Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by clonal blood cells that are deficient in glycosylphosphatidylinositol-anchored proteins because of somatic mutations of the PIG-A gene. Many patients with PNH have more than one PNH clone, but it is unclear whether a single PNH clone remains dominant or minor clones eventually become dominant. Furthermore, it is unknown how many hematopoietic stem cells (HSCs) sustain hematopoiesis and how long a single HSC can support hematopoiesis in humans. To understand dynamics of HSCs, we reanalyzed the PIG-A gene mutations in 9 patients 6 to 10 years after the previous analyses. The proportion of affected peripheral blood polymorphonuclear cells (PMNs) in each patient was highly variable; it increased in 2 (from 50% and 65% to 98% and 97%, respectively), was stable in 4 (changed less than 20%), and diminished in 3 (94%, 99%, and 98% to 33%, 57%, and 43%, respectively) patients. The complexity of these results reflects the high variability of the clinical course of PNH. In all patients, the previously predominant clone was still present and dominant. Therefore, one stem cell clone can sustain hematopoiesis for 6 to 10 years in patients with PNH. Two patients whose affected PMNs decreased because of a decline of the predominant PNH clone and who have been followed up for 24 and 31 years now have an aplastic condition, suggesting that aplasia is a terminal feature of PNH.

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by intravascular hemolytic anemia.1-3 Abnormal blood cells are deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs).4,5 In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective.4 At least 5 genes are involved in this reaction step,6 and one of them, an X-linked gene termedPIG-A, is mutated in affected cells.7-9 ThePIG-A gene is mutated in every patient with PNH reported to date, and deficiency of GPI in PNH has thus been considered to result solely from the PIG-A mutation(s).4,9 

Many patients with PNH have more than one PNH clone.10-15How the affected stem cell clone comes to dominate hematopoiesis is a current issue. It is also unclear whether the predominant clone is maintained or a minor clone eventually overcomes. It is unknown how many hematopoietic stem cells (HSCs) sustain hematopoiesis and how long one HSC could support hematopoiesis in humans. To understand the dynamics of HSCs, we re-analyzed the PIG-Agene mutations in 9 patients 6 to 10 years after the previous analyses.9,11,15,16 

Patients, materials, and methods

Patients and blood samples

We examined blood samples from 9 patients with PNH. All the patients were described elsewhere and had somatic PIG-A mutations.9,15,16 Diagnoses of PNH and aplastic anemia (AA) were made by the diagnostic criteria of the Ministry of Health and Welfare of Japan. Patients who had AA before clinically apparent PNH developed received a diagnosis of AA/PNH syndrome. The diagnosis of PNH aplasia (PNH progressed to aplasia in 2 patients) was made by our own criteria in the patients whose tri-lineage blood cell counts were reduced more than 20% from the original counts. With the informed consent of the patients, peripheral blood (PB) was obtained from each patient, and a bone marrow (BM) sample was also obtained from patient J19 6 to 10 years after the previous analysis. Polymorphonuclear cells (PMNs) and mononuclear cells (MNCs) were separated by sedimentation in 6% dextran and Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifugation. Approval was obtained from the Institutional Review Board at Osaka University for these studies. Informed consent was provided according to the Declaration of Helsinki.

Fluorescence-activated cell sorter analysis

The cells in the fractions of PBPMNs and BMMNCs were stained with fluorescein isothiocyanate– or phycoerythrin-conjugated anti-CD59 monoclonal antibodies (PharMingen, San Diego, CA) for the detection of GPI-APs. After staining, cells were analyzed with a FacsCalibur (Becton Dickinson, Bedford, MA). Cell types were determined by their forward and right scatter, similarly to the previous analysis.9 In 3 patients, the change of percentage GPI-AP PMNs from previous analysis was within 20% (categorized as stable). Two patients, in whom percentage GPI-AP PMNs increased more than 40%, were categorized as increased. Three patients, in whom the percentage GPI-AP PMNs decreased more than 40%, were categorized as decreased.

PIG-A gene analysis

DNA was isolated from the fractions of PBPMNs and BMMNCs. The coding regions of PIG-A were amplified by polymerase chain reaction (PCR) in 5 fragments using the primer sets described previously15 and were cloned into pBluescript II. Subcloned products containing PIG-A fragments were then amplified again by PCR using the same primer sets for heteroduplex analysis with mutation detection enhancement gel (Hydrolink; AT Biochem, Malvern, PA).15 If a region containing a mutation was suspected, clones were sequenced using dideoxy chain termination and a model 377 DNA sequencer (Applied Biosystems, Foster City, CA). In each patient, the mutation ratio was counted as mutant amplified subclones (AS) out of analyzed AS.

