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Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis

The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or a...

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Autores principales: Garcia-Llorens, Guillem, Lopez-Navarro, Sergi, Jaijo, Teresa, Castell, Jose V., Bort, Roque
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9322025/
https://www.ncbi.nlm.nih.gov/pubmed/35887608
http://dx.doi.org/10.3390/jpm12071111
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author Garcia-Llorens, Guillem
Lopez-Navarro, Sergi
Jaijo, Teresa
Castell, Jose V.
Bort, Roque
author_facet Garcia-Llorens, Guillem
Lopez-Navarro, Sergi
Jaijo, Teresa
Castell, Jose V.
Bort, Roque
author_sort Garcia-Llorens, Guillem
collection PubMed
description The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or alterations in intron sequences. Notwithstanding, elucidation of the pathogenicity of a specific variant might be critical for an accurate diagnosis. In this study, we described a novel intronic variant c.2597 + 5G > T in the donor splice sequence of the PHKA2 gene. To investigate PHKA2 mRNA splicing, as well as the functional consequences on glycogen metabolism, we generated hepatocyte-like cells from a proband’s fibroblasts by direct reprogramming. We demonstrated an aberrant splicing of PHKA2, resulting in the incorporation of a 27 bp upstream of intron 23 into exon 23, which leads to an immediate premature STOP codon. The truncated protein was unable to phosphorylate the PYGL protein, causing a 4-fold increase in the accumulation of glycogen in hepatocyte-like cells. Collectively, the generation of personalized hepatocyte-like cells enabled an unequivocal molecular diagnosis and qualified the sister’s proband, a carrier of the same mutation, as a candidate for a preimplantation genetic diagnosis. Additionally, our direct reprogramming strategy allows for an unlimited source of “diseased” hepatocyte-like cells compatible with high-throughput platforms.
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spelling pubmed-93220252022-07-27 Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis Garcia-Llorens, Guillem Lopez-Navarro, Sergi Jaijo, Teresa Castell, Jose V. Bort, Roque J Pers Med Article The diagnosis of inherited metabolic disorders is a long and tedious process. The matching of clinical data with a genomic variant in a specific metabolic pathway is an essential step, but the link between a genome and the clinical data is normally difficult, primarily for new missense variants or alterations in intron sequences. Notwithstanding, elucidation of the pathogenicity of a specific variant might be critical for an accurate diagnosis. In this study, we described a novel intronic variant c.2597 + 5G > T in the donor splice sequence of the PHKA2 gene. To investigate PHKA2 mRNA splicing, as well as the functional consequences on glycogen metabolism, we generated hepatocyte-like cells from a proband’s fibroblasts by direct reprogramming. We demonstrated an aberrant splicing of PHKA2, resulting in the incorporation of a 27 bp upstream of intron 23 into exon 23, which leads to an immediate premature STOP codon. The truncated protein was unable to phosphorylate the PYGL protein, causing a 4-fold increase in the accumulation of glycogen in hepatocyte-like cells. Collectively, the generation of personalized hepatocyte-like cells enabled an unequivocal molecular diagnosis and qualified the sister’s proband, a carrier of the same mutation, as a candidate for a preimplantation genetic diagnosis. Additionally, our direct reprogramming strategy allows for an unlimited source of “diseased” hepatocyte-like cells compatible with high-throughput platforms. MDPI 2022-07-07 /pmc/articles/PMC9322025/ /pubmed/35887608 http://dx.doi.org/10.3390/jpm12071111 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Garcia-Llorens, Guillem
Lopez-Navarro, Sergi
Jaijo, Teresa
Castell, Jose V.
Bort, Roque
Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title_full Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title_fullStr Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title_full_unstemmed Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title_short Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis
title_sort modeling a novel variant of glycogenosis ixa using a clonal inducible reprogramming system to generate “diseased” hepatocytes for accurate diagnosis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9322025/
https://www.ncbi.nlm.nih.gov/pubmed/35887608
http://dx.doi.org/10.3390/jpm12071111
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