Culpan D, Standen G, Wood N, Mazurier C, Gaucher C, Bidwell J
University of Bristol, Department of Transplantation Sciences, Cotham, U.K.
Br J Haematol. 1997 Mar;96(3):464-9. doi: 10.1046/j.1365-2141.1997.d01-2054.x.
Patients with type 2A von Willebrand's disease (VWD) commonly have missense mutations in the A2 domain of the von Willebrand factor (VWF) protein. This domain is encoded by the 3' region of VWF gene exon 2 8 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA-based diagnostic technique which enables at least 10 previously described mutations to be rapidly identified. The method involves polymerase chain reaction (PCR) amplification of two exon 28 gene segments between codons 717-788 and 803-893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletions contiguous to the sites of known mutations and, following hybridization, allele-specific heteroduplexes are generated which can be detected by simple polyacrylamide gel electrophoresis and ethidium bromide staining. A small panel of UHG molecules covering the 3' region of exon 28 should enable the large majority of type 2A VWD patients to be rapidly diagnosed by genotype.
2A型血管性血友病（VWD）患者的血管性血友病因子（VWF）蛋白A2结构域通常存在错义突变。该结构域由VWF基因外显子28的3'区域编码，绝大多数患者的杂合突变集中在密码子742至909之间的序列中。我们描述了一种基于DNA的诊断技术，该技术能够快速识别至少10种先前描述的突变。该方法包括分别对密码子717 - 788和803 - 893之间的两个外显子28基因片段进行聚合酶链反应（PCR）扩增。然后将每个片段与一个大小相似的合成互补DNA分子杂交，该分子称为通用异源双链生成器（UHG）。UHG在已知突变位点附近包含碱基缺失，杂交后会产生等位基因特异性异源双链，可通过简单的聚丙烯酰胺凝胶电泳和溴化乙锭染色进行检测。一小套覆盖外显子28 3'区域的UHG分子应能使绝大多数2A型VWD患者通过基因型快速得到诊断。
