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Microsatellite Instability and Mutation of DNA
Mismatch Repair Genes in Gliomas
Suet Yi Leung,* Tsun Leung Chan,*
Lap Ping Chung,*
†
Annie S. Y. Chan,*
Yiu Wah Fan,
‡
Kwan Ngai Hung,
‡
Wai Kay Kwong,
§
Judy W. C. Ho,
†‡
and Siu Tsan Yuen*
†
From the Departments of Pathology,*Surgery,
‡
and Radiation
Oncology,
§
and the Hereditary Gastrointestinal Cancer Registry,
†
Queen Mary Hospital, The University of Hong Kong, Hong Kong
Microsatellite instability (MSI) has been identified in
various human cancers, particularly those associated
with the hereditary nonpolyposis colorectal cancer
syndrome. Although gliomas have been reported in a
few hereditary nonpolyposis colorectal cancer syn-
drome kindred, data on the incidence of MSI in glio-
mas are conflicting, and the nature of the mismatch
repair (MMR) defect is not known. We established the
incidence of MSI and the underlying MMR gene mu-
tation in 22 patients ages 45 years or less with spo-
radic high-grade gliomas (17 glioblastomas, 3 ana-
plastic astrocytomas, and 2 mixed gliomas, grade III).
Using five microsatellite loci, four patients (18%) had
high level MSI, with at least 40% unstable loci. Germ-
line MMR gene mutation was detected in all four pa-
tients, with inactivation of the second allele of the
corresponding MMR gene or loss of protein expres-
sion in the tumor tissue. Frameshift mutation in the
mononucleotide tract of insulin-like growth factor
type II receptor was found in one high-level MSI gli-
oma, but none was found in the transforming growth
factor
b
type II receptor and the Bax genes. There was
no family history of cancer in three of the patients,
and although one patient did have a family history of
colorectal carcinoma, the case did not satisfy the Am-
sterdam criteria for hereditary nonpolyposis colorec-
tal cancer syndrome. Three patients developed meta-
chronous colorectal adenocarcinomas, fitting the
criteria of Turcot’s syndrome. Thus, MSI and germline
MMR gene mutation is present in a subset of young
glioma patients, and these patients and their family
members are at risk of developing other hereditary
nonpolyposis colorectal cancer syndrome-related tu-
mors, in particular colorectal carcinomas. These
results have important implications in the genetic
testing and management of young patients with
glioma and their families. (Am J Pathol 1998,
153:1181–1188)
Microsatellite instability (MSI) is characterized by the ex-
pansion and contraction of small repeat sequences dur-
ing DNA replication and is present in the majority of
tumors in the hereditary nonpolyposis colon cancer
(HNPCC) syndrome.
1
HNPCC is characterized by familial
occurrence of cancer in various sites, including the co-
lon, endometrium, and urinary tract, at an early age.
2
The
mechanism leading to MSI is related to a defect in the
DNA mismatch repair (MMR) system, of which more than
5 DNA mismatch repair (MMR) genes are now known.
3–12
Mutation of the hMSH2 and hMLH1 genes accounts for
more than 60% of individuals with the syndrome.
13
Many sporadic cancers have also been found to show
MSI.
14
For gliomas, relatively few studies have been per-
formed, and the results are conflicting. Izumoto et al
15
and Dams et al
16
demonstrated the presence of MSI in 20
to 45% of glioblastomas and anaplastic astrocytomas
and in no low-grade astrocytomas. Zhu et al
17
found MSI
in 17% of oligodendrogliomas and 3% of astrocytic tu-
mors. Wooster et al
18
and Amariglio et al
19
found MSI in
1.9% of brain tumors and in no brain tumors, respectively.
In most of these series, MSI was considered positive if
there was an allelic shift in a single locus only. The status
of the MMR gene, be it germline or somatic, is largely
unknown for these MSI-positive gliomas. On the other
hand, there have been reports of increased risk for brain
tumors in HNPCC kindred,
20–22
and a few patients with
Turcot’s syndrome, characterized by the development of
both colorectal and brain cancers, have been shown to
have MSI and to harbor germline mutation in the MMR
genes.
23,24
We have previously reported an unusually high inci-
dence of colorectal carcinoma in the young Hong Kong
population.
25
Coincidentally, there have been several
studies reporting an unexpected tendency for the occur-
rence of glioblastomas and anaplastic astrocytomas in
young Chinese in Taiwan, the People’s Republic of
China, and Hong Kong,
26–28
when compared with the
West.
