Jiangsu Institute of Hematology, Soochow University, First Affiliated Hospital, Suzhou, China.
PLoS One. 2012;7(3):e33263. doi: 10.1371/journal.pone.0033263. Epub 2012 Mar 27.
von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.
血管性血友病因子（VWF）对于正常止血至关重要。VWF 基因突变导致血管性血友病（VWD）出血。本研究中，一名 9 岁男孩因异常出血、血浆 VWF 抗原和活性降低、血浆因子 VIII 活性降低和缺乏血浆高分子量（HMW）VWF 多聚体而被诊断为 2A 型 VWD。测序分析发现其 VWF 基因外显子 28 中存在 6bp 缺失，导致 VWF A2 结构域中缺失 D1529V1530 残基的突变体。该突变也存在于有异常出血的家族成员中，但不存在于 >60 例正常对照中。在转染的 HEK293 细胞中，重组 VWF ΔD1529V1530 蛋白在条件培养基中的水平显著降低（野生型（WT）VWF 的 42±4%，p<0.01）。培养基中的突变型 VWF 具有较少的 HMW 多聚体。相比之下，转染细胞中突变型 VWF 的细胞内水平明显高于 WT（174±29%，p<0.05），表明突变型 VWF 在内质网（ER）中滞留。在共转染实验中，突变型 VWF 降低了 WT VWF 从细胞中的分泌。通过免疫荧光染色，鉴定到突变型 VWF 在内质网中滞留。综上，我们鉴定了一个独特的 VWF 突变，该突变导致一个 2A 型 VWD 患者家族的出血表型。该突变损害了 VWF 通过 ER 的运输，从而阻止了 VWF 从细胞中分泌。我们的结果说明了 VWF 基因突变的多样性，这导致了 VWD 的广泛谱。
