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Thromb Haemost 2013; 109(04): 652-660
DOI: 10.1160/TH12-10-0737
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients

Firdos Ahmad
1   Department of Haematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
2   Center for Translational Medicine, School of Medicine Temple University, Philadelphia, Pennsylvania, USA
,
Ulrich Budde
3   Medilys Laboratory, Coagulation, Asklepios Hospital Altona, Hamburg, Germany
,
Rifat Jan
1   Department of Haematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
,
Florian Oyen
4   Department of Pediatric Haematology Oncology, University Medical Centre Hamburg Eppendorf, Hamburg, Germany
,
Meganathan Kannan
1   Department of Haematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
,
Renu Saxena
1   Department of Haematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
,
Reinhard Schneppenheim
4   Department of Pediatric Haematology Oncology, University Medical Centre Hamburg Eppendorf, Hamburg, Germany
› Author Affiliations Financial support: This work was supported by grants from Prof. Gunther Landbeck Stipendium (University Medical Center Hamburg, Germany) and Senior Research Fellowship (Indian Council of Medical Research, New Delhi) to Firdos Ahmad.
Further Information

Publication History

Received: 09 October 2012

Accepted after major revision: 17 January 2013

Publication Date:
22 November 2017 (online)

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Summary

Severe type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14–15, 80,830bp (−41510_657+7928A*del) and 2,231bp [1534–2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

Keywords

Type 3 VWD - Phenotype and genotype - VWF mutation - Large deletion - Non-homologous recombination

 

