"There is broad parent interest in a genetic/epigenetic test for ASD"

The quote heading this post - "There is broad parent interest in a genetic/epigenetic test for ASD [autism spectrum disorder]" - comes from the findings reported by Kayla Wagner and colleagues [1]. As per the observation that most parents of those diagnosed with an ASD, or developmental delay (DD) or asymptomatic controls "had positive perceptions toward genetic/epigenetic research in ASD", far from being the 'danger' that it is sometimes portrayed as, the science of genetics seems to have an important standing among many parents.

The Wagner results came about as a result of this research group publishing studies "that demonstrate the utility of RNA sequencing technology (non-coding RNA) to identify children with ASD." They did what few genetic studies have done: asked parents participating in their studies whether what they were doing/finding was of interest to them. This was achieved via the use of a questionnaire which included six themes: "(1) reasons for participating in the epigenetic study; (2) prior knowledge of genetics/epigenetics, and the source of this information; (3) overall interest in genetic/epigenetic testing for ASD; (4) concerns about genetic/epigenetic testing for ASD; (5) preferences about the approach for genetic/epigenetic testing (including age of administration and biofluid of choice); and (6) extent of results to be returned." You'll probably have noted that alongside their use of the term 'genetics' they also talk about a still up-and-coming branch of genetics called epigenetics. I've talked about epigenetics and autism before on this blog (see here and see here) but the description Wager et al use just about says it all: "changes in gene expression, where the changes are not due to modification of the actual DNA sequence, but instead result from modifications that regulate DNA structure and expression." Gene expression seems to be particularly important to the science of epigenetics as per the notion that we don't all walk around with all our genes permanently switched to the 'on' or 'off' position.

Results: quite a few important points emerged from the obtained data. Most parents understood at least a little bit about genetics and some of the processes involved. Epigenetics wasn't as well known about or understood (something which is probably not so surprising). Other points also emerged: "There were no parents (0%, 0/244) concerned about a lack of scientific evidence supporting genetic and epigenetics." But that's not to say that some parents weren't concerned about the implications of genetics and epigenetics, as mention about issues like privacy and insurance status were raised during the Wagner study.

Also: "Nearly all parents (96%, 235/244) indicated that if there were genetic testing for ASD, they were interested in learning results about their child’s risk for ASD." And when it came to results, over three-quarters of all parents expressed a preference for "all epigenetic/genetic results, regardless of whether they were implicated in health and disease" and not just an overview or interpretation of any results. People want data not overviews.

And then to some important but potentially controversial findings: "The majority of parents (71%, 164/231) desired results of a genetic/epigenetic test for ASD when their child was 12 months of age or younger. Over half of parents were interested in receiving results at conception (34%, 78/231) or at birth (37%, 86/231), while fewer requested results at 12 months (17%, 40/231) or at 2 years of age (12%, 27/231)." You can perhaps see where this might be going - particularly 'receiving results at conception' - even if the authors seem to have chosen not to pursue it any further in their discussions.

I'm no bioethicist and so am nowhere near qualified to talk about the ins-and-outs of genetic testing in the context of autism and what implications this could have. I note other commentators have approached this subject previously based on some of the peer-reviewed science in this area (see here) and various points have been raised. One of the important things to bear in mind is that, as it currently stands, there is no single genetic test for all autism. Indeed, allied to the idea that the concept of 'a universal autism gene' is fast becoming a distant memory, genetic studies are serving to further highlight how complex autism actually is.

But there is always the possibility that some day someone will potentially deliver a genetic/epigenetic 'test for some autism'. The question then is how will it be used? What checks and balances will be in place to ensure that it is not misused?

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[1] Wagner KE. et al. Parent Perspectives Towards Genetic and Epigenetic Testing for Autism Spectrum Disorder. Journal of Autism & Developmental Disorders. 2019. March 22.

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"The current study provides the first analysis of potential transgenerational impacts of glyphosate in mammals"

'Carefully' is a word that applies to any science in terms of 'meaning' and 'generalisation'. Don't get me wrong, I'm a big fan of science and scientific evidence when it comes to life, the universe and everything. But I also understand that science can be used/manipulated in many, many different ways, and rarely, if at all, do we see something that could be considered perfect science. Hence my use of the word 'carefully' in opening this blog post. With all that in mind, I bring the findings reported by Deepika Kubsad and colleagues [1] to the blogging table and the proposal that: "glyphosate can induce the transgenerational inheritance of disease and germline (e.g. sperm) epimutations."

Why am I talking about such a study on a blog primarily hosting autism research? Well, a few reasons (see here and see here), predominantly based around the observations that (a) herbicides and insecticides whilst very useful, are not seemingly without a risk-benefit profile, and (b) autism (and other related labels) have been 'talked about' in the context of risk and 'exposure' to such compounds. I emphasised the words 'talked about' to ensure that the current peer-reviewed science base is not contorted into something that it is currently not (i.e. cause-and-effect). I might also add that 'transgenerational inheritance' is something that has also been discussed in the peer-reviewed science literature in the context of autism too (see here).

The basics of the Kubsad paper: "There are an increasing number of conflicting reports regarding the direct exposure toxicity (risk) of glyphosate, but no rigorous investigations on the generational actions." So, using a rodent (rat) model of study, researchers exposed mummy rats (F0 'gestating female') to glyphosate - "daily intraperitoneal injections of glyphosate" - at doses that probably would be considered 'excessive', and then followed successive generations of rats (F1, F2, F3) looking for any "transgenerational pathologies observed... including prostate disease, obesity, kidney disease, ovarian disease, and parturition (birth) abnormalities." They also included a set of control animals - "F0 generation gestating females administered vehicle control dimethyl sulfoxide (DMSO) or phosphate buffered saline (PBS)" - and whilst they were at it, checked for any germline transmission issues/differences via the inspection of sperm; specifically looking for differential DNA methylation regions (DMRs). Those DMRs are important when it comes to whether specific genes are expressing or not ('switched on' or 'off').

Results: with that word 'carefully' in mind, we are told that there were: "negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology." That's important because it suggests that those directly exposed and their immediate offspring don't seemingly show adverse effects.

