Studies from laboratories across the world have described the occurrence of significant alterations in the level of redox/methylation metabolites in autistic patients, indicating that these pathways are central contributors to autism spectrum disorders (ASDs). However, to date no studies have evaluated the dynamic regulation of enzymes involved in these pathways at the transcriptional level. Changes in mRNA levels might be related to the alterations of metabolic biomarkers and may represent adaptive responses to the oxidative stress (decreased GSH/GSSG) and reduced methylation capacity (decreased SAM/SAH) observed in autistic patients.
Objectives: The purpose of this study is to evaluate the transcriptional status of enzymes involved in redox/methylation and transsulfuration pathways using a qPCR assay. Such measurements may prove useful for the clinical diagnosis of ASD
Methods:
Blood samples:Blood samples were obtained from autistic and non autistic controls (n=40) in PAXgene tubes™ according to an IRB-approved protocol.
Isolation of the RNA:RNA was isolated using the PAXgene Blood RNA extraction ® kit. cDNA was synthesized using the First Strand cDNA synthesis® kit from Roche.
qRT-PCR: qRT-PCR was performed in a 96 well plate using a Roche LightCycler® 480. 5 μL of cDNA template, primers, SYBR Green I Master®, and dH20 in a final volume of 20 μL was used. Thermal parameters included, incubation for 5 min at 95°C, 45 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec, followed by a single cycle of 95ºC for 5 sec, 1 min at 65ºC and 97ºC for the melting curve, followed by cooling at 40ºC for 90 sec. Data was analyzed using the Roche relative quantification method and normalized to GAPDH.
Results:
Our preliminary results indicate increased expression several redox/methylation genes in autistic subjects, including EAAT3, the transporter responsible for the uptake of cysteine. Cysteine is the rate limiting precursor for GSH synthesis, and GSH levels are significantly decreased in autistic subjects. In addition, mRNA levels of the folate and vitamin B12-dependent enzyme methionine synthase were increased approximately 2-fold. Methionine synthase regulates levels of methionine and homocysteine, and both these metabolites have been reported to be decreased in autistic patients. Lastly, a significant increase in mRNA levels of the retrotransposon LINE-1 was also observed, an indicator of decreased DNA methylation.
Conclusions:
These preliminary results show that abnormal levels of metabolites in the redox and methylation pathways are associated with altered transcriptional levels of key enzymes, as monitored in blood samples from autistic patients. These mRNA changes could potentially be useful a clinical marker for ASD. Follow up studies will utilize a focused qRT-PCR array to assess all the enzymes involved in the redox, methylation and transsulfuration pathways. Alterations observed at the transcriptional level, along with intracellular thiol data, will be incorporated into a mathematical model of the relevant pathways, developed using the Simbiology toolbox from MATLAB. This model will allow dynamic simulation of redox and methylation pathways in neurons, including the changes that accompany autism.