The Children’s Autism Metabolome Project (CAMP) reports: "Amino acid dysregulation metabotypes"

The Children’s Autism Metabolome Project (CAMP), mentioned in the title of this post, is an initiative that aims to develop "a diagnostic blood test for autism." It's a project that grabbed my attention for a few reasons; not least the reliance on the science of metabolomics and the use of some pretty amazing technology headed under the term 'mass spectrometry' (see here for another example) to try and accomplish their goal.

The recent results published by Alan Smith and colleagues [1] provide some of the first results to come from the CAMP, and the observation that: "Identification and utilization of metabotypes of ASD [autism spectrum disorder] can lead to actionable metabolic tests that support early diagnosis and stratification for targeted therapeutic interventions." Just in case you were wondering: "A metabotype is a subpopulation defined by a common metabolic signature that can be differentiated from other members of the study population." Inevitable lay media headlines have also followed on from this work (see here).

The nuts and bolts? Well: "Dysregulation of AA metabolism was identified by comparing plasma metabolites from 516 children with ASD with those from 164 age-matched typically-developing (TYP) children recruited into CAMP." AA refers to amino acids, the biological building blocks of proteins, and how, yet again (see here and see here) these compounds might be quite important to at least some autism. Researchers were able to analyse blood (plasma) samples from the participants, pertinent to detecting various amino acids and looking at how levels might differ as a function of a diagnosis of autism. Interestingly and importantly, the words "Training and Test Sets" are also used in the Smith paper, denoting how: "A training set was used to identify metabotypes associated with ASD and a test set was used to evaluate the reproducibility of the metabotypes." Similar methodological processes have been noted in other autism metabolomic studies (see here). I'm not going to bore you with the technological details of the "Triple Quadrupole LC-MS/MS Method" used (I'm more inclined to q-ToF mass spec myself) but suffice to say that such technology did yield some pretty accurate and important results, and it wasn't all just about autism vs. not-autism either.

"A simple analysis of the mean concentrations of free plasma amines did not reveal meaningful differences between the ASD and TYP populations of children." This is an important point. It suggests that within this cohort, there was no significant difference in the biological profiles following a straight 'autism vs not-autism' analysis. Something perhaps not entirely unexpected given the significant heterogeneity under the behaviourally-defined label called autism. But... "scatterplots of amine levels indicated that there were subsets of children with ASD with amine levels at the extreme upper or lower end of the abundance distribution." Researchers then began zooming in on different sub-groups of their autistic cohort as part of their "Amino Acid Dysregulation Metabotype (AADM)" description. Such analysis revealed a few AADMs based on the ratios between various amino acids. Further: "Taken together, all AADMs identified an altered metabolic phenotype of imbalanced BCAA [branched chain amino acid] metabolism in 16.7% of CAMP ASD subjects with a specificity of 96.3% and PPV [positive predictive value] of 93.5%." This *could* be interpreted as suggesting that about 15% of kids with autism *could* be correctly identified via their amino acid profile.

Caveats? Well yes, a few. This was work, for example, based on a single blood sample from each participant, in effect, providing a snapshot of each person at a particular point in time. There are lots and lots of different variables that will affect our metabolome including health/illness, diet, exercise, any medicines taken, comorbidity, et al. It's not beyond the realms of possibility that any or all of those factors could have influenced the results both in the short- or longer-term. Indeed, I'd like to see a lot more research on the consistency of individual sample results across different time frames before any big claims about a diagnostic test for autism are made. Also, the term 'biomaker for autism': I again get the impression that there needs to be lots more 'cross-linking' discussion between groups committed to this research agenda (see here and see here for examples).

But I don't want to take anything away from this work and (hopefully) future publications to come the CAMP. And I do also want to mention a couple of other interesting snippets of information garnered from the current study. So, in one of the write-ups of the study, one of the authors who is not stranger to the concept of 'biomarkers for autism' (see here) discusses: "Amaral points to phenylketonuria (PKU) as a possible template. PKU is a rare disease in which the amino acid phenylalanine builds up, causing brain damage. However, relatively small dietary adjustments can make a big difference." PKU as a template for autism? I think I've heard that somewhere before (see here). And that's also to acknowledge that PKU and autism can very much exist together (see here) in the context that various inborn errors of metabolism seem to be able to produce autistic signs and symptoms (see here). And some of them are very treatable...

Also alongside, I must quickly mention about those branched-chain amino acids (BCAAs) highlighted by Smith et al. How, in the context of other previous important research talking for example, about a 'new form of autism found' (see here), there are lots and lots of research (and clinical) possibilities to come from the analysis of these types of amino acids in the context of autism (see here). And yes, this includes intervention...

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[1] Smith AM. et al. Amino acid dysregulation metabotypes: potential biomarkers for diagnosis and individualized treatment for subtypes of autism spectrum disorder. Biological Psychiatry. 2018. Sept 6.

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Autism and phenylketonuria: a double syndrome

I want to briefly talk about the letter to the editor from Esra Demirci [1] (open-access) today and a continuation of some rather important research/clinical chatter about the inborn error of metabolism called phenylketonuria (PKU) intersecting with cases of autism (see here).

The author describes a case report of a child who was diagnosed with an autism spectrum disorder (ASD) "after performing a clinical assessment that included the Autism Behavior Checklist (ABC) and Childhood Autism Rating Scale (CARS)" and then subsequently diagnosed with PKU following some important metabolic investigations. They also highlight how instigation of a low phenylalanine diet - the treatment of choice for PKU - seemed to impact on the presentation of autism: "Eight months after the phenylalanine intake diet was initiated, he began to make eye contact, look when his name was said, and form two word sentences. His ABC scores fell from 57 to 46, and his CARS scores fell from 48 to 42."

