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New Somatic Mutation in the PIG-A Gene Emerges at Relapse
of Paroxysmal Nocturnal Hemoglobinuria
By Khédoudja Nafa, Monica Bessler, H. Joachim Deeg, and Lucio Luzzatto
We report a detailed longitudinal study of the first patient to
be treated (in 1973) for paroxysmal nocturnal hemoglobinuria (PNH) with syngeneic bone marrow transplantation
(BMT). The patient subsequently relapsed with PNH in 1983,
and still has PNH to date. Analysis of the PIG-A gene in a
recent blood sample showed in exon 6 an insertionduplication causing a frameshift. Polymerase chain reaction
(PCR) amplification of the PIG-A exon 6 from bone marrow
(BM) slides obtained before BMT showed that the duplica-
P
AROXYSMAL NOCTURNAL hemoglobinuria (PNH) is
an acquired chronic disorder associated with intravascular
hemolysis, increased tendency to venous thrombosis, and
cytopenia due to bone marrow (BM) failure.1 PNH is associated
with somatic mutations in the X-linked PIG-A gene in an early
hematopoietic stem cell.2 The biochemical defect in PNH has
been localized to an early step in the glycosyl phosphatidylinositol (GPI) anchor biosynthetic pathway. Consequently, PNH
cells are deficient in GPI-anchored proteins, including the
Decay Accelerating Factor (DAF or CD55) and the Membrane
Inhibitor of Reactive Lysis (CD59), both of which are involved
in the regulation of complement activity on the cell surface.3
The only curative therapy available for PNH is BM transplantation (BMT). A first report of the successful application of
BMT for PNH associated with severe aplastic anemia (AA)
appeared in 1973.4 Subsequently, out of 17 PNH patients who
had BMT, 11 received human leukocyte antigen (HLA) identical sibling marrow with conditioning, 1 received HLAhaploidentical marrow with conditioning, and 5 received syngeneic marrow without conditioning. Long-term follow up of
these 17 patients showed that only the 5 patients who had been
transplanted with syngeneic BM, without immunosuppressive
conditioning therapy relapsed, although some were doing well
without need of transfusion.5-10 Here, we show that relapse of
PNH after BMT can result from the expansion of new PNH
clones rather than from the persistence of the original PNH
clone.
From the Department of Human Genetics, Memorial Sloan-Kettering
Cancer Center, New York, NY; Department of Internal Medicine,
Washington University School of Medicine, St Louis, MO; and Fred
Hutchinson Cancer Research Center, University of Washington, Seattle,
WA.
Submitted April 3, 1997; accepted June 21, 1998.
Supported by the NIH grant ROI-HL-56778, the DeWitt Wallace
Clinical Research Fund, and the Kleberg Foundation.
Address correspondence to Khédoudja Nafa, Department of Human
Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave,
New York, NY 10021; e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9209-0019$3.00/0
3422
tion was not present; instead, we found several single base pair
substitutions in exons 2 and 6. Thus, relapse of PNH in this
patient was not due to persistence of the original clones; rather,
it was associated with the emergence of a new clone. These
findings support the notion that the BM environment may
create selective conditions favoring the expansion of PNH
clones.
r 1998 by The American Society of Hematology.
PATIENT
R.S. (MSK13) was investigated at the age of 18 because of
anemia (date of birth, 03/13/54). In September 1972, based on a
positive Ham test, a diagnosis of PNH was made. In June 1973,
the patient had an infusion of BM from his syngeneic twin,
without conditioning.11 The patient had a good clinical and
hematological response (see Fig 1), but his white cell count
remained rather low, and after 4 years the platelet count started
declining. Ten years after BMT (in 1983) the complement lysis
test was again positive (10.7% lysis) and the patient could be
regarded as having a lab relapse of PNH6; by 1987, with the
recurrence of anemia, the patient had to be regarded as having
clinical relapse of PNH. The patient also developed pancreatitis
and splenic vein thrombosis and underwent splenectomy in
1989. Subsequently, he had gastrointestinal bleeding from
esophageal varices requiring sclerotherapy. In 1992, tests for
hepatitis C were positive and a liver biopsy showed evidence of
hepatitis but no fibrosis or cirrhosis. Currently, the patient is
mildly pancytopenic and is being managed conservatively.
