Biomarkers

Advances in Autism Research
April 2010

1. Mechanistic biomarkers for autism treatment
Hendren RL, Bertoglio K, Ashwood P, Sharp F.
Med Hypotheses. 2009 Dec;73(6):950-4.
$ http://tinyurl.com/y8g3n25

OBJECTIVE: Autism is a syndrome with a number of etiologies with differing mechanisms that lead to abnormal development. This review highlights the need to identify autism subgroups as they each might require unique approaches for prevention or treatment. METHODS: Targeting treatments to specific mechanisms and utilizing biomarkers can more rapidly advance our understanding of how to classify and treat autism subgroups based on translational mechanisms. We illustrate this approach using mechanisms that may influence the course of autism and provide rationale for selected biomarkers that could guide treatments targeted anywhere from DNA to symptom expression. CONCLUSIONS: The use of potential biomarkers that point to specific mechanisms of disordered neurodevelopment will help identify meaningful subtypes of autism and will help tailor treatment or prevention strategies for each mechanism rather than solely to a symptom category.

2. Novel plasma phospholipid biomarkers of autism: mitochondrial dysfunction as a putative causative mechanism
Pastural E, Ritchie S, Lu Y, Jin W, Kavianpour A, Khine Su-Myat K, Heath D, Wood PL, Fisk M, Goodenowe DB.
Prostaglandins Leukot Essent Fatty Acids. 2009 Oct;81(4):253-64.
http://www.ncbi.nlm.nih.gov/pubmed/19608392

Autism is a neurological disorder that manifests as noticeable behavioral and developmental abnormalities predominantly in males between the ages of 2 and 10. Although the genetics, biochemistry and neuropathology of this disease have been extensively studied, underlying causal factors to this disease have remained elusive. Using a longitudinal trial design in which three plasma samples were collected from 15 autistic and 12 non-autistic age-matched controls over the course of 1 year, universal and unambiguous alterations in lipid metabolism were observed. Biomarkers of fatty acid elongation and desaturation (poly-unsaturated long chain fatty acids (PUFA) and/or saturated very long chain fatty acids (VLCFA)-containing ethanolamine phospholipids) were statistically elevated in all autistic subjects. In all 8 of the affected/non-affected sibling pairs, the affected sibling had higher levels of these biomarkers than the unaffected sibling. Exposure of neurons, astrocytes and hepatocytes in vitro to elevated extracellular glutamate levels resulted in lipid biomarker changes indistinguishable from those observed in autistic subjects. Glutamate stress also resulted in in vitro decreased levels of reduced glutathione (GSH), methionine and cysteine, in a similar way to the decreases we observed in autism plasma. Impaired mitochondrial fatty acid oxidation, elevated plasma VLCFAs, and glutamate toxicity as putative causal factors in the biochemistry, neuropathology, and gender bias in autism are discussed.

3. Serum protein profiling and proteomics in autistic spectrum disorder using magnetic bead-assisted mass spectrometry
Taurines R, Dudley E, Conner AC, Grassl J, Jans T, Guderian F, Mehler-Wex C, Warnke A, Gerlach M, Thome J.
Eur Arch Psychiatry Clin Neurosci. 2009 Sep 27. [Epub ahead of print]
http://www.ncbi.nlm.nih.gov/pubmed/19784855

The pathophysiology of autistic spectrum disorder (ASD) is not fully understood and there are no diagnostic or predictive biomarkers. Proteomic profiling has been used in the past for biomarker research in several non-psychiatric and psychiatric disorders and could provide new insights, potentially presenting a useful tool for generating such biomarkers in autism. Serum protein pre-fractionation with C8-magnetic beads and protein profiling by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-ToF-MS) were used to identify possible differences in protein profiles in patients and controls. Serum was obtained from 16 patients (aged 8-18) and age-matched controls. Three peaks in the MALDI-ToF-MS significantly differentiated the ASD sample from the control group. Sub-grouping the ASD patients into children with and without comorbid Attention Deficit and Hyperactivity Disorder, ADHD (ASD/ADHD+ patients, n = 9; ASD/ADHD- patients, n = 7), one peak distinguished the ASD/ADHD+ patients from controls and ASD/ADHD- patients. Our results suggest that altered protein levels in peripheral blood of patients with ASD might represent useful biomarkers for this devastating psychiatric disorder.

