Advancing grandparental age and autism risk

The paper by Emma Frans and colleagues* looking at autism risk across the generations is the focus of this post. Published in the journal JAMA Psychiatry alongside a provocative article by Andrea Roberts and colleagues** on maternal exposure to child abuse being "associated" with elevated risk for offspring autism (see here and here), the theme is transgenerational effects and quote: "that your father's and grandfather's lifestyle choices can affect you" as per some of media on this paper.

Ο Κακός Εγγονός @ Wikipedia  

I'm not going to head too heavily into the Frans study because others have already discussed it far better than I ever could (see here and here). Indeed NHS Choices carries a particularly good run-down of the study which is well worth a read (see here).

The main details were that based on an analysis of nearly 6000 cases of autism spectrum disorder (ASD) in Sweden, grandfathers who had fathered their daughter when aged 50 or above were 1.79 times more likely to have a grandchild diagnosed with autism than younger fathering grandfathers.

If grandfathers fathered a son when aged 50 or above, they were 1.67 times more likely to have a grandchild diagnosed with autism (again compared to grandfathers having children when they were younger).

The magic word 'epigenetics' is also mentioned to potentially account for results alongside the mutation side of things. I should also point out that Frans has published on similar things before with schizophrenia in mind***.

I'm interested in studies like the current Frans one despite their reliance on association and relatively limited increased risk of autism. Interested because alongside the 'older dads and autism risk' research (see here), I have actually talked about grandparents and risk of autism and schizophrenia previously on this blog (see here) based partly on some interesting data derived from ALSPAC published by Jean Golding and colleagues**** (open-access) and also that 2011 Frans study. In particular was the emphasis on the Golding 3M - meiotic mismatch methylation - hypothesis used to account for their results on grandmother's age as potentially being relevant to grandchild autism risk (please read the Golding article for more information on 3M complete with nice diagram).

I know it might sound a little far-fetched that the lives of our grandparents might so profoundly be able to affect the lives of subsequent generations but before we put this down to mere coincidence, let me draw your attention to some work that was done on a dark period of quite recent history: the Hongerwinter. The basics: the Dutch famine of 1944, where a Nazi blockade led to the deaths of thousands. As per the often cruel twists of fate, science actually learned something from the suffering of the Dutch people in these dark days. Not only the confirmation that wheat was tied into coeliac (celiac) disease but also the suggestion that famine exposure during a critical period of gestation *might* potentially affect offspring physical and mental health. I've kinda talked about something similar before with 'thin-fat bodies' and David Barker in mind (see here).

Granted in the current Frans study we are heading back even further through the germline as potentially hosting some effect, but to all intents and purposes, the theory is the same as per the intergenerational effects noted in other conditions like depression*****. I suppose one could ask whether specific types of autism might be more related to this grandparental age hypothesis over others. So for example, older grandparents at time of fathering or mothering impacting on the genome of their offspring - themselves then expressing certain traits associated with autism or the broader phenotype (not necessarily hitting the diagnostic threshold) - which are then transmitted (amplified?) to the next generation. Perhaps even some link to things like assortative mating theory too? I'm not saying that this is the only scenario and such 'transmission' works on its own to elevate risk of an autism diagnosis but the theory is an interesting one; even more so if we assume for example, that the autism and schizophrenia spectrums might not necessarily be poles apart (see here).

By the same token one might also extend such a hypothesis to include other variables other than just parental age at offspring conception. The availability of and exposure to certain food in these 'olden days', exposure events to pollutants, pharmaceuticals (yes, we did have them then) or lifestyle factors such as smoking and drinking habits, various psychological and somatic stressors; the list is seemingly endless. In at least some of these factors, there are subtle clues to potential future directions for autism research and beyond already being examined (see here and here). Assuming also that epigenetics might be tied into all of this, we also open up the concept of epigenetic reprogramming of the germline as per the very interesting article by Petra Hajkova****** (open-access); something which I think might be/have been discussed at the recent Environmental Epigenetics symposium hosted by the MIND Institute.

But let's not get too carried away with this area of inquiry or where it potentially leads in terms of the autism 'blame game'. Although I've not been able to find specific figures, I assume the actual numbers of grandfathers who fathered their children aged 50+ years is probably not going to be all that frequent if more current rates, at least here in the UK, are anything to go by (see this paper by Bray and colleagues*******). And then we have to wonder whether other variables might come into play such as the effect of paternal age at conception on birth factors such as birth weight or time of gestation even fecundity itself and how that might relate to autism risk.

Such transgenerational effects whilst interesting, should also not detract research attention away from other more here-and-now possibilities which might affect autism risk as per the recent valproate work or indeed all that immune activation research currently on-going. Neither should it deflect attention from the fact that the autism numbers are really starting to get quite serious - 1 in 50 US kids (with caveats) - and what needs to be done (a) asking why there is such an increase in cases and (b) to ensure the relevant help and support is available to all who need it.

