The 'oral microbiota' and autism: the power of a spit sample

Although not everyone's cup of tea there's a lot that can be learned from a humble spit (saliva) sample. Buccal epithelial cells from saliva samples provide a medium for the collection and analysis of DNA for example, and alongside, make for a much less invasive collection method than DNA capture from blood samples for example. Saliva also has it's own metabolome, meaning that one can potentially get quite a bit of information on quite an extensive library of small molecules linked to various genetic and biological processes.

Now add the oral microbiota to the list and, as per the findings reported by Yanan Qiao and colleagues [1], how the oral cavity (i.e. the mouth) is home to a complex network of bacteria and relations that might provide some important clues pertinent to various diagnoses.

Autism was the particular diagnosis in the research spotlight this time around, as authors "collected samples from two distinct intraoral habitats, including saliva and dental plaques, in children with and without ASD [autism spectrum disorder]." Analysing over 100 samples provided by 32 children with ASD and 27 not-autism controls, a few interesting things were noted in the results published by Qiao et al.

Bearing in mind this was a cross-sectional study which relied on a 'snapshot' sample over only one testing occasion, authors reported that: (a) data on bacterial richness and diversity showed no significant differences in salivary samples across the groups, but a difference was reported based on examination of those dental plaque samples; (b) "the phylum Proteobacteria was more abundant in ASD patients (both in salivary and dental samples) compared to controls" whilst other phyla predominated in controls; (c) "increased amounts of potential pathogens, including Haemophilus, Corynebacterium, Cardiobacterium, Kingella, Streptococcus and Rothia, were observed in ASD patients" some of which correlated with the measurement of the severity of autism (via parental report on the Aberrant Behavior Checklist (ABC) questionnaire); and (d) "diagnostic models based on key microbes were constructed, with 96.3% accuracy in saliva."

Of course, there is still some way to go in this research area, not least starting with independent replication of the Qiao results perhaps also relying on multiple samples provided across a range of times and situations. Y'know, assuming that medication for example, could be part and parcel of the profile with autism in mind and indeed remembering that autism as a stand-alone label is probably less like 'real-life autism' than many people realise (see here). I'm also a little unsure as to why the Aberrant Behavior Checklist (ABC) was employed "to preliminarily evaluate the severity of ASD" when both so many controls were employed for inclusion/exclusion on the study and so many other more 'autism-specific' instruments are quite freely available (see here for one example). There are things that could have been done differently for sure...

But, this is a good first attempt. It puts the oral microbiome on the research map with autism in mind, added to the more well-known relation: the gut microbiome (see here). It also provides us with a more generalised view of how the bacteria and various other miniature beasties around us, are probably more intricately involved in our lives than we ever previously thought possible...

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[1] Qiao Y. et al. Alterations of oral microbiota distinguish children with autism spectrum disorders from healthy controls. Scientific Reports; 8: 1597.

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Yet more on potential biomarkers and chronic fatigue syndrome

'Thick and fast' is probably the best way that I can describe the flurry of peer-reviewed scientific papers recently appearing (see here and see here for examples) talking about how chronic fatigue syndrome (CFS) (also linked to the diagnosis of myalgic encephalomyelitis, ME) might have some important biological processes attached to it.

Now we can add the findings reported by Federica Ciregia and colleagues [1] (open-access) to the list and their observations that "the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS."

The Ciregia paper is open-access but well-worth a few inches of discussion on this blog. Not least because (a) the word 'mitochondria' is part and parcel of the their findings in line with other research in this area, (b) one of the gold standards of analytical chemistry - liquid chromatography mass spectrometry -  was used, and (c) some of the findings are based on a study of twins: "a patient suffering from CFS in comparison with his healthy monozygotic twin." This mirrors other similar published work from this authorship group [2].

So, using a discovery/training and validation approach similar to other biomarker studies in other areas, researchers initially set out to "study the mitochondria extracted from platelets of the twins" using "nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS)." They were looking for evidence of different compounds being presented/expressed in those twins diagnosed with CFS compared with their non-affected twin and eventually came up with 41 proteins - "34 were upregulated in CFS and 7 were downregulated" (see here for the list of compounds).

Using a process called Ingenuity Pathway Analysis (IPA) "to retrieve the known functions of each protein" authors were able to visualise where each compound 'fitted' in terms of specific biological functions. The top three included: "metabolism of isocitric acid..., metabolism of NADH... and metabolism of nucleic-acid component or derivative." Certainly NADH has some 'history' when it comes to CFS/ME (see here).

