The genetics of self-injurious behaviour accompanying autism? Not quite...

I'd like to start by making one thing abundantly clear about today's post: I am not insinuating that self-injurious behaviour (SIB) accompanying autism is solely under genetic (or epigenetic) control.

As I've discussed before on this blog, there are potentially many, many reasons why SIB under the umbrella of the so-called 'challenging behaviours' occurs (see here). As and when it does happen, the onus is on those significant others to turn detective before anyone immediately reaches for something like the anti-challenging behaviour meds (see here) or indeed makes any sweeping generalisations about it 'just being part of their autism'. I say this mindful that sometimes it can seemingly be the smallest things that can trigger such episodes...

The findings reported by Matthew Shirley and colleagues [1] (open-access) do however require some attention with the idea that certain genetic issues *might* "contribute to the etiology of SIB." The specific genetic issues under the research spotlight were copy number variants (CNVs) and authors were looking at quite a precise cohort of children/young adults diagnosed with "autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage" (N=14). I might add that CNVs with autism and learning disability in mind have some history (see here).

Based on quite a thorough work-up (including a functional behavioural assessment), researchers zoomed in on 4 children (29%) where they identified "a CNV likely to have a causal role" in SIB. I'm afraid my very limited knowledge of genetics precludes any critical discussion about the nature or role of any individual genetic issues reported but I might backtrack slightly based on something the authors write regarding 'causality': "the present findings are not able to indicate definitively that any of these variants is causal." Apparently we need to wait for more data from additional patients with the same/similar clinical phenotype before much more can be said on this issue. Indeed: "it is likely that exome or genome sequencing will greatly increase the diagnostic yield of the cohort we are studying."

Perhaps just as important as the question of whether genetics plays a role in a complex behaviour pattern like SIB are the authors' observations of what might have triggered SIB in their participants. The authors talk about the results of the very important functional behaviour assessments as revealing some common themes: "SIB was multiply maintained by escape from demands and access to preferred toys... SIB was multiply maintained by access to preferred foods and access to attention... Head-banging was found to be maintained by access to preferred foods... Head-hitting, self-biting, and head-banging against hard surfaces were observed to be maintained by automatic reinforcement." What these excerpts tell me is that SIB could potentially be a communicative act, bearing in mind details of language and communication 'level' of participants are fairly scant in the Shirley paper. As I've talked about previously (see here), issues such as fatigue and setting event are also potentially important parameters when getting to the bottom of SIB and other challenging behaviours.

The final question, and perhaps an important one when one realises just how extreme an effect SIB can exert (see here), is 'what can be done about reducing levels of SIB' when they present.  Well, working out the hows and whys of such behaviour should be the first strategy, and can sometimes yield impressive results. Although I suggested at the beginning of this post that the 'anti-challenging behaviour meds' should be a further-down-the-list resort, there is evidence that they can be helpful for some people in some situations assuming appropriate medicines management and monitoring for potential side-effects (see here). With no medical or clinical advice given or intended, I'd also be minded to direct readers to some research looking at adjuvant therapies such as the use of N-acetylcysteine (NAC) where issues like irritability might show some connection to SIB (see here and see here) or even something of particular interest to me, the use of naltrexone (see here). More research is indicated and indeed, quite a lot more with much greater participant numbers before anyone starts on about having identified the genetics of SIB in autism...

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[1] Shirley MD. et al. Copy Number Variants Associated with 14 Cases of Self-Injurious Behavior. PLoS ONE. 2016; 11: e0149646.

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Shirley, M., Frelin, L., López, J., Jedlicka, A., Dziedzic, A., Frank-Crawford, M., Silverman, W., Hagopian, L., & Pevsner, J. (2016). Copy Number Variants Associated with 14 Cases of Self-Injurious Behavior PLOS ONE, 11 (3) DOI: 10.1371/journal.pone.0149646

HERVs as a mechanism of genetic deletion formation: relevance to some autism?

My stark lack of knowledge in the area of genetics and specifically that linked to the human endogenous retroviruses (HERVs) that litter the genome is likely to shine through in this post so be ready with that pinch of salt.

