Annals of Hematology

, Volume 97, Issue 6, pp 1061–1069 | Cite as

Variants in FIX propeptide associated with vitamin K antagonist hypersensitivity: functional analysis and additional data confirming the common founder mutations

  • Behnaz Pezeshkpoor
  • Katrin J. Czogalla
  • Michael Caspers
  • Ann-Cristin Berkemeier
  • Kerstin Liphardt
  • Suvoshree Ghosh
  • Marco Kellner
  • Silvia Ulrich
  • Anna Pavlova
  • Johannes Oldenburg
Original Article


One of the most common and unwanted side effects during oral anticoagulant therapy (OAT) is bleeding complications. In rare cases, vitamin K antagonist (VKA)-related bleeding events are associated with mutations affecting the F9 propeptide at amino acid position 37 due to a substitution of alanine to either valine or threonine. Based on our actual cohort of 18 patients, we update the knowledge on this rare phenotype and its origin. A founder mutation for both variants was reconfirmed by haplotype analysis of intronic and extragenic short tandem repeat (STR) polymorphisms with a higher prevalence in Switzerland than in other regions of Europe. Screening of healthy individuals for the presence of these F9 gene mutations did not identify any of these variants, thus proving the rare occurrence of this genotype. Furthermore, both variants were expressed in vitro and warfarin dose responses were studied. Our warfarin dose response analysis confirmed higher sensitivity of both variants to warfarin with the effect being more apparent for Ala37Thr. Thus, although F9 propeptide mutation-associated hypersensitivity to VKA is a rare phenomenon, awareness towards this bleeding phenotype is important to identify patients at risk.


FIX Propeptide F9 Thr37 F9 Val37 Hypersensitivity to VKA 


Author contributions

BP, KJC, and JO designed the study. MC, MK, AC, and KL performed the experiments. BP, KJC, MC, MK, SG, KL, AP, and JO analyzed and interpreted the data. SU revised the manuscript and provided the clinical data of the patients. BP, KJC, and MC wrote the manuscript.

Funding information

This work was supported by a grant from the Stiftung Hämotherapie-Forschung to Michael Caspers.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individuals participating in the study.