Then came the validation side of the study where "the most promising biomarkers were validated by western blot [WB] analysis in a big cohort of patients, using whole saliva (WS)." Here some 45 patients diagnosed with CFS ("based on the classification criteria of Fukuda et al") were recruited alongside 45 not-CFS controls and spit samples from all were analysed for "aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM)." Two proteins, ACON and ATPB. were replicated or at least "consistent with the results from nano-LC-MS."

Finally, researchers looked at whether presented clinical features as described in various questionnaires delivered to participants might play a role in the presentation of their biological results. They did see something (see here) - "For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin" - but I would be minded to suggest that quite a bit more work is needed before anyone reads too much into this as the results stand.

So, there you have it. A little bit more evidence to suggest that science is edging a little closer to potentially identifying some of the biology behind (or least associated with) at least some CFS (and ME). A little bit more peer-reviewed evidence moving the discussions away from 'psychosomatic' [3] to something a little more testable/analysable with CFS/ME in mind (I'll be coming to the paper by Geraghty & Esmail soon enough on this blog by the way). Independent replication is the next step, onwards to potentially "developing tailored treatments." That bearing in mind, we already have some emerging data in this area too (see here) (with no medical advice given or intended).

And just in case you want yet more potential biomarker research for CFS, here's another paper that has just been published [4]. Thick and fast people, thick and fast.

So, there is a new trailer for Rogue One (A Star Wars story)...

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[1] Ciregia F. et al. Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome. Transl Psychiatry. 2016 Sep 27;6(9):e904.

[2] Ciregia F. et al. A multidisciplinary approach to study a couple of monozygotic twins discordant for the chronic fatigue syndrome: a focus on potential salivary biomarkers. J Transl Med. 2013 Oct 2;11:243.

[3] Geraghty KJ. & Esmail A. Chronic fatigue syndrome: is the biopsychosocial model responsible for patient dissatisfaction and harm? Br J General Practitioners. 2016. Aug 1.

[4] Yamano E. et al. Index markers of chronic fatigue syndrome with dysfunction of TCA and urea cycles. Scientific Reports. 2016; 6: 34990.

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Ciregia F, Kollipara L, Giusti L, Zahedi RP, Giacomelli C, Mazzoni MR, Giannaccini G, Scarpellini P, Urbani A, Sickmann A, Lucacchini A, & Bazzichi L (2016). Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome. Translational psychiatry, 6 (9) PMID: 27676445

High risk for autism = shortened telomeres?

I don't want to spend too long discussing the paper by Charles Nelson and colleagues [1] suggesting that: "Families of children with ASD [autism spectrum disorder] who have an infant show shortened telomeres relative to families with no history of ASD" but it is worth blogging about.

As per a previous entry on telomeres and autism (see here), telomeres - the biological equivalent of plastic aglets on shoelace tips to prevent fraying - are starting to enter the autism [peer-reviewed] research psyche on top of their more traditional role suggested in ageing and cancer for example (see here). Indeed, telomeres and cellular ageing are getting quite a bit of press these days with psychiatry in mind [2] as per the goings-on with schizophrenia in mind [3] and psychotic symptoms [4].

The Nelson study started from the angle that: "Exposure to psychological stress is associated with accelerated telomere shortening, and a well-established body of evidence indicates that families with a child with autism spectrum disorder (ASD) experience heightened levels of psychological stress." They also make mention of the words 'oxidative stress' and 'DNA methylation' as also potentially impacting on telomere length and at the same time having some research 'form' when it comes to autism (see here and see here respectively).

With that all in mind, saliva samples were analysed for family members designated as 'high risk for ASD (HRA)' or 'low risk for ASD (LRA)' as a function of "older siblings' diagnostic status." Relative average telomere length was the chosen variable analysed by a "real-time polymerase chain reaction (PCR) telomere assay."

Results: "HRA families demonstrated significantly shorter telomere length relative to LRA families." This was noted across the board when it came to family members analysed (infants, older siblings parents) although the group data comparing fathers between the groups were not significantly different. The authors conclude that: "such "high-risk" families should be monitored for the physical and mental health consequences that are often associated with accelerated telomere shortening."

