Page 278 - Haematologica April 2020

Coagulation & its Disorders
Ferrata Storti Foundation
Haematologica 2020 Volume 105(4):1120-1128
Cryptic non-canonical splice site activation is part of the mechanism that abolishes multimer organization in the c.2269_2270del von Willebrand factor
Viviana Daidone,1 Eva Galletta,1 Luigi De Marco2 and Alessandra Casonato1
1University of Padua Medical School, Department of Medicine, First Chair of Internal Medicine, Padua and 2IRCCS, C.R.O. Aviano, Department of Translational Research, Stem Cells Unit, Aviano, Italy
ABSTRACT
We report a new pathogenic mechanism in von Willebrand disease involving the use of a non-canonical splicing site. The proband, carrying the homozygous c.2269_2270del mutation previously classified as a type 3 mutation, showed severely reduced plasma and platelet von Willebrand factor antigen levels and functions, and no factor VIII binding capacity. A particular von Willebrand factor multimer pattern emerged in plasma, characterized by the presence of only two oligomers: the dimer and an unusually large band, with no intermediate components. There were von Willebrand factor multimers in platelets, but each band ran more slowly than the normal counterpart. No anti-von Willebrand factor antibodies were detectable. The proband was classified as having severe type 1 von Willebrand disease. Seeking the relationship between pheno- type and genotype, we found the c.2269_2270del mutation associated with three different RNA: r.2269_2270del (RNAI), giving a truncated von Willebrand factor protein; r.[2269_2270del;2282_2288del] (RNAII), resulting from activation of a cryptic “AG” splicing site; and r.[2269_2270del;2281_2282insAG] (RNAIII), where the wild-type “AG” acceptor of exon 18 was retained due to the non-canonical 2279-2280 “CG” acceptor splicing site being used. The aberrant RNAII and RNAIII caused the alteration of the furin cleavage and binding sites, respectively, both resulting in a von Willebrand factor protein characterized by the persistence of von Willebrand factor propeptide, as confirmed by western blot analysis of the recombinant mutated von Willebrand factor molecules produced in vitro. Taken together, these findings explain the residual von Willebrand factor synthesis, slower-running multimers, and absent factor VIII binding capacity. The apparently pure gene null mutation c.2269_2270del pro- foundly alters von Willebrand factor gene splicing, inducing a complex RNA expression pattern.
Introduction
Von Willebrand factor (vWF) is a multimeric glycoprotein involved in the early stages of the hemostatic process. Synthesized by endothelial cells and megakary- ocytes, vWF has two main functions: (i) it guarantees platelet recruitment and aggre- gation at the site of vascular injury; and (ii) it transports factor VIII (FVIII) in the blood circulation.1-3 vWF function depends on its proper multimeric organization, compris- ing a set of discrete oligomers ranging from 500,000 to more than 20 million Daltons in size, the larger forms having the greater hemostatic capacity.4 vWF is synthesized as a monomer of 2,813 amino acids, including a signal peptide of 22 amino acids, a propeptide of 741 amino acids, and a mature molecule of 2,050 amino acids. After dimerization in the endoplasmic reticulum (ER), vWF is multimerized in the Golgi apparatus by an inter-chain disulfide bond forming between cysteine residues 1099 and 1142 of two vWF dimers, thanks to the contribution of vWF propeptide (vWFpp). The propeptide is then cleaved by furin, and the mature vWF molecule is either
Correspondence:
ALESSANDRA CASONATO
sandra.casonato@unipd.it
Received: April 2, 2019. Accepted: July 10, 2019. Pre-published: July 18, 2019.
doi:10.3324/haematol.2019.222679
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/4/1120
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