The 'double syndrome' mentioned in the title of this post refers to the idea that there may be those on the autism spectrum who also have "an already described medical condition" and findings of autism and PKU comorbid fall into that category. I have to say that I'm a fan of this kind of thinking given the range of particularly metabolic conditions that do see to have 'an autistic element' to them (see here for another example). Screening is yet again implied (bearing in mind that PKU is already fairly routinely examined in all newborns in many countries). The idea, also yet again, that use of a low phenylalanine diet might also affect some of the signs and symptoms of autism in such cases remains a point for further investigation into hows and whys...

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[1] Demirci E. Autism Spectrum Disorder and Phenylketonuria: Dyzygotic Twins with Double Syndrome. Noro Psikiyatr Ars. 2017 Mar;54(1):92-93.

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Demirci E (2017). Autism Spectrum Disorder and Phenylketonuria: Dyzygotic Twins with Double Syndrome. Noro psikiyatri arsivi, 54 (1), 92-93 PMID: 28566968

Presenting with the symptoms of autism and then diagnosed with phenylketonuria (PKU)

The case report from Betül Mazlum and colleagues [1] (open-access available here) illustrates once again that (a) the plural 'autisms' exist (see here) and (b) screening for inborn errors of metabolism (IEM) should be an important part of any autism assessment (see here). Indeed, screening for IEM should really be part of assessments for many different labels...

Detailing a case report wherein a 3-year old child came to clinical attention for "speech delay and social problems", the authors describe how following a diagnosis of "autism according to DSM-IV criteria" further investigations were undertaken. Said investigations included analysis of blood and urine amino acid levels and, voilà, high levels of phenylalanine were detected and a diagnosis of phenylketonuria (PKU) made. Initiation of a low phenylalanine diet (the treatment of choice for PKU) followed and was accompanied by some important [positive] changes to behaviour and cognition. Of particular note to the presentation of autism we are told that: "At 4 months follow-up improvement was noticed in his eye contact, joined attention and speech."

The authors further note: "This case was not at particular risk for PKU at first thought, being born to non-consanguineous parents and during a period when newborn screening with Guthrie test was widely applied in Turkey. Although the child had a heel prick in the hospital where he was delivered, the results are unavailable and therefore whether his sample was analyzed is questionable."

OK, this was a case report and whilst an important 'N=1' is not necessarily generalisable to all autism (or rather all autisms). Insofar as the methods talked about for establishing raised phenylalanine - "Blood and urine amino acid chromatography" - I would have liked to have seen a little more detail in relation to the specific 'chromatography' methods used and any results related to another aromatic amino acid (tyrosine). We don't also have any data on follow-up either (repeat biological testing)...

PKU is an important but quite rare IEM. This is not however the first time that PKU has been linked to autism or the presentation of autistic traits (see here) particularly in cases of 'untreated' PKU. Aside from PKU providing quite a good template for how diet - certain aspects of diet - can affect behaviour and mental state for some (see here) there are other potential implications and 'correlations' on the back of this work. Not least is the intersection between another intervention measure potentially indicated for PKU - tetrahydrobiopterin (sapropterin or BH4) - and research suggesting that the 'mopping up phenylalanine' properties of this compound might be potentially effective for some cases and facets of autism too (see here) based on double-blind, placebo-controlled trial results [2].

"The possibility of a metabolic disorder including PKU should be considered in any child presenting with symptoms of autism, learning or speech problems and PKU should be tested unless the newborn screening results are available." I wouldn't argue with those sentiments [3], allowing for the fact that other correlates should also be considered (see here for example) particularly it seems, when autism appears alongside something like intellectual (learning) disability. The question of whether the quite restrictive low phenylalanine diet typically indicated for PKU might also impact autistic signs and symptoms is something that science still perhaps needs to look into...

Music to close, and how about something lively from The King?

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[1] Mazlum B. et al. A late-diagnosed phenylketonuria case presenting with autism spectrum disorder in early childhood. Turk J Pediatr. 2016;58(3):318-322.

[2] Klaiman C. et al. Tetrahydrobiopterin as a treatment for autism spectrum disorders: a double-blind, placebo-controlled trial. J Child Adolesc Psychopharmacol. 2013 Jun;23(5):320-8.

[3] Bilder DA. et al. Neuropsychiatric comorbidities in adults with phenylketonuria: A retrospective cohort study. Mol Genet Metab. 2017 Mar 6. pii: S1096-7192(17)30052-5.

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Mazlum B, Anlar B, Kalkanoğlu-Sivri HS, Karlı-Oğuz K, Özusta Ş, & Ünal F (2016). A late-diagnosed phenylketonuria case presenting with autism spectrum disorder in early childhood. The Turkish journal of pediatrics, 58 (3), 318-322 PMID: 28266201

Autism and inborn errors of metabolism

I'd like to think that the review article by Annik Simons and colleagues [1] (open-access) highlights some pretty strong evidence to suggest there being at least some connection between some autism and some of the collected inborn errors of metabolism. Indeed, when people generally talk about 'not knowing what causes autism' if we perhaps consider a more plural view of 'the autisms', there is a case to be made to say we might know what causes 'some' autism and some of it might lie in this area...

Inborn errors of metabolism (IEM) cover a whole host of different conditions "in which there is an accumulation of toxic and/or complex compounds or energy problems within the cells due to enzymatic defects or other protein dysfunction." The absolutely magnificent work of people like Robert Guthrie who's name is synonymous with the neonatal heel prick test offered to newborns to screen for various IEMs and a jobbing physician called Ivar Asbjørn Følling who lent his name to a condition that was eventually called phenylketonuria (PKU), have proved to be some of the real successes of modern medicine.