The starting point for today's post is the paper by Ines Quintela and colleagues [1] detailing a case report of "a 9-year-old female patient with autistic disorder, total absence of language, intellectual disability, anxiety disorder and disruptive, and compulsive eating behaviors." Following some genetic analysis of this young girl researchers reported on "the identification of a de novo recurrent 3q13.2-q13.31 deletion encompassing 25 genes." This in itself is interesting and adds to a growing tide of research suggesting that there may be lots of different genetic influences acting in different cases of autism; all pertinent to a more plural view of the label: the autisms.

One sentence however took my specific interest in this paper insofar as: "a 3.4 Mb recurrently altered region at 3q13.2-q13.31 has been recently described and non-allelic homologous recombination (NAHR) mediated by flanking human endogenous retrovirus (HERV-H) elements has been suggested as the mechanism of deletion formation."

As I indicated at the start of this post, the finer details of genetics are not really my forte so be warned. I was however really interested in the suggestion that the process of NAHR - when "highly similar portions of the genome wrongly recombine, deleting and sometimes duplicating a portion of the genome that lies between them" - might be linked to the presence of all/some of those fossil viruses that make us who we are [2].

The long-and-short of it is the idea that some of those little variants that we ALL have in our genome might not be all due to just chance if described as de novo (as in not inherited from mum or dad). Take CNVs (copy number variants), small alterations to the genome characterised by gains and losses in segments of DNA (I think!), as the starting point. The idea is that the location of said CNVs in relation to HERVs and other transposable elements means that there may be some kind of relationship between the two. Preliminary research has suggested that HERVs flanking particular parts of the genome might be involved in the formation of CNVs [3]. Other research has noted this process as potentially being relevant in other case reports [4] similar to that described by Quintela and colleagues where behaviour and autism have been mentioned.

Just to make the whole process even more complicated (and interesting) is the idea that CNVs when talked about with autism in mind might tend to be concentrated in 'hypomethylated' regions of the genome [5]. This brings in the potentially important process of DNA methylation (yes, epigenetics yet again) into proceedings, made further interesting by suggestions that hypomethylation of DNA = more genomic instability [6] and that certain HERVs might also to some degree be 'kept in check' by methylation means [7]. Add in some evidence of methylation issues associated with some autism (see here) and preliminary evidence of certain HERVs expression correlating with autism (see here) and comorbidity (see here), and there is the making of some potentially important hypotheses ripe for further testing.

But please, don't take my word for it.

Music: Purple Rain - Prince.

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[1] Quintela I. et al. Female patient with autistic disorder, intellectual disability, and co-morbid anxiety disorder: Expanding the phenotype associated with the recurrent 3q13.2-q13.31 microdeletion. Am J Med Genet A. 2015 Aug 29.

[2] Nelson PN. et al. Demystified . . . Human endogenous retroviruses. Molecular Pathology. 2003;56(1):11-18.

[3] Campbell IM. et al. Human endogenous retroviral elements promote genome instability via non-allelic homologous recombination. BMC Biol. 2014 Sep 23;12:74.

[4] Shuvarikov A. et al. Recurrent HERV-H-mediated 3q13.2-q13.31 deletions cause a syndrome of hypotonia and motor, language, and cognitive delays. Hum Mutat. 2013 Oct;34(10):1415-23.

[5] Li J. et al. Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome. PLoS Genetics. 2012: 8: e1002692.

[6] Wilson AS. et al. DNA hypomethylation and human diseases. Biochimica et Biophysica Acta. 2007; 1775: 138–162.

[7] Lavie L. et al. CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2). J. Virol. 2005; 79: 876-883

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Quintela I, Gomez-Guerrero L, Fernandez-Prieto M, Resches M, Barros F, & Carracedo A (2015). Female patient with autistic disorder, intellectual disability, and co-morbid anxiety disorder: Expanding the phenotype associated with the recurrent 3q13.2-q13.31 microdeletion. American journal of medical genetics. Part A PMID: 26332054

Autism, CNVs and sensitivity to maternal infection?

An intriguing quote to begin today's post: "Our findings support a gene-environment interaction model of autism impairment, in that individuals with ASD-associated CNVs are more susceptible to the effects of maternal infection and febrile episodes in pregnancy on behavioral outcomes and suggest that these effects are specific to ASD [autism spectrum disorder] rather than to global neurodevelopment."

