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

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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.

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Author notes

  1. Behnaz Pezeshkpoor and M. Gazorpak contributed equally to this work.

Authors and Affiliations

  1. Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, Sigmund-Freud str. 25, 53127, Bonn, Germany

    Behnaz Pezeshkpoor, M. Gazorpak, A-C. Berkemeier, H. Singer, A. Pavlova, A. Biswas & J. Oldenburg

  2. Center for Rare Diseases Bonn (ZSEB), University Clinic Bonn, Bonn, Germany

    J. Oldenburg

Authors
  1. Behnaz Pezeshkpoor
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  2. M. Gazorpak
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  3. A-C. Berkemeier
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  4. H. Singer
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  5. A. Pavlova
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  6. A. Biswas
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  7. J. Oldenburg
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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.

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Correspondence to Behnaz Pezeshkpoor.

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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.

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Pezeshkpoor, B., Gazorpak, M., Berkemeier, AC. et al. In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies. Ann Hematol 98, 1855–1865 (2019). https://doi.org/10.1007/s00277-019-03691-1

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  • DOI: https://doi.org/10.1007/s00277-019-03691-1

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