For quite a few years, peer-reviewed science has suggested some potentially important 'associations' between various behavioural and psychiatric labels manifesting in both treated and untreated IEMs (see here and see here for examples). Simons and colleagues decided to look through the collected research on this topic to provide "child and adolescent psychiatrists with an overview of metabolic disorders associated with child psychiatric symptoms, their main characteristics and recommendations for further investigations."

So after boiling down the available peer-reviewed literature to some 71 articles (and in so doing making an important distinction between an inborn error of metabolism and the 'metabolic syndrome'!) authors summarise some of the key IEM associated with labels such as autism, attention deficit hyperactivity disorder (ADHD), learning disability, psychosis and eating disorders. Given that (a) the paper is open-access and (b) this blog tends to favour autism research, I'm gonna focus in on some of the details pertinent to the autism spectrum. I do however recognise that when it comes to the term 'over-represented comorbidity' in autism some of the other diagnostic labels covered by Simons et al might also come into the frame.

Long quote coming up: "Known metabolic disorders in autism are phenylketonuria, disorders in purine metabolism (such as adenosine deaminase deficiency, adenylosuccinate lyase deficiency, dihydropyrimidine dehydrogenase and dihydropyrimidinase deficiencies), organic acidurias (such as propionic academia, 3-methylcrotonyl-CoA carboxylase deficiency and pyridoxine dependency), disorders of branched-chain amino acids creatine deficiency, biotinidase deficiency, cerebral folate deficiency, succinic semialdehyde dehydrogenase deficiency, Smith–Lemli–Opitz syndrome (SLOS), late infantile ceroid lipofuscinosis, histidinemia, Sanfilippo disease, glucose 6-phosphate dehydrogenase deficiency, urea cycle disorders, X-linked ichthyosis, and mitochondrial disorders." I've popped in a few links to other occasions where a specific IEM has been associated with autism and covered on this blog.

Simons and colleagues also cover some of the important research findings where specific amino acids have been analysed and found in unusual levels in cases of autism as potentially being important too. This is relevant because disordered amino acid levels as noted in the case of phenylketonuria (PKU) and the aromatic amino acids phenylalanine (and tyrosine) can be an important finding in relation to some IEM. That they specifically focus on some of the research looking at homocysteine levels and autism is rather interesting (see here) and something that I am going to be discussing in future posts.

What's more to say? Well, I think it is also important to highlight how Simons and colleagues talk about 'other signs and symptoms of the metabolic disease' [IEM] alongside the presentation of autism. This is important in the context that science is starting to more fully understand how a diagnosis of autism rarely exists in some sort of diagnostic vacuum (see here) and quite a lot of different types of comorbidity seem to be 'over-represented'. I'd be inclined to suggest that this detail provides even stronger evidence for how IEM and at least some autism represent an important partnership.

Finally, I refer back to one of the statements made by the authors on "recommendations for further investigations." They suggest that those presenting with: "A positive family history of metabolic disease... Symptoms or signs are triggered by food intake (esp high protein content foods), fever, fasting, surgery (catabolism)... Feeding difficulties, food refusal, failure to thrive, eating disorders combined with symptoms of myopathy or fatigue... Mental retardation and/or regression... Epilepsy, episodes of lethargy or confusion... Dysmorphic feature" should be considered for further investigations. Yes, some of the language is not what I would use and yes, that covers quite a bit of clinical ground but screening is the first part to ruling out such a potential organic correlate of some autism and may in some cases, yield potentially important insights (see here)...

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[1] Simons A. et al. Can psychiatric childhood disorders be due to inborn errors of metabolism? European Child & Adolescent Psychiatry. 2016. Sept 30.

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Simons A, Eyskens F, Glazemakers I, & van West D (2016). Can psychiatric childhood disorders be due to inborn errors of metabolism? European child & adolescent psychiatry PMID: 27695954

Autism in phenylketonuria (PKU)

"Autism has been reported in untreated patients with phenylketonuria."

Indeed it has, as the paper by Sameh Khemir and colleagues [1] revisits something of a long known about association whereby the archetypal inborn error of metabolism that is phenylketonuria (PKU) has been linked to the presentation of autism or autistic traits [2].

Looking at 18 participants diagnosed with PKU, Khemir et al "report their clinical, biochemical and molecular peculiarities" (authors words not mine) and how 15 of the 18 presented with autism as per assessment with "The Childhood Autism Rating Scale and the Autism Diagnostic Interview-Revised." Following some molecular biological analysis specifically with the "phenylalanine hydroxylase gene" in mind (a key player in PKU), the authors reported on various potentially important issues but "no correlation between autism and mutations affecting the phenylalanine hydroxylase gene."

I have a lot of time for PKU on this blog. Not only because PKU represents one of the best examples of how certain foods for some can affect development and onwards mental health (see here) but also because some of the other intervention options for PKU (outside of low phenyalanine diet) might hold some promise for some autism too (see here). Indeed, the idea that tetrahydrobiopterin (BH4) - an important cofactor for phenyalanine hydroxylase and related aromatic amino acid hydroxylase enzymes - might be quite good at helping to mop up excess phenylalanine and other compounds continues to find favour in some autism research circles. Dare I also mention the effects of BH4 on tryptophan and 5-HTP as potentially being relevant to some autism too? (see here)

In many parts of the world, the advent of the newborn screening program (built on the genius of people like Robert Guthrie and others) has all but eradicated untreated PKU and perhaps impacted on the number of people presenting with autism too. There remain however, challenges in certain areas of the globe, where people are not so fortunate to have such screening measures in place. Indeed, Khemir and colleagues report their results based in Tunisia and Algeria; other geographically related areas might also benefit from the implementation of such screening practices [3].    

