Paroxysmal Nocturnal Hemoglobinuria Phenotype Research Articles

Decreased susceptibility of leukemic cells with PIG-Amutation to natural killer cells in vitro

AbstractThe cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or withoutPIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2–activated NK cells betweenPIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

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.

Red Cells with Paroxysmal Nocturnal Hemoglobinuria-phenotype in Patients with Acute Leukemia

CD55 and CD59 are complement regulatory proteins that are linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. They are reduced mainly in paroxysmal nocturnal hemoglobinuria (PNH) and in other hematological disorders. However, there are very few reports in the literature concerning their expression in patients with acute leukemias (AL). We studied the CD55 and CD59 expression in 88 newly diagnosed patients with AL [65 with acute non-lymphoblastic leukemia (ANLL) and 23 with acute lymphoblastic leukemia (ALL)] using the sephacryl gel test, the Ham and sucrose lysis tests and we compared the results with patients' clinical data and disease course. Eight patients with PNH were also studied as controls. Red cell populations deficient in both CD55 and CD59 were detected in 23% of ANLL patients (especially of M0, M2 and M6 FAB subtypes), 13% of ALL and in all PNH patients. CD55-deficient erythrocytes were found in 6 ANLL patients while the expression of CD59 was decreased in only 3 patients with ANLL. No ALL patient had an isolated deficiency of these antigens. There was no correlation between the existence of CD55 and/or CD59 deficiency and the percentage of bone marrow infiltration, karyotype or response to treatment. However no patient with M3, M5, M7 subtype of ANLL and mature B- or T-cell ALL showed a reduced expression of both antigens. The deficient populations showed no alteration after chemotherapy treatment or during disease course. This study provides evidence about the lower expression of CD55 and CD59 in some AL patients and the correlation with their clinical data. The possible mechanisms and the significance of this phenotype are discussed.

Proliferative capacity of single isolated CD34+ hematopoietic stem/progenitor cells in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations of the X-linked PIG-A (phosphatidylinositol glycan-class A) gene, which occurs on a hematopoietic stem cell level, leading to a proportion of blood cells being deficient in all glycosylphosphatidylinositol (GPI)-anchored surface proteins. Although these GPI-deficient cells can explain many of the clinical symptoms of PNH, the pathogenesis of PNH is still somewhat obscure and many questions remain. To assess the hematopoietic defect involved in PNH, CD34+ CD59+ (normal phenotype hematopoietic stem/progenitor) and CD34+ CD59- (PNH phenotype) cells from PNH patients (n = 16) and CD34+ CD59+ cells from healthy volunteers (n = 10) were sorted as single cells into 96-well flat-bottom culture plates containing culture medium supplemented with stem cell factor, interleukin (IL)-3, erythropoietin, granulocyte-macrophage-colony-stimulating factor (GM-CSF), G-CSF, IL-6, thrombopoietin, and Flt-3 ligand. We found that the single PNH CD34+ CD59- cells had a growth advantage over the single CD34+ CD59+ cells to some extent, but they both had impaired growth abilities compared with CD34+ cells from healthy volunteers.

Relative increase of granulocytes with a paroxysmal nocturnal haemoglobinuria phenotype in aplastic anaemia patients: the high prevalence at diagnosis.

To clarify the pathologic significance of granulocytes exhibiting the paroxysmal nocturnal haemoglobinuria (PNH) phenotype in patients with aplastic anaemia (AA), we examined peripheral blood from 100 patients with AA for the presence of granulocytes deficient in glycosylphosphatidylinositol (GPI)-anchored proteins using a sensitive flow cytometric assay. A significant increase in the frequency of CD55-CD59-CD11b+ granulocytes (>0.003%) compared to normal individuals was observed in 31 of 35 (88.6%) patients with untreated AA at diagnosis. The proportions of patients showing increased PNH granulocytes in treated AA patients with a short (<5 yr) and long (>5 yr) disease duration were 68.6% (11/16) and 20.4% (10/49), respectively. When 19 patients showing increased frequency of PNH granulocytes before therapy were studied 6-12 months after antithymocyte globulin plus cyclosporin A therapy, the frequency decreased to 0.01-90% of pretreatment values in 15 recovering patients. These findings suggest that a relative increase in the number of PNH granulocytes is a common feature of AA at diagnosis, and that it may represent the presence of immunologic pressure to normal haematopoietic stem cells as a cause of AA.

