A review of candidate urinary biomarkers for autism spectrum disorder

Context: Autism is a complex, heterogeneous neurodevelopmental condition with a strong genetic component potentially impacted by various environmental factors influencing susceptibility. There are no reliable laboratory tests available to confirm an autism diagnosis.

Objective: To examine the published literature and identify putative urinary biomarkers of autism.

Methods: A comprehensive literature search was conducted using electronic bibliographic databases.

Results: Putative autism biomarkers were identified that could be categorized according to the key theories that exist regarding the etiology of autism: gastrointestinal factors, immune dysregulation, heavy metal toxicity, neurotransmitter abnormalities, and oxidative stress.

Conclusion: There is scope for specific urinary biomarkers to be useful for identification of autistic metabolic phenotypes.     

Level of Trace Elements (Copper,Zinc, Magnesium and Selenium) and Toxic Elements (Lead and Mercury) in the Hair and Nail of Children with Autism

Autism is a multi-factorial pathology observed in children with altered levels of essential and elevated levels of toxic elements. There are also studies reporting a decrease in nutritional trace elements in the hair and nail of autistic children with healthy controls; moreover, bioelements have been shown to play an important role in the central nervous system. Therefore, the purpose of the present study was to assess the levels of trace elements like copper (Cu), zinc (Zn), magnesium (Mg), and selenium (Se) and toxic elements like mercury (Hg), and lead (Pb) in the hair and nail samples of autistic children and to evaluate whether the level of these elements could be correlated with the severity of autism. The subjects of the study were 45 autistic children with different grades of severity (low (LFA), medium (MFA), and high (HFA) functioning autism) according to Childhood Autism Rating Scale, n = 15 children in each group and 50 healthy children (age and sex matched). The boys and girls ratio involved in this study was 4:1, and they were 4-12 years of age. The study observed a valid indication of Cu body burden in the autistic children. The children with different grades of autism showed high significance (p < 0.001) in the level of copper in their hair and nail samples when compared to healthy controls. The level of Cu in the autistic children could be correlated with their degree of severity (more the Cu burden severe is autism). The study showed a significant elevation (p < 0.001) in the levels of toxic metals Pb and Hg in both hair and nail samples of autistic children when compared to healthy control group. The elevation was much pronounced in LFA group subjects when compared among autistic groups MFA and HFA. The levels of trace elements Mg and Se were significantly decreased (p < 0.001) in autistic children when compared to control. The trace element Zn showed significant variation in both hair and nails of LFA group children when compared to control group and other study groups. The significant elevation in the concentration of Cu, Pb, and Hg and significant decrease in the concentration of Mg and Se observed in the hair and nail samples of autistic subjects could be well correlated with their degrees of severity.     

A genomic point-of-view on environmental factors influencing the human brain methylome

The etiologic paradigm of complex human disorders such as autism is that genetic and environmental risk factors are independent and additive, but the interactive effects at the epigenetic interface are largely ignored. Genomic technologies have radically changed perspective on the human genome and how the epigenetic interface may impact complex human disorders. Here, I review recent genomic, environmental and epigenetic findings that suggest a new paradigm of “integrative genomics” in which genetic variation in genomic size may be impacted by dietary and environmental factors that influence the genomic saturation of DNA methylation. Human genomes are highly repetitive, but the interface of large-scale genomic differences with environmental factors that alter the DNA methylome such as dietary folate is under-explored. In addition to obvious direct effects of some environmental toxins on the genome by causing chromosomal breaks, non-mutagenic toxin exposures correlate with DNA hypomethylation that can lead to rearrangements between repeats or increased retrotransposition. Since human neurodevelopment appears to be particularly sensitive to alterations in epigenetic pathways, a further focus will be on how developing neurons may be particularly impacted by even subtle alterations to DNA methylation and proposing new directions towards understanding the quixotic etiology of autism by integrative genomic approaches.Key words: DNA methylation, copy number variation, autism, neurodevelopment, genomics, epigenomics, epigenetics, folate, folic acid, environmental exposures, Alu, MeCP2, LINE-1     

Novel plasma phospholipid biomarkers of autism: Mitochondrial dysfunction as a putative causative mechanism

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.     