Just before I go, there is one last comment to make on something discussed by Khemir and colleagues: "age of diet onset was the determining factor in autistic symptoms' evolution." Diet, as I've mentioned, refers to the low phenylalanine (low protein) diet commonly used to manage PKU. It appears that there might be more to see in terms of how long PKU goes untreated and the progression of autistic traits similar to other descriptions, particularly the findings reported by Baieli and colleagues [4]: "None out of 62 patients with classic PKU diagnosed early met criteria for autism. In the group of 35 patients diagnosed late, two boys (5.71%) ages 16 and 13 years fulfilled the diagnostic criteria for autism."

Diet potentially affecting the presentation of autism eh? I'll be coming to the paper by Oyarzabal and colleagues [5] soon enough built on some related research...

Music: Led Zeppelin - Rock And Roll.

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[1] Khemir S. et al. Autism in Phenylketonuria Patients: From Clinical Presentation to Molecular Defects. J Child Neurol. 2016 Jan 12. pii: 0883073815623636.

[2] Miladi N. et al. Phenylketonuria: an underlying etiology of autistic syndrome. A case report. J Child Neurol. 1992 Jan;7(1):22-3.

[3] Saad K. et al. ADHD, autism and neuroradiological complications among phenylketonuric children in Upper Egypt. Acta Neurol Belg. 2015 Dec;115(4):657-63.

[4] Baieli S. et al. Autism and phenylketonuria. J Autism Dev Disord. 2003 Apr;33(2):201-4.

[5] Oyarzabal A. et al. Mitochondrial response to the BCKDK-deficiency: Some clues to understand the positive dietary response in this form of autism. Biochim Biophys Acta. 2016 Jan 22. pii: S0925-4439(16)30003-5.

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Khemir S, Halayem S, Azzouz H, Siala H, Ferchichi M, Guedria A, Bedoui A, Abdelhak S, Messaoud T, Tebib N, Belhaj A, & Kaabachi N (2016). Autism in Phenylketonuria Patients: From Clinical Presentation to Molecular Defects. Journal of child neurology PMID: 26759449

Paediatric congenital heart disease and autism risk?

"Children aged 2-17 with CHD [congenital heart disease] were more likely than those without CHD to have had a diagnosis of autism spectrum disorder (crude OR, 4.6; 95% CI, 1.9-11.0) or intellectual disability (Crude OR, 9.1; 95% CI, 5.4-15.4)".

The traveller @ Wikipedia 

That was a key conclusion reported in the study by Hilda Razzaghi and colleagues [1] based on their analysis of data from "the 1997-2011 National Health Interview Survey", a US initiative which aims to provide "information on the health of the U.S. civilian noninstitutionalized population through confidential interviews conducted in households" (see here).

I have to say that I was pretty interested in these findings (even though this is not the first time that neurodevelopmental outcomes have been tied to CHD); a view it seems, that was shared by the authors of the paper, bearing in mind the very wide confidence intervals (CIs) detailed for both conditions and the reliance on second-hand reporting over actual independent screening results. I can't readily offer any one 'smoking gun' explanation for the findings specifically in relation to the autism spectrum but will draw your attention to some discussion about the potential causes of congenital heart disease provided by the NHS Choices websites and how they might tie in.

So: various genetic conditions including Down's syndrome are linked to CHD [2] and the net seems to be closing in on some of the underlying genetics around the association [3]. Although still in need of quite a bit more study, there is a growing appreciation that a diagnosis of autism / autistic traits can coexist alongside a diagnosis of Down's syndrome (see here). As per a recent conversation (thanks Marilyn), one also wonders how this autism - Down's syndrome link might also play out with regards to other areas of the autism research landscape such as dietary effects for example...

Next up is maternal diabetes. As per the NHS Choices entry: "It is estimated that 3 to 6% of women with diabetes who become pregnant will give birth to a baby with a heart defect". Autism and maternal diabetes is an interesting topic which has again cropped up in the autism research literature [4]. If you want my take on the Xu meta-analysis paper and related literature, look no further...

After that is a role for alcohol consumption during pregnancy and mention of the condition called foetal alcohol syndrome (FAS). I have talked about FAS and foetal alcohol spectrum disorder (FASD) before on this blog in the context of autism (see here) but can't readily conclude that the links are overly strong between FAS/FASD and autism presentation.

"A rubella infection can cause multiple birth defects, including congenital heart disease". An interesting association given the history of rubella and autism based to quite a large extent on the work from Stella Chess [5]. I don't know enough about the rate of rubella infection in pregnant women to make any informed statement about how this risk factor might tie into both CHD and autism but the numbers of non-immune women do seem to be quite alarming here in the UK [6].

Influenza during pregnancy? "Women who get flu during the first trimester (three months) of pregnancy are twice as likely to give birth to a baby with congenital heart disease than the general population" according to the NHS Choices website. Regular readers of this blog might already know that I tend to talk quite a bit about how infection - immune system response to infection - during pregnancy seems to have some link to subsequent offspring psychology and development (see here). The link is not altogether straight forward with autism in mind and potentially better related to concepts like fever or fever control [7] but still, there might be more to do here.

The final links - certain medications taken during pregnancy, phenylketonuria (PKU) and exposure to organic solvents during pregnancy - have also, to various extents, been linked to autism (see here and see here for example).

I may very well just be plucking at straws trying to link the causes of CHD with some of the suspected 'causes' of autism (some autism at least) but it strikes me that if one was to further pursue the findings reported by Razzaghi et al those would be the places to start (assuming some shared effect). As per the reports of other physiological findings potentially manifesting alongside a diagnosis of autism (see here), some additional screening of children on the autism spectrum where one or more of the correlates of CHD are suspected might be indicated.

