Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.
Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.
Department of Pathology and Molecular Medicine and.
Lindsey
L
Hawke
Department of Medicine, Queen's University, Kingston, ON, Canada;
Mackenzie L
ML
Bowman
Department of Medicine, Foothills Medical Centre, Alberta Health Services, University of Calgary, Calgary, AB, Canada;
Man-Chiu
MC
Poon
Division of Hematology/Oncology, Health Sciences Centre, St. Johns, NL, Canada; and.
Mary-Frances
MF
Scully
Clinique d'Hematologie, Centre Hospitalier Universitaire St. Justine, Montréal, QC, Canada.
Georges-Etienne
GE
Rivard
Department of Medicine, Queen's University, Kingston, ON, Canada;
Paula D
PD
James
D005091:Exons; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D017354:Point Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D056725:von Willebrand Disease, Type 1 / Q000235:genetics* / Q000378:metabolism; D056729:von Willebrand Disease, Type 3 / Q000235:genetics* / Q000378:metabolism; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000235:genetics*
D022821:RNA Splice Sites; D012333:RNA, Messenger; D014841:von Willebrand Factor