Results

Changes in proportion of GPI-AP cells

To investigate the pattern of clonal dominance, we re-analyzed the surface expression of GPI-APs on PBPMNs from 9 patients with PNH 6 to 10 years after the previous analysis. We described all the patients elsewhere9,15,16 and summarized the previous data (Tables1 and2). The percentage of PBPMNs deficient in CD59 (a measure of clonal hematopoiesis) was 74.5% ± 24.5% (medium ± range) at the previous analysis and 65.5% ± 32.5% at the current analysis (difference not significant;P = .2, Wilcoxon-signed rank test) (Figure1). However, a change in the proportion of CD59-deficient PMNs was highly variable from patient to patient; it increased in 2 (50% to 98% in J4 and 65% to 97% in J5), was stable in 4 (97% to 93% in J3, 83% to 82% in J11, 87% to 73% in J13, and 78% to 61% in J19), and diminished in 3 patients (94% to 33% in J12, 99% to 57% in J15, and 98% to 43% in J16) (Table 2).

Table 1.

Summary of laboratory data and clinical course in 9 patients with PNH at the previous analysis and the current analysis

PatientSex/age (y)Laboratory data (Previous/current)Clinical course
Hb (g/dL)WBC (/μL)PMN (/μL)(CD59PMN) (/μL)Plt (× 109/L)LDH (U/L)Duration (y)
J4 7.8 6300 5090 2545 313 1840 11 
 56 9.4 5610 3574 3503 214 4342  
J5 12.3 6500 3835 2453 198 3260 
 39 8.5 5100 3162 3067 194 1830  
J3 6.9 3100 1783 1730 270 4359 11 
 53 8.0 3400 2108 1960 271 2281  
J11 4.6 1870 822 652 128 1084 12 A-P 
 46 3.8 1820 791 649 108 1785  A-P 
J13 7.8 2900 1102 758 289 4872 
 69 4.9 2600 468 342 333 2816  
J19 11.5 5780 3109 2425 248 2423 14 
 70 9.0 5130 1615 985 288 1689  
J12 10.5 4930 3879 3646 175 862 17 A-P → P 
 50 13.5 5010 3181 1050 118 383  
J15 7.1 3460 1647 1631 109 2772 31 
 58 4.2 1830 805 459 59 ND  PA 
J16 7.5 4400 2948 2889 90 5364 24 A → P 
 62 5.8 1900 1178 507 287  PA 
PatientSex/age (y)Laboratory data (Previous/current)Clinical course
Hb (g/dL)WBC (/μL)PMN (/μL)(CD59PMN) (/μL)Plt (× 109/L)LDH (U/L)Duration (y)
J4 7.8 6300 5090 2545 313 1840 11 
 56 9.4 5610 3574 3503 214 4342  
J5 12.3 6500 3835 2453 198 3260 
 39 8.5 5100 3162 3067 194 1830  
J3 6.9 3100 1783 1730 270 4359 11 
 53 8.0 3400 2108 1960 271 2281  
J11 4.6 1870 822 652 128 1084 12 A-P 
 46 3.8 1820 791 649 108 1785  A-P 
J13 7.8 2900 1102 758 289 4872 
 69 4.9 2600 468 342 333 2816  
J19 11.5 5780 3109 2425 248 2423 14 
 70 9.0 5130 1615 985 288 1689  
J12 10.5 4930 3879 3646 175 862 17 A-P → P 
 50 13.5 5010 3181 1050 118 383  
J15 7.1 3460 1647 1631 109 2772 31 
 58 4.2 1830 805 459 59 ND  PA 
J16 7.5 4400 2948 2889 90 5364 24 A → P 
 62 5.8 1900 1178 507 287  PA 

Nine patients were subdivided into 3 groups: increased (J4, J5); stable (J3, J11, J13, J19); and decreased (J12, J15, J16).

In each patient, the previous laboratory data and the clinical diagnosis were described in the upper row, and the current laboratory data and the clinical diagnosis were described in the lower row.

M indicates Male; F, Female; Hb, hemoglobin; WBC, white blood cell; Plt, platelet; LDH, lactate dehydrogenase; Duration, the period between the diagnosis and the current analysis; P, PNH; A-P, AA-PNH syndrome; A, aplastic anemia; ND, not done; PA, PNH aplasia.

Table 2.

