Paroxysmal Nocturnal Hemoglobinuria Cells Research Articles

Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the Glycosylphosphatidylinositol-Binding Toxin Aerolysin

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59+ cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

The use of flow cytometry in the diagnosis and monitoring of malignant hematological disorders

Flow cytometry is a modality with ever increasing application in modern hematological practice. This is due to the rapidity of obtaining results, ease of use and increasing power to detect abnormal populations of cells. The major uses of flow cytometry in malignant hematology are in the diagnosis, classification and monitoring of diseases such as leukemia, lymphoma and myeloma. The technique is now used also to detect disease-specific populations of cells in paroxysmal nocturnal hemoglobinuria. This review describes the use of flow cytometry in many disease states.

Circulating Primitive Stem Cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) Are Predominantly Normal in Phenotype But Granulocyte Colony-Stimulating Factor Treatment Mobilizes Mainly PNH Stem Cells

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia resulting from a somatic mutation in a hemopoietic stem cell. In most cases of hemolytic PNH, the majority of the marrow cells are derived from the PNH clone. Recent evidence has indicated, however, that the majority of the most primitive peripheral blood stem cells (PBSCs) in PNH appear to be of normal phenotype. This has led to tentative suggestions that normal PBSCs could be collected and used for autologous transplantation. We have investigated this possibility in four PNH patients by treating them with granulocyte colony-stimulating factor (G-CSF) in an attempt to mobilize normal progenitors. The expression of glycosylphosphatidylinositol (GPI)-linked proteins was analyzed by flow cytometry on mature neutrophils, late stem cells (CD34+/CD38+), and primitive stem cells (CD34+/CD38−). The phenotyping and stem cell quantitation was performed in steady-state blood and post–G-CSF administration. The most primitive PBSCs (CD34+/CD38−) were almost all normal before G-CSF treatment, even when the patients' neutrophils were mainly PNH. However, after G-CSF, the cells that were mobilized into the peripheral blood were of a similar phenotype to the mature neutrophils, ie, mainly PNH. It is possible that PNH-stem cells are preferentially destroyed by complement in the peripheral blood leaving only normal cells in the circulation. After G-CSF, the PNH cells in the marrow are released into the blood. Our findings suggest that it would be difficult to collect sufficient numbers of normal stem cells for autologous transplantation.

Circulating Primitive Stem Cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) Are Predominantly Normal in Phenotype But Granulocyte Colony-Stimulating Factor Treatment Mobilizes Mainly PNH Stem Cells

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia resulting from a somatic mutation in a hemopoietic stem cell. In most cases of hemolytic PNH, the majority of the marrow cells are derived from the PNH clone. Recent evidence has indicated, however, that the majority of the most primitive peripheral blood stem cells (PBSCs) in PNH appear to be of normal phenotype. This has led to tentative suggestions that normal PBSCs could be collected and used for autologous transplantation. We have investigated this possibility in four PNH patients by treating them with granulocyte colony-stimulating factor (G-CSF) in an attempt to mobilize normal progenitors. The expression of glycosylphosphatidylinositol (GPI)-linked proteins was analyzed by flow cytometry on mature neutrophils, late stem cells (CD34+/CD38+), and primitive stem cells (CD34+/CD38−). The phenotyping and stem cell quantitation was performed in steady-state blood and post–G-CSF administration. The most primitive PBSCs (CD34+/CD38−) were almost all normal before G-CSF treatment, even when the patients' neutrophils were mainly PNH. However, after G-CSF, the cells that were mobilized into the peripheral blood were of a similar phenotype to the mature neutrophils, ie, mainly PNH. It is possible that PNH-stem cells are preferentially destroyed by complement in the peripheral blood leaving only normal cells in the circulation. After G-CSF, the PNH cells in the marrow are released into the blood. Our findings suggest that it would be difficult to collect sufficient numbers of normal stem cells for autologous transplantation.