Increased excretion of a lipid peroxidation biomarker in autism

It is thought that autism could result from an interaction between genetic and environmental factors with oxidative stress as a potential mechanism linking the two. One genetic factor may be altered oxidative-reductive capacity. This study tested the hypothesis that children with autism have increased oxidative stress. We evaluated children with autism for the presence of two oxidative stress biomarkers. Urinary excretion of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-isoprostane-F2alpha (8-iso-PGF2alpha) were determined in 33 children with autism and 29 healthy controls. 8-iso-PGF2alpha levels were significantly higher in children with autism. The isoprostane levels in autistic subjects were variable with a bimodal distribution. The majority of autistic subjects showed a moderate increase in isoprostane levels while a smaller group of autistic children showed dramatic increases in their isoprostane levels. There was a trend of an increase in 8-OHdG levels in children with autism but it did not reach statistical significance. There was no significant correlation between the levels of the biomarkers and vitamin intake, dietary supplements, medicine, medical disorders, or history of regression. These results suggest that the lipid peroxidation biomarker is increased in this cohort of autistic children, especially in the subgroup of autistic children.     

Pyrosequencing study of fecal microflora of autistic and control children

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention.     

Genetic determinants of autism in individuals with deletions of 18q

Previous research has suggested that individuals with constitutional hemizygosity of 18q have a higher risk of autistic-like behaviors. We sought to identify genomic factors located on chromosome 18 as well as other loci that correlate with autistic behaviors. One hundred and five individuals with 18q- were assessed by high-resolution oligo aCGH and by parental ratings of behavior on the Gilliam Autism Rating Scale. Forty-five individuals (43%) had scores within the “possibly” or “very likely” categories of risk for an autism diagnosis. We searched for genetic determinants of autism by (1) identifying additional chromosome copy number changes (2) Identifying common regions of hemizygosity on 18q, and (3) evaluating four regions containing candidate genes located on 18q (MBD1, TCF4, NETO1, FBXO15). Three individuals with a “very likely” probability of autism had a captured 17p telomere in addition to the 18q deletion suggesting a possible synergy between hemizygosity of 18q and trigosity of 17p. In addition, two of the individuals with an 18q deletion and a “very likely” probability of autism rating had a duplication of the entire short arm of chromosome 18. Although no common region of hemizygosity on 18q was identified, analysis of four regions containing candidate genes suggested that individuals were significantly more likely to exhibit autistic-like behaviors if their region of hemizygosity included TCF4, NETO1, and FBXO15 than if they had any other combination of hemizygosity of the candidate genes. Taken together, these findings identify several new potential candidate genes or regions for autistic behaviors.     

The maternal effect in infantile autism: Elevated DNA damage degree in patients and their mothers

Infantile autism is a common disorder of mental development; besides hereditary predisposition, various environmental factors, including the condition of the mother’s body during pregnancy (“maternal effect”), have a significant impact on its appearance. Oxidative stress is suggested to play a key role in the pathogenesis of infantile autism. It causes a prominent genotoxic effect, which is realized through appearance of single and double strand breaks of the nuclear DNA. In patients with infantile autism and their mothers we evaluated the degree of DNA damage by using the DNA comet assay and determining the comet tail moment and DNA percent ratio in the tail. These two parameters demonstrated strong correlation (r = 0.90). Mean and median values of both parameters were significantly higher in samples from autistic children, than in agematching healthy controls. Interestingly, these parameters were also higher in healthy mothers of autistic children and they did not differ from the values in the group of autistic children. In the control group of healthy women of reproductive age, who had no children with autism, the mean value of the DNA comet tail moment significantly differed from the group of mothers of autistic children, but not from the control group of healthy children. The results suggest that mentally healthy mothers of autistic children have some genotoxic factors, which can determine development of the pathological process in the fetus via the environmental “maternal effect” during gestation.     

线粒体功能障碍与孤独症

孤独症谱系障碍（ASDs）患儿中约有5%伴有线粒体功能紊乱.线粒体功能紊乱会损害对能量高度依赖的生理进程,如神经发育和神经可塑性,从而导致孤独症.本文综述了孤独症个体中线粒体过量的活性氧（reactive oxygen species,ROS）产生及其抗氧化系统减弱、呼吸链复合物异常、线粒体基因突变及与线粒体功能相关的基因组DNA编码的蛋白质异常等方面的研究,旨在阐述线粒体系统多方面的紊乱在孤独症个体中均有所体现,希望能够对孤独症的发病机制和治疗提供帮助.     