MATERIALS AND METHODS
Flow cytometric analysis. Analysis of GPI-anchored protein on red
blood cells (RBCs), polymorphonuclear neutrophils (PMN), and mononuclear cells (MNC) was performed by flow cytometry (FACscan;
Becton Dickinson, Mountain View, CA) using monoclonal antibodies
towards GPI-anchored protein.12
DNA extraction. DNA was extracted separately from PMN and
from MNC by dodecyl sulfate proteinase K-method.13 Archival Wrightstained BM smear slides were processed as follows. After soaking for 3
hours in xylene, the coverslips were removed and the slides were then
soaked further overnight to remove residual mounting medium. The
xylene was then removed by evaporation. Cells were scraped from
slides into a 1.5 mL Eppendorf tube containing lysis buffer (100 mmol/L
Tris-HCl, pH 7.4; 5 mmol/L EDTA, pH 8; 0.2% sodium dodecyl sulfate
[SDS]; 200 mmol/L NaCl; and 100 mg/mL proteinase K) using a sterile
scalpel. After overnight incubation at 55°C, DNA was extracted twice
by equal volume of phenol, and twice with an equal volume of
chloroform-isoamyl alcohol (24:1).13 The DNA was precipitated with
an equal volume of isopropanol, mixed by inverting the tube, and then
incubated 1 hour at 270°C. After centrifugation, the pellet was washed
once with 70% ethanol and left to dry at room temperature. Invisible
pellet was resuspended in 10 µL of TE (TE510 mmol/L Tris-HCl, pH
7.4; 1 mmol/L EDTA, pH 8).
Polymerase chain reaction (PCR) amplification. The PIG-A coding
region was PCR amplified in 4 fragments from genomic PMN DNA as
previously described.14,15 For slide-stripped DNA analysis, the first
round of PCR amplification did not give any visible PCR product.
Therefore, the primary PCR products (5 µL) were reamplified by nested
Blood, Vol 92, No 9 (November 1), 1998: pp 3422-3427
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RELAPSE OF PNH WITH NEW PIG-A MUTATION
3423
Fig 2. Flow cytometry analysis with anti-CD59 of PMN from a
normal control (broken line) and PNH patient MSK13 (full line).
RESULTS
Fig 1.
Long history of PNH in patient MSK13.
PCR for exons 2, 3, and 6, and seminested PCR for exons 4 and 5 by
using internal oligonucleotides (Table 1).
Characterization of PIG-A gene mutations. Single-strand conformation analysis (SSCA) and heteroduplex analysis (HA) were performed,
as previously described,14,15 on the primary and on the secondary PCR
products obtained from the post-transplant and the pre-BMT samples,
respectively. Nucleotide (nt) sequencing was performed by Sequenase
Version 2.0 DNA sequencing kit (US Biochemical Corp, Cleveland,
OH), after cloning the abnormal fragments into phage M13.
Molecular analysis of PNH relapse. FACscan analysis of a
recent blood sample (1995) showed that 2% of the patient’s
RBCs were deficient in CD59, and 90% of his PMN were
deficient in CD59 (Fig 2), CD24, and CD16 (data not shown),
consistent with PNH. HA and SSCA showed no abnormalities
in exons 2-5 of the PIG-A gene. Analysis of a BstNI restriction
enzyme digest obtained from PCR-amplified exon 6 (474 bp)
showed an abnormally large double-stranded fragment (Fig 3).
