Stefanie Jernigan

Metabolic endophenotype and related genotypes are associated with oxidative stress in children with 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 glutathione‐S‐transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.

Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism

BACKGROUND Metabolic abnormalities and targeted treatment trials have been reported for several neurobehavioral disorders but are relatively understudied in autism. OBJECTIVE The objective of this study was to determine whether or not treatment with the metabolic precursors, methylcobalamin and folinic acid, would improve plasma concentrations of transmethylation/transsulfuration metabolites and glutathione redox status in autistic children. DESIGN In an open-label trial, 40 autistic children were treated with 75 microg/kg methylcobalamin (2 times/wk) and 400 microg folinic acid (2 times/d) for 3 mo. Metabolites in the transmethylation/transsulfuration pathway were measured before and after treatment and compared with values measured in age-matched control children. RESULTS The results indicated that pretreatment metabolite concentrations in autistic children were significantly different from values in the control children. The 3-mo intervention resulted in significant increases in cysteine, cysteinylglycine, and glutathione concentrations (P < 0.001). The oxidized disulfide form of glutathione was decreased and the glutathione redox ratio increased after treatment (P < 0.008). Although mean metabolite concentrations were improved significantly after intervention, they remained below those in unaffected control children. CONCLUSION 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.

Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children;however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/ GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.— James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D. W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism. FASEB J. 23, 2374–2383 (2009)

Mechanisms of DNA Damage, DNA Hypomethylation, and Tumor Progression in the Folate/Methyl-Deficient Rat Model of Hepatocarcinogenesis

Using the folate/methyl-deficient rat model of hepatocarcinogenesis, we obtained evidence that may provide new insights into a major unresolved paradox in DNA methylation and cancer research: the mechanistic basis for genome-wide hypomethylation despite an increase in DNA methyltransferase activity and gene-specific regional hypermethylation. Previous studies revealed that the methyltransferase binds with higher affinity to DNA strand breaks, gaps, abasic sites, and uracil than it does to its cognate hemimethylated CpG sites, consistent with its ancestral function as a DNA repair enzyme. These same DNA lesions are an early occurrence in models of folate and methyl deficiency and are often present in human preneoplastic cells. We hypothesized that the high-affinity binding of the maintenance DNA methyltransferase to unrepaired lesions in DNA could sequester available enzyme away from the replication fork and promote passive replication-dependent demethylation. In support of this possibility, we found that lesion-containing DNA is less efficiently methylated than lesion-free DNA from folate/methyl-deficient rats and that an increase in DNA strand breaks precedes DNA hypomethylation. Despite an adaptive increase in DNA methyltransferase activity, hemimethylated DNA from folate/methyl-deficient rats is progressively replaced by double-stranded unmethylated DNA that is resistant to remethylation with dietary methyl repletion. In promoter regions, the inappropriate binding of the DNA methyltransferase to unrepaired lesions or mispairs may promote local histone deacetylation, methylation, and regional hypermethylation associated with tumor suppressor gene silencing. These insights in an experimental model are consistent with the possibility that DNA lesions may be a necessary prerequisite for the disruption of normal DNA methylation patterns in preneoplastic and neoplastic cells.

Abnormal transmethylation/transsulfuration metabolism and DNA hypomethylation among parents of children with autism

An integrated metabolic profile reflects the combined influence of genetic, epigenetic, and environmental factors that affect the candidate pathway of interest. Recent evidence suggests that some autistic children may have reduced detoxification capacity and may be under chronic oxidative stress. Based on reports of abnormal methionine and glutathione metabolism in autistic children, it was of interest to examine the same metabolic profile in the parents. The results indicated that parents share similar metabolic deficits in methylation capacity and glutathione-dependent antioxidant/detoxification capacity observed in many autistic children. Studies are underway to determine whether the abnormal profile in parents reflects linked genetic polymorphisms in these pathways or whether it simply reflects the chronic stress of coping with an autistic child.

A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism

The biologic basis of autism is complex and is thought to involve multiple and variable gene–environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate‐dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one‐carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell‐specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population‐based sample of autism case‐parent triads. In case–control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S‐adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism.

Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status

Treatments targeting metabolic abnormalities in children with autism are limited. Previously we reported that a nutritional treatment significantly improved glutathione metabolism in children with autistic disorder. In this study we evaluated changes in adaptive behaviors in this cohort and determined whether such changes are related to changes in glutathione metabolism. Thirty-seven children diagnosed with autistic disorder and abnormal glutathione and methylation metabolism were treated with twice weekly 75 µg/Kg methylcobalamin and twice daily 400 µg folinic acid for 3 months in an open-label fashion. The Vineland Adaptive Behavior Scale (VABS) and glutathione redox metabolites were measured at baseline and at the end of the treatment period. Over the treatment period, all VABS subscales significantly improved with an average effect size of 0.59, and an average improvement in skills of 7.7 months. A greater improvement in glutathione redox status was associated with a greater improvement in expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills. Age, gender, and history of regression did not influence treatment response. The significant behavioral improvements observed and the relationship between these improvements to glutathione redox status suggest that nutritional interventions targeting redox metabolism may benefit some children with autism.

Developmental exposure to trichloroethylene promotes CD4+ T cell differentiation and hyperactivity in association with oxidative stress and neurobehavioral deficits in MRL+/+ mice

The non adult immune system is particularly sensitive to perinatal and early life exposures to environmental toxicants. The common environmental toxicant, trichloroethylene (TCE), was shown to increase CD4+ T cell production of the proinflammatory cytokine IFN-gamma following a period of prenatal and lifetime exposure in autoimmune-prone MRL+/+ mice. In the current study, MRL+/+ mice were used to further examine the impact of TCE on the immune system in the thymus and periphery. Since there is considerable cross-talk between the immune system and the brain during development, the potential relationship between TCE and neurobehavioral endpoints were also examined. MRL+/+ mice were exposed to 0.1 mg/ml TCE (~ 31 mg/kg/day) via maternal drinking water or direct exposure via the drinking water from gestation day 1 until postnatal day (PD) 42. TCE exposure did not impact gross motor skills but instead significantly altered social behaviors and promoted aggression associated with indicators of oxidative stress in brain tissues in male mice. The immunoregulatory effects of TCE involved a redox-associated promotion of T cell differentiation in the thymus that preceded the production of proinflammatory cytokines, IL-2, TNF-alpha, and IFN-gamma by mature CD4+ T cells. The results demonstrated that developmental and early life TCE exposure modulated immune function and may have important implications for neurodevelopmental disorders.