Characterization of variants of uncertain significance in isovaleryl-CoA dehydrogenase identified through newborn screening: An approach for faster analysis.

Autor: D'Annibale OM; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA., Koppes EA; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA., Alodaib AN; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Department of Clinical Genomics, Centre for Genomic Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia., Kochersperger C; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA., Karunanidhi A; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA., Mohsen AW; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA., Vockley J; Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA. Electronic address: gerard.vockley@chp.edu.
Jazyk: angličtina
Zdroj: Molecular genetics and metabolism [Mol Genet Metab] 2021 Sep-Oct; Vol. 134 (1-2), pp. 29-36. Date of Electronic Publication: 2021 Aug 30.
DOI: 10.1016/j.ymgme.2021.08.012
Abstrakt: Introduction: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS.
Methods: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity.
Results: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants.
Conclusions: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.
Competing Interests: Declaration of Competing Interest The authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.
(Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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