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The Journal of
The American Society of Hematology
BLOOD
VOL 94, NO 9
NOVEMBER 1, 1999
COMMENTARY
Paroxysmal Murine Hemoglobinuria(?): A Model for Human PNH
By Lucio Luzzatto
W
ITH 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 antigenantibody 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
Blood, Vol 94, No 9 (November 1), 1999: pp 2941-2944
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 highcontribution 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 GPInegative 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
From the Department of Human Genetics, Memorial Sloan-Kettering
Cancer Center, New York, NY.
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9409-0011$3.00/0
2941
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2942
LUCIO LUZZATTO
Table 1. Two Mouse Models of PNH
No. of mice studied
Sex
PNH phenotype, %
(Range) mean PNH RBC, %‡
(Range) mean PNH PMN, %‡
Mean PNH T cells, %‡
(Range) mean PNH B cells, %‡
Murakami et al19
Tremml et al20
32
M*
100
(20-30)
(20-30)
85
(20-30)
185
F and M
24†
4.1
10.5
18.6
11.0
*From female donors.
†Defined as having at least 5% PNH cells.
‡At 32 weeks.
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;
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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PNH IN MICE AND IN HUMANS
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.
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
2943
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
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2944
LUCIO LUZZATTO
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.
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1999 94: 2941-2944
Paroxysmal Murine Hemoglobinuria(?): A Model for Human PNH
Lucio Luzzatto
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