|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Department of Immunoregulation/Research
Institute for Microbial Diseases and the Research Foundation for
Microbial Diseases, Osaka University; the Department of Hematology and
Oncology, Osaka University Medical School; Ikoma General Hospital,
Nara; Osaka Red Cross Hospital; Second Department of Internal Medicine,
Hyogo College of Medicine; Osaka Medical Center for Cancer; Asiya
Municipal Hospital, Hyogo; Southern Wakayama National Hospital; and
Osaka National Hospital, Japan.
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.
(Blood. 2002;99:2748-2751) 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 termed PIG-A, is mutated in affected cells.7-9 The
PIG-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.10-15
How 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-A
gene mutations in 9 patients 6 to 10 years after the previous
analyses.9,11,15,16
Patients and blood samples
Fluorescence-activated cell sorter analysis
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.
Changes in proportion of GPI-AP
We also investigated the absolute numbers of CD59 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 (Table 2). 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.
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 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 mCD34
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.
Submitted August 17, 2001; accepted November 26, 2001.
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.
Reprints: 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{at}acpub.duke.edu.
1. Ware RE, Hall SE, Rosse WF. Paroxysmal nocturnal hemoglobinuria with onset in childhood and adolescence. N Engl J Med. 1991;325:991-996[Abstract].
2.
Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV.
Natural history of paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
1995;333:1253-1258 3. Socie G, Mary JY, de Gramont A, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. Lancet. 1996;31:573-577. 4. Kinoshita T, Inoue N, Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria. Adv Immunol. 1995;60:57-103[Medline] [Order article via Infotrieve]. 5. Luzzatto L, Bessler M, Rotoli B. Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell. 1997;88:1-4[CrossRef][Medline] [Order article via Infotrieve]. 6. Watanabe R, Murakami Y, Marmor MD, et al. Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2. EMBO J. 2000;19:4402-4411[CrossRef][Medline] [Order article via Infotrieve].
7.
Miyata T, Takeda J, Iida Y, et al.
Cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science.
1993;259:1318-1320 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. 1993;73:703-711[CrossRef][Medline] [Order article via Infotrieve].
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.
1994;330:249-255 10. Bessler M, Mason P, Hillmen P, Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. Lancet. 1994;343:951-953[CrossRef][Medline] [Order article via Infotrieve].
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.
1995;85:885-892
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.
1995;86:4650-4655
13.
Nagarajan S, Brodsky RA, Young NS, Medof ME.
Genetic defects underlying paroxysmal nocturnal hemoglobinuria that arises out of aplastic anemia.
Blood.
1995;86:4656-4661
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.
1996;87:2546-2557
15.
Nishimura J, Inoue N, Wada H, et al.
A patient with paroxysmal nocturnal hemoglobinuria bearing four independent PIG-A mutant clones.
Blood.
1997;89:3470-3476
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.
1992;4:1263-1271
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.
1998;92:3422-3427
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.
1994;83:2323-2328
19.
Griscelli-Bennaceur A, Gluckman E, Scrobohaci ML, et al.
Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link.
Blood.
1995;85:1354-1363 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. 1995;23:81-87[Medline] [Order article via Infotrieve]. 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. 1999;104:523-529[CrossRef][Medline] [Order article via Infotrieve].
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.
1964;51:29-36 23. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996;273:242-245[Abstract].
24.
Ema H, Takano H, Sudo K, Nakauchi H.
In vitro self-renewal division of hematopoietic stem cells.
J Exp Med.
2000;192:1281-1288
25.
Sudo K, Ema H, Morita Y, Nakauchi H.
Age-associated characteristics of murine hematopoietic stem cells.
J Exp Med.
2000;192:1273-1280
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  R. Hu, G. L. Mukhina, S. Piantadosi, J. P. Barber, R. J. Jones, and R. A. Brodsky PIG-A mutations in normal hematopoiesis Blood, May 15, 2005; 105(10): 3848 - 3854. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||||
 |
 |
 |
 |
 |
 |
 |
 |
||||
| |||||||||||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||||
Top
Abstract
Introduction
PMNs from
previous analysis was within 20% (categorized as stable). Two
patients, in whom percentage GPI-AP