  • References

  • 1 Sadler JE, Mannucci PM, Berntorp E. et al. Impact, diagnosis and treatment of von Willebrand disease. Thromb Haemost 2000; 84: 160-174.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 3 Mannucci PM, Bloom AL, Larrieu MJ. et al. Atherosclerosis and von Willebrand factor. I. Prevalence of severe von Willebrand’s disease in western Europe and Israel. Br J Haematol 1984; 57: 163-169.
    CrossrefPubMedGoogle Scholar
  • 4 Gupta PK, Ahmed RP, Sazawal S. et al. Relatively high frequency of VWD types 3 and 2 in a cohort of Indian patients: the role of multimeric analysis. J Thromb Haemost 2005; 03: 1321-1322.
    CrossrefPubMedGoogle Scholar
  • 5 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.
    CrossrefPubMedGoogle Scholar
  • 6 Sadler JE. New concepts in von Willebrand disease. Annu Rev Med 2005; 56: 173-191.
    CrossrefPubMedGoogle Scholar
  • 7 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.
    CrossrefPubMedGoogle Scholar
  • 8 Schneppenheim R, Budde U, Obser T. et al. Expression and characterization of von Willebrand factor dimerization defects in different types of von Willebrand disease. Blood 2001; 97: 2059-2066.
    CrossrefPubMedGoogle Scholar
  • 9 Zhang ZP, Blomback M, Egberg N. et al. Characterization of the von Willebrand factor gene (VWF) in von Willebrand disease type III patients from 24 families of Swedish and Finnish origin. Genomics 1994; 21: 188-193.
    CrossrefPubMedGoogle Scholar
  • 10 Schneppenheim R, Krey S, Bergmann F. et al. Genetic heterogeneity of severe von Willebrand disease type III in the German population. Hum Genet 1994; 94: 640-652.
    PubMedGoogle Scholar
  • 11 Baronciani L, Cozzi G, Canciani MT. et al. Molecular defects in type 3 von Willebrand disease: updated results from 40 multiethnic patients. Blood Cells Mol Dis 2003; 30: 264-270.
    CrossrefPubMedGoogle Scholar
  • 12 Ginsburg D, Sadler JE. von Willebrand disease: a database of point mutations, insertions, and deletions. For the Consortium on von Willebrand Factor Mutations and Polymorphisms, and the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1993; 69: 177-184.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Eikenboom JC. Congenital von Willebrand disease type 3: clinical manifestations, pathophysiology and molecular biology. Best Pract Res Clin Haematol 2001; 14: 365-379.
    CrossrefPubMedGoogle Scholar
  • 14 Schneppenheim R, Castaman G, Federici AB. et al. A common 253-kb deletion involving VWF and TMEM16B in German and Italian patients with severe von Willebrand disease type 3. J Thromb Haemost 2007; 05: 722-728.
    CrossrefPubMedGoogle Scholar
  • 15 Xie F, Wang X, Cooper DN. et al. A novel Alu-mediated 61-kb deletion of the von Willebrand factor (VWF) gene whose breakpoints co-locate with putative matrix attachment regions. Blood Cells Mol Dis 2006; 36: 385-391.
    CrossrefPubMedGoogle Scholar
  • 16 Bernardi F, Patracchini P, Gemmati D. et al. In-frame deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease. Hum Mol Genet 1993; 02: 545-548.
    CrossrefPubMedGoogle Scholar
  • 17 Ngo KY, Glotz VT, Koziol JA. et al. Homozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease. Proc Natl Acad Sci USA 1988; 85: 2753-2757.
    CrossrefPubMedGoogle Scholar
  • 18 Mohl A, Boda Z, Jager R. et al. Common large partial VWF gene deletion does not cause alloantibody formation in the Hungarian type 3 von Willebrand disease population. J Thromb Haemost 2011; 09: 945-952.
    CrossrefPubMedGoogle Scholar
  • 19 Cabrera N, Casana P, Cid AR. et al. First application of MLPA method in severe von Willebrand disease. Confirmation of a new large VWF gene deletion and identification of heterozygous carriers. Br J Haematol 2011; 152: 240-242.
    CrossrefPubMedGoogle Scholar
  • 20 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.
    CrossrefPubMedGoogle Scholar
  • 21 Ahmad F, Jan R, Kannan M. et al. Characterisation of mutations and molecular studies of type 2 von Willebrand disease. Thromb Haemost 2013; 109: 39-46.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Ruggeri ZM, Zimmerman TS. The complex multimeric composition of factor VIII/von Willebrand factor. Blood 1981; 57: 1140-1143.
    PubMedGoogle Scholar
  • 23 Schneppenheim R, Plendl H, Budde U. Luminography--an alternative assay for detection of von Willebrand factor multimers. Thromb Haemost 1988; 60: 133-136.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 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.
    CrossrefPubMedGoogle Scholar
  • 25 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a Laboratory Manual. Cold Spring Harbor NY. 1989 Cold Spring Harbor Laboratory.
    PubMedGoogle Scholar
  • 26 Schneppenheim R, Brassard J, Krey S. et al. Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease. Proc Natl Acad Sci USA 1996; 93: 3581-3586.
    CrossrefPubMedGoogle Scholar
  • 27 Purvis AR, Gross J, Dang LT. et al. Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi. Proc Natl Acad Sci USA 2007; 104: 15647-15652.
    CrossrefPubMedGoogle Scholar
  • 28 Yadegari H, Driesen J, Hass M. et al. Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification. J Thromb Haemost 2011; 09: 1083-1086.
    CrossrefPubMedGoogle Scholar
  • 29 Adzhubei IA, Schmidt S, Peshkin L. et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 07: 248-249.
    CrossrefPubMedGoogle Scholar
  • 30 Cheng J, Randall A, Baldi P. Prediction of protein stability changes for single-site mutations using support vector machines. Proteins 2006; 62: 1125-1132.
    PubMedGoogle Scholar
  • 31 Mathe E, Olivier M, Kato S. et al. Computational approaches for predicting the biological effect of p53 missense mutations: a comparison of three sequence analysis based methods. Nucleic Acids Res 2006; 34: 1317-1325.
    CrossrefPubMedGoogle Scholar
  • 32 Sim NL, Kumar P, Hu J. et al. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res 2012; 40 (Web Server issue) W452-457.
    CrossrefPubMedGoogle Scholar
  • 33 Reese MG, Eeckman FH, Kulp D. et al. Improved splice site detection in Genie. J Comput Biol 1997; 04: 311-323.
    CrossrefPubMedGoogle Scholar
  • 34 Desmet FO, Hamroun D, Lalande M. et al. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 2009; 37: e67.
    CrossrefPubMedGoogle Scholar
  • 35 Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 2004; 11: 377-394.
    CrossrefPubMedGoogle Scholar
  • 36 Smith PJ, Zhang C, Wang J. et al. An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet 2006; 15: 2490-2508.
    CrossrefPubMedGoogle Scholar
  • 37 Hebsgaard SM, Korning PG, Tolstrup N. et al. Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res 1996; 24: 3439-3452.
    CrossrefPubMedGoogle Scholar
  • 38 Eikenboom J, Hilbert L, Ribba AS. et al. Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study. J Thromb Haemost 2009; 07: 1304-1312.
    CrossrefPubMedGoogle Scholar
  • 39 Hashemi Soteh M, Peake IR, Marsden L. et al. Mutational analysis of the von Willebrand factor gene in type 1 von Willebrand disease using conformation sensitive gel electrophoresis: a comparison of fluorescent and manual techniques. Haematologica 2007; 92: 550-553.
    CrossrefPubMedGoogle Scholar
  • 40 Goodeve A, Eikenboom J, Castaman G. et al. Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD). Blood 2007; 109: 112-121.
    CrossrefPubMedGoogle Scholar
  • 41 Schneppenheim R, Budde U, Batlle J. et al. Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D’-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study. Blood. 2012 120. Abstract 99
    PubMedGoogle Scholar
  • 42 Haberichter SL, Balistreri M, Christopherson P. et al. Assay of the von Willebrand factor (VWF) propeptide to identify patients with type 1 von Willebrand disease with decreased VWF survival. Blood 2006; 108: 3344-3351.
    CrossrefPubMedGoogle Scholar
  • 43 Gupta PK, Saxena R, Adamtziki E. et al. Genetic defects in von Willebrand disease type 3 in Indian and Greek patients. Blood Cells Mol Dis 2008; 41: 219-222.
    CrossrefPubMedGoogle Scholar
  • 44 Yadegari H, Driesen J, Pavlova A. et al. Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients. Thromb Haemost 2012; 108: 662-671.
    Article in Thieme ConnectPubMedGoogle Scholar