The findings come from the paper by Varvara Mazina and colleagues [1] who sought to "explore the impact of ASD-associated copy number variants (CNVs) and prenatal maternal infection on clinical severity of ASD." Based on "data from the Simons Simplex Collection sample including 1971 children with a diagnosis of ASD aged 4 to 18 years" who underwent genetic screening for the presence of CNVs, researchers cross-referenced genetic information with parent reported maternal infection and/or febrile episodes during pregnancy alongside autism severity in cases. They found something of a potentially important relationship: "individuals with CNVs and history of maternal infection demonstrated increased rates of social communicative impairments and repetitive/restricted behaviors."

Of course let us not forget that it is not beyond the realms of possibility that CNV load and maternal infection during pregnancy might just be a fluke finding when it comes to the presentation or severity of autistic traits. Indeed, outside of just looking at core autistic traits I'm taken to wonder whether other behaviours might show a similar relationship for example, based on some of the heightened comorbidity seemingly connected to autism such as attention-deficit hyperactivity disorder (ADHD) (see here).

That being said, I do think there is more to see in this "gene-environment interaction model" specifically focused on what could bridge maternal infection and CNV load in cases of autism. CNVs and autism have something of a research history albeit rather complicated (see here). Maternal infections during pregnancy and autism have a similarly interesting history too (see here). In a previous post, I've covered CNV load and autism focused on behavioural presentation (see here) and the idea that some of the genes linked to cases of autism might also affect immune function  (see here and see here) including response to infection.

Without also trying to speculate too much and bearing in mind the 'epigenetic' slant to the Mazina paper, I was wondering whether there may be additional merit in looking at something like the HERVs (human endogenous retroviruses) in future work in this area. I say this acknowledging the preliminary research forays that have already been taking into this area with autism (see here), ADHD (see here) and even myalgic encephalomyelits / chronic fatigue syndrome (ME/CFS) in mind (see here). HERVs by the way are perhaps best described as fossil viruses which we carry with us as badges of honour following our evolutionary war against such microscopic invaders (see here). The idea that HERVs could 'mediate' genetic variations for example, as per the findings reported by Shuvarikov and colleagues [2] is still quite a new one but interesting in light of the suggestion of 'reactivation' under certain circumstances including stress and/or infection [3]. The contribution of 'hypomethylation' to genomic instability [4] might also be important to such HERV research in light of the preliminary suggestions of methylation issues associated with cases of autism (see here).

But enough of me speculating (on a blog in a very non-peer reviewed manner). The findings from Mazina et al stand as testament to our very limited understanding of how genes and environment [variably] interact when it comes to autism but offer something of a new area ripe for lots more investigation...

Music: Ash and Girl from Mars.

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[1] Mazina V. et al. Epigenetics of Autism-related Impairment: Copy Number Variation and Maternal Infection. J Dev Behav Pediatr. 2015 Jan 27.

[2] Shuvarikov A. et al. Recurrent HERV-H-mediated 3q13.2-q13.31 deletions cause a syndrome of hypotonia and motor, language, and cognitive delays. Hum Mutat. 2013 Oct;34(10):1415-23.

[3] Schulz WA. et al. Methylation of endogenous human retroelements in health and disease. Curr Top Microbiol Immunol. 2006;310:211-50.

[4] Li J. et al. Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome. PLoS Genetics. 2012: 8: e1002692.

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Mazina V, Gerdts J, Trinh S, Ankenman K, Ward T, Dennis MY, Girirajan S, Eichler EE, & Bernier R (2015). Epigenetics of Autism-related Impairment: Copy Number Variation and Maternal Infection. Journal of developmental and behavioral pediatrics : JDBP PMID: 25629966

Common variation and the genetics of autism

The paper by Trent Gaugler and colleagues [1] reporting that the genetic architecture of the autism spectrum disorders (ASDs) seems in the most part to be due to "common variation" over and above "rare variants or spontaneous glitches" adds to the quite voluminous literature in this area.

Everything in proportion? @ Wikipedia 

Based on an analysis of "a unique epidemiological sample from Sweden" researchers looked at DNA variations in some 3000 individuals with autism and asymptomatic controls. They were able to model their findings "based mostly on combined effects of multiple genes and non-shared environmental factors" including some "synthesis of results from other studies".

