Implication of PIGA genotype on erythrocytes phenotype in Paroxysmal Nocturnal Hemoglobinuria (original) (raw)
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Glycosyl phosphatidylinositol-linked blood group antigens and paroxysmal nocturnal hemoglobinuria
Transfusion Clinique et Biologique, 1995
Human erythrocyte cell surface molecules that are attached to the cell membrane by glycosyl-phosphatidylinositol (GPI) anchors include the complement regulatory proteins decay accelarating factor (DAF, CD55) and membrane inhibitor of reactive 1ysis (MIRL, CD59), as well as the proteins that bear the Cartwright, Dombrock, and JMH blood group antigens. The acquired hematopoietic stem cell disorder paroxysmal nocturnal hemoglobinuria {PNH) results from the absence or marked deficiency in expression of GPI-anchored proteins in affected hematopoietic cells. PNH usually if not always results from a somatic mutation of an X-linked gene called PIG-A; the product of the PIG-A gene is a glycosyl transferase necessary for construction * This review contains material extracted from previously published reviews by the author and updated with the addition of recently acquired information.
Proceedings of the National Academy of Sciences, 1999
In paroxysmal nocturnal hemoglobinuria (PNH), acquired somatic mutations in the PIG-A gene give rise to clonal populations of red blood cells unable to express proteins linked to the membrane by a glycosylphosphatidylinositol anchor. These proteins include the complement inhibitors CD55 and CD59, and this explains the hypersensitivity to complement of red cells in PNH patients, manifested by intravascular hemolysis. The factors that determine to what extent mutant clones expand have not yet been pinpointed; it has been suggested that existing PNH clones may have a conditional growth advantage depending on some factor (e.g., autoimmune) present in the marrow environment of PNH patients. Using f low cytometric analysis of granulocytes, we now have identified cells that have the PNH phenotype, at an average frequency of 22 per million (range 10-51 per million) in nine normal individuals. These rare cells were collected by f low sorting, and exons 2 and 6 of the PIG-A gene were amplified by nested PCR. We found PIG-A mutations in six cases: four missense, one frameshift, and one nonsense mutation. PNH red blood cells also were identified at a frequency of eight per million. Thus, small clones with PIG-A mutations exist commonly in normal individuals, showing clearly that PIG-A gene mutations are not sufficient for the development of PNH. Because PIG-A encodes an enzyme essential for the expression of a host of surface proteins, the PIG-A gene provides a highly sensitive system for the study of somatic mutations in hematopoietic cells.
The Hematology Journal, 2000
The association of paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) raises the yet unresolved questions as t o whether these two disorders are different forms of the same disease. We compared two groups of patients with respect t o cytogenetic features, glycosylphosphatidylinositol (GPII-linked protein expression, protein C/ protein Slthrombomodulinlantithrombin 111 activity, and PIG-A gene expression. The first group consisted of eight patients with PNH (defined as positive Ham and sucrose tests at diagnosis), and the second, 37 patients with AA. Twelve patients with AA later developed a PNH clone. Monoclonal antibodies used t o study GPI-linked protein expression (CD14 [on monocytesl, CD16 [on neutrophils], CD48 [on lymphocytes and monocytes], CD67 [on neutrophils and eosinophils], and, more recently, CD55, 0 5 8 , and CD59 [on erythrocytesl) were also tested on a cohort of 20 normal subjects and five patients with constitutional AA. Ham and sucrose tests were performed on the same day as flow-cytometric analysis. Six of 12 patients with A A , who secondarily developed a PNH clone, had clinical symptoms, while all eight patients with PNH had pancytopenia and/or thrombosis andlor hemolytic anemia. Cytogenetic features were normal in all but t w o patients. Proteins C and S, thrombomodulin, and antithrombin 111 levels were within the normal range in patients with PNH and in those with AA (with or without a PNH clone). In patients with PNH, CD16 and CD67 expression were deficient in 78% t o 98% of the cells and CD14 in 7696 t o 100Y0. By comparison, a GPI-linked defect was detected in 13 patients with AA, affecting a mean of 32% and 33% of CD16/CD67 and CD14 cell populations, re-CQUIRED APLASTIC ANEMIA (AA) is a heterogeneous disease, in which several pathophysiologic factors are involved.' In contrast to patients who undergo bone marrow transplantation (BMT), those who are successfully treated with immunosuppressive therapy (IST) are at risk for subsequently developing paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes, and acute myeloid l e~k e m i a .~.~ D e novo PNHh-' is an acquired clonal disorderY"" characterized by complement-mediated hemolysis and the expansion of affected cells of various hematopoietic lineages. The most typical manifestation of PNH is intravascular hemolysis due to abnormal sensitivity of red blood cells
Blood, 1999
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or a few clones of mutant hematopoietic stem cells defective in glycosylphosphatidylinositol (GPI) synthesis as a result of somatic mutation in the X-linked gene PIG-A. The mutant stem cell clone dominates hematopoiesis by a mechanism that is unclear. To test whether a lack of multiple GPI-anchored proteins results in dysregulation and expansion of stem cells, we generated mice in which GPI-anchor negative cells are present only in the hematopoietic system. We transplanted lethally irradiated mice with female fetal liver cells bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages contained GPI-anchor negative cells. The percentage of GPI-anchor negative cells was much higher in T lymphocytes includin...