This is interesting work (isn't is always?) but I'm going to advise a little caution before anyone goes assuming that telomere length is the be-all-and-end-all of autism research. The inevitable hype that has followed telomere research down the years has done some real damage to the credibility of some of the findings on telomeres in other areas so one treads a little carefully. That telomere length seems also to correlate with quite a few other interesting concepts such as inflammation for example [5] is also of potential interest, particularly when inflammation seems to crop up time and time again with [some] autism in mind (see here). I dare say that future studies of telomere length and autism might want to take quite a wide view of any association including the analysis of telomerase too.

Music: The Charlatans - The Only One I Know.

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[1] Nelson CA. et al. Shortened Telomeres in Families With a Propensity to Autism. J Am Acad Child Adolesc Psychiatry. 2015 Jul;54(7):588-94.

[2] Lindqvist D. et al. Psychiatric disorders and leukocyte telomere length: Underlying mechanisms linking mental illness with cellular aging. Neurosci Biobehav Rev. 2015 May 18;55:333-364.

[3] Polho GB. et al. Leukocyte telomere length in patients with schizophrenia: A meta-analysis. Schizophr Res. 2015 Jul;165(2-3):195-200.

[4] Pawelczyk T. et al. Telomere length in blood cells is related to the chronicity, severity, and recurrence rate of schizophrenia. Neuropsychiatr Dis Treat. 2015 Jun 22;11:1493-503.

[5] Jurk D. et al. Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nat Commun. 2014 Jun 24;2:4172.

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Nelson CA, Varcin KJ, Coman NK, DeVivo I, & Tager-Flusberg H (2015). Shortened Telomeres in Families With a Propensity to Autism. Journal of the American Academy of Child and Adolescent Psychiatry, 54 (7), 588-94 PMID: 26088664

Analysing the salivary proteome in autism

The paper from Armand Ngounou Wetie and colleagues [1] (open-access here) reporting pilot results from a mass spectrometry based proteomic analysis of saliva in cases of autism or autism spectrum disorder (ASD) compared with asymptomatic controls is served up for your reading delight today. There has already been some media attention about this paper (see here).

It's an interesting paper for quite a few reasons; not least the continuing voyage of the analytical technique known as mass spectrometry into autism research (see here) and further beyond [2]. Mass spec by the way, represents a rather advanced way of looking at biological samples for potential biomarkers or compounds of interest to specific states or conditions (among other things). Ngounou Weite et al have some research form in this area as per a previous paper titled: 'Mass spectrometry as a tool for studying autism spectrum disorder' [3] which I would encourage you to peruse for some background reading.

Their latest study delves into an interesting analytical medium, saliva, something we all generally have and importantly, something pretty non-invasive when it comes to collecting a sample [4]. "Using nano liquid chromatography-tandem mass spectrometry, we found statistically significant differences in several salivary proteins" the authors report, comparing saliva samples from those with autism vs. asymptomatic controls. The sorts of differences detected between the groups - quite small groups (N=6 per) - tended to fall into the domain of 'immune function' as per issues with neutrophil elastase and various antigen binding sites of immunoglobulin. Indeed, the authors conclude: "Our results indicate that this is an effective method for identification of salivary protein biomarkers, support the concept that immune system and gastrointestinal disturbances may be present in individuals with ASDs." I would agree with those sentiments.

Aside from the small participant groups and the fact that all participants were male and many carried some comorbidity (ADHD, epilepsy) alongside their autism label, 4 of the participants with autism were taking some kind of pharmaceutic/nutraceutical compared with none of the controls. As per other biomarker studies of autism, one always needs to be a little mindful of any effects from these factors particularly when looking at functional biofluids. In terms of the mass spec method, it all looks pretty comprehensive including the use of Q-ToF as the detector of choice and pooled group samples run in triplicate to ensure some kind of reproducibility in results. The authors did subject saliva samples to some preparation before analysis as per their focus on peptide content and a "full MS scan [that] covered the m/z range from 400 to 1,350". What this might mean is that some very low molecular weight compounds and indeed, potentially important larger compounds might have escaped their attention. But certainly I'm not going to quibble about this for now.

Of course this is not the first time that saliva has been used as an analytical medium in autism research (see here) outside of just looking at parameters such as cortisol (see here). And I assume it won't be the last either...

Music to close: Jane's Addiction and Stop.

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[1] Ngounou Wetie AG. et al. A Pilot Proteomic Analysis of Salivary Biomarkers in Autism Spectrum Disorder. Autism Res. 2015 Jan 27. doi: 10.1002/aur.1450.