Nt sequence analysis showed (in 19 out of 23 M13 clones) an
insertion of 2 nucleotides (AA) at nt position 1355, followed by
a duplication of 32 nucleotides encompassing nt 1324-1355,
(Fig 4). This insertion-duplication will cause a frameshift
resulting in a truncated PIG-A protein of 462 instead of 484
amino acids (aa), in which aa 453-to-462 are abnormal. The
PIG-A protein will be functionally inactivated; therefore, we
Table 1. Details of Oligonucleotide Primers Used in This Study
Exon
2
3
4§
5§
6
Primer
Orientation*
External
Primer†
F
R
F
R
F
R
F
R
2I1
I2
2I2
I3b‡
2I3
I5
2I5
q‡
Internal Primer
A: 58-GAGAtctagaTTTTGTTTCTGAGCTG-38
B: 58-CAAgaattcAACAGCTTTCTATAG-38
b: 58-ATAAGTGaATTCTCAGTCGTTCTGGTGA-38
I3†
e: 58-CTCAGGAATTCCACCAAC-38
s: 58-ATTCTGCATGGCGATCGTGG-38
2I5b: 58-TTTATAAAATGTTCCTGCAGGATC-38
f†
D\: 58-AGTCATCCATCTCAATTTC-38
Fragment
Size
(bp)
891
309
414
532
415
Lowercase indicates non–PIG-A sequence designed to introduce restriction enzyme sites.
*F, forward; R, reverse.
†Oligonucleotide sequence as reported by Nafa et al.15
‡New external primers: ‘‘I3b’’: 58-gggaaTTCCTATTTATATAAAAATT-38 and ‘‘q’’: 58-AAAATATTGAATGATATAGAGGTAGCATAAC-38 at position nt
1595-1565 in exon 6.
§In the primary PCR, exons 4 1 5 PCR amplified in one fragment by using primers ‘‘2I3’’ and ‘‘I5.’’15 In the secondary PCR, exons 4 and 5 are
individually amplified by using primer ‘‘e’’ (position nt 1014-994 in exon 5) and primer ‘‘s’’ (position nt 938-958 in exon 4).
\Specific oligonucleotide that spans the insertion (underlined).
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3424
NAFA ET AL
ful, but it failed to produce the double-stranded fragment of
abnormal size observed in the recent sample (Fig 3). Nevertheless, 36 M13 clones were sequenced. None of them had the
exon 6 duplication; but in 13 of them we found a 1442 C=T
point mutation, causing a 481 ser=phe amino acid replacement
(Table 2). To test the possibility that the exon 6 duplication
might have been present already in 1973 in a very small
percentage of cells, we made efforts to increase the sensitivity
of our detection method.16 For this purpose, we designed a
reverse primer called D that spans the insertion at the 38 end of
the duplication (Table 1 and Fig 5A). By using primer D and
primer -I5b, and the primary PCR product as template (because
no more DNA extracted from BM slides was left), we amplified
the abnormal fragment very efficiently from the post-transplant
sample, but not at all from the pre-BMT sample (Fig 5B).
Hybridization of the Southern-blot of the same gel with a PIG-A
cDNA probe did not show any signal (data not shown). All
others exons were also amplified by nested-PCR from the
pre-BMT slides and an abnormal fragment in exon 2 was
observed by HA and SSCA. Nt sequence analysis of the
appropriate DNA fragments showed several single bp substitutions (Table 2). In exon 2, 50% of the M13 clones sequenced
had a 211A=C base change, causing a 71 thr=ala amino acid
replacement; and 28% of these clones also have 251 C=T,
causing 84 thr=ile (suggesting that the latter mutation arose in
a cell belonging to the clone that had the former mutation). Still
Fig 3. Analysis of exon 6 of the PIG-A gene before BMT and after
relapse. (A) Heteroduplex Analysis (HA); Two extra bands (235 bp,
mutant homoduplex; H, heteroduplex) in patient MSK13 are clearly
observed after relapse (lane 4) but not before BMT (lane 2). (B)
Single-Strand Conformation Analysis (SSCA). Along with doublestranded fragments DNA, an altered electrophoretic mobility (shift)
of the single-stranded DNA (SS) is only present after relapse (lane 4).
Normal controls (lanes 1, 3, 5); normal homoduplex, 201 bp.
regarded this mutation as responsible for PNH in this patient at
this time. The duplicated DNA element was flanked by a 4 bp
TTGA direct repeat (Fig 5A); the same repeat is found four
times in the mutant sequence. Therefore, we presume that a
nonhomologous recombination event (by sister chromatid exchange) must have occurred between TTG in the normal
sequence at nt position 1357 and the duplicated sequence at nt
position 1324 (Fig 5A). It appears that this recombinational
event was either preceded or followed by the insertion of two A
at position nt 1355. For brevity, we will refer to this abnormality
simply as an exon 6 duplication.
