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In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies

  • Behnaz PezeshkpoorEmail author
  • M. Gazorpak
  • A-C. Berkemeier
  • H. Singer
  • A. Pavlova
  • A. Biswas
  • J. Oldenburg
Original Article
  • 8 Downloads

Abstract

Haemophilia A (HA) is caused by a lack or reduced amount of factor VIII protein (FVIII). About one-third of patients with non-severe HA carrying specific missense mutations show discrepant results between FVIII activity (FVIII:C), measured by one-stage or chromogenic two-stage assays. The aim of this study was to elucidate the mechanism underlying the assay discrepancy in vitro and in silico. Thirteen missense mutations in the Factor 8-gene associated with discrepant results in patients were transiently expressed. FVIII:C of the mutations was determined using two one-stage assays (FVIII:C1st, FVIII:CBonn) and a two-stage chromogenic assay (FVIII:Cchr). Furthermore, thrombin generation test (TGT) and in silico analysis were performed to investigate the haemostatic potential as well as the structural impact of the variants, respectively. For the majority (9/13) of the analysed mutations, the discrepancy was confirmed. Moreover, we established a modified TGT protocol for in vitro characterization of FVIII. Hence, TGT parameters were significantly impaired in the group of variants associated with higher chromogenic values. Additionally, in silico analysis revealed the impact of the mutations on FVIII protein structure leading to assay discrepancy. Moreover, the data shows that also among one-stage clotting assays, assay discrepancy is observed. Our results show that for the majority of mutations, application of a global assay like TGT method could help to improve diagnosis or correct assessment of the severity of HA.

Keywords

FVIII:C One-stage assay Chromogenic assay Discrepancy Haemophilia A 

Notes

Author contributions

BP, JO and MG designed the study; MG and ACB performed research; BP and MG analysed the data; AB performed in silico analysis; AP and JO revised the manuscript; and BP and MG wrote the paper.

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.

Supplementary material

277_2019_3691_MOESM1_ESM.pdf (662 kb)
ESM 1 (PDF 661 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Experimental Hematology and Transfusion MedicineUniversity of BonnBonnGermany
  2. 2.Center for Rare Diseases Bonn (ZSEB)University Clinic BonnBonnGermany

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