipopolysaccharide (LPS), and control animals received sterile saline. Three weeks later, extracellular recordings from hippocampal slices revealed enhanced field EPSP slopes after Schaffer collateral stimulation and increased epileptiform burst-firing activity in CA1 after 4-aminopyridine application. Six to 8 weeks after postnatal LPS injection, seizure susceptibility was assessed in response to lithium–pilocarpine, kainic acid, and pentylenetetrazol. Rats treated with LPS showed significantly greater adult seizure susceptibility to all convulsants, as well as increased cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. These persistent increases in seizure susceptibility occurred only when LPS was given during a critical postnatal period (P7 and P14) and not before (P1) or after (P20). This early effect of LPS on adult seizures was blocked by concurrent intracerebroventricular administration of a tumor necrosis factor (TNF) antibody and mimicked by intracerebroventricular injection of rat recombinant TNF. Postnatal LPS injection did not result in permanent changes in microglial (Iba1) activity or hippocampal cytokine [IL-1β (interleukin-1β) and TNF] levels, but caused a slight increase in astrocyte (GFAP) numbers. These novel results indicate that a single LPS injection during a critical postnatal period causes a long-lasting increase in seizure susceptibility that is strongly dependent on TNF.
Another, very similar study, Viral-like brain inflammation during development causes increased seizure susceptibility in adult reports:
Viral infections of the CNS and their accompanying inflammation can cause long-term neurological effects, including increased risk for seizures. To examine the effects of CNS inflammation, we infused polyinosinic:polycytidylic acid, intracerebroventricularly to mimic a viral CNS infection in 14 day-old rats. This caused fever and an increase in the pro-inflammatory cytokine, interleukin (IL)-1beta in the brain. As young adults, these animals were more susceptible to lithium-pilocarpine and pentylenetetrazol-induced seizures and showed memory deficits in fear conditioning. Whereas there was no alteration in adult hippocampal cytokine levels, we found a marked increase in NMDA (NR2A and C) and AMPA (GluR1) glutamate receptor subunit mRNA expression. The increase in seizure susceptibility, glutamate receptor subunits, and hippocampal IL-1beta levels were suppressed by neonatal systemic minocycline. Thus, a novel model of viral CNS inflammation reveals pathophysiological relationships between brain cytokines, glutamate receptors, behaviour and seizures, which can be attenuated by anti-inflammatory agents like minocycline.
If we look closely here, we can see that either viral or bacterial mimics were able to generate similar physiological outcomes, outcomes that have strong correlations to the autism realm, namely increased rates of epilepsy, associations with seizures during infancy, and abnormal EEGs. But importantly for the decision tree in the case of childhood infections in the studies above, taken together, we can see that it didn’t matter if the trigger was bacterial or viral, just that there was an innate immune response at all. This is further evidenced by the fact that in both instances, different anti-inflammatory agents were capable of attenuating the changes. Our mechanism of action does not mandate pathogen specific interactions, in many cases, the cut off is whether or not you generate an innate immune response or not, regardless of the specific trigger. Another way of putting this would be, if an immune response for any pathogen were capable of initiating an cascade responsible for development of autistic behaviors, what would a pattern of hospitalization look like? [Children admitted to the hospital for any infectious disease displayed an increased rate of ASD diagnoses (HR, 1.38 [95% confidence interval, 1.31-1.45]). This association was found to be similar for infectious diseases of bacterial and viral origin.]
If you ask the wrong question even the right answer might not be useful in understanding a mystery.
All that being said, I have begun to see why Denmark makes such an attractive location for this kind of study. They have amassed an impressive set of data that could yield important clues if we can use it wisely.
I also noted that there is a P. Thorsen listed. I, for one, could care less.
– pD
Intriguing Findings – Maternal Obesity, Inflammation, and Consequent Priming of Microglia, Immune Alterations, and Spatial Processing in Offspring (!)