But... "In contrast, the F2 generation grand-offspring, derived from a direct exposure F1 generation germline, had significant increases in testis disease, kidney disease, obesity, and multiple diseases in males" and: "The F2 generation females had significant increases in ovary disease, obesity, mammary gland tumors, parturition abnormalities, and multiple disease susceptibility." Researchers also reported seeing a few things in their F3 generation too, as the words "generational toxicology" entered discussions.

As to the DMRs results, well, there were some interesting findings there too: "the glyphosate lineage sperm were found to have altered DNA methylation in direct exposure F1 and F2 generations, as well as the transgenerational F3 generation." This led Kubsad et al to conclude that glyphosate exposure could "promote germline epigenetic alterations in DNA methylation" but more needs to be done on what this actually means for gene expression and whether it tied into the disease risk(s) noted in their exposed cohort and subsequent generations.

The authors mention how their study had limitations including the use of "an environmentally relevant exposure of twice the allowed industry exposure" and the use of an animal model (bearing in mind the logical fallacies that can follow). Lots more investigation is indicated before any big headlines are generated.

But... the Kubsad results are not to brushed under the scientific carpet. They add to the range of other compounds that seemingly carry transgenerational biological correlates and imply further inspection of glyphosate in that same potential category. Whether also there may be additive or interactive effects with other compounds could also be part of the research agenda.

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[1] Kubsad D. et al. Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology. Scientific Reports. 2019; 9: 6372.

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Regression and autism: "The regression group was significantly more functionally impaired..."

The quote forming part of the title of today's post - "The regression group was significantly more functionally impaired..." - comes from the findings reported by Lucy Thompson and colleagues [1] (open-access available here). It continues something of an important theme in autism research circles whereby regression, as in a regression of previously acquired skills, is being seen as important not just for a few but for many (see here).

There were a few aims to the Thompson study such as establishing "the relative prevalence of regression in autism" and "possible predictors, mediators and moderators of regression in autism, including pre- and perinatal factors." The data for the study were derived from "two community-based cohorts" in Sweden totalling just over 300 participants (children) diagnosed with an autism spectrum disorder (ASD) who were observed over two different time points (T1 and T2 2 years later). Another important detail is mentioned by Thompson et al: "Given the lack of previous systematic representative studies in the field, our study sets out to be descriptive rather than hypothesis-driven." Figures and details on regression in the cohort(s) were obtained via specific questioning on this topic "defined as loss of expressive language skills (loss of 5 or more words that had been used communicatively) in connection with the onset of autism." This data was also combined with other medical records information to determine 'consistency'.

Results: "Just over 20% (62/303) of the combined sample of children had regressive autism." That's 1 in 5 children with autism experiencing some kind of regression in relation to language skills. When looking at those who regressed (n=62) compared with those with no regression (n=241), a few details emerged: "Those with regressive autism had a younger age when they first walked... had a more severe language impairment at T1... and more often intellectual disability... [and] lower mean VAB [Vineland Adaptive Behaviour Scales-II] scores." Also: "Severity of autism was higher in the regressive group, with a higher proportion of children with autism... (as opposed to autistic-like condition)."

This is important data. It kinda tallies with other studies of regression accompanying autism suggesting that those who regress tend to have a more 'severe' form of autism with accompanying learning (intellectual) disability. The diagnostic issues - as in more likely to be diagnosed with Kanner's autism rather than other diagnoses - similarly ties in with other findings.

Caveats? Well, a few: "We have chosen to focus on language regression specifically (rather than social, play or motor regression) as communication is by far the most common skill to be lost or diminished in regressive autism." That being said, regression accompanying autism seems to take many, many forms and does not always just mean a loss or partial loss of vocal communication (see here).

Also: "There was also a similar level of maternal disease in pregnancy in the regressive and non-regressive groups, suggesting that prenatal exposure via maternal disease does not seem to be a key feature in the development of regressive autism." I have to question why the authors stuck to looking at just pre- and perinatal factors as possibly being *linked* to regression when regression is likely to occur quite some time after such a developmental window. Surely it would have made more sense to ask a few further questions about the timing of regression - "The average age at regression was 20.13 months... with 54 children (88.5%) showing regression by the age of 24 months" - and whether one or more event might have proceeded such regression in a similar time frame. Y'know whether infection might be a feature (see here and see here for examples) or whether other events might require further investigation (see here). I know this might take such research down some uncomfortable paths, but temporality is surely an important factor for some regression in some cases of autism? Or am I being too unreasonable?

I might also advance the idea that the time to start asking questions about the biology of regression accompanying autism is fast approaching. We've already had some clues in the recent (at the time of writing) peer-reviewed research literature (see here) but lots more needs to be done in this area. Are there important genetic and/or epigenetic variables to consider? Do mitochondrial issues play a role in some regressive autism (see here)? We just don't know enough yet. And yes, this does mean also asking about whether regression in behaviour or cognitive skills was also accompanied by any changes to somatic variables too (see here).

And then there is another question to ask/answer: does regression mean that certain 'therapeutic' options might be particularly useful? I'm thinking back to some research a few years back talking about corticosterioid therapy *potentially* being indicated for some cases of regressive autism (see here). No, I'm not making any medical or clinical claims or giving any advice on such an issue. Merely mentioning that regressive autism needs to be more of a research priority than it currently is. To quote Thompson and colleagues again: "Children with a regressive developmental trajectory, with or without autism, always need a careful neuropediatric work-up to investigate possible neurological disorders that may lead to developmental regression, taking into account possible treatable conditions." Who would argue with that?

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[1] Thompson L. et al. Autism With and Without Regression: A Two-Year Prospective Longitudinal Study in Two Population-Derived Swedish Cohorts. J Autism Dev Disord. 2019 Feb 4.

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"Progesterone hormone treatment significantly increased the risk of ASD"

The quote titling this post - "Progesterone hormone treatment significantly increased the risk of ASD [autism spectrum disorder]" - comes from the findings reported by Michael Davidovitch and colleagues [1].

Researchers set out to examine whether there was any *association* between the receipt of infertility treatments and offspring risk of a diagnosis of autism spectrum disorder (ASD). They concluded that use of IVF treatment - in vitro fertilisation, where a woman's egg(s) are removed and fertilised outside of the body then 'put back in' - probably doesn't show any significantly enhanced risk for offspring autism. When however it came to the use of progesterone hormone treatment complementing such assisted reproductive methods, there may be something to see...