Pathogenesis and management of paroxysmal nocturnal haemoglobinuria

Over the past 30 years, our understanding of the pathogenesis of paroxysmal nocturnal haemoglobinuria (PNH) has increased dramatically. During that time, the events during complement activation and regulation have been described, the molecular basis for the exaggerated complement sensitivity of PNH cells has been uncovered, and the responsible gene mutation has been identified. It is now possible to relate almost all the protean manifestations of PNH to a single gene mutation in a haematopoietic stem cell. Unfortunately, our ability to manage these patients has not kept pace, and, with the exception of bone marrow transplantation, our major efforts are still directed toward control of complications rather than interruption of the disease process.

Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome

AbstractBone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-α, interferon-γ, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.

Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome

Bone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-alpha, interferon-gamma, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.

Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a- ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH.

Resistance to apoptosis caused by PIG-A gene mutations in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene, PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found that PIG-A mutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34(+) (CD59(-)) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIG-A gene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting from PIG-A mutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.

Expression of blood group i antigen and fetal hemoglobin in paroxysmal nocturnal hemoglobinuria.

The mechanism by which the paroxysmal nocturnal hemoglobinuria (PNH) clone progressively takes over normal hematopoietic cells remains unknown. The respective in vivo differentiation of normal and PNH erythroid progenitors was investigated through the expression of two fetal erythroid markers (i antigen and fetal hemoglobin [HbF]) whose expression in adult red cells is associated with altered erythropoiesis. Murine monoclonal antibodies directed against HbF and i and CD59 antigens were used to phenotype red cells of 10 PNH patients. A multiparametric flow cytometry assay of red cells and reticulocytes was designed to assess a possible association of i and HbF with PNH or normal red cells. Most patients exhibited greater expression of i and HbF than did normal controls. In each case, the percentages of i-positive or HbF-positive cells within CD59-deficient and CD59-positive red cells were very close, clearly showing a lack of preferential association of these markers with normal or PNH cells. In PNH patients, normal and PNH erythroid progenitors have the same ability to promote HbF and i antigen expression, which suggests that normal and PNH erythroid progenitors (burst-forming units-erythroid, colony-forming units-erythroid, erythroblasts) behave similarly in response to bone marrow stress.

Megakaryocyte progenitors in paroxysmal nocturnal haemoglobinuria are sensitive to complement.

We have investigated growth in vitro of bone marrow megakaryocytic progenitors (CFU-Mk) in 7 patients with paroxysmal nocturnal haemoglobinuria (PNH) to determine the sensitivity of CFU-Mk to complement. Bone marrow light density mononuclear cells were exposed to fresh or heat-inactivated AB human serum in the presence of medium or isotonic sucrose solution. We found that the proliferative activity of bone marrow CFU-Mk in PNH patients was significantly lower than in controls. In addition, the number of CFU-Mk in PNH bone marrow cells exposed to isotonic sucrose and complement was reduced to 25% of that in PNH cells exposed to isotonic sucrose without complement. In conclusion, our finding showed an increased sensitivity of CFU-Mk in PNH bone marrow cells to complement, supporting the hypothesis that the PNH defect is present at the level of CFU-Mk.

A Novel Class of Cell Surface Glycolipids of Mammalian Cells: FREE GLYCOSYL PHOSPHATIDYLINOSITOLS

Glycosyl phosphatidylinositol (GPI) lipids function as anchors of membrane proteins, and free GPI units serve as intermediates along the path of GPI-anchor biosynthesis. By using in vivo cell surface biotinylation, we show that free GPIs: 1) can exit the rough endoplasmic reticulum and are present on the surface of a murine EL-4 T-lymphoma and a human carcinoma cell (HeLa), 2) arrive at the cell surface in a time and temperature-dependent fashion, and 3) are built on a base-labile glycerol backbone, unlike GPI anchors of surface proteins of the same cells. The free GPIs described in this study may serve as a source of hormone-sensitive phosphoinositol glycans. The absence of free GPIs from the cell surface may also account for the growth advantage of blood cells in paroxysmal nocturnal hemoglobinuria.

