Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.

Centre de Référence Maladie de Willebrand Hématologie Hôpital Cardiologique Lille France; and

Department of Biomedical Sciences University of Hull Hull United Kingdom

Department of Diabetes Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Environmental Science and Biomedicine Kristianstad University Kristianstad Sweden

Einthoven Laboratory for Vascular and Regenerative Medicine Section of Thrombosis and Hemostasis Department of Internal Medicine Leiden University Medical Center Leiden The Netherlands

Haemostasis Research Group Department of Infection Immunity and Cardiovascular Disease University of Sheffield Sheffield United Kingdom

Hämostaseology Medilys Laborgesellschaft mbH Hamburg Germany

See more in PubMed

Sadler JE, Budde U, Eikenboom JCJ, et al. ; Working Party on von Willebrand Disease Classification . Update on the pathophysiology and classification of von Willebrand disease: a report of the subcommittee on von Willebrand factor. J Thromb Haemost. 2006;4(10):2103-2114. PubMed

Hampshire DJ, Goodeve AC. The molecular basis of von Willebrand disease: the under investigated, the unexpected and the overlooked. Haematologica. 2011;96(6):798-800. PubMed PMC

Goodeve AC. The genetic basis of von Willebrand disease. Blood Rev. 2010;24(3):123-134. PubMed

Acquila M, Bottini F, DI Duca M, Vijzelaar R, Molinari AC, Bicocchi MP. Multiplex ligation-dependent probe amplification to detect a large deletion within the von Willebrand gene. Haemophilia. 2009;15(6):1346-1348. PubMed

Cabrera N, Casaña P, Cid AR, et al. . First application of MLPA method in severe von Willebrand disease. Confirmation of a new large VWF gene deletion and identification of heterozygous carriers. Br J Haematol. 2011;152(2):240-242. PubMed

Yadegari H, Driesen J, Hass M, Budde U, Pavlova A, Oldenburg J. Large deletions identified in patients with von Willebrand disease using multiple ligation-dependent probe amplification. J Thromb Haemost. 2011;9(5):1083-1086. PubMed

Yadegari H, Driesen J, Pavlova A, Biswas A, Hertfelder H-J, Oldenburg J. Mutation distribution in the von Willebrand factor gene related to the different von Willebrand disease (VWD) types in a cohort of VWD patients. Thromb Haemost. 2012;108(4):662-671. PubMed

Ahmad F, Budde U, Jan R, et al. . Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients. Thromb Haemost. 2013;109(4):652-660. PubMed

Bowman M, Tuttle A, Notley C, et al. ; Association of Hemophilia Clinic Directors of Canada . The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles. J Thromb Haemost. 2013;11(3):512-520. PubMed PMC

Hampshire DJ, Abuzenadah AM, Cartwright A, et al. . Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort. Thromb Haemost. 2013;110(2):264-274. PubMed PMC

Kasatkar P, Shetty S, Ghosh K. Genetic heterogeneity in a large cohort of Indian type 3 von Willebrand disease patients. PLoS One. 2014;9(3):e92575. PubMed PMC

Obser T, Ledford-Kraemer M, Oyen F, et al. . Identification and characterization of the elusive mutation causing the historical von Willebrand disease type IIC Miami. J Thromb Haemost. 2016;14(9):1725-1735. PubMed PMC

Veyradier A, Boisseau P, Fressinaud E, et al. ; French Reference Center for von Willebrand disease . A laboratory phenotype/genotype correlation of 1167 French patients from 670 families with von Willebrand disease: a new epidemiologic picture. Medicine (Baltimore). 2016;95(11):e3038. PubMed PMC

Vangenechten I, Smejkal P, Zapletal O, et al. . Analysis of von Willebrand disease in the South Moravian population (Czech Republic): results from the BRNO-VWD study. Thromb Haemost. 2019;119(4):594-605. PubMed

Sutherland MS, Cumming AM, Bowman M, et al. . A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3. Blood. 2009;114(5):1091-1098. PubMed

Starke RD, Paschalaki KE, Dyer CEF, et al. . Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells. Blood. 2013;121(14):2773-2784. PubMed PMC

Casari C, Pinotti M, Lancellotti S, et al. . The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation. Blood. 2010;116(24):5371-5376. PubMed