Posted on: May 4, 2010
Hello friends –
I’ve been forced to modify my pubmed alerts so that I don’t miss abstracts like this:
Enduring consequences of maternal obesity for brain inflammation and behavior of offspring
Obesity is well characterized as a systemic inflammatory condition, and is also associated with cognitive disruption, suggesting a link between the two. We assessed whether peripheral inflammation in maternal obesity may be transferred to the offspring brain, in particular, the hippocampus, and thereby result in cognitive dysfunction. Rat dams were fed a high-saturated-fat diet (SFD), a high-trans-fat diet (TFD), or a low-fat diet (LFD) for 4 wk prior to mating, and remained on the diet throughout pregnancy and lactation. SFD/TFD exposure significantly increased body weight in both dams and pups compared to controls. Microglial activation markers were increased in the hippocampus of SFD/TFD pups at birth. At weaning and in adulthood, proinflammatory cytokine expression was strikingly increased in the periphery and hippocampus following a bacterial challenge [lipopolysaccharide (LPS)] in the SFD/TFD groups compared to controls. Microglial activation within the hippocampus was also increased basally in SFD rats, suggesting a chronic priming of the cells. Finally, there were marked changes in anxiety and spatial learning in SFD/TFD groups. These effects were all observed in adulthood, even after the pups were placed on standard chow at weaning, suggesting these outcomes were programmed early in life.
WOW. [All emphasis is mine]
You may note that there isn’t any mention of autism per se here, but we do seem to hit a lot of sweet spots that immediately grabbed my attention for a couple of reasons. While my primary persona as Some Jerk On The Internet is a self appointed autism investigator, somewhere along the line in real life I’ve been trying some relatively strange (for the US) dietary practices; a ‘veganesque’ ingredient selection and the move to a diet based on whole foods, organic when possible. What I’ve noticed during this timeframe is just how fat so many Americans are. The obesity epidemic is real, folks, and doesn’t have the fuzzy nature of ‘increased awareness’ to allow us to (pretend) hope that there isn’t something real happening; we have been getting fatter and fatter for the past few decades. And here we have evidence that obesity can create physiological and behavioral changes in offspring through our mediator de jour, inflammation.
So, why am I blogging about this paper on an autism blog? Creepily enough, a lot of the differences listed here (well, all of them, actually), have similarities to findings in the autism realm.
At weaning and in adulthood, proinflammatory cytokine expression was strikingly increased in the periphery and hippocampus following a bacterial challenge [lipopolysaccharide (LPS)] in the SFD/TFD groups compared to controls.
With human subjects, it is a bit problematic to determine if there are ‘striking increases’ in proinflammatory cytokine expression in the hippocampus following bacterial challenge, but in vitro, we have scads of evidence that the autism population creates an exaggerated innate immune response when compared to ‘normal’. The most recent example of this is, Differential monocyte responses to TLR ligands in children with autism spectrum disorders, by Enstrom, which I also blogged about. We also have Ashwood, and several by Jyonouchi showing similar findings; increased production of proinflammatory cytokines TNF-alpha, IL-6, and IL1-B to some TLR agonists, including TLR4.
Microglial activation within the hippocampus was also increased basally in SFD rats, suggesting a chronic priming of the cells.
Of course, the seminal paper in this regard was Vargas, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism, which found increased levels of microglial activation in an autism cohort; their focus seemed to be areas other than the hippocampus. Similar findings of an ongoing immune response within the CNS in autism population can be found in Elevated immune response in the brain of autistic patients, and Immune transcriptome alterations in the temporal cortex of subjects with autism. The concept of ‘primed microglia’ is touched on in another paper by Bilbo, Early-life programming of later-life brain and behavior: a critical role for the immune system, which I’d like to get to eventually, but haven’t had the time for yet, but essentially suggests that there are time critical periods during which the microglia are vulnerable to persistent immunological modification, changing their resting state and response to future immune challenges.
Finally, there were marked changes in anxiety and spatial learning in SFD/TFD groups.