Detection of CD55- and/or CD59-deficient red cell populations in patients with lymphoproliferative syndromes.

Paroxysmal nocturnal hemoglobinuria is an acquired clonal stem cell disorder characterized by the decrease or absence of glycosylphosphatidylinositol-anchored molecules from the surface of the affected cells, such as CD55 and CD59, resulting in chronic intravascular hemolysis, cytopenia and increased tendency to thrombosis. PNH-phenotype has been described in various hematological disorders, mainly in aplastic anemia and myelodysplastic syndromes, while it has been reported that complete deficiency of CD55 and CD59 has also been found in patients with lymphoproliferative syndromes, like non-Hodgkin's lymphomas. The presence of CD55- and/or CD59-defective red cell populations was evaluated in 217 patients with lymphoproliferative syndromes. The study population included 87 patients with NHL, 55 with HD, 49 with CLL, 22 with ALL and four with hairy cell leukemia. One hundred and twenty-one healthy blood donors and seven patients with PNH were also studied as control groups. The sephacryl gel microtyping system was performed for the detection of CD55- and CD59-deficient red cell populations. Ham and sucrose lysis tests were also performed in all samples with CD55 or CD59 negative populations. Red cell populations deficient in both CD55 and CD59 molecules were detected in 9.2% of patients with lymphoproliferative syndromes (more often in ALL and nodular sclerosis type of HD) and in all PNH patients. CD55-deficient red cell populations were found in 8.7% of LPS patients (especially in low grade B-cell NHL), while CD59-deficient populations were found in only two patients with low grade B-cell NHL. These data indicate a possible association between paroxysmal nocturnal hemoglobinuria phenotype and lymphoproliferative syndromes, while further investigation is necessary to work out the mechanisms and the significance of the existence of this phenotype in these patients.

Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

Coexistence of normal and clonal haemopoiesis in aplastic anaemia patients treated with immunosuppressive therapy

Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow-up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI-AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG-AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG-AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively). This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow-up of 4 years.

The PNH phenotype cells that emerge in most patients after CAMPATH-1H therapy are present prior to treatment.

Paroxysmal nocturnal haemoglobinuria (PNH) cells are deficient in glycosylphosphatidylinositol (GPI) linked antigens due to a somatic mutation of the PIG-A gene in a haemopoietic stem cell. It appears that a PNH clone reaches detectable proportions only when there is selection in its favour. GPI-deficient T lymphocytes have been identified in patients treated with CAMPATH-1H, a monoclonal antibody against the GPI-linked CD52 molecule. CAMPATH-1H selects for cells that are deficient in CD52 (such as PNH-like cells) promoting the development of a PNH-like clone (analogous to PNH). We report that 10/15 patients with chronic lymphocytic leukaemia developed PNH-like lymphocytes after therapy with CAMPATH-1H. The remaining five patients developed no PNH-like cells at any stage, including one patient who received 12 weeks of therapy. The inactivating PIG-A mutation has been identified in one patient. This mutation was detectable by an extremely sensitive mutation-specific PCR-based analysis in the patient's mononuclear cells prior to CAMPATH-1H therapy. The frequency and phenotype of GPI-deficient lymphocytes after CAMPATH-1H and the detection of a PIG-A mutation in the lymphocytes prior to CAMPATH-1H therapy indicated that such mutations were present in a very small proportion of cells prior to selection in their favour by CAMPATH-1H. This suggests that a large proportion of individuals have cells with PIG-A mutations that are not detectable by flow cytometry and thus may have the potential to develop PNH.