Before going further into these findings, I want to hark back to some discussions (see here) on the previous research in this area. The paper by Liang Liu and colleagues [2] meta-analysing the peer-reviewed literature on the use of assisted reproductive technology (ART) and offspring risk of autism did suggest there might be something more to see; albeit concluding that: "The complexity of ART treatment renders the identification of individual risk factors extremely challenging." I say this on the basis that various different 'pregnancy and birth' factors have been *correlated* with offspring autism risk down the years (see here for example) and teasing apart which ones are the more 'important' is difficult to say the least.

The Davidovitch study relied on data for over 100,000 children (males) born between 1999 and 2008. Authors identified approaching one thousand children diagnosed with an ASD representing about 1% of the total cohort. As well as looking at IVF, they also examined "five hormone treatments" pertinent to ART, singling out progesterone hormone treatment as part of their observations.

There is lots more work to be done in this area before too many sweeping generalisations are made. Aside from the issue of teasing out exactly what conception/pregnancy variables *might* be related to offspring risk of autism (including reproduction itself!) there is a question of mechanism(s) to also consider. The authors refer to the term "epigenetic modification by progesterone" but only few details are provided. I might also at this point introduce a recent piece from Jill Escher [3] who provided some really interesting commentary on how synthetic hormone drugs *might* have played a role in her own family circumstances as a template for some further studies. I say this bearing in mind that progesterone should not be too readily confused with progestins [4].

To close, I also read some other work from Michael Davidovitch recently [5] talking about cell (mobile) phone use and "the development of joint attention in infants" pertinent to "the development of autistic features among a vulnerable subgroup of infants." I'm slightly less enthralled about such suggestions and the strength of any 'technology use cause autism' sentiments but am willing to keep an open mind on how our fascination with mobile phones *might* impact on parent-child interactions (but not necessarily in the context of autism)...

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[1] Davidovitch M. et al. Infertility treatments during pregnancy and the risk of autism spectrum disorder in the offspring. Progress in Neuro-Psychopharmacology and Biological Psychiatry. 2018. June 1.

[2] Liu L. et al. Association between assisted reproductive technology and the risk of autism spectrum disorders in the offspring: a meta-analysis. Scientific Reports. 2017; 7: 46207.

[3] Escher J. Bugs in the program: can pregnancy drugs and smoking disturb molecular reprogramming of the fetal germline, increasing heritable risk for autism and neurodevelopmental disorders? Environ Epigenet. 2018 Apr 26;4(2):dvy001.

[4] Spark MJ. Progesterone or progestogen or progestin; which is it? BMJ. 2009; 339: b4380.

[5] Davidovitch M. et al. The Role of Cellular Phone Usage by Parents in the Increase in ASD Occurrence A Hypothetical Framework. Medical Hypotheses. 2018. June 7.

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"DNA methylation data from neonatal blood spots can be used to accurately predict age and maternal smoking status"

There were two primary reasons why I wanted to blog about the findings reported by Eilis Hannon and colleagues [1]: first, the focus of the study was "to identify DNA methylation biomarkers of ASD [autism spectrum disorder] detectable at birth", and second, the authors actually "identified robust epigenetic signatures of gestational age and prenatal tobacco exposure, confirming the utility of DNA methylation data generated from neonatal blood spots."

The first reason, looking at DNA methylation biomarkers in relation to autism, follows other similar initiatives down the years (see here for example) on the back of some significant interest in how "epigenetic variation induced by non-genetic exposures" might complement/fill in some gaps left by more traditional genetic studies. Epigenetics by the way, seemingly means different things to different people, but is currently summarised as "the study of heritable changes in gene function that do not involve changes in the DNA sequence." DNA methylation reflects one epigenetic process (there are others). The second reason - robust epigenetic signatures linked to gestational age and prenatal tobacco exposure - actually turned out to be the more important finding, or at least the more significant finding, reported by Hannon et al hence the title of this post...

'Guthrie cards' are mentioned as the starting material for the Hannon paper, and yet another hat-tip to a true medical pioneer, Robert Guthrie (and his team), who has saved multitudes of lives with his cards used to collect and store neonatal blood spots. As well as being used to screen for various potential inborn errors of metabolism (some of which seem to have something of a relationship with some autism), those archived blood spot cards have also proved to be important research fodder too (see here).

Hannon garnered neonatal methylomic data for approaching 1300 individual - "comprising equal numbers of ASD cases and matched controls, 50% male/female" - derived from "the iPSYCH case–control sample" based in Denmark. We are told that: "DNA methylation was quantified across the genome using the Infinium HumanMethylation450k array" bearing in mind this technique/system "only assays ~ 3% of CpG sites in the genome." Alongside: "Matched genome-wide single nucleotide polymorphism (SNP) genotyping data from the same individuals enabled us to undertake an integrated genetic–epigenetic analysis of ASD, exploring the extent to which neonatal methylomic variation at birth is associated with elevated polygenic burden for ASD."

Results: with regards to a neonatal methylomic 'signature' for autism or ASD, nothing significant was detected. Obviously one has to bear in mind the limitations of the assay/method used and the fact that blood from bloodspots represents only one type of tissue (DNA methylation patterns are not necessarily the same across different tissues). I was also interested to see the authors talk about "the chronology of sample collection prior to ASD diagnosis" and the 'plausability' that they were "looking too early on in the disease process." No, autism isn't 'a disease', but this work might provide some support for the idea that the processes and onset of autism is not always set either during conception nor during gestation (see here). Dangerous thinking for some people of a sweeping generalisation ilk...

Having said all that, researchers did talk about a "significant association between increased polygenic burden for autism and methylomic variation at specific loci" but I'd like to see replication of this effect before any big claims are made.

Then to those other findings of "robust epigenetic signatures of gestational age and prenatal tobacco exposure" and what that could mean to several different areas of research, autism and beyond. I'm a little surprised that the authors didn't make more of their 'robust findings' in their discussion of these results. I appreciate that their primary aim to "identify DNA methylation biomarkers of ASD detectable at birth" was not met with startling success but the suggestion of a tell-tale epigenetic sign of exposure to maternal smoking during pregnancy for example is, I would have thought, an important advance. Certainly one that could be at least relevant to various other studies, including those related to an important comorbidity that seems to be 'over-represented' in relation to some autism (see here) and perhaps more.

