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

The importance of genetic diagnosis for hemophilia patients 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 the patient 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 the patient and used genome editing to insert the EF1α promoter to express F8 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 BDD F8 cDNA suppressed 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.