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

Abstract

SummaryMolecular 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.

High-throughput molecular diagnosis of von Willebrand disease by next generation sequencing methods

“…Consequently, genomic DNA in equimolar concentrations from every 5 patients, including one control patient per pool, was mixed and subjected to the conventional short PCR amplification described above. 4 After amplification, normalization, and pooling of amplicons, the process yielded 8 tubes, each representing 5 patients. Illumina's library construction protocol was performed for each of the 8 tubes, inserting 8 different pentanucleotide sequences as index tags (i.e.…”

“…4 All the coding Figure 1A). Normalization of amplicons was carried out by gel quantification performed with ImageJ (Version 1.43u), a public domain Java image processing program (http://rsbweb.nih.gov/ij/).…”

“…4 The sequences obtained were assembled and aligned against the consensus wild-type VWF sequence using SeqScape software (v2.7) (Applied Biosystems, Foster City, CA, USA).…”

“…Current sequencing technology has allowed only some expert laboratories to perform VWF sequencing in patients with VWD. [2][3][4] The cost of traditional sequencing based on the Sanger method remains a serious obstacle for most diagnostic laboratories. However, perspectives in the molecular diagnosis of monogenic diseases have radically changed over recent years with the introduction of new massively parallel next-generation sequencing (NGS) platforms that are considered alternatives or complementary to traditional sequencing.…”

“…[7][8][9] To take advantage of this new technology for molecular diagnosis of VWD, we designed a procedure for fast, low-cost characterization of a large number of samples, derived from a highly optimized method developed in our laboratory. 4 A proof-of-concept study was performed using an NGS platform. The results demonstrate a high performance DNA sequencing of VWF in a large number of VWD patients and their families, and establishes the basis for performing a large-scale sequencing study in an affected population.…”