Determination of GPI-anchored protein expression in bone marrows of normal individuals and patients with paroxysmal nocturnal hemoglobinuria (PNH)

正常人和阵发性睡眠性血红蛋白尿症 (PNH) 患者骨髓中 GPI 锚定蛋白表达的测定

• 批准号: 10022067
• 负责人: Raul Braylan
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10022067
• 关键词: Antibodies; Aplastic Anemia; Apoptosis; Aspirate substance; B-Lymphocytes; Binding; Biological Assay; Blood Cells; Bone Marrow; Bone Marrow Cells; Bone Marrow Diseases; CD14 gene; Cell Maturation; Cell membrane; Cell surface; Cells; Clinical; Complement; Data; Defect; Detection; Diagnosis; Diagnostic; Disease; Dysmyelopoietic Syndromes; Elements; Erythrocytes; Evaluation; FCGR3B gene; Failure; Flow Cytometry; GPI Membrane Anchors; Gene Mutation; Genes; Glycoproteins; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemolysis; Hemolytic Anemia; Human Pathology; ITGAM gene; Immune; Individual; Inositol; International; Jordan; Label; Laboratories; Leukocytes; Lymphocyte; Lymphoid; Marrow; Mediating; Methods; Mutate; Normal Cell; PI-Glycan; PTPRC gene; Pancytopenia; Patients; Play; Population; Proteins; Role; Sampling; Sensitivity and Specificity; Serum; Somatic Mutation; Spain; Specimen; Stem cells; Structure; Suggestion; Surface; Testing; Therapeutic; Variant; aerolysin; alanine aminopeptidase; base; bone marrow failure syndrome; cell type; comparative; cytopenia; follow-up; gene product; granulocyte; individual patient; link protein; longitudinal analysis; meetings; monocyte; neutrophil; peripheral blood; posters; protein expression; rat Piga protein; symposium; volunteer