To finish a very catchy tune from Jake Bugg - Lightning Bolt.

----------

* Frans EM. et al. Autism risk across generations. A population-based study of advancing grandpaternal and paternal age. JAMA Psychiatry. March 2013.

** Roberts AL. et al. Association of maternal exposure to childhood abuse With elevated risk for autism in offspring. JAMA Psychiatry. March 2013.

*** Frans EM. et al. Advanced paternal and grandpaternal age and schizophrenia: a three-generation perspective. Schizophr Res. 2011; 133: 120-124.

**** Golding J. et al. Parental and grandparental ages in the autistic spectrum disorders: a birth cohort study. PLoS ONE. 2010; 5: e9939.

***** Warner V. et al. Grandparents, parents, and grandchildren at high risk for depression: a three-generation study. J Am Acad Child Adolesc Psychiatry. 1999; 38: 289-296.

****** Hajkova P. Epigenetic reprogramming in the germline: towards the ground state of the epigenome. Phil. Trans. R. Soc. B. 2011; 366: 2266-2273.

******* Bray I. et al. Advanced paternal age: How old is too old? J Epidemiol Community Health. 2006; 60: 851–853.

----------

Frans, E. (2013). Autism Risk Across GenerationsA Population-Based Study of Advancing Grandpaternal and Paternal AgeAutism Risk JAMA Psychiatry DOI: 10.1001/jamapsychiatry.2013.1180

1 in 50 children with parent-reported autism in the US

An estimated 1 in 50 US children aged between 6 - 17 years old present with an autism spectrum disorder (ASD). Attention-grabbing isn't it?

Today's post is based on the source of that soundbite, the publication by Stephen Blumberg and colleagues* (open-access) describing results from data mining of the 2007 and 2011-2012 US National Survey of Children's Health (NSCH) relevant to the numbers of cases of ASD.

Testing 1, 2, 3 @ Wikipedia  

The autism-numbers game is something that has been discussed previously on this blog; most recently with the CDC 1 in 88 estimate in mind (see here) and the even more recent data from New Jersey (see here). The direction of the figures seems only one way - up - but the reason(s) for the increase still remain the source of discussion.

The Blumberg report is open-access, but a few pointers might be useful:

• Based on 91,642 telephone interviews in 2007 and 95,677 interviews between 2011-2012, parental reports of receipt of an ASD diagnosis (autism, Asperger syndrome, PDD) in offspring were noted, alongside other variables such as age of the child, severity of presentation (mild, moderate, severe) and aged when first diagnosed.
• Actually in the age range 6-17 years old, data were collected from 63,967 interviews (2007) and 65,556 (2011-2012).
• Results: "based on parental reports, the prevalence of diagnosed ASD in 2011-2012 was estimated to be 2.00% for children aged 6-17". This compared with 1.16% or 1 in 86 for 6-17 year olds in 2007.
• The increase in prevalence was noted across the age ranges when they were sub-categorised and perhaps not surprisingly, there was a greater increase in prevalence in boys (2007: 1.8% vs. 2011-2012: 3.23%) than girls (2007: 0.49% vs. 2011-2012: 0.70%). Reported severity also shifted between the various data points (and age ranges) indicating that there was a trend towards less severe presentation (milder ASD) post 2008 diagnosis.
• The authors were able to some degree, rule out "survey-based measurement error" as being a major contributor to the prevalence increase and there is some discussion about the data not necessarily reflecting "factors that exist prior to or occur just after birth". Indeed the authors very firmly suggest that the changes are a consequence of either "recognition of ASD by health professionals or survey-based measurement changes over time". They also conclude that increases in the prevalence of parent-reported ASD especially for children aged 6-13 "was the result of diagnoses of children with previously unrecognized ASD".
• As per the report on this story in USA Today "15% to 20% of children who were once diagnosed with autism no longer have the condition". Which raises similar questions as to that of the Fein study on 'outgrowing autism' and 'optimal outcomes' which created so many column inches recently (see here and here).

Bearing in mind that this was a prevalence study not an incidence study (see here for the difference) and issues with regards to response rates (2007: 46.7% vs. 2011-2012: 23.0%), and the sole reliance on parental judgement of variables like severity, the data being presented are indeed stark.

That the rates of autism have seen an inordinate shift from what was once considered a rare condition to something which theoretically should [almost] appear in every school classroom at least once is an eye-opener. As mentioned, the debates rumble on about factors such as better awareness of autism, better case ascertainment, diagnostic switching and broadening, etc. as being the source of the increase. A real increase in cases? Hardly a mention in this latest data. The implication that for example our screening methods and skilled professionals have been able to miss or mis-diagnoses a staggering number of children presenting with an ASD is truly mind-boggling and worthy of an inquiry or two in the US and beyond. Indeed better be quick with that investigation with the DSM-V revision deadline fast approaching and the potential impact that might have on the autism numbers game (including adult numbers**).

