Another blood test for autism?

"These results form the foundation for the development of a biochemical test for ASD [autism spectrum disorder] which promises to aid diagnosis of ASD and provide biochemical understanding of the disease, applicable to at least a subset of the ASD population."

OK, use of the word 'disease' in the context of autism is really, really not OK in this day and age. Researchers, peer reviewers and their publishing journals should be doing something about this kind of language. There are however some potentially important aspects to the work published by Daniel Howsmon and colleagues [1] worth talking about. Not least is their observation on how "folate‐dependent one carbon metabolism (FOCM) and transsulfuration (TS) pathways" that have been quite readily *associated* with autism might be linked to quite a bit more than just uncovering the biochemistry of at least some autism (see here for example).

Before progressing further into these findings, I note there has already been some media interest in them (see here) with a byline reading: "First physiological test for autism proves high accuracy in second trial." We'll see about that...

So, after quite a long introduction about 'biomarkers for autism' and how they "come with their own set of challenges before they reach clinical translation", authors report further results building on some of their previous work in this area [2] that I've already covered on this blog (see here). On that previous research occasion, the suggestion was that between 5 and 7 metabolites linked to folate and/or transsulfuration pathways provided a 'best fit' when it came to picking out children diagnosed with autism from those not diagnosed with autism.

This time around, there was an 'extension' to that work: "(a) By comparing univariate analysis with four different multivariate methods on FOCM/TS data for ASD biomarker development to ensure that the identified results are not restricted to FDA [Fisher Discriminant Analysis] and (b) to test and validate multivariate FOCM/TS biomarkers on data collected from a new cohort of ASD participants." The words 'training data' and 'validation data' are used quite a bit throughout the Howsmon article, illustrating how different statistical classification methods were initially applied to training data from the cohort used in their first paper, which were then tested on a new cohort of participants (n=154) diagnosed with an ASD. Given some of the names included on the authorship list, it's no surprise that participant data with regards to the metabolites being looked at were drawn from other studies looking at the possible clinical value of preparations like folinic acid (see here) and sapropterin (see here) with autism in mind.

When those different statistical classification methods were applied and data was crunched, a few observations were made. The headline result was that one model/method produced the best 'potential' biomarker results and it was the same/similar method to that previously discussed by the authors. To quote: "An FDA model using five variables was shown to slightly outperform the other models on this new validation data set." That being said, the accuracy rates (including false positive and false negative rates) hovering around the high 80%s have to take into account that two of the metabolites thought to be important on the last research occasion - % DNA methylation and 8‐OHG - "were not present in the validation set" on this research occasion. This is a pity and a weakness of the current study.

So, do we at last have a 'physiological test' with 'high accuracy' for picking out autism from not-autism? Erm, not quite yet. With all due respect to the authors, their data is interesting and does partially back up their original findings, but we're not quite there yet with regards to rolling out any sort of biological test for autism. Indeed, in these days of the plural 'autisms' (see here) and acknowledging that the diagnosis of autism rarely presents in some sort of diagnostic vacuum (see here) it could be worthwhile re-evaluating whether we're ever likely to see a 'one biological test to diagnose them all' situation.

Further investigations are however indicated and of course, this more recent information does add to the quite rich data already generated suggesting that quite a bit more focus on things like methionine, homocysteine, cysteine and glutathione in relation to autism could be an important research path to follow. I'm also minded to suggest that different research teams taking on a 'possible biomarker for autism' type research perhaps need to talk more to each other (see here) pooling findings, resources and perhaps participant groups too...

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[1] Howsmon DP. et al. Multivariate techniques enable a biochemical classification of children with autism spectrum disorder versus typically‐developing peers: A comparison and validation study. Bioengineering & Translational Medicine. 2018. May 14.

[2] Howsmon DP. et al. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS Comput Biol. 2017 Mar 16;13(3):e1005385.

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Risperidone administration affects homocysteine levels in first episode schizophrenia

Of the many themes that seem to quite consistently surface on this blog looking at the peer-reviewed science in relation to various behavioural/psychiatric labels, the idea that various pharmacological interventions might do quite a bit more than 'what they say on the tin' is a recurring one. Take one case in point (see here)...

Today's offering adds to that theme as the results reported by Ning Fan and colleagues [1] are presented for your reading pleasure and the particular finding that: "Serum Hcy [homocysteine] levels were significantly decreased in schizophrenia patients after risperidone treatment."

OK, a few points need explaining first. Homocysteine is a compound that has also received considerable attention on this blog as per it's involvement in a particular group of intersecting biological cycles with relevance to various diagnostic labels including schizophrenia (see here for example). Alongside other independent pieces of research (see here), the diagnosis of schizophrenia seems to have some important connections to homocysteine although the precise details still need to be properly elucidated.

High levels of homocysteine are generally not thought to be a good thing for quite a few reasons [2] and that goes as much for persons diagnosed with schizophrenia as it does for everyone else. So when elevated plasma levels of homocysteine are recorded, medicine really does need to do something to (a) identify why and (b) try and bring levels back to within a more typical range.

