A New Aspect of the Molecular Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder which is manifest by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Complement-mediated hemolysis in PNH is explained by the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59 on erythrocyte surfaces. All the PNH patients had phosphatidylinositol glycan-class A (PIG-A) gene abnormalities in various cell types, indicating that PIG-A gene mutations cause the defects in GPI-anchored proteins that are essential for the pathogenesis of PNH. In addition, a PIG-A gene abnormality results in a PNH clone. Bone marrow failure causes cytopenias associated with a proliferative decrease of its hematopoietic stem cells and appears to be related to a pre-leukemic state. Although it is unclear how a PNH clone expands in bone marrow, it is considered that the most important hypothesis implicates negative selection of a PNH clone, but it does not explain the changes in the clinical features at the terminal stage of PNH. Recently, it has been suggested that an immune mechanism, in an HLA-restricted manner, plays an important role in the occurrence or selection of a PNH clone and GPI may be a target for cytotoxic-T lymphocytes. Also, it has been indicated that the Wilms' tumor gene (WT1) product is related to a PNH clone, but the significance of WT1 expression is not clear because of the functional diversity of the gene. To elucidate this problem, it is important to know the pathophysiology of bone marrow failure in detail and how bone marrow failure affects hematopoietic stem cells and immune mechanisms in bone marrow failure syndromes.     

Heterogeneity in the Molecular Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria (PNH) Syndromes and Expansion Mechanism of a PNH Clone

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure and is one disorder of acquired bone marrow failure syndromes that include as aplastic anemia and myelodysplastic syndrome. Nowadays, acquired PNH should be understood as one of the disorders of PNH syndromes. These syndromes include congenital PNH (such as inherited complete CD59 deficiency and PNH with PIG-M mutations), because complement-mediated hemolysis and thrombosis are observed in association with defects of various factors associated with the complement regulatory pathway, including biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. At present, how a "true" PNH clone in acquired PNH expands in the bone marrow remains unclear. Although several candidate genes, including the Wilms tumor gene, the early growth response gene, anti-apoptotic genes, and the high mobility group AT-hook 2 gene, that target corresponding proteins (excluding GPI-related proteins) have been reported, the evidence is insufficient to completely explain the diversity of the clinical and basic pathophysiology in acquired PNH. However, the hypothesis of expansion of a PNH clone due to the WT1 gene may explain various features of PNH.     

Molecular Genetics of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by the clonal expansion of glycosylphosphatidylinositol (GPI)-deficient cells that leads to complement-mediated hemolysis. A somatic mutation in the PIG-A gene involved in GPI biosynthesis causes a deficiency of GPI-anchored proteins. However, it is evident that the clonal expansion of GPI-deficient cells is not caused by only the PIG-A mutation and that other changes should be involved in the development of PNH. Some patients with aplastic anemia (AA) develop PNH. Furthermore, it has been reported that most patients with AA and refractory anemia (RA) who carry HLA-DRB1*15 and show a good response to immunosuppressive therapies have an expanded population of GPI-deficient clones. This finding, together with recent data showing resistance of GPI-deficient cells to cytotoxic cells, suggests that GPI-deficient cells escape immunologic attack and are positively selected in the autoimmune environment. However, GPI-deficient clones found in AA and RA are generally small and do not increase to near-complete dominance. Therefore, 1 or more additional genetic abnormalities that confer the growth phenotype on GPI-deficient cells are probably required for fully developed PNH or so-called florid PNH. The next 10 years should witness the discovery of the molecular mechanisms of immunologic selection and the identification of abnormalities involved in the further clonal expansion of PNH cells.     

