Investigating the role of RHOBTB2 in DEE to develop novel therapies

ABSTRACT The past decade has seen an explosion of gene discovery in the developmental and epileptic encephalopathies (DEEs) due to rapid advances in sequencing technologies. CurrenUy, nearly 50% of patients will receive a precise genetic diagnosis, yet few diagnoses have an effective, targeted treatment. To develop a novel therapy requires an understanding of the disease mechanism, including molecular and cellular consequences of the pathogenic variants and whether variants result in gain or loss of function. De novo heterozygous variants in the RHOBTB2 gene cause a severe developmental disorder characterized by intractable epilepsy, severe developmental impairment, hypotonia, movement disorder, and postnatal microcephaly, referred to here as RHOBTB2-DEE. Although previously studied in the context of tumor suppression, the role of RHOBTB2 in neurodevelopment and the impact of RHOBTB2-DEE pathogenic variants has yet to be investigated in a human cellular model. Pathogenic variants cluster in the evolutionarily conserved BTB-domain, with three recurrent variants accounting for -65% of affected individuals. Transient overexpression experiments show that mutant RHOBTB2 protein is more abundant than wildtype RHOBTB2, suggesting hyper-stability and a potential dominant negative effect. We postulate that the hyper-stable mutant gene products in dominant RHOBTB2-DEE may interfere with wildtype RHOBTB2 functions in the brain and/or exhibit deleterious neomorphic functions that result in disease phenotypes. Therefore, effective treatment will likely require decreasing or eliminating the mutant form of the protein. lmportanUy, a milder, recessive disorder due to biallelic loss-of-function (LOF) variants in RHOBTB2 has been described, and parents carrying heterozygous LOF alleles are unaffected. This suggests that (i) haploinsufficiency is unlikely to be the mechanism for the de novo. dominant RHOBTB2- DEE. and (ii) selective knockdown of the mutant allele may be an effective therapy for RHOBTB2-DEE. In this study, we will investigate the consequences of de novo pathogenic variants in the RHOBTB2 gene that cause DEE in patient-derived cells, including 3D organoid models. We will then investigate potential targeted therapeutic approaches, bridging the gap between genetic diagnosis and precision therapy.