Daidone V, Saga G, Barbon G, et al. . The p.R1819_C1948delinsS mutation makes von Willebrand factor ADAMTS13-resistant and reduces its collagen-binding capacity. Br J Haematol. 2015;170(4):564-573. PubMed

Goodeve A, Eikenboom J, Castaman G, et al. . Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD). Blood. 2007;109(1):112-121. PubMed

Caron C, Mazurier C, Goudemand J. Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents. Br J Haematol. 2002;117(3):716-718. PubMed

Federici AB, Canciani MT, Forza I, et al. . A sensitive ristocetin co-factor activity assay with recombinant glycoprotein Ibalpha for the diagnosis of patients with low von Willebrand factor levels. Haematologica. 2004;89(1):77-85. PubMed

Eikenboom J, Van Marion V, Putter H, et al. . Linkage analysis in families diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 VWD. J Thromb Haemost. 2006;4(4):774-782. PubMed

Tosetto A, Rodeghiero F, Castaman G, et al. . A quantitative analysis of bleeding symptoms in type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1 VWD). J Thromb Haemost. 2006;4(4):766-773. PubMed

Eikenboom J, Federici AB, Dirven RJ, et al. ; MCMDM-1VWD Study Group . VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 von Willebrand disease. Blood. 2013;121(12):2336-2339. PubMed PMC

Budde U, Schneppenheim R, Eikenboom J, et al. . Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD). J Thromb Haemost. 2008;6(5):762-771. PubMed

Schindelin J, Arganda-Carreras I, Frise E, et al. . Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682. PubMed PMC

Nielsen H. Predicting secretory proteins with SignalP. Methods Mol Biol. 2017;1611(1):59-73. PubMed

Lek M, Karczewski KJ, Minikel EV, et al. ; Exome Aggregation Consortium . Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536(7616):285-291. PubMed PMC

Verweij CL, Diergaarde PJ, Hart M, Pannekoek H. Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein considerably larger than the mature vWF subunit. EMBO J. 1986;5(8):1839-1847. PubMed PMC

Carvalho CMB, Lupski JR. Mechanisms underlying structural variant formation in genomic disorders. Nat Rev Genet. 2016;17(4):224-238. PubMed PMC

Pruss CM, Golder M, Bryant A, et al. . Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models. Blood. 2011;117(16):4358-4366. PubMed PMC

Legendre P, Navarrete A-M, Rayes J, et al. . Mutations in the A3 domain of von Willebrand factor inducing combined qualitative and quantitative defects in the protein. Blood. 2013;121(11):2135-2143. PubMed

Ewenstein BM, Inbal A, Pober JS, Handin RI. Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease. Blood. 1990;75(7):1466-1472. PubMed

Valentijn KM, Eikenboom J. Weibel-Palade bodies: a window to von Willebrand disease. J Thromb Haemost. 2013;11(4):581-592. PubMed

Gustafsson MGL, Shao L, Carlton PM, et al. . Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J. 2008;94(12):4957-4970. PubMed PMC

Torisu T, Torisu K, Lee IH, et al. . Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor. Nat Med. 2013;19(10):1281-1287. PubMed PMC

McKinnon TAJ, Goode EC, Birdsey GM, et al. . Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor. Blood. 2010;116(4):640-648. PubMed

Purvis AR, Sadler JE. A covalent oxidoreductase intermediate in propeptide-dependent von Willebrand factor multimerization. J Biol Chem. 2004;279(48):49982-49988. PubMed

von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985;184(1):99-105. PubMed

Groeneveld DJ, Wang J-W, Mourik MJ, et al. . Storage and secretion of naturally occurring von Willebrand factor A domain variants. Br J Haematol. 2014;167(4):529-540. PubMed

Castaman G, Lethagen S, Federici AB, et al. . Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD. Blood. 2008;111(7):3531-3539. PubMed

Huang R-H, Wang Y, Roth R, et al. . Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor. Proc Natl Acad Sci USA. 2008;105(2):482-487. PubMed PMC

Wang J-W, Groeneveld DJ, Cosemans G, et al. . Biogenesis of Weibel-Palade bodies in von Willebrand’s disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation. Haematologica. 2012;97(6):859-866. PubMed PMC

Lopes da Silva M, O’Connor MN, Kriston-Vizi J, et al. . Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells. J Cell Sci. 2016;129(10):2096-2105. PubMed PMC