Strategic validation of variants of uncertain significance in ECHS1 genetic testing

ECHS1 is the causative gene for mitochondrial short-chain enoyl-CoA hydratase 1 deficiency. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. Therefore, we constructed an assay system to verify VUS function. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. The functional validation of VUS identified novel variants causing loss of ECHS1 function. Moreover, we identified cases with functional ECHS1 defects through multi-omics analysis. We identified a synonymous substitution, p.P163=, and candidate pathogenic variants in the above validation experiments. In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to functional evaluation of other genes associated with mitochondrial disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by a grant for the Practical Research Project for Rare/Intractable Diseases from AMED to H.O., K.M., Y.O. and A.O. (Fund ID: JP21im0210625, JP21ek0109511, JP22ek0109485, JP22ek0109468, JP22gk0110038, JP19ek0109273), Program for Promoting Platform of Genomics based Drug Discovery to Y.O. (Fund ID: JP22kk0305015), the Research Center Network for Realization of Regenerative Medicine (The Acceleration Program for Intractable Diseases Research utilizing Disease-specific iPS cells, JP21bm0804018), and JSPS KAKENHI JP19H03624 to Y.O. and JP20H03648 to H.O. ### 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 studies were approved by the regional Ethics Committees at Juntendo University, Saitama Medical University, Chiba Children's Hospital and Kindai University. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the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 Raw data are available from the corresponding author upon reasonable request. ECHS1 knockout cells can also be distributed. Some genomic information that could be used to identify individuals cannot be shared due to ethical restrictions.