Their results: "Most genetic risk for autism comes from common inherited gene variations that can be found in many individuals without the disorder" as per one write-up of the study results. Spontaneous mutations - those so-called de novo mutations which seem to be of growing interest to autism research - were reported to only 'modestly' increase risk of the condition (2.6% of the total risk). About 40% of the risk was unaccounted for, but combined with those common inherited gene variations, made up about 90% of the total risk or liability for ASD.

Quite a lot of the discussion about these results has focused on the issue of tiny genetic effects which many people not on the autism spectrum have present in their genome adding up into something with "substantial impact" when present together. Other research has hinted at similar things as for example, in the paper by St Pourcain and colleagues [2] looking at the genetics of social communication issues.

Whilst I do think that the Gaugler paper is an important one, I am minded to suggest a few words of caution. First and foremost is the reliance on observed genetic variation in the current paper. Although no expert in genetics, my very basic knowledge is that such variations are structural in nature as per issues like single-nucleotide polymorphisms (SNPs). The presence of such mutations (which we all have by the way, dotted around our genomic landscape) whilst of interest, don't actually though tell you an awful lot about the function of particular genes as a consequence of those point mutations unless further studies are conducted. Genes for example expressing protein can be affected by such mutations but, as we've come to realise in the past few decades, gene expression is also to some degree affected by other variables, as per the rise and rise of the science of epigenetics and the focus on non-structural effects on the genome. It's beyond the scope of this post to go too heavily into epigenetics and autism, but the research forays so far have provided some interesting data on issues like DNA methylation and autism (see here) and potential knock-on effects (see here). Importantly, structural variations might not necessarily be the same, or have the same effects, as epigenetic variations although the two may work synergistically.

Second, and I hate to bang on about this, but autism or ASD does not normally appear in some sort of diagnostic vacuum. As per the Gillberg work on the ESSENCE of autism (see here) or the 'big data' studies from the likes of Kohane and colleagues (see here), not only is autism an extremely heterogeneous condition in terms of presentation, but also a condition more than likely to co-exist alongside some heightened risk of certain comorbidity. It's all well and good saying that cumulative common genetic variants raise the risk of autism but, as per other biomarker discussions, we might very well replace the word autism with something like attention-deficit hyperactivity disorder (ADHD) or epilepsy or even something more somatic along the lines of the various work looking at autoimmune conditions appearing alongside autism. In short, genetic risk might be related to other things outside of just autism or its individual traits, and as I was reminded recently: "correlation is not the same as causation" (thanks Natasa). Oh, and then there is the RDoC initiative to consider...

Finally, it is a glaring omission in quite a bit of the coverage of this paper that the 41% of risk "unaccounted for" does not receive more interest than it has. I don't want to speculate on what might be included in the array of factors involved in this category (outside of my previous chatter on possible epigenetic factors) but will again draw your attention to other work on the old genetics-environment relationship with autism in mind and the question of heritability (see here and see here). That also one media piece talking about the Gaugler study is quoted as saying: "On their own, none of these common variants will have sufficient impact to cause autism" is an important detail which implies both cumulative effects and possibly the input of some external force(s). And those effects may very well cross the nature-nuture debate in some instances as per the results from Mitchell and colleagues talked about in a previous post.

Deciphering the genetic architecture of autism is still very much a work in progress. This latest contribution to the issue is important not least for the conclusions arrived at with talk of an additive model and it's intersection with common genetic mutations present in the general population. That being said, I still want to see more from the discipline. I'd like to see a more comprehensive analysis taking into account both genetic and epigenetic factors crossing environmental contributions too. I'd also like to see more focus on smaller groups on the autism spectrum as a function of things like developmental trajectory (see here) or response to certain interventions (see here). And for those who seem to be using this work as a hammer against environment being related to cases of autism, just remember, there may be many, many routes towards a clinical diagnosis...

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[1] Gaugler T. et al. Most genetic risk for autism resides with common variation. Nature Genetics. 2014. July 20.

[2] St Pourcain B. et al. Common variation contributes to the genetic architecture of social communication traits. Mol Autism. 2013 Sep 18;4(1):34.

