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Wednesday 21 October 2015

Biomarkers in Autism






This post has been sitting unfinished for a while, so I decided to publish it before I forget all about it

The two papers discussed today really confirm much of what we have already established in this blog, but they are very useful as a recap and for those with limited time.

The first paper is extremely comprehensive and, if you go through it very slowly, really tells you much of what you need to know about the biology of autism.  It is some wonder that so few clinicians are aware of these findings.



Abstract

Autism spectrum disorders (ASDs) are complex, heterogeneous disorders caused by an interaction between genetic vulnerability and environmental factors. In an effort to better target the underlying roots of ASD for diagnosis and treatment, efforts to identify reliable biomarkers in genetics, neuroimaging, gene expression, and measures of the body’s metabolism are growing. For this article, we review the published studies of potential biomarkers in autism and conclude that while there is increasing promise of finding biomarkers that can help us target treatment, there are none with enough evidence to support routine clinical use unless medical illness is suspected. Promising biomarkers include those for mitochondrial function, oxidative stress, and immune function. Genetic clusters are also suggesting the potential for useful biomarkers.



Here are the key parts; I do suggest you read the full text of the paper.


Metabolic Biomarkers


There are no autism-defining, metabolic biomarkers, but examining the biomarkers of pathways associated with ASD can point to potentially treatable metabolic abnormalities and provide a baseline that can be tracked over time. Each child may have different metabolic pathologies related to SNPs, nutrient deficiencies, and toxic exposures. Examples of metabolic disorders that can lead to an autistic-like presentation include phenylketonuria (PKU) (37), disorders of purine metabolism (38), biotinidase deficiency (39), cerebral folate deficiency (40), creatine deficiency (41), and excess propionic acid (which is produced by Clostridium) (42, 43).

A recent review assessed the research on physiological abnormalities associated with ASD (44). The authors identified four main mechanisms that have been increasingly studied during the past decade: immunologic/inflammation, oxidative stress, environmental toxicants, and mitochondrial abnormalities. In addition, there is accumulating research on the lipid, GI systems, microglial activation, and the microbiome, and how these can also contribute to generating biomarkers associated with ASD.

The brain is highly vulnerable to oxidative stress (51), particularly in children (52) during the early part of development (47). As environmental events and metabolic imbalances affect oxidative stress and methylation, they also can affect the expression of genes.

Several studies have detected altered levels of a large collection of substances in body-based fluids from ASD subjects compared to controls (e.g., serum, whole-blood, and CSF) (53). These findings encompass either of two main disease-provoking mechanisms: a CNS disorder that is being detected peripherally [e.g., serotonin and its metabolites, sulfate (54), low platelet levels of gamma-aminobutyric acid (GABA) (55), low oxytocin (which affects social affiliation) (56), and low vitamin D levels (57, 58)] or a systemic abnormality that has repercussions in the brain (59).

Oxidative stress markers

Oxidative stress can be detected by studying antioxidant status, antioxidant enzymes, lipid peroxidation, and protein/DNA oxidation, all of which have been found to be elevated in children with autism (Table (Table2).2). Different subgroups of children with ASD have different redox abnormalities, which may arise from various sources



Measurements of antioxidant status include measurement of glutathione, the primary antioxidant in the protection against oxidative stress, neuroinflammation, and mitochondrial damage (68, 69). Glutathione is instrumental in regulating detoxification pathways and modulates the production of precursors to advanced glycation end products (AGEs) (70). Measuring reduced glutathione, oxidized glutathione, or the ratio of reduced glutathione to oxidized glutathione helps determine the patient’s oxidation status. In many patients with ASD, the ratio of reduced glutathione to oxidized glutathione is decreased, indicating a poor oxidation status

The enzyme glutathione peroxidase has been used as a marker and is typically reduced. There are mixed results concerning the enzyme levels of superoxide dismutase (SOD) (72). Other markers for glutathione inadequacy include alpha hydroxybutyrate, pyroglutamate, and sulfate, which can be assessed in an organic acid test. Lipid peroxidation refers to the oxidative degradation of cell membranes. There is a significant correlation between the severity autism and urinary lipid peroxidation products (67), which are increased in patients with ASD

Plasma F2t-Isoprostanes (F2-IsoPs) are the most sensitive indicator of redox dysfunction and are considered by some to be the gold standard measure of oxidative stress (73). They are increased in patients with ASD and are even higher when accompanied by gastrointestinal dysfunction (73).