Oh yeah. Everyone has heard the story about the kid with autism who can paint New York City after a helicopter ride, or seeing colors in sound, or whatever, but there does also seem to be a lot of applied research involving specific kinds of visual tests that people with autism seem to do better at than people without. Curiously, we even have a knock out model in rodents that show superior spatial processing skills. Anyone who knows a couple of kids with autism knows one that has anxiety problems; there are some evaluations of this, but honestly, this type of thing suffers a bit from my mind because in order to ask the right question, ‘Are you anxious?’, you’ve eliminated a chunk of the autism population. If we break down to the chemical level and start looking at known biomarkers for the stress response, the HPA-Axis, we’ve got tons of evidence that something is amiss.
Here are some of the juicier parts of the paper. From the introduction:
Obesity and insulin resistance are also strongly linked to cognitive dysfunction, including Alzheimer’s disease (13, 14). Neuroinflammation is independently linked to cognitive disruption (15); brain IL-1 expression, in particular, is implicated in Alzheimer’s disease pathogenesis (16). However, a direct mechanism linking these diverse factors is lacking; that is, whether peripheral inflammation in obesity contributes directly to inflammation/ cytokine production in cognitive regions of the brain, and thus cognitive disruption, remains unclear. Moreover, whether maternal obesity may program inflammation within the brains of offspring long term, particularly in regions important for cognition, such as the hippocampus, is virtually unknown. Tozuka et al. (17) recently reported long-term impairments in neurogenesis within the dentate gyrus as a consequence of being born to obese mouse dams, although the researchers did not explore a potential role for inflammation. Notably, White et al. (18) reported increased glial activation and oxidative stress in the cortex of high-fat-diet-fed rats that were also born to high-fat dams. Glia are the primary immunocompetent cells of brain; thus, long-term changes in their activity as a consequence of diet could be critical in long-term programming of neural function.
The whole ‘long term programming of neural function’ theory is beautiful and terrifying. From the discussion section:
A central question in this study was to identify whether systemic inflammation is transferred to cognitive regions of the brain. The answer to that question is clearly yes, especially in the SFD rats. These animals exhibited increased peripheral cytokines (in liver, fat,and serum) and hippocampal IL-1 responses to an LPS challenge. At P20 and in adults, rats from SFD dams exhibited a very large increase in hippocampal IL-1 following a moderate dose of LPS (Fig. 4). Males exhibited a larger response than females in adulthood, although the diet effect was significant in both sexes. The exaggerated response in adults was particularly striking, given that these animals had been fed a low-fat diet since weaning, a period of time longer in duration (9 wk) than the total time they were exposed to the high-fat diets during development (6 wk). Notable as well was the increase in basal levels of IL-1 in high-fat diet groups in adulthood. These data indicate a basal shift in the expression of this cytokine.
It is almost as if being male provides risks every time they bother to look. Oh well. This would seem to be an illustration of ‘long term programming’, what happened during development was more important than what happened afterwards. There were some differences found in the transfat group compared to the saturated fat group, primarily observed in differences in neural and peripheral response to LPS; the authors theorize that this is related to the deposition locations of the different kinds of fats; i.e., saturated fats make it into the brain more easily than trans fats, which are stored in the liver (where they measured perfipheral inflammation).
We explored the influence of a trans-fat-rich vs. saturated-fat-rich diet independently in this study. This comparison yielded surprising findings as well, as the SFD appeared to be much more deleterious for body weight, leptin, and IL-1 compared to the TFD, especially in males. Conversely, CRP expression in the liver, a reliable risk factor for heart disease in humans, was significantly increased in the TFD groups following LPS compared to the SFD and LFD groups. It should be noted, however, that the dams consumed less of the TFD than of the SFD, suggesting it may have been less palatable, and therefore, induced fewer changes. Furthermore, the role of CRP as an acute-phase protein in rodents is controversial (52); support for this idea is the lack of increase in response to LPS in every diet group. Thus, the role of CRP in this experimental model, if any, remains to be further explored.