Targeting of Glycine Site on NMDA Receptor as a Possible New Strategy for Autism Treatment

The exact pathophysiology of the neurodevelopment disorder of autism is not clear and there is not any curative approach for it. There is only one FDA-approved medication for its management. Therefore, providing of novel treatments is highly required. The hypofunction of GABAergic system and glutamate toxicity are generally believed to have a causal role for autism. The antagonist of the N-methyl-d-aspartic acid (NMDA) glutamate receptor improves autism. Glycine is required for the activation of NMDA receptor. The antagonist of glycine site decreases NMDA receptor conductance. It is hypothesis that glycine site antagonists can be tested as a new strategy for the management of autism.     

Maternal and offspring methylenetetrahydrofolate‐reductase genotypes interact in a mouse model to induce autism spectrum disorder‐like behavior

Individuals with autism constitute a variable population whose members are spread along the autism spectrum. Subpopulations within that spectrum exhibit other conditions, such as anxiety, intellectual disabilities, hyperactivity and epilepsy, with different severities and co‐occurrences. Among the genes associated with the increased risk for autism is the methylenetetrahydrofolate‐reductase (MTHFR) 677C>T polymorphism, which impairs one‐carbon (C1) metabolic pathway efficiency. The frequency of the MTHFR677TT homozygote is markedly higher among autism patients and their mothers than in the general population. Here, we report on the Mthfr heterozygous knockout (KO) mouse as a rodent model of autism that shows the contributions of maternal and offspring genotypes to the development of autistic‐like behaviors. Maternal Mthfr‐deficiency was associated with developmental delays in morphogenic features and sensory‐motor reflexes in offspring. In the adult male mouse, behaviors representing core autism symptoms, such as repetitive behavior and restricted interest, were affected by maternal genotype while social behaviors were affected by both maternal and offspring genotypes. In females and males, behaviors associated with autism such as memory impairment, social aggression and anxiety were affected by both the maternal and offspring Mthfr genotypes, with sex‐dependent differences. Mthfr‐deficient male mice with observable impacts on behavior presented a particular laminar disturbance in parvalbumin interneuron density and innervation in superficial and deep layers of the cingulate cortex. This mouse model of autism will help to elucidate the molecular mechanisms that predispose a significant subgroup of autistic patients to abnormal development and to distinguish between the in‐utero and autonomous factors involved in autism.     

Reduced blood levels of reelin as a vulnerability factor in pathophysiology of autistic disorder

1. Autism is a severe neurodevelopmental disorder with potential genetic and environmental etiologies. Recent genetic linkage reports and biochemical analysis of postmortem autistic cerebellum point to Reelin, an important secretory extracellular protein, as being involved in the pathology of autism.2. We hypothesized that blood levels of Reelin and its isoforms would be altered in autistic twins, and their first degree relatives versus normal controls.3. We measured blood levels of unprocessed Reelin (410 kDa) and its proteolytic cleavage products (Reelins 330 and 180 kDa) as well as albumin and ceruloplasmin in 28 autistic individuals, their parents (13 fathers, 13 mothers), 6 normal siblings, and 8 normal controls using SDS-PAGE and western blotting.4. Results indicated significant reductions in 410 kDa Reelin species in autistic twins (–70%, p < 0.01), their fathers (–62%, p < 0.01), their mothers (–72%, p < 0.01), and their phenotypically normal siblings (–70%, p < 0.01) versus controls. Reelin 330 kDa values did not vary significantly from controls. Reelin 180 kDa values for parents (fathers –32% p < 0.05 vs. controls, mothers –34%) declined when compared to controls. In contrast autistic Reelin 180 kDa increased, albeit nonsignificantly versus controls. Albumin and ceruloplasmin values for autistics and their first degree relatives did not vary significantly from controls. There were no significant meaningful correlations between Reelin, albumin and ceruloplasmin levels, age, sex, ADI scores, or age of onset.5. These results suggest that Reelin 410 deficiency may be a vulnerability factor in the pathology of autism.     