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Gaugler T, Klei L, Sanders SJ, Bodea CA, Goldberg AP, Lee AB, Mahajan M, Manaa D, Pawitan Y, Reichert J, Ripke S, Sandin S, Sklar P, Svantesson O, Reichenberg A, Hultman CM, Devlin B, Roeder K, & Buxbaum JD (2014). Most genetic risk for autism resides with common variation. Nature genetics PMID: 25038753

DNA methylation patterns and autism: buccal up

"The results indicate the presence of a mosaic subpopulation of epigenetically-dysregulated, ectodermally-derived cells in subjects with ASD [autism spectrum disorder]". That was the very clinical primary conclusion reached in the study by Esther Berko and colleagues [1] (open-access here). They looked at DNA methylation patterns in cheek cell samples for a small sample of children diagnosed with an ASD (n=47) "born to mothers aged 35 and over" compared with samples from an asymptomatic control group (n=48) and found some potentially important differences.

Cheek of it.. @ Wikipedia 

Some of the accompanying press for this paper can be found here. As per the headline: "Study shows environmental influences may cause autism in some cases" the results have been suggested to further add to the notion that genetics may not be the be-all-and-end-all of autism, at least some cases of autism. We'll see about that. That alongside the body of research which seems to suggest that offspring of older mums might be at increased risk of being diagnosed with autism (see here) makes for some interesting reading.

Before going on, I should perhaps define a few terms which have or will crop up in this post. So, buccal swabs refers to a way of collecting cells and DNA [non-invasively] from the inside of a person's cheek. Ectoderm refers to the outer layer of germ cells which eventually give rise to hair, skin, the lens of the eye and importantly, the nervous system including neurons and glial cells, as well as the mucus membrane of the mouth. DNA methylation is something which has been covered a few times on this blog (see here and see here) and is part of that wonderful concept of epigenetics and one of the ways that genes are turned on or off depending on their methylation status.

The Berko paper is open-access but a few pointers might be useful:

• Buccal cells were examined as a sort of proxy for brain cells (which generally aren't harvested from live donors). As per the description previously, the ectodermal origins of buccal cells cross that proxy bridge. DNA was extracted from the buccal cells and subjected to various genetic and epigenetic analyses.
• Researchers used the Mosaic Alteration Detection (MAD) algorithm [2] to test "for abnormal chromosome numbers as well as other chromosomal defects". No evidence of chromosome aneuploidy was detected in either cases or controls which kinda ruled out more traditional genetic effects being related to advancing maternal age.
• Methylation was then the name of the game, and as I've talked about on a previous post (see here) various ways and means of looking at the methylation status of those little genetic islands: CpG sites. 
• Results:  "a first pass" approach looking at DNA methylation patterns between the groups identified "3560 differentially methylated GCs". Researchers then further refined their analysis by means of something called bump-hunting which took into account age and ancestry as potential effectors of DNA methylation and something called co-methylation network analysis. They were then left with 15 differentially methylated regions (DMRs) on 14 genes "distinguishing the ASD and TD [typically developing] samples". Even further refinement taking into account the overlapping presence of copy number variations (CNVs) (see here for an explanation) boiled the total down and looked at which genes might be associated with DMRs.
• "The candidate DMRs from the genome-wide analysis were associated with genes, of which many have already been implicated in previous studies with ASD" shown in Table 1 of the paper. Further: "The model that results is of mosaic epigenetic dysregulation affecting the same networks and pathways targeted by mutational mechanisms, creating comparable deleterious effects on neuronal function". In other words, the more traditional genetic mutations noted in other work on some genes in relation to autism might also be complemented by epigenetic changes affecting those gene functions too. 

I found the results of this paper to be absolutely fascinating. Not only that the emphasis was on cells which share some source commonality with those found in the grey-pink matter which has been a primary focus of autism research but also that genetic mutations and epigenetic changes may potentially work synergistically in cases of autism. The added detail that this tied into the seemingly enhanced risk of autism in older mothers [3] (at conception) adds further interest to this work. As the authors noted: "The epigenetic dysregulation observed in these ASD subjects born to older mothers may be associated with aging parental gametes, environmental influences during embryogenesis or could be the consequence of mutations of the chromatin regulatory genes increasingly implicated in ASD". Chromatin by the way, is something of an upcoming star in autism research [4] (open-access here).

