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Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 2941-2944
Paroxysmal Murine Hemoglobinuria(?): A Model for Human PNH
By
Lucio Luzzatto
From the Department of Human Genetics, Memorial Sloan-Kettering
Cancer Center, New York, NY.
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ARTICLE |
WITH THE INCREASING integration of
molecular genetics into the study of human diseases we have come to
almost expect that, whenever a new gene is cloned, the next paper will
report the consequences of `knocking out' the gene. In a way, we are thus witnessing the logical updating of a century-old approach to the
study of physiology. To define the function of an organ, a simple way
was to remove it surgically; today, targeted homologous recombination
has proven a powerful micro-surgical technique to remove the function
of one gene at a time.1 However, lest we lose sight of our
purpose, it is useful to draw a distinction. When a gene is isolated by
positional cloning, we often have no idea of its normal function:
therefore, knocking it out can suddenly provide an entirely new
insight. When a gene is isolated instead on the basis of its known
biochemical function, and the abnormality of that function is already
known to cause a certain disease, no immediate surprise is to be
expected. However, the knock-out approach can be still used to identify
subtle pathophysiological features of the disease concerned, and
especially to obtain an animal model lending itself to experimental
manipulations and ultimately to investigating new forms of
therapy.2
The PIG-A gene was cloned by the group of Miyata et al3 in
1993 by an elegant approach: the functional complementation of a cell
line with a known defect in the biosynthesis of glycosyl phosphatidyl
inositol (GPI) anchors. At the time it was also already known that the
biochemical lesion in paroxysmal nocturnal hemoglobinuria (PNH) cells
was in this metabolic pathway,4 and it became immediately clear that the `PNH gene' had been really identified.5,6 We did not need to wait for any knock-out experiment to understand that
a frameshift mutation of PIG-A would completely abrogate GPI
biosynthesis,7 which would prevent CD59 from appearing on
the surface of red blood cells (RBCs), which would cause these RBCs to
be highly vulnerable to the final stage of the complement activation
cascade. Complement activation can be triggered by an antigen-antibody
reaction or, through the alternative pathway, by the flimsiest of
excuses: if CD59 is lacking this ends up in a large number of RBCs
being destroyed. However, there are of course two important differences
between PNH and other hemolytic anemias due to intracorpuscular
abnormalities of the RBCs: (1) Because PNH is caused by a somatic
mutation in a hematopoietic stem cell,8 rather than by an
inherited mutation, the other cells in the body are not affected. (2)
Even within the hematopoietic system, because only one or very
few9 stem cells can be expected to have PIG-A mutations,
there is always a residual population of cells with normal PIG-A and,
therefore, normal GPI-linked molecules. Indeed, as originally suggested
a decade ago,10 and as more recently highlighted in several
recent reviews,11-15 a central issue to understanding PNH
is to pinpoint the factors that determine the balance between the
normal and the PNH hematopoietic populations. Thus, knock-out animals
were needed not so much to prove that PIG-A mutations cause
PNH, or to define more precisely the function of PIG-A, but
very much to provide an animal model in which other components of the
disease process could be investigated.
Three groups promptly targeted pig-a in male mouse embryonic
stem (ES) cells and, thanks to the fact that this gene is X-linked, they promptly obtained ES cells that were genetically and
phenotypically GPI-negative and that proved viable.16-18
However, when these cells were used to produce pig-a null mice,
the problem arising was similar to that confronting an
over-enthusiastic physiologist who, a century ago, having successfully
defined the function of the pancreas and of the pituitary gland by
surgically removing them, decided to do the same with the liver or the
heart. We all found that high-contribution chimeric embryos died in
utero and, therefore, germ-line transmission could not be obtained.
Low-contribution pig-a null mice were born, but their low
numbers of GPI-negative blood cells failed to provide a valid model of
the human disease PNH.