Molecular analysis of original PNH cells from archival
material. To determine whether in this patient the relapse of
PNH took place because of resurgence of the same PNH clone
that had originally caused his disease, we needed to analyze
pretransplant DNA. The only available archive material consisted of two BM slides from the time of diagnosis, before BMT
(1973). Amplification of PIG-A exon 6 from these slides
(performed by the so called nested-PCR approach) was success-
Fig 4. Insertion duplication in exon 6 of the PIG-A gene in patient
MSK13. The sequence of 32 nt starting by 58-TCAA-3 until 58-CAA-3 in
the normal control is replicated in MSK13, but after the insertion of
two thymidines. The insertion-duplication introduces a frameshift at
codon 452 and leads to the production of truncated PIG-A protein of
only 462 AA.
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RELAPSE OF PNH WITH NEW PIG-A MUTATION
3425
B
Fig 5. High-sensitivity analysis of the 1355insAA-duplication nt 1324-1355
before and after BMT. (A) Schematic representation of the insertion-duplication
in exon 6 of the PIG-A gene. At the top, two copies of the normal sequence are
aligned to show direct-sequence homology (underlined) that may favor nonhomologous exchange below the sequence in the recent sample of patient MSK13.
Identical bases are indicated by a vertical line. The short repeat of 4 nt flanking
the duplication are boldface. The 19 mer D has been designed to match
completely the mutant sequence, whereas the last 4 nt (58-. . . .ttTC-38) are
mismatched to the normal sequence (see arrow bent at the left). The fragment
amplified with primers -I5b and D and -I5b and f are expected to be 292 bp and
415 bp in length, respectively. (B) Gel electrophoretic analysis of nested-PCR
products of exon 6 of PIG-A gene from PMN in the pretransplant sample. A
fragment of 292 bp amplified with the primers -I5b and D was present in the
post-transplant sample (lane 2); by contrast no amplification was obtained in the
pretransplant sample (lane 1) and in a normal control sample (lane 3). When the
3 primers -I5b, D, and f were used in the same PCR reaction, a fragment of 415 bp
only was amplified in the pretransplant sample and in a normal control (lanes 4
and 6), and a fragment of 292 bp only was amplified in the posttransplant sample
(lane 5).
13/36 (36)
was found in 9 out of 10 M13 clones from the nested-PCR
product (Table 3, line 5); and in all 12 M13 clones from BM
slides (Table 3, line 3). Therefore, we regarded this frameshift
mutation as responsible for PNH in patient MSK11. Some of
these clones had the PNH-related mutation plus another point
mutation (different in each clone, as shown in column 5 of
Table 3). In the non-PNH 24 year old BM slide we did not find
more than two M13 clones with the same nt change (Table 3,
line 2). These control experiments indicate that, not surprisingly, Taq I polymerase errors do occur, but they are always
different in different M13 clones, and they are not increased by
using a high number of PCR cycles. There is no difference in
Taq I polymerase errors between primary and secondary PCR
(see Table 3, lines 4 and 5) . By contrast, in the pre-BMT sample
from patient MSK13 the same nucleotide changes were found
consistently in several independently isolated M13 clones,
indicating that they reflect true mutations (Table 3, line 1,
column 4 and 5).
Analysis of the PIG-A gene in the BM donor. Given that the
PIG-A mutation in the relapse sample was completely different
from those existing in this patient before BMT, it was possible
that the former was in fact of donor origin. HA and SSCA of all
exons of the donor’s PIG-A gene failed to show any abnormality. The highly sensitive duplication-specific nested-PCR technique that we developed (see Materials and Methods) failed to
amplify exon 6 of the donor’s PIG-A gene. Thus, there is no
evidence of this mutation being of donor origin. The donor
remains clinically and hematologically normal.
19/23 (83)
DISCUSSION
in exon 2, 14% of the M13 clones sequenced have a 16 G=T,
causing 6 gly=stop (Fig 6).
