Cross-level analysis of molecular and neurobehavioral function in a prospective series of patients with germline heterozygous PTEN mutations with and without autism
Authors: Frazier Thomas W. , Jaini Ritika , Busch Robyn M. , Wolf Matthew , Sadler Tammy , Klaas Patricia , Hardan Antonio Y. , Martinez-Agosto Julian A. , Sahin Mustafa , Eng Charis ,   the Developmental Synaptopathies Consortium, Warfield Simon K. , Scherrer Benoit , Dies Kira , Filip-Dhima Rajna , Gulsrud Amanda , Hanson Ellen , Phillips Jennifer M. 
Published: January 2021

Abstract
Background
PTEN is a well-established risk gene for autism spectrum disorder (ASD). Yet, little is known about how PTEN mutations and associated molecular processes influence neurobehavioral function in mutation carriers with (PTEN-ASD) and without ASD (PTEN no-ASD). The primary aim of the present study was to examine group differences in peripheral blood-derived PTEN pathway protein levels between PTEN-ASD, PTEN no-ASD, and idiopathic macrocephalic ASD patients (macro-ASD). Secondarily, associations between protein levels and neurobehavioral functions were examined in the full cohort.

Methods
Patients were recruited at four tertiary medical centers. Peripheral blood-derived protein levels from canonical PTEN pathways (PI3K/AKT and MAPK/ERK) were analyzed using Western blot analyses blinded to genotype and ASD status. Neurobehavioral measures included standardized assessments of global cognitive ability and multiple neurobehavioral domains. Analysis of variance models examined group differences in demographic, neurobehavioral, and protein measures. Bivariate correlations, structural models, and statistical learning procedures estimated associations between molecular and neurobehavioral variables. To complement patient data, Western blots for downstream proteins were generated to evaluate canonical PTEN pathways in the PTEN-m3m4 mouse model.

Results
Participants included 61 patients (25 PTEN-ASD, 16 PTEN no-ASD, and 20 macro-ASD). Decreased PTEN and S6 were observed in both PTEN mutation groups. Reductions in MnSOD and increases in P-S6 were observed in ASD groups. Elevated neural P-AKT/AKT and P-S6/S6 from PTEN murine models parallel our patient observations. Patient PTEN and AKT levels were independently associated with global cognitive ability, and p27 expression was associated with frontal sub-cortical functions. As a group, molecular measures added significant predictive value to several neurobehavioral domains over and above PTEN mutation status.

Limitations
Sample sizes were small, precluding within-group analyses. Protein and neurobehavioral data were limited to a single evaluation. A small number of patients were excluded with invalid protein data, and cognitively impaired patients had missing data on some assessments.

Conclusions
Several canonical PTEN pathway molecules appear to influence the presence of ASD and modify neurobehavioral function in PTEN mutation patients. Protein assays of the PTEN pathway may be useful for predicting neurobehavioral outcomes in PTEN patients. Future longitudinal analyses are needed to replicate these findings and evaluate within-group relationships between protein and neurobehavioral measures.

Trial registration
ClinicalTrials.gov Identifier NCT02461446