Characterisation of mutations and molecular studies of type 2 von Willebrand disease

Firdos Ahmad; Rifat Jan; Meganathan Kannan; Tobias Obser; Md Imtaiyaz Hassan; Florian Oyen; Ulrich Budde; Renu Saxena; Reinhard Schneppenheim

doi:10.1160/TH12-07-0475

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2013; 109(01): 39-46
DOI: 10.1160/TH12-07-0475

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Characterisation of mutations and molecular studies of type 2 von Willebrand disease

Authors

Firdos Ahmad

¹Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India

⁴Center for Translational Medicine, Temple University, Philadelphia, Pennsylvania, USA
Rifat Jan

¹Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
Meganathan Kannan

¹Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
Tobias Obser

³Department of Pediatric Haematology Oncology, University Medical Centre Hamburg Eppendorf, Hamburg, Germany
Md Imtaiyaz Hassan

²Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
Florian Oyen

³Department of Pediatric Haematology Oncology, University Medical Centre Hamburg Eppendorf, Hamburg, Germany
Ulrich Budde

⁵Medilys Laboratory, Coagulation, Asklepios Hospital Altona, Hamburg, Germany
Renu Saxena

¹Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
Reinhard Schneppenheim

³Department of Pediatric Haematology Oncology, University Medical Centre Hamburg Eppendorf, Hamburg, Germany

Abstract

Summary

Type 2 von Willebrand disease (VWD) is characterised by qualitative defects in von Willebrand factor (VWF). Exon 28 of the VWF gene is known to be a hot spot for type 2 VWD mutations. The goal of this study was to characterise the mutations in VWF exon 28 and understand the molecular basis of phenotypes through in vitro and in silico studies. Mutation screening was performed in 56 type 2 VWD patients through direct sequencing. Expression vectors for five mutations were transiently expressed in 293-EBNA cells to understand the mutations pathology. Furthermore, in silico structure analysis was performed for 13 missense mutations.A total of 16 including eight novel mutations were detected in 23 (41%) patients. Of these, 15 were missense (including seven V1439M, A1464P, M1495L, I1509V, R1527Q, N1635I and A1647D novel ones) and one was a novel gene conversion. Expression studies and characterisation of recombinant VWF suggested the loss of VWF function for mutants P1266Q, V1439M and N1635I and gain of function for mutant R1308C. No apparent defect was seen in mutant N1231S. In silico structure analysis suggested the probable gain or loss of hydrogen/van der Waals interactions in 10 mutant proteins. In conclusion, type 2A mutations and gene conversion were found to be a common cause of type 2 VWD. Expression studies suggest the mutations N1635I for type 2A(II), P1266Q and V1439M for type 2M, R1308C for type 2B VWD and N1231S as a non-causative variant. Moreover, in silico studies of the mutants show the probable cause of respective phenotypes.

Keywords

Type 2 VWD mutation - gene conversion - phenotype genotype - in vitro expression - molecular modelling