In this issue of Blood two papers report success in producing
such a model, based on the technique of conditional knock-out, using
the system known in jargon as cre-lox. Murakami et
al19 have crossed male mice, in which two lox sequences had
been engineered to flank exon 6 of pig-a, with female mice
transgenic for cre driven by the universally active
cytomegalovirus (CMV) promoter. The resulting females, heterozygous for
a pig-a gene vulnerable to inactivation, died late in fetal
life with brain abnormalities: but before they died, hematopoietic
cells from their liver were used to reconstitute hematopoiesis in
syngeneic irradiated recipient mice. Because, following X chromosome
inactivation, these liver cells were a mosaic of cells with a normal
and a knocked out pig-a, the transplanted animals had both
normal and PNH cells in their blood. Instead, Tremml et
al20 crossed mice (both males and females) in which two lox
sequences had been engineered to flank exon 2 of pig-a, with
mice (females and males) transgenic for cre driven by the EIIa
promoter, which is expected to drive gene expression only in the very
early, preimplantation stage of embryonic life. In this case the
offspring were born live, and the proportion of PNH cells in their
blood presumably reflected the proportion of stem cells arising from
cells in which, in the early embryo, cre expression had knocked
out pig-a. Thus, both of these two different approaches have
yielded the desired animal model; and the first and gratifying
observation from reading the two papers is that the results are in good
agreement (see Table 1).
In fact, the two approaches differ in a non-trivial way (see Fig
1). Because the EIIa promoter is not
tissue-specific, the mice produced by Tremml et al must be mosaics for
pig-a inactivation in all tissues: unlike the case of patients
with PNH, in whom only the hematopoietic tissue is involved. (However,
in a separate report21 the same group shows that in most
tissues in the majority of cells the X chromosome with the normal
pig-a gene is the active one, indicating negative selection
against the GPI-negative cells early in organogenesis.) In this respect
the mice produced by Murakami et al are more like PNH patients, because
their nonhematopoietic tissues have not been manipulated. On the other
hand, the transplantation procedure required the use of irradiation,
and the authors themselves point out that in theory this might affect
the fate of the transplanted cells: for instance, through an effect on
stromal cells. No irradiation was used in Tremml's experiments. The
fact that the results obtained are similar virtually eliminates a
significant role of these potentially confounding variables, and the
data in the two papers further corroborate one another. Therefore I
will choose to consider the two papers together in looking at them as a
model of human PNH.
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| Fig 1.
PNH in mice and humans. The two upper panels represent in
cartoon form the mouse models constructed in refs 19 and 20, respectively. Although all cells have the same genotype,
their phenotype differs depending on which X chromosome has been
inactivated: white circles indicate cells in which the X chromosome
with the normal pig-a gene is active; gray circles indicate
cells in which the X chromosome with the inactivated pig-a gene
(pig-a°) is active. As a result, in the peripheral blood there is a
mixture of normal (white) and PNH (gray) blood cells. The lower
proportion of PNH cells in the model by Tremml et al is caused by the
fact that cre, driven by Ella, does not knock out pig-a
in every cell (Tremml et al have produced also male mice with PNH [see
text].) In both models the proportion of PNH cells in the peripheral
blood depends on the proportion of cells in which cre-mediated
recombination has taken place: there is no evidence of selection
thereafter. The bottom panel represents in cartoon form what happens in
the human disease, PNH. Here, unlike in the mice, the entire PNH
population arises usually from a single stem cell in which a somatic
mutation has taken place: therefore, selection must take place at the
level of stem cells in order for a large proportion of the blood cells
to have the PNH phenotype. Here only one allele of the PIG-A
gene is shown because the entire process takes place after
X-inactivation, when somatic cells are functionally haploid for most
X-linked genes in both males and females.
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Three main points have emerged. First, the mice have two discrete
populations of RBCs, granulocytes, monocytes, and lymphocytes: in first
approximation, the flow cytometry patterns are remarkably similar to
those seen in patients with PNH. Second, the mice are not anemic. This
may be at first sight disappointing: however, in vitro the RBCs display
a perfect mimicry of the classical PNH phenotype, with a marked shift
in susceptibility to complement. Indeed, although the proportions of
RBCs are lower in the mice studied by Tremml et al (Table 1), they have
been able to produce a positive micro-Ham test, and they show
convincingly, by analyzing reticulocytes, that the life span of the PNH
RBC population is significantly shortened in vivo. Thus, although, by
looking at their cages, it does not seem the mice really have
hemoglobinuria, the RBC phenotype of the human disease is fully
reproduced (and, who knows, perhaps hemoglobinuria would turn up if the
mice were allowed out of the sheltered environment of a high-class
research animal house). Third, and perhaps least expected, both reports show a remarkably high proportion of PNH B cells and T cells. In fact,
Murakami et al find that almost all the T cells are PNH, and their
detailed analysis suggests that the takeover occurs after the cells
have entered the thymus, implying a paradoxical advantage of T cells
lacking GPI-linked proteins at this particular stage in development.
