Strategic validation of variants of uncertain significance inECHS1genetic testing

AbstractECHS1is the causative gene for mitochondrial short-chain enoyl-CoA hydratase 1 deficiency. While genetic analysis studies have diagnosed numerous cases withECHS1variants, 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 usingECHS1knockout 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 functionalECHS1defects 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 newECHS1cases based on VUS validation and omics analysis; these analyses are applicable to functional evaluation of other genes associated with mitochondrial disease.