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3.
Leukemia ; 16(11): 2243-8, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12399968

ABSTRACT

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.


Subject(s)
Hematopoiesis , Hemoglobinuria, Paroxysmal/physiopathology , Adolescent , Adult , Bone Marrow/pathology , CD59 Antigens/metabolism , Child , Clone Cells , Erythrocytes/pathology , Female , Flow Cytometry , Granulocytes/pathology , Hematopoietic Stem Cells/chemistry , Humans , Longitudinal Studies , Male , Middle Aged
4.
Br J Haematol ; 115(2): 360-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11703336

ABSTRACT

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of a haematopoietic stem cell clone with a PIG-A mutation (the PNH clone) in an environment in which normal stem cells are lost or failing: it has been hypothesized that this abnormal marrow environment provides a relative advantage to the PNH clone. In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal, unlike in myelodysplastic syndrome (MDS), another clonal condition in which cytogenetic abnormalities are regarded as diagnostic. In a retrospective review of 46 patients with a PNH clone, we found a karyotypic abnormality in 11 (24%). Upon follow-up, the proportion of cells with abnormal karyotype decreased significantly in seven of these 11 patients. Abnormal morphological bone marrow features reminiscent of MDS were common in PNH, regardless of the karyotype. However, none of our patients developed excess blasts or leukaemia. We conclude that in patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukaemia.


Subject(s)
Chromosome Aberrations , Hemoglobinuria, Paroxysmal/genetics , Adolescent , Adult , Female , Follow-Up Studies , Hematopoietic Stem Cells/pathology , Hemoglobinuria, Paroxysmal/pathology , Hemoglobinuria, Paroxysmal/therapy , Humans , Karyotyping , Male , Middle Aged , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Retrospective Studies , Treatment Outcome
5.
Br J Haematol ; 115(4): 1010-4, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11843843

ABSTRACT

There is mounting evidence to suggest that T-cell-mediated suppression of haemopoiesis is a pathogenetic mechanism in three bone marrow failure syndromes: aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplasia (MDS). T-cell microclones can be detected by sensitive polymerase chain reaction (PCR)-based methods in all three disorders. Recently, larger clonal populations of T-cell large granular lymphocytes (T-LGLs) have been observed in some patients with AA and MDS. Here, we report the development of a large clonal T-LGL population in a patient with bona fide PNH. In this patient, we defined part of the sequence of the T-cell receptor (TCR) beta-chain gene, and we have shown that the large T-LGL population emerged from a background of multiple smaller T-cell clones. Thus, T-LGL clones in AA, MDS and PNH probably expand as a result of antigenic stimulation. It is postulated that the antigen driving clonal T-cell proliferations in these disorders exists on haemopoietic stem cells.


Subject(s)
Hemoglobinuria, Paroxysmal/immunology , Leukemia, T-Cell/complications , T-Lymphocytes/pathology , Adult , Anemia, Aplastic/immunology , Cell Division , Clone Cells , Coculture Techniques , Female , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/pathology , Hemoglobinuria, Paroxysmal/genetics , Humans , Leukemia, T-Cell/genetics , Leukemia, T-Cell/immunology , Polymerase Chain Reaction/methods
6.
Blood ; 96(7): 2613-20, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-11001919

ABSTRACT

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in the PIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R(2) values 0.82 vs 0.91, P <.001). The mean frequency of skewed families per individual was increased by more than 2-fold in patients with PNH, compared with controls (28% +/- 19.6% vs 11.4% +/- 6%, P =.002). In addition, several TCR BV families were significantly more frequently skewed in patients with PNH than in controls. These findings provide experimental support for the concept that PNH, like IAA, has an immune pathogenesis. In addition, the identification of expanded T-cell clones by CDR3 size analysis will help to investigate the effect of HSC-specific T cells on normal and PNH HSCs.


Subject(s)
Genes, T-Cell Receptor beta , Hemoglobinuria, Paroxysmal/immunology , T-Lymphocytes/immunology , Adolescent , Adult , Female , Hematopoietic Stem Cells/immunology , Hemoglobinuria, Paroxysmal/genetics , Humans , Immunoglobulin Variable Region/genetics , Male , Middle Aged , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/chemistry , Sequence Analysis, RNA
8.
Proc Natl Acad Sci U S A ; 96(9): 5209-14, 1999 Apr 27.
Article in English | MEDLINE | ID: mdl-10220445

ABSTRACT

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 flow 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 flow 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.


Subject(s)
Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/physiology , Hemoglobinuria, Paroxysmal/genetics , Hemoglobinuria, Paroxysmal/pathology , Membrane Proteins/genetics , Adult , Aged , Clone Cells , Flow Cytometry , Hemoglobinuria, Paroxysmal/blood , Humans , Male , Middle Aged , Mutation , Phenotype
9.
Transplantation ; 55(1): 76-82, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8420068

ABSTRACT

An adult recipient of an HLA-DR, DQ-mismatched, T cell-depleted bone marrow graft, who remains without graft versus host disease and who is not maintained on immunosuppressive therapy, was studied at 23 months posttransplantation for in vitro reactivity against the mismatched antigens of the host. The donor's PBMC's proliferated vigorously against the recipient's stimulators in the pretransplant mixed lymphocyte cultures (MLC). After transplant reconstitution, MLCs demonstrated that the in vitro response of engrafted donor T cells against host MHC class II antigens was equivalent to control allogeneic responses, while there was no detectable response against the donor's antigens. Posttransplantation limiting dilution analysis showed no difference between the precursor frequencies of antihost responders among populations of fresh donor PBMCs and among the engrafted cells of donor origin that are found circulating in the patient. This result suggests that clonal deletion is, at best, incomplete and that peripheral tolerance is essential in protecting this patient from GVHD. These findings also support the conclusion that bone marrow-derived thymic elements may be important for clonal deletion in human chimeras.


Subject(s)
B-Lymphocytes/immunology , Bone Marrow Transplantation/immunology , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , Histocompatibility/immunology , Immune Tolerance/immunology , Leukemia, Promyelocytic, Acute/immunology , Transplantation Immunology/immunology , Adult , HLA-DQ Antigens/immunology , HLA-DR Antigens/immunology , Haplotypes , Humans , In Vitro Techniques , Leukemia, Promyelocytic, Acute/surgery , Lymphocyte Culture Test, Mixed , Male
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