Thus, there is no doubt that we have a good model of the hematological
picture of PNH at a particular point in time. But what is the clinical
course of the mice patients (or patient mice)? On this point, we get
two important messages: (1) Once the mice are adult, the proportion of
PNH cells is remarkably stable. (2) At least until now, the mice have
not developed leukemia. For a whole generation we have been
inspired by William Dameshek's classic 1967 editorial22 in
this journal, in which, while giving full credit to Dacie and his
colleagues23 for first outlining the close links between
PNH and acquired aplastic anemia (AAA), he explored himself what may be
in common between PNH, AAA, and "hypoplastic leukemia." Two years
later, as editor of Blood, Dameshek succeeded in bringing
together in one special issue of the journal (published `to whittle
down our rather impressive backlog' of manuscripts) 3 individual case
reports of PNH that `evolved' to acute myeloid leukemia (AML). In a
second editorial24 in that issue he developed clearly the
notion that, although PNH was traditionally classified among hemolytic
anemias, it was `a disorder of the entire bone marrow,' and he
suggested it was "a candidate" myeloproliferative disorder (MPD).
This notion seemed to become corroborated when it became clear that PNH
was a clonal disease25: indeed, because leukemias are
clonal par excellence, the word `clonal' is often used as
though it were synonymous to neoplastic, or malignant.
Dameshek made no secret of his distaste for "pigeonholes," and he
was more sympathetic to `lumpers' than to `splitters.' Therefore, bringing together AAA, PNH, MPD, hypoplastic leukemia (now
myelodysplastic syndrome [MDS]?), and Di Guglielmo's
disease (now AML-M6) appealed to him. As a credit to his
vision, it is now abundantly confirmed that PNH shares with MDS, MPD,
and AML the feature of being clonal; and with AAA, the feature of bone
marrow failure. However, one generation later, we have several reasons
to say that the link of PNH to AAA is closer than its link to MPD: (1)
Evolution to AML is common in MPD and in MDS; but, contrary to
Dameshek's expectation, it is rare in human PNH,26 and it
has not been a feature of murine PNH. (2) `Clonal expansion' is
progressive and usually inexorable in MDS or in MPD: by contrast, PNH
clones expand up to a point, and then tend to be stable. The mouse data
are remarkably similar in this respect. (3) In a number of
well-documented patients PNH has evolved to AAA: this rarely happens in
MDS and practically never in MPD. All these data are in keeping with
the notion that MDS and MPD clones (let alone AML clones) have an
unconditional or absolute growth advantage, whereas PNH clones have a
relative or conditional growth advantage, which is made prominent by
the marrow environment of AAA.
On this third point we do not yet have help from the mouse models,
because the mice do not have AAA. In fact, in the model mice the PNH
cell population arises from a substantial number of mutated stem cells,
and the size of the PNH population depends on the number of cells that
were transplanted, or from the number of cells in which cre
activation has caused inactivation of pig-a (see Fig 1). By
contrast, in human PNH the PNH cell population arises from the
expansion of a single mutant stem cell. It has been suggested that this
expansion results from the fact that PNH stem cells are able to escape
from damage inflicted to normal stem cells by a specific pathogenic
mechanism27: the best candidate being an autoimmune
mechanism.10,11 Thus, PNH cells have a conditional growth
advantage if, and only if, such a process is operating. This
pathogenetic model has recently received support from the finding that
PNH microclones are present in normal people: but in normal people they
fail to expand.28 The logical implication of this model
with respect to management is that the best way to treat PNH is to
treat the underlying AAA. In fact, treatment with antilymphocyte
globulin has been beneficial in individual cases29,30; and
there is evidence that PNH does not recur after an allogeneic bone
marrow transplant (BMT) even without a fully myeloablative
conditioning.31 This suggests that BMT helps the patient by
providing transient intensive immune suppression together with new stem
cells: eradication of the PNH clone(s) is not necessary. By contrast,
recurrence has occurred after a syngeneic BMT performed without any
conditioning,32 because this treatment, although it
provided new stem cells, did not provide immune suppression.