So 1 in 50 children with an ASD. What happens next? Sure, many children were described as falling into the mild and moderate ability ranges but as I've said before, terms like 'high-functioning' don't necessarily mean 'can function' with regards to daily living skills, quality of life and onwards translating into positive outcomes in adulthood. Certainly society has to play its role in helping people with autism reach their potential (I have a post scheduled on job interviews and autism coming up soon) and changes are indeed on-going (e.g. the implementation of the Autism Act here in the UK). But let's not be too proud of the achievements done in this area, as still many people on the autism spectrum, their families and concerned others have to fight daily for appropriate recognition, provisions and services.

Another important issue also springs to my mind on the basis of the new prevalence figure. Comorbidity, of which autism is by no means immune from, are not mentioned. The realisation that autism is often very much more than the sum of its triad - soon to be dyad - in terms of comorbidity must surely factor into the potential impact of the latest figures. Not least because of the quite startling health inequalities which seem to be present when an ASD is diagnosed and how as was very recently detailed in the CIPOLD report, such inequality can in some cases, have the most profound and far-reaching effects. I'm not trying to scare anyone; just sayin' that we need to be mindful of the whole person not just their autism.

----------

* Blumberg SJ. et al. Changes in prevalence of parent-reported autism spectrum disorder in school-aged U.S. children: 2007 to 2011–2012. National Health Statistics Reports. 2013: 65.

** Wilson CE. et al. Comparison of ICD-10R, DSM-IV-TR and DSM-5 in an adult autism spectrum disorder diagnostic clinic. J Autism Dev Disord. March 2013.

----------

Wilson, C., Gillan, N., Spain, D., Robertson, D., Roberts, G., Murphy, C., Maltezos, S., Zinkstok, J., Johnston, K., Dardani, C., Ohlsen, C., Deeley, P., Craig, M., Mendez, M., Happé, F., & Murphy, D. (2013). Comparison of ICD-10R, DSM-IV-TR and DSM-5 in an Adult Autism Spectrum Disorder Diagnostic Clinic Journal of Autism and Developmental Disorders DOI: 10.1007/s10803-013-1799-6

Folic-ing around in schizophrenia

I seem to have been focusing a lot on folic acid (vitamin B9) and vitamin B12 these days. It's not that I'm in anyway choosing the direction taken, it just happens that the published papers are appearing that way.

And as if to prove my point, enter the paper by Joshua Roffman and colleagues* (open-access) reporting results from a gold-standard, randomised, double-blind, placebo-controlled study on the addition of folic acid and vitamin B12 supplement to antipsychotic medication for a group of adults with chronic schizophrenia.

MTHFR, FOLH = word score @ Wikipedia  

The paper is open-access but a few summary points are worth mentioning bearing in mind Dr Emily Deans has already discussed this research:

• One hundred and forty participants diagnosed with schizophrenia but psychiatrically stable were included for initial study. They were all taking an antipsychotic for 6 months "but displayed persistent symptoms despite antipsychotic treatment".
• As per the study protocol, participants were randomly split into folate-vitamin B12 supplementation or placebo. Actually whilst it was random, it was stratified random, meaning that randomisation took into account serum folate levels which were measured at baseline and formed an important part of the study outcomes. Indeed, the split was also not 50:50 in each group; instead weighted towards the supplementing group. 
• On most other variables the groups showed no significant difference (age, gender splits, medications, SES). That is aside from serum vitamin B12 levels, where the experimental group showed a significantly higher mean level at baseline compared to the placebo group (631 pg/ml vs. 511 pg/ml respectively).
• The primary outcome measure was the change in negative symptoms as judged by the SANS. Not being an expert on schizophrenia, I was interested to read about the characterisation of positive and negative symptoms in schizophrenia and, as the authors put it, "considerable disability is associated with negative symptoms and cognitive deficits, for which effective treatment is not available".
• Results: after 16 weeks of study, there were lots and they were mixed in with some DNA genotyping data pertinent to genes involved in the folate metabolism cycle. So our old Scrabble friend MTHFR (see here) got a look in, as did MTR (methionine synthase) - as per my previous post. 
• One gene in particular seemed to get quite well caught up in the study results: FOLH1 - which among other things is involved in folate transfer and absorption. Mention of the words 'glutamate excitotoxicity' alongside FOLH1 also stirs up some interesting thoughts. 
• So, yadda, yadda, "folate and vitamin B12 improves negative symptoms of schizophrenia" but only modestly given the "15% difference in SANS scores" between the experimental and placebo groups. Importantly in these days of personalised medicine, the FOLH1 gene was the focus, in that FOLH1 484C>T variant seemed to tie into treatment response. This was slightly at odds with what had been noted on another occasions**.
• So, if a participants was homozygous - as in identical copies of the same allele - for FOLH1 484T,  they were more likely to show greater benefit from the supplements. I'll come back to this shortly.
• That's not also to say that there weren't other gene related findings tied into intervention response. As the authors note about MTHFR 677C>T "only T allele carriers exhibited a significant benefit for active treatment over placebo for negative symptoms". Thankfully in line with what has previously been discussed***.

