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Mutations in the\n                      <jats:italic>ECHS1<\/jats:italic>\n                      gene 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 with\n                      <jats:italic>ECHS1<\/jats:italic>\n                      variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Here, we constructed an assay system to verify VUS function for\n                      <jats:italic>ECHS1<\/jats:italic>\n                      gene. A high-throughput assay using\n                      <jats:italic>ECHS1<\/jats:italic>\n                      knockout 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The functional validation of VUS identified novel variants causing loss of\n                      <jats:italic>ECHS1<\/jats:italic>\n                      function. 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      In summary, this study uncovered new\n                      <jats:italic>ECHS1<\/jats:italic>\n                      cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jmg-2022-109027","type":"journal-article","created":{"date-parts":[[2023,4,13]],"date-time":"2023-04-13T13:28:15Z","timestamp":1681392495000},"page":"1006-1015","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":10,"title":["Strategic validation of variants of uncertain significance in\n                    <i>ECHS1<\/i>\n                    genetic testing"],"prefix":"10.1136","volume":"60","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4978-1680","authenticated-orcid":false,"given":"Yoshihito","family":"Kishita","sequence":"first","affiliation":[{"name":"Department of Life Science, Faculty of Science and Engineering, Kindai University, Higashiosaka, Osaka, Japan"},{"name":"Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4776-1478","authenticated-orcid":false,"given":"Ayumu","family":"Sugiura","sequence":"additional","affiliation":[{"name":"Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Bunkyo-ku, Tokyo, 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Children's Hospital, Midori-ku, Chiba, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takuya","family":"Fushimi","sequence":"additional","affiliation":[{"name":"Department of Metabolism, Chiba Children's Hospital, Midori-ku, Chiba, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7009-8299","authenticated-orcid":false,"given":"Noriko","family":"Ichino","sequence":"additional","affiliation":[{"name":"Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshie","family":"Nagatakidani","sequence":"additional","affiliation":[{"name":"Department of Life Science, Faculty of Science and Engineering, Kindai University, Higashiosaka, Osaka, 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