Rapid molecular diagnosis of von Willebrand disease by direct sequencing. Detection of 12 novel putative mutations in VWF gene

Summary

Molecular diagnosis of von Willebrand Disease (VWD) is particularly complex. The autosomal von Willebrand factor gene (VWF) is large and highly polymorphic, and there is a highly homologous (>96%) partial pseudogene in chromosome 22. Because of these difficulties, application of molecular study of VWD to the clinical routine has been considerably delayed. Recent advances in sequencing technology and bioinformatics could convert direct sequencing of the complete VWF into a routine diagnostic tool for VWD, which is especially desirable in types 1 and 3. This study describes a highly optimized procedure in which all the coding and intronic flanking regions of VWF are amplified under identical thermocycling parameters in a ready-to-use PCR plate format. The entire sequencing procedure, from blood extraction to mutation identification, can be done within 24 hours, resulting in a simple, versatile, cost-effective strategy with little hands-on time requirements. To validate the method, we performed full-length VWF sequencing of 21 index cases including seven of each VWD type. A total of 30 VWF genetic variations were identified. Twelve of these sequence variations are new, including four missense, one nonsense, one insertion, the first insertion-deletion described in VWF, and 5 potential splice site mutations. To our knowledge, this is the fastest and most efficient protocol designed to date for complete sequencing of the VWF coding region in the molecular diagnosis of VWD.

Keywords

Automated DNA sequencing - molecular diagnosis - mutation analysis - von Willebrand disease - von Willebrand factor

• References

• 1 Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 1998; 67: 395-424.
• 2 Zhang Z, Lindstedt M, Blomback M. et al. Effects of the mutant von Willebrand factor gene in von Willebrand disease. Hum Genet 1995; 96: 388-394.
• 3 Castaman G, Eikenboom JC, Bertina RM. et al. Inconsistency of association between type 1 von Willebrand disease phenotype and genotype in families identified in an epidemiological investigation. Thromb Haemost 1999; 82: 1065-1070.
• 4 Wahlberg TB, Blomback M, Magnusson D. Influence of sex, blood group, secretor character, smoking habits, acetylsalicylic acid, oral contraceptives, fasting and general health state on blood coagulation variables in randomly selected young adults. Haemostasis 1984; 14: 312-319.
• 5 Abildgaard CF, Suzuki Z, Harrison J. et al. Serial studies in von Willebrand’s disease: variability versus “variants”. Blood 1980; 56: 712-716.
• 6 Blombäck M, Eneroth P, Andersson O. et al. On laboratory problems in diagnosing mild von Willebrand’s disease. Am J Hematol 1992; 40: 117-120.
• 7 Pruthi RK. A practical approach to genetic testing for von Willebrand disease. Mayo Clin Proc 2006; 81: 679-691.
• 8 Millar CM, Riddell AF, Brown SA. et al. Survival of von Willebrand factor released following DDAVP in a type 1 von Willebrand disease cohort: influence of glycosylation, proteolysis and gene mutations. Thromb Haemost 2008; 99: 916-924.
• 9 Eikenboom J, Van Marion V, Putter H. et al. Linkage analysis in families diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 VWD. J Thromb Haemost 2006; 4: 774-782.
• 10 James PD, Paterson AD, Notley C. et al. Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study. J Thromb Haemost 2006; 4: 783-792.
• 11 Kakela JK, Friedman KD, Haberichter SL. et al. Genetic mutations in von Willebrand disease identified by DHPLC and DNA sequence analysis. Mol Genet Metab 2006; 87: 262-271.
• 12 Michiels JJ, Berneman Z, Gadisseur A. et al. Classification and characterization of hereditary types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (unclassifiable) von Willebrand disease. Clin Appl Thromb Hemost 2006; 12: 397-420.
• 13 Cumming A, Grundy P, Keeney S. et al. An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease. Thromb Haemost 2006; 96: 630-641.
• 14 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.
• 15 James PD, Notley C, Hegadorn C. et al. The mutational spectrum of type 1 von Willebrand disease: Results from a Canadian cohort study. Blood 2007; 109: 145-154.
• 16 Sadler JE, Budde U, Eikenboom JC. et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006; 4: 2103-2114.
• 17 Vidal F, Julia A, Altisent C. et al. Von Willebrand gene tracking by single-tube automated fluorescent analysis of four short tandem repeat polymorphisms. Thromb Haemost 2005; 93: 976-981.
• 18 Wu X, Munroe DJ. EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 2006; 5: 119-120.
• 19 Gaucher C, DiÈval J, Mazurier C. Characterization of von Willebrand factor gene defects in two unrelated patients with type IIC von Willebrand disease. Blood 1994; 84: 1024-1030.
• 20 Houdayer C, Dehainault C, Mattler C. et al. Evaluation of in silico splice tools for decision-making in molecular diagnosis. Hum Mutat 2008; 29: 975-982.
• 21 Nitu-Whalley IC, Riddell A, Lee CA. et al. Identification of type 2 von Willebrand disease in previously diagnosed type 1 patients: a reappraisal using phenotypes, genotypes and molecular modelling. Thromb Haemost 2000; 84: 998-1004.
• 22 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.
• 23 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; 5: 722-728.
• 24 Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31: 3812-3814.
• 25 Tchernitchko D, Goossens M, Wajcman H. In silico prediction of the deleterious effect of a mutation: proceed with caution in clinical genetics. Clin Chem 2004; 50: 1974-1978.
• 26 Huizinga EG, Tsuji S, Romijn RA. et al. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain. Science 2002; 297: 1176-1179.
• 27 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.
• 28 Chen JM, Cooper DN, Chuzhanova N. et al. Gene conversion: mechanisms, evolution and human disease. Nat Rev Genet 2007; 8: 762-775.
• 29 Baronciani L, Federici AB, Castaman G. et al. Prevalence of type 2B ‘Malmo/New York’ von Willebrand disease in Italy: the role of von Willebrand factor gene conversion. J Thromb Haemost 2008; 6: 887-890.