Fan et al reported a few important things in their study of 56 participants "first-episode and drug-naïve inpatients with schizophrenia" compared with a similar number of sex- and age-matched asymptomatic controls. First, plasma homocysteine levels were elevated in those with schizophrenia. Nothing new there. They also reported on some potentially interesting *associations* between elevated homocysteine levels and specific symptoms relevant to schizophrenia: "a significant positive correlation between Hcy levels and PANSS negative sub-score was observed." Finally, they reported that risperidone use seemed to affect homocysteine levels in their clinical group. Interesting.

That last point is rather intriguing. Risperidone is a treatment of choice as per its second generation antipsychotic label and accompanying properties. Despite quite a lot of focus on the side-effects associated with such a medicine (see here for example) it does serve an important purpose for many people. It's mode of action is still under debate but it is generally thought to have some action in relation to effects on one or more neurotransmitter receptors. But... other potential effects have also been noted in the peer-reviewed research literature pertinent to mode of action including some in relation to immune function (see here) and how schizophrenia (some schizophrenia?) *might* have a significant immune component attached to it (see here for example). The Fan results add another potential mode of action to risperidone use and why it may impact on symptom presentation for some diagnosed with schizophrenia and related conditions. I'm not by any means saying that homocysteine is 'causative' of schizophrenia but the Fan results do suggest that quite a few more investigations may be warranted on the connection between schizophrenia, homocysteine and risperidone. Indeed, whether also other homocysteine-lowering compounds may provide some novel intervention options for some diagnosed with schizophrenia (see here) (with no medical or clinical advice given or intended).

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[1] Fan N. et al. Effect of Risperidone on Serum Homocysteine Levels in First-episode, Drug-naïve Patients with Schizophrenia. Neurosci Lett. 2017 Apr 15. pii: S0304-3940(17)30326-9.

[2] Zhang D. et al. Elevated Homocysteine Level and Folate Deficiency Associated with Increased Overall Risk of Carcinogenesis: Meta-Analysis of 83 Case-Control Studies Involving 35,758 Individuals. PLoS ONE. 2015; 10(5): e0123423. 

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Fan N, Tan Y, Yang F, Tian L, Chen S, Li J, Wang Z, & Zhang X (2017). Effect of Risperidone on Serum Homocysteine Levels in First-episode, Drug-naïve Patients with Schizophrenia. Neuroscience letters PMID: 28419824

Folate-dependent one carbon metabolism and transsulfuration pathways: biomarkers for autism?

I found it a little unusual that the findings reported by Daniel Howsmon and colleagues [1] (open-access) talking about "multivariate statistical analysis presented herein [provided] unprecedented quantitative classification results for separating participants into ASD [autism spectrum disorder] and NEU [neurotypical] cohorts based solely on biochemical data" merited a rapid reply in a prominent science magazine pouring cold water on the results (see here). Not least because one of the commentators interviewed in said science magazine article is also not seemingly immune when it comes to sweeping claims being made on the basis of preliminary research findings about autism (see here as per the previous headline: 'Super-parenting' improves children's autism).

No mind, the Howsmon paper - including a notable research name on the authorship list - mentions a few important compounds and biological processes in their discussions on: "Stepping towards this goal of incorporating biochemical data into ASD diagnosis." The sorts of things covered included various biological 'markers' pertinent to folate-dependent one-carbon metabolism (FOCM) and transsulfuration (TS) some of which have been fodder for this blog previously (see here and see here for examples). Researchers looked at these various compounds in blood samples from some 80 children diagnosed with an ASD and compared levels with 47 siblings and 76 age-matched controls. They applied some nifty statistics to try and determine whether any combination of the 24 analytes examined might be potential biomarker-material for an autism diagnosis. You'll note that once again the quite problematic binary description of 'neurotypical' was used to define 'not-autism' leading onwards to the inevitable questions: 'what is neurotypical?' and 'what are the boundaries of being neurotypical?' Sensible [evidence-based] answers on a postcard please.

Results: "FDA [Fisher Discriminant Analysis] on seven metabolites allows sufficient separation such that a linear classifier can correctly resolve 96.9% of participants." But actually this was not the whole story as the authors also report that five compounds/variables - GSSG, tGSH/GSSG, Nitrotyrosine, Tyrosine, and fCysteine - provided the best 'fit' when it came to potentially picking out children with autism. You might note that some of those 'famous five' have some autism research history (see here). The authors similarly note that: "these variables are affected by high quality vitamin supplementation that also decreases ASD severity in at least a subset of cases." Mmm.

There is definitely more science to do in this area. Biomarkers in relation to autism have come and gone down the years (see here for example) and I'm not altogether sure that using the label 'autism' as a starting point for this kind of research is necessarily the best idea (see here). Outside of just the heterogeneity and plurality - the autisms - associated with the label autism, there are other considerations to take on board such as the impact of all that over-represented comorbidity too (something that continues to 'mess around' with various 'autism is linked to..' studies).

But that shouldn't stop further efforts in this area including those also looking to expand into the 'genetics' of folate metabolism alongside the biochemistry, as per everyone's favourite scrabble word 'MTHFR' (see here) and its [meta-analysed] potential contribution to some autism. I agree that we are not quite there when it comes to folate metabolism as providing a generic biomarker or set of biomarkers for autism, but there again, the authors never said that it definitively did: "it should be noted that these studies should be replicated and empirically tested on a wider scale before more definite conclusions can be drawn." Too true but the Howsmon results represent an interesting first attempt...