As mentioned earlier, we already knew that the PNH abnormality resulted
from a PIG-A mutation. However, having reproduced the phenotype
by targeting the pig-a gene has provided formal proof that such
a mutation is necessary and sufficient to produce that phenotype: a
clear analogy to Koch's postulate for the etiology of an infectious
disease. We now need to reproduce in the mice those conditions which,
in humans, are able to select for the cells with the PNH abnormality.
It would not be right to second-guess the two groups that have
developed these models, but I would be surprised if they were not
already hard at work in finding ways to induce AAA in their mice,
probably by some immunological approach.
| |
ACKNOWLEDGMENT |
I thank Dr R. Notaro for help with the illustration; and I am most
grateful to him, to Dr D. Araten, and to Dr A. Karadimitris for much
thought and much work on PNH.
| |
FOOTNOTES |
Submitted June 30, 1999; accepted July 7, 1999.
Supported in part by Grant No. 5RO1 HL56778-04 from NHLBI, NIH.
Address reprint requests to Lucio Luzzatto, MD, PhD, Department of
Human Genetics, Memorial Sloan Kettering Cancer Center, 1275 York Ave,
Box 110, New York, NY 10021.
| |
REFERENCES |
1.
Capecchi MR:
The new mouse genetics: Altering the genome by gene targeting.
Trends Genet
5:70, 1989[Medline]
[Order article via Infotrieve]
2.
He LZ, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, Zelent A, Pandolfi PP:
Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL.
Nat Genet
18:126, 1998[Medline]
[Order article via Infotrieve]
3.
Miyata T, Takeda J, Ilda J, Yamada N, Inoue N, Takahashi M, Maeda K, Kitani T, Kinoshita T:
The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science
259:1318, 1993[Abstract/Free Full Text]
4.
Hillmen P, Bessler M, Mason PJ, Watkins WM, Luzzatto L:
Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.
Proc Natl Acad Sci USA
90:5272, 1993[Abstract/Free Full Text]
5.
Takeda J, Miyata T, Kawagoe K, Kinoshita T:
Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.
Cell
73:703, 1993[Medline]
[Order article via Infotrieve]
6.
Bessler M, Mason PJ, Hillmen P, Miyata T, Yamada N, Luzzatto L, Kinoshita T:
Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.
EMBO J
13:110, 1994[Medline]
[Order article via Infotrieve]
7.
Rosse WF, Ware RE:
The molecular basis of paroxysmal nocturnal hemoglobinuria.
Blood
86:3277, 1995[Free Full Text]
8.
Nafa K, Bessler M, Castro-Malaspina H, Jhanwar S, Luzzatto L:
The spectrum of somatic mutations in the PIG-A gene in paroxysmal nocturnal hemoglobinuria includes large deletions and small duplications.
Blood Cells Mol Dis
24:370, 1998[Medline]
[Order article via Infotrieve]
9.
Bessler M, Mason PJ, Hillmen P, Luzzatto L:
Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria.
Lancet
343:951, 1994[Medline]
[Order article via Infotrieve]
10.
Rotoli B, Luzzatto L:
Paroxysmal nocturnal hemoglobinuria.
Semin Hematol
26:201, 1989[Medline]
[Order article via Infotrieve]
11.
Young NS:
The problem of clonality in aplastic anemia: Dr Dameshek's riddle, restated [see comments].
Blood
79:1385, 1992[Free Full Text]
12.
Kinoshita T, Inoue N, Takeda J:
Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria.
Adv Immunol
60:57, 1995[Medline]
[Order article via Infotrieve]
13.
Luzzatto L, Bessler M:
The dual pathogenesis of paroxysmal nocturnal hemoglobinuria.
Curr Opin Hematol
3:101, 1996[Medline]
[Order article via Infotrieve]
14.
Dunn DE, Ware RE, Parker CJ, Mishoe HO, Young NS:
Research directions in paroxysmal nocturnal hemoglobinuria.
Immunol Today
20:168, 1999[Medline]
[Order article via Infotrieve]
15.
Luzzatto L, Bessler M, Rotoli B:
Somatic mutations in paroxysmal nocturnal hemoglobinuria: A blessing in disguise?
Cell
88:1, 1997[Medline]
[Order article via Infotrieve]
16.
Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa M, Watanabe T, Kinoshita T, Takeda J:
Glycosylphosphatidylinositol-anchor-deficient mice: Implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria.
Blood
87:3600, 1996[Abstract/Free Full Text]
17.
Dunn DE, Yu J, Nagarajan S, Devetten M, Weichold FF, Medof ME, Young NS, Liu JM:
A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a( ) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.
Proc Natl Acad Sci USA
93:7938, 1996[Abstract/Free Full Text]
18.
Rosti V, Tremml G, Soares V, Pandolfi PP, Luzzatto L, Bessler M:
Embryonic stem cells without pig-a gene activity are competent for PNH- like hematopoiesis but not for clonal expansion.
J Clin Invest
100:1028, 1997[Medline]
[Order article via Infotrieve]
19.
Murakami Y, Kinoshita T, Maeda Y, Nakano T, Kosaka H, Takeda J:
Different roles of glycosylphosphatidylinositol in various hematopoietic cells as revealed by model mice of paroxysmal nocturnal hemoglobinuria.
Blood
94:2963, 1999[Abstract/Free Full Text]
20.
Tremml G, Dominguez C, Rosti V, Zhang Z, Pandolfi PP, Keller P, Bessler M:
Increased sensitivity to complement and a decreased red cell life span in mice mosaic for a non-functional Piga gene.
Blood
94:2945, 1999[Abstract/Free Full Text]
21.
Keller P, Tremml G, Rosti V, Bessler M:
X inactivation and somatic cell selection rescues female mice carrying a Piga-null mutation.
Proc Natl Acad Sci USA
96:7479, 1999[Abstract/Free Full Text]
22.
Dameshek W:
Riddle: What do aplastic anemia, paroxysmal nocturnal hemoglobinuria and `hypoplastic leukemia' have in common?
Blood
30:251, 1967[Free Full Text]
23.
Dacie JV:
Paroxysmal nocturnal haemoglobinuria, in
Dacie JV
(ed):
The Haemolytic Anaemias, vol 4 (ed. 2). London, UK, Churchill, 1967, p 1128
24.
Dameshek W:
Foreward and a proposal for considering paroxysmal nocturnal hemoglobinuria (PNH) as a "candidate" myeloproliferative disorder.
Blood
33:263, 1969[Free Full Text]
25.
Oni SB, Osunkoya BO, Luzzatto L:
Paroxysmal nocturnal hemoglobinuria: Evidence for monoclonal origin of abnormal red cells.
Blood
36:145, 1970[Abstract/Free Full Text]
26.
Harris JW, Koscick R, Lazarus HM, Eshleman JR, Medof ME:
Leukemia arising out of paroxysmal nocturnal hemoglobinuria.
Leuk Lymphoma
32:401, 1999[Medline]
[Order article via Infotrieve]
27.
Rotoli B, Luzzatto L:
Paroxysmal nocturnal haemoglobinuria.
Baillieres Clin Haematol
2:113, 1989[Medline]
[Order article via Infotrieve]
28.
Araten D, Nafa K, Pakdeesuwan K, Luzzatto L:
Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals.
Proc Natl Acad Sci USA
96:5209, 1999[Abstract/Free Full Text]
29.
Kusminsky GD, Barazzutti L, Korin JD, Blasetti A, Tartas NE, Sanchez Avalos JC:
Complete response to antilymphocyte globulin in a case of aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome [letter].
Am J Hematol
29:123, 1988[Medline]
[Order article via Infotrieve]
30.
Rosse W:
Paroxysmal nocturnal hemoglobinuria, in
Handin RI LS,
Stossel TP
(eds):
Blood Principles and Practice of Hematology. Philadelphia, PA, Lippincott, 1995, p 367
31.
Antin JH, Ginsburg D, Smith BR, Nathan DG, Orkin SH, Rappeport JM:
Bone marrow transplantation for paroxysmal nocturnal hemoglobinuria: Eradication of the PNH clone and documentation of complete lymphohematopoietic engraftment.
Blood
66:1247, 1985[Abstract/Free Full Text]
32.
Nafa K, Bessler M, Deeg HJ, Luzzatto L:
New somatic mutation in the PIG-A gene emerges at relapse of paroxysmal nocturnal hemoglobinuria.
Blood
92:3422, 1998[Abstract/Free Full Text]
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