Biallelic null variants in PNPLA8 cause microcephaly through the reduced abundance of basal radial glia

PNPLA8, one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. However, little is known about its role in brain development. Here, we report 12 individuals from 10 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide spectrum of clinical features ranging from developmental and epileptic-dyskinetic encephalopathy (DEDE) to progressive movement disorders. Complete loss of PNPLA8 was associated with the severe end of the spectrum, showing DEDE manifestations and congenital or progressive microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 reduced the number of basal radial glial cells (bRGCs) and upper-layer neurons. By spatial transcriptomic analysis targeting apical radial glial cells (aRGCs), we found the downregulation of bRGC-related gene sets in patient-derived cerebral organoids. Lipidomic analysis revealed a decrease in the amount of lysophosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid, indicative of the disturbed phospholipid metabolism in PNPLA8 knockout neural progenitor cells. Our data suggest that PNPLA8 has a critical role in the bRGC-mediated expansion of the developing human cortex by regulating the fate commitment of aRGCs. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by grants from the Japan Society for the Promotion of Science (Grant Numbers JP21463512, JP20K07907, JP21K07869, JP20K21584, and JP21K07803) and the Japan Agency for Medical Research and Development (AMED) under grant numbers, JP21wm0425007, JP20ek0109488, JP22ek0109486, JP22ek0109549, and JP22ek0109493. This study was also supported by grants from the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, and the Ono Medical Research Foundation to YN, the Daiko Foundation, The Hori Science and Arts Foundation, the Mochida Foundation for Medical and Pharmaceutical Research, the Takeda Science Foundation, iPS Academia Japan Inc., Leave a Nest Co. Ltd. (Ikeda Rika award), and a Grand-in-Aid for research in Nagoya City University (2013009) to ISS, and the Takeda Science Foundation to YK. HSD is supported by the Cologne Clinician Scientist Program/Faculty of Medicine/University of Cologne and funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Project No. 413543196) as well as the Koeln Fortune Program / Faculty of Medicine, University of Cologne, (Project No. 371/2021, 243/2022). R.D.S.P. and M.F. are supported by a Medical Research Council (UK) Clinician Scientist Fellowship (MR/S002065/1) and Medical Research Council (UK) award MC\_PC\_21046 to establish a National Mouse Genetics Network Mitochondria Cluster (MitoCluster). R.D.S.P. is supported by Medical Research Council (UK) strategic award MR/S005021/1 to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) and is grateful to The Lily Foundation for funding. ### 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: Genetic and functional analysis using samples from study participants was approved by the institutional review boards at Nagoya City University (Approval Number: 70-00-0200), Yokohama City University (Approval Number: A170525011), and University College London (Approval Number: #310045/1571740/37/598). Parents and legal guardians of all affected individuals consented to the publication of clinical and genetic information, including video and photographs, and the study was approved by the respective local ethics committees. The generation and application of the iPSCs were approved by the Nagoya University Ethics Committee (Approval Number: 2012-0184) and Nagoya City University Ethics Committee (Approval Number: 19-143). Written informed consent was obtained from patient guardians. The analysis using skin fibroblasts derived from the patient with Leigh syndrome was approved by the institutional review board at Jichi Medical University (Approval number: J21-014). 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.