Toward the Development of an ASD Biomarker: Altered Activity of Kinase Signaling Pathways in Blood

Background:  A high priority in autism spectrum disorder (ASD) research is the identification of a robust and reliable biomarker. Biomarkers can enable early detection and hence early intervention leading to better outcomes, and may help in the development and stratification of precision therapeutics. Recent progress in the genetics of ASD suggests that the signaling of two key kinase pathways, RAS-MAPK and AKT-PI3K, may be important to ASD pathogenesis. We previously demonstrated that the degree of activation of components of these Ras-MAPK and AKT-PI3K pathways as measured in the brain correlate with social behavior in an ASD-related mouse strain. We also demonstrated that the degree of this kinase activation in the brain correlated with that observed in lymphocytes. These findings motivated our current study, suggesting we can indirectly measure the state of activation of these signaling pathways in the brains of ASD patients and controls from measurements in peripheral blood.

Objectives:  The aim of this study is to assess whether there is over and/or under activation of kinase signaling in children with ASD versus controls by using freshly isolated peripheral blood mononuclear cells (PBMCs) as a model. Our initial biopanel measures total expression and activity level (phosphorylated isoforms) of three groups of proteins, p44/42 MAPK (ERK1/2), PTEN and AKT (1-3)

Methods:  A cohort of 57 idiopathic ASD children and 66 typically-developing control children (matched for age and gender) were enrolled, clinically assessed and had blood drawn. PBMCs were isolated from whole blood using Ficoll separation. After cell lysis the cytoplasmic fraction was used for Western Blot analysis. Total and phosphorylated form of ERK (ERK and p-ERK), PTEN (PTEN and p-PTEN) and AKT (AKT and p-AKT) were detected and quantified, corrected by Actin levels, and the ratios of the phospho to the total for each protein (p-ERK/ERK, p-AKT/AKT, and p-PTEN/PTEN) were calculated. These values were then z-scored using the mean and standard deviation across the entire cohort to measure the deviation of each individual from a standard value. The absolute value z-scored ratios were separately inserted into a generalized linear model (GLM) with group, age, gender, and gel set as independent variables. A statistically significant p-value (p<0.05) associated with the group coefficient was used to determine group differences.

Results:  We found that the absolute value of the z-score for the p-ERK/ERK ratio is statistically significantly increased (p<0.017) as it is for the p-PTEN/PTEN ratio (p<0.032) in ASD children compared to controls, while we found a trend-level increase in p-AKT/AKT in our cohort (p<0.19). These comparisons were most significant through the use of |z-score|, demonstrating that both over and under activation of these pathways are found in ASD.

Conclusions:  Our results show for the first time that steady state activation levels (both over and under) in the blood of key signaling pathways are significantly altered in idiopathic ASD children compared to controls. Our next steps are to understand how the degree of deviation can predict clinical severity and importantly how these and other measures can serve as a blood-based biomarker for ASD.