Peter Gabriel and Kate Bush with a beautiful song to close: Don't Give Up.

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[1] Razzaghi H. et al. Long Term Outcomes in Children with Congenital Heart Disease: National Health Interview Survey. J Pediatr. 2014 Oct 8. pii: S0022-3476(14)00820-8.

[2] Laursen HB. Congenital heart disease in Down's syndrome. Br Heart J. Jan 1976; 38(1): 32–38.

[3] Ramachandran D. et al. Contribution of copy-number variation to Down syndrome-associated atrioventricular septal defects. Genet Med. 2014 Oct 23. doi: 10.1038/gim.2014.144.

[4] Xu G. et al. Maternal diabetes and the risk of autism spectrum disorders in the offspring: a systematic review and meta-analysis. J Autism Dev Disord. 2014 Apr;44(4):766-75.

[5] Chess S. Follow-up report on autism in congenital rubella. J Autism Child Schizophr. 1977 Mar;7(1):69-81.

[6] Skidmore S. et al. Is the MMR vaccination programme failing to protect women against rubella infection? Epidemiol Infect. 2014 May;142(5):1114-7.

[7] Zerbo O. et al. Is maternal influenza or fever during pregnancy associated with autism or developmental delays? Results from the CHARGE (CHildhood Autism Risks from Genetics and Environment) study. J Autism Dev Disord. 2013 Jan;43(1):25-33.

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Razzaghi H, Oster M, & Reefhuis J (2014). Long Term Outcomes in Children with Congenital Heart Disease: National Health Interview Survey. The Journal of pediatrics PMID: 25304924

Phenylalanine and schizophrenia: new directions for intervention?

As regular readers might already have noticed, amino acids are a bit of a obsession of mine on this blog. Out of all of them - and there are quite a few - I'm particularly interested in the aromatic amino acids and the their various connections to health and wellbeing. I've talked at length about some of the proposed connections made between amino acids such as tryptophan, tyrosine and phenylalanine to all manner of conditions but specifically with the autism spectrum in mind (see here).

The conversion. Matthews (2007) J Nutr. 137: 15495-15555.

Phenylalanine (or Phe) has been a particular favourite on this blog, not least because of its connection to that most classical 'diet can affect mental health' condition known as Phenylketonuria (PKU). As per other research chatter however, the connection between phenylalanine and PKU might just be the tip of the iceberg (see here). Indeed, today that iceberg just got a little bigger as I discuss the paper by Olaoluwa Okusaga and colleagues* (open-access) and their observations of elevated blood levels of phenylalanine in cases of schizophrenia. Such findings might indeed have some important management consequences as you'll see shortly when it comes to the use of something called BH4.

The Okusaga paper is open-access but a few of the important details:

• Well, one can't say that this was an under-powered study from a participant number point of view, as blood samples from 950 adult participants with a confirmed diagnosis of schizophrenia via the SCID were compared with 1000 asymptomatic controls for levels of phenylalanine and tyrosine.
• Analysis of samples was via HPLC with fluorescence detection, which whilst OK as a separative-detection method is not exactly the gold-standard that is mass spectrometry (MS) or nuclear magnetic resonance (NMR). 
• From the measures of phenylalanine and tyrosine, an estimate of the activity of phenylalanine hydroxylase (PAH) was also calculated and expressed as a phenylalanine: tyrosine ratio**. PAH represents an important step in the conversion of phenylalanine to tyrosine, which then proceeds down a metabolic pathway to eventually end up as dopamine. It's worth pointing out that dopamine has some important research history when it comes to the presentation of schizophrenia or at least, that's the suggestion (see here).
• Results: well bearing in mind some issues with the matching of the two sample groups in terms of age and BMI (a higher BMI in the schizophrenia group bearing in mind that these were not medication-naive participants), the schizophrenia group "had significantly higher Phe (geometric mean difference 1.26 µmol/L; CI 1.18 to 1.36, p<0.0001) and Phe:Tyr ratio (geometric mean difference 1.41; CI 1.33 to 1.48, p<0.0001) compared to healthy controls and this finding persisted after controlling for gender, age, education, and BMI differences between the 2 groups". As a group however, there was no significant differences for the schizophrenia and control groups when it came to measures of tyrosine although "lower levels of Tyr are more common among schizophrenia patients".
• The authors conclude that alongside further, more controlled study with regards to sample collection (including looking at measures of inflammation), there may also be some merit in looking at the potential effects of "Phe-lowering interventions in schizophrenia".

As I mentioned before, Phe-lowering interventions very much includes the use of BH4, but could also mean the rather more invasive use of a low phenylalanine diet (and then tyrosine supplements?) more commonly indicated for cases of PKU. I should point out that this does not mean I am in any way endorsing such a dietary change or pharmacological action at this time as a function of my caveat on this blog about not giving medical or clinical advice. That being said, the research gauntlet has been thrown down by the results of the Okusaga study so I'll be keeping my eyes open for future work in this area. 

There is other evidence suggestive of issues with the availability of BH4 in cases of schizophrenia as per the results from Richardson and colleagues*** which also extended to related schizoaffective disorder too****. Given that BH4 provides an important support service to a variety of enzymes relevant to the metabolism of aromatic amino acids (think tryptophan hydroxylase, TPH, for example), lower levels are probably not all that desirable. This might be particularly important to ensuring phenylalanine does not build up to too higher levels and the effects that can have*****.

