Functional analysis of rare genetic variants in Autism Spectrum Disorder: an oligonenic mode of inheritance?

Abstract

Recent whole-genome-based analyzes have revealed a large number of potentially deleterious genetic variants in patients with Autism Spectrum Disorder (ASD), most of them rare and private. One of the major current challenge is to determine which of these variants are involved in the etiology of the disease and how many variants are required for the complete penetrance of the disorder in each patient. Recently, we performed whole-exome sequencing in Brazilian patients with ASD and identified in one of the patients, referred to as F2688, rare and potentially pathogenic variants in the RELN and CACNA1H genes; and in another patient, referred to as F10129, rare and potentially deleterious variants in the CACNA1H and MTOR genes. The RELN gene encodes Reelin, a large secreted glycoprotein that controls neuronal migration and plasticity of synapses. The CACNA1H gene encodes the ±1-subunit of the T-type low voltage dependent calcium (Ca2+) channel Cav3.2. The MTOR gene encodes the mTOR protein kinase, whose signaling pathway can be regulated by intracellular Ca2+ levels, controls several essential cellular processes and negatively regulates Reelin signaling pathway. Using induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs) from patient F2688, we have recently shown that the variants identified in the RELN gene are deleterious and lead to diminished Reelin secretion and impaired Reelin signal transduction. Also, our results suggest that mTOR signaling is overactivated in F2688 NPCs and function as a "second hit" event contributing to downregulation of the Reelin cascade. We do not yet know whether the CACNA1H variant identified in this patient contributes to overactivation of the mTOR pathway and to downregulation of the Reelin signaling. We also do not know whether the variants identified in patient F10129 are deleterious and interact, leading to mTOR and Reelin signaling pathways dysfunction. The present project aims to verify whether the rare variants identified in the CACNA1H gene in patients F2688 and F10129 and whether the rare variant identified in the MTOR gene in patient F10129 are functional, lead to changes in Ca2+ influx, in the activity of mTOR and Reelin signaling pathways and in the migration of neural cells derived from these patients and, thus, act in an oligogenic mode of inheritance for ASD. In order to achieve this, we will use as an experimental model iPSCs-derived NPCs from patients F2688 and F10219, from other patients with idiopathic ASD and from heath controls, and we will perform several functional studies that address abnormalities in the Cav3.2 channel, and in the mTOR and Reelin signaling pathways. In addition, we intend to use a heterologous expression system to confirm the results. Experimental validation of interactions between the different rare and potentially risk variants found in patients is an essential step forward in understanding the complex inheritance of ASD. (AU)