Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project

Abstract

The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study ‘Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)’ has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.

Update on Molecular Testing in von Willebrand Disease

“…NGS is based on simultaneous and parallel sequencing of thousands or millions of amplicons or captured regions allowing highthroughput molecular characterization of a high number of patients and relatives. Its potential in VWD diagnosis was revealed in a preliminary study 45 by means of an innovative protocol based on the amplification of all exons, intron-exon boundaries, and promoter regions using microfluidics technology and NGS [46][47][48][49][50] followed by the analysis of 556 patients from the "Molecular and clinical profile of VWD in Spain (PCM-EVW-ES)" project. This protocol 47 was also used in the molecular study of a smaller Portuguese cohort (92 patients).…”

“…2 The Spanish "PCM-EVW-ES" Project Experience This is an ongoing centralized study of VWD initiated in 2010 incorporating phenotypic and molecular VWF analysis for all recruited patients. [46][47][48][49][50] Analysis of VWF was conducted in 556 individuals by NGS, and multiplex ligation-dependent probe amplification in cases where no mutations were detected in individuals with a clear VWD phenotype. 15,28 In this regard, VWF (exons 1-52, adjacent intronic regions, and $1,300 bp of the promoter region) was analyzed by using a MiSeq Illumina Sequencer and a previously published protocol.…”

“…Occasionally, a dominant phenotype masked a second abnormality. [46][47][48][49][50] A total of 704 variants (237 different variants) along VWF were identified. One hundred and fifty-five of these had not been recorded previously in relevant international mutation databases.…”

“…Molecular testing is especially useful in type 2 VWD, being crucial to establish an accurate and definitive diagnosis particularly when some clinical assays are not available. 2,23,[46][47][48][49][50]71 This is the case of the multimeric analysis for types 2A and 2M classification, the VWF:FVIIIB to differentiate between a mild hemophilia A (HA) and a VWD type 2N, as well as the RIPA test, essential to discriminate between types 2A and 2B VWD, and to differentiate 2B VWD from PT-VWD. 71 Molecular testing may also be helpful for the distinction of type 2B VWD from immune thrombocytopenia (ITP), which is also probably true for PT-VWD.…”

“…The future of molecular studies in the setting of VWD will undoubtedly move toward universal methods such as the socalled clinical exome sequencing, whole exome sequencing Table 3 Main conclusions from the Spanish PCM-EVW-ES study [46][47][48][49][50] 1. The sensitivity and specificity of NGS compared with the conventional method were 99 and 98%, respectively.…”