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Polymorphism of Human Platelet Membrane Glycoprotein IIb Associated With the
Bak"/Bakb Alloantigen System
By Suzanne Lyman, Richard H. Aster, Gian P. Visentin, and Peter J. Newman
The human Bak"/Bakb alloantigen system has been implicated in the pathogenesis of post-transfusion purpura and
neonatal alloimmune thrombocytopenic purpura. Human
alloantisera specific for either the Bak' or Bakb allele have
been shown t o react exclusively with the heavy chain of
membrane glycoprotein (GP) Ilb. To investigate the structure of the Bak epitopes, w e used the polymerase chain
reaction (PCR) t o amplify GPllb cDNA synthesized from
platelet RNA samples prepared from individuals of known
serologic phenotype. Subsequent DNA sequence analysis
of amplified GPllb cDNAs derived from one Bak' homozygous individual and one Bakb homozygous individual revealed a single nucleotide base difference near the 3'end of
the mRNA encoding the GPllb hefivy chain. Short 13 base
allele-specific oligonucleotides (ASO) containing the putative phenotype-specific base in the middle were then
synthesized, end-labeled with digoxigenin-1 1-dUTP using
terminal transferase, and used as probes in subsequent
dot-blot hybridization experiments. Platelet RNA was prepared from a panel made up of four Bak"", three BakbIb,and
t w o BakaIbindividuals, and the mRNA encoding GPllb was
amplified using PCR and spotted onto nylon membranes.
AS0 hybridization showed that the nucleotide base difference identified above segregated with Bak phenotype in all
nine individuals examined ( P = .002).The base pair substitution results in an amino acid polymorphism at residue 843
of the mature heavy chain. The Bak" form of GPllb encodes
an isoleucine at this position, whereas the Bakb allele
contains a serine. Identification of the polymorphism associated with this clinically important alloantigen system
should permit new therapeutic and diagnostic approaches
for treating and managing patients with alloimmune thrombocytopenic disorders.
0 1990by The American Society of Hematology.
P
systems," and these predictions appear to have been borne
out by more recent physical mapping studies using pulse-field
gel electrophoresisz0
Recent developments have now made it possible to examine the molecular basis of the Bak"/Bakb alloantigen system.
First, the nucleotide sequence of GPIIb has been fully
determined." Subsequently, it was reported that the polymerase chain reaction (PCR) could be adapted for the
amplification and subsequent analysis of platelet-specific
mRNA sequences from single individuals.22 We have recently used this approach to elucidate the molecular nature
of the PIA' and PIA' p ~ l y m o r p h i s m .In
~ ~this report, we
provide evidence that a single base difference at nucleotide
position 2622 of GPIIb mRNA is associated with the
Baka/Bakballoantigen system. This base change results in an
isoleucine/serine polymorphism at amino acid 843 near the
C-terminus of the GPIIb heavy chain.
LATELET MEMBRANE glycoproteins bear serologically defined determinants that can elicit an alloimmune response leading to platelet destruction in such clinical
settings as post-transfusion purpura (PTP) and neonatal
alloimmune thrombocytopenia (NATP).' These well-characterized syndromes are associated with the development of
platelet-reactive alloantibodies as a result of platelet incompatibility in blood transfusion recipients (PTP) and in
maternal sensitization to paternal antigens on fetal platelets
(NATP).' While the incidence of PTP is not precisely
known, the incidence of NATP has been estimated to be as
high as 1 in 2,000 live birth^,^ and delivery can result in
significant morbidity and mortality to the n e ~ b o r n . ~ ~ '
Nine different platelet alloantigen systems have thus far
been described. The PlA'/PlA2antigen system, located on
membrane glycoprotein (GP) IIIa, is perhaps the most
important clinically, accounting for a majority of reported
cases of PTP and NATP. However, other antigen systems
have clearly been implicated in these two immune disorders
as well. These include, in order of clinical significance, the