The finding of more than one clone in PNH is not unusual.15,17-20 However, we were concerned about the possibility
that the two-stage amplification procedure we used might be
associated with a higher probability of artifacts. Therefore, we
performed control experiments by applying exactly the same
nested-PCR amplification protocol to a 24-year-old BM slide
from a non-PNH subject and to a relatively recent BM slide
from another PNH patient (MSK11). In addition, we similarly
analyzed a PMN DNA from a normal subject. In the normal
control several different single bp substitutions were found;
however, we never found more than one M13 clone with the
same nt change (Table 3, line 6). In the regular DNA sample
from patient MSK11 we found an abnormal exon 2 fragment,
and direct sequencing showed a deletion of cytidine at nt
position 259. After cloning in M13 this mutation was confirmed
in 11 out of 12 M13 clones (Table 3, line 4). The same mutation
Table 2. Somatic Mutations in the PIG-A Gene of Patient
MSK13 Before and After BMT
DNA
Sample
1973
Exon
2
2
2
6
1995
6
bp Change
aa Change
16 GGA = TGA
211 ACC = GCC
211 ACC = GCC
251 ACC = ATC
1442 TCT = TTT
3
4
1355insAA-dupl3241355
*Predicted stop at codon 463.
M13 Clones
Mutant/Total
(%)
6 gly = stop
71 thr = ala
71 thr = ala
84 thr = ile
481
ser = phe
Frameshift*
3
4
5/36 (14)
13/36 (36)
5/36 (14)
The study of this patient has been informative in two
respects. First, duplications in the PIG-A gene must be very
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3426
Fig 6. Point mutations in exon 2 of the PIG-A gene in patient
MSK13 before BMT. (A) Exon 2 of the PIG-A gene was first amplified
by external primers -I1 and I2, and then reamplified with the internal
primers (see Table 1). (B) Nt sequence of M13 clones containing exon
2 fragment of patient MSK13. All (G) reactions were loaded adjacent
to each other, followed by the (A, T, and C) reactions. Two of the 3 M13
clones with substitution 211 T 8 C (clones 21, 22, 24) also have the
251 G 8 A substitution (clones 22 and 24).
rare. Until now, only one has been reported.21 Second, we have
found that over the long history of this patient PNH was caused
by clones with different PIG-A mutations at different times in
his clinical course (Fig 1). Identifying the duplication mutation
that currently underlies PNH in this patient was straightforward.
For the pre-BMT phase of this disease, only a few BM slides
were available, and PCR-amplification of all exons was performed successfully. Because of the very small amount of
material available, we went to considerable lengths to adapt our
methodology and to avoid being misled by PCR artifacts. Thus,
we regarded a mutation as significant only if it was found in at
least two independently isolated M13 clones. Of course, if a
Taq I polymerase error occurs early during amplification, we
might mistake a PCR artifact for a true mutation, especially if
the DNA template consists only of very few copies. However,
the comparison we have performed between the MSK13 slide
and a non-PNH slide from the same year was clear cut; this
NAFA ET AL
control makes it unlikely that fixation, staining, and storage
time contributed to create artifacts.
To our surprise, numerous mutations were found in the
pre-BMT MSK13 sample. Although we cannot say with certainty which one or which ones were responsible for the
patient’s original PNH phenotype, their representation amongst
the M13 clones we sequenced was well above the threshold we
had set. On the other hand, the duplication observed in the
relapse sample gives such a characteristic pattern in HA and
SSCA (Fig 3), that it could not be missed; and despite
developing a highly sensitive customized nested-PCR methodology, we could not detect the exon 6 duplication in the
pre-BMT sample. Of course it is impossible to rule out that the
clone with the duplication may have existed in the patient’s BM
in a site other than the one that was aspirated. With this proviso
we feel confident that at the time the patient originally
presented, the duplication could not account for clinical PNH
(see Results and Table 2). Thus, we have provided proof that
relapse of PNH in this patient was not due to failure of
eliminating the original PNH clone, but rather to the emergence
of a new clone.