Obviously there is quite a bit more work to do in this area not least to test whether the Berko findings are also applicable to children with autism born to younger mothers and any influence from the various overlapping conditions which can and do occur in cases of autism such as epilepsy (see here) or intellectual disability. I also hark back to some interesting work presented at IMFAR this year (2014) by Feinberg and colleagues who talked about age-related methylation changes found in semen as being something to watch when it comes to the older dads and autism risk work (see here) which might point to a cumulative effect. Combined with what is already being reported about methylation status and something like those HERVs in relation to autism (see here) and even ADHD (see here), epigenetics is certainly continuing its rise and rise in autism research [5].

Music then to close. Hey Ya!

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[1] Berko ER. et al. Mosaic epigenetic dysregulation of ectodermal cells in autism spectrum disorder. PLoS Genet. 2014 May 29;10(5):e1004402.

[2] González JR. et al. A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data. BMC Bioinformatics 2011, 12:166

[3] Gillberg C. Maternal age and infantile autism. J Autism Dev Disord. 1980 Sep;10(3):293-7.

[4] Lasalle JM. Autism genes keep turning up chromatin. OA Autism. 2013 Jun 19;1(2):14.

[5] Siniscalco D. et al. Epigenetic Findings in Autism: New Perspectives for Therapy. Int. J. Environ. Res. Public Health. 2013; 10: 4261-4273.

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Berko ER, Suzuki M, Beren F, Lemetre C, Alaimo CM, Calder RB, Ballaban-Gil K, Gounder B, Kampf K, Kirschen J, Maqbool SB, Momin Z, Reynolds DM, Russo N, Shulman L, Stasiek E, Tozour J, Valicenti-McDermott M, Wang S, Abrahams BS, Hargitai J, Inbar D, Zhang Z, Buxbaum JD, Molholm S, Foxe JJ, Marion RW, Auton A, & Greally JM (2014). Mosaic epigenetic dysregulation of ectodermal cells in autism spectrum disorder. PLoS genetics, 10 (5) PMID: 24875834

Autism and the methylome

Q: When is an identical twin not an identical twin? A: Pretty much all the time (at least according to some people) as our increasing understanding of the complexity of genetics stretches and modifies long-held beliefs about the building blocks of life and their role in our health and wellbeing.

DNA methylation differences? @ Wikipedia  

Today I'm talking about identical (monozygotic) twins - siblings derived from one fertilised egg - and how the science of epigenetics might have some interesting implications for autism research as per the study by Chloe Wong and colleagues* (open-access). I should perhaps also direct you to some other interesting research recently discussed in this area too (see here) which might be relevant.

Regular readers will probably already know about my amateur interest in epigenetics (see here and here) and how the area of the epigenome - those chemical marks which have the ability to influence the expression of the genome - has started to yield some potentially important observations. At times I'll admit to being slightly too over-excited at the possibilities of epigenetics. Subsequently brought back down to earth by more sobering accounts (see here**) but not yet ready to poo-poo the whole science just yet***.

I don't want to rehash the whole epigenetic story in this one post, so instead am going to concentrate on the particular area covered by Wong et al and their analysis of the methylome (yes, another -ome for you) which is concerned with the addition of methyl groups to various regions of the genome and how that subsequently alters the expression of genes. DNA methylation has been a sort of peripheral topic in relation to cases of autism for quite some time now; brought to the forefront by all that folic acid (see here) and MTHFR research (see here) and the availability of those lovely methyl groups. Suffice to say that we are still very much at the beginning when it comes to looking at the relationship between all these elements and very complicated conditions like autism.