Every paper covered on this blog is a learning journey for me and this one is no exception. Likewise, it is always interesting to see when results don't exactly pan out as they are predicted to. In the case of the Roffman paper, it was the FOLH1 gene findings which didn't go to plan, and how contrary to the expected role of the 484C variant, the so-called low-functioning variant which one would expect to have reduced folate absorption - as was demonstrated in a separate asymptomatic cohort - it was actually the presence of the high-functioning variant (484T) which governed a positive treatment response.

In light of these findings, and the fact that red blood cell (RBC) levels of folate grew and grew in the experimental group over the course of the trial (although not significantly related to the change in negative symptoms), one starts to ponder other explanations to account for the results.

I've gone over MTHFR so won't say much more on that. The authors touch upon one potentially pertinent issue - DNA methylation - which is where I always seem to end up back to when talking about folate and MTHFR and the like. That for example, the supplementation of folic acid and vitamin B12 might, just might, impact on important reactions such as the recycling of homocysteine back to methionine onward to the production of SAMe is one possible effect. Indeed, it is a shame that elements of the methionine cycle were not measured over the course of the current trial.

I could go on. I could ask what kind of vitamin B12 was used as a supplement, whether the oral dosage form is the ideal way to get vitamin B12 into the body, whether outside of the reported symptoms, there may have been other variables affected by the results and whether despite increasing levels of folate, there were corresponding increases to levels of the active form of folic acid, 5-methyltetrahydrofolate? Indeed on that last point apparently there are plans afoot to look at the use of 5-methyltetrahydrofolate (or as the authors call it 1-methylfolate)...

Please stop now... and so I shall.

----------

* Roffman JL. et al. Randomized multicenter investigation of folate plus vitamin B12 supplementation in schizophrenia. JAMA Psychiatry. March 2013.

** Roffman JL. et al. Genetic variation throughout the folate metabolic pathway influences negative symptom severity in schizophrenia. Schizophr Bull. 2013; 39: 330-338.

*** Hill M. et al. Folate supplementation in schizophrenia: a possible role for MTHFR genotype. Schizophr Res. 2011; 127: 41-45.

----------

Roffman JL, Lamberti JS, Achtyes E, Macklin EA, Galendez GC, Raeke LH, Silverstein NJ, Smoller JW, Hill M, & Goff DC (2013). Randomized Multicenter Investigation of Folate Plus Vitamin B12 Supplementation in Schizophrenia. JAMA psychiatry (Chicago, Ill.), 1-9 PMID: 23467813

Autism, maternal immune activated mice and suramin

Avid followers of the autism research circuit must have noticed the increasing tide of studies looking at a possible role for maternal immune activation (MIA) in relation to risk of offspring autism spectrum disorder (ASD). It's a topic I've covered more than once on this blog; predominantly in relation to the work of people like Paul Patterson and his colleagues (see here), observations on things like C-reactive protein (see here) and the various ways to experimentally mimic such MIA in the mouse model of autism / schizophrenia / other for example (see here).

Squeakers @ Wikipedia  

So it is in this post that I'm serving a double helping of the MIA model of autism as per the publication of studies from Jared Schwartzer and colleagues from the MIND Institute* (open-access) and Robert Naviaux and colleagues** (open-access).

Both studies looked at the effects of artificial induction of MIA in the mouse model following poly I:C use as an immunostimulant. Thereafter the two studies went their separate ways as Schwartzer looked at the variable of mouse strain on the after-effects of MIA on offspring and Naviaux looked at the role of purinergic signaling.

I'll say right now that I am neither qualified nor experienced enough to go into these papers with any great detail. So I won't; instead a brief overview of each - bearing in mind their open-access status - and some interesting factoids which have already been mentioned in the autism research peer-reviewed domain which might tie into results.

The work of Schwartzer and colleagues basically "indicate[s] the need to consider how genetic predisposition may exacerbate or protect against the effects of environmental insults in the etiology of ASD". In other words, based on a mouse model looking at different strains of mouse, the specific genetic make-up of that mouse model might impact on offspring presentation after an artificial MIA event.