Bak"/Bakb system that has been localized to the heavy chain
of GPIIb,6.7the recently described Bra/Brb alloantigen system that has been localized to the GPIa/IIa c0mplex,8.~the
Pen," alternatively known as Yuk," alloantigen system,
located on GPIIIa," and the PIE antigen, which has been
localized to the heavy chain of G P 1b.l3
The Bak antigen system was first described in 1980 by von
dem Borne et a1 when they reported a new specificity present
in a maternal alloantibody in a case of NATP.6 Subsequently, an antibody termed anti-Leka was described by
Boizard and Wautier,I4 and the antigenic determinant for
this antibody was localized to 76 and 60 Kd fragments of the
heavy chain of GPIIb." Shortly thereafter, the Bak" antigen
was also localized to GPIIb,' and the Baka and Lek" antigens
were shown to be identical.l6-" The first characterization of
the allele for Bak", Bakb, was published only recently," and it
too was localized to the heavy chain of GPIIb. Interestingly,
Letellier et a1 predicted, on the basis of family studies, a close
genetic linkage between the P I A and Bak alloantigen
Blood, Vol 75, No 12 (June 15). 1990: pp 2343-2348
MATERIALS AND METHODS
Platelet PCR. Platelet RNA from a panel of nine unrelated
normal volunteers, including four BakaIa,three BakbIb,and two
Barib individuals, was prepared according to the procedure of
Chomczynski and SacchiZ4except that the final RNA pellet was
From The Blood Center of Southeastern Wisconsin, Milwaukee,
WI.
Submitted November 29,1989; accepted February 23,1990.
Supported by Grants No. HL-38166 (to P.J.N.) and HL-I 3629 (to
R.H.A.)from the National Institutes of Health.
G.P.V. is the recipient of a Scholarship in Biomedicine from the
Lynde and Harry Bradley Foundation.
Address reprint requests to Peter J. Newman. PhD, The Blood
Center of Southeastern Wisconsin, I701 W Wisconsin Ave. Milwaukee, WI 53233.
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.
0 1990 by The American Society of Hematology.
0006-4971/90/7512-0013$3.00/0
2343
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2344
subjected to one additional phenol/chloroform extraction and ethanol precipitation necessary to achieve reproducible gene amplification of platelet cDNA. Bak" and Bakb phenotype was assessed using
well-characterized human alloantisera in a standard antigen capture
a s ~ a y . ' ~The
. ' ~ C-terminal end of the GPIIb heavy and light chain
message from base 1988 to 2821 was selected for sequence analysis
and comparison, and two 24-base oligonucleotide primers flanking
833 base pairs of this region were synthesized on a Gene Assembler
(Pharmacia Fine Chemicals, Piscataway, NJ). The antisense primer
(5'-CAGGAAGGCCAGCACCGTGACCATG-3')from base 2821
to 2797 was used to prime the synthesis of cDNA from platelet
RNA, as previously d e ~ c r i b e d . ~The
~ , ~second
,
strand was generated
by the sense primer (5'-GACCTGCAGATGGACGCAGCCAAC3') from base 1988 to 2011 during the first round of PCR.
Amplification was carried out in a DNA Thermal Cycler (PerkinElmer Cetus, Norwalk, CT) programmed to permit denaturation at
94°C for 1l/2 minutes, annealing at 50°C for 1]/2 minutes, and chain
extension at 72°C for 3 minutes. The reaction was allowed to proceed
for 30 cycles, followed by a final incubation at 72°C for 7 minutes to
allow completion of strand synthesis.
Analysis of PCR products. PCR samples were analyzed on
1.8% Seakem GTG agarose gels (FMC BioProducts, Rockland,
ME), and the appropriate bands were excised and recovered by
electroelution. The plasmid vector pCEM-SZf (Promega Biotech,
Madison, WI) was prepared for ligation by restriction digestion with
EcoRV (New England Biolabs, Beverly, MA) to yield blunt ends,
and ligated to purified amplification products, followed by transformation into Escherichia coli strain NM522 competent cells (Stratagene Cloning Systems, San Diego, CA). Two clones representing
each Bak homozygous phenotype were selected for direct sequence
analysis by the dideoxy sequencing method using T7 DNA polymerase (USB, Cleveland, OH). Four 24 to 25 base oligonucleotides
corresponding to internal GPIIb cDNA sequences were synthesized
and used as sequencing primers.
