Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains without disease trait associations. Using exome sequencing and family-based rare variant analysis, we identified 20 individuals with de novo , ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative HPO analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from a patient with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.(Gly411Glu) and p.(Arg761Gln), altered DHX9 ATPase activity. The severe NDD-associated variant p.(Arg141Gln) did not impact DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9 -/-mice exhibit hypoactivity in novel environments, tremor, and sensorineural hearing loss. Taken together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported in part by the U.S. National Human Genome Research Institute (NHGRI) and National Heart Lung and Blood Institute (NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542, J.R.L); NHGRI grant as part of the GREGoR Consortium (U01 HG011758 to J.E.P., J.R.L., and R.A.G.); NHGRI grant to Baylor College of Medicine Human Genome Sequencing Center (U54HG003273 to R.A.G.); U.S. National Institute of Neurological Disorders and Stroke (NINDS) (R35NS105078 to J.R.L), Muscular Dystrophy Association (MDA) (512848 to J.R.L.), and Spastic Paraplegia Foundation Research Grant to J.R.L. This study was also supported by the General Research Fund from Research Grants Council of Hong Kong (24101921 to S.G.) and National Natural Science Foundation of China (82202045 to S.G.). D.M. was supported by a Medical Genetics Research Fellowship Program through the United States National Institute of Health (T32 GM007526-42). T.M. is supported by the Uehara Memorial Foundation. D.P. was supported by a NINDS 1K23 NS125126-01A1 and Rett Syndrome Research Trust fellowship award from International Rett Syndrome Foundation (IRSF grant #3701‐1). J.E.P. was supported by NHGRI K08 HG008986. D.G.C. was supported by NIH – Brain Disorders and Development Training Grant (T32 NS043124), NIH Medical Genetics Research Fellowship Program (T32 GM007526), the Chao Physician Scientist Award, and MDA Development Grant (873841). This study was supported by the CHU de Dijon Bourgogne and by a grant from the French Ministry of Health DIS-SEQ – Evaluation medico-economique des differentes strategies de technologies de sequencage par haut debit dans le diagnostic des patients atteints de deficience intellectuelle, Clinical Trial [NCT03287206][1]. The Deciphering Developmental Disabilities (DDD) study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003], a parallel funding partnership between Wellcome and the Department of Health, and the Wellcome Sanger Institute [grant number WT098051]. The views expressed in this publication are those of the author(s) and not necessarily those of Wellcome or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network. The work by Tomi Pastinen and Isabelle Thiffault was made possible by the generous gifts to Childrens Mercy Research Institute and Genomic Answers for Kids program at Childrens Mercy Kansas City. Tomi Pastinen holds the Dee Lyons/Missouri Endowed Chair in Pediatric Genomic Medicine. Research reported in this manuscript was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under Award Number(s) [UO1HG007672-Shashi]. ### 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: The study was approved by Baylor College of Medicine Institutional Review Board (IRB) protocol H-29697. The work by Tomi Pastinen and Isabelle Thiffault were approved by the Children's Mercy Institutional Review Board (study # 11120514). 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 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03287206&atom=%2Fmedrxiv%2Fearly%2F2023%2F03%2F05%2F2023.03.01.23286475.atom