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

GPI-锚定合成基因 PIG-A 的克隆及其临床意义

• 批准号: 05102006
• 负责人: KINOSHITA Taroh
• 金额: $ 104.96万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Specially Promoted Research
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05102006/
• 关键词: GPI Anchor; X-chromosome; Somatic Mutation; Complement; Hematopoietic Stem Cell; Glycogenes; Genetic Disease; Paroxysmal Nocturnal Hemoglobinuria; ×染色体

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.

1.阵发性夜间血红蛋白尿的分子基础。两个事件对PNH至关重要；1)猪- a在造血干细胞中的体细胞突变和2)突变细胞的克隆扩增。我们分析了63例患者，发现所有患者都有猪- a异常，这表明猪- a是大多数（如果不是全部）患者GPI缺乏的原因。突变在基因内广泛分布，表明突变是随机发生的。大约20%的患者有两个或更多的突变克隆。因此，PNH是一种寡克隆疾病。猪- a位于x染色体上。这将是负责基因一致性的基础。事实上，我们证明了猪- f和-B是常染色体。为了测试猪-a突变是否也会导致克隆扩增，我们敲除了雄性胚胎干细胞中的小鼠猪-a，并用这些缺乏GPI的细胞产生嵌合小鼠。6例在造血细胞中嵌合，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。在一个嵌合体中，突变红细胞在12月龄时开始增加，在17月龄时达到30%。我们将产生更多的嵌合体来复制这个。2。gpi锚定合成基因的结构与功能。我们克隆了六个新的哺乳动物基因。加上之前克隆的3个基因，现在有9个基因可用。我们发现，参与第一步的PIG-A和-H蛋白都在内质网的细胞质侧形成复合物，而参与第三甘露糖转移的PIG-B蛋白在内质网的管腔侧起作用。这些结果部分阐明了GPI合成的膜拓扑结构。

项目成果

Kinoshita, T.: "Expression cloning of genes for GPI-anchor biosynthesis." Brazilian J. Med. Biol. Res.,. 27. 127-132 (1994)

Kinoshita, T.：“GPI 锚定生物合成基因的表达克隆。”

Kinoshita, T., N.Inoue and J.Takeda.: "Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria." Adv.Immunol.60. 57-103 (1995)

Kinoshita, T.、N.Inoue 和 J.Takeda.：“糖基磷脂酰肌醇锚合成缺陷和阵发性睡眠性血红蛋白尿。”

Ohishi, K., Y.Kurimoto, N.Inoue, Y.Endo, J.Takeda and T.Kinoshita.: "Cloning and Characterization of the murine GPI-anchor synthesis gene Pigf, a homologue of the human PIGF gene." Genomics. (in press).

Ohishi, K.、Y.Kurimoto、N.Inoue、Y.Endo、J.Takeda 和 T.Kinoshita.：“鼠 GPI 锚定合成基因 Pigf（人类 PIGF 基因的同源物）的克隆和表征。”

Inoue,N.,T.Kinoshita,T.Orii,and J.Takeda.: "Cloning of a human gene,PIG-F,a component of glycosylphosphatidylinositol-anchor biosynthesis,by a novel expression cloning strategy." Journal of Biological Chemistry. 268. 6882-6885 (1993)

Inoue, N.、T.Kinoshita、T.Orii 和 J.Takeda.：“通过一种新的表达克隆策略克隆人类基因 PIG-F，它是糖基磷脂酰肌醇锚定生物合成的一个组成部分。”

Bessler M.,P.J.Mason,P.Hillmen,T.Miyata,N.Yamada,J.Takeda,L.Luzzatto and T.Kinoshita.: "Paroxysmal nocturnal haemoglobinuria(PNH)is caused by somatic mutations in the PIG-A gene." EMBO Journal. 13. 110-117 (1994)

Bessler M.、P.J.Mason、P.Hillmen、T.Miyata、N.Yamada、J.Takeda、L.Luzzatto 和 T.Kinoshita.：“阵发性睡眠性血红蛋白尿 (PNH) 是由 PIG-A 基因的体细胞突变引起的