Molecular and Cellular Analyses across Three Autism-Associated Genetic Disorders

Background: The Developmental Synaptopathies Consortium (DSC) is studying three rare genetic disorders associated with high risk for autism, specifically Phelan-McDermid Syndrome (PMS), Tuberous sclerosis complex (TSC), and PTEN-hamartoma tumor syndrome (PHTS). Each of these disorders are being studied extensively using biomarkers and clinical assessments, while, in parallel, molecular analyses are being carried out on patient derived samples, in the context of ongoing preclinical research.

Objectives: The purpose of this study was to map out genotype–phenotype correlations, to collect and characterize patient-derived stem cells, to look for genetic modifier loci, and to analyze gene expression data from blood samples and correlate this with phenotypes.

Methods: The study samples comprised 98 participants in the PMS group, 98 with TSC, and 67 with PHTS. Mean age was 8.74 years (SD = 4.74) with no group differences observed. Blood and skin samples were collected where feasible, and induced pluripotent stem cells were derived. Whole exome sequencing and RNAseq in blood samples were carried out in a subset of cases and family controls. Protein lysates from peripheral leucocytes collected from PHTS patients were subjected to Western blotting with antibodies against the signaling proteins of the PTEN-mTOR pathway.

Results: Induced stem cells were derived from over 30 participants. Gene expression analyses in PMS-derived neurons indicate that specific pathways are dysregulated in these human neurons in vitro. Some of these findings suggest potential therapeutic approaches to PMS. Similar studies in TSC and PHTS are ongoing. The greatest genetic variation across the 3 disorders is found in PMS, were point mutations and deletions can contribute to the phenotype. We demonstrate that there is a strong overlap of phenotypes observed with point mutations as compared to deletions; however, the severity of the phenotype is typically greater in the presence of a deletion. For example, language abnormalities are more profound in the presence of a deletion, compared to a point mutation. RNAseq in over 70 blood samples identifies significantly dysregulated genes in cases versus controls. Interestingly, the dysregulation correlates with the severity of the phenotype, as measured by the Aberrant Behavioral Checklist, and particularly the lethargy/social withdrawal subscale. No genetic modifiers were identified to date, although we have identified individuals with more than one deleterious likely pathogenic change. Western blotting was performed for 54 PHTS subjects, which demonstrate altered expression of PTEN-mTOR signaling molecules.

Conclusions: In the first iteration of the DSC, we have made significant advances in relating preclinical research to clinical findings. We have an extensive repository of patient-derived stem cells for ongoing and future studies, and we continue to expand our sample that undergo RNAseq and Western blotting. While still quite preliminary, we will present how some of these first findings suggest pathways that might be therapeutic drug targets in these rare genetic disorders.