Allele-specific hybridization. Amplified cDNA from four individuals with Bak"/" phenotype, three with BakbIb,and two heterozygous for Bak was subjected to hybridization with 13-base allelespecific oligonucleotides (ASO). Probe A (TGCCCATCCCCAG)
corresponds to the published sequence from base 2616 to 2628, and
probe B (TGCCCAGCCCCAG) differs only in the middle base, a G
instead of a T, and corresponds to a single base difference observed in
the region sequenced. The oligos (200 ng) were end-labeled with
digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) in
25 pL 100 mmol/L potassium cacodylate, 2 mmol/L CoCl,, 0.2
mmol/L DTT, pH 7.2 containing 1 U terminal transferase
(Boehringer Mannheim), and the probes were used for hybridization
without purification. Amplified DNA was used directly for blotting,
or, in some cases, appropriate bands were recovered from agarose
gels using Gene Clean (Bio 101, La Jolla, CA). The samples were
eluted in 20 pL water, diluted 1/10,000, and 10 pL was used for
reamplification using the same oligos and PCR conditions. Amplified
or reamplified DNA was denatured in 0.25 N NaOH, 1.5 mol/L
NaCl at room temperature for 5 minutes, followed by neutralization
in 2 mol/L NaCI, 1.3 mol/L Tris, pH 7.4 at room temperature for 15
minutes. Each sample was divided between two wells of a Minifold
dot blot apparatus (Schleicher and Schuell, Keene, NH) and
transferred to Magnagraph nylon membrane (MSI, Westboro, MA)
by vacuum suction. The filter was exposed to UV irradiation
(Fotodyne, New Berlin, WI) for 5 minutes, followed by baking at
8OoC for 15 minutes. The membrane was prehybridized in 5x
Denhardt's, 5 x standard sodium citrate (SSC), 5 mmol/L EDTA,
I O mmol/L Na2HP04,pH 7.0 at 68°C for 1 hour and then cut into
two strips that were hybridized to either probe A or probe B in 4 mL
l o x Denhardt's, 5 x SSC, 5 mmol/L EDTA, 7% sodium dodecyl
sulfate (SDS), SOpg/mL salmon sperm DNA, 20 mmol/L Na2HP04,
LYMAN ET AL
pH 7.0 at 42°C overnight. The filters were washed in two changes of
6 x SSC for 30 minutes each at room temperature, followed by two
changes of 3 mol/L tetramethylammoniumchloride (Aldrich Chemical, Milwaukee, WI), 2 mmol/L EDTA, 1% SDS, 50 mmol/L Tris,
pH 8.0 for 20 minutes each at 42°C. Positive hybridizations were
visualized using the Genius kit (Boehringer Mannheim, Indianapolis, IN), which uses an alkaline phosphatase-conjugated antidigoxigenin antibody.
Flow cytometric analysis. Platelets were washed in Ringer's
citrate dextrose buffer (RCD:IO8 mmol/L NaCI, 38 mmol/L KCI,
1.7 mmol/L NaHCO,, 21.2 mmol/L Na citrate, 27.8 mmol/L
dextrose, 1.1 mmol/L MgCI,, 2 mmol/L EDTA, and 50 ng/mL
PGE,, pH 6.5). Approximately 5 x lo7 cells were resuspended in
either 200 pL anti-Bakb(provided by the Platelet Antibody Laboratory of the Blood Center of Southeastern Wisconsin, Milwaukee,
WI) antisera diluted 1:lO or 1:25, or the anti-GPIIb murine
monoclonal antibody PMI-1 (provided by Dr Mark Ginsberg,
Research Institute of Scripps Clinic, La Jolla, CA) diluted to 1 to 5
pg/mL in RCD buffer, and incubated for 1 hour at room temperature. After three washes, Bak-labeled cells were incubated with
affinity purified F(ab'), fragments of fluorescein isothiocyanate
(FITC)-conjugated goat anti-human IgG (Jackson Immunoresearch, West Grove, PA), and PMI-1-labeled cells were incubated
with affinitypurified F(ab')2 fragments of R-phycoerythrin (rPH)conjugated goat anti-mouse IgG (Tago Immunological, Burlingame,
CA) for 30 minutes at room temperature. Labeled cells were washed
three times and subjected to flow analysis. To assess potential
competition between PMI-1 and anti-Bakbantibodies, platelets were
incubated with anti-Bakbat a 1:25 dilution (95% saturation) in the
presence of PMI-1 at 5 pg/mL, which was in excess of saturation.