Remarkably, the authors found that low fat diet rats performed better in some tasks, and the authors speculate that a high fat diet may produce some cognitive gains.
A very intriguing possibility as well is that increased basal IL-1 in the high-fat-diet groups facilitated cognition. A growing body of evidence suggests a role for IL-1 in normal, nonpathological, synaptic plasticity mechanisms within the brain, including memory (44). IL-1 is critical for long-term potentiation (LTP) maintenance during learning (45, 46). However, exaggerated IL-1 within the brain is also strongly associated with memory impairment, providing support for an inverted-U function for optimal IL-1 and cognition(46, 47).
The inveterd U function is, I believe, similar to the concept of hormesis, wherein exposure to an agent and physiological response does not necessarily follow a straight linear response. A good example of this that may be the Pessah studies, which found that for some types of PCBs, low level exposure caused more problems than high levels of exposure. [Note: Beware of anyone who wants to use the ‘poison is in the dose’ cannard, which might be meaningful if the measurement endpoints are mortality, but increasingly less worthwhile if you want to measure subtle effects.]
Here is the closing paragraph:
In closing, it is clear that maternal high-fat diet has a profound influence on the innate immune response of the offspring, in both the periphery and the brain, and that this has enduring consequences for cognition and affect in both males and females. Future studies are needed to assess whether peripheral signals such as leptin vs. central targets such as microglia may be driving the responses in brain, and whether immune targeting (e.g., TLR4 signaling) may be sufficient to prevent exaggerated CNS inflammation in high-fatdiet-exposed pups.
Great idea at the end! I’d love to see a paper where they replicated these groups, but one group of rats also got fish oil or other anti-inflammatories to see if the effect of the inflammation was attenuated. TLR4 knockout mice might also be neat to see. This is a cool study that tells us just how much we still have to learn about how our choices can have very difficult to predict effects.
One of the authors of this paper, Staci Bilbo, has been on a bit of a tear lately regarding the effect of early life immune challenges and subsequent immune and behavioral differences in the treatment animals, including recent hits like Early-life infection is a vulnerability factor for aging-related glial alterations and cognitive decline, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain, and Early-life programming of later-life brain and behavior: a critical role for the immune system, all of which may have implications for everyone’s favorite environmental agent. I’ll be tackling those papers, and several others that have come out with similar methodologies soon enough, but the entire Frontline debacle has left me a little exhausted on the issue.
– pD
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 on: April 25, 2010
- In: Autism | BCL-2 | Brain | Epigenetics | Epigenome | Fatemi | Genetics | Hu | Immunology | Inflammation | Intriguing | Oxidative Stress | Purkinje | RORA | Some Jerk On The Internet | The Fairytale | Uncategorized
- 4 Comments
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
Neat Study: “Increased serum levels of high mobility group box 1 protein in patients with autistic disorder”
Posted on: March 27, 2010
- In: Autism | Immunology | Inflammation | Intriguing | Seizures | Toll Like Receptors | Uncategorized
- 1 Comment
Hello friends – The other day a pretty neat abstract hit my inbox: Increased serum levels of high mobility group box 1 protein in patients with autistic disorder
BACKGROUND: High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein that functions as an activator for inducing the immune response and can be released from neurons after glutamate excitotoxicity. The objective of the present study was to measure serum levels of HMGB1 in patients with autistic disorder and to study their relationship with clinical characteristics. METHODS: We enrolled 22 adult patients with autistic disorder (mean age: 28.1+/-7.7years) and 28 age- and gender-matched healthy controls (mean age: 28.7+/-8.1years). Serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with healthy subjects, serum levels of HMGB1 were significantly higher in patients with autistic disorder (10.8+/-2.6ng/mL versus 5.6+/-2.5ng/mL, respectively, P<0.001). After adjustment for potential confounders, serum HMGB1 levels were independently associated with their domain A scores in the Autism Diagnostic Interview-Revised, which reflects their impairments in social interaction. CONCLUSIONS: These results suggest that HMGB1 levels may be affected in autistic disorder. Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder.