Anyhow, after that very long introduction (I am only an amateur science blogger after all), a few details from the Wong paper might be in order, bearing in mind it is open-access:

• Part of the UK TEDS initiative and partly sponsored by Autism Speaks who were involved in that recent environmental epigenetics symposium (see here), researchers looked at 50 monozygotic (MZ) twin pairs. It wasn't just a case of 25 twin pairs where one twin was diagnosed with an autism spectrum disorder (ASD) compared with 25 twin pairs where both were concordant for ASD. No, instead the authors looked at a variety of phenotypic combinations based on concordance/discordance for ASD and various core traits based on the Childhood Autism Symptom Test (CAST) schedule. Supplementary table 1 shows the combinations (see here).
• A genome-wide analysis of DNA methylation was undertaken on blood samples provided by participants. I can't pretend to intimately know all the techniques that were employed so won't profess to do so. What I can glean from the paper is that both DNA methylation differences between MZ twin pairs discordant for ASD and analyses between groups scoring high and low on the various core symptom areas were completed with "the aim of identifying real, biologically relevant within-twin and between group DNA methylation differences".
• Results: "ASD is not associated with systemic differences in global DNA methylation". In other words, within the twins, there is quite a high degree of similarity when it comes to patterns of DNA methylation. This might tie in with other work looking at methylation in cases of autism and other family members (see here). 
• When looking at DNA methylation patterns between twin pairs discordant for ASD, specific sites of the genome however seemed to show some variability as a function of ASD diagnosis or not. The authors list the top 50 "differentially methylated CpG sites" (see here) showing gene and position, with a combination of hyper- and hypo-methylated regions identified. Top of the methylation differences pops was the NFYC promoter which was "consistently hypermethylated in affected individuals" (see here for some additional papers on this gene). Hypermethylation by the way, generally means gene silencing as per its function when it comes to transposons (think HERVs). 
• Various other data are presented based on either syndrome or trait specific differences across the twins. I'm not going to go through all of these because, well because that's called plagiarism. I will draw your attention to one particular finding which might be important as they identified "one MZ twin pair, concordant for a very severe autistic phenotype, that appear to represent epigenetic outliers at multiple CpG sites across the genome". I'm immediately drawn back to my autism or autisms post and that all-important phenotypic variability as potentially being relevant here too. That and the tie-up with more structural changes to the genome as per the mention of CNVs and hotspots.

I know I've gone on a bit in the post but this is potentially a very important paper. Not only does it put the epigenome, or at least one part of the epigenome, firmly on the autism research map, but it offers something of a partial explanation for [some of] that 'missing heritability' which was talked about not so many years ago (see here).

The fact also that methylation patterns might be variable both intra-twins and intra-ASD is also important; suggesting that as with more traditional genomic findings in relation to autism, there isn't going to be just one epigenomic factor affecting risk or presentation, but rather a plethora of sites which are hyper- or hypo-methylated, potentially also linked to (affecting?) more structural changes to the genome in cases of ASD.

Don't get me wrong, the same questions remain as are seen in other areas of biological functioning with autism in mind: the heterogeneity, the reliance on diagnosis by observation and note-taking, the (elevated) risk of comorbidities, etc. All of which cloud the waters of association. Distinct however from the question of whether there is anything that can be done when structural changes to the genome are observed (outside of gene therapy for example), when talking about methylation, one speculates that this might be something that lends itself to pharmacotherapy as per the already use of DNA methyltransferase inhibitors for example. Yes, going back to that folic acid-autism link work, the possibility also that environment might help shape gene function and some new light on other external factors (noting that I am not advocating anything at the current time).

So endth the lesson for today.

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* Wong CC. et al. Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits. Molecular Psychiatry. April 2013.

** Ptashne M. Epigenetics: core misconcept. PNAS. April 2013.

*** Fraga MF. et al. Epigenetic differences arise during the lifetime of monozygotic twins. PNAS. 2005; 102: 10604-10609.

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Wong, C., Meaburn, E., Ronald, A., Price, T., Jeffries, A., Schalkwyk, L., Plomin, R., & Mill, J. (2013). Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits Molecular Psychiatry DOI: 10.1038/mp.2013.41

CNV duplication load, hotspots and autism

Viewers here in the UK might remember the catchphrase of one Michael Barrymore on the show 'Strike It Lucky': "What is a hotspot not? A good spot". It is with a rather different kind of hotspot in mind, that I'm posting about today: genomic hotspots and autism with a specific focus on copy number variants.

An intriguing paper by Santhosh Girirajan and colleagues* (open-access) popped up on my Twitter radar recently discussing copy number variant (CNV) load in relation to autism spectrum disorders. Whilst only being an amateur enthusiast when it comes to all things genes and genomic, I can't offer an expert opinion on what CNVs are, just that fairly similar to single-nucleotide polymorphisms (SNPs), we're talking physical alterations to the genome and in particular gains/losses to segments of DNA (I think!)