To close, it's not the first time that the Star Wars universe has been subject to peer-reviewed 'science' but the paper by Hatters Friedman and colleagues is an interesting one...

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[1] Hannon E. et al. Elevated polygenic burden for autism is associated with differential DNA methylation at birth. Genome Med. 2018 Mar 28;10(1):19.

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"findings suggest a protective effect of CRP" on schizophrenia risk?

Science is often a puzzling endeavour. Sometimes, just when you think that you've got something nailed down, scientific results appear that 'trash' long held, cherished beliefs. So it was with the publication of the results by Fernando Pires Hartwig and colleagues [1] who presented findings looking at "the effect of inflammatory markers on schizophrenia risk" based on the use of "a mendelian randomization (MR) design."

MR, by the way, is a interesting technique based on the principle that "genetic variants that either alter the level of, or mirror the biological effects of, a modifiable environmental exposure that itself alters disease risk should be related to disease risk to the extent predicted by their influence on exposure to the environmental risk factor." It's a technique that has already been applied to inflammatory markers in the context of schizophrenia on more than one occasion (see here and see here). Those inflammatory markers studied have included ones which were covered by the Hartwig paper. Specifically: "Genetically elevated circulating levels of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra), and soluble interleukin-6 receptor (sIL-6R)." I should also point out that Hartwig and colleagues have some research form in the area of applying MR to various aspects of medical science (see here).

As per an accompanying editorial on the Hartwig paper [2], the long-and-short of it was that researchers "used 2-sample MR to test for a potentially causal relationship between inflammation and schizophrenia and to improve inference for the association between genes, inflammatory biomarkers, and risk of developing schizophrenia." I can't claim any specific expertise in the use of MR (see here for a good overview [3]) but it appears that data on single-nucleotide polymorphisms (SNPs) in relation to those inflammatory markers was used to test whether said markers might be linked 'causally' to risk of schizophrenia. Their results were interesting: "we did not find strong evidence that lifelong exposure to increased action of these proinflammatory cytokines increases schizophrenia risk, as previously hypothesized" and indeed that: "blockade of IL-6 effects and low CRP levels might instead increase schizophrenia risk." This is contrary to quite a lot of other research in this area (see here for example).

There are a few words of caution to attach to the Hartwig results that need mentioning not least the primary tenet on which analyses are based: genetic variants (SNPs) affecting something like CRP are of primary importance to schizophrenia. I don't for example, see anything in the data looking at gene function/expression being affected as a result of non-structural changes to the genome via something like epigenetic 'alterations' for example (and there is such a thing as epigenetic Mendelian randomization y'know). I say this on the basis that other genes involved potentially involved in processes linked to DNA methylation have also been *associated* with cases of schizophrenia (see here). The authors also caution that their analyses are based on "lifelong exposure to elevated cytokine and CRP levels" and that exposure during 'critical windows' might be the important issue when it comes to any change in schizophrenia risk. Similarly they note that "it is possible that IL-6 and CRP effects on schizophrenia risk are related to a maternal effect (eg, maternal susceptibility to infections during pregnancy), so that our findings are explained by the correlation between maternal and offspring genotypes." This final point is based on the idea that maternal infection during pregnancy (or the biological consequences of) seems to be quite a big risk factor for at least some presentations of schizophrenia (see here) as well as [cautiously] other labels (see here). Here, the importance of a reprogrammed immune system during pregnancy might also come into play alongside any maternal 'susceptibility'.

Personally I'm not yet ready to totally trash the idea that the immune system, and specifically elevations in inflammatory markers such as CRP and other pentraxins, might not be important to some schizophrenia risk in a more detrimental way. I appreciate that one has to be careful when talking about immune system markers and their inflammatory direction (see here for some chatter on IL-6 and its pro- and anti-inflammatory natures) but the existing data is too evident to just discard on the basis of one new study, despite it's scientific prowess. I don't however doubt that there may be several confounding variables linked to increases in CRP in schizophrenia; not least the impact of something like increased body mass index (BMI) that seems to follow some cases of schizophrenia [4]. These variables need to be further explored, particularly in the context of what side-effects pharmacological management of schizophrenia might have (see here). And I also hat-tip the paper by Manu and colleagues [5] applying the Bradford Hill's guidelines on 'causation' to this area and concluding that (upto 2014) "there is insufficient evidence that the replicated, strong association between schizophrenia and elevated inflammatory markers has etiopathological relevance"...

For now however, the Hartwig findings reiterate that science is an ever-changing, ever-evolving process...

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[1] Hartwig FP. et al. Inflammatory Biomarkers and Risk of Schizophrenia: A 2-Sample Mendelian Randomization Study. JAMA Psychiatry. 2017. Nov 1.

[2] Byrne E. et al. Inference in Psychiatry via 2-Sample Mendelian Randomization—From Association to Causal Pathway? JAMA Psychiatry. 2017. Nov 1.

[3] Sheehan N. et al. Mendelian Randomisation and Causal Inference in Observational Epidemiology. PLoS Med. 2008; 5(8): e177.

[4] Fernandes BS. et al. C-reactive protein is increased in schizophrenia but is not altered by antipsychotics: meta-analysis and implications. Mol Psychiatry. 2016 Apr;21(4):554-64.

[5] Manu P. et al. Markers of inflammation in schizophrenia: association vs. causation. World Psychiatry. 2014 Jun; 13(2): 189–192.

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"we found no association between maternal folic acid supplementation and offspring ASD" but...

The findings reported by Marit Strøm and colleagues [1] observing "no association between maternal folic acid supplementation and offspring ASD [autism spectrum disorder]" throw yet another research 'spanner in the works' when it comes to the [very generalised] idea that pregnancy folic acid supplementation might affect risk of offspring autism.

Don't get me wrong, I appreciate all the data suggesting that folic acid supplementation during pregnancy is a useful thing for helping to reduce the risks of neural tube defects (NTDs) for example. But when it comes to pregnancy folic acid (folate) potentially impacting on offspring risk of autism, I've always been a little cautious of the collected data so far and how its been interpreted/generalised in certain quarters (see here and see here for examples).