Aside from the phenylalanine-lowering interventions call made from the Okusaga study, a few other questions are floating round my mind. So, at what point do phenylalanine levels become elevated in some cases of schizophrenia? I'd assume that as per the quite comprehensive use of the Guthrie test these days, we aren't talking about participants reaching the cut-off points for PKU in early infancy, so when does this issue present itself in cases of schizophrenia and why? I'm also interested in the cognitive effects of [chronic] elevated phenylalanine levels and how this might also map onto similar elevations noted in cases of schizophrenia too. Noting the increasing interest in cognition and schizophrenia (see this paper****** for example) and the growing  importance of cognitive impairment to cases, could the elevated phenylalanine results merely reflect this one facet of schizophrenia?

So you can see that there is a lot more to do in this area. Given also that schizophrenia, like autism, is probably better represented on a spectrum model, the question is also whether hyperphenylalaninemia in relation to cases of schizophrenia might represent one particular part of that schizophrenia spectrum? At least one other study suggests possibly******* (open-access) with quite a novel alternative method for detecting phenylalanine used. A lot more to do in this area methinks.

Music to close. Driftwood by Travis.

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* Okusaga O. et al. Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality? PLoS ONE 2014. 9(1): e85945.

** Matthews DE. An Overview of Phenylalanine and Tyrosine Kinetics in Humans. J. Nutr. 2007; 137: 15495-15555.

*** Richardson MA. et al. Evidence for a tetrahydrobiopterin deficit in schizophrenia. Neuropsychobiology. 2005;52(4):190-201.

**** Richardson MA. et al. Analysis of plasma biopterin levels in psychiatric disorders suggests a common BH4 deficit in schizophrenia and schizoaffective disorder. Neurochem Res. 2007 Jan;32(1):107-13.

***** Pascucci T. et al. Behavioral and neurochemical characterization of new mouse model of hyperphenylalaninemia. PLoS One. 2013 Dec 20;8(12):e84697.

****** Keefe RS. & Harvey PD. Cognitive impairment in schizophrenia. Handb Exp Pharmacol. 2012;(213):11-37.

******* Teraishi T. et al. 13C-phenylalanine breath test detects altered phenylalanine kinetics in schizophrenia patients. Translational Psychiatry 2012; 2: e119.

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Olaoluwa Okusaga, Olesja Muravitskaja, Dietmar Fuchs, Ayesha Ashraf, Sarah Hinman, Ina Giegling, Annette M. Hartmann, Bettina Konte, Marion Friedl, Jason Schiffman, Elliot Hong, Gloria Reeves, & et al (2014). Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality? PLoS ONE, 9 DOI: 10.1371/journal.pone.0085945

BH4 for autism?

I've talked about tetrahydrobiopterin (sapropterin or BH4) a few times on this blog with reference to autism (see here and here) and also some interesting suggestions about it being a potential intervention for the archetypal 'diet can affect behaviour' condition, PKU (see here).

The Nubian Giraffe @ Wikipedia 

A quick recap: BH4 is a hold-my-hand cofactor involved in some pretty important biochemical reactions; notably quite a few utilising those interesting aromatic amino acids (see here) and their neurotransmitter relations. Deficiency of BH4 has a few consequences as one might imagine; one of the important ones being a build up of the amino acid phenylalanine, which as seen in PKU and perhaps other conditions, is not necessarily a great position to be in.

Going back to the autism connection, the paper by Cheryl Klaiman and colleagues* caught my eye, as they reported on the results of a gold-standard double-blind, placebo-controlled trial of BH4 in young children (3-7 years old) diagnosed with an autism spectrum disorder (ASD). Actually, you can see a little bit more about their trial from their entry in the ClinicalTrials.gov database (see here) with the requirement for the study authors to update their study details!! (as of July 2013).

Anyhow, in their fairly small participant group they looked at children taking BH4 - 20mg/Kg body weight per day - compared with those taking a placebo for 16 weeks and examined various autism and related behaviours. They reported no statistically significant difference on their primary outcome measure (the CGI-I and CGI-S) but..... there were a number of significant improvements noted on some of the secondary measures used including behaviours related to social awareness, hyperactivity and aspects of language. Importantly too, reported side-effects from BH4 were minimal and on a par with those reported by the placebo group. They conclude: "These results indicate that BH4 offers promise in reducing symptoms of ASD".

These are interesting results both insofar as what is reported and the speculations about what BH4 might be doing. Unfortunately, this study did not report on any specific biochemical measures so it's slightly difficult to add anything further even though just some simple measures of things like blood phenylalanine levels** or even nitric oxide (NO) metabolites*** would, I dare say, have been quite revealing.

I'm not going quibble however about this paper because it adds to the already interesting evidence base on BH4 for at least some cases of autism. That and the quite impressive record on few and far between side-effects of BH4 makes for another interesting potential therapeutic agent (and its targets) should anyone wish to take up the research gauntlet further.

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* Klaiman C. et al. Tetrahydrobiopterin as a treatment for autism spectrum disorders: a double-blind, placebo-controlled trial. J Child Adolesc Psychopharmacol. 2013 Jun;23(5):320-8. doi: 10.1089/cap.2012.0127.

** Burton BK. et al. Sapropterin therapy increases stability of blood phenylalanine levels in patients with BH4-responsive phenylketonuria (PKU). Mol Genet Metab. 2010 Oct-Nov;101(2-3):110-4. doi: 10.1016/j.ymgme.2010.06.015.

*** Frye RE. et al. Metabolic effects of sapropterin treatment in autism spectrum disorder: a preliminary study. Transl Psychiatry. 2013 Mar 5;3:e237. doi: 10.1038/tp.2013.14.

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Klaiman C, Huffman L, Masaki L, & Elliott GR (2013). Tetrahydrobiopterin as a treatment for autism spectrum disorders: a double-blind, placebo-controlled trial. Journal of child and adolescent psychopharmacology, 23 (5), 320-8 PMID: 23782126

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.