I had not heard of “high mobility group box 1” before, but as being described as an ‘activator for inducing the immune response’, my interest was definitely piqued! The author, Emanuele Enzo, was extremely gracious in providing me a copy of his manuscript. Below are the juicy parts: From the introduction:
In recent years, many different mechanisms have been suggested to play a role in the pathophysiology of ASD, including impaired neurotransmission, genetic mutations, viral infections, gastrointestinal factors, and excitotoxicity (Levy et al., 2009; Rapin and Tuchman, 2008). Growing evidence has also suggested that inflammation (Cohly and Panja, 2005), neuroinflammation (Pardo et al., 2005), and oxidative stress (McGinnis, 2004) may be involved in the pathogenesis of ASD. High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein released from inflammatory cells that functions as a signal for inducing inflammation and as an activator for inducing the immune response (Klune et al., 2008; Bianchi and Manfredi, 2007). The action of extracellular HMGB1 appears to be dependent on interaction with several cell surface receptors, including toll-like receptors 2/4 (TLRs-2/4) (Yu et al., 2006) and the receptor for advanced glycation endproducts (RAGE) (Rauvala and Rouhiainen, 2007). RAGE is a member of the immunoglobulin superfamily of cell surface receptors that is activated by HMGB1 but also by advanced glycation end products and S100 proteins (Yan et al., 2009), all of which have been shown to be altered in autism (Boso et al., 2006; Junaid et al., 2004). In addition, HMGB1 seems to be released from neurons after glutamate excitotoxicity (Kim et al., 2006; Kim et al., 2008). [emphasis and links are mine]
Some familiar players here , namely, neuroinflammation [Vargas, Li, Garbett], and TLR2 and TLR4 [Engstrom, Jyounouci]. I read Klune this afternoon and it is a very good review paper regarding HMGB1, which essentially illuminates on its description as ‘a signal for inducing inflammation’ involved with TLR2 and TLR4. In it, HMGB1 is termed an ‘alarmin’, an endogenous immune adjuvant, or more plainly, a homegrown danger signal. There is mention of synergistic effects in promoting an inflammatory response in conjunction with tnf-alpha, a presence in autoimmunity, cancer and other nasty conditions, as well as potential restorative effects. Anyone who has been paying attention to the ‘abnormal immune response’ findings in autism is going to see a lot of crosstalk here. [Interested by the semantics, I would encourage readers to take a look at paperwork on resolvins as potential mediators of inflammation]. The RAGE stuff is another paper when Enzo is an author that I haven’t read yet, but mean to. From the results section:
After allowance for age, BMI, and Raven’s Progressive Matrices scores, we found a positive independent association between HMGB1 levels and the ADI-R Social Scores (HMGB1, the worse the social interaction β=0.39, t=2.81, P < 0.01, Fig. 2); the higher the serum level of HMBG1, the worse the social interaction.
Now, this is pretty interesting, because it is another instance where we observe a correlation between immunomodulators and autism severity; Grigorenko found that MIF, a known upregulator of the innate immune response, was positively associated with autism severity, and Ashwood found an inverse relationship between the immune regulating cytokine, TGF-Beta1 and autism severity. It would seem that by several measurements, a propensity towards an inflammatory state seems to be able to affect the degree of impairment.