[2] Dumas M-E. & Davidovic L. Metabolic Profiling and Phenotyping of Central Nervous System Diseases: Metabolites Bring Insights into Brain Dysfunctions. Journal of Neuroimmune Pharmacology. 2015. Jan 24.

[3] Wood AG. et al. Mass spectrometry as a tool for studying autism spectrum disorder. Journal of Molecular Psychiatry 2013; 1: 6.

[4] Wormwood KL. et al. Salivary proteomics and biomarkers in neurology and psychiatry. Proteomics Clin Appl. 2015 Jan 29.

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Ngounou Wetie AG, Wormwood KL, Russell S, Ryan JP, Darie CC, & Woods AG (2015). A Pilot Proteomic Analysis of Salivary Biomarkers in Autism Spectrum Disorder. Autism research : official journal of the International Society for Autism Research PMID: 25626423

Autism and saliva

This is a little bit of an unusual post in that I want to discuss some of the collected data looking at a bit of a forgotten biological fluid, saliva, specifically with autism spectrum conditions in mind. The idea for doing this post stemmed in part from a quite recent paper by Soukup and colleagues* (full-text) which suggested that monitoring salivary uric acid might be a good idea in terms of cardiometabolic risk. Exactly how this finding itself might translate to some work on purines and autism linked to uric acid is food for thought.

Whilst pretty essential for lots of things, saliva or spit is not exactly dinner table conversation so I promise to try and keep this scientific. I hasten to add that I am not covering things like the excessive production of saliva (and onward things like drooling) or anything like that, accepting that such issues have been reported primarily as a side-effect to certain medications.

Certain areas on the autism research landscape have examined saliva as a functional biofluid. So looking at levels of cortisol in saliva is a bit of a favourite, as is some interest in salivary testosterone levels. A very recent paper by Rai and colleagues suggested that salivary antioxidant levels in autism were significantly reduced compared to asymptomatic controls which might tie in with some other findings on important markers of antioxidant health. Molecular biologists out there will already know about the value of buccal (cheek) samples as a way of collecting DNA but cheek cells are slightly different from plain old saliva.

The study I want to focus on is this one from Castagnola and colleagues** published in 2008. They reported results for a group of children diagnosed with an autism spectrum condition (n=27) compared to controls (n=23) based on a chemical analysis of naturally occurring salivary peptides. With my metabolomics hat on, this kind of study makes so much sense. Here you have a biofluid which most people produce in pretty copious amounts (during waking hours), easily available and relatively non-invasive to capture which contrasts against other research on blood as an analyte medium for example. The technology nowadays is such that you can get a lot of information from a spit sample.

Evidence suggests that little if any difference is present in the basic salivary parameters between people with autism and asymptomatic controls. The results produced by Castagnola, based on mass spectrometric analysis, suggested that hypo-phosphorylation (reduced) of certain salivary peptides was more common in the samples from children with autism compared to controls. Phosphorylation means the addition of a phosphate group to a molecule which can have various effects on a compound and its actions. The authors speculated that such hypo-phosphorylation might be a marker for other issues with phosphorylation in other tissues, using Rett syndrome as an example. Interestingly, they also draw a comparison with some of their other work on phosphorylation in preterm and term babies, suggesting that delayed peptide phosphorylation in the early days ".. may cause asynchrony or timing deregulation in some process involved in neuronal maturation, development or differentiation resulting in ASD phenotype". A few ideas there to digest possibly.

I'd like to think that as the technology gets more sensitive, analysis of saliva might be a growth area for at least some parameters related to autism currently only measurable via invasive collection of blood and plasma. Let's face it children in particular don't like having blood drawn, and children with autism are no exception to that rule. I note that studies have already reported in the area of vitamin D - measuring salivary 25-hydroxyvitamin D(3), for example; vitamin D having cropped up more than once in relation to autism. One wonders how many more biological parameters might also be measurable from a humble sample of spit.

To finish, how about a red hot chilli pepper to get us all salivating?

* Soukup M. et al. Salivary uric acid as a noninvasive biomarker of metabolic syndrome. Diabetology & Metabolic Syndrome. April 2012.
DOI: 10.1186/1758-5996-4-14

** Castagnola M. et al. Hypo-phosphorylation of salivary peptidome as a clue to the molecular pathogenesis of autism spectrum disorders. Journal of Proteome Research. 2008; 7: 5237-5232.