Hawke Lindsey, Bowman Mackenzie L, Poon Man-Chiu, Scully Mary-Frances, Rivard Georges-Etienne, James Paula D
Department of Pathology and Molecular Medicine and.
Department of Medicine, Queen's University, Kingston, ON, Canada;
Blood. 2016 Jul 28;128(4):584-93. doi: 10.1182/blood-2015-10-678052. Epub 2016 Jun 17.
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.
约10%的血管性血友病因子（VWF）基因突变被认为通过破坏共有剪接位点来改变信使核糖核酸（mRNA）剪接。这种机制可能未得到充分认识，且会受到共有剪接位点以外的突变影响。在VWF合成过程中，剪接异常会导致VWF出现质量缺陷或数量不足。本研究使用患者来源的血液生成内皮细胞（BOECs）和异源人胚肾（HEK 293(T)）细胞模型，研究了3个疑似存在剪接突变的血管性血友病（VWD）家系中的病理机制。外显子突变c.3538G>A导致3种框内剪接变体（23del、26del和23/26del），这些变体无法结合血小板、凝血因子VIII或胶原蛋白，通过共表达的野生型（WT）VWF的显性负性细胞内滞留以及增加向溶酶体的转运导致VWD。c.5842+1G>C突变的杂合个体产生外显子33跳跃、外显子33 - 34跳跃和WT VWF转录本。缺乏外显子33 - 34的VWF的致病性细胞内滞留导致他们患VWD。分支位点突变c.6599 - 20A>T通过外显子38跳跃转录本的mRNA降解导致1型VWD。暴露于剪切应力的BOECs中异常转录本与共表达的WT的剪接比率发生了改变。本研究提供了共有剪接位点以外的突变破坏剪接的证据，并引入了VWF剪接受内皮细胞剪切应力影响的概念。
