OC 27.1 - Dominant negative effects of Gln50Arg mutation in the EGF-1 domain of factor IX causes severe Hemophilia B affecting factor IX replacement therapy

Sunday, June 23, 2024

14:45 – 15:00 ICT

Room: 210 A-D

Presenting Author(s)

T. T. van Duijl, T.T.

Post-doctoral researcher
Molecular Hematology, Sanquin Research, Asterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands

Co-Author(s)

KS

Kyra N. Smit

Clinical chemist
Department of Laboratory medicine, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
NF

Nadia Freato

Guest scientist
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
JM

Joost C.M. Meijers

Professor
Department of Experimental Vascular Medicine, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands.
Amsterdam, Noord-Holland, Netherlands
AK

Alette Kooiker

Master student
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
Marlene Beijlevelt, MSc

Advanced Nurse Practitioner
Amsterdam UMC
Amsterdam, Noord-Holland, Netherlands
HH

Henrike M. Hamer

Clinical chemist
Department of Laboratory medicine, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
Mv

Maartje van den biggelaar

Principal investigator
Molecular Hematology, Sanquin Research, Amsterdam, The Netherlands
Amsterdam, Noord-Holland, Netherlands
SG

Samantha C. Gouw, MD PhD

Pediatric hematologist
Department of Pediatric Hematology, Emma Children’s Hospital, Amsterdam UMC location University of Amsterdam, Amsterdam, the Netherlands
Amsterdam, Noord-Holland, Netherlands

Background: We investigated two brothers with Hemophilia B with residual factor IX (FIX) activity of 5%, who had frequent joint bleeding despite high-dose prophylactic FIX replacement therapy with high trough levels >15%.

Aims: To identify mechanisms for the suboptimal clinical response to prophylactic treatment by studying the biochemical defect of the Gln50Arg variant in F9.

Methods: Blood and DNA samples were drawn from two brothers with Hemophilia B. FIX activity and antigen and thrombin generation tests were performed with plasma collected before and after Alprolix replacement therapy. Plasma was in vitro spiked with Alprolix, Rixubis, Benefix or FEIBA. We determined platelet function, clotting factor activities and screened for FIX inhibitors. In addition, laboratory analyses were complemented with plasma proteomics and in-silico mutagenesis.

Results: Hemostatic screening revealed normal platelet aggregation and coagulation factor activities, except for FIX activity levels of 5 IU/dL in both brothers and a FXI activity of 39 IU/dL in one boy. FIX antigen concentrations were in the normal range in both boys and their mother, FIX inhibitor screening was negative (Figure 1). After bolus infusion of Alprolix, FIX activity in vivo recovery was within expected range, but thrombin generation indicated weak response to FIX substitution therapies. Bypassing activation of FIX with FEIBA resulted in enhanced thrombin generation. Gln50Arg resulted in the introduction of a positively charged residue in the calcium ion binding cavity of the EGF1 domain that is involved in the interaction with tissue factor (TF) for TF-FVIIa complex mediated FIX activation (Figure 2).

Conclusion(s): Our findings suggest that Gln50Arg is as a dominant-negative FIX proteoform that competes with FIX replacement therapy and impairs coagulation via the extrinsic pathway. These data argue for careful evaluation of qualitative defects in FIX in hemophilia B patients with a discordant activity/antigen ratio in relation to a poor response to replacement therapy.