The converse experiment was also done, in which platelets were
incubated with PMI-1 at 1 pg/mL (90% of saturation) in the
presence of anti-Bakb diluted to l:lO, which was in excess of
saturation. Preliminary studies were done to show that FITC-goatanti-human IgG did not crossreact with mouse IgG, and rPH-goatanti-mouse IgG did not crossreact with human IgG (data not
shown). The samples were analyzed on a FACStarp'"' Fluorescence
Activated Cell Sorter (Becton Dickinson, Mountain View, CA)
using an argon laser source at 488 nm. Fluorescein and rPhycoerythrin were detected through a 530+ / - 30 nm band-pass
filter, and 10,000events were acquired per sample.
RESULTS
Amplijcation and analysis of the C-terminal region of
GPIIb platelet mRNA. T o compare the nucleotide sequence of GPIIb m R N A derived from one Bak" and one Bakb
homozygous individual, four different pairs of oligonucleotide primers were designed such t h a t overlapping segments
of the molecule could be sequentially amplified a n d their
sequence determined. Nucleotide sequence analysis of a n
833 base pair region positioned near the 3' end of G P I I b
c D N A (Fig 1) revealed a single nucleotide difference a t base
2622 of t h e GPIIb m R N A (Fig 2). T h e Bak"'" individual has
a T a t this position, whereas t h e Bakb/bindividual has a G.
This results in t h e substitution of a serine for a n isoleucine at
amino acid residue 843 of t h e G P I I b heavy chain.
Correlation of Ne,,, t--* Ser,,, polymorphism with Bak
allotype. In order t o determine whether t h e T c* G
polymorphism was associated with Bak phenotype, as opposed t o a Taq polymerase error or a naturally occurring
polymorphism unrelated to Bak phenotype, platelet R N A
was prepared from a total of nine unrelated individuals of
known Bak phenotype, and then amplified using P C R to
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B u ALLOANTIGEN SYSTEM
2346
Fig 1. Amplifiatkr, .nrt.gY 8nd 8M)y.k of th. Gt.nnhul
r.gkn of the pbtdet OPllb mRNA md.cule. The anthomo PCR
ptimer was positionedwithin the 8equmceencodingthe N-terminaI
end of the l i h t chain. and the aen8e primer was positioned such
that the total region nmpli6ed -8 833 bp. Thi8 interval emompun8 exons spanning more than 3.400 base8 of genomic DNA.”
and thus could only repre8ent GPllb mRNA.
yield the same 833 bp product. We then performed allelespecific hybridization analysis, using two 13-base probes
containing the putative phenotype-specific base in the middle, toscreen the amplified cDNAs. Allele-specificoligonucleotides will hybridize to the exact complementary sequence;
however. a single base mismatch will prevent annealing of
the probe at a specified temperature that is dependent on the
length of the probe.I6As shown in Fig 3, probe A (containing
a T in the middle) was positive with the four Bak”’
homozygous individuals, and probe B (containing a G in the
middle) was positive with three BakbIbindividuals, whereas
both oligonucleotides hybridized with amplified cDNA derived from the two heterozygous individuals. Thus, all nine
individuals examined by DNA typing. representing the three
possible Bak phenotypes, yielded the genotypes expected
(P = .002) Based upon these results, wc conclude that the
observed base change is directly associated with the Bak’/
Bakballoantigen system.
Physical relationship of the Bok and PMI-I epitopes.
Comparison of the location of the Bak polymorphism to the
amino acid sequence of GPllb (Fig 4) revealed that this
B A K ~
T G C A T
amino acid is just outside of a 17-residue sequence that
contains the epitope for the anti-GPllb murine monoclonal
antibody, PMI-I.” To explore the physical relationship
between these two epitopes, a series of competitive binding
studies were done. In one set of experiments, PMI-I was
present in limiting concentrations (approximately 904% of
saturation). while anti-Bakb was in excess. The converse
experiments. in which anti-Bakb was present in limiting
amounts (95% saturation) and PMI-Iwas present in excess.
were also done. Platelets containing one or both sets of
antibodies in various ratios were analyzed by flow cytometry,
and the results are shown in Fig 5. As can be seen, no
competition between the two antibodies was observed, despite their apparent physical proximity.