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[1] Howsmon DP. et al. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS Comput Biol. 2017 Mar 16;13(3):e1005385.

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Howsmon DP, Kruger U, Melnyk S, James SJ, & Hahn J (2017). Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS computational biology, 13 (3) PMID: 28301476

L-methylfolate administration and autism: a case report

I should have really titled this post 'another case report' given yesterday's entry on this blog talking about a case of [untreated] PKU and autistic behaviours/diagnosis. Here I am again talking about another N=1 with autism in mind and specifically the findings reported by Kim Siscoe & David Lohr [1] on how: "L-methylfolate supplementation improved symptoms of aggression and disruptive behavior in a child with autism who tested positive for the C677TT allele of the methyltetrahydrofolate reductase enzyme gene."

First things first. This was a case report; please keep that in mind. Second, I am not a medical doctor and don't provide medical or clinical advice on this blog. Within those caveats I am however very interested in the Siscoe/Lohr observations.

Why? Well, methylene tetrahydrofolate reductase (MTHFR) (gene and enzyme) has featured quite a bit on this blog in light of findings linking gene and enzyme to cases of autism (see here and see here for examples). The idea is that MTHFR serves a primary function in reducing the compound 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate - another name for L-methylfolate -  the reduced and methylated form of folic acid, is an important methyl group donor for the recycling of homocysteine back to methionine utilising vitamin B12 along the way (see here for a nice hand drawn graphic). The implications of disruptions to MTHFR (gene and enzyme) are potentially multiple but include effects on methyl group donor ability (methyl groups potentially linked to things like DNA methylation as part of all that epigenetics jazz that you hear so much about these days) and effects on downstream metabolites such as those related to homocysteine metabolism (see here).

So Siscoe & Lohr present data on what happened when the active form of folate was supplemented following the identified genetic issue with the MTHFR gene potentially affecting typical production of L-methlyfolate.

Where next with this work? Well, it stands to reason that in these days of personalised medicine percolating through to autism research and practice (see here), knowledge about a potential genetic issue identified in [some] cases of autism should be further investigated. We have other examples (see here). I'd like to see larger and more controlled trials of L-methlyfolate supplementation in relation to autism for example, based on screening for issues with the MTHFR gene. I'd like to see a few more biological measures incorporated in such study looking at other aspects of the folate and related cycles too (see here). I'd also like to see more discussion about any long-term implications and/or adverse effects associated with such supplementation along the lines of: should we really be tinkering with mechanisms linked to DNA methylation? Also in relation to some of the other diagnoses associated with issues with MTHFR there is similarly important work emerging [2] which could be quite important in certain instances...

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[1] Siscoe KS. & Lohr WD. L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele. Psychiatr Genet. 2017 Mar 7.

[2] Roffman JL. et al. Biochemical, physiological and clinical effects of l-methylfolate in schizophrenia: a randomized controlled trial. Mol Psychiatr. 2017. Mar 14.

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Siscoe, K., & Lohr, W. (2017). L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele Psychiatric Genetics DOI: 10.1097/YPG.0000000000000170

Hyperhomocysteinemia as a significant risk factor for autism?

The findings reported by Naushad Shaik Mohammad and colleagues [1] provide some blogging fodder today and the suggestion of a link between some of the genetics of the folate pathway and the finding of elevated levels of homocysteine with [some] autism in mind.

OK, from the start, the genetics of folate metabolism mentioned in the context of autism typically means reference to the quite well replicated finding of issues with the gene methylenetetrahydrofolate reductase (MTHFR) (see here for some background). This gene (product) serves an important purpose in relation to the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate; the latter helping to convert the amino acid homocysteine to methionine. Outside of the importance of methionine to the process of DNA methylation (yep, some of that epigenetics stuff that you keep hearing about), there is quite a body of literature emerging to suggest that elevated levels of homocysteine might also have some important health effects.

For quite a few years now, a specific genetic issue with MTHFR - MTHFR C677T - has been reported in quite a few people on the autism spectrum (see here). This allied to other independent research suggesting that the downstream effects of issues with MTHFR linked to elevations in levels of homocysteine may not also be an uncommon finding (see here). Shaik Mohammad et al therefore set about looking at the relationship between genetic issues with MTHFR and hyperhomocysteinemia in the context of autism.

They did this by use of an "artificial neural network (ANN) model" where data initially from "138 autistic and 138 nonautistic children" on various genetic issues linked to folate metabolism (including MTHFR) were used as potential "predictors of autism risk." We are also told that: "Meta-analyses were carried out on 1361 ASD children and 6591 nonautistic children to explore the association of MTHFR C677T and homocysteine with the risk for ASD [autism spectrum disorder]."

Results: well, the model wasn't exactly brilliant at predicting the risk of autism (63.8% accuracy). The authors call this a 'moderate' finding but I'd probably suggest that their results are yet another very good example of how heterogeneous the autism spectrum actually is. The idea of not using the term 'autism' as a research starting point in this context (see here) also receives support. Perhaps of greater importance were their findings linked to homocysteine and autism and how: "Hyperhomocysteinemia was observed in autistic children" to a greater extent that controls. They did also confirm that the MTHFR C677T genetic polymorphism was linked to 'inflating homocysteine levels' alongside another genetic issue called MTRR A66G (methionine synthase reductase). This is not an unusual finding in the context of what is already known about MTRR and homocysteine. The MTRR bit potentially linked to autism is however, something that this research group have previously suggested to 'reduce the risk' of autism [2].

