Comprehensive Genetic Analysis of Hemophilia A in a Chinese Cohort: Insights from Next-Generation and Long-Read Sequencing

Objective: Hemophilia A is a congenital bleeding disorder caused by F8 gene variants, leading to factor Ⅷ deficiency. Genetic testing has become essential for precise diagnosis and personalized treatment strategies. However, the detection of F8 gene variants remains challenging due to their heterogeneity. This study aims to investigate the diagnostic significance of next-generation sequencing (NGS) and long-read sequencing (LRS) in hemophilia A patients.
Methods: This study included 118 hemophilia A patients. A two-step strategy was adopted: NGS was first used to screen for F8 gene variants, and LRS or long-distance PCR (LD-PCR) was subsequently performed in cases with negative NGS results. LRS or LD-PCR was specifically employed to detect complex structural variants.
Results: NGS identified pathogenic F8 variants in 74 of 118 (62.7%) hemophilia A patients, while the remaining 44 (37.3%) tested negative. Among the 74 positive cases, 53 carried single-nucleotide variants (SNVs), including 7 novel SNVs; 9 had small deletions, 5 had large deletions, and 7 had duplications or insertions. Inversion analysis detected intron 22 inversions in 44 patients, with LD-PCR confirming 38 cases and LRS subclassifying the other 6 as type I. No type II inversions were detected.
Conclusion: In summary, this study offers novel strategies for hemophilia A genetic diagnosis, confirming that NGS alone is inadequate and requires complementary methods in specific cases. Our results highlight the utility of LRS in comprehensively identifying various F8 gene variants, particularly in precisely subtyping intron 22 inversions.

Keywords: Hemophilia A, F8 gene, Long-read sequencing, Genetic testing