A Case study of severe hemophilia B due to pathogenic C.676C>T missense mutation in the F9 gene

¹ Faculty of Medicine, Chulalongkorn University, 10330 Bangkok, Thailand
² Faculty of Biology, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia
³ Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, 55281 Yogyakarta, Indonesia

^* Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.

Abstract

The diversity and frequency of F9 gene mutations in Indonesia remain largely undocumented. In this study, long-read sequencing was performed using the PromethION24 platform developed by Oxford Nanopore Technologies plc (ONT). The research subject was an individual diagnosed with severe hemophilia B. Amplification of the F9 gene was carried out using the forward primer F (5′-CGGAGCCAAATGTTCTTTTC-3′) and the reverse primer R (5′-CACCAACTGCTCATCTCTGG-3′). The three-dimensional conformation of the mutant coagulation factor IX protein was modeled using SWISS-MODEL, and the resulting structure was examined with Discovery Studio and UCSF Chimera. Long-read sequencing analysis revealed a missense mutation (c.676C>T) in the F9 gene, which was subsequently validated through Sanger sequencing. This pathogenic substitution (p.Arg226Trp) results in an amino acid change at residue 226 within exon 6, located in the activation peptide region of the serine protease domain. Structural modeling indicated an alteration in the coil configuration at residue 226, which may interfere with atomic interactions and compromise the activation process of factor IX, thereby contributing to the clinical manifestations of hemophilia B.