Decreased levels of major antioxidant serum proteins transferrin (iron-binding protein) and ceruloplasmin (copper binding protein) have been observed in patients with ASD. The levels of reduction in these proteins correlate with loss of previously acquired language (47) although there are mixed reviews of the significance of this (66).


Plasma 3-chlortyrosine (3CT), a measure of reactive nitrogen species and myeloperoxidase activity, is an established biomarker of chronic inflammatory response. Plasma 3CT levels reportedly increased with age for those with ASD and mitochondrial dysfunction but not for those with ASD without mitochondrial dysfunction (65).

3-Nitrotyrosine (3NT) is a plasma measure of chronic immune activation and is a biomarker of oxidative protein damage and neuron death. This measure correlates with several measures of cognitive function, development, and behavior for subjects with ASD and mitochondrial dysfunction but not for subjects with ASD without a mitochondrial dysfunction (65).


Mitochondrial dysfunction markers

Mitochondrial dysfunction is marked by impaired energy production. Some children with ASD are reported to have a spectrum of mitochondrial dysfunction of differing severity (44) (Table (Table3).3). Mitochondrial dysfunction, most likely an early event in neurodegeneration (76), is one of the more common dysfunctions found in autism (77) and is more common than in typical controls (78). There is no reliable biomarker to identify all cases of mitochondrial dysfunction (79). It is possible that up to 80% of the mitochondrial dysfunction in patients with both ASD and a mitochondrial disorder are acquired rather than inherited (44).

Mitochondrial dysfunction can be a downstream consequence of many proposed factors including dysreactive immunity and altered calcium (Ca2+) signaling (80), increased nitric oxide and peroxynitrite (68), propionyl CoA (81), malnutrition (82), vitamin B6 or iron deficiencies (83), toxic metals (83), elevated nitric acid (84, 85), oxidative stress (86), exposure to environmental toxicants, such as heavy metals (8789), chemicals (90), polychlorinated biphenyls (PCBs) (91), pesticides (92, 93), persistent organic pollutants (POPs) (94), and radiofrequency radiation (95). Other sources of mitochondrial distress include medications such as valproic acid (VPA), which inhibits oxidative phosphorylation (96) and neuroleptics (97, 98).

Markers of mitochondrial dysfunction include lactate, pyruvate and lactate-to-pyruvate ratio, carnitine (free and total), quantitative plasma amino acids, ubiquinone, ammonia, CD, AST, ALT, CO2 glucose, and creatine kinase (CK) (44). Many studies of ASD report elevations in lactate and pyruvate, others report a decrease in carnitine, while others report abnormal alanine in ASD patients (44) or elevations in aspartate aminotransferase and serum CK (99). Increases in lactate are not specific and may only occur during illness, after exercise or struggling during a blood draw (100).


Rossignol and Frye (44) recommend a mitochondrial function screening algorithm. This includes fasting morning labs of lactate, pyruvate, carnitine (free and total), acyl carnitine panel, quantitative plasma amino acids, ubiquinone, ammonia, CK, AST/ALT, CO2, and glucose (44). The interpretation of such a panel and the indications for specific treatments has not yet been established.