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals.

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.

Paroxysmal nocturnal haemoglobinuria clones in patients with myelodysplastic syndromes.

Among acquired stem cell disorders, pathological links between myelodysplastic syndromes (MDS) and aplastic anaemia (AA), and paroxysmal nocturnal haemoglobinuria (PNH) and AA, have been often described, whereas the relationship between MDS and PNH is still unclear. We analysed blood cells of patients with MDS to determine the incidence of the PNH clone, and analysed the PIG-A gene to find mutations characteristic of the PNH clone in MDS. In four (10%) of 40 patients with MDS, flow cytometry showed affected erythrocytes and granulocytes negative for decay-accelerating factor (DAF) and CD59. The population of affected erythrocytes was smaller in MDS patients with PNH clone (MDS/PNH) than in patients with de novo PNH, and haemolysis was milder in the MDS/PNH patients. PIG-A mutations were found in granulocytes of all patients with MDS/PNH. In type and site, the PIG-A mutations were heterogeneous, similar to that observed in de novo PNH; i.e. no mutation specific to MDS/PNH was identified. Of note, three of four patients with MDS/PNH each had two PNH clones with different PIG-A mutations, suggesting that PIG-A is mutable in patients with MDS/PNH. In a MDS/PNH patient with trisomy 8, FISH detected a distinct karyotype in a portion of granulocytes with PNH phenotype, indicating that PNH and MDS partly shared affected cells. Thus, MDS predisposes to PNH by creating conditions favourable to the genesis of PNH clone. Considering the increasing prevalence and incidence of MDS, these disorders could be useful for investigating the mechanism by which PIG-A mutation is induced.

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.

Antibody selection against CD52 produces a paroxysmal nocturnal haemoglobinuria phenotype in human lymphocytes by a novel mechanism.

The CD52 antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.

ANALYSIS OF THE EXPRESSION OF GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORED PROTEINS ON PLATELETS FROM PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal disorder is manifested by failure of hematopoietic cells to express phosphatidylinositol glycan-anchored proteins (PIG-AP). Since the PIG-A mutation is present at the stem cell level, all cell lines may be affected. Although the pathogenesis of hemolytic anemia in PNH is related to the absence of CD55 and CD59 molecules on the membrane of red cells, the mechanism responsible for the increased incidence of thrombotic events in PNH is not clear. In this study we measured two glycosylphosphatidylinositol (GPI)-linked molecules on platelets (CD55 and CD59) and two GPI-linked proteins on neutrophils (CD14 and CD16), comparing their expression on normal and PNH patients. Using two-color flow cytometric analysis with antibodies directed against CD42b and CD41a, we found that CD55 and CD59 were constitutively expressed by normal fresh platelets, but that the expression levels decreased during the five day storage of platelets. A substantial population of platelets lacking the GPI-linked proteins were detected in most cases. We demonstrated varying degrees of deficiency in the expression of GPI-anchored molecules with neutrophils, monocytes and platelets with the highest proportion of deficient cells found within monocytic lineage. Similar numbers of platelets with the PNH phenotype and normal platelets expressed activation markers before and after exposure to platelet agonists. Flow cytometry is more sensitive than Ham's test in monitoring expression of PNH in platelets. Differences in the numbers of circulating GPI-deficient platelets and myeloid cells suggest that either the survival of platelets and mature myeloid cells differs or megakaryocytopoeisis is abnormal within the GPI-deficient clone.