4. Metabolic biomarkers related to energy metabolism in Saudi autistic children
Al-Mosalem OA et al.
Clin Biochem. 2009 Jul;42(10-11):949-57. Epub 2009 Apr 17.
$ http://tinyurl.com/y9obtgc

OBJECTIVES: Energy metabolism is usually manipulated in many neurodegenerative diseases. Autism is considered a definable systemic disorder resulting in a number of diverse factors that may affect the brain development and functions both pre and post natal. The increased prevalence of autism will have enormous future public implications and has stimulated intense research into potential etiologic factors. This study aims to establish a connection between autism and the deterioration accompanied it, especially in the brain cognitive areas through a postulation of energy manipulation. MATERIALS AND METHODS: The biochemical changes in activities of enzymes and pathways that participate in the production of ATP as the most important high-energy compound needed by the human brain were measured in Saudi autistic children. Na(+)/K(+)ATPase, ectonucleotidases (NTPDases) (ADPase and ATPase) and creatine kinase (CK), were assessed in plasma of 30 Saudi autistic patients and compared to 30 age-matching control samples. In addition, adenosine mono, di and trinucleotides (ATP, ADP, and AMP) were measured calorimetrically in the red blood cells of both groups and the adenylate energy charge (AEC) was calculated. Moreover, lactate concentration in plasma of both groups was monitored. RESULTS: The obtained data recorded 148.77% and 72.35% higher activities of Na(+)/K(+)ATPase and CK respectively in autistic patients which prove the impairment of energy metabolism in these children compared to age and sex matching healthy controls. While ADPase was significantly higher in autistic patients, ATPase were non-significantly elevated compared to control. In spite of the significant increase of Na(+)/K(+)ATPase activity in autistic patients, there was no significant difference in the levels of ATP, ADP, and AMP in both groups and the calculated AEC values were 0.814+/-0.094 and 0.806+/-0.081 for autistic and control groups respectively. The unchanged AEC value in autistic patients was easily correlated with the induced activity of CK and ADPase as two enzymes playing a critical role in the stabilization of AEC. Lactate as an important energy metabolite for the brain was significantly higher in autistic patients compared to control showing about 40% increase. CONCLUSION: The present study confirmed the impairment of energy metabolism in Saudi autistic patients which could be correlated to the oxidative stress previously recorded in the same investigated samples. The identification of biochemical markers related to autism would be advantageous for earlier clinical diagnosis and intervention.

5. Family analysis of immunoglobulin classes and subclasses in children with autistic disorder
Spiroski M et al.
Institute of Immunobiology and Human Genetics, Faculty of Medicine, 1109 Skopje, PO Box 60, Macedonia.
Bosn J Basic Med Sci. 2009 Nov;9(4):283-9.

Autistic disorder is a severe neurodevelopment disorder characterized by a triad of impairments in reciprocal social interaction, verbal and nonverbal communication, and a pattern of repetitive stereotyped activities, behaviours and interests. There are strong lines of evidence to suggest that the immune system plays an important role in the pathogenesis of autistic disorder. The aim of this study was to analyze quantitative plasma concentration of immunoglobulin classes, and subclasses in autistic patients and their families. The investigation was performed retrospectively in 50 persons with autistic disorder in the Republic of Macedonia. Infantile autistic disorder was diagnosed by DSM-IV and ICD-10 criteria. Plasma immunoglobulin classes (IgM, IgA, and IgG) and subclasses (IgG1, IgG2, IgG3, and IgG4) were determined using Nephelometer Analyzer BN-100. Multiple comparisons for the IgA variable have shown statistically significant differences between three pairs: male autistic from the fathers (p = 0,001), female autistic from the mothers (p = 0,008), as well as healthy sisters from the fathers (p = 0,011). Statistically significant differences found between three groups regarding autistic disorder (person with autistic disorder, father/mother of a person with autistic disorder, and brother/sister) independent of sex belongs to IgA, IgG2, and IgG3 variables. Multiple comparisons for the IgA variable have shown statistically significant differences between children with autistic disorder from the fathers and mothers (p < 0,001), and healthy brothers and sisters from the fathers and mothers (p < 0,001). Comparison between healthy children and children with autistic disorder from the same family should be tested for immunoglobulin classes and subclasses in order to avoid differences between generations.