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* 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.

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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

Stop that phenylalanine now!

I'm going slightly off-piste with this post not strictly related to autism; however remaining true to my interest in all things amino acids, and in particular one of those most interesting aromatic amino acids, phenylalanine.

Phenylalanine @ Wikipedia

A few months back Chemistry World carried a very interesting article by Jon Evans* on how the amino acid phenylalanine might very well have the capacity to form amyloid-like fibrils classically related to conditions like Alzheimers disease. The article was based on this study by Adler-Abramovich and colleagues** which reported on a few pretty important observations.

I've done amyloid or rather amyloid precursor protein (in relation to autism) on this blog before (see here). Fibrils as their name suggests, are fibre-like structures. Without trying to plagiarise the article or study, a few points are worth noting:

• The starting point was the metabolic condition phenylketonuria (PKU), probably the most famous of the inborn errors of metabolism.
• PKU... the problems with metabolising phenylalanine as a result of issues with phenylalanine hydroxylase leads to a build up of the amino acid which has some particularly nasty effects on the developing body and brain. Said phenylalanine levels normally controlled by lifetime dietary restriction of phenylalanine but also potentially another intervention (BH4).
• Adler-Abramovich et al observed that phenylalanine in solution tended to 'clump' together forming something that looked like amyloid-like fibrils. Further when such phenylalanine fibrils were added to cell lines, the fibrils showed some degree of toxicity. Even further, rabbits injected with said fibrils started to generate antibodies against them. Finally, after imaging the phenylalanine fibrils, there was a match between what they saw in the laboratory and what they saw in an engineered mouse model of PKU and indeed in real people diagnosed with PKU.
• Ipso facto: phenylalanine can form amyloid-like fibrils and such fibrils might account for the tissue damage observed in PKU.

I admit to being really quite excited about this work. Modern science knows quite a bit about PKU following the very astute observations made by Dr. Asbjørn Følling. One area however that has always required a little more study was exactly how the build-up of phenylalanine caused the problems that it does; maybe part of that question has been answered with this work.

My mind also goes back to those other aromatic amino acids like tryptophan and tyrosine and whether similar fibrils could be formed from these compounds. The answer, according to the published scientific literature so far, is a very possible yes; as per studies like this one from Cecchini and colleagues*** although quite a lot of the investigations in this area goes well above my state of knowledge.

So, once again amino acids do their stuff. It makes you wonder whether Alzheimer's disease, at least some cases, might also reflect issues with phenylalanine. Logically also whether interventions like a low phenylalanine diet or even BH4 supplementation (see here) might reflect possible areas of intervention requiring much further study?

To finish, I'm sure many of you had the same 'could try harder' reports from school as I did when I was a kid. But to get something like this.... from your Dad?

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* Evans J. Anti-social amino acids gang up. Chemistry World. August 2012.

** Adler-Abramovich L. et al. Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nat Chem Biol. 8: 701-706.

*** Cecchini P. et al. The role of tryptophan in protein fibrillogenesis: relevance of Trp7 and Trp14 to the amyloidogenic properties of myoglobin. PEDS. 2012; 25: 199-203.

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Adler-Abramovich L, Vaks L, Carny O, Trudler D, Magno A, Caflisch A, Frenkel D, & Gazit E (2012). Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nature chemical biology PMID: 22706200

BCKDK mutations: a treatable form of autism?

The Nature headline reads: Amino-acid deficiency underlies rare form of autism. My interest is piqued. The paper by Novarino and colleagues* suggests "autism presenting with intellectual disability and epilepsy caused by BCKDK mutations represents a potentially treatable syndrome". My interest is most definitely piqued. Hence therefore this very quick post (yeah right!) on some potentially very important results.

Let's rewind a bit first. I've talked about amino acids and autism (and schizophrenia) quite a bit already on this blog as per this Godzilla post and this one on those very, very interesting aromatic amino acids. Generally speaking, issues with specific amino acids related to paediatric mental health fall into the category of an in-born error of metabolism; where phenylketonuria (PKU) represents the archetypal 'diet can affect mental health' condition. I say generally but am willing to concede if I am wrong.

Anyway, I'm still in the process of trying to get the full-text paper by Gaia Novarino et al and indeed get my head around the findings, but from what I gather this was a multi-faceted study:

• To start with they sequenced the genomes (exome sequencing) of six children with autism from three families where mum and dad were first cousins and focused in on mutation related to the BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) gene. The gene ties into a protein which inactivates an enzyme, or complex of enzymes called branched chain ketoacid dehydrogenase (BCKDH) related to the degradation of a certain family of amino acids, called branched-chain amino acids including valine, leucine and isoleucine. Issues with the BCKDHA gene (one of the enzyme subunit genes) is linked to another inborn error of metabolism, maple syrup urine disease, so named because of the distinctive smell of the urine of those with the condition as a result of the build up of these branched-chain amino acids.
• Anyway, these amino acids are essential in that they cannot be synthesised endogenously so must be derived from food or other nutrient sources. The suggestion being that with BCKDH running unchecked as a result of issues with BCKDK, functional levels of these branched chain amino acids are also going to be reduced.
• Sure enough those participants who presented homozygously (see zygosity) with the BCKDK mutation/s showed "reductions in BCKDK mRNA and protein, E1-α phosphorylation, and plasma branched chain amino acids".
• Next, the authors working with an engineered strain of mouse with the same issue with the BCKDK gene (knockout mice) reported that the mice showed certain traits similar to those noted in the human participants with regards to seizures and importantly also showed low levels of the branched-chain amino acids.
• A diet enriched with said amino acids seemed to 'abate' some of these symptoms in the mice; or as the authors put it they "respond to dietary supplementation". Ipso facto, autism and epilepsy associated with issues with BCKDK might also be treatable by such amino acid supplementation.

