Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII AssaysFunding M.B. was supported by funds from Stockholm County Council.
Background Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them.
Methods The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential.
Results Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays.
Conclusion There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.
Keywordsclinical laboratory techniques - coagulation factor VIII - DNA mutational analysis - hemophilia A - diagnostic errors
A.S. analyzed and interpreted the data and wrote the manuscript. M.B. was involved in the recruitment of patients, interpretation of the data, and contributed to the writing of the manuscript. D.M. was involved in the recruitment of patients. L.O. and M.B. took part in designing the study. N.M.H.S. performed laboratory measurements and critically revised the manuscript. R.C. and M.H. were involved in the recruitment of patients and critically revised the manuscript. D.E.S. interpreted data and critically revised the manuscript. J.A. took part in designing the study, interpreted data, and critically revised the manuscript.
Received: 12 February 2020
Accepted: 02 July 2020
13 August 2020 (online)
© 2020. Thieme. All rights reserved.
Georg Thieme Verlag KG
Stuttgart · New York
- 1 Mannucci PM, Tuddenham EG. The hemophilias–from royal genes to gene therapy. N Engl J Med 2001; 344 (23) 1773-1779
- 2 White II GC, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. ; Factor VIII and Factor IX Subcommittee. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001; 85 (03) 560-560
- 3 Bowyer AE, Duncan EM, Antovic JP. Role of chromogenic assays in haemophilia A and B diagnosis. Haemophilia 2018; 24 (04) 578-583
- 4 Duncan EM, Duncan BM, Tunbridge LJ, Lloyd JV. Familial discrepancy between the one-stage and two-stage factor VIII methods in a subgroup of patients with haemophilia A. Br J Haematol 1994; 87 (04) 846-848
- 5 Parquet A, Boneu B, Bosser C. , et al. Clinical and biological survey of haemophilia A and B patients infused with French heat-treated concentrates. Nouv Rev Fr Hematol 1988; 30 (04) 205-207
- 6 Keeling DM, Sukhu K, Kemball-Cook G, Waseem N, Bagnall R, Lloyd JV. Diagnostic importance of the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954–>Leu substitution in the factor VIII A3 domain. Br J Haematol 1999; 105 (04) 1123-1126
- 7 Hathaway WE, Christian MJ, Jacobson LJ. Variant mild hemophilia - discrepancy in one stage and 2-stage factor-VIII assays. Thromb Haemost 1983; 50 (01) 357-357
- 8 Rudzki Z, Duncan EM, Casey GJ, Neumann M, Favaloro EJ, Lloyd JV. Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods. Br J Haematol 1996; 94 (02) 400-406
- 9 Mumford AD, Laffan M, O'Donnell J. , et al. A Tyr346–>Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy. Br J Haematol 2002; 118 (02) 589-594
- 10 Cid AR, Calabuig M, Cortina V. , et al. One-stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype. Haemophilia 2008; 14 (05) 1049-1054
- 11 Trossaërt M, Regnault V, Sigaud M, Boisseau P, Fressinaud E, Lecompte T. Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype. J Thromb Haemost 2008; 6 (03) 486-493
- 12 Bowyer AE, Van Veen JJ, Goodeve AC, Kitchen S, Makris M. Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A. Haematologica 2013; 98 (12) 1980-1987
- 13 Pavlova A, Delev D, Pezeshkpoor B, Müller J, Oldenburg J. Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost 2014; 111 (05) 851-861
- 14 Duncan EM, Rodgers SE, McRae SJ. Diagnostic testing for mild hemophilia a in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays. Semin Thromb Hemost 2013; 39 (03) 272-282
- 15 Castaldo G, D'Argenio V, Nardiello P. , et al. Haemophilia A: molecular insights. Clin Chem Lab Med 2007; 45 (04) 450-461
- 16 Potgieter JJ, Damgaard M, Hillarp A. One-stage vs. chromogenic assays in haemophilia A. Eur J Haematol 2015; 94 (Suppl. 77) 38-44
- 17 Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 1999; 93 (01) 176-183
- 18 Pipe SW, Saenko EL, Eickhorst AN, Kemball-Cook G, Kaufman RJ. Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa. Blood 2001; 97 (03) 685-691
- 19 Rodgers SE, Duncan EM, Barbulescu DM, Quinn DM, Lloyd JV. In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results. Br J Haematol 2007; 136 (01) 138-145
- 20 Antovic JP, Mikovic D, Elezovic I. , et al. Two global haemostatic assays as additional tools to monitor treatment in cases of haemophilia A. Thromb Haemost 2012; 108 (01) 21-31
- 21 Gilmore R, Harmon S, Gannon C, Byrne M, O'Donnell JS, Jenkins PV. Thrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancy. Haemophilia 2010; 16 (04) 671-674
- 22 Trossaert M, Lienhart A, Nougier C. , et al. Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements. Haemophilia 2014; 20 (04) 550-558
- 23 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 2019; 98 (08) 1855-1865
- 24 Ljungkvist M, Berndtsson M, Holmström M. , et al. Correlation to FVIII:C in two thrombin generation tests: TGA-CAT and INNOVANCE ETP. Mediterr J Hematol Infect Dis 2017; 9 (01) e2017064
- 25 Antovic A. Screening haemostasis–looking for global assays: the overall haemostasis potential (OHP) method–a possible tool for laboratory investigation of global haemostasis in both hypo- and hypercoagulable conditions. Curr Vasc Pharmacol 2008; 6 (03) 173-185
- 26 Trossaërt M, Boisseau P, Quemener A. , et al. Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity. J Thromb Haemost 2011; 9 (03) 524-530
- 27 Provaznikova D, Houskova K, Radovska A, Salaj P, Hrachovinova I. Novel mutations associated with a discrepancy between one-stage and chromogenic FVIII activity assays. Haemophilia 2015; 21 (04) e330-e332
- 28 Poulsen AL, Pedersen LH, Hvas AM, Poulsen LH, Thykjaer H, Ingerslev J. Assay discrepancy in mild haemophilia A: entire population study in a National Haemophilia Centre. Haemophilia 2009; 15 (01) 285-289
- 29 Schwaab R, Oldenburg J, Kemball-Cook G. , et al. Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family. Br J Haematol 2000; 109 (03) 523-528
- 30 Cutler JA, Mitchell MJ, Smith MP, Savidge GF. The identification and classification of 41 novel mutations in the factor VIII gene (F8C). Hum Mutat 2002; 19 (03) 274-278
- 31 Lannoy N, Abinet I, Bosmans A, Lambert C, Vermylen C, Hermans C. Computational and molecular approaches for predicting unreported causal missense mutations in Belgian patients with haemophilia A. Haemophilia 2012; 18 (03) e331-e339
- 32 Casaña P, Martínez F, Cabrera N, Cid AR, Aznar JA. Founder haplotype associated with the factor VIII Asp1241Glu polymorphism in a cohort of mild hemophilia A patients. J Thromb Haemost 2008; 6 (08) 1428-1430
- 33 van Moort I, Meijer P, Priem-Visser D. , et al. Analytical variation in factor VIII one-stage and chromogenic assays: Experiences from the ECAT external quality assessment programme. Haemophilia 2019; 25 (01) 162-169