论文部分内容阅读
运用Southern blot,对来自10个家系的11例血小板无力症患者DNA进行限制性内切酶酶切,并与~(32)P标记的GPⅡb全长cDNA杂交,在一个家系(渐江黄岩)的2个同胞患者中发现了一TaqⅠ酶切的2.3kb异常区带。进一步与三个缩短了的GPⅡb cDNA探针杂交,证实在GPⅡb 基因第15～17外显子区域存在一杂合子突变。通过PCR扩增对该区域进行序列分析,表明GPⅡb 584位精氨酸密码子(CGA)突变为终止密码子(TGA)。Western blot检测表明患者血小板GPⅡb 与GPⅢa均缺乏,推测患者GPⅢa不能与缩短的GPⅡb形成复合物在巨核细胞内迅速降解。另外,还应用2.3kb异常基因片段作为特异的遗传标记对该家系的其他成员进行遗传追踪研究。Southernblot与PCR结果表明该异常基因片段来自母方,临床表现与GPⅡb/Ⅲa正常的同胞妹妹亦存在此缺陷基因,诊断为携带者;父亲则携带另一尚未发现的GPⅡb缺陷基因,推测患者为带有两种不同的GPⅡb缺陷基因的双重杂合子。
Southern blot was used to detect the DNA of 11 cases of platelet inattentiveness from 10 pedigrees and then hybridized with ~ (32) P-labeled full-length cDNA of GP Ⅱ b in a pedigree A TaqI digested 2.3kb abnormal band was found in 2 sibling patients. Further hybridization to three shortened GP IIb cDNA probes confirmed the presence of a heterozygous mutation in the region of exon 15-17 of the GP IIb gene. Sequence analysis of this region by PCR amplification indicated that codon 584 arginine codon (GPG) was mutated to a stop codon (TGA). Western blot showed that patients with platelet GP Ⅱ b and GP Ⅲ a lack of speculated that patients with GP Ⅲ a can not be shortened with GP Ⅱ b complex formed in the rapid degradation of megakaryocytes. In addition, 2.3kb abnormal gene fragments were also used as specific genetic markers of other members of the family genetic tracing study. Southernblot and PCR results showed that the abnormal gene fragment from the mother, the clinical manifestations and GP Ⅱ b / Ⅲ a normal siblings also exist this defect gene diagnosis of carriers; father is carrying another yet undetected GP Ⅱ b defective gene, presumably patients with Double heterozygotes for two different GP IIb-deficient genes.