Methylation

The methylation pathway provides methyl groups for many functions, including the methylation of genes, which can result in the epigenetic changes of turning genes on and off (Table (Table4).4). This transfer occurs when S-adenosylmethionine (SAM) donates a methyl group and is transformed to S-adenosylhomocysteine (SAH). SAH can be transferred to homocysteine, which can either be re-methylated to methionine or be transferred by the sulfuration pathway to cysteine to create glutathione. With increased oxidative stress, SAH might be diverted away from the methylation pathway to the sulfuration pathway in order to make more glutathione. This will result in less methionine and less methylation ability.

A marker of methylation dysfunction is decreased SAM/SAH ratio in patients with ASD. Fasting plasma methionine decreases since through SAM it is the main methyl donor. Fasting plasma cysteine, a sulfur containing amino acid is the rate-limiting step in the production of glutathione and is significantly decreased. Plasma sulfate is decreased, which may impair detoxification pathways. Homocysteine is generally increased, but the studies are mixed (66). Vitamin B12 and folate are required for the methylation pathway. The MTHFR genetic SNP is reported to heavily influence the methylation pathway (66).

Immune dysregulation


Cytokine evaluation


Chronic inflammation and microglia cell activation is present in autopsied brains of people with ASD (101, 102) (Table (Table5).5). Factors that increase the risk of activating brain microglia include traumatic brain injury (TBI) (103) reactive oxygen species (104) and a dysfunctional blood brain barrier (105). The blood brain barrier can be compromised by oxidative stress (106), acutely stressful situations (107), elevated homocysteine (108), diabetes (109), and hyperglycemia (110). Cytokines can pass through a permeable blood brain barrier and start this process (111). Hence, cytokines can serve as a marker of the immune dysregulation, which can further complicate ASD.


Autoimmunity and maternal antibodies


Autoimmune autistic disorder is proposed as a major subset of autism (118), and autoimmunity may play a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders (119). There are many autoantibodies found in the nervous system of children with ASD who have a high level of brain antibodies (120, 121). These can be measured as biomarkers in this subset of ASD patients. The anti ganglioside M1 antibodies (122), antineuronal antibodies (123), and serum anti-nuclear antibodies (123, 124) correlate with the severity of autism. Other autoantibodies postulated to play a pathological role in autism include: anti neuron-axon filament protein (anti-NAFP) and glial fibrillary acidic protein (anti-GFAP) (125), antibodies to brain endothelial cells and nuclei (119), antibodies against myelin basic protein (126, 127), and anti myelin associated glycoprotein, an index for autoimmunity in the brain (128). BDNF antibodies were found higher in ASD (129), and low BDNF levels may be involved in the pathophysiology of ASD (130).

Antibodies in patients with autism are found to cells in the caudate nucleus (131), cerebellum (132, 133), hypothalamus and thalamus (121), the cingulate gyrus (134), and to cerebral folate receptors (135). Children with cerebellar autoantibodies had lower adaptive and cognitive function as well as increased aberrant behaviors compared to children without these antibodies (132).

Mother’s immune status


Research studies indicate an association between viral or bacterial infections in expectant mothers and their ASD offspring (136, 137). Maternal antibodies cross the underdeveloped blood brain barrier of the fetus (138) leading to impaired fetal neurodevelopment and long-term neurodegeneration, neurobehavioral, and cognitive difficulties (139).

Dysbiosis


When the gut becomes inflamed, it breaks down and becomes permeable, sometimes referred to as dysbiosis. Dysbiosis is reported to be an upstream contributing factor to autoimmune conditions and inflammation. Markers under consideration include circulating antibodies against tight junction proteins, LPS, actomyosin (145) calprotectin (146), and lactoferrin (147). Dysbiosis was found in 25.6% of patients with ASD (148). It is proposed to have a direct effect on the brain as it is a hypothesized source of inflammation (149151) and autoimmunity (152, 153), possibly through molecular mimicry (154). Diet is one source of dysbiosis (155).