6. Urinary Excretion of 5-Hydroxyindoleacetic Acid, Serotonin and 6-Sulphatoxymelatonin in Normoserotonemic and Hyperserotonemic Autistic Individuals
Mulder EJ, Anderson GM, Kemperman RF, Oosterloo-Duinkerken A, Minderaa RB, Kema IP.
University Medical Center Groningen, The Netherlands.
Neuropsychobiology. 2009 Nov 13;61(1):27-32. [Epub ahead of print]
$ http://content.karger.com/produktedb/produkte.asp?typ=fulltext&file=000258640

Objective: A substantial proportion of individuals with autism have elevated levels of platelet serotonin (5-HT). We examined whether platelet hyperserotonemia is associated with increased gut 5-HT synthesis, altered 5-HT catabolism or altered melatonin production. Methods: Urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA) and 5-HT was compared in 10 normoserotonemic and 10 hyperserotonemic age-matched autistic individuals. The relationship of urinary 6-sulfatoxymelatonin (6-SM) excretion to platelet 5-HT, and to urinary 5-HT and 5-HIAA excretion, was also examined. Results: In the hyperserotonemic group, significant increases at trend level in urinary excretion of 5-HIAA (p = 0.061) and 5-HT (p = 0.071) and a significant decrease for 6-SM were found (p = 0.027). The urinary 5-HIAA:5-HT ratio was similar in the normo- versus the hyperserotonemic groups. Conclusions: The catabolism of 5-HT does not differ in the groups, but greater exposure of the platelet to 5-HT cannot be ruled out as a cause of the platelet hyperserotonemia of autism. Although only trend level significant, the data point to a need for larger studies to examine more thoroughly the relationships between platelet hyperserotonemia, gut 5-HT synthesis and melatonin production.

7. Visual attention in autism families: 'unaffected' sibs share atypical frontal activation
Belmonte MK, Gomot M, Baron-Cohen S.
Cornell University
J Child Psychol Psychiatry. 2009 Nov 13. [Epub ahead of print]
$ http://www3.interscience.wiley.com/cgi-bin/fulltext/122683869/PDFSTART

Background: In addition to their more clinically evident abnormalities of social cognition, people with autism spectrum conditions (ASC) manifest perturbations of attention and sensory perception which may offer insights into the underlying neural abnormalities. Similar autistic traits in ASC relatives without a diagnosis suggest a continuity between clinically affected and unaffected family members. Methods: We applied fMRI in the context of a non-social task of visual attention in order to determine whether this continuity persists at the level of brain physiology. Results: Both boys with ASC and clinically unaffected brothers of people with ASC were impaired at a visual divided-attention task demanding conjunction of attributes from rapidly and simultaneously presented, spatially disjoint stimuli and suppression of spatially intervening distractors. In addition, both groups in comparison to controls manifested atypical fronto-cerebellar activation as a function of distractor congruence, and the degree of this frontal atypicality correlated with psychometric measures of autistic traits in ASC and sibs. Despite these resemblances between the ASC and sib groups, an exploratory, hypothesis-generating analysis of correlations across brain regions revealed a decrease in overall functional correlation only in the ASC group and not in the sibs. Conclusions: These results establish a neurophysiological correlate of familial susceptibility to ASC, and suggest that whilst abnormal time courses of frontal activation may reflect processes permissive of autistic brain development, abnormal patterns of functional correlation across a wider array of brain regions may relate more closely to autism's determinants.

8. Developmental coordination disorder and other motor control problems in girls with autism spectrum disorder and/or attention-deficit/hyperactivity disorder
Kopp S, Beckung E, Gillberg C.
Göteborg University, Institute of Neuroscience and Physiology, Sweden.
Res Dev Disabil. 2009 Nov 11. [Epub ahead of print]

Examine the rate, predictors, and effect on daily life skills of developmental coordination disorder (DCD) and other motor control difficulties in school age girls with autism spectrum disorder (ASD) and/or attention-deficit/hyperactivity disorder (ADHD), in preschool age girls with ASD referred to a neuropsychiatric clinic, and in a community sample of school age girls. The girls (131 in total) were examined with standardised test of motor function and parent interviews and questionnaires. The school girls were compared with 57 age-and IQ-matched girls from the community. DCD was diagnosed in 25% of clinic school girls with ASD, in 32% of those with ADHD, and in 80% of the clinic preschool girls with ASD. Parents reported more motor problems in the school age clinic group. Agreement between a brief motor screening test and a full comprehensive motor examination was moderate to good in the clinic group. Young age, autistic symptomatology, and low performance IQ predicted more motor coordination problems. Motor coordination problems were related to lower ability in daily life skills even when the effect of PIQ was controlled for. A large minority of school girls with ASD and/or ADHD, and a majority of preschool girls with ASD meet full diagnostic criteria for DCD. Their motor problems contribute to reduced activity in daily life even when the effects of IQ have been partialled out.

