<em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants
Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the <i>APOB</i> gene, a condition associated with...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/8/4281 |
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| author | Xavier Vanhoye Alexandre Janin Amandine Caillaud Antoine Rimbert Fabienne Venet Morgane Gossez Wieneke Dijk Oriane Marmontel Séverine Nony Charlotte Chatelain Christine Durand Pierre Lindenbaum Jennifer Rieusset Bertrand Cariou Philippe Moulin Mathilde Di Filippo |
| author_facet | Xavier Vanhoye Alexandre Janin Amandine Caillaud Antoine Rimbert Fabienne Venet Morgane Gossez Wieneke Dijk Oriane Marmontel Séverine Nony Charlotte Chatelain Christine Durand Pierre Lindenbaum Jennifer Rieusset Bertrand Cariou Philippe Moulin Mathilde Di Filippo |
| author_sort | Xavier Vanhoye |
| collection | DOAJ |
| description | Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the <i>APOB</i> gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry <i>APOB</i> missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous <i>APOB</i>-VUS (p.Leu351Arg), in a FHBL family. We generated <i>APOB</i> knock-out (KO) and <i>APOB</i>-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the <i>APOB</i> expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The <i>APOB</i> expression was decreased by 70% in the heterozygous <i>APOB</i>-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The <i>APOB</i>-p.Leu351Arg homozygous cells presented with a 40% decreased <i>APOB</i> expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of <i>APOB</i>-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients. |
| first_indexed | 2024-03-09T13:33:37Z |
| format | Article |
| id | doaj.art-e2ca63f47c0e4450bc327462cfb59715 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T13:33:37Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-e2ca63f47c0e4450bc327462cfb597152023-11-30T21:15:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01238428110.3390/ijms23084281<em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel VariantsXavier Vanhoye0Alexandre Janin1Amandine Caillaud2Antoine Rimbert3Fabienne Venet4Morgane Gossez5Wieneke Dijk6Oriane Marmontel7Séverine Nony8Charlotte Chatelain9Christine Durand10Pierre Lindenbaum11Jennifer Rieusset12Bertrand Cariou13Philippe Moulin14Mathilde Di Filippo15Service de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceService de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceInstitut du Thorax, Nantes Université, CHU Nantes, CNRS, INSERM, F-44000 Nantes, FranceInstitut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, FranceLaboratoire d’Immunologie, Edouard Herriot Hospital, Hospices Civils de Lyon, F-69437 Lyon, FranceLaboratoire d’Immunologie, Edouard Herriot Hospital, Hospices Civils de Lyon, F-69437 Lyon, FranceInstitut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, FranceService de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceService de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceService de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceCarMen Laboratory, INSERM, INRA, INSA Lyon, Université Claude Bernard Lyon 1, Pierre-Bénite, F-69364 Lyon, FranceInstitut du Thorax, Nantes Université, CNRS, INSERM, F-44000 Nantes, FranceCarMen Laboratory, INSERM, INRA, INSA Lyon, Université Claude Bernard Lyon 1, Pierre-Bénite, F-69364 Lyon, FranceInstitut du Thorax, Nantes Université, CHU Nantes, CNRS, INSERM, F-44000 Nantes, FranceCarMen Laboratory, INSERM, INRA, INSA Lyon, Université Claude Bernard Lyon 1, Pierre-Bénite, F-69364 Lyon, FranceService de Biochimie et de Biologie Moléculaire, Laboratoire de Biologie Médicale MultiSites, Hospices Civils de Lyon, F-69677 Bron, FranceHypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the <i>APOB</i> gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry <i>APOB</i> missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous <i>APOB</i>-VUS (p.Leu351Arg), in a FHBL family. We generated <i>APOB</i> knock-out (KO) and <i>APOB</i>-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the <i>APOB</i> expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The <i>APOB</i> expression was decreased by 70% in the heterozygous <i>APOB</i>-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The <i>APOB</i>-p.Leu351Arg homozygous cells presented with a 40% decreased <i>APOB</i> expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of <i>APOB</i>-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.https://www.mdpi.com/1422-0067/23/8/4281primary hypobetalipoproteinemiaapolipoprotein Bgenome editingvariants of uncertain significancefunctionalitysecretion |
| spellingShingle | Xavier Vanhoye Alexandre Janin Amandine Caillaud Antoine Rimbert Fabienne Venet Morgane Gossez Wieneke Dijk Oriane Marmontel Séverine Nony Charlotte Chatelain Christine Durand Pierre Lindenbaum Jennifer Rieusset Bertrand Cariou Philippe Moulin Mathilde Di Filippo <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants International Journal of Molecular Sciences primary hypobetalipoproteinemia apolipoprotein B genome editing variants of uncertain significance functionality secretion |
| title | <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants |
| title_full | <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants |
| title_fullStr | <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants |
| title_full_unstemmed | <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants |
| title_short | <em>APOB</em> CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants |
| title_sort | em apob em crispr cas9 engineering in hypobetalipoproteinemia a promising tool for functional studies of novel variants |
| topic | primary hypobetalipoproteinemia apolipoprotein B genome editing variants of uncertain significance functionality secretion |
| url | https://www.mdpi.com/1422-0067/23/8/4281 |
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