So, drawing on data from the "entire DNBC [Danish National Birth Cohort]" initially including nearly 100,000 singleton, live born children, researchers set out to find female "users" of folic acid supplements either just before conception or during the earliest stages of their pregnancy. Not just content with folic acid, they also looked at available data on "periconceptional vitamin B12" use too on the basis of some connection between the two vitamins. They then searched connected databases to find those offspring with a diagnosis of autism spectrum disorder (ASD): "identified by International Classification of Diseases (ICD)-10 diagnosis codes F840, F841, F845, F848, and F849; ‘childhood autism’ by diagnosis code F840." Analyses of these collected variables were undertaken, as well as adjusting for potentially confounding variables such as maternal age, parity, education level and the like.

Results: well, as per the title of this post, researchers reported finding very little when it came to pre-pregnancy or early pregnancy folate use: "There was no detectable association between maternal folic acid supplementation in the periconceptional period and offspring ASD" and: "Results from the analyses using midpregnancy exposure data were similar: there was no association with ASD/childhood autism neither for folic acid supplementation nor for dietary folate intake." Such results held when various 'corrections' were made for variables such as "sex specific effects" and cases where intellectual (learning) disability was present for example.

I have to say that the authors do seem genuinely surprised that their results did not tally with other large, population studies on this topic: "At present we are not able to present any viable explanation for these discrepant results." They do mention one particularly important point insofar as the usefulness of looking at small changes to something called the methylenetetrahydrofolate reductase (MTHFR) gene in the context of autism and folic acid as other authors have done [2]. This, on the basis that MTHFR plays an important role in folate metabolism (see here) and issues with this gene are no stranger to the autism research landscape (see here). I'm also minded to refer readers back to another potentially important issue identified in relation to some autism that might also affect folate metabolism: folate receptor autoantibodies (FRAAs) (see here).

I still think there is a place for further investigations on folic acid use during pregnancy and offspring autism risk. But like many things in the context of the plural 'autisms' (see here), it perhaps makes more sense to zoom in on potentially relevant sub-groups on the autism spectrum rather than treating all autism as being homogeneous in either aetiology or presentation. I might add that folic acid use as part of wider range of nutritional supplements potentially used during early pregnancy remains an important area of research attention in the context of offspring autism [3].

And also just to complicate things even further, the results from Wang and colleagues [4] add: "this comprehensive meta-analysis suggested that maternal use of folic acid supplements during pregnancy could significantly reduce the risk of ASD in children regardless of ethnicity, as compared to those women who did not supplement with folic acid." I don't think the debate is finished yet on this topic.

Music to close, and since my brood and I are competing again today, Sia (again) and some brilliant kata (hopefully our Heian Sandan will be as good).

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[1] Strøm M. et al. Research Letter: Folic acid supplementation and intake of folate in pregnancy in relation to offspring risk of autism spectrum disorder. Psychol Med. 2017 Sep 26:1-7.

[2] Schmidt RJ. et al. Maternal periconceptional folic acid intake and risk of autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks from Genetics and Environment) case-control study. Am J Clin Nutr. 2012 Jul;96(1):80-9.

[3] DeVilbiss EA. et al. Antenatal nutritional supplementation and autism spectrum disorders in the Stockholm youth cohort: population based cohort study. BMJ 2017; 359: j4273.

[4] Wang M. et al. The association between maternal use of folic acid supplements during pregnancy and risk of autism spectrum disorders in children: a meta-analysis. Molecular Autism. 2017; 8: 51.

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"early medical events are associated with clinical ASD phenotypes"

The paper by Charlotte Willfors and colleagues [1] (open-access) provides some food for thought today and the observation that various individual and cumulative medical events - "early medical events likely to be caused by environmental factors" - may be important to at least some autism.

Researchers "scrutinized the early medical histories of a rare and informative sample of 13 MZ [monozygotic] twin pairs discordant for clinical ASD [autism spectrum disorder]" also including "13 MZ typically developing (TD) control pairs (n=52) matched for sex" as an 'exploratory step. Discordant for autism means that one twin had autism and the other did not.

This research first step looked at medical events (likely to be caused by environmental factors!) included things like delivery and neonatal variables (e.g. foetal distress, hypoxia), minor and frequent infections (e.g. ear infections), allergy and epilepsy to name a few. Data was acquired from a few sources including medical records and medical history "assessed from a parent reported questionnaire." They examined exposure to the medical events "in relation to either quantitative or qualitative discordance for ASD." Qualitative discordance referred to when "only one twin within a pair meeting the diagnostic criteria of ASD." A 'confirmatory' study was also carried out whereby a larger, independent cohort of 100 twin pairs "quantitatively discordant for autistic traits" were also quizzed and findings cross-validated.

Results: a few non-shared environmental (NSE) events seemed to be important based on their analysis. So: "Single early medical factors, likely to be caused by NSE, that discriminated between twins in qualitative ASD discordant pairs were dysregulation during the first year of life (comprising feeding and sleeping problems, excessive crying and worrying) and birth weight." Authors also reported that cumulatively, the appearance of early medical events were significantly different in MZ twins with autism compared with their non-ASD co-twin. It's worth mentioning that some of those 'dysregulation' events have been talked about in the earliest descriptions of autism (see here). Birth weight too has something of a long-standing connection to [some] autism (see here). When it came to analysis based on autistic traits (the confirmatory study) it seemed that "early dysregulation and the cumulative load of a variety of early adverse medical events" continued to be important variables (although birth weight linked to ASD traits lost its significance).

These are important findings. The focus on MZ twins (who share a common structural genetic blueprint) means that the genetics side of things is to a large extent 'controlled for' and the results are more likely to reflect some environmental or, more accurately, non-genetic influence. There is a caveat to this though, as per the authors recognition: "with the exception of putative post-twinning de novo mutations." I might also add that MZ twins are also not necessarily epigenetically the same too so gene expression can (and does) differ. What causes these epigenetic differences is still the source of some debate but I might chime in with one idea (see here) out of many possibilities.

"Our data indicate that taking into account the cumulative load of early medical factors might strengthen or discourage a suspicion of ASD, at least in a minority of cases." This is an interesting thought provided by the authors based on their findings. It ties in well with the idea that although behavioural presentation is core to autism presentation and diagnosis, behaviour might not be the only important feature present in relation to autism. I do have to express a degree of caution however with such an approach based on the idea that various types of regression have been noted in the peer-reviewed literature to accompany some autism (see here) and with it, the concept of 'acquired autism' should really be properly recognised (see here for example) in these days of the plural 'autisms'. Indeed, there's a research study idea for anyone out there: looking at MZ twins discordant for autism with onset of said autism tied into a regression of skills?

