Abstract
Background: Hemophilia A(HA) is an X-linked recessive coagulation disorder associated with factor VIII (F8) gene mutation. F8 gene mutation analysis is important for confirming the diagnosis, understanding genotype-phenotype correlations, and genetic counseling and family studies. According to the Korea Hemophilia Foundation (KHF) registry, a total of 1,835 patients with hemophilia A were recorded in Korea in 2024. Among them, 71% have severe disease, 15% had moderate disease, and 14% had mild disease. There is a lack of large-scale genomic data on HA in the Korean population. The purpose of this study is to identify causative mutations in the F8 gene in Korean patients with HA and to correlate mutation types with disease severity and the risk of inhibitor development.
Methods: HA was diagnosed based on factor VIII (FVIII) activity. Severe HA was defined as FVIII activity less than 1%, moderate HA as 1-5%, and mild HA as FVIII activity between 5% and 40%. The Bethesda assay was used to determine the presence of an inhibitor. A positive history of FVIII inhibitor was defined as >0.6 Bethesda units (BU) on two separate occasions, and a high-titre inhibitor was defined as > 5 BU. Molecular analysis of the F8 gene was performed using a combination of techniques, including long-distance inversion PCR (for intron 1 and 22), direct F8 gene sequencing, next-generation sequencing (NGS), and the multiplex ligation-dependent probe amplification (MLPA) method.
Results: A total of 926 Korean patients with severe HA, representing over 50% of patients with HA in Korea, were enrolled in this study from 15 medical centers across the country. Among them, 17 were female: 13 had mild HA, 2 had moderate disease, and the remaining 2 had severe disease. Pathogenic mutations were identified in 902 patients. Among them, 11 patients carried more than two types of pathogenic mutations, and in 24 patients, no causative mutation was identified. Mutation analysis revealed that missense mutations were the most common (33%), followed by intron 22 inversion (29%), frameshift mutations (15%), nonsense mutations (12%), large deletion (4%), and splicing mutations (3%). Disease severity was correlated with mutation type: most of patients with large deletions (100%), intron 22 or intron 1 inversions (99%), frameshift mutations (96%), and nonsense mutations (97%) had severe disease, whereas 84 % of those with splicing mutations and only 42% of patients with missense mutations had severe disease. A total of 181 patients (20%) developed FVIII inhibitors at least once, and high-titre inhibitors were observed in 86 patients (9.2%). The risk of developing FVIII inhibitors in patients with intron 22 inversion was 22%, highest among those with large deletions (58%) and lowest among those with missense mutations (12%). Interestingly, only 18% of patients with more than two pathogenic mutations, including large deletions, developed FVIII inhibitors.
Conclusions: This study is the first to provide large-scale genetic data on Korean patients with HA. Molecular diagnostic strategies, including Sanger sequencing, NGS, inversion analysis, and dosage analysis using MLPA, identified causative mutations in the majority of patients. The type of F8 gene mutation was a strong predictor of the clinical phenotype. Both disease severity and the risk of developing FVIII inhibitors were associated with mutation type. These findings are expected to enhance genetic counseling and medical care for HA patients in Korea.