From the very lightweight discussion section:
HMGB1 has been shown to function as a proinflammatory cytokine-like involved in both excitotoxicity (Kim et al., 2006) and glial activation (Pedrazzi et al., 2007) . Of note, growing evidence has suggested a pathophysiological role for excitotoxicity (Blaylock and Strunecka, 2009) and glutamatergic dysregulation (Blaylock, 09; Shinohe et al., 2006) in ASD. In addition, neuroinflammation may be an important feature in some patients with autistic disorder (Pardo et al., 2005; Vargas et al., 2005). Recently, Pedrazzi et al. (2007) have shown that HMGB1 promotes a specific proinflammatory program in primary astrocytes. Increased oxidative stress and immune dysregulation are other important feature in ASD (McGinnis, 2004; Cohly and Panja, 2005; Blaylock, 2009), and HMGB1 protein plays important roles in both processes (Bianchi and Manfredi, 2007; Klune et al., 2008). Interestingly, HMGB1 may induce a prooxidative state through interaction with its cell-surface receptor RAGE (Rauvala and Rouhiainen, 2007), a molecule previously implicated in the pathogenesis of ASD (Boso et al., 2006). An interesting observation in this study is that raised HMGB1 levels in our patient group were correlated with disturbances in social function as assessed with ADI–R, suggesting that this molecule may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder. This finding is intriguing, but needs to be confirmed with further studies.
You don’t see Blaylock get referenced too often, I need to read those papers, he does seem to have an online credibility problem that I can’t figure out. Anyways, the statement that HMGB1 and astrocytes is particularly interesting, because we can see from the seminal Vargas paper, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism that astrocytes were the primary producer of the increased cytokine IL-6 and chemokine MCP-1 in the brains of autistics. A link to increased oxidative stress doesn’t surprise me too much, though again, I haven’t read anything about RAGE, so seeing another pathway towards increased oxidative stress is a nice touch. There is a section on the weaknesses of the study including smaller study sizes and uncertainty towards the source of HMGB1. As always, there is a call for additional study.
Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1b (IL-1b), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy.
Unequal distributions of scary chemicals and (possible) implications for autism clusters, a static rate of autism, and why some of us may be more doomed than others
Posted on: March 12, 2010
- In: Autism | BPA | Impending Doom | Intriguing | PBDE | Pesticides | Prevalance | Some Jerk On The Internet | Synthetics | The Fairytale | The Pretzel
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Hello friends –
Recently there have been a few studies that tackled the issue of apparent autism clusters in California, The spatial structure of autism in California, 1993-2001, and Geographic distribution of autism in California: A retrospective birth cohort analysis. A nice overview and some discussion of these papers can be found at LBRB, here, and here. One of the arguments we see made there is that the rates of autism diagnosis are, in fact, a reflection of the available services in an area, as opposed to an actual difference in the number of children with autism; essentially that an undiagnosed child with autism who lives far from a center of autism services will not get a diagnosis, but a child born relatively close to such services, will be appropriately diagnosed. We are measuring diagnosis, as opposed to autism. I have no doubt that there is some validity to this, but have many doubts that we can, or should, assign all of our observed increases in autism as consequences of this type of artifact.
There have been several other studies that looked at things like mercury emissions, or airborne pollutants, or Superfund sites and autism rates at larger scales. However, on a macro level, these types of studies have, so far, been unable to design around a feature of reality; the likelihood that things like Superfund sites or airborne pollution are situated in relative proximity to an urban center, and as such, autism diagnosis services. In effect, the argument that these observations are diagnostic only is the same; without a controlling factor for diagnostic availability, we can not assume that other parameters are actually responsible. And again, I have no doubt that this is a force that contributes to the findings of these studies.
But.
At the end of the day, I’m just not satisfied with a God of the Gaps explanation; what we seem to be seeing is just too goddamned important to explain away with the spongy soft and ultimately unmeasurable forces of greater awareness et all. (The Fairytale, 20##).
