Variant mapping using mass spectrometry–based proteotyping as a diagnostic tool in von Willebrand disease

Journal of Thrombosis and Haemostasis, vol. 22, pp. 1894–1908

Abstract

BACKGROUND: von Willebrand disease (VWD) is the most common inherited bleeding disorder, characterized by either partial or complete von Willebrand factor (VWF) deficiency or by the occurrence of VWF proteoforms of altered functionality. The gene encoding VWF is highly polymorphic, giving rise to a variety of proteoforms with varying plasma concentrations and clinical significance. OBJECTIVES: To address this complexity, we translated genomic variation in VWF to corresponding VWF proteoforms circulating in blood. METHODS: VWF was characterized in VWD patients (n = 64) participating in the Willebrand in the Netherlands study by conventional laboratory testing, DNA sequencing and complementary discovery, and targeted mass spectrometry-based plasma proteomic strategies. RESULTS: Unbiased plasma profiling combined with immune enrichment of VWF verified VWF and its binding partner factor VIII as key determinants of VWD and revealed a remarkable heterogeneity in VWF amino acid sequence coverage among patients. Subsequent VWF proteotyping enabled identification of both polymorphisms (eg, p.Thr789Ala, p.Gln852Arg, and p.Thr1381Ala), as well as pathogenic variants (n = 16) along with their corresponding canonical sequences. Targeted proteomics using stable isotope-labeled peptides confirmed unbiased proteotyping for 5 selected variants and suggested differential proteoform quantities in plasma. The variant-to-wild-type peptide ratio was determined in 6 type 2B patients heterozygous for p.Arg1306Trp, confirming the relatively low proteoform concentration of the pathogenic variant. The elevated VWF propeptide/VWF ratio indicated increased clearance of specific VWF proteoforms. CONCLUSION: This study highlights how VWF proteotyping from plasma could be the first step to bridge the gap between genotyping and functional testing in VWD.

Authors 14

1. Iris charlotte Kreft corresponding

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands. Electronic address: i.kreft@sanquin.nl

2. Tirsa T van Duijl

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

3. Calvin B. van Kwawegen

   Erasmus MC

   Affiliation as printed

   Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands

4. Ferdows Atiq

   Erasmus MC

   Affiliation as printed

   Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands

5. Winny Phan

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

6. Margo B. P. Schuller

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

7. Mariëtte Boon-Spijker

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

8. Carmen van der Zwaan

   Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

9. Alexander B. Meijer

   Utrecht University · Sanquin

   Affiliation as printed

   Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands; Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the Netherlands

10. Arie Johan Hoogendijk

    Sanquin

    Affiliation as printed

    Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands

11. Ruben Bierings

    Erasmus MC

    Affiliation as printed

    Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands

12. Jeroen C.J. Eikenboom Aachen Department of Internal Medicine Division of Thrombosis and Hemostasis

    Leiden University Medical Center

    Affiliation as printed

    Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands

13. Frank W.G. Leebeek

    Erasmus MC

    Affiliation as printed

    Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands

14. Maartje van den Biggelaar corresponding

    Sanquin

    Affiliation as printed

    Laboratory of Proteomics, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands. Electronic address: m.vandenbiggelaar@sanquin.nl