Summary of CD59 expressions and somatic mutations of PIG-A in 9 patients with PNH at the previous analysis and the current analysis

PatientMutationPrevious analysisCurrent analysisDuration (y)
PB PMNBM Colonies/bursts DNA (AS)PB PMNBM MNC DNA (AS)
CD59(%)mRNA (AS)CD59 (%)DNA (AS)
J4 298C to T 50 11/22 ND 98 3/10 ND 
 273C to A  0/10 ND  2/10 ND  
J5 1309C del 65 10/20 ND 97 9/9 ND 7  
J3 383A to G 97 18/20 ND 93 4/10 ND 7  
J11 408T del 83 15/20 ND 82 7/18 ND 7  
J13 116C to A 87 18/20 ND 73 7/10 ND 7  
J19 987 T ins 78 14/27 13/25 61 5/10 10/10 8  
 338 T to C  ND 2/25  0/15 1/16  
 1003 G to T  2/27 4/25  0/10 0/10  
 1028 AA del  0/27 2/25  0/10 0/10  
J12 936A del 94 14/20 ND 33 4/16 ND 6  
 322A del  1/12 ND  2/11 ND  
J15 Int 5 3′splice site G to A 99 (Major) ND 57 4/10 ND 
 368A ins  (Minor) ND  1/9 ND  
J16 Int 5 5′splice site T del 98 5/5 ND 43 4/18 ND 10 
PatientMutationPrevious analysisCurrent analysisDuration (y)
PB PMNBM Colonies/bursts DNA (AS)PB PMNBM MNC DNA (AS)
CD59(%)mRNA (AS)CD59 (%)DNA (AS)
J4 298C to T 50 11/22 ND 98 3/10 ND 
 273C to A  0/10 ND  2/10 ND  
J5 1309C del 65 10/20 ND 97 9/9 ND 7  
J3 383A to G 97 18/20 ND 93 4/10 ND 7  
J11 408T del 83 15/20 ND 82 7/18 ND 7  
J13 116C to A 87 18/20 ND 73 7/10 ND 7  
J19 987 T ins 78 14/27 13/25 61 5/10 10/10 8  
 338 T to C  ND 2/25  0/15 1/16  
 1003 G to T  2/27 4/25  0/10 0/10  
 1028 AA del  0/27 2/25  0/10 0/10  
J12 936A del 94 14/20 ND 33 4/16 ND 6  
 322A del  1/12 ND  2/11 ND  
J15 Int 5 3′splice site G to A 99 (Major) ND 57 4/10 ND 
 368A ins  (Minor) ND  1/9 ND  
J16 Int 5 5′splice site T del 98 5/5 ND 43 4/18 ND 10 

AS indicates amplified subclones; Duration, the period between the previous analysis and the current analysis; ND, not done.

Fig. 1.

Scattergraph for the fraction of CD59PBPMNs from 9 patients with PNH during the previous analysis and the current analysis.

Fig. 1.

Scattergraph for the fraction of CD59PBPMNs from 9 patients with PNH during the previous analysis and the current analysis.

We also investigated the absolute numbers of CD59 PMNs in each patient (Table 1). In the groups with an increased (J4 and J5) and a diminished percentage GPI-AP (J12, J15, and J16), the absolute numbers of affected PMNs were increased and decreased, respectively. In the group with a stable percentage GPI-AP, the numbers of affected PMNs were stable in J3 and J11, whereas they were decreased in J13 and J19. In the latter 2 patients, proportions of affected PMNs were stable because of a decline of PNH and normal clones.

Changes in proportion of PIG-A mutant clones

We then analyzed the PIG-A gene in PBPMNs after the amplification and subcloning of appropriate regions. In all patients, the previously predominant mutant clone was still dominant (Table 2). A proportion of affected PMNs in patient J5 increased because of the expansion of the predominant clone (1309delC). In patient J4, whose proportion of affected PMNs increased from 50% to 98%, the predominant clone (298 C-to-T) was detected in 3 of 10 AS of DNA, and the second clone (273 C-to-A) was newly detected in 2 of 10 AS (Table2). Because this patient is female, the result indicates that 2 mutant clones almost completely occupied the hematopoiesis and that the first clone supported approximately half the hematopoiesis for 7 years.

In 4 patients with stable proportions of affected PMNs (J3, J11, J13, and J19), the dominant clones were still dominant 7 to 8 years later. Blood samples from J19, bearing 4 independent PIG-A mutant clones, were analyzed in detail. Sixty percent of PBPMNs and 90% of BMMNCs were defective in CD59 expression (Table 2). The predominant clone (987insT) was detected in 5 of 10 AS from PBPMNs and in 10 of 10 AS from BMMNCs, indicating that proportions of abnormal phenotype and genotype are well correlated. A minor type mutation (388 T-to-C) was only detected in 1 of 16 AS from BMMNCs, and 2 other minor mutations and any additional mutations were not detected (Table 2). These 2 minor clones might have declined spontaneously, or the predominant clone might have superseded them. In every other year for 8 years, we analyzed the PBPMNs of J19 using FACS and found that 60% to 80% had complete deficiency and that none or few had partial deficiency. Because the minor clone (338 T-to-C) causes partial deficiency,15,16 the results suggest that the major clone, which has complete deficiency, maintained dominance for the 8 years.

Proportions of affected PMNs in 3 patients (J12, J15, and J16) decreased because of a decline of the major PNH clone. In patient J12 the predominant clone (936delA), which was previously detected in 14 of 20 AS, was detected in only 4 of 16 AS. A minor clone (322delA), previously detected in 1 of 12 AS, was found in 2 of 11 AS (Table 2). In patient J15, the predominant clone (3′ splice site of intron 5, G-to-A), previously detected as dominant, was detected in only 4 of 10 AS. A minor clone (368insA) was found in 1 of 9 AS (Table 2). Thus, the minor clones in 2 patients (J12 and J15) remained minor, and the decreases in affected PMNs were attributed to declines in the predominant clones.

Discussion

In all patients, the previously predominant clone was still dominant, indicating that the predominant PNH clone mainly supported hematopoiesis and manifested disease for years. However, the minor clone in 2 patients (J4 and J12) is now competing with the previously predominant clone and may overcome it in the future.

Indeed, Nafa et al17 reported a patient with PNH who underwent syngeneic bone marrow transplantation without conditioning. The patient's condition improved, but PNH relapsed 10 years later. They found that current and original PNH clones have different PIG-A mutations. Thus, the proportion of CD59-deficient PMNs was highly variable with time, indicating that the pattern of abnormal clonal expression in PNH may change quantitatively (clones increase or decrease in proportion) and qualitatively (generation of new clones, disappearance of clones). The complexity of these results reflects the marked variability of the clinical course of PNH. A demographic study of the correlation between clonal expansion or diminution and the development of or recovery from aplasia will help our understanding of the pathogenesis and natural history of PNH.

We especially investigated the clinical course in 3 patients (J12, J15, and J16), whose affected PMN levels decreased in proportion. Interestingly, de novo PNH progressed to aplasia in 2 patients (J15 and J16), and their conditions are clinically poor despite the decrease in affected PMNs. In addition, these 2 patients have been followed up significantly longer (31 and 24 years) than other patients with increased or stable proportions of GPI-AP PMNs (8-14 years), though there is no significant disparity in age between these groups (Table 1). Thus, aplasia may be a terminal feature of PNH given that the abnormal clone declines in hematopoietic capacity, probably because of its lifespan, and other residual clones could not take its place, probably because of sustained suppression by an autoimmune mechanism thought to operate in AA. Indeed, PNH hematopoietic cells emerge in patients with severe AA at high frequency (29%-52%) during long-term survival,18-21 and the evolution to pancytopenia is associated with poor survival in patients with PNH.3 In other words, active hematopoiesis of the PNH stem cell clone, though it would result in desperate hemolysis, is better than aplasia because of a decline in the PNH stem cell clone.5 Therefore, our observations suggest that immunosuppressive therapy, such as antithymocyte or antilymphocyte globulin, may be effective in these patients. In patient J12, the normal hematopoietic stem cell clone could have enough activity to support hematopoiesis instead of the PNH stem cell clone, possibly because suppression by an autoimmune mechanism has disappeared.

Taken together, our observations provide convincing evidence that the predominant PNH stem cell clone can support hematopoiesis and manifest disease for 6 to 10 years. Furthermore, the active life of a PNH stem cell clone can be estimated to be approximately 15 years. HSCs supply all blood cells throughout life by their self-renewal and multilineage differentiation capabilities. CD34+ is a marker of human HSCs, and clinical transplantation studies that used enriched CD34+ BM cells indicated the presence of HSCs with long-term BM reconstitution ability within this fraction.22 Unlike in human, mouse primitive BM HSCs, individually having self-renewal and multilineage differentiation, were detected in the mCD34low to mCD34fraction.23-25 Thus, the nature of HSCs is still not well characterized, especially in humans, and it is a current issue. It is also unclear how many HSCs sustain hematopoiesis and how long one HSC could support hematopoiesis in humans. Whether the long-lasting hematopoiesis of one stem cell clone found in patients with PNH is also true for normal HSCs is yet to be determined.