In terms of what these results mean in the context of autism, there are a few possibilities. First, screening. Knowing what we now seem to know about MTHFR and homocysteine in relation to quite a lot of people with autism, I would have thought it would be good practice to screen genetics/biochemistry. Minus any scaremongering or sweeping generalisations, the observation that hyperhomocysteinemia 'may' have links to cardiovascular disease and other adverse states for example, also perhaps implies screening save any further charges of health inequality when it comes to the label of autism. Next management. Far from being a 'nothing can be done about it' state, there is some good evidence that small adjustments to nutrition can potentially have positive effects on some of these parameters. With no medical or clinical advice given or intended, high levels of homocysteine seem in some cases, to be reactive to certain vitamin supplementation. The focus on vitamin B12 could also be set in the context of other recent studies of this vitamin (and its vitamers) with autism in mind (see here) (but I am careful not to link the two parameters just yet). And just recently there is news that there is a new way of assaying for vitamin B12 on the horizon which could also be useful. Finally, more research is indicated. As per my discussions not so long ago about another potentially important link to folate metabolism and autism (see here), there does appear to be quite a bit more to see when it comes to the folate cycle intersecting with homocysteine metabolism (and it's downstream effects). Yes, we can talk about whether folate is 'protective' or not when it comes to 'risk' of autism (see here and see here) but what this latest work suggests is that this area is complicated and potentially includes many genetic/epigenetic/biochemical variables that need to be taken into account.

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[1] Shaik Mohammad N. et al. Clinical utility of folate pathway genetic polymorphisms in the diagnosis of autism spectrum disorders. Psychiatr Genet. 2016 Oct 17.

[2] Mohammad NS. et al. Aberrations in folate metabolic pathway and altered susceptibility to autism. Psychiatr Genet. 2009 Aug;19(4):171-6.

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Shaik Mohammad N, Sai Shruti P, Bharathi V, Krishna Prasad C, Hussain T, Alrokayan SA, Naik U, & Radha Rama Devi A (2016). Clinical utility of folate pathway genetic polymorphisms in the diagnosis of autism spectrum disorders. Psychiatric genetics PMID: 27755291

NAC for 'social impairment' in youth with autism... probably not

"The results of this trial indicate that NAC [N-acetylcysteine]  treatment was well tolerated, had the expected effect of boosting GSH [glutathione] production, but had no significant impact on social impairment in youth with ASD [autism spectrum disorder]."

So said the results reported by Logan Wink and colleagues [1] (open-access) who, continuing an autism research theme, looked at whether this important L-cysteine prodrug might have more to give when it comes to at least some facets of some autism. Their trial registration entry can be found here. Reporting results from "a 12-week randomized, double-blind, placebo-controlled trial of oral NAC in youth with ASD" researchers followed some 30 children (aged 4-12 years) diagnosed with autism based on their use of NAC or a placebo. Alongside various biological measures including blood levels of "reduced and oxidized glutathione (GSH and GSSG)" and the big 'H' (homocysteine), the primary outcome was the effect on "the CGI-I scale anchored to study physician assessment of core social impairment considering the individuals’ overall level of cognitive, adaptive, and social functioning." I might add that other secondary behavioural outcomes were also included for study.

Bearing in mind the loss (dropping out) of some participants during the study period including details that "three withdrew due to irritability (NAC), diarrhea and encopresis (placebo), and defiant and self-injurious behavior (placebo), respectively" the authors report data on 25 study completers. Titrated doses of NAC depending on tolerance and body weight of participants did seem to do what they were supposed to in terms of an effect on levels of glutathione: "had the expected effect of boosting GSH production in peripheral blood" at 12 weeks.

But... on every other measure - biological and behavioural - there were no significant differences between the NAC and placebo groups leading the authors to conclude that their results "do not support the use of NAC for treatment of core social impairment of ASD." The research door does however remain open as they also comment that: "the health impact of the resultant increase on GSH remains unclear."

These are interesting results. NAC has seemingly found something of a research place in quite a few areas of psychiatry including 'some' autism (see here) and 'some' schizophrenia (see here). The focus has tended to be more on the 'irritability' side of things (see here) which makes it all the more surprising that one of the participants in the Wink trial receiving NAC actually withdrew 'due to irritability'. That being said, the very controlled nature of the Wink study cannot be readily ignored assuming no influence from the placebo [2].

I would like to see a little more on the whole NAC -- cysteine -- glutathione connection given previous discussions with autism in mind (see here). This includes the idea that blood levels of the various compounds involved might not be the same as 'brain levels' and taking into 'level of functioning' into account (see here). But in the context of the Wink data, one has to perhaps realise that NAC is not likely to be a panacea when it comes to autism...

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[1] Wink LK. et al. A randomized placebo-controlled pilot study of N-acetylcysteine in youth with autism spectrum disorder. Molecular Autism. 2016; 7:26.

