Skip to main content

Advertisement

SpringerLink
Log in

Structural and functional exploration of three newly identified coagulation factor IX mutations in Chinese hemophilia B patients

  • Original Article
  • Published:
International Journal of Hematology Aims and scope Submit manuscript

Abstract

Aim

To structurally and functionally characterize three newly identified F9 missense mutations, C268Y, I316F, and G413V, in Chinese hemophilia B patients.

Methods

FIX mutants were expressed in vitro by transient transfection of Chinese hamster ovary (CHO) cells. One-stage activated partial thromboplastin time (APTT) based assay and enzyme-linked immunosorbent assay (ELISA) were used to measure the coagulation activity and antigen level of FIX in conditioned medium. Western blot analysis was also used to evaluate interference of the mutations with synthesis and secretion of FIX. A structural model of the FIX G413V mutant was constructed and structural disturbance caused by the mutation was determined by molecular dynamics simulations.

Results

Both C268Y and I316F impaired expression of FIX. However, the I316F mutant degraded quickly, whereas the C268Y mutant mostly accumulated intracellularly. The G413V mutant could be synthesized and secreted normally, but procoagulant activity was almost completely lost. This loss is likely mostly due to the impact on the catalytic residue cS195.

Conclusion

The three FIX mutations identified in Chinese hemophilia B patients either impaired the expression of FIX, as was seen with the I316F and C268Y mutants, or impaired the function of FIX, as was seen with the G413V mutant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Peyvandi F, Garagiola I, Young G. The past and future of haemophilia: diagnosis, treatments, and its complications. Lancet. 2016;388(10040):187–97.

    Article  PubMed  Google Scholar 

  2. Stonebraker JS, Bolton-Maggs PH, Michael Soucie J, Walker I, Brooker M. A study of variations in the reported haemophilia B prevalence around the world. Haemophilia. 2012;18(3):e91–4.

    Article  CAS  PubMed  Google Scholar 

  3. Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood. 2019;133(5):407–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Franchini M, Marano G, Pati I, Candura F, Profili S, Veropalumbo E, et al. Emicizumab for the treatment of haemophilia A: a narrative review. Blood Transfus. 2019;17(3):223–8.

    PubMed  PubMed Central  Google Scholar 

  5. Rallapalli PM, Kemball-Cook G, Tuddenham EG, Gomez K, Perkins SJ. An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B. J Thromb Haemost. 2013;11(7):1329–40.

    Article  CAS  PubMed  Google Scholar 

  6. Lu Z, Zhang H, Chen C, Wu W, Wei H. The novel mutation p.Asp315Tyr causes severe hemophilia B by impairing coagulation factor IX expression. Thromb Res. 2021;198:23–5.

    Article  CAS  PubMed  Google Scholar 

  7. Lu Y, Wu X, Dai J, Ding Q, Wu W, Wang X. The characteristics and spectrum of F9 mutations in Chinese sporadic haemophilia B pedigrees. Haemophilia. 2019;25(2):316–23.

    Article  CAS  PubMed  Google Scholar 

  8. Case DA, Cheatham TE 3rd, Darden T, Gohlke H, Luo R, Merz KM Jr, et al. The Amber biomolecular simulation programs. J Comput Chem. 2005;26(16):1668–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput. 2015;11(8):3696–713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Horn HW, Swope WC, Pitera JW, Madura JD, Dick TJ, Hura GL, et al. Development of an improved four-site water model for biomolecular simulations: TIP4P-Ew. J Chem Phys. 2004;120(20):9665–78.

    Article  CAS  PubMed  Google Scholar 

  11. Ryckaert J-P, Ciccotti G, Berendsen HJ. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys. 1977;23(3):327–41.

    Article  CAS  Google Scholar 

  12. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103(19):8577–93.

    Article  CAS  Google Scholar 

  13. Berendsen HJ, Postma JV, Van Gunsteren WF, DiNola A, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81(8):3684–90.

    Article  CAS  Google Scholar 

  14. Gotz AW, Williamson MJ, Xu D, Poole D, Le Grand S, Walker RC. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized born. J Chemical Theory Comput. 2012;8(5):1542–55.

    Article  CAS  Google Scholar 

  15. Roe DR, Cheatham TE 3rd. PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput. 2013;9(7):3084–95.

    Article  CAS  PubMed  Google Scholar 

  16. Lechtenberg BC, Murray-Rust TA, Johnson DJ, Adams TE, Krishnaswamy S, Camire RM, et al. Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis. Blood. 2013;122(16):2777–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stenson PD, Mort M, Ball EV, Chapman M, Evans K, Azevedo L, et al. The human gene mutation database (HGMD(®)): optimizing its use in a clinical diagnostic or research setting. Hum Genet. 2020;139(10):1197–207.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Crudele JM, Finn JD, Siner JI, Martin NB, Niemeyer GP, Zhou S, et al. AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood. 2015;125(10):1553–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mancuso ME, Mahlangu JN, Pipe SW. The changing treatment landscape in haemophilia: from standard half-life clotting factor concentrates to gene editing. Lancet. 2021;397(10274):630–40.

    Article  CAS  PubMed  Google Scholar 

  20. Goodeve AC. Hemophilia B: molecular pathogenesis and mutation analysis. J Thromb Haemost. 2015;13(7):1184–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Carter P, Wells JA. Dissecting the catalytic triad of a serine protease. Nature. 1988;332(6164):564–8.

    Article  CAS  PubMed  Google Scholar 

  22. Kraut J. Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem. 1977;46:331–58.

    Article  CAS  PubMed  Google Scholar 

  23. Vadivel K, Schreuder HA, Liesum A, Schmidt AE, Goldsmith G, Bajaj SP. Sodium-site in serine protease domain of human coagulation factor IXa: evidence from the crystal structure and molecular dynamics simulations study. J Thromb Haemost. 2019;17(4):574–84.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to patients participating in the study, and the technical staff of Shanghai Center for Clinical Laboratory for assisting with the experiments.

Funding

This study was supported by a grant from the General Program of National Natural Science Foundation of China (81670130, 81970127, 82070137, 81900139, 82270129 and 31770772).

Author information

Author notes

  1. Rui Wang and Shifeng Jiang have contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No.197 Ruijin Second Road, Shanghai, 200025, China

    Rui Wang, Xuefeng Wang & Wenman Wu

  2. College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Rui Wang, Xuefeng Wang & Wenman Wu

  3. Shanghai Center for Clinical Laboratory, Shanghai, China

    Rui Wang

  4. State Key Laboratory of Microbial Metabolism and Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Ye-Jiequan building 412, 800 Dongchuan Road, Shanghai, 200240, China

    Shifeng Jiang, Dongqing Wei & Qin Xu

  5. Collaborative Innovation Center of Hematology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Xuefeng Wang & Wenman Wu

Authors
  1. Rui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shifeng Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xuefeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dongqing Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qin Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenman Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RW: designed experiments and performed research. SJ: performed molecular modeling and MD simulations. QX: analyzed the results and wrote the manuscript of molecular modeling and MD simulations. WW supervision of the entire project and provision of critical feedback on the manuscript. XW and DW supervise the project. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Qin Xu or Wenman Wu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 49 KB)

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, R., Jiang, S., Wang, X. et al. Structural and functional exploration of three newly identified coagulation factor IX mutations in Chinese hemophilia B patients. Int J Hematol 118, 201–209 (2023). https://doi.org/10.1007/s12185-023-03616-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12185-023-03616-9

Keywords

Search

Navigation