Scientific replication is the name of the [future] game in this area of study, drawing on larger cohorts and perhaps based in other geographical areas outside of Sweden. We also need to find out what mechanisms might be potentially associating something like 'early dysregulation' with the onset of autism, taking into account how factors such as early feeding practices/issues for example, might provide at least one avenue for future study (see here).

To close, in light of some recent media headlines about the 'myth' that autism rates are on the up (and quite significantly so over past two decades), I offer some past posts suggesting that the word 'myth' should be reserved for other [non-peer-reviewed] matters (see here and see here and see here) and not this particular branch of epidemiological science. As to what may be 'causing' the upswing in numbers of diagnosed cases, well, it's likely to be very, very, very complicated (and without any need for sweeping generalisations please)...

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[1] Willfors C. et al. Medical history of discordant twins and environmental etiologies of autism. Transl Psychiatry. 2017 Jan 31;7(1):e1014.

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Willfors C, Carlsson T, Anderlid BM, Nordgren A, Kostrzewa E, Berggren S, Ronald A, Kuja-Halkola R, Tammimies K, & Bölte S (2017). Medical history of discordant twins and environmental etiologies of autism. Translational psychiatry, 7 (1) PMID: 28140403

Acetylation focus over methylation in autism epigenetics?

The paper by Wenjie Sun and colleagues [1] (open-access) provides the blogging fodder for today's post and although based on the science of epigenetics, the usual suspect - DNA methylation - gives way to another concept: histone acetylation with autism in mind. Before heading into the paper myself, I'll draw your attention to some other write-ups of the study including a hat-tip to Jeff Craig and his piece on the topic (see here).

So histone acetylation... I've covered the subject before on this blog (see here) but basically DNA, the stuff that carries the genetic blueprint, complexes with histones to form something called nucleosomes. It's a combination likened to thread wrapped around a spool. Continuing that thread wrapped around a spool analogy, protruding threads called histone tails can be modified in a chemical sense (via processes such as acetylation where an acetyl group is added or deacetylation where one is removed) which can subsequently affect genetic transcription.

Still with me? Good. Sun and colleagues set out to look at histone acetylation in the context of autism; specifically in post-mortem brain samples donated from those deceased who were diagnosed with autism and whether they might show some important changes based on the use of "a histone acetylome-wide association study (HAWAS)."

Specific areas of the brain were assessed using the HAWAS approach - "prefrontal cortex (PFC), temporal cortex (TC), and cerebellum (CB)" - and researchers were looking for a specific type of acetylation mark called H3K27ac linked to gene activation. Based on brain samples from 94 participants ("45 ASD [autism spectrum disorder], 49 control"), a few details emerged:

• Despite the expected heterogeneity across the presentation of autism in terms of whether the diagnosis of autism was syndromic (secondary to an existing condition) or non-syndromic (idiopathic), the authors reported that approaching 70% of the autism cases "shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex." In other words, a not uncommon molecular signature in relation to histone acetylation seemed to be present in quite a few of the participant samples included for study.
• Although one needs to be a little cautious about making grand, sweeping claims about how such an 'acetylome signature' comes about, the authors reported "that ASD-specific differential acetylation is driven mostly by.. factors such as environmental influences, SNPs in trans (at a different locus), indels, and larger chromosomal variants." Note the term 'environmental influences' (something I'll come back to shortly).
• When it came to what types of genes were potentially being 'affected' by acetylation, the authors report on quite a diverse spread "involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity." Epilepsy and autism is a recurrent theme in the research and clinical literature (see here for example) so there are no great surprise there. 'Immunity' and autism is something else that keeps cropping time and time and time again (see here).
• I appreciate that the authors also acknowledge that whilst autism was the focus on the current work, they do also mention: "By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases." This opens up the idea that various different psychiatric/behavioural labels might show 'overlap' when it comes to the histone acetylome too.

Interesting stuff by all accounts. I do like the idea that autism research is continuing to look at other areas of gene expression outside of just structural issues to the genome being linked to the condition (or should that be plural). Aside from the fact that people don't walk around with their genes permanently stuck in the 'on or off position' in every tissue all the time, the whole epigenetics field is a welcome complement to more traditional genomics. The focus on gene expression being potentially 'modifiable' might also reunite genetics and environment too (see here).

Criticisms of the Sun study? Well, brain samples from the deceased are a precious resource but not without complications when it comes their use for science (see here). I appreciate that we don't have the technology to look at histone acetylation in real-time or real-life yet with the brain in mind but one has to be cautious about the results from the brains of the deceased who may have passed away for many different reasons. There is also the temptation to move the whole epigenetics 'thing' towards acetylation on the basis of such research, but the methylome still remains potentially important (see here) and probably for more than one reason (see here). Perhaps soon we'll see a study looking at more than one epigenetic factor with autism in mind?

Going back to the concept of 'environmental influences' mentioned in the Sun paper, there are some potentially important repercussions from study results such as these. As with the concept of DNA methylation, one of the important concepts linked to the science of epigenetics is that such chemical alterations affecting the expression of DNA are potentially modifiable. This could mean that particular environmental factors working at critical periods might affect acetylation and methylation patterns and onward the expression of certain genes pertinent to the presentation of something like autism or at least facets of autism. The other scenario is that certain 'conditions' or 'interventions' might 'reverse such changes. On that last point, I might bring in some previous discussions on this blog in relation to something called HDAC (histone deacetylase) inhibitors (see here) that, as their name suggests, have the ability to inhibit the action of histone deacetylases (they remove acetyl groups). Various classes of medicines are classed at HDAC inhibitors including something called valproic acid which has some autism research history (see here for example). It's not therefore beyond the realms of possibility that the actions of certain medicines or other non-genetic factors with an influence on acetylation could be a source for further research in this area.

Independent replication is the next stage in the research process here. Alongside marrying acetylation trends with methylation trends, I do also wonder whether more functional analysis of other tissue(s) outside of just the brain might also be revealing too. I might add that traditional structural genomic issues (all those SNPs and CNVs that are talked about) can still play a role and indeed, might show some association with epigenetic issues too (see here).