[2] Masi A. et al. Predictors of placebo response in pharmacological and dietary supplement treatment trials in pediatric autism spectrum disorder: a meta-analysis. Transl Psychiatry. 2015 Sep 22;5:e640.

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Wink, L., Adams, R., Wang, Z., Klaunig, J., Plawecki, M., Posey, D., McDougle, C., & Erickson, C. (2016). A randomized placebo-controlled pilot study of N-acetylcysteine in youth with autism spectrum disorder Molecular Autism, 7 (1) DOI: 10.1186/s13229-016-0088-6

Meta-meta-analysing MTHFR and autism

"In conclusion, [the] present meta-analysis strongly suggested a significant association of the MTHFR C677T polymorphism with autism."

So said the findings reported by Vandana Rai [1] as yet more discussion emerges on the possible role of issues with the methylenetetrahydrofolate reductase (MTHFR) gene in relation to at least some autism. The reason I've titled this post as a 'meta-meta-analysis' is because we've previously seen meta-analysis done on this polymorphism (SNP) in relation to autism as per other entries on this blog (see here). That and other entries will also provides readers with a little more background on what MTHFR does and why it might be so important to some autism.

This time around Rai looked at 13 studies hitting the criteria for study entry covering nearly 2000 people diagnosed on the autism spectrum compared with over 7000 asymptomatic - not autism - controls. Looking at the various genetic combinations based on zygosity, the author concluded that in both Caucasian and Asian populations, there was an association between the C677T polymorphism and autism.

Where next you might ask? Well, a few possible directions are potentially indicated. First is the idea that screening for the MTHFR C677T SNP does seem to be indicated when a diagnosis of autism is received. By saying that, I'm not suggesting that science has discovered 'a gene for autism' or anything like that (not unless you, for example, count some cases of schizophrenia within that definition of autism as per other work from Rai [2]) but alongside other genetic issues associated with some autism (see here), there is a possible target gene to look at/for. Additional screening of extended family members such as parents and siblings for this SNP might also be similarly indicated, not least because of the various other 'conditions' linked to this SNP [3] and implications for preventative treatment targeting elevated homocysteine for example.

Second, as and when issues with MTHFR are detected, there are some potentially important implications for things like folic acid metabolism (yes, that has been linked to some autism too with some caveats) and potentially onwards the availability of things like methyl groups for important processes such as DNA methylation (again, something looked at with autism in mind). I might also mention some of the literature on homocysteine specifically related to autism might also be something linked here (see here) given the positioning of this compound in relation to the folate and methionine metabolic cycles. Although still in its infancy, talk of MTHFR issues when identified as being part of a 'personalised medicine' approach to autism (see here) provide an important thinking/talking point.

I'd like to think that the MTHFR C677T SNP might offer some quite important clues to at least some types of autism as and when further research is undertaken. With the continuing advances being made using the CRISPR-Cas9 gene editing tool and the ever-increasing CRISPR zoo, the modelling of the MTHFR C677T SNP is set to become quite a bit easier in times to come, and may eventually provide some important advances pertinent to autism and beyond.

Yet again, screening is the first port of call as and when a diagnosis of autism is [eventually] received...

To close, following the very sad news that Ronnie is now with Ronnie, I think it's appropriate to light four candles...

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[1] Rai V. Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility. Metab Brain Dis. 2016 Mar 8.

[2] Yadav U. et al. Role of MTHFR C677T gene polymorphism in the susceptibility of schizophrenia: An updated meta-analysis. Asian J Psychiatr. 2016 Apr;20:41-51.

[3] Rajagopalan P. et al. Common folate gene variant, MTHFR C677T, is associated with brain structure in two independent cohorts of people with mild cognitive impairment. NeuroImage : Clinical. 2012;1(1):179-187.

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Rai, V. (2016). Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility Metabolic Brain Disease DOI: 10.1007/s11011-016-9815-0

Methyl B12 for autism? Placebo-controlled results say maybe...

"Methyl B12 treatment improved clinician-rated symptoms of ASD [autism spectrum disorder] that were correlated with improvements in measures of methionine metabolism and cellular methylation capacity."

Those were the very encouraging results published by Robert Hendren and colleagues [1] who can now update their ClinicalTrials.gov study entry (see here). Building on the ideas that: "Children with autism spectrum disorder (ASD) have been reported to have reduced ability to methylate DNA and elevated markers of oxidative stress" (topics that have been covered on this blog before), researchers undertook a gold-standard trial - randomised, placebo-controlled - to ascertain the effect (if any) of "8 weeks of treatment with methyl B12 (75 μg/kg) or saline placebo every 3 days in a subcutaneous injection." The success of the treatment was measured by "the Clinical Global Impressions-Improvement (CGI-I) score" accompanied by "changes in the Aberrant Behavior Checklist (ABC) and the Social Responsiveness Scale (SRS)" scores. At the same time, researchers also looked at various biochemical parameters pertinent to "methionine methylation and antioxidant glutathione metabolism."

