Prenatal Biomarkers of Oxidative Stress and Reduced Methylation Capacity in An Autism High-Risk Pregnancy Cohort

Background:   In two independent cohorts of post-pregnant mothers of autistic children, molecular abnormalities in folate-dependent one-carbon metabolism were identified. Specifically, glutathione redox/antioxidant status was significantly more oxidized and associated with protein/DNA oxidative damage and DNA hypomethylation in these mothers.  These observations suggest the possibility that metabolic imbalance during gestation may influence the epigenetic and redox microenvironment of the fetus to promote abnormal neurodevelopment resulting in autism.

Objectives:    In collaboration with the EARLI Network, the goal of the present investigation is to assess the prevalence of molecular abnormalities in folate-dependent one-carbon metabolism in samples collected from pregnant mothers who already have an older child with autism.   This will set the stage for future analyses exploring the association of these maternal prenatal metabolic and molecular biomarkers of epigenetic dysregulation and oxidative stress/damage with autism related phenotypes in this high risk cohort. 

 Methods:   Serial prenatal plasma and urine samples from 120 autism high risk pregnancies and maternal and offspring DNA from the EARLI study will be analyzed.   To date, plasma samples from the first 30 pregnancies have been evaluated.  HPLC with electrochemical detection was used to quantify plasma levels of S-adenosylmethionine (SAM; major methylation donor), S-adenosylhomocysteine (SAH; methylation product inhibitor), SAM/SAH (cellular methylation capacity), homocysteine (established risk factor for neurodevelopmental abnormalities), glutathione (GSH: major intracellular antioxidant); GSSG (oxidized form of glutathione), GSH/GSSG (antioxidant/detoxification capacity), and 3-nitrotyrosine (marker of protein oxidative damage).  DNA samples will be assessed for global methylation (% 5-methylcytosine/total cytosine) and oxidative damage (8-oxo-deoxyguanine).  Urine samples will be assayed for lipid peroxidation.

 Results:   Over 10% of all samples available from each trimester had functionally significant alterations in the assessed metabolites.  The metabolite imbalance was generally greatest during the 2^nd and 3^rd trimesters which are the periods of greatest brain growth and maturation.  For example, while multiple studies have shown that homocysteine levels normally drop during normal pregnancy to ~6 umol/L, these preliminary findings indicate that 33% of autism high-risk mothers have homocysteine levels >10.5 umol/L during third trimester accompanied by significant decreases in SAM/SAH methylation capacity and GSH/GSSG antioxidant capacity in the second and third trimester.

 Conclusions: These results demonstrate for the first time that abnormalities in folate-dependent methylation metabolism and glutathione-dependent antioxidant/detoxification capacity are present during gestation in a significant percent of mothers at risk for a second child with autism.  We will be able to verify this in a larger sample size and incorporate analyses of biomarkers of DNA oxidative damage and hypomethylation.   Eventually, these data can be considered as potential risk biomarkers for autism-related endpoints within the enriched-risk cohort.