The dual pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease with distinct and rather peculiar characteristics that have puzzled hematologists for more than a century. PNH cells are deficient in a set of membrane proteins that have in common a glycolipid anchor. We refer to this combination of deficiencies as the PNH abnormality or the PNH phenotype. Biochemical analysis has recently made it possible to pinpoint the metabolic block in PNH cells to an early step in the biosynthesis of the glycolipid anchor. This block is due in turn to the deficiency of a protein, called PIG-A, which is encoded by an X-linked gene. Expression cloning of the PIG-A gene has been followed by the identification in patients with PNH of somatic mutations in this gene that inactivate or impair the function of the PIG-A protein. These findings explain in full the molecular basis of the PNH abnormality, but they do not explain how the PNH clone, which is biochemically defective, can expand to the extent of contributing a substantial proportion of the patient's hematopoiesis. Thus a second factor is required to explain the pathogenesis of PNH. This is most likely the coexistence of an element of bone marrow failure that produces, paradoxically, a survival or growth advantage for the PNH clone. The notion that the injury causing failure of normal stem cells spares selectively cells with the PNH phenotype is supported by a number of observations, including the finding of multiple independently arisen PNH clones in patients with PNH.

Emergence of CD52-, Phosphatidylinositolglycan-Anchor-Deficient T Lymphocytes After In Vivo Application of Campath-1H for Refractory B-Cell Non-Hodgkin Lymphoma

CD52 is a phosphatidylinositolglycan (PIG)-anchored glycoprotein (PIG-AP) expressed on normal T and B lymphocytes, monocytes, and the majority of B-cell non-Hodgkin lymphomas. We observed the emergence of CD52- T cells in 3 patients after intravenous treatment with the humanized anti-CD52 monoclonal antibody Campath-1H for refractory B-cell lymphoma and could identify the underlaying mechanism. In addition to the absence of CD52, the PIG-AP CD48 and CD59 were not detectable on the CD52- T cells in 2 patients. PIG-AP-deficient T-cell clones from both patients were established. Analysis of the mRNA of the PIG-A gene showed an abnormal size in the T-cell clones from 1 of these patients, suggesting that a mutation in the PIG-A gene was the cause of the expression defect of PIG-AP. An escape from an immune attack directed against PIG-AP+ hematopoiesis has been hypothesized as the cause of the occurrence of PIG-AP-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia. Our results support the hypothesis that an attack against the PIG-AP CD52 might lead to the expansion of a PIG-anchor-deficient cell population with the phenotypic and molecular characteristics of PNH cells.

PIG-A, DAF and proto-oncogene expression in paroxysmal nocturnal haemoglobinuria-associated acute myelogenous leukaemia blasts.

Failed surface expression of the complement decay-accelerating factor (DAF) due to mutation of the PIG-A gene is a hallmark of affected paroxysmal nocturnal haemoglobinuria (PNH) blood elements. Previous findings that acute myelogenous leukaemia (AML) blasts evolving in a PNH patient differed from idiopathic AML blasts in that they exhibited DAF negativity suggested that the leukaemic blasts derived from an affected PNH cell. To investigate whether these cells differ from untransformed PNH cells in PIG-A genetic alterations or in DAF mRNA processing, or are distinguishable from conventional AML blasts in proto-oncogene activation or chromosomal structure, their DNA and RNA were examined using PIG-A, DAF and proto-oncogene probes and their karyotype was analysed. Analyses of the PIG-A genome revealed dual exchanges of A1110-->G and T1130-->A resulting in conversions of T370 to R and I377 to N in the coding region but no deletions or rearrangements. Investigations of DAF mRNA processing showed mRNA species differing in 3' UT regions from those in untransformed cells but similar to those in DAF-positive leukaemia cell lines. Studies of c-myb, c-myc, c-fos and c-fms showed no gross genetic alterations, amplifications or variations in mRNA transcripts deriving from these genes. Karyotypic analysis showed no alterations. The results indicate that in AML blasts evolving in PNH: (1) the PIG-A genome exhibits multiple point mutations but no gross genetic changes; (2) DAF mRNA transcripts exhibit differentiation-dependent variations that do not affect GPI-anchoring; (3) c-myb, c-myc, c-fos and c-fms activation show no differences from idiopathic AML; and (4) no karyotypic abnormalities are associated with AML transformation.