9. Childhood serum anti-fetal brain antibodies do not predict autism
Morris CM, Zimmerman AW, Singer HS.
Johns Hopkins University School of Medicine
Pediatr Neurol. 2009 Oct;41(4):288-90.
$ http://linkinghub.elsevier.com/retrieve/pii/S0887-8994%2809%2900212-4

Autoimmune hypotheses for autism include in utero transplacental exposure to maternal antibodies and acquired postnatal insults. Previous work demonstrated that some mothers of children with autistic disorder have specific antibodies against human fetal brain that differentiate them from mothers with typical children. In the present study, Western immunoblotting was used to determine whether children with autistic spectrum disorders (n = 29) have serum reactivity against human fetal brain that differs from that of controls (n = 14). There was no significant difference in reactivity, corrected for serum immunoglobulin G content and brain actin content and with special attention to reactive bands at 36, 39, 61, and 73 kDa, between autistic children and normal control subjects. Thus, in contrast to mothers, antibody reactivity against human fetal brain as measured in children ages 3-12 years does not appear to be a useful biomarker for autism.

10. Pain reactivity and plasma beta-endorphin in children and adolescents with autistic disorder
Tordjman S et al.
PLoS One. 2009 Aug 26;4(8):e5289.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005289

BACKGROUND: Reports of reduced pain sensitivity in autism have prompted opioid theories of autism and have practical care ramifications. Our objective was to examine behavioral and physiological pain responses, plasma beta-endorphin levels and their relationship in a large group of individuals with autism. METHODOLOGY/PRINCIPAL FINDINGS: The study was conducted on 73 children and adolescents with autism and 115 normal individuals matched for age, sex and pubertal stage. Behavioral pain reactivity of individuals with autism was assessed in three observational situations (parents at home, two caregivers at day-care, a nurse and child psychiatrist during blood drawing), and compared to controls during venepuncture. Plasma beta-endorphin concentrations were measured by radioimmunoassay. A high proportion of individuals with autism displayed absent or reduced behavioral pain reactivity at home (68.6%), at day-care (34.2%) and during venepuncture (55.6%). Despite their high rate of absent behavioral pain reactivity during venepuncture (41.3 vs. 8.7% of controls, P<0.0001), individuals with autism displayed a significantly increased heart rate in response to venepuncture (P<0.05). Moreover, this response (Delta heart rate) was significantly greater than for controls (mean+/-SEM; 6.4+/-2.5 vs. 1.3+/-0.8 beats/min, P<0.05). Plasma beta-endorphin levels were higher in the autistic group (P<0.001) and were positively associated with autism severity (P<0.001) and heart rate before or after venepuncture (P<0.05), but not with behavioral pain reactivity. CONCLUSIONS/SIGNIFICANCE: The greater heart rate response to venepuncture and the elevated plasma beta-endorphin found in individuals with autism reflect enhanced physiological and biological stress responses that are dissociated from observable emotional and behavioral reactions. The results suggest strongly that prior reports of reduced pain sensitivity in autism are related to a different mode of pain expression rather than to an insensitivity or endogenous analgesia, and do not support opioid theories of autism. Clinical care practice and hypotheses regarding underlying mechanisms need to assume that children with autism are sensitive to pain.

11. Vision in autism spectrum disorders
Simmons DR, Robertson AE, McKay LS, Toal E, McAleer P, Pollick FE.
Vision Res. 2009 Nov;49(22):2705-39. Epub 2009 Aug 12.

Autism spectrum disorders (ASDs) are developmental disorders which are thought primarily to affect social functioning. However, there is now a growing body of evidence that unusual sensory processing is at least a concomitant and possibly the cause of many of the behavioural signs and symptoms of ASD. A comprehensive and critical review of the phenomenological, empirical, neuroscientific and theoretical literature pertaining to visual processing in ASD is presented, along with a brief justification of a new theory which may help to explain some of the data, and link it with other current hypotheses about the genetic and neural aetiologies of this enigmatic condition.

