Genetic and pathophysiological mechanisms in mendelian neurodevelopmental disorders: investigation of the genes AHDC1 and EHMT2

Abstract

Neurodevelopmental disorders encompass a range of conditions that involve changes in brain development, including intellectual disability (ID) and autism spectrum disorder, among others. This study aims to investigate the genetic and pathophysiological mechanisms underlying alterations in two genes as potential causes of ID: AHDC1, associated with an autosomal dominant form of syndromic ID (known as Xia-Gibbs syndrome - XGS), and EHMT2, a gene previously identified by our research group as a candidate for an autosomal recessive syndromic ID condition. We are developing cellular models for functional studies, with a focus on evaluating the hypothesis of EHMT2 as a causal gene and also to elucidate molecular mechanisms that trigger the phenotypes of patients with mutations in both genes (AHDC1 and EHMT2).In a previous project conducted by our group (FAPESP 2018/08486-3 and 2013/08028-1), we established iPSC (induced pluripotent stem cells) of three XGS patients; in the same project, we identified a potentially deleterious splice site EHMT2 homozygous variant in a syndromic ID patient. More recently, 10 additional unrelated XGS patients were recruited by our research group. This project will proceed with the following steps: (1) detailed clinical characterization of a Brazilian series of 13 XGS patients to expand the clinical spectrum and establish genotype-phenotype relationships, given the variable expression of the condition; (2) expansion of the number of cell lines from XGS patients for functional study, including collecting PBMCs (peripheral blood mononuclear cells) from at least seven additional patients and reprogramming them into iPSC, thus reaching a minimum of 10 cell lines; (3) construction of a peripheral blood DNA bank from XGS patients for the purpose of characterizing epigenetic changes and developing a specific epigenetic signature for the condition; (4) investigation of the hypothesis of EHMT2 as a candidate gene for a new neurodevelopmental syndrome by episignature analysis of peripheral blood DNA from a patient; (5) collection and reprogramming of cells from a patient with a homozygous EHTM2 splice site variant; (6) obtaining by CRISPR-Cas9 three lines with biallelic mutations in this same gene; (7) establishment of three control iPSC lines (totaling six) from PBMC of neurotypical individuals; (8) differentiation of iPSC strains with ADHC1 and EHTM2 mutations, in addition to the control cells, into cortical neurons; (9) utilizing neural stem cells (NSC) and mature neurons (60 days of differentiation) for studying the transcriptome (RNAseq); and (10) cellular morphology; (11) study of subcellular localization of the AHDC1 protein by immunofluorescence; (12) electrophysiology study of SXG neurons to assess whether there is a relationship between the position of the AHDC1 variants of patients with or without epilepsy in a cellular model. Expected contributions: (1) elucidation of molecular aspects of SXG and EHMT2 biallelic mutations; (2) generate data that contribute to the understanding of the epigenome of patients with AHDC1 mutations, with the potential development of an epigenetic signature; (3) evaluation of the hypothesis that EHMT2 may be a causal gene in a new autosomal recessive neurodevelopmental syndrome. (AU)