And with all this talk of epigenetics and the like, due credit needs to be given to those who've been talking about this for quite a while in the peer-reviewed domain [2]...

To close, we have one final look at Rogue One before touchdown...

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[1] Sun W. et al. Histone Acetylome-wide Association Study of Autism Spectrum Disorder. Cell. 2016. Nov 17.

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

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Sun, W., Poschmann, J., Cruz-Herrera del Rosario, R., Parikshak, N., Hajan, H., Kumar, V., Ramasamy, R., Belgard, T., Elanggovan, B., Wong, C., Mill, J., Geschwind, D., & Prabhakar, S. (2016). Histone Acetylome-wide Association Study of Autism Spectrum Disorder Cell, 167 (5), 1385-2147483647 DOI: 10.1016/j.cell.2016.10.031

On HERV-H, autism, ADHD and methylphenidate?

Today's post is a bit of a mash-up including two paper: the first from Emanuela  Balestrieri and colleagues [1] (open-access available here) talking about "increased HERV-H [Human Endogenous Retroviruses - H] transcriptional activity in all autistic patients" included in their cohort (author's words not mine) and the second from D'Agati and colleagues [2] (open-access available here) describing "the reduction of HERV-H expression and the significant improvement of ADHD [attention-deficit hyperactivity disorder] symptoms after 6 months of methylphenidate treatment."

Taken together, both papers provide some potentially important information on how those fossil viruses that litter the human genome might not be as redundant as we might have first thought. Also how some of the commonly used medications to treat/manage certain psychiatric labels might have quite a few more effects than those listed on the package insert. A shocker indeed.

I've covered HERVs a few times on this blog in relation to quite a few labels (see here and see here and see here). If you've clicked on that first link, you'll know that this is not the first time that Balestrieri et al have talked about HERVs with autism in mind [2]. On that first occasion, they even went as far as proposing that "HERV-H expression be explored in larger samples of individuals with autism spectrum in order to determine its utility as a novel biological trait of this complex disorder." This time around "the transcriptional activity of three human endogenous retrovirus (HERV) families, in peripheral blood mononuclear cells (PBMCs)" was examined in 30 children diagnosed with autism spectrum disorder (ASD) and 30 asypmtomatic controls. Quantitative real-time PCR was the analytical weapon of choice, as "transcriptional levels of env of HERV families were quantitatively evaluated." As I've already mentioned, HERV-H expression showed some interesting trends compared to the not-autism controls. The authors note that this data from Albanian children is pretty much the same as what they found in Italian children diagnosed with autism.

The D'Agati findings - also including Balestrieri on the authorship list - although discussing a case report on what happened to HERV-H expression following use of methlyphenidate (MPH) in relation to ADHD, might also have some implications for [some] autism. Reiterating that this was a case report where both before and after HERV-H expression levels were measured, it potentially offers a road map for how HERV-H expression might be 'affected' by the use of certain medicines. Yes, I know that researchers only measured one variable (HERV-H) and one variable/measurement does not a link make. But given the quite significant overlap between ADHD and autism (see here) and the insinuation that over-expression of HERV-H might not necessarily be a 'good thing', one could see how further [independent] studies might be informative in this area.

Although slightly complicated by the fact that we are only beginning to realise how important HERVs might be to things like stem cells for example or even potentially being involved in the process of genetic deletion (see here), what is becoming clear is that these fossil viruses might be something to watch when it comes to health and wellbeing at different times of development. I've tried not to be too enthusiastic about HERVs and autism / ADHD / other (delete as appropriate) on this blog given our lack of understanding on any connection, specifically the hows and whys of any effect on either aetiology or symptoms. But it is getting harder not to wonder what role these and other mobile elements might play in development and behaviour, particularly in the context of HERVs being implicated in autoimmunity [3] (yes, that might also show a connection to some autism) and a possible role for the still emerging science of epigenetics in both HERV expression [4] and also [some] autism. There is lots more research to be done on this topic.

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[1] Balestrieri E. et al. Transcriptional activity of human endogenous retrovirus in Albanian children with autism spectrum disorders. New Microbiol. 2016 Sep;39(3):228-31.

[2] D'Agati E. et al. First evidence of HERV-H transcriptional activity reduction after methylphenidate treatment in a young boy with ADHD. New Microbiol. 2016 Sep;39(3):237-9.

[3] Tugnet N. et al. Human Endogenous Retroviruses (HERVs) and Autoimmune Rheumatic Disease: Is There a Link? The Open Rheumatology Journal. 2013;7:13-21.

[4] Lavie L. et al. CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2). J Virol. 2005 Jan;79(2):876-83.

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Balestrieri E, Cipriani C, Matteucci C, Capodicasa N, Pilika A, Korca I, Sorrentino R, Argaw-Denboba A, Bucci I, Miele MT, Coniglio A, Alessandrelli R, & Sinibaldi Vallebona P (2016). Transcriptional activity of human endogenous retrovirus in Albanian children with autism spectrum disorders. The new microbiologica, 39 (3), 228-31 PMID: 27602423


D'Agati E, Pitzianti M, Balestrieri E, Matteucci C, Sinibaldi Vallebona P, & Pasini A (2016). First evidence of HERV-H transcriptional activity reduction after methylphenidate treatment in a young boy with ADHD. The new microbiologica, 39 (3), 237-9 PMID: 27602426

A prenatal 'unhealthy' diet and offspring ADHD?

'Scientists study link between unhealthy pregnancy diet and ADHD' went one media headline covering the paper by Jolien Rijlaarsdam and colleagues [1].

The name of the research game was again to draw on data derived from ALSPAC (Avon Longitudinal Study of Parents and Children) (yes, again) to look-see "the degree to which prenatal high-fat and -sugar diet might relate to ADHD [attention-deficit hyperactivity disorder] symptoms via IGF2 DNA methylation for early-onset persistent (EOP) versus low CP [conduct problems] youth." In other words: "Experts examine how a diet high in fat and sugar could alter baby’s DNA in a way that might cause behavioural problems."