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) is an acquired disorder of the hematopoietic stem cell. PNH is the result of a somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene. The product of this gene is crucial in the initial stage of synthesis of the glycosylphosphatidyl-inositol (GPI) anchor, a structure which attaches numerous important proteins to cell membranes. Because PIG-A gene mutation originates in a multipotent hematopoietic stem cell capable of differentiation, all progenitor cells deriving from the mutated HSC harbor the GPI defect, and have a complete or partial loss of expression of all GPI-anchored proteins (GPI-APs) on the cell surface. Some of these GPI-anchored proteins, such as CD55 and CD59, play important roles in the control of complement. Activated serum complement is known to play a crucial role in the hemolytic anemia. In normal individuals, red blood cells are protected from complement-mediated destruction, whereas those deficient in anchored proteins CD55 and CD59 undergo hemolysis, predominantly intravascular. Expansion of PNH type clones is commonly associated with idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS), conditions of immune-mediated failure of normal hematopoiesis. In was shown that in AA, up to 60% of patients harbor PNH-type cells. In MDS, approximately 15% of patients harbor PNH-type cells. Diagnosis and follow-up of PNH patients is currently done by flow cytometry (FC)-based assays involving evaluation of deficient expression of GPI-APs in PB cells. Besides testing the expression of the two GPI-APs CD55 and CD59 on both red cells and neutrophils, a newer and more sensitive approach for PNH testing emerged, involving fluorescently labeled inactive variant of the protein aerolysin (FLAER). PB red and white cells have been extensively studied in PNH. Less has been done to assess the abnormalities of BM populations in these patients. BM specimens are generally considered less suitable than PB owing to variable expression of GPI-APs during the various stages of hematopoietic cell maturation and for that reason, are rarely studied for PNH evaluation. However, BM aspirates from patients with unexplained cytopenias, including bone marrow failure syndromes, are frequently submitted to laboratories for general diagnostic purposes. Specific goals of this project: 1. Analysis of fluorescently labeled aerolysin (FLAER) binding to bone marrow (BM) cell populations in normal volunteers and patients with BM failure conditions such as aplastic anemia (AA) or myelodysplastic syndrome (MDS) who have detectable PNH in the peripheral blood (PB). 2. Comparison of PNH clone size in BM and PB within the same cell types. 3. Detection of PNH in BM by combinations of antibodies routinely used for diagnosis of BM disorders in clinical laboratories. 4. Assessment of proliferation and apoptosis of PNH cells and normal cells in the same BM samples. Methods: Flow cytometry analysis of FLAER binding, and expression of CD55/CD59 in conjunction with combinations of other antibodies in BM cells. Proliferation and apoptosis assays by flow cytometry. Results: FLAER binds to all normal BM cells, except for erythrocytes, which bind CD55 and CD59. Within each lineage except for RBCs, FLAER binding increases with cell maturation and reaches the highest level on mature elements. In PNH, the defect (clone size) is smaller in lymphocytes than in other BM cells. PNH clone sizes in BM and PB are very similar, making BM equally suitable for PNH detection and quantitation. With combination of antibodies routinely used for general diagnosis (anti CD45, CD13, CD11b, CD64 and the anchored proteins CD16 and CD14) PNH clones can also be detected in BM mature neutrophils and monocytes with high specificity and sensitivity. Currently, data continue to be analyzed to answer the question of predicting variation in clone size (which has therapeutic implications)based on longitudinal analysis of clone sizes at prior values. PRESENTATIONS 1. Dulau Florea A, Young NS, Maric I, Braylan RC. Human Pathology (Poster Abstract). Expression of Glycosylphosphatidylinositol (GPI) Anchor Protein (AP) in Bone Marrows of Normal Subjects and Aplastic Anemia Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).Human Pathology (Poster abstract).The USCAP Annual Meeting 2016, Seattle, WA (March 12-18, 2016). 2. Dulau Florea A, Young NS, Maric I, Jordan EK, Jiang C, Ahmad F, Braylan R. Immunophenotypic Abnormalities Highly Suggestive of Paroxysmal Nocturnal Hemoglobinuria (PNH) can be Detected with Routine Flow Cytometric Analysis of Bone Marrow (BM). Human Pathology (Poster abstract).The USCAP Annual Meeting 2017, San Antonio, TX (March 4-10, 2017). 3. Dulau Florea A, Young NS, Jordan E, Maric I, Braylan R. 154 Bone Marrow Aspirate Samples are Equal to Peripheral Blood for the Detection of Paroxysmal Nocturnal Hemoglobinuria (PNH). Leuk Res. April 2017, Vol.55: S96 (Poster abstract). The 14th International Symposium on Myelodysplastic Syndromes (MDS 2017), Valencia, Spain (May 3-6, 2017).

阵发性夜间血红蛋白尿（PNH）是一种获得性造血干细胞疾病。PNH是由磷脂酰肌醇聚糖a （PIG-A）基因的体细胞突变引起的。该基因的产物在糖基磷脂酰肌醇（GPI）锚蛋白合成的初始阶段至关重要，GPI锚蛋白是一种将许多重要蛋白质附着在细胞膜上的结构。由于猪-a基因突变起源于能够分化的多能造血干细胞，所有源自突变的造血干细胞的祖细胞都含有GPI缺陷，并且细胞表面所有GPI锚定蛋白（GPI- aps）的表达完全或部分丧失。其中一些gpi锚定蛋白，如CD55和CD59，在补体的控制中起重要作用。活化的血清补体在溶血性贫血中起重要作用。在正常人中，红细胞受到保护，免受补体介导的破坏，而那些缺乏锚定蛋白CD55和CD59的红细胞则发生溶血，主要发生在血管内。PNH型克隆的扩增通常与特发性再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）相关，这是免疫介导的正常造血功能失败的情况。研究表明，在AA患者中，高达60%的患者携带pnh型细胞。在MDS中，大约15%的患者携带pnh型细胞。