Supplementary material

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277_2018_3264_MOESM4_ESM.docx (50 kb)
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  1. 1.
    Palareti G, Leali N, Coccheri S, Poggi M, Manotti C, D’Angelo A, Pengo V, Erba N, Moia M, Ciavarella N, Devoto G, Berrettini M, Musolesi S (1996) Bleeding complications of oral anticoagulant treatment: an inception-cohort, prospective collaborative study (ISCOAT). Italian Study on Complications of Oral Anticoagulant Therapy Lancet 348(9025):423–428PubMedGoogle Scholar
  2. 2.
    Palareti G, Cosmi B (2009) Bleeding with anticoagulation therapy—who is at risk, and how best to identify such patients. Thromb Haemost 102(2):268–278. PubMedGoogle Scholar
  3. 3.
    Hansson K, Stenflo J (2005) Post-translational modifications in proteins involved in blood coagulation. J Thromb Haemost 3(12):2633–2648. CrossRefPubMedGoogle Scholar
  4. 4.
    Stenflo J (1999) Contributions of Gla and EGF-like domains to the function of vitamin K-dependent coagulation factors. Crit Rev Eukaryot Gene Expr 9(1):59–88PubMedGoogle Scholar
  5. 5.
    Berkner KL (2000) The vitamin K-dependent carboxylase. J Nutr 130(8):1877–1880. CrossRefPubMedGoogle Scholar
  6. 6.
    Berkner KL (2008) Vitamin K-dependent carboxylation. Vitam Horm 78:131–156. CrossRefPubMedGoogle Scholar
  7. 7.
    Berkner KL, Pudota BN (1998) Vitamin K-dependent carboxylation of the carboxylase. Proc Natl Acad Sci U S A 95(2):466–471. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Quenzel EM, Hertfelder HJ, Oldenburg J (1997) Severe bleeding in two patients due to increased sensitivity of factor IX activity to phenprocoumon therapy. Ann Hematol 74(6):265–268. CrossRefPubMedGoogle Scholar
  9. 9.
    Harbrecht U, Oldenburg J, Klein P, Weber D, Rockstroh J, Hanfland P (1998) Increased sensitivity of factor IX to phenprocoumon as a cause of bleeding in a patient with antiphospholipid antibody associated thrombosis. J Intern Med 243(1):73–77. CrossRefPubMedGoogle Scholar
  10. 10.
    Oldenburg J, Kriz K, Wuillemin WA, Maly FE, von Felten A, Siegemund A, Keeling DM, Baker P, Chu K, Konkle BA, Lammle B, Albert T (2001) Genetic predisposition to bleeding during oral anticoagulant therapy: evidence for common founder mutations (FIXVal-10 and FIXThr-10) and an independent CpG hotspot mutation (FIXThr-10). Thromb Haemost 85(3):454–457CrossRefPubMedGoogle Scholar
  11. 11.
    Ulrich S, Brand B, Speich R, Oldenburg J, Asmis L (2008) Congenital hypersensitivity to vitamin K antagonists due to FIX propeptide mutation at locus -10: a (not so) rare cause of bleeding under oral anticoagulant therapy in Switzerland. Swiss Med Wkly 138(7–8):100–107. 2008/07/smw-12022Google Scholar
  12. 12.
    Van den Veyver IB (2001) Skewed X inactivation in X-linked disorders. Semin Reprod Med 19(2):183–191. CrossRefPubMedGoogle Scholar
  13. 13.
    Chu K, Wu SM, Stanley T, Stafford DW, High KA (1996) A mutation in the propeptide of factor IX leads to warfarin sensitivity by a novel mechanism. J Clin Invest 98(7):1619–1625. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Oldenburg J, Quenzel EM, Harbrecht U, Fregin A, Kress W, Muller CR, Hertfelder HJ, Schwaab R, Brackmann HH, Hanfland P (1997) Missense mutations at ALA-10 in the factor IX propeptide: an insignificant variant in normal life but a decisive cause of bleeding during oral anticoagulant therapy. Br J Haematol 98(1):240–244. CrossRefPubMedGoogle Scholar
  15. 15.
    Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3):1215. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Goodeve AC, Reitsma PH, McVey JH Nomenclature of genetic variants in hemostasis. J Thromb Haemost 9(4):852–855Google Scholar
  17. 17.
    Fregin A, Czogalla KJ, Gansler J, Rost S, Taverna M, Watzka M, Bevans CG, Muller CR, Oldenburg J (2013) A new cell culture-based assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay. J Thromb Haemost 11(5):872–880. CrossRefPubMedGoogle Scholar
  18. 18.
    Bowen DJ (2002) Haemophilia A and haemophilia B: molecular insights. Mol Pathol 55(2):127–144. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Winship PR, Nichols CE, Chuansumrit A, Peake IR (1993) An MseI RFLP in the 5′ flanking region of the factor IX gene: its use for haemophilia B carrier detection in Caucasian and Thai populations. Br J Haematol 84(1):101–105. CrossRefPubMedGoogle Scholar