To do this meant looking at around 80 youth presenting with early-onset conduct problems compared with around 80 youths who didn't have the same magnitude of issues. Data on maternal nutrition captured for the two groups (ALSPAC did a lot of data collecting!) was cross-referenced with group status and also epigenetic - methylation - status of the insulin-like growth factor 2 gene (IGF2). IGF2 is a gene that seems to be pretty active during the nine months that made us but less so as we enter the big, wide world. As the name suggests it seems to have a 'growth' role which is probably why issues with this gene have also been associated with the development of a number of cancers.

Results: "Prenatal ‘unhealthy diet’ was positively associated with IGF2 methylation at birth for both the EOP and low CP youth." Minus any 'blame', such results suggest that maternal diet might be important for the developing child. I know this is it not exactly a shock, but in these days of more and more research and clinical focus on the special time called pregnancy, the idea that particular epigenetic changes might come from a chosen diet is a potentially important one.

Further: "For EOP only: (a) higher IGF2 methylation predicted ADHD symptoms; and (b) prenatal ‘unhealthy diet’ was associated with higher ADHD symptoms indirectly via higher IGF2 methylation." The higher IGF2 methylation - higher ADHD symptoms is an interesting association. Methylation - the addition of a methyl group - is normally taken to mean gene silencing suggesting that a malfunction of the the IGF2 gene and/or lower levels of its protein product might have some important implications. At this point I might add that whilst there is a bit of a research gap when it comes to IGF2 and behaviour specifically linked to ADHD, there is some interesting animal research looking at mice engineered to show low levels of the protein product in terms of behaviours such as anxiety [2] alongside "a role for the placenta in long-term programming of emotional behaviour." Cutting edge stuff to be sure.

"At present, this is not a study that would change my clinical practice, but if intervention studies resulting from this work show nutritional support in pregnancy can have an effect then we should take any opportunity we can to help." That was one comment from a physician discussing the results of the Rijlaarsdam study that I would agree with. The fact that the study focuses on just one gene in amongst the thousands potentially linked to conduct problems and ADHD is something to bear in mind.

Set however within the context that food can seemingly also affect behaviour and psychiatry as well as physiology (see here for example) I'd like to think that more studies on nutrition during pregnancy would be forthcoming and quickly on this important topic.

To close, thanks to Gene for the laughter...

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[1] Rijlaarsdam J. et al. Prenatal unhealthy diet, insulin-like growth factor 2 gene (IGF2) methylation, and attention deficit hyperactivity disorder symptoms in youth with early-onset conduct problems. J Child Psychol Psychiatry. 2016 Aug 18.

[2] Mikaelsson MA. et al. Placental programming of anxiety in adulthood revealed by Igf2-null models. Nat Commun. 2013;4:2311.

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Rijlaarsdam J, Cecil CA, Walton E, Mesirow MS, Relton CL, Gaunt TR, McArdle W, & Barker ED (2016). Prenatal unhealthy diet, insulin-like growth factor 2 gene (IGF2) methylation, and attention deficit hyperactivity disorder symptoms in youth with early-onset conduct problems. Journal of child psychology and psychiatry, and allied disciplines PMID: 27535767

No association between [current] mycotoxin exposure and autism

Although research stories stating a link (an association if you will) between condition A and factor X make for interesting reading, not all science is so blessed with such news-worthy findings. That's not to say that 'negative findings' are any less important than the 'hey, we found this...' studies, just that they don't perhaps tend to grab the headlines as much as those finding something.

In saying all that I'm standing up for negative findings today and some rather interesting science reported by Jennifer Duringer and colleagues [1] (open-access available here) on the lack of any significant relationship between current urinary mycotoxin content and their cohort of 25 young people diagnosed with an autism spectrum disorder (ASD).

Just in case you didn't already know, mycotoxin refers to "“natural” environmental contaminants, defined as secondary metabolites produced by fungi that reside in our food supply and on every surface in our environment." There are various types of mycotoxins (lots!) that occur as a consequence of various exposure patterns including fungi/moulds growing on foods or being present in our environment. Generally speaking, mycotoxins are something to be avoided (being careful with any sweeping generalisations there).

Duringer et al started their research journey on the basis that weather conditions pertinent to mycotoxin production have been linked to autism (see here) and some small-scale study that "suggested that individual exposure to mold increased the severity of neurophysiological abnormalities seen in autistic children" [2]. I know that furrowed brows will ensue when one talks about precipitation correlating with autism as per 'other correlations' but that was the basis for their study and should so be respected.

The technology used to test participants' urine sample for any presence of mycotoxin is not to be sniffed at as yet again (see here) tandem mass spectrometry steps up to the analytical plate. Some 87 mycotoxins were assayed for based on some previously published work and some important details such as the fact that urine samples were run in triplicate (even six times if you count the fact that each replicate was run in positive and negative ion modes) are included as part of the study protocol.

Results: given the fact that the cohort consisted of 25 young people with autism and 29 age-matched controls, one has to be a little cautious about making too many sweeping generalisations. What we can say is this: 20% of the participants with autism (5/25) came up with a 'positive' sample defined as 'any mycotoxin' being present. This contrasted with 14% of controls (4/29). The amounts detected were low; in some cases very close to or below the limit of quantitation where suitable standards could be obtained and used as comparators. When pooled together, those 9 participants with a positive sample were compared with the participants with a negative result to see if any potential correlates might show statistical significance. They didn't.

There are caveats to this work outside of the small participant group included for study. The authors highlight a particularly important one insofar as their focus on young adults "well after the in utero or infant-toddler exposure window when ASD develops and is diagnosed." In other words, the burden of current exposure to mycotoxins with autism in mind is probably low but that does not mean that "exposure windows" at other times might not exert an important effect. Indeed, other authors have speculated on other correlates [2] linked to the science of epigenetics for example, as perhaps being another area ripe for further study. This should be added to the list.

For now however, we can only base conclusions on the available (peer-reviewed) evidence...

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[1] Duringer J. et al. No Association between Mycotoxin Exposure and Autism: A Pilot Case-Control Study in School-Aged Children. Toxins (Basel). 2016 Jul 20;8(7). pii: E224.

[2] Mezzelani A. et al. Ochratoxin A as possible factor trigging autism and its male prevalence via epigenetic mechanism. Nutr Neurosci. 2016;19(1):43-6.

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Duringer J, Fombonne E, & Craig M (2016). No Association between Mycotoxin Exposure and Autism: A Pilot Case-Control Study in School-Aged Children. Toxins, 8 (7) PMID: 27447670