Full Text

References

References
1 Sadler JE, Mannucci PM, Berntorp E. et al. Impact, diagnosis and treatment of von Willebrand disease. Thromb Haemost 2000; 84: 160-174.
2 Werner EJ, Broxson EH, Tucker EL. et al. Prevalence of von Willebrand disease in children: a multiethnic study. J Pediatr 1993; 123: 893-898. DOI:10.1016/S0022-3476(05)80384-1.
3 Ahmad F, Kannan M, Ranjan R. et al. Inherited platelet function disorders versus other inherited bleeding disorders: an Indian overview. Thromb Res 2008; 121: 835-841. DOI:10.1016/j.thromres.2007.07.015.
4 Budde U. Diagnosis of von Willebrand disease subtypes: implications for treatment. Haemophilia 2008; 14 (Suppl. 05) 27-38. DOI:10.1111/j.1365-2516.2008.01849.x.
5 Emsley J, Cruz M, Handin R. et al. Crystal structure of the von Willebrand Factor A1 domain and implications for the binding of platelet glycoprotein Ib. J Biol Chem 1998; 273: 10396-10401. DOI:10.1074/jbc.273.17.10396.
6 Zhang Q, Zhou YF, Zhang CZ. et al. Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor. Proc Natl Acad Sci USA 2009; 106: 9226-9231. DOI:10.1073/pnas.0903679106.
7 Matsushita T, Sadler JE. Identification of amino acid residues essential for von Willebrand factor binding to platelet glycoprotein Ib. Charged-to-alanine scanning mutagenesis of the A1 domain of human von Willebrand factor. J Biol Chem 1995; 270: 13406-13414. DOI:10.1074/jbc.270.22.13406.
8 Stepanian A, Ribba AS, Lavergne JM. et al. A new mutation, S1285F, within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease. Br J Haematol 2003; 120: 643-651. DOI:10.1046/j.1365-2141.2003.04168.x.
9 Sutherland JJ, O’Brien LA, Lillicrap D. et al. Molecular modeling of the von Willebrand factor A2 Domain and the effects of associated type 2A von Willebrand disease mutations. J Mol Model 2004; 10: 259-270. DOI:10.1007/s00894-004-0194-9.
10 Kannan M, Ahmad F, Yadav BK. et al. Molecular defects in ITGA2B and ITGB3 genes in patients with Glanzmann thrombasthenia. J Thromb Haemost 2009; 07: 1878-1885. DOI:10.1111/j.1538-7836.2009.03579.x.
11 Ahmad F, Kannan M, Biswas A. et al. Impact of 789Ala/Ala genotype on quantitative type of von Willebrand disease. Ann Hematol 2009; 88: 479-483. DOI:10.1007/s00277-008-0623-4.
12 Ruggeri ZM, Zimmerman TS. The complex multimeric composition of factor VIII/von Willebrand factor. Blood 1981; 57: 1140-1143.
13 Schneppenheim R, Plendl H, Budde U. Luminography--an alternative assay for detection of von Willebrand factor multimers. Thromb Haemost 1988; 60: 133-136.
14 Budde U, Schneppenheim R, Eikenboom J. et al. Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD). J Thromb Haemost 2008; 06: 762-771. DOI:10.1111/j.1538-7836.2008.02945.x.
15 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a Laboratory Manual. Cold Spring Harbor NY; 1989. (Cold Spring Harbor Laboratory)
16 Gupta PK, Adamtziki E, Budde U. et al. Gene conversions are a common cause of von Willebrand disease. Br J Haematol 2005; 130: 752-758. DOI:10.1111/j.1365-2141.2005.05660.x.
17 Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; 60: 2126-2132. DOI:10.1107/S0907444904019158.
18 Zhou AQ, O’Hern CS, Regan L. Revisiting the Ramachandran plot from a new angle. Protein Sci 2011; 20: 1166-1171. DOI:10.1002/pro.644.
19 Lill MA, Danielson ML. Computer-aided drug design platform using PyMOL. J Comput Aided Mol Des 2011; 25: 13-19. DOI:10.1007/s10822-010-9395-8.
20 Cheng J, Randall A, Baldi P. Prediction of protein stability changes for single-site mutations using support vector machines. Proteins 2006; 62: 1125-1132. DOI:10.1002/prot.20810.
21 Schneppenheim R, Federici AB, Budde U. et al. Von Willebrand Disease type 2M „Vicenza“ in Italian and German patients: identification of the first candidate mutation (G3864A; R1205H) in 8 families. Thromb Haemost 2000; 83: 136-140.
22 Lavergne JM, De Paillette L, Bahnak BR. et al. Defects in type IIA von Willebrand disease: a cysteine 509 to arginine substitution in the mature von Willebrand factor disrupts a disulphide loop involved in the interaction with platelet glycoprotein Ib-IX. Br J Haematol 1992; 82: 66-72. DOI:10.1111/j.1365-2141.1992.tb04595.x.
23 O’Brien LA, Sutherland JJ, Weaver DF. et al. Theoretical structural explanation for Group I and Group II type 2A von Willebrand disease mutations. J Thromb Haemost 2005; 03: 796-797. DOI:10.1111/j.1538-7836.2005.01219.x.
24 Ruggeri ZM. Type IIB von Willebrand disease: a paradox explains how von Willebrand factor works. J Thromb Haemost 2004; 02: 2-6. DOI:10.1111/j.1538-7836.2003.00523.x.
25 Ruggeri ZM, Zimmerman TS. Variant von Willebrand’s disease: characterization of two subtypes by analysis of multimeric composition of factor VIII/von Willebrand factor in plasma and platelets. J Clin Invest 1980; 65: 1318-1325. DOI:10.1172/JCI109795.
26 Kroner PA, Friedman KD, Valentino LA. et al. Type 2 M von Willebrand disease due to linked P1266Q and V1279I substitutions in the von Willebrand factor A1 domain. J Thromb Haemost 2003; 01: OC351a.
27 Auton M, Sedlak E, Marek J. et al. Changes in thermodynamic stability of von Willebrand factor differentially affect the force-dependent binding to platelet GPIbalpha. Biophys J 2009; 97: 618-627. DOI:10.1016/j.bpj.2009.05.009.

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