GPI ANCHOR DEFICIENCY IN HEMATOPOIESIS

阵发性睡眠性血红蛋白尿(PNH)是一种克隆性造血干细胞疾病，由 猪A基因的体细胞突变。PIG-A突变的生化后果是全球范围内的 糖基磷脂酰肌醇锚定蛋白(GPI-AP)。两个GPI-AP(CD55和CD59)很重要 补体调节蛋白的缺失解释了补体介导的血管内溶血 和血栓形成，发生在PNH患者中；然而，PNH细胞实现克隆的机制 支配地位还没有完全被理解。所有PNH患者都被证明携带PIG-A基因突变； 然而，在健康对照受试者中也可以发现罕见的PIG-A突变；因此，PIG-A基因的生物学相关性 健康对照中的PIG-A基因突变仍有待确定。这些研究的总体目标是 研究GPI锚定缺陷与PNH、正常和恶性淋巴造血细胞的相关性。 我们的初步数据表明，造血细胞可以获得PNH表型(没有细胞表面 GPI-AP)，无PIG-A突变。这些细胞中GPI-AP缺乏的机制是通过 GPI锚生物合成所需基因的转录沉默。我们还建立了一个PIGAnull 在可诱导启动子控制下的含有PIG-A基因的CD34细胞株。该细胞系起到了 为研究PNH的克隆显性机制提供了有价值的模型。我们的初步数据显示，PNH 细胞对T细胞介导的凋亡具有相对抵抗力，PNH细胞的生长优势是 在免疫攻击的背景下放大了。PNH细胞对T细胞介导的杀伤具有抵抗力的原因尚不清楚。 但我们的初步数据表明，这可能与通过膜筏和 产生促凋亡的第二信使神经酰胺的能力降低。具体来说，我们会： 1)探讨正常人GPI-AP缺陷细胞的相关性。 假设1.1：正常人淋巴细胞的GPI-AP缺陷可能是一种新的机制导致的 不涉及PICA突变。 假设1.2：在健康对照组中，HSPC的一个子集是GPI-Apio/neg，但不携带PIG-A 突变。 2)研究GPI锚定缺陷在细胞抗凋亡中的作用。 假设2.1：GPI-Apio/neg细胞通过全局抵抗凋亡来抵抗免疫攻击。 假设2.2：GPI-锚的表达在正常细胞对凋亡刺激的反应中发挥作用。 假设2.3：缺乏GPI-AP的表达扰乱了脂筏信号。

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder that is caused by a somatic mutation of the PIG-A gene. The biochemical consequence of PIG-A mutations is a global loss of glycosylphosphatidyl-inositol anchored proteins (GPI-AP). Two GPI-AP (CD55 and CD59) are important complement regulatory proteins, their absence explains the complement-mediated intravascular hemolysis and thrombosis and that occur in PNH patients; however, the mechanism by which PNH cells achieve clonal dominance is not completely understood. All PNH patients have been shown to harbor PIG-A mutations; however, rare PIG-A mutations can also be found in healthy control subjects; thus, the biologic relevance of PIG-A mutations in healthy controls remains to be determined. The overall objective of these studies is to study the relevance of GPI-anchor deficiency in PNH, normal, and malignant lymphohematopoietic cells. Our preliminary data demonstrates that hematopoietic cells can acquire a PNH phenotype (no cell surface GPI-AP) without PIG-A mutations. The mechanism of GPI-AP deficiency in these cells is through transcriptional silencing of genes required for GPI anchor biosynthesis. We have also established a PIGAnull CD34+ cell line with the PIG-A gene under the control of an inducible promoter. This cell line serves as a valuable model to study the mechanism of clonal dominance in PNH. Our preliminary data shows that PNH cells are relatively resistant to T cell mediatied apoptosis, and that the growth advantage of the PNH cells is amplified in the setting of an immune attack. Why PNH cells are resistant to T cell mediated killing is unclear, but our preliminary data suggest it may be related to perturbed signaling through membrane rafts and reduced ability to generate the proapoptotic second messenger, ceramide. Specifically, we will: 1) Investigate the relevance of GPI-AP deficient cells in normals. Hypothesis 1.1: GPI-AP deficiency in lymphocytes from normals can result from a novel mechanism that does not involve PICA mutations. Hypothesis 1.2: In healthy controls, a subset of HSPC in are GPI-APIo/neg, but do not harbor PIG-A mutations. 2) Study the role of GPI-anchor deficiency in cellular resistance to apoptosis. Hypothesis 2.1: GPI-APIo/neg cells are resistant to immune attack via global resistance to apoptosis. Hypothesis 2.2:GPI-anchor expression plays a role in the response of normal cells to apoptotic stimuli. Hypothesis 2.3: Lack of GPI-AP expression disrupts lipid raft signaling.