New Insights into Molecular Pathogenesis of Bone Marrow Failure in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by the clonal expansion of hematopoietic stem cells with mutations of the phosphatidylinositol glycan-class A gene (PIGA). PNH clones then fail to generate glycosylphosphatidylinositol (GPI) or to express a series of GPI-linked membrane proteins including complement-regulatory proteins, resulting in complement-mediated intravascular hemolysis and thrombosis. Bone marrow failure is another characteristic feature of PNH. It is currently considered that immune-mediated injury of hematopoietic cells is implicated in PNH marrow failure as well as in aplastic anemia, a well-known PNH-related disorder. There is increasing evidence that the autoimmune attack allows PNH clones to selectively survive in the injured marrow, leading to clinical manifestations characteristic of PNH. As candidate molecules that trigger the immune attack on marrow cells, stress-inducible membrane proteins and Wilms’ tumor protein WT1 have been proposed. Among the stress-inducible proteins, GPI-linked proteins, such as cytomegalovirus glycoprotein UL16-binding protein, are distinct candidates that not only induce immune attack, but also allow PNH clones to survive the attack. Here, we overview the current understanding of the molecular pathogenesis of bone marrow failure in PNH.     

PIG-A gene mutations in Four Taiwanese Patients with Paroxysmal Nocturnal Haemoglobinuria Following Aplastic Anaemia

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by deficient biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor in haemopoietic stem cells. PIG-A , an X-linked gene that participates in the first step of GPI-anchor synthesis, is responsible for PNH. Various abnormalities of the PIG-A gene have been demonstrated in all patients with PNH so far examined. In this study we characterized the somatic mutations in PIG-A gene in four Taiwanese patients with PNH. We identified five novel mutations in the PIG-A gene, three single nucleotide substitution mutations (−342, C → G, codon 335, GGT → AGT and codon 405, GCT → GTT) and two frameshift mutations (codon 22, GGA → G-A and codon 356, TGT → TGTT) in the PIG-A gene. The −342 mutation was judged to be a polymorphism. Furthermore, three patients had previous clinicopathologic evidence which suggested aplastic anaemia (AA), before the development of PNH. One of these was found to have thrombocytopenia during follow-up. We suggest that the somatic PIG-A gene mutations highlight a subgroup of AA having a pathogenetic link with PNH.     

阵发性睡眠性血红蛋白尿患者外周血细胞CD47的表达

目的:通过研究阵发性睡眠性血红蛋白尿症(PNH)患者外周血细胞CD47的表达,探讨CD47在PNH发病机制中的作用。方法:选取PNH患者8例,正常对照组15例,以逆转录-聚合酶链反应异源双链分析银染法进行PIG-A基因检测,流式细胞术检测外周血细胞表面CD47、CD59的表达。结果:6例PNH患者PIG A基因第2外显子有碱基变异,其余2例未发现异常;PNH患者外周血红细胞、粒细胞CD47表达阳性率及平均荧光强度比正常对照组均减低(P<0．05)。结论:PNH患者外周血细胞表面CD47表达减少,CD47的表达与PNH发病机制可能有关。     

Microvascular Thrombosis in the Hepatic Vein of a Patient with Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. In May 2003, a 33-year-old man was admitted to a hospital with right hypochondralgia and fever. He had a history of aplastic anemia. The patient's diagnosis of diffuse microvessel thrombosis in the hepatic vein due to an unknown cause was derived from the findings of a contrast-enhanced computed tomography examination of the abdominal region, angiographic evaluation of abdominal vessels, and pathohistologic examination of a liver biopsy sample. The patient was subsequently treated with warfarin. The abdominal pain and fever continued, however, and anemia gradually appeared. In April 2004, the patient was referred to our hospital to examine the cause of the thrombosis. On admission, slight anemia and a low serum haptoglobin level were observed. A flow cytometry evaluation of CD55 and/or CD59, CD59, and CD48 expression in erythrocytes, granulocytes, and monocytes, respectively, showed that the respective proportions of negative populations were 5.6%, 97.1%, and 96.2%. The patient then received a diagnosis of aplastic anemia/PNH syndrome, which had caused the hemolytic anemia and thrombosis, although no hemoglobinuria had been observed during his clinical course. This patient is, to our knowledge, the first reported case of a PNH patient with thrombosis present only in hepatic microvessels and not in hepatic large vessels, in spite of the presence of few hemolytic events.     

