Doruelo A L, Haberichter S L, Christopherson P A, Boggio L N, Gupta S, Lentz S R, Shapiro A D, Montgomery R R, Flood V H
Department of Pediatrics, Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI, USA.
Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA.
J Thromb Haemost. 2017 Aug;15(8):1559-1566. doi: 10.1111/jth.13742. Epub 2017 Jun 23.
Essentials The pathophysiology of type 2M von Willebrand disease (VWD) is poorly understood. Sequence variations in type 2M VWD subjects were characterized. A high degree of clinical and laboratory variability exists within type 2M VWD variants. Some type 2M variants may share features of type 2A VWD.
Background von Willebrand factor (VWF) is a multimeric coagulation factor that tethers platelets to injured subendothelium. Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in VWF with preserved multimer distribution. Objectives Through the Zimmerman Program for the Molecular and Clinical Biology for VWD, five VWF sequence variations were studied in subjects diagnosed with type 2M VWD. Methods Bleeding phenotype was assessed using the ISTH bleeding assessment tool. Full-length VWF gene sequencing was performed for each subject. Each variant was placed into a recombinant VWF vector using site-directed mutagenesis and expressed in HEK293T cells as homozygous or heterozygous VWF. Variant expression, collagen binding and platelet GPIbα binding were studied through ELISA assays. Multimer analysis was performed by gel electrophoresis. Results Bleeding scores were elevated for all subjects except for the p.P1162L and p.R1374C variants. Although all had reduced VWF ristocetin cofactor activity/VWF antigen ratios on plasma testing, recombinant VWF did not show a classic type 2M phenotype for any of the five variants. Homozygous expression of variants p.D1283Y, p.R1349C, p.R1374C and p.I1453N was consistent with type 2A VWD, although all had normal expression as heterozygous recombinant VWF. Variant p.P1162L had normal VWF expression and function, consistent with the lack of bleeding symptoms. Conclusions Although originally classified as type 2M VWD, these homozygous recombinant VWF variants do not fulfill complete 2M VWD diagnostic criteria. A better classification schema and improved testing for putative type 2M variants is needed in order to effectively diagnose and treat affected patients.
要点 2M型血管性血友病（VWD）的病理生理学了解甚少。对2M型VWD患者的序列变异进行了特征分析。2M型VWD变异体存在高度的临床和实验室变异性。一些2M型变异体可能具有2A型VWD的特征。
背景 血管性血友病因子（VWF）是一种多聚体凝血因子，可将血小板与受损的内皮下组织相连。2M型血管性血友病（VWD）的特征是VWF存在定性缺陷，但其多聚体分布保持不变。目的 通过血管性血友病分子与临床生物学齐默尔曼计划，对诊断为2M型VWD的患者中的5种VWF序列变异进行了研究。方法 使用国际血栓与止血学会（ISTH）出血评估工具评估出血表型。对每个受试者进行全长VWF基因测序。使用定点诱变将每个变异体放入重组VWF载体中，并在HEK293T细胞中作为纯合或杂合VWF进行表达。通过酶联免疫吸附测定（ELISA）分析研究变异体表达、胶原结合和血小板糖蛋白Ibα（GPIbα）结合。通过凝胶电泳进行多聚体分析。结果 除p.P1162L和p.R1374C变异体外，所有受试者的出血评分均升高。虽然所有受试者血浆检测时VWF瑞斯托霉素辅因子活性/VWF抗原比值均降低，但重组VWF对这5种变异体中的任何一种均未表现出典型的2M型表型。p.D1283Y、p.R1349C、p.R1374C和p.I1453N变异体的纯合表达与2A型VWD一致，尽管所有变异体作为杂合重组VWF时表达均正常。p.P1162L变异体具有正常的VWF表达和功能，这与无出血症状一致。结论 尽管这些纯合重组VWF变异体最初被归类为2M型VWD，但并不符合完整的2M型VWD诊断标准。为了有效诊断和治疗受影响的患者，需要更好的分类方案和改进对假定的2M型变异体的检测。
