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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3422-3427
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
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.
 |
ABSTRACT |
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
insertion-duplication 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 duplication 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.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
PAROXYSMAL 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 HLA-haploidentical 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.
| |
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.
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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 Wright-stained 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  70°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 (TE=10 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 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.
| |
RESULTS |
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 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.
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| Fig 2.
Flow cytometry analysis with anti-CD59 of PMN from a
normal control (broken line) and PNH patient MSK13 (full line).
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| 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 double-stranded
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.
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| Fig 4.
Insertion duplication in exon 6 of the PIG-A gene
in patient MSK13. The sequence of 32 nt starting by 5 -TCAA-3
until 5-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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| 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
(5-... .ttTC-3) 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).
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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 successful,
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 serphe 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
3 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 211AC base change,
causing a 71 thrala amino acid replacement; and 28% of these
clones also have 251 CT, causing 84 thrile
(suggesting that the latter mutation arose in a cell belonging to the
clone that had the former mutation). Still in exon 2, 14% of the M13
clones sequenced have a 16 GT, causing 6 glystop
(Fig 6).
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| 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
C (clones 21, 22, 24) also have the 251 G A substitution
(clones 22 and 24).
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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 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.
| |
DISCUSSION |
The study of this patient has been informative in two respects. First,
duplications in the PIG-A gene must be very 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 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.
| |
FOOTNOTES |
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: k-nafa{at}ski.mskcc.org.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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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L. Luzzatto
Paroxysmal Murine Hemoglobinuria(?): A Model for Human PNH
Blood,
November 1, 1999;
94(9):
2941 - 2944.
[Full Text]
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D. J. Araten, K. Nafa, K. Pakdeesuwan, and L. Luzzatto
Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals
PNAS,
April 27, 1999;
96(9):
5209 - 5214.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract
