the overlap of idiopathic aplastic anemia (AA) and paroxys- ... Since aplastic anemiaâparoxysmal nocturnal hemoglobinuria syndrome was reported in 1967, the ...
International Journal of
Progress in Hematology
HEMATOLOGY
Relationship Between Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria Taroh Kinoshita,a Norimitsu Inoueb a
b
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, and Department of Molecular Genetics, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Received December 7, 2001; accepted December 11, 2001
Abstract 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. Int J Hematol. 2002;75:117-122. ©2002 The Japanese Society of Hematology Key words: Aplastic anemia; Paroxysmal nocturnal hemoglobinuria; Hematopoietic stem cell; Somatic mutation; Autoimmune disease
PNH is an acquired hematopoietic stem cell disorder characterized by the PNH clone that appears in various hematopoietic lineages. Clonal PNH erythrocytes are abnormally sensitive to complement, leading to intravascular hemolysis and hemoglobinuria [7]. PNH clones appearing in patients with AA actively participate in hematopoiesis that is beneficial to these patients [8]. Here, we summarize the molecular basis of PNH, review reports dealing with linkage between AA and PNH, and discuss possible mechanistic links between AA and PNH.
1. Introduction Since the 1967 report by Lewis and Dacie [1] on aplastic anemia–paroxysmal nocturnal hemoglobinuria syndrome, the overlap of idiopathic aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) has been well known. The link between these 2 diseases became even more evident when survival of patients with severe AA was improved by immunosuppressive therapy [2-4]. Idiopathic AA is acquired pancytopenia with a fatty bone marrow in that the number of hematopoietic stem cells is severely reduced. The most likely mechanism of the bone marrow failure is immunologically mediated destruction of the hematopoietic stem cell pool [5,6]. Although the immune mechanism of AA is supported by various lines of clinical and laboratory evidence, the immune effectors and autoantigens responsible have yet to be determined.
2. Molecular Basis of PNH PNH is an acquired hematopoietic stem cell disorder characterized by the presence of clonal population of abnormal cells in multiple hematopoietic lineages. The abnormal cells are defective in the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. CD59 and decayaccelerating factor (DAF or CD55) are widely distributed GPI-anchored proteins that inhibit activation of complement on the host cell surface, thereby protecting cells from the destructive action of complement. Abnormal erythrocytes lacking CD59 and DAF are very sensitive to complement
Correspondence and reprint requests: Taroh Kinoshita, PhD, Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; 81-6-6879-8328; fax: 81-6-6875-5233.
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and are lysed during infections and other events that activate complement. The lack of GPI-anchored proteins is due to defective biosynthesis of GPI in the endoplasmic reticulum. In affected blood cells from patients with PNH (PNH cells), the first reaction of GPI biosynthesis is defective, resulting in a lack of attachment of GPI anchor to various proteins and subsequent defective surface expression of those proteins [7]. The first reaction of GPI biosynthesis, an addition of N-acetylglucosamine to phosphatidylinositol, is mediated by GPI N-acetylglucosamine transferase, which consists of 6 proteins: PIG-A, PIG-C, PIG-H, PIG-P, GPI1, and DPM2 [9]; among them, PIG-A is a catalytic component [10]. In PNH cells, activity of this enzyme is absent or greatly decreased. The defect in the first enzyme is due to an abnormality in PIG-A that is caused by somatic mutation in the PIG-A gene [11]. PIG-A is X-linked; therefore, one inactivating somatic mutation in PIG-A should cause defective GPI biosynthesis in male cells and in female cells as well because of X-chromosome inactivation [11]. The PIG-A somatic mutation would occur in the long-lasting multipotential hematopoietic stem cells because the same mutation is found in granulocytes and lymphocytes [11], and because the same PNH clone supports hematopoiesis for at least 6 years [12].
3. PIG-A Mutation Alone Does Not Cause Clonal Expansion In patients with PNH, clonal GPI-anchor–deficient cells occupy a significant fraction of peripheral blood cells and bone marrow cells. Nearly complete occupancy of granulocytes by PNH clone is not rare. Dominance of GPIanchor–deficient cells is seen in CD34+ and CD38– cells in bone marrow, implying that the clonal expansion occurs at the stem cell level [13]. The clonal expansion is an essential step in pathogenesis of PNH because the PIG-A mutant clone would not cause clinical symptoms without expansion. Several lines of evidence support the idea that PIG-A mutation alone does not cause the clonal expansion. First, the extent of clonal expansion varies among patients who have similar null mutations in PIG-A [14]. Therefore, the extent to which the PIG-A mutant clone expands is influenced by a factor other than PIG-A mutation. Second, somatic mutation of PIG-A is found in a very small fraction (average of 0.002%) of granulocytes from most healthy individuals [15], suggesting that some factor required for the clonal expansion does not exist in healthy individuals. Third, GPI-anchor–deficient mouse hematopoietic stem cells generated by disrupting Pig-a gene, the orthologue of PIG-A, did not expand [16-21]. Therefore, the factor responsible for the clonal expansion is also essential for development of PNH.
4. Overlap of AA and PNH A number of years after the introduction of immunosuppressive therapy to treat patients with severe AA, a late complication with PNH was noted. Three reports published
in 1988-1990 indicated that 4% to 9% of more than 700 patients with AA developed PNH diagnosed by classical hemolytic tests (Table 1) [2-4]. Studies with flow cytometric analysis of GPI-anchored proteins published in 1994-1995 reported that 35% to 52% of AA patients had PNH cells (granulocytes and/or erythrocytes) at a level of 1% or higher [22-24]. Similar studies published in 1998-1999 reported 15% to 29% [25-27]. More recent studies designed to detect a smaller population of GPI-anchored protein–deficient cells demonstrated that 67% to 89% of untreated patients with AA had granulocytes of PNH phenotype [28,29]. Somatic mutation of PIG-A gene was also demonstrated in GPIanchor–deficient cells in patients with AA, showing the common genetic basis [24,26,30].
5. Possible Mechanistic Link Between AA and PNH: Hypotheses Based on the highly frequent overlap of PNH and AA, and the marrow failure commonly seen in patients with PNH, Rotoli and Luzzatto [31] proposed that PNH clones may survive pathologic bone marrow conditions in patients with AA. Young [32] proposed that the responsible pathologic conditions that positively select the PNH clone may be cytotoxic lymphocytes suspected to play a role in development of AA. As suggested by the fact that normal individuals have GPI-anchor–deficient granulocytes bearing the PIG-A mutation [15], somatic mutation of PIG-A occurring in hematopoietic stem cells, resulting in generation of GPIanchor–deficient hematopoietic stem cell clones, may not be so rare. When this mutation happens in normal individuals, the GPI-deficient clone would remain as a very minor population because conditions would not select for the clone, and because hematopoiesis is supported by many clones of the hematopoietic stem cells. When this mutation happens in individuals having autoreactive cytotoxic lymphocytes, such as patients with AA, and if the GPI-anchor–deficient hematopoietic stem cells are more resistant to cytotoxic lymphocytes than are normal hematopoietic stem cells, then the PIG-A mutant clone would become a large enough population to be detected by laboratory tests and even to have clinical significance. Recently, Young and Maciejewski [33] and Karadimitris and Luzzatto [34] summarized and discussed hypothetical mechanisms of resistance of GPI-anchor–deficient cells to cytotoxic lymphocytes. Here, incorporating some new information, we discuss all the possible mechanisms. 1. GPI-anchored proteins on hematopoietic stem cells may be important for cytotoxic lymphocytes to effectively kill them; some GPI-anchored proteins are important for effector-to-target interaction or are ligands of costimulatory molecules on effector T lymphocytes. Figure 1A shows this model. If the presence of some GPI-anchored protein is important for efficient stimulation of effector T lymphocytes, GPI-anchor–deficient hematopoietic stem cells should be more resistant to cell death. However, results of experiments that compared GPI-positive and GPI-negative cells for their sensitivities to cytotoxic lymphocytes and for their abilities to stimulate T lymphocytes are controversial. GPI-positive and GPI-negative clones of JY B-lymphoblastoid cell line were
Relationship Between Aplastic Anemia and PNH
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Table 1. Presence of GPI-Deficient Cells in Patients With Aplastic Anemia No. of Patients 137 486 156 29
52
37 111 73 33 82 115 35 49 16 100 18 23
Cell Types Studied*
Therapy†
Total
No. of Patients With GPI-Deficient Cells (%) Lab Test 0.003%-1% >1% Positive
13 (9) 19 (4) 13 (8) G M Either G M L E Either Either G and E G
G
G E
13 (9) 15 (3) 13 (8)
12 (41)
27 (52) 17 (15) 21 (29) Untreated After IST Total Untreated After IST (>5 yrs) After IST (0.003% to 1% of granulocytes being GPI negative [28] because of a decreased number of hematopoietic stem cells. Step 2 is selection by immunologic attack on the hematopoietic stem cells. When this immunologic attack occurs in normal individuals having a PIG-A mutant stem cell, the number of GPI-negative cells would increase, and the number of normal stem cells would decrease. If GPI-negative red cells exceed several percent, they may be detectable by the Ham test. If the number of GPI-negative cells increases to 10% to 20%, the patient may show clinical PNH. In patients with AA, if an immunologic attack is still active, a PIG-A mutant would immediately be subjected to selection. Because of the severely decreased stem cell number, the number of GPI-negative cells may reach 10% to 30%, a common range for the number of PNH cells seen in patients with AA [26,50]. If the immunologic attack has already disappeared or is reduced at the time of somatic PIG-A mutation, the extent of expansion should be lower. We think that selection may not be sufficient to account for so-called “florid PNH,” namely, nonbicytopenic PNH with a highly expanded PIG-A mutant clone. We imagine that a PNH clone supporting the major part or nearly all of hematopoiesis is like a benign tumor having a growth phenotype. Because PIG-A mutation alone does not seem to confer a growth phenotype, such a PNH clone should have an additional mutation relevant to a growth phenotype. Under conditions of decreased hematopoiesis, the remaining stem cells would be forced to proliferate. This proliferation would increase the chance of additional genetic alteration occurring in the PIG-A mutant clone. Step 3 in development of PNH would be the second genetic change that generates a subclone having a growth phenotype (Figure 2). Consistent with this idea, upregulated expression of EGR-1 gene, one of
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the zinc finger nuclear transcription factor genes, has been found in all PNH cases analyzed [51]. Identification of the second somatic mutation is critical to prove this model.
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