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Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malab...

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Autores principales: Bordat, Claire, Vairo, Donato, Cuerq, Charlotte, Halimi, Charlotte, Peiretti, Franck, Penhoat, Armelle, Vieille-Marchiset, Aurélie, Gonzalez, Teresa, Michalski, Marie-Caroline, Nowicki, Marion, Peretti, Noël, Reboul, Emmanuelle
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9921550/
https://www.ncbi.nlm.nih.gov/pubmed/36771214
http://dx.doi.org/10.3390/nu15030505
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author Bordat, Claire
Vairo, Donato
Cuerq, Charlotte
Halimi, Charlotte
Peiretti, Franck
Penhoat, Armelle
Vieille-Marchiset, Aurélie
Gonzalez, Teresa
Michalski, Marie-Caroline
Nowicki, Marion
Peretti, Noël
Reboul, Emmanuelle
author_facet Bordat, Claire
Vairo, Donato
Cuerq, Charlotte
Halimi, Charlotte
Peiretti, Franck
Penhoat, Armelle
Vieille-Marchiset, Aurélie
Gonzalez, Teresa
Michalski, Marie-Caroline
Nowicki, Marion
Peretti, Noël
Reboul, Emmanuelle
author_sort Bordat, Claire
collection PubMed
description Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (−57.0 ± 2.6% to −83.9 ± 1.6%) and cholesterol (−35.3 ± 4.4% to −60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (−41.5 ± 3.7% to −97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.
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spelling pubmed-99215502023-02-12 Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias Bordat, Claire Vairo, Donato Cuerq, Charlotte Halimi, Charlotte Peiretti, Franck Penhoat, Armelle Vieille-Marchiset, Aurélie Gonzalez, Teresa Michalski, Marie-Caroline Nowicki, Marion Peretti, Noël Reboul, Emmanuelle Nutrients Article Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (−57.0 ± 2.6% to −83.9 ± 1.6%) and cholesterol (−35.3 ± 4.4% to −60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (−41.5 ± 3.7% to −97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism. MDPI 2023-01-18 /pmc/articles/PMC9921550/ /pubmed/36771214 http://dx.doi.org/10.3390/nu15030505 Text en © 2023 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
Bordat, Claire
Vairo, Donato
Cuerq, Charlotte
Halimi, Charlotte
Peiretti, Franck
Penhoat, Armelle
Vieille-Marchiset, Aurélie
Gonzalez, Teresa
Michalski, Marie-Caroline
Nowicki, Marion
Peretti, Noël
Reboul, Emmanuelle
Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title_full Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title_fullStr Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title_full_unstemmed Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title_short Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias
title_sort validation of knock-out caco-2 tc7 cells as models of enterocytes of patients with familial genetic hypobetalipoproteinemias
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9921550/
https://www.ncbi.nlm.nih.gov/pubmed/36771214
http://dx.doi.org/10.3390/nu15030505
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