{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,8]],"date-time":"2026-01-08T09:37:21Z","timestamp":1767865041686,"version":"3.49.0"},"reference-count":33,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2022,3,11]],"date-time":"2022-03-11T00:00:00Z","timestamp":1646956800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 DK7877"],"award-info":[{"award-number":["R01 DK7877"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 DK109907"],"award-info":[{"award-number":["R01 DK109907"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 DK78755"],"award-info":[{"award-number":["R01 DK78755"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Inher Metab Disea"],"published-print":{"date-parts":[[2022,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Very long\u2010chain acyl\u2010CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from mutations in the <jats:italic>ACADVL<\/jats:italic> gene and is among the disorders tested for in newborn screening (NBS). Confirmatory sequencing following suspected VLCADD NBS results often identifies variants of uncertain significance (VUS) in the <jats:italic>ACADVL<\/jats:italic> gene, leading to uncertainty of diagnosis and providing effective treatment regimen. Currently, <jats:italic>ACADVL<\/jats:italic> has &gt;300 VUSs in the ClinVar database that requiring characterization to determine potential pathogenicity. In this study, CRISPR\/Cas9 genome editing was used to knock out <jats:italic>ACADVL<\/jats:italic> in HEK293T cells, and targeted deletion was confirmed by droplet digital polymerase chain reaction (PCR). No VLCAD protein was detected and an 84% decrease in enzyme activity using the electron transfer flavoprotein fluorescence reduction assay and C21\u2010CoA as substrate was observed compared to control. Plasmids containing control or variant <jats:italic>ACADVL<\/jats:italic> coding sequence were transfected into the <jats:italic>ACADVL<\/jats:italic> null HEK293T. While transfection of control <jats:italic>ACADVL<\/jats:italic> restored VLCAD protein and enzyme activity, cells expressing the VLCAD Val283Ala mutant had 18% VLCAD enzyme activity and reduced protein compared to control. VLCAD Ile420Leu, Gly179Arg, and Gln406Pro produced protein comparable to control but 25%, 4%, and 5% VLCAD enzyme activity, respectively. Leu540Pro and Asp570_Ala572dup had reduced VLCAD protein and 10% and 3% VLCAD enzyme activity, respectively. VLCADD fibroblasts containing the same variations had decreased VLCAD protein and activity comparable to the transfection experiments. Generating <jats:italic>ACADVL<\/jats:italic> null HEK293T cell line allowed functional studies to determine pathogenicity of <jats:italic>ACADVL<\/jats:italic> exonic variants. This approach can be applied to multiple genes for other disorders identified through NBS.<\/jats:p>","DOI":"10.1002\/jimd.12492","type":"journal-article","created":{"date-parts":[[2022,2,26]],"date-time":"2022-02-26T13:32:50Z","timestamp":1645882370000},"page":"529-540","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Characterization of exonic variants of uncertain significance in very long\u2010chain acyl\u2010CoA dehydrogenase identified through newborn screening"],"prefix":"10.1002","volume":"45","author":[{"given":"Olivia M.","family":"D'Annibale","sequence":"first","affiliation":[{"name":"Division of Genetic and Genomic Medicine, Department of Pediatrics University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh  Pittsburgh Pennsylvania USA"},{"name":"Department of Human Genetics University of Pittsburgh Graduate School of Public Health  Pittsburgh Pennsylvania 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