PB0268 - Severe hemophilia B caused by aberrant splicing induced by a deep intronic insertion of LINE-1

Background: With current conventional genetic analysis, about 1% of hemophilia B (HB) patients remain genetically unresolved. In such cases, the underlying causes might be attributed to deep intronic variants or complex rearrangements.

Aims: This study aims to investigate the pathogenic variant of a severe HB patient in whom no genetic variants were found using conventional genetic analysis.

Methods: Given the limited size of the F9 gene (less than 40,000 base pairs), large structural variant screening was carried out using LR-PCR combined with Sanger sequencing to further detect specific nucleic acid sequence changes. For candidate variants, in silico analysis was conducted to predict their effect on splicing. Finally, the potential pathogenic variant was identified using a minigene assay.

Results: A large structural variant was detected by LR-PCR, and Sanger sequencing revealed a deep intronic insertion of full-length LINE-1 in intron 3 of F9. According to in silico analysis, the insertion may utilize the donor splice site of the LINE-1 sequence and the cryptic acceptor splice site near the insertion site, resulting in the formation of pseudo-exons. Furthermore, the minigene assay revealed that the insertion led to an almost completely aberrant splicing pattern.

Conclusion(s): A novel deep intronic variant was found in a severe HB patient with negative results of conventional methods, and the nearly completely abnormal splicing pattern was implicated as the underlying cause of the severe phenotype through the minigene assay.