Based on the 50 children ("mean age 5.3 years") who completed the study, researchers reported a trend of improvement in autistic and related behaviours following the methyl B12 injections. Importantly, the primary outcome measure - the CGI-I scores - rated by clinicians, showed a trend of being "statistically significantly better (lower) in the methyl B12 group (2.4) than in the placebo group (3.1) (0.7 greater improvement in the methyl B12 group, 95% CI 1.2-0.2, p = 0.005)." Biological parameters also showed changes: "increases in plasma methionine (p = 0.05), decreases in S-adenosyl-l-homocysteine (SAH) (p = 0.007) and improvements in the ratio of S-adenosylmethionine (SAM) to SAH (p = 0.007), indicating an improvement in cellular methylation capacity" following the use of methyl B12 compared with placebo.

Accepting that 'subcutaneous injection' of methyl B12 is hardly a 'user-friendly' option and may very well scupper plans to use this particular intervention option for quite a few, these are potentially important results. I'm really quite interested in how vitamin B12 'vitamers' might show some links to at least some 'types' of autism (see here) including the measurement of 'brain levels' of the stuff (see here). The Hendren results suggest that quite a few more research resources might be needed in this area. I wonder also if this future research agenda would include the 'baby and bathwater' compound that is methylmalonic acid in relation to autism too (see here)?

I do also have to point out that previous research from members of this research team has not been so complimentary about the use of methyl B12 in cases of autism [2] despite the idea that there may be 'responders' to this type of intervention. To quote: "methyl B12 may alleviate symptoms of autism in a subgroup of children, possibly by reducing oxidative stress. An increase in glutathione redox status (GSH/GSSG) may provide a biomarker for treatment response to methyl B12." Such differences in reported results are not unfamiliar to autism research (the rule rather than the exception) but perhaps provides a further focus for clarification of effect.

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[1] Hendren RL. et al. Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. J Child Adolesc Psychopharmacol. 2016 Feb 18.

[2] Bertoglio K. et al. Pilot study of the effect of methyl B12 treatment on behavioral and biomarker measures in children with autism. J Altern Complement Med. 2010 May;16(5):555-60.

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Hendren RL, James SJ, Widjaja F, Lawton B, Rosenblatt A, & Bent S (2016). Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. Journal of child and adolescent psychopharmacology PMID: 26889605

Decreased brain levels of vitamin B12 in autism

I have to thank Dr Malav Trivedi for bringing my attention to some recent findings reported by Yiting Zhang and colleagues (including Malav) [1] (open-access) suggesting that: "levels of vitamin B12, especially its MeCbl [methylcobalamin] form, decrease with age in frontal cortex of control human subjects."

Further, researchers reported: "abnormally lower total Cbl [cobalamin] and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls." Some media on the findings can also be read here.

Working from the lab of Dr Richard Deth (quite a familiar name to this blog), researchers initially analysed a most precious sample medium (postmortem brain samples) obtained from various biobanks and including various patient groups. So alongside samples from 12 children with autism were samples from 9 people diagnosed with schizophrenia and some 43 'controls' with ages ranging between 19 weeks old and 80 years old. "Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites" accompanied by data derived from a knock-out mouse model: "the influence of decreased GSH [glutathione] production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70%."

Looking at postmortem frontal cortex brain samples, researchers reported that finding on levels of vitamin B12 - particularly the MeCbl vitamer -  decreasing with age. Bearing in mind the relatively small participant numbers included, the idea that lower brain tissue levels of total cobalamin and methylcobalamin were also present (almost unanimously) in the autism and schizophrenia groups could be important. I might at this point direct readers to previous discussions on vitamin B12 and autism on this blog (see here) including the research idea of supplementing (see here) with no medical advice given or intended.

There are a few other details worth pointing out from the Zhang findings. Analysis of thiols in brain samples across the autism vs control group revealed some potentially interesting data. So, methionine levels were quite a bit lower in the autism group [significantly lower] as were levels of "the methyl donor S-adenosylmethionine (SAM)." Both these compounds form an important part of the whole 'methylation of DNA' process (see here) among other things.

Glutathione, a compound that has seen its fair share of speculation with autism in mind (see here), was also on the research menu in the Zhang study. Interestingly and again bearing mind the small participant numbers, brain levels of this stuff were lower in the autism group as a whole but not significantly so when compared to controls. This finding might map on to other brain studies with autism in mind (see here). Likewise, cysteine (another potentially relevant compound to some autism) produced a similar finding.

I would encourage readers to take some time looking at the Zhang paper. In conjunction with other results reporting on some important elements to the emerging story (see here) I believe there are further studies to be done applicable to the notion that: "impaired methylation may be a critical pathological component" for at least some autism (see here). Indeed, other research papers have also discussed this issue [2]. The idea that studies about human ageing may likewise be informative to autism (and schizophrenia) research also carries quite a lot of traction too.

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[1] Zhang Y. et al. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797.

[2] Keil KP. & Lein PJ. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders? Environmental Epigenetics. 2016; 1-15.

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Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, & Deth RC (2016). Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PloS one, 11 (1) PMID: 26799654

Serum folate levels in schizophrenia meta-analysed

So: "In conclusion, the present meta-analysis found that folate deficiency is associated to SZ [schizophrenia], and subgroups which did not reach enough statistical power need further investigation in the future."

That was the research bottom line discussed in the paper by Dan Wang and colleagues [1] on a topic that has been of some interest to this blog down the years (see here and see here for example). Folate (folic acid if you will) is a pretty vital nutrient that, among other things, plays an important role in a particular cycle linked to the methylation of DNA. With schizophrenia specifically in mind, there is a body of peer-reviewed evidence emerging suggesting that folate levels might provide some important information about at least some cases bearing in mind the concept of plurality (see here).

