Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): comprehensive genetic analysis by next-generation sequencing of 480 patients

Abstract

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

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.…”