Anyways, the other day pubmed alerted me to the publication of this interesting study:
BACKGROUND: Levels of brominated flame retardants are increasing in US populations, yet little data are available on body burdens of these and other persistent hormonally active agents (HAAs) in school-aged children. Exposures to such chemicals may affect a number of health outcomes related to development and reproductive function. OBJECTIVE: Determine the distribution of biomarkers of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and organo-chlorinated pesticides (OCPs), such as DDT/DDE, in children, and their variation by key descriptor variables. METHODS: Ethnically diverse cohorts of girls 6-8y old at baseline are being followed for growth and pubertal development in a multi-site, longitudinal study. Nearly 600 serum samples from the California and Ohio sites were analyzed for lipids, 36 PCB congeners, 11 PBDE congeners, and 9 OCPs. The biomarker distributions were examined and geometric means compared for selected analytes across categories of age, race, site, body mass index (BMI), parental education, maternal age at delivery, and breast feeding in adjusted models. RESULTS: Six PBDE congeners were detected among greater than 70% of samples, with BDE-47 having the highest concentration (median 42.2, range 4.9-855ng/g lipid). Girls in California had adjusted geometric mean (GM) PBDE levels significantly higher than girls in Ohio. Furthermore, Blacks had significantly higher adjusted GMs of all six PBDE congeners than Whites, and Hispanics had intermediate values. GMs tended to be lower among more obese girls, while other variables were not strongly associated. In contrast, GMs of the six PCB congeners most frequently detected were significantly lower among Blacks and Hispanics than Whites. PCBs and the three pesticides most frequently detected were also consistently lower among girls with high BMI, who were not breast-fed, whose mothers were younger, or whose care-givers (usually parents) were less educated. Girls in California had higher GMs than in Ohio for the pesticides and most PCB congeners, but the opposite for CB-99 and -118. CONCLUSIONS: Several of these potential HAAs were detected in nearly all of these young girls, some at relatively high levels, with variation by geographic location and other demographic factors that may reflect exposure pathways. The higher PBDE levels in California likely reflect differences in fire regulation and safety codes, with potential policy implications.
The environmental impact argument usually focuses on vaccines, or in some instances, similarly widespread environmental pollutants (i.e., mercury emissions); external forces which tend to operate more or less evenly across large geographic swaths, and also largely independent of things like culture or race. But with this paper we can observe the counter-intuitive opposite, chemicals that have achieved widespread distribution in society and the environment, seem to be bioaccumulating differentially according to factors such as geography, race, body type, and education levels. The paper here mentions fire regulation as a possible factor in state by state differences, but taking things a bit further, it can quickly be seen how socio-economic factors might play a role in why we might observe different levels of chemicals. It takes a lot of crazy chemistry to make a baby onesie not catch on fire, but at a high level, it involves dousing the material with a bunch of exotic chemicals. Politically correct or not the facts on the ground are that the well to do white woman has baby showers where she gets a bewildering array of freshly minted, ‘extra safe’ baby clothes more often than, say, the not so well to do Latina woman. We have already established a connection between having older parents and a diagnosis of autism, it would seem, there is also a correlation between having older parents and your bodies burden of these molecular mimics; and again, white women tend to have babies at later stages in life than their Black or Latina counterparts; especially the ones that happen to be residing near the trendy autism diagnosis hubs (i.e., the wealthier white women). The ability for these types of chemicals to cause a variety of difficult to predict developmental trajectories is too long, and terrifying to go into detail in this post; for purposes of this discussion, it is sufficient to understand that we have a growing body of evidence that endocrine disrupting compounds can have wide ranging effects; including epigenetic changes, changes in immune profiles, altered behaviors and neuroanatomical structures known to be abnormal in autism.
I found the finding of BDE-47 particularly intriguing, considering it was used as a primer for immunological response measurement by Ashwood, who found in vitro differences in immune responses in the autism population (an exaggerated innate immune response was observed).
Of course, this study does not present sufficient evidence for us to draw conclusions about the geographic distribution of autism rates in the two California studies above; but it should give us enough to pause before we take the comforting road out and assume that our observation are the result of diagnostic artifact alone; such assumptions feel good (except for the guilt), but ultimately require that we ignore our growing knowledge of how unpredictable endocrine disruptors affect the body, and how much more we have to learn.
– pD
passionless Droning about autism 