We thank Drs Russell E. Ware and Wendell F. Rosse for their critical reading and discussion. We also thank Yuki Murakami, Keiko Kinoshita, Fumiko Ishii, and Keiko Yamamoto for the excellent technical assistance.

Supported in part by a grant from the Japan Intractable Diseases Research Foundation and the Osaka Medical Research Foundation for Incurable Diseases.

J.N. and T.H. contributed equally to this work.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Ware
RE
Hall
SE
Rosse
WF
Paroxysmal nocturnal hemoglobinuria with onset in childhood and adolescence.
N Engl J Med.
325
1991
991
996
2
Hillmen
P
Lewis
SM
Bessler
M
Luzzatto
L
Dacie
JV
Natural history of paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
333
1995
1253
1258
3
Socie
G
Mary
JY
de Gramont
A
et al
Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors.
Lancet.
31
1996
573
577
4
Kinoshita
T
Inoue
N
Takeda
J
Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria.
Adv Immunol.
60
1995
57
103
5
Luzzatto
L
Bessler
M
Rotoli
B
Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise?
Cell.
88
1997
1
4
6
Watanabe
R
Murakami
Y
Marmor
MD
et al
Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2.
EMBO J.
19
2000
4402
4411
7
Miyata
T
Takeda
J
Iida
Y
et al
Cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science.
259
1993
1318
1320
8
Takeda
J
Miyata
T
Kawagoe
K
et al
Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.
Cell.
73
1993
703
711
9
Miyata
T
Yamada
N
Iida
Y
et al
Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
330
1994
249
255
10
Bessler
M
Mason
P
Hillmen
P
Luzzatto
L
Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria.
Lancet.
343
1994
951
953
11
Yamada
N
Miyata
T
Maeda
K
Kitani
T
Takeda
J
Kinoshita
T
Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria.
Blood.
85
1995
885
892
12
Nafa
K
Mason
PJ
Hillmen
P
Luzzatto
L
Bessler
M
Mutations in the PIG-A gene causing paroxysmal nocturnal hemoglobinuria are mainly of the frameshift type.
Blood.
86
1995
4650
4655
13
Nagarajan
S
Brodsky
RA
Young
NS
Medof
ME
Genetic defects underlying paroxysmal nocturnal hemoglobinuria that arises out of aplastic anemia.
Blood.
86
1995
4656
4661
14
Endo
M
Ware
RE
Vreeke
TM
et al
Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria.
Blood.
87
1996
2546
2557
15
Nishimura
J
Inoue
N
Wada
H
et al
A patient with paroxysmal nocturnal hemoglobinuria bearing four independent PIG-A mutant clones.
Blood.
89
1997
3470
3476
16
Ueda
E
Nishimura
J
Kitani
T
et al
Deficient surface expression of glycosylphosphatidylinositol-anchored proteins in B cell lines established from patients with paroxysmal nocturnal hemoglobinuria.
Int Immunol.
4
1992
1263
1271
17
Nafa
K
Mason
PJ
Hillmen
P
Luzzatto
L
Bessler
M
New somatic mutation in the PIG-A gene emerges at relapse of paroxysmal nocturnal hemoglobinuria.
Blood.
92
1998
3422
3427
18
Schubert
J
Vogt
HG
Zielinska-Skowronek
M
et al
Development of the glycosylphosphatidylinositol-anchoring defect characteristic for paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia.
Blood.
83
1994
2323
2328
19
Griscelli-Bennaceur
A
Gluckman
E
Scrobohaci
ML
et al
Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link.
Blood.
85
1995
1354
1363
20
Schrezenmeier
H
Hertenstein
B
Wagner
B
Raghavachar
A
Heimpel
H
A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins.
Exp Hematol.
23
1995
81
87
21
Azenishi
Y
Ueda
E
Machii
T
et al
CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia.
Br J Haematol.
104
1999
523
529
22
Till
JE
McCulloch
EA
Siminovitch
L
A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells.
Proc Natl Acad Sci U S A.
51
1964
29
36
23
Osawa
M
Hanada
K
Hamada
H
Nakauchi
H
Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Science.
273
1996
242
245
24
Ema
H
Takano
H
Sudo
K
Nakauchi
H
In vitro self-renewal division of hematopoietic stem cells.
J Exp Med.
192
2000
1281
1288
25
Sudo
K
Ema
H
Morita
Y
Nakauchi
H
Age-associated characteristics of murine hematopoietic stem cells.
J Exp Med.
192
2000
1273
1280

Author notes

Jun-ichi Nishimura, Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail:junnishi@acpub.duke.edu.