Transcriptomic Modeling of Phelan-Mcdermid Syndrome Using Glutamatergic Neurons Generated from Patient iPSCs

Background:  Autism spectrum disorder (ASD) has high heritability and a prevalence of nearly 1% worldwide, but heterogeneity of patients has made identifying the underlying etiology difficult. By focusing on monogenic disorders with high penetrance for causing ASD, specific pathways might be identified that are common to a broader group of ASD patients with mechanistically related etiology. Phelan-McDermid syndrome (PMS) is one such ASD-associated syndrome that is caused by haploinsufficiency of the gene SHANK3, which encodes for a scaffolding protein of the post-synaptic density at glutamatergic synapses. While animal models provide great insight into pathways involved in PMS, induced pluripotent stem cells (iPSCs) generated from patient tissue samples retain the full genetic background of the individual and can be expanded extensively and differentiated into neural progenitor cells (NPCs) and neurons. Since it isn't possible to obtain brain tissue from living ASD patients, having a source of human neural cells in incredibly valuable.

Objectives: The overarching goal of this study is to improve our mechanistic understanding of PMS and identify candidate genes and pathways that can be targeted pharmacologically to alleviate neurological deficits of the syndrome. To accomplish this, we applied a multi-step approach that aimed to: 1) generate high-quality iPSC clones from PMS patients and siblings; 2) differentiate them into NPCs, followed by neurons, to capture the neurodevelopmental phenotype of PMS in patients; 3) identify PMS-associated differential gene expression in iPSC-derived neurons by RNA sequencing; and, 4) identify candidate drug targets and FDA-approved drug compounds for repositioning in PMS.

Methods:  Blood samples from patients with PMS and unaffected siblings are collected for 14 PMS patient/sibling pairs and reprogrammed using a modified non-integrating Sendai virus to express reprogramming factors. For each individual, 2-3 clones are expanded for quality control (QC) and banking, and those that pass QC are differentiated into NPCs using a monolayer approach with PSC Neural Induction Media from Fisher. NPC lines are then transfected with doxycycline-inducible Ngn2 lentiviruses with puromycin selection and grown for 3 weeks to allow for maturation. RNA is isolated from NPC and neuron samples and subjected to RNA sequencing, and the PMS-associated changes in gene expression are then compared to known gene expression profiles of FDA-approved drugs and other ASD-associated syndromes.

Results:  Twelve PMS patient/sibling pairs have been reprogrammed with 2-3 clones per individual having been used for NPC generation followed by neuronal induction. Initial NPC samples have been subjected to RNA sequencing for preliminary investigation into the early developmental transcriptional profile of PMS. Differentially expressed genes between PMS patient-derived NPCs compared to those from their healthy siblings were found to be enriched for components of the Wnt signaling pathway and were upregulated in the PMS-associated cells.

Conclusions: iPSCs from PMS patients offer a powerful tool for disease characterization, drug identification, and screening. Generating an expression profile for these patient-derived NPCs and neurons will provide a unique perspective on the transcriptional signature of PMS that can be used in conjunction with other models of the disease and known drug expression profiles to identify new therapeutics.