Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We performed saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identified 3,432 functionally abnormal variants, in three distinct classes. We trained a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants could account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X - driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols [https://github.com/HurlesGroupSanger/Saturation\_Genome\_Editing][1] ### Funding Statement This work was supported by core Wellcome funding to the Wellcome Sanger Institute (grant Grant reference number: 108413/A/15/D), and for the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This work was also funded by a Clinical Lecturer Starter Grant from the Academy of Medical Sciences, the Wellcome Trust, the Medical Research Council, the British Heart Foundation, Versus Arthritis, Diabetes UK, the British Thoracic Society (Helen and Andrew Douglas bequest), and the Association of Physicians of Great Britain and Ireland to E.J.R. [SGL023\1060]. E.J.G. and J.R.B.P. are funded by the Medical Research Council (Unit programs: MC\_UU\_12015/2, MC\_UU\_00006/2, MC\_UU\_12015/1, and MC\_UU\_00006/1). The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant no. HICF-1009-003). The full acknowledgements can be found online (www.ddduk.org/access.html). This study makes use of data generated by the DECIPHER community. A full list of centres who contributed to the generation of the data is available from and via email from contact{at}deciphergenomics.org. Funding for the DECIPHER project was provided 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: We have both openly public data and non-public data in our study. Public data: 1)gnomAD () 2) ClinVar () 3) DECIPHER () 4) Kaplanis, Samocha et al (doi: 10.1038/s41586-020-2832-5) 5) Lennox et al (doi: 10.1016/j.neuron.2020.01.042) 6) Nicola et al (doi: 10.1002/ajmg.a.61061) 7) TCGA (https://www.cancer.gov/tcga). Non-public data which required registration: 1) UKBiobank --Research Ethics Committee of the United Kingdom Health Research Agency - North West Haydock gave ethical approval for this work (Ref: 21/NW/0157) 2) Deciphering Developmental Disorders (DDD) --Research Ethics Committee of the United Kingdom - Cambridge South gave ethical approval for this work (Ref: 10/H0305/83) --Research Ethics Committee of the Republic of Ireland gave ethical approval for this work (Ref: GEN/284/12) 3) Genomics England (GEL) --Research Ethics Committee of the Health Research Agency East of England - Cambridge South gave ethical approval for this work (Ref: 14/EE/1112) 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 sequencing data generated in this study were deposited to ENA with accession number PRJEB52929. [1]: https://github.com/HurlesGroupSanger/Saturation_Genome_Editing