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.