Developmental vitamin D deficiency and schizophrenia: the role of animal models

Schizophrenia is a debilitating neuropsychiatric disorder that affects 1% of the US population. Based on twin and genome‐wide association studies, it is clear that both genetics and environmental factors increase the risk for developing schizophrenia. Moreover, there is evidence that conditions in utero, either alone or in concert with genetic factors, may alter neurodevelopment and lead to an increased risk for schizophrenia. There has been progress in identifying genetic loci and environmental exposures that increase risk, but there are still considerable gaps in our knowledge. Furthermore, very little is known about the specific neurodevelopmental mechanisms upon which genetics and the environment act to increase disposition to developing schizophrenia in adulthood. Vitamin D deficiency during the perinatal period has been hypothesized to increase risk for schizophrenia in humans. The developmental vitamin D (DVD) deficiency hypothesis of schizophrenia arises from the observation that disease risk is increased in individuals who are born in winter or spring, live further from the equator or live in urban vs. rural settings. These environments result in less exposure to sunlight, thereby reducing the initial steps in the production of vitamin D. Rodent models have been developed to characterize the behavioral and developmental effects of DVD deficiency. This review focuses on these animal models and discusses the current knowledge of the role of DVD deficiency in altering behavior and neurobiology relevant to schizophrenia.     

Bacopa monniera (L.) Wettst ameliorates behavioral alterations and oxidative markers in sodium valproate induced autism in rats

Early prenatal or post natal exposure to environmental insults such as valproic acid (VPA), thalidomide and ethanol could induce behavioral alterations similar to autistic symptoms. Bacopa monniera, a renowned plant in ayurvedic medicine is useful in several neurological disorders. The purpose of the present study was to evaluate the effect of B. monniera on VPA induced autism. On 12.5 day of gestation the female pregnant rats were divided into control and VPA treated groups. They were administered saline/VPA (600 mg/kg, i.p.) respectively and allowed to raise their own litters. Group I—male pups of saline treated mothers. On postnatal day (PND) 21 VPA induced autistic male pups were divided into two groups (n = 6); Group II—received saline and Group III—received B. monniera (300 mg/kg/p.o.) from PND 21–35. Behavioral tests (nociception, locomotor activity, exploratory activity, anxiety and social behavior) were performed in both adolescence (PND 30–40) and adulthood (PND 90–110) period. At the end of behavioral testing animals were sacrificed, brain was isolated for biochemical estimations (serotonin, glutathione, catalase and nitric oxide) and histopathological examination. Induction of autism significantly affected normal behavior, increased oxidative stress and serotonin level, altered histoarchitecture of cerebellum (decreased number of purkinje cells, neuronal degeneration and chromatolysis) when compared with normal control group. Treatment with B. monniera significantly (p < 0.05) improved behavioral alterations, decreased oxidative stress markers and restored histoarchitecture of cerebellum. In conclusion, the present study suggests that B. monniera ameliorates the autistic symptoms possibly due to its anti-anxiety, antioxidant and neuro-protective activity.     

Bisphenol A induces oxidative stress and mitochondrial dysfunction in lymphoblasts from children with autism and unaffected siblings

Autism is a behaviorally defined neurodevelopmental disorder. Although there is no single identifiable cause for autism, roles for genetic and environmental factors have been implicated in autism. Extensive evidence suggests increased oxidative stress and mitochondrial dysfunction in autism. In this study, we examined whether bisphenol A (BPA) is an environmental risk factor for autism by studying its effects on oxidative stress and mitochondrial function in the lymphoblasts. When lymphoblastoid cells from autistic subjects and age-matched unaffected sibling controls were exposed to BPA, there was an increase in the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential in both groups. A further subdivision of the control group into two subgroups—unaffected nontwin siblings and twin siblings—showed significantly higher ROS levels without any exposure to BPA in the unaffected twin siblings compared to the unaffected nontwin siblings. ROS levels were also significantly higher in the autism vs the unaffected nontwin siblings group. The effect of BPA on three important mtDNA genes—NADH dehydrogenase 1, NADH dehydrogenase 4, and cytochrome b—was analyzed to observe any changes in the mitochondria after BPA exposure. BPA induced a significant increase in the mtDNA copy number in the lymphoblasts from the unaffected siblings group and in the unaffected twin siblings group vs the unaffected nontwin siblings. In all three genes, the mtDNA increase was seen in 70% of the subjects. These results suggest that BPA exposure results in increased oxidative stress and mitochondrial dysfunction in the autistic subjects as well as the age-matched sibling control subjects, particularly unaffected twin siblings. Therefore, BPA may act as an environmental risk factor for autism in genetically susceptible children by inducing oxidative stress and mitochondrial dysfunction.     