Relationship Between Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Since aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome was reported in 1967, the overlap of idiopathic aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) has been well known. The link between the 2 diseases became even more evident when immunosuppressive therapy improved survival of patients with severe AA. More than 10% of patients with AA develop clinically evident PNH. Moreover, flow cytometric analysis demonstrates that the majority of patients with AA have a subclinical percentage of granulocytes with PNH phenotype. Some of them have clearly recognizable PNH clones. Granulocytes with a PNH phenotype are also often found in normal individuals, though at much smaller percentages of cells. This finding suggests that a PNH clone is expanded in AA. consistent with a hypothesis that blood cells from patients with PNH are more resistant to an autoimmune environment. Survival of PNH clones in pathologic bone marrow may account for limited expansion of PNH clones; however, additional genetic change(s) that confers cells with growth phenotype may be required for the full development of PNH.     

蛇毒因子溶血试验在阵发性睡眠性血红蛋白尿症中的诊断意义

对４１例阵发性睡眠性血红蛋白尿症，３３例再障和１６例阵发性睡眠性血红蛋白尿─再障综合征等２４５例各种贫血患者，进行蛇毒因子溶血试验，并与糖水及Ｈａｍｓ试验进行比较，认为蛇毒因子溶血试验敏感性及特异性都较好，可作为诊断阵发性睡眠性血红蛋白尿症的一个较好方法。     

Recent Advances in Biological and Clinical Aspects of Paroxysmal Nocturnal Hemoglobinuria

The unique feature of paroxysmal nocturnal hemoglobinuria (PNH), a chronic disease with severe hemolytic anemia, is the presence of a population of blood cells that, being deficient in surface proteins tethered to the membrane through a glycosylphosphatidylinositol molecule, are said to have the PNH phenotype. Therefore, the diagnosis of PNH is based on the demonstration that a substantial proportion of red cells and granulocytes have this phenotype. Diagnosis is currently best done by flow cytometry analysis, most appropriately by using anti-CD59 and anti-CD55 antibodies. Flow cytometry can also quantitate these cells and monitor their numbers as a function of time, thereby aiding clinical management. The most important advance in management has been the introduction of a human monoclonal antibody (eculizumab) that is directed against the C5 component of complement. Because hemolysis in PNH is mostly intravascular and complement dependent, periodic administration of anti-C5 produces complement blockade. This agent is the first to substantially reduce the rate of hemolysis in patients with PNH. Because very small PNH clones have been known for some years to exist in healthy people, it is clear that a crucial factor in causing PNH as a clinical disease is a marked expansion of the PNH clones themselves. Several lines of evidence from studies of mouse models and patients suggest that the process of expansion is probably the result of 2 phenomena: (1) damage to normal hematopoietic stem cells and (2) the sparing of PNH hematopoietic stem cells. This process of somatic cell selection may have an autoimmune basis, and the most likely agents are cells belonging to the natural killer-like subset of T-cells.     

Clinical Manifestations of Paroxysmal Nocturnal Hemoglobinuria: Present State and Future Problems

The clinical pathology of paroxysmal nocturnal hemoglobinuria (PNH) involves 3 complications: hemolytic anemia, thrombosis, and hematopoietic deficiency. The first 2 are clearly the result of the cellular defect in PNH, the lack of proteins anchored to the membrane by the glycosylphosphatidylinositol anchor. The hemolytic anemia results in syndromes primarily related to the fact that the hemolysis is extracellular. Thrombosis is most significant in veins within the abdomen, although a number of other thrombotic syndromes have been described. The hematopoietic deficiency may be the same as that in aplastic anemia, a closely related disorder, and may not be due to the primary biochemical defect. The relationship to aplastic anemia suggests a nomenclature that emphasizes the predominant clinical manifestations in a patient. This relationship does not explain cases that appear to be related to myelodysplastic syndromes or the transition of some cases of PNH to leukemia. Treatment, except for bone marrow transplantation, remains noncurative and in need of improvement.     