Preferential Hematopoiesis by Paroxysmal Nocturnal Hemoglobinuria Clone Engrafted in SCID Mice

In paroxysmal nocturnal hemoglobinuria (PNH), little is known about the molecular events leading to the clinical manifestations except for the hemolysis. To unfold the complex pathophysiology, it is necessary to elucidate the nature of the PNH clone. PNH exhibits an acquired stem cell disorder, a clonal expansion of affected cells, concomitant depression of normal hematopoiesis in bone marrow (BM), and, although infrequently, the development of leukemia. The PNH clone is thus expected to exhibit some neoplastic features. We report here that CD34+ hematopoietic progenitor cells of PNH-BM yielded blood cells of three lineages with PNH phenotype alone when transplanted into sublethally irradiated severe combined immunedeficient mice. The hematopoiesis persisted for more than 10 months and did not always need human cytokines. In contrast, the hematopoiesis by control grafts obtained from healthy volunteers required an intense cytokine treatment. This in vivo model defines the preferential hematopoiesis of pluripotent PNH progenitor cells, indicating the intrinsic growth abnormality of PNH clone.

Acute leukaemia in paroxysmal nocturnal haemoglobinuria. Case report and review of the literature

Paroxysmal nocturnal haemoglobinuria (PNH) terminating in acute leukaemia (AL) is an infrequent condition. In several cases, flow cytometric analysis of glycosylphosphatidylinositol anchored membrane proteins such as DAF and CD59/MACIF has suggested the leukaemic cells to be derived from the PNH clone, thereby implicating PNH as a potential preleukaemic disease. In the present paper, we review the data for one patient treated in our hospital and 20 cases reported in the literature from 1969 to 1993. The sex ratio is 1 female/2 males, mean age at diagnosis of PNH was 46 years and the mean interval between the diagnoses of PNH and AL was 53 months. AL type was AML M6 in 8 patients, other types of AML in 12 and ALL in one, with a mean survival of 7.1 months following diagnosis of AL. In all cases analyzed, the PNH phenotype of erythrocytes disappeared with progression of AL, whereas reappearance of this phenotype with complete remission of AL was inconstant. PNH would thus appear to be a potential preleukemic disease. When this disorder terminates in AL, the type is often AML M6, although ALL is also possible. The prognosis of AL in PNH is poor as for other secondary leukaemias. Apart from marrow aplasia, leukaemic transformation is another life threatening complication of PNH which may justify allogeneic bone marrow transplantation (allo-BMT) and potential leukaemic transformation can therefore be an additional argument in favour of allo-BMT when pancytopenia develops in PNH patients.

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.

Clonal hematopoiesis as defined by polymorphic X-linked loci occurs infrequently in aplastic anemia

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.

Clonal Hematopoiesis as Defined by Polymorphic X-Linked Loci Occurs Infrequently in Aplastic Anemia

We evaluated the methylation status of the X-linked gene phosphoglycerate kinase (PGK1) and the DXS 255 locus detected by probe M27 beta to study clonality in acquired aplastic anemia (AA). A total of 30 females were suitable for clonal analysis of peripheral blood polymorphonuclear cells (PMN) and mononuclear cells using a polymerase chain reaction-based procedure in 24 patients and Southern blotting in 9. Overall, 10 of 30 patients exhibited an imbalanced X-inactivation pattern. However, in 4 patients, analysis of constitutional DNA suggested a skewed methylation pattern and 2 further cases had to be excluded because of the lack of an appropriate control. A truly clonal pattern was thus established in 4 of 30 (13%) patients. In 7 patients who later developed clonal disorders of hematopoiesis, X-inactivation analysis did not predict this event in any case. In patients with a paroxysmal nocturnal hemoglobinuria phenotype, there was no correlation between the proportion of phosphatidylinositol glycan anchored protein (PIG-AP)-deficient blood cells and the corresponding X-inactivation pattern. X-inactivation analysis detected clonal hematopoiesis in only 3 of 10 patients with a deficiency in PIG-AP in the cell population under study, but sorting of nucleated cells on the basis of PIG-AP expression showed the clonal nature of PIG-AP-deficient cells. We conclude that the majority of patients with AA show polyclonal hematopoiesis using X-linked clonal analysis, but that minor clonal populations, such as PIG-AP-deficient cells, may not be detected unless sorted cell populations are separately analyzed.