Resistance to change and vulnerability to stress: autistic‐like features of GAP43‐deficient mice

There is an urgent need for animal models of autism spectrum disorder (ASD) to understand the underlying pathology and facilitate development and testing of new treatments. The synaptic growth‐associated protein‐43 (GAP43) has recently been identified as an autism candidate gene of interest. Our previous studies show many brain abnormalities in mice lacking one allele for GAP43 [GAP43 (+/?)] that are consistent with the disordered connectivity theory of ASD. Thus, we hypothesized that GAP43 (+/?) mice would show at least some autistic‐like behaviors. We found that GAP43 (+/?) mice, relative to wild‐type (+/+) littermates, displayed resistance to change, consistent with one of the diagnostic criteria for ASD. GAP43 (+/?) mice also displayed stress‐induced behavioral withdrawal and anxiety, as seen in many autistic individuals. In addition, both GAP43 (+/?) mice and (+/+) littermates showed low social approach and lack of preference for social novelty, consistent with another diagnostic criterion for ASD. This low sociability is likely because of the mixed C57BL/6J 129S3/SvImJ background. We conclude that GAP43 deficiency leads to the development of a subset of autistic‐like behaviors. As these behaviors occur in a mouse that displays disordered connectivity, we propose that future anatomical and functional studies in this mouse may help uncover underlying mechanisms for these specific behaviors. Strain‐specific low sociability may be advantageous in these studies, creating a more autistic‐like environment for study of the GAP43‐mediated deficits of resistance to change and vulnerability to stress.     

Does microglial dysfunction play a role in autism and Rett syndrome?

Autism spectrum disorders (ASDs) including classic autism is a group of complex developmental disabilities with core deficits of impaired social interactions, communication difficulties and repetitive behaviors. Although the neurobiology of ASDs has attracted much attention in the last two decades, the role of microglia has been ignored. Existing data are focused on their recognized role in neuroinflammation, which only covers a small part of the pathological repertoire of microglia. This review highlights recent findings on the broader roles of microglia, including their active surveillance of brain microenvironments and regulation of synaptic connectivity, maturation of brain circuitry and neurogenesis. Emerging evidence suggests that microglia respond to pre- and postnatal environmental stimuli through epigenetic interface to change gene expression, thus acting as effectors of experience-dependent synaptic plasticity. Impairments of these microglial functions could substantially contribute to several major etiological factors of autism, such as environmental toxins and cortical underconnectivity. Our recent study on Rett syndrome, a syndromic autistic disorder, provides an example that intrinsic microglial dysfunction due to genetic and epigenetic aberrations could detrimentally affect the developmental trajectory without evoking neuroinflammation. We propose that ASDs provide excellent opportunities to study the influence of microglia on neurodevelopment, and this knowledge could lead to novel therapies.     

Increased BDNF levels and NTRK2 gene association suggest a disruption of BDNF/TrkB signaling in autism

The brain‐derived neurotrophic factor (BDNF), a neurotrophin fundamental for brain development and function, has previously been implicated in autism. In this study, the levels of BDNF in platelet‐rich plasma were compared between autistic and control children, and the role of two genetic factors that might regulate this neurotrophin and contribute to autism etiology, BDNF and NTRK2, was examined. We found that BDNF levels in autistic children (n = 146) were significantly higher (t = 6.82; P < 0.0001) than in control children (n = 50) and were positively correlated with platelet serotonin distribution (r = 0.22; P = 0.004). Heritability of BDNF was estimated at 30% and therefore candidate genes BDNF and NTRK2 were tested for association with BDNF level distribution in this sample, and with autism in 469 trio families. Genetic association analysis provided no evidence for BDNF or NTRK2 as major determinants of the abnormally increased BDNF levels in autistic children. A significant association with autism was uncovered for six single nucleotide polymorphisms (SNPs) [0.004 (Z_(1df) = 2.85) < P < 0.039 (Z_(1df) = 2.06)] and multiple haplotypes [5 × 10^?4(χ_(3df) = 17.77) < P < 0.042 (χ_(9df) = 17.450)] in the NTRK2 gene. These results do not withstand correction for multiple comparisons, however, reflect a trend toward association that supports a role of NTRK2 as a susceptibility factor for the disorder. Genetic variation in the BDNF gene had no impact on autism risk. By substantiating the previously observed increase in BDNF levels in autistic children in a larger patient set, and suggesting a genetic association between NTRK2 and autism, this study integrates evidence from multiple levels supporting the hypothesis that alterations in BDNF/tyrosine kinase B (TrkB) signaling contribute to an increased vulnerability to autism.