Proceedings of the National Academy of Sciences, 1993
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cel...
Brazilian Journal of Medical and Biological Research, 2001
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal syndrome characterized by intravascular hemolysis mediated by complement, thrombotic events and alterations in hematopoiesis. Basically, the molecular events which underlie the complexity of the syndrome consist of the absence of the glycosylphosphatidylinositol (GPI) anchor as a consequence of somatic mutations in the PIG-A gene, located on the X chromosome. The GPI group is responsible for the attachment of many proteins to the cytoplasmic membrane. Two of them, CD55 and CD59, have a major role in the inhibition of the action of complement on the cellular membrane of blood cells. The absence of GPI biosynthesis can lead to PNH. Since mutations in the PIG-A gene are always present in patients with PNH, the aim of this study was to characterize the mutations in the PIG-A gene in Brazilian patients. The analysis of the PIG-A gene was performed using DNA samples derived from bone marrow and peripheral blood. Conformationsensitive gel electrophoresis was used for screening the mutation and sequencing methods were used to identify the mutations. Molecular analysis permitted the identification of three point mutations in three patients: one G®A transition in the 5 portion of the second intron, one T®A substitution in the second base of codon 430 (Leu430®stop), and one deletion DA in the third base of codon 63. This study represents the first description of mutations in the PIG-A gene in a Brazilian population.
Transfusion, 2008
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG-A) gene. In this study, the expression of a high number of GPI-APs on different subsets of peripheral blood (PB) cells from 14 PNH patients and their potential association with underlying genetic abnormalities has been analyzed. This study confirms the existence of variable patterns of expression of different GPI-APs on both major and minor PB-cell subsets from PNH patients. The size of the PNH clone within PB neutrophils and monocytes was systematically higher than that of other cell populations. Genetic changes were detected in the PIG-A gene in 5 of 13 cases analyzed. Interestingly, the reactivity for many GPI-APs was significantly higher on different subsets of normal PB cells from PNH patients than those observed on healthy volunteers. The best combination of markers for the diagnostic screening of PNH would include evaluation of CD14 on monocytes and of CD16 on neutrophils, although further analysis of CD55 and CD59 expression may contain additional clinically useful information. Clear association between the genetic changes detected in the PIG-A gene in 5 of 13 cases analyzed, and the phenotypic profile of PNH cells has not been found. Additionally, an abnormally higher expression of several GPI-APs among normal residual cells from PNH patients in comparison to healthy donors was observed, suggesting that factors other than the PIG-A mutation could determine the phenotypic profile of PB cells in PNH.
Leukemia, 2002
PNH is characterized by expansion of one or more stem cell clones with a PIG-A mutation, which causes a severe deficiency in the expression of glycosylphosphatidylinositol (GPI)-anchored proteins. There is evidence that the expansion of PIG-A mutant clones is concomitant with negative selection against PIG-A wild-type stem cells by an aplastic marrow environment. We studied 36 patients longitudinally by serial flow cytometry, and we determined the proportion of PNH red cells and granulocytes over a period of 1-6 years. We observed expansion of the PNH blood cell population(s) (at a rate of over 5% per year) in 12 out of 36 patients; in all other patients the PNH cell population either regressed or remained stable. The dynamics of the PNH cell population could not be predicted by clinical or hematologic parameters at presentation. These data indicate that in most cases the PNH cell expansion has already run its course by the time of diagnosis. In addition, since in most cases no further expansion takes place, we can infer that the tendency to overgrow normal cells is not an intrinsic property of the PNH clone.