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Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A

The importance of genetic diagnosis for patients with hemophilia has been recently demonstrated. However, the pathological variant cannot be identified in some patients. Here, we aimed to identify the pathogenic intronic variant causing hemophilia A using induced pluripotent stem cells (iPSCs) from...

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Autores principales: Hiramoto, Takafumi, Inaba, Hiroshi, Baatartsogt, Nemekhbayar, Kashiwakura, Yuji, Hayakawa, Morisada, Kamoshita, Nobuhiko, Nishimasu, Hiroshi, Nureki, Osamu, Kinai, Ei, Ohmori, Tsukasa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society of Hematology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10690555/
https://www.ncbi.nlm.nih.gov/pubmed/37792826
http://dx.doi.org/10.1182/bloodadvances.2023010838
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author Hiramoto, Takafumi
Inaba, Hiroshi
Baatartsogt, Nemekhbayar
Kashiwakura, Yuji
Hayakawa, Morisada
Kamoshita, Nobuhiko
Nishimasu, Hiroshi
Nureki, Osamu
Kinai, Ei
Ohmori, Tsukasa
author_facet Hiramoto, Takafumi
Inaba, Hiroshi
Baatartsogt, Nemekhbayar
Kashiwakura, Yuji
Hayakawa, Morisada
Kamoshita, Nobuhiko
Nishimasu, Hiroshi
Nureki, Osamu
Kinai, Ei
Ohmori, Tsukasa
author_sort Hiramoto, Takafumi
collection PubMed
description The importance of genetic diagnosis for patients with hemophilia has been recently demonstrated. However, the pathological variant cannot be identified in some patients. Here, we aimed to identify the pathogenic intronic variant causing hemophilia A using induced pluripotent stem cells (iPSCs) from patients and genome editing. We analyzed siblings with moderate hemophilia A and without abnormalities in the F8 exon. Next-generation sequencing of the entire F8 revealed 23 common intron variants. Variant effect predictor software indicated that the deep intronic variant at c.5220-8563A>G (intron 14) might act as a splicing acceptor. We developed iPSCs from patients and used genome editing to insert the elongation factor 1α promoter to express F8 messenger RNA (mRNA). Then, we confirmed the existence of abnormal F8 mRNA derived from aberrant splicing, resulting in a premature terminal codon as well as a significant reduction in F8 mRNA in iPSCs due to nonsense-mediated RNA decay. Gene repair by genome editing recovered whole F8 mRNA expression. Introduction of the intron variant into human B-domain–deleted F8 complementary DNA suppressed factor VIII (FVIII) activity and produced abnormal FVIII lacking the light chain in HEK293 cells. Furthermore, genome editing of the intron variant restored FVIII production. In summary, we have directly proven that the deep intronic variant in F8 results in aberrant splicing, leading to abnormal mRNA and nonsense-mediated RNA decay. Additionally, genome editing targeting the variant restored F8 mRNA and FVIII production. Our approach could be useful not only for identifying causal variants but also for verifying the therapeutic effect of personalized genome editing.
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spelling pubmed-106905552023-12-02 Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A Hiramoto, Takafumi Inaba, Hiroshi Baatartsogt, Nemekhbayar Kashiwakura, Yuji Hayakawa, Morisada Kamoshita, Nobuhiko Nishimasu, Hiroshi Nureki, Osamu Kinai, Ei Ohmori, Tsukasa Blood Adv Thrombosis and Hemostasis The importance of genetic diagnosis for patients with hemophilia has been recently demonstrated. However, the pathological variant cannot be identified in some patients. Here, we aimed to identify the pathogenic intronic variant causing hemophilia A using induced pluripotent stem cells (iPSCs) from patients and genome editing. We analyzed siblings with moderate hemophilia A and without abnormalities in the F8 exon. Next-generation sequencing of the entire F8 revealed 23 common intron variants. Variant effect predictor software indicated that the deep intronic variant at c.5220-8563A>G (intron 14) might act as a splicing acceptor. We developed iPSCs from patients and used genome editing to insert the elongation factor 1α promoter to express F8 messenger RNA (mRNA). Then, we confirmed the existence of abnormal F8 mRNA derived from aberrant splicing, resulting in a premature terminal codon as well as a significant reduction in F8 mRNA in iPSCs due to nonsense-mediated RNA decay. Gene repair by genome editing recovered whole F8 mRNA expression. Introduction of the intron variant into human B-domain–deleted F8 complementary DNA suppressed factor VIII (FVIII) activity and produced abnormal FVIII lacking the light chain in HEK293 cells. Furthermore, genome editing of the intron variant restored FVIII production. In summary, we have directly proven that the deep intronic variant in F8 results in aberrant splicing, leading to abnormal mRNA and nonsense-mediated RNA decay. Additionally, genome editing targeting the variant restored F8 mRNA and FVIII production. Our approach could be useful not only for identifying causal variants but also for verifying the therapeutic effect of personalized genome editing. The American Society of Hematology 2023-10-05 /pmc/articles/PMC10690555/ /pubmed/37792826 http://dx.doi.org/10.1182/bloodadvances.2023010838 Text en © 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Thrombosis and Hemostasis
Hiramoto, Takafumi
Inaba, Hiroshi
Baatartsogt, Nemekhbayar
Kashiwakura, Yuji
Hayakawa, Morisada
Kamoshita, Nobuhiko
Nishimasu, Hiroshi
Nureki, Osamu
Kinai, Ei
Ohmori, Tsukasa
Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title_full Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title_fullStr Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title_full_unstemmed Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title_short Genome editing of patient-derived iPSCs identifies a deep intronic variant causing aberrant splicing in hemophilia A
title_sort genome editing of patient-derived ipscs identifies a deep intronic variant causing aberrant splicing in hemophilia a
topic Thrombosis and Hemostasis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10690555/
https://www.ncbi.nlm.nih.gov/pubmed/37792826
http://dx.doi.org/10.1182/bloodadvances.2023010838
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