Expression of recombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria B cells confers resistance to human complement

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by complement-mediated hemolytic anemia, pancytopenia, and venous thrombosis. These clinical manifestations arise from an underlying molecular defect of bone marrow stem cells. Specifically, somatic mutations in the phosphatidylinositol glycan class A gene result in the ability of blood cells to anchor complement- regulatory proteins (CD59 and DAF) to the cell surface via glycosyl phosphatidylinositol (GPI). In an attempt to circumvent the functional defect in PNH cells, a recombinant transmembrane form of CD59 (CD59-TM) was analyzed for the ability to regulate complement activity. Balb/3T3 stable transfectants expressing similar levels of either CD59-TM or native CD59 (CD59-GPI) were equally protected against human complement- mediated membrane damage. Treatment of these cells with phosphatidylinositol-specific phospholipase C failed to release CD59-TM from the cell surface. Retroviral transduction of GPI-anchoring deficient mouse L cells with CD59-TM resulted in surface expression of the protein and rendered these cells resistant to human complement- mediated membrane damage. Conversely, L cells transduced with CD59-GPI failed to express this protein on the cell surface. A GPI-anchoring deficient complement-sensitive B-cell line derived from a PNH patient was successfully transduced with CD59-TM, resulting in surface expression of the protein. The PNH B cells expressing CD59-TM were protected against classical complement-mediated membrane damage by human serum. Taken together, these data establish that a functional recombinant transmembrane form of CD59 can be expressed on the surface of GPI-anchoring deficient PNH cells and suggest that retroviral gene therapy with this molecule could provide a treatment for PNH patients.

Expression of Recombinant Transmembrane CD59 in Paroxysmal Nocturnal Hemoglobinuria B Cells Confers Resistance to Human Complement

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by complement-mediated hemolytic anemia, pancytopenia, and venous thrombosis. These clinical manifestations arise from an underlying molecular defect of bone marrow stem cells. Specifically, somatic mutations in the phosphatidylinositol glycan class A gene result in the ability of blood cells to anchor complement-regulatory proteins (CD59 and DAF) to the cell surface via glycosyl phosphatidylinositol (GPI). In an attempt to circumvent the functional defect in PNH cells, a recombinant transmembrane form of CD59 (CD59-TM) was analyzed for the ability to regulate complement activity. Balb/3T3 stable transfectants expressing similar levels of either CD59-TM or native CD59 (CD59-GPI) were equally protected against human complement-mediated membrane damage. Treatment of these cells with phosphatidylinositol-specific phospholipase C failed to release CD59-TM from the cell surface. Retroviral transduction of GPI-anchoring deficient mouse L cells with CD59-TM resulted in surface expression of the protein and rendered these cells resistant to human complement-mediated membrane damage. Conversely, L cells transduced with CD59-GPI failed to express this protein on the cell surface. A GPI-anchoring deficient complement-sensitive B-cell line derived from a PNH patient was successfully transduced with CD59-TM, resulting in surface expression of the protein. The PNH B cells expressing CD59-TM were protected against classical complement-mediated membrane damage by human serum. Taken together, these data establish that a functional recombinant transmembrane form of CD59 can be expressed on the surface of GPI-anchoring deficient PNH cells and suggest that retroviral gene therapy with this molecule could provide a treatment for PNH patients.

Peripheral Blood Cells Are Predominantly Chimeric of Affected and Normal Cells in Patients With Paroxysmal Nocturnal Hemoglobinuria: Simultaneous Investigation on Clonality and Expression of Glycophosphatidylinositol-Anchored Proteins

To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59- and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP- or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins

To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59-and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP-or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

Paroxysmal nocturnal hemoglobinuria: Correction of abnormal phenotype by somatic cell hybridization

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disorder thought to result from a somatic mutation in a hemopoietic stem cell. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in proteins attached to the cell membrane via a glycosylphosphatidylinositol structure, called the GPI anchor, and the primary lesion in PNH is thought to be a defect in the biosynthesis of the GPI anchor. We have recently established permanent lymphoblastoid cell lines that have the PNH phenotype and we report now the isolation of human-human somatic cell hybrid clones obtained by fusing them with normal lymphoblastoid cells. In all of 21 hybrid clones, obtained from five different patients, the expression of three different GPI-linked proteins on the hybrid cells was normal. These findings indicate that the PNH mutant gene is recessive with respect to the normal allele and that a recessive mutation can cause a clonal preneoplastic disorder.