In their case they looked at the C57BL/6J and BTBR T+tf/J inbred mouse strains and concluded that the dangermouse that is the BTBR strain combined with the poly I:C stressor seemed to "be synergistic resulting in greater behavioral impairment than from either factor alone" when compared with the C57BL/6J mouse strain. Some interesting variables are noted including elevations in cytokines like IL-6 (see here) and IL-17 (see here) in the BTBR offspring mice compared to C57BL/6J mice alongside some sex specific behavioural differences. All in all, some very interesting observations; and on that sex-specific notion, not completely at odds with other work in this area (see here).

The work of Naviaux and colleagues - summarised quite well here - has definitely taken the interest of the media as per headlines such as 'New drug that may help reverse autism' or should that be 'Century old drug could beat autism'. I'm confused. The long-and-short of it is that based on the analysis of the MIA mouse model - C57BL/6J mice - there was a suggestion that "hyperpurinergia is a fundamental and treatable feature of the multisystem abnormalities in the poly(IC) mouse model of autism spectrum disorders". Treatable via "antipurinergic therapy (APT)" which in this study was via the drug suramin. The observant reader should immediately be comparing Schwartzer and Naviaux and the MIA mouse models chosen and results obtained.

Anyhow, Naviaux et al continue in their observations on how MIA affected offspring mice and how the administration of suramin seemed to have some pretty wide-ranging effects on offspring mice. Alongside various behavioural effects on social and coordination issues, suramin administration was reported to show important effects such as "the preservation of cerebellar Purkinje cells", which as I discussed in a recent post, have more than a token link to cases of autism. "Suramin treatment strongly increased the expression of the nicotinic acetylcholine receptor subunit α7 (nAchRα7) in cerebral synaptosomes of MIA animals" was another potentially important finding in view of other work in this area. In all, "16 multisystem features of this model were either corrected or improved by suramin treatment".

Impressive stuff I hear you say. Indeed all the more impressive given that the authors on purpose did not start suramin treatment until 6 weeks because they "wished to test the hypothesis that many of the autism-like features of the MIA model were treatable after they appear". And apparently there is more to come according to the authors, with the promise of human trials of suramin...

But just before you pop down to your local doctor or pharmacist to ask for suramin (off-label), it might be worth pointing out a few things. Mice. Yep, this was a study of mice and as per the Schwartzer study, not necessarily the best and only mouse model of autism from an MIA point of view. Indeed if I needed to go back to the BTBR mouse and its overlap with autism, I might also recall some work looking at that most forgotten of autism research parameters, sulphate (sulfate) and findings related to the BTBR model (see here). Mice are not humans and suramin is to be added to a growing list of mouse findings with an autism slant (see here and here).

That the US National Cancer Institute holds an entry for suramin should also give you some idea as to what uses the drug has and why bearing in mind it was injected into the study mice. Alongside its anti-parasitic effects related to things like sleeping sickness, the activity of suramin has been linked to its blocking of various growth factor binding which might yet hold some clue to other effects of the drug outside of competitive inhibiting of purinergic signalling (see here and here). As with most medicines, there are other effects to keep in mind which might also tie into results. And then there are the reported side-effects...

I'm not by any means trying to belittle the Naviaux results of suramin in the MIA mouse model of autism so please do not take this post as such. I am very keen to see some replication studies done in other mouse and other animal models, just to see if the results stack up before progressing to human trials with the all-important focus on 'first do no harm' and whether other meds have similar actions. As such I'll keep my eye open for suramin and autism and perhaps post some updates.

In the meantime, the maternal immune activated hypothesis grinds forward...

----------

* Schwartzer JJ. et al. Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice. Translational Psychiatry. 2013; 3: e240.

** Naviaux RK. et al. Antipurinergic therapy corrects the autism-like features in the poly(IC) mouse model. PLoS ONE. 2013; 8: e57380.

----------

Schwartzer JJ, Careaga M, Onore CE, Rushakoff JA, Berman RF, & Ashwood P (2013). Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice. Translational psychiatry, 3 PMID: 23481627

Naviaux, R., Zolkipli, Z., Wang, L., Nakayama, T., Naviaux, J., Le, T., Schuchbauer, M., Rogac, M., Tang, Q., Dugan, L., & Powell, S. (2013). Antipurinergic Therapy Corrects the Autism-Like Features in the Poly(IC) Mouse Model PLoS ONE, 8 (3) DOI: 10.1371/journal.pone.0057380

Just say NO to sapropterin for autism

Actually the title of this post is a bit of a misnomer.

