Cloning of a GPI-anchor synthsis gene, PIG-A and characterization of its clinical significanece

1.阵发性睡眠性血红蛋白尿的分子基础。PNH的发生有两个关键事件：1)PIG-A在造血干细胞(S)中的体细胞突变和2)突变细胞的克隆性扩增。我们分析了63名患者，发现PIG-A全部异常，确定PIG-A是大多数患者(如果不是所有患者)的GPI缺陷的原因。突变在基因内广泛分布，表明是随机发生的。约20%的患者携带两个或更多突变克隆。因此，PNH是一种寡克隆性疾病。PIG-A位于X染色体上。这将是负责任基因一致性的基础。事实上，我们证明了PIG-F和-B是常染色体。为了测试PIG-A突变是否也会导致克隆性扩张，我们在男性胚胎干细胞中敲除了小鼠PIG-A，并使用这些GPI缺陷细胞产生了嵌合小鼠。6个在造血细胞中嵌合，具有0.5-4%的GPI缺陷红细胞。突变红细胞的比例在5到10个月内没有增加，这表明克隆扩增可能需要其他因素(S)。突变红细胞在12个月龄时在一个嵌合体中开始增加，17个月龄时达到30%。我们将产生更多的嵌合体来复制这个。2.GPI-锚合成基因的结构和功能。我们克隆了六个新的哺乳动物基因。在之前克隆的三个基因中，现在有九个基因是可用的。我们发现，参与第一步的PIG-A和-H蛋白在内质网的胞质侧形成一个复合体，而参与第三个甘露糖转移的PIG-B蛋白在内质网的管腔侧起作用。这些结果部分地阐明了GPI合成的膜拓扑结构。

1.Molecular basis of paroxysmal nocturnal hemoglobinuria (PNH). Two events are essential for PNH ; 1) Somatic mutation of PIG-A in a hematopoietic stem cell (s) and 2) Clonal expansion of the mutant cells. We analyzed 63 patients and found PIG-A abnormalities in all, establishing that PIG-A is responsible for GPI deficiency in most if not all patients. The mutations were distributed widely within the gene, indicating random occurrence. About 20% of patients bore two or more mutant clones. So, PNH is an oligoclonal disease. PIG-A is located on the X-chromosome. This would be a basis of the uniformity of the responsible gene. In fact, we demonstrated that PIG-F and -B are autosomal. To test if PIG-A mutation causes the clonal expansion as well, we knocked out mouse Pig-a in the male embryonic stem cells and generated chimeric mice using these GPI deficient cells. Six were chimeric in hematopoietic cells, having 0.5-4% of GPI deficient erythrocytes. Proportions of the mutant erythrocytes did not increase for five to ten months, suggesting that other factor (s) may be required for clonal expansion. The mutant erythrocytes began to increase in one chimera at twelve months of age and reached to 30% at seventeen months. We are going to generate more chimeras to reproduce this.2.Structure and function of GPI-anchor synthesis genes. We cloned six new mammalian genes. With three previously cloned ones, nine genes are now available. We found that PIG-A and -H proteins both involved in the first step form a complex on the cytoplasmic side of the ER,and that PIG-B protein that is involved in transfer of the third mannose functions on the luminal side of the ER.These results partially clarified membrane topology of GPI synthesis.