Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders [RNA-seq]

Genetic disruption of chromatin regulators is frequently found in neurodevelopmental disorders (NDDs). While chromatin regulators are attractive therapeutic targets, studies to determine their implication in the etiology of NDDs are limited, preventing advances in diagnosis and treatment strategies. Here, we uncover pathogenic variants in the chromatin modifier Enhancer of Zeste Homologue 1 (EZH1) as the cause of overlapping dominant and recessive NDDs in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 (H3K27) methyltransferases of the Polycomb Repressive Complex 2 (PRC2). Unlike the other PRC2 subunits, which are associated with the pathogenesis of human cancers and developmental disorders with overgrowth, the implication of EZH1 in human development and disease is largely unknown. Using cellular models and biochemical studies, we demonstrate that EZH1 variants identified in our study alter EZH1 molecularly. While recessive variants are EZH1 loss of function (LOF) variants that impair EZH1 expression, dominant variants are all missense mutations that affect evolutionarily conserved aminoacids likely impacting EZH1 structure or function. Accordingly, we found increased H3K27 methyltransferase activity for two EZH1 missense variants we tested, thus generating EZH1 gain of function (GOF) variants. Furthermore, we show that EZH1 is necessary and sufficient to differentiate neural stem cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell (hPSC)-derived neural cultures and forebrain organoids, we demonstrate that EZH1 LOF and GOF variation perturbs cortical projection neuron differentiation. Our work identifies EZH1 LOF and GOF variants as the genetic basis of previously undefined recessives and dominant NDDs and uncovers an essential role of EZH1 in regulating neurogenesis. Overall design: Comparative gene expression profiling analysis of RNA-seq data for 8-week 2D neuron differentiated from isogenic stem cell lines carrying WT EZH1, synonymous variants of EZH1, EZH1+/A678G, and EZH1-/-. week8_H9: 8-week differentiated neurons, EZH1+/+ (WT 1) week8_WTS5: 8-wk differentiated neurons, EZH1+/+ (WT 2, S5: silent mutation) week8_8del: 8-wk differentiated neurons, EZH1-/- (KO clone 1 - 8 bp deletion) week8_16del: 8-wk differentiated neurons, EZH1-/- (KO clone 2- 16 bp deletion) week8_het5: 8-wk differentiated neurons, EZH1+/A678G (Het clone 5) week8_het6: 8-wk differentiated neurons, EZH1+/A678G (Het clone 6)