PIG-A mutations in normal hematopoiesis

Johns Hopkins University, School of Medicine, Division of Hematology, Baltimore, MD 21205, USA.
Blood (Impact Factor: 10.45). 06/2005; 105(10):3848-54. DOI: 10.1182/blood-2004-04-1472
Source: PubMed


Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.

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    • "We have reported that in granulocytes from normal donors [13], the mean frequency of spontaneously arising GPI (−) cells is about 22 × 10 −6 . We have detected PIG-A mutations in these cells, and others have confirmed these finding in bone marrow cultures from normal individuals [19]. A further advantage of using PIG-A as a mutation reporter gene is that pre-existing mutants can be eliminated by flow cytometry, which allows the measurement of as well as f. "
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    ABSTRACT: The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼ 9.4×10(-7) (range ∼ 3.6 to 23×10(-7)) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis.
    Full-text · Article · Jun 2013 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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    • "Moreover, alkaline phosphatase and renal dipeptidase are also known as catalytically active enzymes [4] [5]. Due to their importance, loss of GPI-protein expression, either by defects in expression of single proteins (decay accelerating factor, DAF) or malfunctioning biosynthesis of GPI-anchors, lead to drastic patterns of disease or lethality in several systems [6] [7]. Attachment of proteins to the membrane by GPI versus transmembrane domains may provide several advantages. "
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    ABSTRACT: Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.
    Full-text · Article · Jan 2012 · Biochimica et Biophysica Acta
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    • "Paroxysmal nocturnal hemoglobinuria (PNH), first described in 1882, is a hematological disorder of complex physiopathology which leads to multiple clinical disorders and complications. Its exact incidence is unknown but it equally affects both genders and occurs at any age, but mainly in young adults with a mean age of 35 years old.(1,2) "
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    ABSTRACT: Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59). To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009. Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens. Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years. These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59).
    Full-text · Article · Mar 2011 · Revista Brasileira de Hematologia e Hemoterapia
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