Pramoonjago P, Wanachiwanawin W, Chinprasertsuk S, Pattanapanyasat K, Takeda J, Kinoshita T
Research Institute for Microbial Diseases, Osaka University, Japan.
Southeast Asian J Trop Med Public Health. 1995;26 Suppl 1:322-4.
Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.
血细胞中糖基磷脂酰肌醇（GPI）锚的生物合成缺陷与阵发性夜间血红蛋白尿（PNH）的发病机制有关。异常克隆细胞出现在各种造血细胞谱系中，这表明PNH是多能造血干细胞阶段发生体细胞突变的结果。我们之前克隆了一个与PNH相关的基因。该基因被称为PIG-A（磷脂酰肌醇聚糖A类），参与GPI锚生物合成的早期步骤。对PNH患者的细胞系和粒细胞的研究表明，在目前已鉴定的所有病例中，PIG-A是体细胞突变的靶点。在本研究中，我们分析了14例泰国PNH患者粒细胞中的PIG-A异常情况。将PIG-A RNA进行逆转录，通过聚合酶链反应扩增编码区并克隆到质粒中。对由此获得的cDNA和基因组DNA进行突变检测增强凝胶电泳和测序分析。对PIG-A cDNA功能的评估基于转染后纠正PIG-A缺陷细胞系表型的能力。结果显示所有患者均存在PIG-A异常。3例患者因基因组DNA水平剪接位点的突变导致PIG-A转录本大小异常。11例患者的PIG-A转录本大小正常，但编码区存在包括小缺失和插入在内的突变。结合日本和英国患者的结果，PNH患者中的PIG-A体细胞突变是广泛分布于编码区和剪接位点的小突变。
