Kinase and Phosphatase Signaling As Biomarkers for ASD Detection

Background: A high priority in autism spectrum disorder (ASD) research is the identification of robust and reliable biomarkers. Biomarkers can enable early detection and intervention, and etiologic stratification leading to the development of precision therapeutics and, ultimately for both approaches, to better outcomes. Recent progress in the genetics of both syndromic and idiopathic ASD suggests that aberrant signaling within two key kinase pathways, RAS-MAPK and PI3K-PTEN-AKT, may be important for ASD pathogenesis (Figure 1). Reports in the literature identify both over and under activation in these pathways (as in Rasopathies and in PTEN associated macrocephaly/autism syndrome) and in genetically mediated disorders that intersect with these pathways, such as Fragile X syndrome and disorders with mutations in ASD genes MECP2 and CHD8. In support of this hypothesis, we previously demonstrated that, in the ASD-related BTBR mouse strain, the degree of kinase activation in the frontal cerebral cortex correlated with the degree of social impairment. We also showed that the degree of activation of kinases in the blood matched those in the brain suggesting that a peripheral blood biomarker may be feasible.

Objectives: We first aim to assess the time-dependent stability of these biomarkers, by assessing kinase/phosphatase measures monthly in a cohort of participants over a three month period (multiple time points; MTP). We principally propose to test whether there is an alteration in kinase signaling in peripheral blood mononuclear cells (PBMC) from ASD patients compared to aged and gender matched neurotypical controls and whether these changes correlate with clinical severity.

Methods: We obtained blood samples and then isolated PBMC from a cohort of 107 children (58 with ADOS confirmed idiopathic ASD and 49 neuro-typical matched controls). We also obtained blood samples from 12 (6 male and 6 female) neurotypical MTP controls. Cognitive and adaptive behavioral assessments were obtained across the cohort. A cytosolic fraction from these PBMC was purified, an equal abundance of protein samples were separated by poly-acrylamide electrophoresis and the abundance of kinases and phosphatases, and their degree of activation were measured by Western blot analysis using monoclonal antibodies targeted to three groups of proteins, p44/42 MAPK (ERK1/2), PTEN and AKT(1-3) and their key activation-based phosphoepitopes.

Results: We found that these kinase/phosphatase measures are highly stable, with a coefficient of variation ranging from 4% to 8% across nine measures assessed thrice. We also found that these key signaling pathways are significantly altered (increased or decreased, by taking the absolute value of the Z-score of each kinase/phosphatase measure) in ASD children versus controls (pERK/ERK, P=0.005; pPTEN/PTEN, P=0.001). Young children separately tested were equally affected; for p-ERK/ERK (P=0.017); for pPTEN/PTEN (P=0.003) and, in this subgroup also for pAKT/AKT (P=0.02). From amongst these three ratios, we found the strongest positive correlation between the degree of the pERK/ERK ratio and the SRS-2 (P=0.0034). A full model analysis of these biomarkers showed an upper bound AUC of 0.86 and a lower bound of 0.73 (figure 2).

Conclusions: These findings demonstrate that blood-based kinase and phosphatase measures enable biomarker development in idiopathic ASD.