Pseudoexon inclusion induced by three deep intronic variants in hemophilia B and correction achieved through an...

Although studies have identified deep intronic variants associated with hemophilia B in patients undiagnosed by conventional genetic testing, knowledge in this field remains limited. Long range-PCR of entire F9 gene was used to screen variants of three unrelated genetically unresolved severe hemophilia B patients. In silico analysis and minigene assay with two minigene plasmid construction methods were conducted to explore the effects of candidate deep intronic variants on splicing. Three novel putative pathogenic deep intronic variants (c.278-765_278-764ins6.1kb, c.392-903A>G and c.724-751T>G) and one variant of uncertain significance (c.723+2708del) in F9 were identified in three patients. All the three putative pathogenic variants created de novo donor splice site and utilized cryptic acceptor splice site to generate pseudoexon. We successfully developed antisense oligonucleotide-mediated exon-skipping correction strategies for the three identified putative pathogenic variants and the previously reported c.392-864T>G. However, the results obtained from minigene assay revealed certain discrepancies with the clinical phenotype. It is still challenging to accurately characterize the pathogenicity of deep intronic variants in vitro. Overall, our results highlight the complementary role of whole-gene sequencing of F9 to conventional genetic diagnosis. Furthermore, we provide insight into a potential therapeutic approach based on antisense oligonucleotide technology-mediated exon-skipping.