I don't claim to have provided a definitive overview of the findings here but I think this is pretty close to what has been reported. In essence the checks and balances which are supposed to keep the levels of these branched-chain amino acids within tolerance are disrupted, but rather than the maple syrup urine disease scenario, lower levels of these essential amino acids seem also to have some potential effect.

There are bound to be quite a few discussions about this new research and its implications as it filters through both the scientific and lay community. I would stress that at the current time, I advocate nothing based on these results until the appropriate safety and efficacy studies are undertaken on the appropriate group.

If anything however, this study reiterates a few things: (i) autisms is probably a better description than autism, and (ii) don't underestimate the power of the amino acids.

Night-night.

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Novarino G. et al. Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy. Science. September 2012.

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Gaia Novarino,Paul El-Fishawy, Hulya Kayserili, Nagwa A. Meguid, Eric M. Scott, Jana Schroth1, Jennifer L. Silhavy1, Majdi Kara, Rehab O. Khalil, Tawfeg Ben-Omran, A. Gulhan Ercan-Sencicek, Adel F. Hashish, Stephan J. Sanders, Abha R. Gupta, Hebatalla S Hashem, Dietrich Matern, Stacey Gabriel, Larry Sweetman, Yasmeen Rahimi, Robert A. Harris, Matthew W. State, & Joseph G. Gleeson (2012). Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy Science : 10.1126/science.1224631

Dried blood spots, cytokines and autism

Thank you Robert Guthrie @ Wikipedia

The Nature website recently carried an interesting headline: "Archived blood spots could be epigenetic jackpot" based on a recent study by Huriya Beyan and colleagues* on the application of DNA methylomics to stored Guthrie cards to study early infant epigenetics.

Just in case this makes no sense to you, the basic suggestion is that all those dried blood spots taken from days old infants to test for various inborn errors of metabolism like PKU, could be used to examine the switching on or off of genes well before most diseases or conditions become apparent. At the very least it would compare what's there at birth with what might be there at later stages in life and focus attention on factors in that intervening period.

I'm interested in this area for lots of different reasons. Not least that infant dried blood spots - whilst a little traumatic to see when blood is taken from this tiny little baby - are indeed a treasure trove of information waiting to be discovered. With regards to a condition like autism(s) which only really manifest in early childhood, there could be several markers, endophenotypic biomarkers, waiting on those little pieces of card and possibly not just all epigentic ones.

Indeed I'm very happy to report that autism research has already taken advantage of the infant dried blood spots available as evidenced by this study by Abdallah and colleagues** on neonatal levels of cytokines in cases of autism. Regular readers will know that I've done cytokines and autism on the blog quite a few times (see here for example). Cytokines, those chemical messengers, linked to interesting things like inflammation are finding a real home in autism research and other areas of investigation but still some questions need to be answered (why? who? what to do about it?).

Abdallah et al looked at several hundred neonatal blood spot samples (n=359) where autism was eventually confirmed compared with a sizable control population (n=741). They found quite a bit of evidence to suggest that children with autism were more likely to present with 'hypoactive immune cell activity' as youngsters than controls including lower levels of everyone's favourite interferon, interferon gamma.

I've talked before about the often conflicting results on immune function in cases of autism and how researchers such as Harumi Jyonouchi have presented similarly 'depressed' immune function related to cases of autism whilst others report immune over-activity or the presence of autoantibodies. I will at this point press a few words home to you: heterogeneity, endophenotypes, comorbidity and gene-environment interactions.

Indeed this recent study by Goines and Ashwood*** (Paul Ashwood that is) kinda summarises how complicated cytokine dysregulation seems to be with autism in mind and how environment combined with genetics may very well play an important role in the presentation of immune function in cases of autism.

Neonatal blood spots offer a fascinating opportunity to explore lots and lots of features potentially related to autism and beyond. Neonatal vitamin D status as per this study by McGrath and colleagues**** using the same Danish neonatal database following on from the recent interest in vitamin D and autism is just one example of where this could go. Indeed given the millions of people around the world who undergo neonatal blood spot screening every year, the possibilities of comparisons by diagnosis, geography, ethnicity, etc. and across different times, are seemingly endless.

To finish a song by another Robert (Smith not Guthrie) about it being Friday and someone being in love? (even though at the time of publication of this post, it is actually still Tuesday where I am).

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* Beyan H. et al. Guthrie card methylomics identifies temporally stable epialleles that are present at birth in humans. Genome Research. August 2012.

** Abdallah MW. et al. Neonatal levels of cytokines and risk of autism spectrum disorders: An exploratory register-based historic birth cohort study utilizing the Danish Newborn Screening Biobank. Journal of Neuroimmunology. August 2012.

*** Goines PE. & Ashwood P. Cytokine dysregulation in autism spectrum disorders (ASD): Possible role of the environment. Neurotoxicology & Teratology. August 2012.

**** McGrath JJ. et al. Neonatal vitamin D status and risk of schizophrenia: a population-based case-control study. Archives of General Psychiatry. 2010; 67: 889-894.

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Abdallah MW, Larsen N, Mortensen EL, Atladóttir HO, Nørgaard-Pedersen B, Bonefeld-Jørgensen EC, Grove J, & Hougaard DM (2012). Neonatal levels of cytokines and risk of autism spectrum disorders: An exploratory register-based historic birth cohort study utilizing the Danish Newborn Screening Biobank. Journal of neuroimmunology PMID: 22917523