阵发性睡眠性血红蛋白尿患者骨髓粒单系祖细胞的体外培养研究

本文采用造血祖细胞体外培养技术，研究了阵发性睡眠性血红蛋白尿（ＰＮＨ）病人骨髓粒单系祖细胞（ＣＦＵ－ＧＭ）的增殖能力；骨髓细胞经酸化ＡＢ型血清处理后ＣＦＵ－ＧＭ的增殖能力；和ＣＦＵ－ＧＭ对粒单系集落刺激因子（ＧＭ－ＣＳＦ）的反应能力，发现ＰＮＨ病人骨髓ＣＦＵ－ＧＭ集落数明显低于正常；骨髓细胞经新鲜酸化ＡＢ型血清处理后培养的ＣＦＵ－ＧＭ集落数明显低于经热灭活酸化ＡＢ型血清处理后培养的集落数；ＣＦＵ－ＧＭ对ＧＭ－ＣＳＦ的剂量反应曲线低平。因此认为ＰＮＨ病人骨髓粒单系祖细胞膜缺陷导致其在酸性条件下对补体的敏感性增高而致损伤或溶解和导致其对粒单系集落刺激因子的敏感性降低。     

Immune Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria

Somatic mutation in the PIG-A gene is the initial event in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), but the pathophysiologic mechanisms leading to clonal expansion remain unclear. The intricate association of PNH with immune-mediated bone marrow failure syndromes, including aplastic anemia (AA), suggests an immunologic selection process for the glycosylphosphatidyl-inositol (GPI)-deficient hematopoietic clone. The mechanism for the growth advantage of PNH cells may be related to the nature of the antigens targeted by the immune response or to the function of immunomodulatory GPI-anchored proteins on the surface of the hematopoietic target cells. Alternative theories of PNH evolution may include intrinsic properties of the mutated cells, but the experimental evidence is largely lacking. Elucidation of the pathogenesis of PNH may provide key information about the causes of idiopathic AA and help understand the regulation of the hematopoietic stem cell compartment.     

Structural and Functional Analysis of the Pig-a Protein That is Mutated in Paroxysmal Nocturnal Hemoglobinuria

ABSTRACT: There is now convincing evidence that thePig-agene is mutated in patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease in which one or more clones of hematopoietic cells have incomplete assembly of glycosylphosphatidylinositol (GPI) anchors and absence of GPI-linked protein expression on the cell surface. Little is known, however, about the Pig-a protein product that is necessary for GPI anchor bioassembly. Relatively few substitution (missense)Pig-agene mutations have been identified, but we noted two apparent clusters at codons 128-129 and 151-156 and hypothesized that these might represent critical regions of the Pig-a protein. We therefore used site-directed mutagenesis to create conservative mutations in the Pig-a protein, then performed structural and functional analysis. EachPig-amutation generated a Pig-a protein of normal size and stability, but certain mutations had substantial deleterious effects on protein function. Conservative mutation of codons histidine 128 (H128), serine 129 (S129), and serine 155 (S155) had greatly diminished function, while mutations of flanking residues had no effect on function. Our results represent the first structure/function analysis of the Pig-a protein, and suggest that codons H128, S129, and S155 represent critical regions of the Pig-a protein. Our results also suggest a means by which transgenic mice with a “partial knock-out” of Pig-a function could be generated, which would allow investigation of PNH in an animal model.     

Proliferative Capacity of Single Isolated CD34+ Hematopoietic Stem/Progenitor Cells in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations of the X-linked PIG-A (phosphatidylinositol glycan-class A) gene, which occurs on a hematopoietic stem cell level, leading to a proportion of blood cells being deficient in all glycosylphosphatidylinositol (GPI)-anchored surface proteins. Although these GPI-deficient cells can explain many of the clinical symptoms of PNH, the pathogenesis of PNH is still somewhat obscure and many questions remain. To assess the hematopoietic defect involved in PNH, CD34+ CD59+ (normal phenotype hematopoietic stem/progenitor) and CD34+ CD59- (PNH phenotype) cells from PNH patients (n = 16) and CD34+ CD59+ cells from healthy volunteers (n = 10) were sorted as single cells into 96-well flat-bottom culture plates containing culture medium supplemented with stem cell factor, interleukin (IL)-3, erythropoietin, granulocyte-macrophage-colony-stimulating factor (GM-CSF), G-CSF, IL-6, thrombopoietin, and Flt-3 ligand. We found that the single PNH CD34+ CD59- cells had a growth advantage over the single CD34+ CD59+ cells to some extent, but they both had impaired growth abilities compared with CD34+ cells from healthy volunteers.     