DISCUSSION
The purpose of the present study was to determine the
molecular basis for the Bak’/Bakb polymorphism of platelet
membrane GPllb. Based on the reported nucleotide sequence of GPllb derived from HEL cells, we designed
oligonucleotide primers and used the PCR to amplify an 833
base pair C-terminal region of GPllb from both a Bak’
homozygous and a Bakb homozygous individual. The sequence of the Bak’ individual proved to be identical with the
published sequence of GPllb, except for a single nucleotide
difference at base 2622 in the sequence from the Bakb
individual. The substitution of a G for a T at this position
resulted in an isoleucine * serine polymorphism at amino
acid 843 of the GPllb heavy chain.
GPllb is composed of a 125 Kd heavy chain that is
disulfide-linked to a 23 Kd light chain.B Earlier studies of the
biosynthesis and processing of GPIlbn”’ provide evidence
that a single mRNA transcript is used to synthesize a
precursor of GPllb that is cleaved post-translationally to
yield the two disulfide-linked chains, and these studies were
substantiated by the recent molecular cloning of GPllb
BAK
M>
T G C A T
GJ - - c ‘
El
CJ
“PI
propM
Fig 2. DNA seqwnw aruly.h of nmplHwd OPNb @NA from Bok.” individual and a Bok”’ individual. The 833 bp PCR prodwt.wore
8ubekmdinto +EM-621 and sequenced usingnn antismso digonucl.otidethat extended 6‘ to 3‘ from base 2779 to 2766. The ..gnnntof
the a u t o r d i r a p h shown here includes the sequence from be808 2616 to 2626. The sequ(~~c.
from the Bok’ indMdual is Mentical to the
previowly publi8hed sequence for GPllb. having a G at base 2622. This base h chenged to a T in the Bokb individual. rowking in an
i8olwcine serine polymorphism.
++
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LYMAN ET AL
2346
PHENOTYPE
Bak*
Bakm
Bak*
of Bok phenotype by eIl.k-.p.ci(ic hybridimtion. Platelet RNA war isolated from nino IndMdwh with known Bok
Fig 3. AMphenotype. The C-terminal region of aPllb from base 1988 to 2821 war reverse transcribed and emplitied by PCR. Aliquot. of each PCR
reaction (4pL)were blottedonto e nylon fikor in duplicate, end the fikor was cut into two parellel strips and hybridized with &her probe A
(top) or probe B (bottom). The serdogically determined Bak phenotypes of the individwlr screened are indkated above the strips.
cDNA.*' Hydropathicity analysis of the cDNA sequence
indicates that GPllb is anchored to the platelet through a
single transmembrane domain located within 20 amino acids
of the C-terminus of the light chain. The studies reported
here localize the amino acid difference associated with the
Bak polymorphism near the C-terminus of the heavy chain
within a proline-rich region.
Further studies are needed to elucidate the exact nature of
the epitope recognized by Bak alloantisera. Although it is
most likely that these two polymorphic amino acids are
responsible for directing the formation of the Bak antigenic
determinants, differences in carbohydrate moieties could also
play a role in antigenicity. Since the amino acid substitution
in Bak involves a serine, it is possible that an 0-linked
carbohydrate could be added to the Bakb molecule. Another
possibility that must be considered is that the Ile
Ser
amino acid difference causes a conformational change on
some other portion of the molecule that forms the actual
epitope. Finally, our findings do not preclude the possibility
of other polymorphisms in the GPllb gene that may be
associated with Bak phenotype.