Amino acids and neuropeptides


Platelet hyperserotonemia is considered one of the most consistent neuromodulator findings in patients with ASD (Table (Table6).6). As for other neuropeptides, a recent review reported approximately 15 components that are altered in ASD compared to controls (53). Among them, interesting research has been done on glutamate, GABA, BDNF, and dopamine and noradrenaline systems. A recent study reported a positive correlation between severity of clinical symptoms and plasma GABA levels in patients with ASD, supporting the idea of a disrupted GABAergic system (156).

 

Fatty acid analysis


Abnormal fatty acid metabolism may play a role in the pathogenesis of ASD and may suggest some metabolic or dietary abnormalities in the regressive form of autism (42, 157). There is evidence of a relationship between changes in brain lipid profiles and the occurrence of ASD-like behaviors using a rodent model of autism (42). Hyperactivity in patients was inversely related to the fluidity of the erythrocyte membrane and membrane polyunsaturated fatty acid (PUFA) levels (158). Imbalances of membrane fatty acid composition and PUFA loss can affect ion channels and opiate, adrenergic, insulin receptors (159) and the modulation of (Na + K)-ATPase activity (160). Analysis of red blood cell membrane fatty acids is a very sensitive indicator of tissue status and may reflect the brain fatty acid composition (161).
Seventeen percent of children with ASD manifest biomarkers of abnormal mitochondrial fatty acid metabolism, the majority of which are not accounted for by genetic mechanisms (162). Patients with ASD had reduced percentages of highly unsaturated fatty acids (163) and an increase in ω6/ω3 ratio (158).

Biomedical Interventions


There are no published studies of interventions for ASD that use neuroimaging or genetic biomarkers in a prospective manner to guide treatment. Biomedical interventions based on body fluid/product biomarkers have been used in a small but growing numbers of well designed, published studies. Several recent reviews summarize these.

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If you have managed to digest all of that information, here is another very interesting paper.

The researchers are, as so often, from Johns Hopkins.  This time they propose an idea to simplify the understanding of the bewildering number of autism sub-types.

I have frequently commented in this blog that in many identified underlying dysfunctions, being hyper (too much) or hypo (two little) causes the same effect, i.e. autism.

They split autism into:-

·        hyper-active pro-growth signaling pathways (e.g. big heads)
·        hypo-active pro-growth signaling pathways  (e.g. small heads)


So the first question is whether the patient is type A or type B.









It is definitely a step forward in simplifying what is going on, so that one day a clinician, without being a Nobel Laureate, could treat autism without just using trial and error.  If the clinician had also read, and understood, the first paper, he/she really would be able to help the patient.






The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.









6 comments:

  1. Hellow Peter , please I urgently need your advice, do you think it is better Dimebon or l-acethy carnitine with cq10 and lipoic acid to treat huntington disease? And do you know where to buy Dimebon? Thank you so much for your answer.

    ReplyDelete
    Replies
    1. Dimebon is in limited supply even in Russia. Some internet companies selling Russian drugs may offer it at a very high price. The clinical trial was not positive.

      An alternative mitochondrial treatment that is available is melatonin, at a higher dose of about 10 mg. There is some solid science behind it. It will induce sleep, so it would need to be taken in the evening.

      Taking carnitine, cq10 and lipoic acid will also help mitochondria.

      Delete
  2. How about testing the possible association between impaired levels and the severity of social and cognitive dysfunctions in individuals with autism spectrum disorders? Is it helpful?

    ReplyDelete
    Replies
    1. If you had a big database on thousands of people with ASD, including details of their autism, behaviors, IQ etc you very likely could find useful correlations and associations. Unfortunately they usually have data on 30 kids and try and draw conclusions.

      Delete
  3. https://pubmed.ncbi.nlm.nih.gov/27904735/
    I think you may already have come across this article.
    It seems to confirm CDR. The sample size was 30 only but they were at young age.

    ReplyDelete
  4. Very informative thanks a lot for this blog

    ReplyDelete

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