Study of molecular and cellular mechanisms involved in the oligogenic inheritance of Autism Spectrum Disorder

Abstract

Recent genomic studies have revealed that the genetic architecture of Autism Spectrum Disorder (ASD) is extremely complex and heterogeneous, and that a portion of patients may carry rare and deleterious variants in two or more risk genes, in an oligogenic inheritance model. Furthermore, a negative correlation was observed between the number of risk variants and the patients' intelligence quotient, which suggests additive or epistatic effects of these variants. However, little is known about how these variants interact with each other and converge on neurobiological pathways underlying ASD. Recently, through Whole Exome Sequencing (WES) of 291 Brazilian individuals with ASD and their parents, we identified in one of the probands, referred to as F2688, two rare missense variants in compound heterozygosity in the RELN gene and a rare de novo variant in a splicing site of the CACNA1H gene. RELN encodes Relin, a secreted glycoprotein that controls neuronal migration and synaptic plasticity, and CACNA1H encodes the T-type calcium channel ±1 subunit (Ca2+) Cav3.2 that controls neuronal excitability. Using neuroprogenitor cells derived from induced pluripotent stem cells (iPSCs) from individual F2688 and from control individuals, we show that variants in RELN and CACNA1H are deleterious and that there is an abnormal crosstalk between these mutated genes through the mTORC1 signaling pathway. Finally, after reanalysis of the WES data from our Brazilian cohort and the whole genome sequencing (WGS) data from the MSSNG cohort (composed of approximately 5000 individuals with ASD and their parents), we verified that there is a significant enrichment of the co-occurrence of variants rare and potentially deleterious in both copies of Relin pathway genes and in one of the copies of Ca2+ channel genes in ASD. However, it is still unknown how these simultaneously mutated genes from the same individual alter brain development and lead to ASD. This project aims to explore the specific types of brain cells and cell phenotypes affected by the synergistic action of deleterious variants in RELN and CACNA1H and those that are unique to each mutated gene. For this, we will use as experimental models neural cells in two-dimensional cultures and three-dimensional brain organoids derived from iPSCs from the individual F2688, which will have the mutated alleles of RELN and CACNA1H corrected by gene editing methodologies (such as CRISPR-Cas9 and/or Prime Editing) . We will perform analyzes of proliferation, differentiation, migration, synaptogenesis and Ca2+ influx. The results of this project will provide further elucidation on the mechanisms of gene interactions and combinations of variants required to cause ASD.