It is of considerable interest that the Bak polymorphism is
immediately adjacent to the 17-amino acid residue region
containing the epitope recognized by the monoclonal antibody PMI-I." Functional studies using the PMI-I antibody
-
-
Fig4. Amino.dd8sqwncopdymorphbma..odmdwiththo
Bok./Bok' p l o t o h alhntigen system. Note that the i.d.ucino
m i n o pdymorphism is located nmr the C-terminua of the GPllb
heevv chain in an extremely prolino-rkh region. The peptide
containing the .pitope for PMCl is immeddi.tely adjacent to the
Bok polymorphk residue. The precise clmvaga site between the
heavy and light chain has not beon experimentally determined, and
the one indicated reprewnts a best estimate besed upon date
obtained using anti-peptide entibody binding analysism and by
compariaon with homologwr requences within the rat GPllb
gena.n
of antWm3 end M . 1 by
Fig 6. Compotitko blndlq OMtwocdor fluoreaceme tiow qtomotry. Slmukar" Mnding of
both antibodies war auossod by uring second antibodies conjugeted to red (anti-mouse) or green (anti-human) fluorophores. Top
quadrent Binding of anti-BakbIgG in the presence of excess PMI-1.
Note that the reletive mean fluor..c.me of neither antibody
(number above the peak) is effected by the binding of the other.
Bottom quadrant: Binding of PMI-1 in the prewncs of e x c e u
anti-BakbIgG.
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2347
BAK ALLOANTIGEN SYSTEM
have previously shown that this region may be involved in
platelet adhesion to collagen.32The effect of anti-Bak alloantisera on platelet/collagen interactions has not been examined to date, nor have the possible consequences of anti-Bak
antibodies on in vivo platelet function in alloimmunized
patients been considered. In order to explore the possible
relationship of these two epitopes, we did cross-blocking
studies using PMI- 1 and anti-Bakb. Somewhat surprisingly,
our data indicate that there is no competition for binding of
these two antibodies. Secondary structure analysis of GPIIb33
indicates that the sequence KRDRRQ, located a t the
C-terminal third of the peptide to which PMI-1 binds, is the
second most likely antigenic site on this molecule. If this
sequence serves as the epitope for PMI-1, then there would
be 11 amino acid residues between this epitope and the likely
Bak antigenic determinant. The proline-rich nature of this
region and its predicted highly convoluted secondary structure may explain the co-occupation of adjacent sites by these
two antibodies.
In addition to its inhibition of platelet-collagen interactions, PMI- 1 has also been shown to bind preferentially to the
GPIIb-IIIa complex after its occupation with either fibrinogen or the Arg-Gly- Asp tripeptide sequence; consequently,
the PMI- 1 epitope has been referred to as a ligand-induced
binding site, or LIBS.34Given the close association between
the BAK and PMI-1 epitopes, it is interesting to note that
anti-Bak" antibodies were found to bind to only 20,000 sites
per platelet at ~aturation,'~
even though 40,000 GPIIb-IIIa
complexes are surface exposed.36 It is tempting to speculate
that the Bak alloantigen may represent another example of
an occupancy-modulated region of the platelet fibrinogen
receptor.
Increasingly, molecular techniques are being used to
define sequence variations responsible for inherited genetic
disorders and naturally occurring polymorphisms. While two
human platelet alloantigen systems, Bak and PIA,have now
been examined at the molecular level, it will be important to
delineate the molecular basis of additional platelet membrane polymorphisms in order to achieve a better understanding of platelet membrane glycoprotein fine structure responsible for generating an alloimmune response. Knowledge of
the amino acids responsible for directing the formation of
these polymorphic determinants may lead to the development of more refined immunodiagnostic assays, for example,
allotype-specific monoclonal antisera. Our findings also provide an alternative to immunologic-based assays, as DNA
typing using allele-specific oligonucleotide hybridization is
able to discriminate between
Bak"', and Bakb/b
individuals with a high degree of precision and reliability.
DNA-based techniques can readily be adapted to establish
diagnostic tests in the transplant or transfusion setting, as
well as cases requiring in utero diagnosis, and may offer
several important advantages over present serologic methods
of phenotyping that rely on a limited supply of alloantisera.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1990 75: 2343-2348
Polymorphism of human platelet membrane glycoprotein IIb
associated with the Baka/Bakb alloantigen system
S Lyman, RH Aster, GP Visentin and PJ Newman
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