Department of Biochemistry, Jichi Medical University School of Medicine, Shimotsuke, Tochigi, Japan.
Department of Laboratory Medicine, Tokyo Medical University, Tokyo, Japan.
Blood Adv. 2023 Nov 28;7(22):7017-7027. doi: 10.1182/bloodadvances.2023010838.
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.
最近已经证明了遗传诊断对于血友病患者的重要性。然而，在一些患者中，无法确定病理变异。在这里，我们旨在使用患者的诱导多能干细胞 (iPSC) 和基因组编辑来鉴定导致血友病 A 的致病内含子变异。我们分析了患有中度血友病 A 且 F8 外显子无异常的兄弟姐妹。对整个 F8 的下一代测序揭示了 23 个常见内含子变异。变体效应预测器软件表明，c.5220-8563A>G（内含子 14）的深内含子变异可能作为剪接受体起作用。我们从患者中开发了 iPSC，并使用基因组编辑插入延伸因子 1α 启动子以表达 F8 信使 RNA (mRNA)。然后，我们证实了异常 F8 mRNA 的存在，该异常 mRNA 源自异常剪接，导致过早的终止密码子以及由于无义介导的 RNA 降解，iPSC 中 F8 mRNA 的显著减少。基因组编辑的基因修复恢复了整个 F8 mRNA 的表达。基因组编辑引入内含子变异抑制了人 B 结构域缺失 F8 cDNA 的因子 VIII (FVIII) 活性，并在 HEK293 细胞中产生缺乏轻链的异常 FVIII。此外，基因组编辑修复了内含子变异，恢复了 FVIII 的产生。总之，我们已经直接证明 F8 中的深内含子变异导致异常剪接，导致异常 mRNA 和无义介导的 RNA 降解。此外，针对变异的基因组编辑恢复了 F8 mRNA 和 FVIII 的产生。我们的方法不仅可用于鉴定因果变异，还可用于验证个性化基因组编辑的治疗效果。
