An increased vulnerability to oxidative stress and a decreased capacity for methylation may contribute to the development and clinical manifestation of autism.

Autism is a behaviorally-defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G>A), transcobalamin II (TCN2 776G>C), catechol-O-methyltransferase (COMT 472G>A), methylenetetrahydrofolate reductase (MTHFR 677C>T and 1298A>C), and GST M1. We propose that an increased vulnerability to

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice.

An elevation in plasma homocysteine is a sensitive but nonspecific biomarker for an imbalance in the integrated pathways of one-carbon metabolism (1, 2). Chronic nutritional deficiencies in folate, choline, methionine, vitamin B 6 , and/or vitamin B 12 can perturb the complex regulatory network that maintains normal one-carbon metabolism and homocysteine homeostasis (3-7). Genetic polymorphisms in these pathways can act synergistically with nutritional deficiencies to accelerate the metabolic pathology associated with chronic disease states (8). Although several hypotheses have been proposed to explain the association between hyperhomocysteinemia and the thrombotic/atherosclerotic process occurring with occlusive cardiovascular disease, as yet none has been definitive (9 -12). Similarly, increases in plasma homocysteine concentrations have been associated with increased risk of certain birth defects (13-16), but the underlying mechanism remains elusive. A major unanswered question is whether direct cellular toxicity of homocysteine is causally involved in pathogenesis or whether homocysteinemia is simply a passive and indirect indicator of a more complex mechanism.Homocysteine is derived solely from methionine metabolism and is significantly recycled to conserve sufficient methionine for protein and S-adenosylmethionine synthesis. The interactive and interdependent pathways of the methionine/homocysteine cycle are diagrammed in Fig. 1 to emphasize the indirect effects of pathway perturbations on cellular methyltransferase reactions. The metabolic generation of homocysteine from methionine is initiated by the ATP-dependent transfer of adenosine to methionine via methionine adenosyltransferase. The product, S-adenosylmethionine (SAM), 1 is a priority for onecarbon metabolism because it is the methyl donor for most cellular methyltransferase reactions. In addition to DNA methylation, SAM-dependent methyltransferase activity is essential for hundreds of other cellular methylation reactions including synthesis of creatine in the liver, membrane phosphatidylcholine synthesis, central nervous system neurotransmittor synthesis, methylation/detoxification, and RNA and protein methylation (17). After transfer of the methyl group, SAM is converted to S-adenosylhomocysteine (SAH) within the active site of the methyltransferase enzyme. Because most methyltransferases bind SAH with higher affinity than SAM, they are subject to potent product inhibition by SAH (18). Thus, the efficiency of methyltransferase reactions is absolutely dependent on efficient product removal of SAH. This is effectively accomplished by SAH hydrolase (SAHH), an enzyme that appears to act in close proximity to the methyltransferases, at least in the nucleus (19). The crystal structure of SAHH has been recently reported, and interestingly, the polypeptide folding pattern at the catalytic domain of SAHH is almost identical to that reported for the DNA methyltransferases and suggests that SAH molecules can travel easily between the catalytic pockets of th...

The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Despite increasing evidence of oxidative stress in the pathophysiology of autism, most studies have not evaluated biomarkers within specific brain regions, and the functional consequences of oxidative stress remain relatively understudied. We examined frozen samples from the cerebellum and temporal cortex (Brodmann area 22 (BA22)) from individuals with autism and unaffected controls ( n =15 and n =12 per group, respectively). Biomarkers of oxidative stress, including reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione redox/antioxidant capacity (GSH/GSSG), were measured. Biomarkers of oxidative protein damage (3-nitrotyrosine; 3-NT) and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG) were also assessed. Functional indicators of oxidative stress included relative levels of 3-chlorotyrosine (3-CT), an established biomarker of a chronic inflammatory response, and aconitase activity, a biomarker of mitochondrial superoxide production. Consistent with previous studies on plasma and immune cells, GSH and GSH/GSSG were significantly decreased in both autism cerebellum ( P <0.01) and BA22 ( P <0.01). There was a significant increase in 3-NT in the autism cerebellum and BA22 ( P <0.01). Similarly, 8-oxo-dG was significantly increased in autism cerebellum and BA22 ( P <0.01 and P =0.01, respectively), and was inversely correlated with GSH/GSSG in the cerebellum ( P <0.01). There was a significant increase in 3-CT levels in both brain regions ( P <0.01), whereas aconitase activity was significantly decreased in autism cerebellum ( P <0.01), and was negatively correlated with GSH/GSSG ( P =0.01). Together, these results indicate that decreased GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain may have functional consequence in terms of a chronic inflammatory response, increased mitochondrial superoxide production, and oxidative protein and DNA damage.

The significant improvements observed in transmethylation metabolites and glutathione redox status after treatment suggest that targeted nutritional intervention with methylcobalamin and folinic acid may be of clinical benefit in some children who have autism. This trial was registered at (clinicaltrials.gov) as NCT00692315.

Autism spectrum disorder (ASD) has been associated with mitochondrial disease (MD). Interestingly, most individuals with ASD and MD do not have a specific genetic mutation to explain the MD, raising the possibility of that MD may be acquired, at least in a subgroup of children with ASD. Acquired MD has been demonstrated in a rodent ASD model in which propionic acid (PPA), an enteric bacterial fermentation product of ASD-associated gut bacteria, is infused intracerebroventricularly. This animal model shows validity as it demonstrates many behavioral, metabolic, neuropathologic and neurophysiologic abnormalities associated with ASD. This animal model also demonstrates a unique pattern of elevations in short-chain and long-chain acyl-carnitines suggesting abnormalities in fatty-acid metabolism. To determine if the same pattern of biomarkers of abnormal fatty-acid metabolism are present in children with ASD, the laboratory results from a large cohort of children with ASD (n=213) who underwent screening for metabolic disorders, including mitochondrial and fatty-acid oxidation disorders, in a medically based autism clinic were reviewed. Acyl-carnitine panels were determined to be abnormal if three or more individual acyl-carnitine species were abnormal in the panel and these abnormalities were verified by repeated testing. Overall, 17% of individuals with ASD demonstrated consistently abnormal acyl-carnitine panels. Next, it was determined if specific acyl-carnitine species were consistently elevated across the individuals with consistently abnormal acyl-carnitine panels. Significant elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines were found in the ASD individuals with consistently abnormal acyl-carnitine panels—a pattern consistent with the PPA rodent ASD model. Examination of electron transport chain function in muscle and fibroblast culture, histological and electron microscopy examination of muscle and other biomarkers of mitochondrial metabolism revealed a pattern consistent with the notion that PPA could be interfering with mitochondrial metabolism at the level of the tricarboxylic-acid cycle (TCAC). The function of the fatty-acid oxidation pathway in fibroblast cultures and biomarkers for abnormalities in non-mitochondrial fatty-acid metabolism were not consistently abnormal across the subgroup of ASD children, consistent with the notion that the abnormalities in fatty-acid metabolism found in this subgroup of children with ASD were secondary to TCAC abnormalities. Glutathione metabolism was abnormal in the subset of ASD individuals with consistent acyl-carnitine panel abnormalities in a pattern similar to glutathione abnormalities found in the PPA rodent model of ASD. These data suggest that there are similar pathological processes between a subset of ASD children and an animal model of ASD with acquired mitochondrial dysfunction. Future studies need to identify additional parallels between the PPA rodent model of ASD and this subset of ASD individuals with this unique pattern of...