Gain and loss of function variants in EZH1 disrupt neurogenesis timing and cause overlapping neurodevelopmental disorders

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 recessive and dominant NDDs in 17 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 (K27) 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, the implication of EZH1 in human development and disease is largely unknown. Using cellular models and biochemical studies, we demonstrate that recessive variants cause EZH1 loss of function (LOF), while dominant variants are all missense mutations that modify the catalytic activity of EZH1, thus generating a gain of function (GOF) effect. Consistent with a pathogenic effect, depletion or overexpression of EZH1 perturbs neuronal differentiation in the developing chick embryo neural tube. Furthermore, using human pluripotent stem cell (hPSC) derived neural cultures and forebrain organoids, we show that EZH1 LOF and GOF variants respectively delay and accelerate the schedule of cortical projection neuron generation. Our work identifies EZH1 LOF and GOF variants as the genetic basis of previously undefined NDDs and uncovers an essential role of EZH1 in regulating the timing of neurogenesis. ### Competing Interest Statement Erin Torti is an employee of LLC: GeneDx, LLC ### Funding Statement This work was supported by the CHOP/UPENN IDDRC-New Program Development award, CHOP-Junior Faculty Pilot Program award, Margaret Q Landerbergen Foundation Award and NIH/NINDS 1R01NS119699-01A1 (to N.A.), BFU2015-69248-P and PGC2018-096082-B-I00 from the Spanish Ministry of Economy to (M.A.M.B.), Alabama Genomic Health Initiative F170303004 through University of Alabama at Birmingham (to A.C.H., M.T.), PID2019-111217RB-I00 Spanish Ministerio de Ciencia e Innovacion (to X.D.L.C.), the King Salman Center For Disability Research Group no RG-2022-010 (to F.S.A.), ID2019-110157RA-I00 (to M.S.), FPU Spanish Ministry of Education and Science predoctoral fellowship (to R.F.) and 2021 FISDU 00400 (to P.E-B.). This research was made possible through access to the data and findings generated by the 100KGP and the DDD. The 100KGP is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100KGP is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100KGP uses data provided by patients and collected by the National Health Service as part of their care and support. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003]. This study makes use of DECIPHER (), which is funded by Wellcome. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All the human subject studies included here were approved by their local IRBs, including the Childrens Hospital of Philadelphia, Boston Childrens Hospital, University College London, Guys Hospital, Kennedy Krieger Institute, King Faisal Specialist Hospital & Research Center and University of Alabama in Birmingham. Informed consent for participation, phenotyping, sample collection and publication of images was obtained through the referring clinical teams. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data generated to support the findings of the study are included in the manuscript or available from the corresponding authors upon reasonable request.