A hotspot indeed... @ Wikipedia  

I'll start by saying that this is not the first time that CNVs have cropped up on this blog either with autism in mind (see here) or with other conditions such as ADHD (see here) and intellectual disability (ID) in mind (see here).

Indeed readers who looked at that ID link will see that we are talking about another paper from Girirajan following the previous suggestion that ID might be particularly prone to a high CNV load. Keep that in mind for now.

Thankfully the latest paper has been very nicely covered by a ScienceDaily entry (see here with a sigh of relief) so as to patch over my considerable non-expertise in this area. Without plagiarising the paper or SD entry, the general gist of the work was to initially look at CNV data from over 500 people with autism (n=274) or asymptomatic controls (n=242) derived from the CHARGE initiative (see here), to ascertain exactly what the CNV load was and how it might link into some of the signs and symptoms of autism. There was also a further testing group to confirm "the increased duplication load" based on a further cohort of autism and control cases but I'm not going to bore you with all the details.

If I'm reading this right, there were some interesting findings to take from this study:

• Children with autism "exhibited a significantly elevated copy number load, represented principally as an increase in duplicated base pairs found in large CNVs". Importantly, this copy number load seemed to include quite a bit of de novo, so not passed from parents to offspring.
• Duplication over deletion seemed to be the important variable for autism cases, which as the authors note "is associated with genomic variants with more modest functional impact". As per that previous CNV work with ID in mind, I think the authors seemed to be suggesting a sort of sliding scale of phenotypes based on CNV profiles: ID at the more severe end of the spectrum (with more deletions also), autism somewhere in the middle and dyslexia at the less severe end of the spectrum in terms of functioning. I could be wrong and I could be over-simplifying the whole thing so accept my apologies if so.
• Copy number load in autism cases also seemed to show some relationship with certain aspects of behaviour. Significant negative correlations for example, were observed between CNV load and VABS scores in core areas of communication and socialisation. That being said, the correlations were not exactly all that great (p=0.048 and p=0.022 respectively) and should be compared with other gold-standard schedules such as the ADOS that did not turn up anything significant. 
• The notion of genomic 'hotspots' is also raised as a consequence of the results, suggesting that parts of the autistic genome already under the spotlight might be more susceptible to such CNVs. Those words-of-the-hour DNA methylation get a mention alongside the folate story (see here). Personally and with my non-expertise caveat in full working order, I'm also wondering about those archived portions of viruses called HERVs - human endogenous retroviruses - (see this post and this post) which dot the genome and whether they might be contributory in any way, shape or form to any genomic instability in target areas.

Putting aside the complexity of the genome when it comes to autism and the suggestion that we might want to be rethinking how we view the condition** (I've a post coming up on this paper fairly soon), there are some interesting themes emerging from the Girirajan paper. That for example CNV load and the type of CNV (duplication or deletion) might roughly fit into phenotypic differences between inter-related conditions including autism and ID is definitely something worth pursuing in future work. That also such CNVs might tie into genomic hotspot areas is also an important point particularly when it comes to things like systems biology. Lest we also forget the potential importance of any genomic instability and how, with the DNA methylation point in mind, this *might* be attenuated via changes to the environment (remember SAMe)?

To close Chuck Berry and a song about Johnny B Goode and his guitar-playing skills.

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* Girirajan S. et al. Global increases in both common and rare copy number load associated with autism. Hum. Mol. Genet. March 2013.

** Moreno-De-Luca A. et al. Developmental brain dysfunction: revival and expansion of old concepts based on new genetic evidence. The Lancet Neurology. 2013; 12: 406-414.

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Girirajan S, Johnson RL, Tassone F, Balciuniene J, Katiyar N, Fox K, Baker C, Srikanth A, Yeoh KH, Khoo SJ, Nauth TB, Hansen R, Ritchie M, Hertz-Picciotto I, Eichler EE, Pessah IN, & Selleck SB (2013). Global increases in both common and rare copy number load associated with autism. Human molecular genetics PMID: 23535821