Wang et al looked at quite a bit of that research literature on folate and schizophrenia and determined that "decreased serum folate was associated with SZ risk" even when taking into account variables such as when serum folate levels were sampled (i.e. in acute schizophrenia) and "measurement after drugs using in SZ patients." Ergo, science needs to know more about this potentially important association.

As per the various research looking at folate levels in relation to autism (see here for example) one has to tread quite carefully so as not to 'big up' any universal relationship between folate and schizophrenia. That for example, the folate cycle intersects with other potentially important compounds such as homocysteine with schizophrenia in mind (see here) is also something to bear in mind.

Music: Jack Garratt - Breathe Life.

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[1] Wang D. et al. Serum folate levels in schizophrenia: A meta-analysis. Psychiatry Res. 2015 Nov 25. pii: S0165-1781(15)30731-9.

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Wang D, Zhai JX, & Liu DW (2015). Serum folate levels in schizophrenia: A meta-analysis. Psychiatry research PMID: 26652840

Maternal folate status and offspring autism risk: where are we up to?

I'd like to briefly draw your attention to the review published by Elizabeth DeVilbiss and colleagues [1] today, covering "what is known about the role of folate in the aetiology of neurodevelopmental disorders."

Folate, is a topic that has graced this blog a few times with autism in mind (see here for example) based on various ideas that folate status during pregnancy might have the ability to modify offspring risk of autism [2] alongside the idea that autoimmune processes might act on folate receptors in some cases of autism (see here) and what this might subsequently mean for pathology / management. The specific idea that folate levels and folate supplementation during pregnancy might influence autism risk has garnered the most research attention, seemingly also crossing geographies too [3].

The DeVilbiss review is quite comprehensive in its scope and material covered, summarising "relevant biological, genetic and epigenetic mechanisms" and the various science that has been done so far on this topic. I would certainly agree with their sentiments that "existing evidence is inconclusive" (as previously indicated) in light of the numerous confounding variables also potentially linked to offspring autism risk. That being said, and acknowledging where folate metabolism sits in terms of areas such as MTHFR genetics (see here) and the whole vitamin B12 story (see here) and perhaps beyond (see here), I do think there is more to see in this area and perhaps outside of autism and related neurodevelopmental conditions (see here). Without jumping on the whole epigenetics bandwagon, the link between the folate cycle and DNA methylation in particular (see here) offers a whole slew of research ideas ripe for further investigation.

Music: Lost Frequencies - Are You With Me.

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[1] DeVilbiss EA. et al. Maternal folate status as a risk factor for autism spectrum disorders: a review of existing evidence. Br J Nutr. 2015 Aug 5:1-10.

[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] Surén P. et al. Association between maternal use of folic acid supplements and risk of autism spectrum disorders in children. JAMA. 2013 Feb 13;309(6):570-7.

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DeVilbiss EA, Gardner RM, Newschaffer CJ, & Lee BK (2015). Maternal folate status as a risk factor for autism spectrum disorders: a review of existing evidence. The British journal of nutrition, 1-10 PMID: 26243379

Homocysteine and autism: yet more...

It's been a while since I've discussed the issue of homocysteine - that's homocysteine not homocystine - with autism in mind, so consider this short blog entry a bit of an update to previous discussions (see here and see here).

In case you need to know it, homocysteine is an important component of the trans-sulfuration pathway intersecting with both the methione cycle and the folate cycle. Collectively, these biological processes have important functions for various aspects of biology including the process of methylation and the issue of oxidative stress leading into the important role of glutathione (see here) for example.

I was brought to this post following the publication of two recent paper from Carmen Puig-Alcaraz and colleagues [1] and from Yu Han and colleagues [2] that both reported on elevated levels of homocysteine to be present in their cohorts of children diagnosed with an autism spectrum disorder (ASD) compared with asymptomatic controls. These findings are pretty much in line with what most other research has reported in this area.

Puig-Alcaraz et al reported that alongside an overall increased level of urinary homocysteine in their cohort, there seemed to be something of a relationship between elevated urinary homocysteine with "the severity of the deficit in communication skills" in their participant group. Something that was not seen when looking at the other core areas of autism (social interaction and repetitive/restricted behaviour). I'm intrigued at the prospect that specific traits may be linked to something like elevated levels of homocysteine although recognise the need for far greater scrutiny of this finding with larger cohorts. That this group only measured homocysteine in urine is another issue that needs to be further explored.

Han et al report results based on Chinese children. This in itself is an important cohort suggesting that issues with homocysteine might cross geography and ethnicity when it comes to autism on the basis of other studies looking at different populations. Alongside reporting on elevations in homocysteine, researchers also noted that total levels of glutathione and cysteine were lower in the autism group; findings that accord with meta-analyses of glutathione and related compounds with autism in mind (see here). Further: "Hcy [homocysteine] levels correlated significantly with increasing CARS [Childhood Autism Rating Scale] scores and GSSG [oxidized glutathione] levels in children with ASD" with the proviso that further investigations are needed in this area.

