DENND5A epileptic encephalopathy features global developmental delay, seizures and ventriculomegaly

Epileptic encephalopathies comprise a clinically and genetically heterogenous group of disorders characterized by global developmental delay and ongoing seizure activity. It is generally considered that seizure activity is the primary pathology and leads to altered cognitive function. However, epileptic encephalopathy can also result from primary defects in neurodevelopment that lead to seizures. Here we examine the symptomology of individuals with epileptic encephalopathy resulting from biallelic pathogenic variants in DENND5A , encoding a protein involved in intracellular membrane trafficking. We established a cohort of 15 individuals from 14 families with 21 unique variants in DENND5A and analyzed phenotypic surveys answered by their treating clinicians. We obtained cells derived from several cohort members and examined DENND5A expression and stability, and we examined the role of DENND5A in symmetric cell division capability. Finally, we generated a mouse line expressing a homozygous mutation found in the cohort and analyzed brain morphology in vivo using a 7 Tesla magnetic resonance imaging system. Our cohort study reveals that global developmental delay, gross abnormalities in brain development including ventriculomegaly and corpus callosum dysgenesis, seizures, microcephaly and hypotonia are found in the majority of the cohort. Patient-derived neural precursor cells from induced pluripotent stem cells, as well as patient-derived lymphoblasts, display loss of DENND5A protein and exhibit problems with symmetric cell division, an essential process in early neural development. The introduction of a homozygous human point mutation into the conserved sequence in mouse leads to an animal with reduced brain volume and widespread regional brain dysgenesis and ventriculomegaly, phenotypes shared with the human cohort. Taken together, our multi-dimensional study establishes DENND5A as a critical gene during neurodevelopment and that biallelic loss of function variants, including missense and truncating variants as well as intronic splice site variants, result in a developmental epileptic encephalopathy. ### Competing Interest Statement KMc is employed by GeneDx, LLC. ### Funding Statement This work was funded by a Foundation Grant from the Canadian Institutes of Health Research to PSM, and a grant from the Alain and Sandra Bouchard Foundation for Intellectual Disabilities to PSM and TMD. This work was also supported by the King Salman Center for Disability Research through Research Group no RG-2022-010 (FSA). This work was also performed under the Care4Rare Canada Consortium funded by Genome Canada and the Ontario Genomics Institute (OGI-147), the Canadian Institutes of Health Research, Ontario Research Fund, Genome Alberta, Genome British Columbia, Genome Quebec, and Childrens Hospital of Eastern Ontario Foundation (DC, ESYG, MO). Research reported in this manuscript was also supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under Award Number(s) \[U01HG007672, U01HG007943\] (HC). ### 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 materials and methods for participant recruitment and clinical data collection was approved by the McGill University Health Centre research ethics board (study 2021-6324) and the McGill Faculty of Medicine and Health Sciences institutional review board (study A12-M66-21B). 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 Supplementary table 1 includes all raw data obtained in the cohort study and describes a comprehensive clinical description of each participant, as well as sums and averages of each phenotype for the cohort as a whole. Raw data concerning in vitro cell experiments and in vivo animal experiments were generated at the Montreal Neurological Institute of McGill University. Derived data supporting the findings of this study are available upon reasonable request from the corresponding author. * 7T : 7-Tesla ASD : autism spectrum disorder EE : Epileptic Encephalopathy DENN : Differentially Expressed in Normal and Neoplastic cells iPSC : induced pluripotent stem cell H& E : hematoxylin and eosin Mut : mutant NPC : neural progenitor cell PLAT : Polycystin-1, Lipoxygenase, Alpha-Toxin RUN : RPIP8 [RaP2 interacting protein 8], UNC-14 and NESCA [new molecule containing SH3 at the carboxyl-terminus] RARE : Rapid Imaging with Refocused Echoes TORCH : Toxoplasmosis, Other agents, Rubella, Cytomegalovirus, and Herpes simplex TR : repetition time WT : wild-type