Recent advances in paroxysmal nocturnal hemoglobinuria: From the biology to the clinic

PNH is now known as an acquired, clonal disorder of the hematopietic stem cells caused by somatic mutation in the X-linked PIG-A gene encoding a protein involved in the synthesis of the glycosylphosphatidylinositol (GPI) anchor by which many proteins are attached to the membrane. Since the past few years, significant advances in the knowledge of the biology of this rare disease have been done. Similarily on the clinical ground, large series of patients with PNH have been published recently, providing estimates of factors affecting survival and of long term follow-up of significant numbers of patients. In this overview we focus on recent advances in the biology and the clinical aspects of this disease, and more importantly try to underline the numerous aspects of yet un-answered questions.     

Markers of Thrombin Generation and Inflammation in Patients with Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) presents with intravascular hemolysis, bone marrow failure and thrombosis. Various studies have reported geographic and ethnic variation in prevalence of thrombosis in PNH. There is limited data on thrombosis in PNH from the Indian subcontinent. In this study we describe disease burden and risk factors for thrombosis in 18 Indian PNH patients. We studied markers of thrombin generation (Thrombin-antithrombin complexes; TAT and D-Dimer), endothelium and platelet activation (soluble P-selectin) and inflammation (interleukin-6; IL-6) in PNH patients and compared their levels with healthy controls. Thrombosis was identified in 17% of PNH patients. TAT, sP-selectin and D-Dimer levels were significantly elevated in PNH patients (TAT: 5.06 ± 1.08 ng/ml; sP-selectin: 80.57 ± 19.5 ng/ml; D-Dimer mean: 936 ng/ml 95% CI 559, 1310) compared to control population (TAT: 3.39 ± 0.769 ng/ml P = 0.016; sP-selectin: 44.67 ± 5.17 ng/ml P = 0.002). Using Youden’s J statistic, the cut-off values for TAT and sP-selectin in our cohort of PNH patients were 2.90 ng/ml and 58.41 ng/ml respectively. TAT, sP-selectin and D-Dimer levels were elevated beyond the cut-off values in PNH patients with thrombosis compared to those without thrombosis. A positive correlation was noted between TAT, sP-selectin and D-Dimer levels. Increased TAT, sP-selectin, and D-Dimer levels may indicate impending thrombosis in PNH.     

Red Cells with Paroxysmal Nocturnal Hemoglobinuria-phenotype in Patients with Acute Leukemia

CD55 and CD59 are complement regulatory proteins that are linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. They are reduced mainly in paroxysmal nocturnal hemoglobinuria (PNH) and in other hematological disorders. However, there are very few reports in the literature concerning their expression in patients with acute leukemias (AL). We studied the CD55 and CD59 expression in 88 newly diagnosed patients with AL [65 with acute non-lymphoblastic leukemia (ANLL) and 23 with acute lymphoblastic leukemia (ALL)] using the sephacryl gel test, the Ham and sucrose lysis tests and we compared the results with patients' clinical data and disease course. Eight patients with PNH were also studied as controls. Red cell populations deficient in both CD55 and CD59 were detected in 23% of ANLL patients (especially of M₀, M₂ and M₆ FAB subtypes), 13% of ALL and in all PNH patients. CD55-deficient erythrocytes were found in 6 ANLL patients while the expression of CD59 was decreased in only 3 patients with ANLL. No ALL patient had an isolated deficiency of these antigens. There was no correlation between the existence of CD55 and/or CD59 deficiency and the percentage of bone marrow infiltration, karyotype or response to treatment. However no patient with M₃, M₅, M₇ subtype of ANLL and mature B- or T-cell ALL showed a reduced expression of both antigens. The deficient populations showed no alteration after chemotherapy treatment or during disease course. This study provides evidence about the lower expression of CD55 and CD59 in some AL patients and the correlation with their clinical data. The possible mechanisms and the significance of this phenotype are discussed.