title: The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation.
abstract: Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded "cysteine knot" domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara.
authors.forename: Caterina
authors.initials: C
authors.lastname: Casari
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.forename: Stefano
authors.initials: S
authors.lastname: Lancellotti
authors.forename: Elena
authors.initials: E
authors.lastname: Adinolfi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Casonato
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
mesh_terms: D002478:Cells, Cultured; D011992:Endosomes; D005799:Genes, Dominant; D006579:Heterozygote; D006801:Humans; D055503:Protein Multimerization; D034741:RNA, Small Interfering / Q000494:pharmacology; D017384:Sequence Deletion; D014842:von Willebrand Diseases / Q000235:genetics* / Q000473:pathology; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014841:von Willebrand Factor