I'm not really asking readers to say 'no' to sapropterin, otherwise known as tetrahydrobiopterin or BH4, for autism as if it were some kind of Zammo-esque drugs in the toilet scenario (note: for anyone born post-Grange Hill golden era or for my non-UK readers, you might want to follow this link to see what I'm going on about). But neither am I saying yes, as per my prime directive on this blog: no medical or clinical advice given or intended (resistance is futile... and all that).

Mr Bronson / Admiral Ozzel @ BBC News

The 'no' actually refers to NO - nitric oxide - and in particular the findings reported by Richard Frye and colleagues* (including Jill James yet again) on the potential involvement of NO metabolism in the behavioural changes noted when BH4 was introduced to a small cohort of children diagnosed with autism. I think we might have seen shadows of this study presented at IMFAR 2012.

OK, a quick description might be in order first. I've covered BH4 previously on this blog (see here) and some of the various roles that it plays; not least in its co-factor duties for the metabolism of some important aromatic amino acids eventually into things like neurotransmitters. Also not forgetting the potential role for BH4 in relation to managing conditions like PKU also (see here). Similarly, NO has also appeared on this blog before (see here). The Frye paper stresses the important role that BH4 has in the production of NO.

A few details from the Frye paper bearing in mind it is open-access:

• Starting with 10 participants (aged 2-6 years) diagnosed with an autism spectrum disorder (ASD) whose parents agreed "to not change any traditional or alternative medical or behavioral therapy during the study", various measures of behaviour and language function were charted over the course of a 16-week open-trial of BH4 (Kuvan).
• Alongside the behavioural and psychometric measures used (which included the VABS and PLS), CSF samples were collected via lumbar puncture (not normally recommended because of its invasiveness) and blood samples used to measure for various marker compounds including BH4, the amino acids L-arginine and L-citrulline and everyone's favourite redox coverboy/covergirl, glutathione.
• Results: bearing in mind that this was an open-trial and that no control group or placebo arm was used, the authors report some interesting changes to various parameters. So language (receptive at least) showed a significant improvement across the group across the testing periods (baseline, 8 weeks, 16 weeks). Some of the VABS subscales also indicated some positive changes (albeit one of them, VABS personal daily living, presented with a p-value of 0.061, I assume to denote Nick Berry style 'we nearly made it').
• The biological stuff: well there was an increase in the reduced-to-oxidised glutathione ratio (good thing) and a decrease in levels of 3-Chlorotyrosine (3CT) (also a good thing) over the course of intervention, positive in terms of oxidative stress (redox status) and the presence of "reactive nitrogen species" respectively (see below).
• Findings also pointed to "a fundamental change in pterin metabolism" coinciding with BH4 supplementation. I won't pretend to know all the ins-and-outs but it all has to do with supplementation modifying the reduced-to-oxidised pterin ratio and degradation of BH4 onwards to the appearance of something called peroxynitrite which is not particularly a good thing. I think this article** (open-access) might explain it a little better than I could.
• The authors also reported that despite no significant change in NO metabolism markers (arginine and citrulline, and their ratio), it did appear that baseline levels of these compounds were allied to behavioural outcomes. Specifically improvements on the behavioural parameters "were related to higher baseline arginine and arginine-to-citrulline ratio".
• Importantly, BH4 supplementation was generally well tolerated with "only one patient discontinuing the medication because of mild adverse effects".

Yes, this was a very small trial, and yes again, there was no control group, no placebo and no blinding. It is preliminary work, of that there is no doubt. I find it a little unusual that the authors also chose HPLC with electrochemical detection when it came to the measurement of important metabolites like CSF levels of BH4. A little bit '80s' if you'll forgive me, given the startlingly increased precision offered by mass spec and NMR techniques as exemplified by papers like this one. Indeed even more odd that LC-MS was used for the analysis of amino acids: why not all metabolites? It should also be noted that Dr Frye is listed as having a potential conflict of interest in this paper via receipt of funding from the producers of Kuvan for this trial; not that this should or did influence the findings in any way, shape or form.

Nevertheless there are a number of interesting observations which might require some follow-up from this paper. That for example, a higher baseline level of arginine seemed to quite strongly correlate (r=0.91) with the PLS total raw score (language) as a result of BH4 supplementation is a point worth following up, particularly in these days emphasising the identification of best- and non-responders to various interventions for the autisms. The implication being that "only some of the participants were able to significantly change their NO metabolism with the dose of Kuvan used in this study" potentially as a result of this correlate - or at least this studied and known about correlate. An endophenotype eh? Or even a biomarker for intervention response?

Given the body of work already published on BH4 supplementation and autism and how BH4 levels might tie into lots of different areas outside of just being a co-factor (see this paper*** open-access) I'd like to see quite a bit more done on this compound and its relations. That it might also overlap with other conditions too - schizophrenia for example**** and other diagnoses***** - is also an important point given the genetic common ground being postulated between quite a few conditions (see here). One also wonders whether that recent vitamin B12-folate supplementation for schizophrenia paper****** by Roffman and colleagues (open-access) might also hint at some involvement of BH4 (more on that paper to come).