Recently, Endo et al10 have reported that, in another patient
who had syngeneic BMT for PNH, a PIG-A mutation present in
one out of six T-cell clones (but not in peripheral blood
leukocytes) before BMT could also be shown in peripheral
blood leukocytes after BMT. Although their case is different
because the follow up was only 14 months, the findings are not
at all incompatible. We suggest that in the patient of Endo et al10
a minor PNH clone simply expanded after BMT (which is not
surprising in the absence of myeloablation); in our patient, a
new clone altogether has emerged.
The occurrence of several clones with independently arisen
PIG-A mutations is well documented in PNH.15,17-20 Our
longitudinal study of this patient, spanning more than 20 years,
provides more compelling evidence that, as we have previously
suggested,1 the PNH clone has a conditional selective advantage
in a particular BM environment. Endo et al10 have argued that in
their patient the clone that expanded must have had an
unconditional advantage; however, we note that if the conditions favoring a PNH clone before treatment are not modified by
a nonablative BMT, the conditional advantage of this clone will
be also unmodified. Indeed, if any of the PNH clones in our
patient had an absolute growth advantage, there is no reason
why any of them should have disappeared after a transplant
procedure was performed without BM ablation. We think that
instead the clone disappeared because the infusion of syngeneic
BM provided a surplus of normal hematopoietic cells. Unfortunately, the aplastic environment must have persisted in the
recipient, and this favored the growth of a clone with a new
PIG-A mutation, whereas the old clones had meanwhile become
exhausted. Because the donor and the recipient are syngeneic,
no genetic marker is available to determine with certainty
whether the relapse PIG-A mutation occurred in a donor cell or
in a recipient cell, but this does not affect our interpretation.
Indeed, if the mutation was in a donor cell, but this cell did not
expand into a detectable clone in the donor BM, this fact would
confirm once again that the pathological BM environment in the
recipient allowed such expansion to take place.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
RELAPSE OF PNH WITH NEW PIG-A MUTATION
3427
Table 3. Extensive Analysis Can Discriminate Pathogenic Mutations From PCR Artifacts
BM slides (nested PCR)
1-MSK13* (1973)
2-Non-PNH (1973)
3-MSK11 (1995)
PMN
4-MSK11 (primary PCR)
5-MSK11 (nested PCR)
6-Normal control
No. of M13
Clones With
Identical Mutation
No. of M13 Clones With
Identical Mutation Plus
Other Point Mutation
17
0
1†
8§
3¶
0
7††
4‡
5\
2#
1**
5‡‡
1
0
6
7††
4‡‡
0
4‡‡
5‡‡
0
No. of M13 Clones
Each Having a Single
Different Point Mutation
M13 Clones
Analyzed
No. of M13
Clones Without
Any Point Mutation
36
6
7
24
12
6
0
12
10
12
0
1
6
*In the pretransplant sample of MSK13, three point mutations (16 G = T, 211 A = G, and 211 A = G 1 251 C = T) are regarded as responsible for PNH.
†This M13 clone had the 16 G = T substitution.
‡Each one of these clones had the 16 G = T substitution (†) plus another point mutation, different in each clone.
§Each one of these clones had the 211 A = G substitution.
\Each one of these clones had the 211 A = G substitution (§) plus another point mutation, different in each clone.
¶Each one of these clones had both mutations 211 A = G and 251 C = T.
#Each one of these clones had the ‘‘211 A = G and 251 C = T’’ (¶) plus another point mutation, different in each clone.
**This M13 clone had two mutations: 101 A = G and 417 T = C. Each one of these was also present in two additional M13 clones together with
another point mutation.
††Each one of these clones had the mutation 259 del C, which we therefore regarded as responsible for PNH in patient MSK11.
‡‡Each one of these clones had the 259 del C mutation (†) plus another point mutation, different in each clone.
ACKNOWLEDGMENT
We are very grateful to the patient and to his twin brother for their
cooperation; to Dr D.P. Miller, the patient’s physician; and to Dr W.F.
Rosse for performing complement lysis studies on the patient’s blood
when a diagnosis of PNH was first made.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1998 92: 3422-3427
New Somatic Mutation in the PIG-A Gene Emerges at Relapse of
Paroxysmal Nocturnal Hemoglobinuria
Khédoudja Nafa, Monica Bessler, H. Joachim Deeg and Lucio Luzzatto
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