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.     

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.     

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。     

Molecular Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH), although named for its marked fluctuations in the visibility of hemoglobinuria, is now classified as an acquired hematopoietic stem cell disorder. The clinical manifestations of PNH are very complicated, and include intravascular hemolytic anemia, venous thrombosis in unusual sites (abdomen, liver, cerebrum), deficient hematopoiesis, evolution to leukemia, and susceptibility to infection [1, 2]. The intravascular hemolysis is attributed to the enhanced susceptibility of erythrocytes to autologous complement [3]. The abnormal sensitivity is explained by a lack of complement regulatory membrane proteins such as decay-accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59), which are covalently linked to the erythrocyte membrane through a glycosylphosphatidylinositol (GPI) anchor. The deficiency of the membrane proteins is caused by a synthetic defect in this anchor caused by impaired transfer of N- acetylglucosamine (GlcNAc) to phosphatidylinositol (PIns) [2]. Mutations of the phosphatidylinositol glycan class A (PIG-A) gene have been shown to contribute this abnormality in nearly all patients with PNH studied to date [4]. Recently, several reviews have been presented on various aspects of PNH [5–10]. This review focuses particularly on the recent elucidation of the molecular pathogenesis of GPI-anchor deficiency on PNH and related hematopoietic stem cell disorders.     

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.     

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.     

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.     

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.     

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.     

Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation

Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare acquired disorder. The aim of this study was to investigate the demographics, clinical manifestations, and outcomes of PNH patients in southern Taiwan. Data on PNH patients diagnosed over a 30-year period (1985–2015) were retrospectively collected from four tertiary medical centers in southern Taiwan. Blood samples were collected for hematologic panel testing and flow cytometry detection of PNH clones. Radiologic studies were performed to assess the frequency of complications. Twenty-four patients were enrolled in this study. The median duration of disease in the study participants was 10.8 years. The median granulocyte PNH clone size was 92.5% (range, 1.3%–99.8%), and the median lactate dehydrogenase (LDH) level was 2920.2 ± 1462.0 IU/L. The incidence of thromboembolism and impaired renal function was 16.7% and 29.2%, respectively. The primary treatment strategies included steroids (79.2%), androgens (42.0%), eculizumab (33.3%), immunosuppressants (16.7%), and anticoagulants (4.2%). In eight patients treated with eculizumab, there was a marked reduction in the LDH levels of 14.89-fold–1.63-fold that of the upper limit of normal; seven patients exhibited decreased transfusion requirements. Twenty-one patients were alive with regular follow-up at the time of publication. Our study demonstrates that PNH patients in southern Taiwan may exhibit different clinical characteristics and outcomes relative to patients in other countries. There was a trend toward a greater PNH granulocyte clone size, which may lead to more hemolysis. In our study, the percentage of patients with impaired renal function, but not the percentage of patients with thrombotic events, was higher than values reported worldwide and in the observational cross-sectional International PNH Registry. More large-scale studies with comprehensive data on the clinical response to different treatments are needed.     

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.     

CASE REPORT: Gastrointestinal Involvement in Paroxysmal Nocturnal Hemoglobinuria: First Report of Electron Microscopic Findings

Thrombotic complications, particularly microthrombi involving intraabdominal veins leading to intestinal ischemia, have remained a major cause of morbidity in patients with paroxysmal nocturnal hemoglobinuria (PNH). While intestinal ischemia has been postulated to be the cause of recurrent bouts of abdominal pain in this population, direct antemortem evidence for this complication is scarcely documented in the literature. We describe a case of PNH in a patient who presented with abdominal distress three years after the initial diagnosis was established. Clinical features and a combination of diagnostic modalities, including radiography, endoscopy, and histology were used to make the prompt diagnosis of intestinal ischemia. This is the first case in which the electronic microscopy of the gastrointestinal lesion is described. Our patient was successfully treated with conservative measures and anticoagulation.     

Notch Signaling in Hematopoietic Stem Cells

The molecular basis of the hematopoietic stem cell (HSC) "niche" has gradually been elucidated. This new knowledge may help us understand how the self-renewal of HSCs is physiologically regulated and may give us clues for developing methods for ex vivo HSC expansion. The Notch pathway is an environmental signaling system that may play an important role in the HSC niche. In this review, we focus on the role of Notch signaling in the regulation of hematopoietic stem and progenitor cells in both embryo and adult hematopoiesis and clarify what is known regarding the self-renewal of HSCs.     

Detection of Erythrocytes Deficient of Glycosylphosphatidyl-inositol Anchored Membrane Proteins in Patients with Paroxysmal Nocturnal Hemoglobinuria by the Toxin HEC Secreted by Aeromonas hydrophila J-1

Objective: To study the feasibility of diagnosing paroxysmal nocturnal hemoglobinuria (PNH) with toxin HEC, the abbreviation of hemolytic, entreotoxigenicity and cytotoxity secreted by Aeromonas hydrophila J-1.

Methods: The crude toxin HEC was extracted from the culture medium of Aeromonas hydrophila J-1 by precipitating with saturated (NH₄)₂SO₄ and then purified through DEAE52. Purified toxin HEC is different from Aerolysin in molecular weight and necessity of activation. Crude toxin is prepared possessed same effect as purified ones. This crude toxin was used to act on red blood cells (RBCs) from patients with PNH, non-PNH anemia, and normal persons. Absorbance at 630 nm was measured to quantitate the extent of hemolysis. Toxin HEC treated and untreated RBCs were both stained with anti-CD59 monoclonal antibody and FITC labeled goat-anti-mouse IgG. The percentage of CD59⁺ cells was detected by flow cytometry (FCM).

Results: After toxin HEC treatment, RBCs from PNH patients showed resistance to the toxin hemolysis, which was negatively related to the percentage of CD59⁺ cells, while RBCs from normal persons and non-PNH anemic patient were nearly totally lysed.

Conclusion: Detection of RBCs resistance to toxin HEC can be used for the diagnosis of PNH.     

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.     

Specificity of Hematopoietic Stem and Progenitor Cell Homing to Bone Marrow: A Perspective

Little is known about the hematopoietic stem and progenitor cell membrane recognition and adhesion molecules which mediate their specific patterns of movement into and out of the marrow compartment during steady state hematopoiesis and during pathological conditions. Implicit in the cellular targeting of these cells to marrow stroma, or “homing”, is a high degree of molecular specificity. Identification of homing determinants and knowledge of their function in conferring specificity to these events may provide new insight into the localization of hematopoietic stem cells within the bone marrow, directly impacting clinical stem cell transplantation. In addition, a homing protein gene/promoter complex, or a stromal counter-receptor gene, may provide a valuable target for driving expression of gene constructs in early hematopoietic cells.