Molecular Diagnostic of von Willebrand Disease
Molecular diagnosis of von Willebrand Disease (VWD) is particularly complex. Systematic identification of the responsible mutations has been hampered because of the large size and complex genomic organization of the VWF gene. The autosomal von Willebrand factor (VWF) gene is large and highly polymorphic, and there is a highly homologous (>96%) partial pseudogene in chromosome 22. Because of these difficulties, molecular study of VWD remains confined to basic investigation; application to the clinical routine has been considerably delayed. Thus, the development of optimized molecular approaches to support the clinical diagnosis is greatly welcomed. Moreover, in many families diagnosed with type 1 VWD, linkage studies have failed to show an association with the VWF gene.

Many type 2 mutations are located in exons 18 to 24 (type 2N) and in exon 28 (types 2A, 2B, and 2M), making them straightforward to DNA sequence analysis, and the molecular defect in many type 2 mutations has been described. However, types 1 and 3 VWD mutations are not restricted to specific exons; hence, study of these mutations, considering the predictable great mutational heterogeneity of VWD, requires analysis of all the essential VWF gene regions. Nonetheless, DNA sequencing of the complete VWF coding region has not yet become routine, and mutations in type 1 VWD are notoriously underrepresented in the International Society on Thrombosis and Hemostasis (ISTH) VWF database.


Numerous methods have been described to detect sequence variations in large genes, ranging from the direct sequencing approach to a variety of screening techniques (CITAS), which generally imply a considerable manipulation effort by highly qualified personnel. Whatever the specific procedure, nucleotide reading is the common final step in all these techniques. Comprehensive analysis of the VWF gene by denaturing high performance liquid chromatography (dHPLC) as well as direct sequencing are both effective techniques for mutation detection in VWD. Nevertheless, completion of the Human Genome Project together with advances in capillary electrophoresis platforms and sequencing reagents have made direct sequencing the gold standard approach for detecting sequencing variations, mutations, and single nucleotide polymorphisms (SNPs).

Advances in sequencing technology and bioinformatics could convert direct sequencing of the VWF gene into a routine diagnostic tool for VWD. This is especially desirable in types 1 and 3 in which mutations are not restricted to specific exons and require analysis of all the essential VWF gene regions.


Complete sequencing of the VWF gene in a large number of patients and relatives, including type 2 cases, will be helpful for establishing the actual contribution of each genetic variation to the disease. Thus, it is likely that procedures currently in use could be adapted to the new emergent massive sequencing platforms, also known as next-generation sequencing (NGS), which are up to 200 times less expensive and faster than traditional Sanger sequencing. These platforms will make possible high-throughput molecular characterization of a high number of patients and relatives to intensify study of the molecular pathophysiological mechanisms of VWD in correlation with molecular defects.