The peer-reviewed evidence is indeed stacking up for something potentially fundamental at work when it comes to autism and homocysteine. At this stage it would be difficult to tease apart homocysteine alone as being 'linked' to [some] autism given the myriad of other compounds/pathways also potentially implicated as per the literature on the B vitamins and their important links to homocysteine. I'm also wondering whether another relation of one of the B vitamins, methylmalonic acid, that has languished in the autism science desert for far too long might also need resurrecting in future studies on the 'big H' and autism?

And yes, screening is important [3]...

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[1] Puig-Alcaraz C. et al. Increased homocysteine levels correlate with the communication deficit in children with autism spectrum disorder. Psychiatry Res. 2015 May 29. pii: S0165-1781(15)00290-5.

[2] Han Y. et al. Abnormal transsulfuration metabolism and reduced antioxidant capacity in Chinese children with autism spectrum disorders. International Journal of Developmental Neuroscience. 2015. July 3.

[3] Ranjan S. & Nasser JA. Nutritional Status of Individuals with Autism Spectrum Disorders: Do We Know Enough? Adv Nutr. 2015 Jul 15;6(4):397-407.

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Puig-Alcaraz C, Fuentes-Albero M, Calderón J, Garrote D, & Cauli O (2015). Increased homocysteine levels correlate with the communication deficit in children with autism spectrum disorder. Psychiatry research PMID: 26070768





Han, Y., Xi, Q., Dai, W., Yang, S., Gao, L., Su, Y., & Zhang, X. (2015). Abnormal transsulfuration metabolism and reduced antioxidant capacity in Chinese children with autism spectrum disorders International Journal of Developmental Neuroscience DOI: 10.1016/j.ijdevneu.2015.06.006

Genomic instability not linked to autism?

An eyebrow was raised upon reading the findings reported by Penelope Main and colleagues [1] concluding that: "it appears unlikely that genomic instability is a feature of the aetiology of autism." Based on results derived in part from "the cytokinesis-block micronucleus cytome (CBMN-cyt) assay" [2] looking at markers of DNA damage, authors reported very little to see in their small cohort of children with autism (n=35) compared with siblings (n=27) and asymptomatic controls (n=25) although with the requirement for: "replication using a larger cohort".

"Nah. I don't need one. I got a Donk".

Of equal interest to this blog was the discovery that there was no significant difference in B vitamins - outside of vitamin B2 - nor homocysteine (the 'big H') levels across the study groups. As regular readers might already know, I've covered homocysteine a few times on this blog with autism in mind (see here for example). Indeed, this authorship group have talked around this topic previously (see here).

Although no expert on the whys and wherefores of the CBMN-cyt assay outside of reading through the Fenech paper [2] and other material around the subject, I gather that this is quite a widely used method for measuring DNA damage covering: "(a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes".

A quick trawl of the other research literature in this area reveals that this is not the first time that members of this group have looked at DNA damage with autism in mind as per another paper by Main and colleagues [3] (including Michael Fenech on the authorship list). On that occasion, lymphoblastoid cell lines (LCLs) from an even smaller group of children with autism and their asymptomatic siblings (N=6 pairs) were analysed for the possible presence of "increased DNA damage events" following artificial challenge to an oxidative stressor (hydrogen peroxide) among other things. They concluded: "(i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally susceptible to DNA damage." The issue of oxidative stress and autism has been discussed quite a bit in the research literature (see here).

I would tend to agree that this is still an area of autism research deserving of further investigations on the basis of that proposed oxidative stress link. I might be further showing my incompetence in this area of endeavour by also referring you back to the paper by Shuvarikov and colleagues [4] and their suggestion that HERV (human endogenous retrovirus) elements may: "mediate other recurrent deletion and duplication events on a genome-wide scale" on the basis of their findings in relation to particular types of de novo deletions including autism as part of the clinical presentation. HERVs are something I've been quite interested in for some time now, with autism (see here), attention-deficit hyperactivity disorder (ADHD) (see here) and myalgic encephalomyelitis (ME) (see here) in mind. Other retrotransposons have also cropped up in more recent times too (see here). The relationship with DNA methylation taps into the rising star discipline that is epigenetics (see here) and potentially back to the reason why homocysteine was included in the most recent Main paper (see here for my lovely hand-drawn picture of the methylation cycle). Certainly with all the recent continued interest in de novo mutations potentially linked to autism [5] it strikes me that further scrutiny of this area is perhaps warranted.

Music then... Emeli Sandé - Next To Me.

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[1] Main PA. et al. Lack of Evidence for Genomic Instability in Autistic Children as Measured by the Cytokinesis-Block Micronucleus Cytome Assay. Autism Res. 2014 Nov 4. doi: 10.1002/aur.1428.

[2] Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc. 2007;2(5):1084-104.

[3] Main PA. et al. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress. Mutagenesis. 2013 Jul;28(4):475-84.

[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] Iossifov I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014 Oct 29. doi: 10.1038/nature13908.

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Main PA, Thomas P, Angley MT, Young R, Esterman A, King CE, & Fenech MF (2014). Lack of Evidence for Genomic Instability in Autistic Children as Measured by the Cytokinesis-Block Micronucleus Cytome Assay. Autism research : official journal of the International Society for Autism Research PMID: 25371234