ABNORMAL PIG A EXPRESSION IN GPI DEFICIENT BLOOD CELLS

GPI 缺陷血细胞中异常猪 A 的表达

• 批准号: 2445264
• 负责人: Russell E Ware
• 金额: $ 24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2445264
• 关键词: DNA repair; DNA replication; T lymphocyte; animal tissue; erythrocytes; flow cytometry; gene expression; gene mutation; hematopoietic stem cells; human subject; lymphocyte proliferation; membrane proteins; messenger RNA; molecular pathology; monoclonal antibody; mutant; nucleotides; paroxysmal nocturnal hemoglobinuria; phosphatidylinositols; protein sequence; tissue /cell culture; transfection

DESCRIPTION (Adapted from investigator's abstract): Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal acquired hematologic disorder which results from the incomplete enzymatic assembly of glycosyl- phosphatidylinositol (GPI) anchors used for surface protein attachment. All PNH patients tested have a complementation Class A defect; a cDNA (Piga) that repairs cell lines with the Class A defect has been cloned. Analysis of the Piga gene is therefore important for the understanding of PNH; genetic mutations of abnormal Piga expression could lead to defective assembly of GPI anchors and ultimately to PNH. The overall goal of the proposed research is an investigation of Piga gene mutations and PNH, with emphasis on its abnormal expression in GPI- deficient hematopoietic cells. The first hypothesis is that Piga gene mutations can be identified in all PNH patients. The investigators will characterize Piga expression in PNH patients at the mRNA and protein level, followed by nucleotide sequencing to identify Piga mutations. The investigators will then correlate each Piga gene mutation (genotype) with the clinical expression of GPI-linked surface proteins (phenotype). Three important questions are posed: (1) Do all patients with Type II (partially GPI-deficient) erythrocytes have missense Piga mutations?; (2) Do patients with both Type II and Type III (completely GPI- deficient) erythrocytes have two distinct mutations?; (3) Can cells gain or lose partial expression of GPI- like proteins in vivo? The second hypothesis is that PNH patients have infidelity of DNA replication and overall genetic instability which leads to mutations in the Piga gene. This hypothesis helps to explain why PNH is an acquired disorder, why the Piga mutations are mostly frameshift and may be multiple, and why patients with PNH have an increased risk of leukemia. The third hypothesis is that abnormal Piga expression leads to a cellular growth advantage. The investigators will focus on PNH lymphocytes, and study mechanisms by which GPI-deficient cells gain a growth advantage. The effects of abnormal Piga expression on lymphocyte growth will also be studied by transfection of Piga mutants and inhibition of normal Piga expression.

描述（改编自研究者摘要）：阵发性夜间 血红蛋白尿症（PNH）是一种克隆获得性血液病， 结果从糖基的不完全酶组装， 磷脂酰肌醇（GPI）锚用于表面蛋白附着。 所有接受检测的PNH患者都有一个互补A类缺陷;一个cDNA （Piga），修复具有A类缺陷的细胞系。 因此，对Piga基因的分析对于理解 PNH; Piga表达异常的基因突变可能导致 GPI锚钉组装缺陷，最终导致PNH。整体 这项研究的目的是调查Piga基因突变 和PNH，重点是其异常表达GPI缺陷 造血细胞第一个假设是Piga基因突变 可以在所有PNH患者中发现。调查人员将 从mRNA和蛋白质水平表征PNH患者中Piga的表达 水平，随后进行核苷酸测序以鉴定Piga突变。 然后，研究人员将每个Piga基因突变（基因型） 与GPI连接的表面蛋白的临床表达（表型）。 提出了三个重要问题：（1）是否所有II型患者 （部分GPI缺陷）红细胞有错义Piga突变？ (2)II型和III型患者（完全GPI- 缺陷）红细胞有两个不同的突变？(3)细胞能否获得 或在体内失去GPI样蛋白的部分表达？第二 假设PNH患者具有DNA复制的不忠实性， 总体遗传不稳定性，导致Piga基因突变。 这一假说有助于解释为什么PNH是一种获得性疾病，为什么 Piga突变大多是移码突变，可能是多重的，为什么 PNH患者患白血病的风险增加。第三 假设Piga表达异常导致细胞生长 优势研究人员将重点关注PNH淋巴细胞， GPI缺陷细胞获得生长优势的机制。的 异常Piga表达对淋巴细胞生长的影响也将被 通过转染Piga突变体和抑制正常Piga研究 表情