Strategic validation of variants of uncertain significance inECHS1genetic testing

Abstract

BackgroundEnoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in theECHS1gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. 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.MethodsHere, we constructed an assay system to verify VUS function forECHS1gene. A high-throughput assay usingECHS1knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis.ResultsThe functional validation of VUS identified novel variants causing loss ofECHS1function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system.ConclusionsIn summary, this study uncovered newECHS1cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.