  20. 20.
    Winship PR, Anson DS, Rizza CR, Brownlee GG (1984) Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms. Nucleic Acids Res 12(23):8861–8872. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bowen DJ, Thomas P, Webb CE, Bignell P, Peake IR, Bloom AL (1991) Facile and rapid analysis of three DNA polymorphisms within the human factor IX gene using the polymerase chain reaction. Br J Haematol 77(4):559–560CrossRefPubMedGoogle Scholar
  22. 22.
    Winship PR, Rees DJ, Alkan M (1989) Detection of polymorphisms at cytosine phosphoguanadine dinucleotides and diagnosis of haemophilia B carriers. Lancet 1(8639):631–634CrossRefPubMedGoogle Scholar
  23. 23.
    Peake IR, Lillicrap DP, Boulyjenkov V, Briet E, Chan V, Ginter EK, Kraus EM, Ljung R, Mannucci PM, Nicolaides K et al (1993) Haemophilia: strategies for carrier detection and prenatal diagnosis. Bull World Health Organ 71(3–4):429–458PubMedPubMedCentralGoogle Scholar
  24. 24.
    Peake I (1990) Carrier detection and prenatal diagnosis of haemophilia. Present and future strategies. Ric Clin Lab 20(3):177–185PubMedGoogle Scholar
  25. 25.
    Bestmann L, Zuger M, Oldenburg J, Buhler D, Maly FE (2001) Coagulation factor IX propeptide mutations causing coumarin hypersensitivity: identification of female alanine-10 valine heterozygotes. Thromb Haemost 85(3):567–568CrossRefPubMedGoogle Scholar
  26. 26.
    Stanley TB, Jin DY, Lin PJ, Stafford DW (1999) The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase. J Biol Chem 274(24):16940–16944. CrossRefPubMedGoogle Scholar
  27. 27.
    Jorgensen MJ, Cantor AB, Furie BC, Brown CL, Shoemaker CB, Furie B (1987) Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell 48(2):185–191. CrossRefPubMedGoogle Scholar
  28. 28.
    Stanley TB, Humphries J, High KA, Stafford DW (1999) Amino acids responsible for reduced affinities of vitamin K-dependent propeptides for the carboxylase. Biochemistry 38(47):15681–15687. CrossRefPubMedGoogle Scholar
  29. 29.
    Weiss P, Soff GA, Halkin H, Seligsohn U (1987) Decline of proteins C and S and factors II, VII, IX and X during the initiation of warfarin therapy. Thromb Res 45(6):783–790. CrossRefPubMedGoogle Scholar
  30. 30.
    Kumar S, Haigh JR, Tate G, Boothby M, Joanes DN, Davies JA, Roberts BE, Feely MP (1990) Effect of warfarin on plasma concentrations of vitamin K dependent coagulation factors in patients with stable control and monitored compliance. Br J Haematol 74(1):82–85. CrossRefPubMedGoogle Scholar
  31. 31.
    Peters J, Luddington R, Brown K, Baglin C, Baglin T (1997) Should patients starting anticoagulant therapy be screened for missense mutations at Ala-10 in the factor IX propeptide? Br J Haematol 99(2):467–468PubMedGoogle Scholar
  32. 32.
    van der Meer FJ, Vos HL, Rosendaal FR (1999) No indication for APTT screening in patients on oral anticoagulant therapy. Thromb Haemost 81(3):364–366CrossRefPubMedGoogle Scholar
  33. 33.
    van der Heijden JF, Rekke B, Hutten BA, van der Meer FJ, Remkes MG, Vermeulen M, Buller HR, Reitsma PH (2004) Non-fatal major bleeding during treatment with vitamin K antagonists: influence of soluble thrombomodulin and mutations in the propeptide of coagulation factor IX. J Thromb Haemost 2(7):1104–1109. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Behnaz Pezeshkpoor
    • 1
    • 2
  • Katrin J. Czogalla
    • 1
    • 2
  • Michael Caspers
    • 1
    • 2
  • Ann-Cristin Berkemeier
    • 1
    • 2
  • Kerstin Liphardt
    • 1
    • 2
  • Suvoshree Ghosh
    • 1
    • 2
  • Marco Kellner
    • 3
  • Silvia Ulrich
    • 4
  • Anna Pavlova
    • 1
    • 2
  • Johannes Oldenburg
    • 1
    • 2
  1. 1.Institute of Experimental Haematology and Transfusion MedicineUniversity Clinic BonnBonnGermany
  2. 2.Center for Rare Diseases Bonn (ZSEB)University Clinic BonnBonnGermany
  3. 3.Zahnarztpraxis Dr. KellnerWürzburgGermany
  4. 4.Clinic of PulmonologyUniversity Hospital of ZürichZürichSwitzerland

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