12. Novel plasma phospholipid biomarkers of autism: mitochondrial dysfunction as a putative causative mechanism
Pastural E et al.
Prostaglandins Leukot Essent Fatty Acids. 2009 Oct;81(4):253-64. Epub 2009 Jul 15.
$ http://www.plefa.com/article/S0952-3278%2809%2900111-2/abstract

Autism is a neurological disorder that manifests as noticeable behavioral and developmental abnormalities predominantly in males between the ages of 2 and 10. Although the genetics, biochemistry and neuropathology of this disease have been extensively studied, underlying causal factors to this disease have remained elusive. Using a longitudinal trial design in which three plasma samples were collected from 15 autistic and 12 non-autistic age-matched controls over the course of 1 year, universal and unambiguous alterations in lipid metabolism were observed. Biomarkers of fatty acid elongation and desaturation (poly-unsaturated long chain fatty acids (PUFA) and/or saturated very long chain fatty acids (VLCFA)-containing ethanolamine phospholipids) were statistically elevated in all autistic subjects. In all 8 of the affected/non-affected sibling pairs, the affected sibling had higher levels of these biomarkers than the unaffected sibling. Exposure of neurons, astrocytes and hepatocytes in vitro to elevated extracellular glutamate levels resulted in lipid biomarker changes indistinguishable from those observed in autistic subjects. Glutamate stress also resulted in in vitro decreased levels of reduced glutathione (GSH), methionine and cysteine, in a similar way to the decreases we observed in autism plasma. Impaired mitochondrial fatty acid oxidation, elevated plasma VLCFAs, and glutamate toxicity as putative causal factors in the biochemistry, neuropathology, and gender bias in autism are discussed.

13. Evidence of Mitochondrial Dysfunction in Autism and Implications for Treatment
Rossignol DA, Bradstreet JJ.
American Journal of Biochemistry and Biotechnology 4(2): 208-217 , 2008
http://www.scipub.org/fulltext/ajbb/ajbb42208-217.pdf

Classical mitochondrial diseases occur in a subset of individuals with autism and are usually caused by genetic anomalies or mitochondrial respiratory pathway deficits. However, in many cases of autism, there is evidence of mitochondrial dysfunction (MtD) without the classic features associated with mitochondrial disease. MtD appears to be more common in autism and presents with less severe signs and symptoms. It is not associated with discernable mitochondrial pathology in muscle biopsy specimens despite objective evidence of lowered mitochondrial functioning. Exposure to environ-mental toxins is the likely etiology for MtD in autism. This dysfunction then contributes to a number of diagnostic symptoms and comorbidities observed in autism including: cognitive impairment, language deficits, abnormal energy metabolism, chronic gastrointestinal problems, abnormalities in fatty acid oxidation, and increased oxidative stress. MtD and oxidative stress may also explain the high male to female ratio found in autism due to increased male vulnerability to these dysfunctions. Biomarkers for mitochondrial dysfunction have been identified, but seem widely under-utilized despite available therapeutic interventions. Nutritional supplementation to decrease oxidative stress along with factors to improve reduced glutathione, as well as hyperbaric oxygen therapy (HBOT) represent supported and rationale approaches. The underlying pathophysiology and autistic symptoms of affected individuals would be expected to either improve or cease worsening once effective treatment for MtD is implemented.

14. Increased serum levels of high mobility group box 1 protein in patients with autistic disorder.
Emanuele E, Boso M, Brondino N, Pietra S, Barale F, di Nemi SU, Politi P.
Prog Neuropsychopharmacol Biol Psychiatry. 2010 Mar 16. [Epub ahead of print]

BACKGROUND: High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein that functions as an activator for inducing the immune response and can be released from neurons after glutamate excitotoxicity. The objective of the present study was to measure serum levels of HMGB1 in patients with autistic disorder and to study their relationship with clinical characteristics. METHODS: We enrolled 22 adult patients with autistic disorder (mean age: 28.1+/-7.7years) and 28 age- and gender-matched healthy controls (mean age: 28.7+/-8.1years). Serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with healthy subjects, serum levels of HMGB1 were significantly higher in patients with autistic disorder (10.8+/-2.6ng/mL versus 5.6+/-2.5ng/mL, respectively, P<0.001). After adjustment for potential confounders, serum HMGB1 levels were independently associated with their domain A scores in the Autism Diagnostic Interview-Revised, which reflects their impairments in social interaction. CONCLUSIONS: These results suggest that HMGB1 levels may be affected in autistic disorder. Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder. {See a list of related articles in Pubmed and more citations here}

This document prepared by
Teresa Binstock
Researcher in Developmental & Behavioral Neuroanatomy
April 2010