To finish, the old Grange Hill intro, including that sausage. Alongside due respect to actor Michael Sheard who played the unfortunate Kendal Ozzel ("he is as clumsy as he is stupid"). Steady on Darth.

----------

* Frye RE. et al. Metabolic effects of sapropterin treatment in autism spectrum disorder: a preliminary study. Transl Psychiatry. 2013; 3: e237.

** Pacher P. et al. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007; 87: 315-424.

*** Frye RE. et al. Central tetrahydrobiopterin concentration in neurodevelopmental disorders. Front Neurosci. 2010; 4: 52.

**** Richardson MA. et al. Evidence for a tetrahydrobiopterin deficit in schizophrenia. Neuropsychobiology. 2005; 52: 190-201.

***** Coppen A. et al. Depression and tetrahydrobiopterin: the folate connection. J Affect Disord. 1989; 16: 103-107.

****** Roffman JL. et al. Randomized multicenter investigation of folate plus vitamin B12 supplementation in schizophrenia. JAMA Psychiatry. March 2013.

----------

Frye RE, Delatorre R, Taylor HB, Slattery J, Melnyk S, Chowdhury N, & James SJ (2013). Metabolic effects of sapropterin treatment in autism spectrum disorder: a preliminary study. Translational psychiatry, 3 PMID: 23462988

Inflammatory bowel disease in autism: distinctive features?

Where to start with this very long post... where to start?

That autism, some cases of autism, also coincide with various comorbidities sometimes including severe gastrointestinal (GI) issues is a relatively undisputed finding these days. I'm actually getting a little bored of saying this myself on this blog and I'm sure some readers are getting bored of hearing it too.

Health inequality

Lymphocytic infiltration @ Wikipedia  

The reason why I continue to keep hammering away at this line however is because there is a substantial gap between what the peer-reviewed literature is saying about GI factors coexisting with cases of autism and the real-world experiences of many people (children and adults) with autism in terms of getting such symptoms/conditions investigated and properly treated.

A health inequality if ever there was one; indeed why else would the folks over at Treating Autism feel compelled to have to produce a document on the medical comorbidities in autism spectrum disorders highlighting such an issue?

An autism-specific IBD variant?
With this in mind, today's post concerns the paper published by Stephen Walker and colleagues* (open-access) which suggests that when it comes to the more GI disease-related aspect of bowel disturbance linked to autism - as in inflammatory bowel disease (IBD) - it appears that such GI disease might have "distinctive features" onwards to either an autism-specific IBD variant or "a prodromal phase of typical inflammatory bowel disease". We had seen a hint that this study was coming to publication based on an abstract presented as IMFAR 2012 (see here).

OK, I know that this paper takes some people into uncomfortable territory. Bowel disease - lymphoid nodular hyperplasia (LNH) and enterocolitis and autism - means 1998, Lancet and retracted paper and Lord Voldemort style 'he who must not be named'. Indeed search through the Walker paper and you will see that 'he' is definitely not named among the references, or not at least as first author on any paper. This despite the fact that other, well-renowned teams have no issue in citing the retracted paper in question (see here). I'll also point out that the authorship list on the Walker paper includes Dr Arthur Krigsman who has published work in this area** (open-access) previously. Just sayin'.

Anyhow, a few details from the Walker paper bearing in mind it is open to all:

• The name of the game was "transcriptome profiling of gastrointestinal mucosal biopsy tissue from ASDGI children and three non-ASD control groups (Crohn's disease, ulcerative colitis, and histologically normal)" to ascertain just how similar/different gene expression was. In other words, whether the molecular signature noted in cases of autism and bowel pathology (autism-GI) matches what it seen in other well-known, and relatively well characterised bowel diseases.
• The focus of analysis was differentially expressed transcripts (DETs) noted in ileal and colonic tissue samples from the various groups under study. The tool of analysis was microarray followed up by quantitative real-time PCR (qPCR) to "validate representative transcripts that showed differential expression by microarray". A sort of scatter gun, see what hits we make approach, followed by a more precise confirmation of said hits. Results were organised according to PCA "to determine similarity among biological replicates". 
• Participants were 25 children diagnosed with an autism spectrum disorder (mean age around 5 years old) who all presented with a regression in previously acquired skills and some kind of inflammatory-related condition of the bowel (ileitis, colitis, ileocolitis). Indeed all presented with LNH. Most of children with ASD were following a gluten- and casein-free (GFCF) diet. Control groups were rather smaller in number and there was a bit of an age difference between them and the autism-GI group.
• Results: Lots. Bearing in mind the authors were looking at tissue from two different parts of the GI tract, across quite a few groups, so this is probably not unexpected. For the autism-GI group, not everyone was included in all the analyses based on the two different regions of the gut because of quality/quantity of the RNA derived from them. 
• For both colonic and ileal mucosa gene expression profiles, the asymptomatic control group (histologically normal) tended to cluster tightly together. Not so when it came to the symptomatic groups, where the autism-GI group in particular showed quite a bit of variability "suggestive of some potential subgroup(s)". Mmm, interesting.
• Various comparisons were made between the groups based on the DETs identified in the two regions. I would be here all day and night listing the results of all this, so I'm cherry-picking a few bearing in mind it was a mix of up-regulated and down-regulated gene expression. (a) Autism-GI vs. asymptomatic controls (ileal mucosa): "1409 DETs unique to ASD-GI samples". DETs tended to link back to things like inflammatory bowel disease and colitis genes and the inflammatory response. Then we get down to genes related to humoral immune response, antibody production and digestive system development and function. (b) Autism-GI vs. asymptomatic controls (colonic mucosa): "1189 DETs unique to ASD-GI samples". DETs were linked to things like again gastrointestinal disease and also "neurological disease" including schizophrenia (50 genes) (see this post) and "hyperactive disorder" (16 genes) (which I take as meaning something like ADHD).
• Again focusing on the autism-GI group and the DETs which overlapped both ileal and colonic tissues (178 transcripts), the top associated biological functions were in relation to inflammatory disease, endocrine system development and function, and digestive system development and function. Interesting also that 3 genes also cropped up in the pathway analysis linked to "Valine, Leucine and Isoleucine Degradation".  Did someone say branched-chain amino acids and autism? 
• A very large quote: "Taken as a whole, the picture that emerges is one in which GI symptomatic children with ASD in whom cellular infiltrate is present in the ileum and colon have a distinct molecular signature that is consistent with the larger disease categories of gastrointestinal disease, and more specifically, overlaps with Crohn's disease, ulcerative colitis, and autoimmunity".

Consistency
What can we surmise from these results? Well potentially quite a bit but with the very strong requirement for replication, replication, replication with a greater number of participants over a longer period of inspection. That all children with ASD included in the Walker study also presented with defined bowel disorder (including LNH) is an important findings. I've talked about inflammatory bowel disease and autism in a previous post based on the paper by Chen and colleagues*** which concluded that LNH was not an inconsistent finding - "apart from intestinal lymphonodular hyperplasia, the majority of these findings were not consistent" - among the various papers looking at bowel disorder in cases of autism. The Walker paper adds to that feeling.

Carbohydrate metabolism
I was interested to read about the potential overlap between the Walker findings and those reported in the Brent Williams paper on carbohydrate metabolism**** in autism (see here and here reloaded). Williams and colleagues reported decreased mRNA expression in cases of autism related to important carbohydrate metabolising enzymes alongside signs and symptoms indicative of things like dysbiosis. Walker says 'yes'; they found similar evidence in their cohort, particularly when it came to the down-regulation of CDX2.

Politics and kingdoms aside, and bearing in mind the heterogeneity present within the autism spectrum (and its fuzzy boundaries), this paper is an important one. Re-opening scientific investigation into how autism is not just the sum of its dyad, and how a diagnosis of autism is seemingly protective of nothing when it comes to comorbidity, are important elements to this work. Realising the very real and very significant pain and discomfort which can occur when such bowel conditions are present alongside autism is a good first step to bringing this particular branch of GI research out of the shadows, and finally asking the question: what can we do about it?

If indeed people want to see real equality when it comes to healthcare access and use for people with autism, continued investigation on the topic of GI disease and autism inclusive of both functional presentation and underlying pathology is absolutely implied.

To close, Blondie and One Way or Another....

----------

* Walker S. et al. Identification of unique gene expression profile in children with regressive autism spectrum disorder (ASD) and ileocolitis. PLoS ONE. 2013; 8: e58058.

** Krigsman A. et al. Clinical presentation and histologic findings at ileocolonoscopy in children with autistic spectrum disorder and chronic gastrointestinal symptoms. Autism Insights. 2010; 2: 11.

*** Chen B. et al. Abnormal gastrointestinal histopathology in children With autism spectrum disorders. J Pediatr Gastroenterol Nutr. February 2011.

**** Williams B. et al. Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances. PLoS ONE. 2011; 6: e24585.

----------

Walker, S., Fortunato, J., Gonzalez, L., & Krigsman, A. (2013). Identification of Unique Gene Expression Profile in Children with Regressive Autism Spectrum Disorder (ASD) and Ileocolitis PLoS ONE, 8 (3) DOI: 10.1371/journal.pone.0058058