Takahashi H, Murata M, Moriki T, Anbo H, Furukawa T, Nikkuni K, Shibata A, Handa M, Kawai Y, Watanabe K
First Department of Internal Medicine, Niigata University School of Medicine, Japan.
Blood. 1995 Feb 1;85(3):727-33.
Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported. The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis. A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha. All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239. Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals. In allele-specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals. Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families. The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).
对先前报道的两个日本家族中4例血小板型血管性血友病（vWD）患者的基因组DNA进行了研究。采用聚合酶链反应（PCR），随后进行直接DNA序列分析，检测了血管性血友病因子（vWF）血小板受体成分血小板糖蛋白（GP）Ibα的整个编码区。在所有患者中均发现了一个单点突变，导致GPIbα第239位残基的甲硫氨酸（ATG）被缬氨酸（GTG）取代。所有患者的该突变均为杂合子，而未受影响的家庭成员在第239位残基均未发生氨基酸替代。由于核苷酸替代破坏了GPIbα上的一个Nla III限制性位点，因此对PCR产物用该酶进行消化；在所有受影响个体中均检测到了来自正常和突变等位基因的DNA片段。在等位基因特异性PCR中，使用含腺嘌呤或鸟嘌呤的引物从患者基因组DNA中扩增DNA，而只有含腺嘌呤的引物成功地从正常个体中扩增出DNA。将扩增的DNA克隆到噬菌体M13mp19中，随后进行DNA序列分析，证实了这些家族中的突变。在检测的正常个体中，GPIbα第239位残基不存在氨基酸替代，以及该替代与这两个家族和最近报道的一个家族中疾病表型表达的连锁关系，表明这种氨基酸变化是血小板型vWD的分子基础，并且该替代可能产生与先前报道的（GPIbα第233位残基的甘氨酸被缬氨酸取代）非常相似的表型。
