passionless Droning about autism

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