29,30
The incidence of MSI is high in cases of spo-
radic colorectal carcinoma in the young of Hong Kong
31
and elsewhere,
32
and germline mutation of the MMR
Supported by Committee on Research and Conference Grant 337/046/
0024 and University Research Committee Grant 344/046/0003 from the
University of Hong Kong and by Croucher Foundation Research Grant
394/046/1238. TLC is a Ph.D. student of the University of Hong Kong.
Accepted for publication July 18, 1998.
Address reprint requests to Dr. Siu Tsan Yuen, Department of Pathol-
ogy, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong
Kong. E-mail: styuen@hkucc.hku.hk.
American Journal of Pathology, Vol. 153, No. 4, October 1998
Copyright © American Society for Investigative Pathology
1181
genes has been found in a high proportion of these
young patients with MSI,
31,32
but little is known of the MSI
status or mutation of the MMR genes in the young pa-
tients with gliomas. Using stringent criteria for MSI,
33,34
we examined a series of local young patients with high-
grade gliomas (grades III to IV by the World Health Or-
ganization system
35
), to look for the presence of MSI and
examined for mutation, both somatic and germline, in the
hMSH2 and hMLH1 genes.
Materials and Methods
Materials
Twenty-two patients, ages 45 years or less, with gliomas
of grades III to IV, were included in this study. The mean
age of the patients was 33 years (range, 13 to 44). The
tumors included 17 glioblastomas, 3 anaplastic astrocy-
tomas, 2 mixed gliomas (grade III), and the histological
classification used was based on the World Health Orga-
nization system.
35
Either frozen or paraffin-embedded
tumor tissue with more than 80% tumor cell content was
used. For normal tissue, either blood leukocytes obtained
by venipuncture with the patients’ consent or normal
brain tissue adjacent to the tumor was used. For all tissue
used for DNA extraction, frozen or paraffin sections were
used to confirm the absence of tumor cell contamination
in the normal tissues and to confirm the percentage of
tumor in tumor blocks. DNA and RNA were extracted
from blood leukocytes and frozen tumor blocks for germ-
line and somatic MMR gene mutational analysis using
standard methods.
MSI Analysis
Paired tumor and normal tissues were amplified by poly-
merase chain reaction (PCR) using 5 microsatellite loci.
These included dinucleotide repeats (Tp53, D18S58, and
D2S123) and polyadenine tracts (Bat40 and Bat26/A26).
Tp53 was purchased from Research Genetics (Hunts-
ville, AL). D18S58, D2S123, and Bat40 were synthesized
according to the sequence published previously.
13
For
the polyadenine tract in intron 5 of the hMSH2 gene, two
pairs of primers were used, including Bat26 as previously
published,
13
and another pair named A26, correspond-
ing to nucleotides 123 to 143 (forward) and nucleotides
222 to 241 (reverse) of the hMSH2 exon 5 genomic
sequence (GenBank accession no. U41210). In all cases,
all five loci were analyzed.
The PCR was performed in a 10-
m
l reaction solution
containing 50 ng of DNA, 10 mmol/L of Tris (ph 8.3), 50
mmol/L of KCl, 2 to 3 mmol/L of Mg
21
, 200
m
mol/L de-
oxynucleotide triphosphate, 1
m
Ci [
a
-
32
P]dCTP, 0.2 to 1
m
mol/L of each primer, and 0.1 U Taq polymerase. A
hot-start reaction was performed by preheating the mix-
ture in the thermocycler at 95°C for 5 minutes, then cool-
ing to 80°C before adding the Taq polymerase. An initial
denaturation step of 95°C for 5 minutes and 25 to 40
cycles, including 95°C for 45 seconds (1 minute), 1
minute (1.5 minutes) in 52 to 64°C annealing temperature
according to the specific primers, and 72°C for 1 minute
(2 minutes) in frozen DNA (paraffin DNA), was performed,
followed by a final extension of 5 minutes at 72°C.
The PCR products were diluted by loading buffer,
heated at 95°C for 5 minutes, and loaded onto 6% vertical
polyacrylamide gel. After electrophoresis, the gels were
fixed, dried, and exposed to X-ray film for 12 hours to 7
days.
The results were interpreted independently by two ob-
servers. Results with discrepancy in interpretation were
discussed and PCR was repeated if necessary. MSI was
defined as the presence of allelic shift or additional
bands in the tumor compared with normal tissue. All
cases with MSI were repeated once. A case was defined
as high-level MSI if there were more than 40% unstable
loci, low-level MSI if less than 40%, and microsatellite
stable if there were no unstable loci.
33,34
MSI in the (A)
10
tract of type II transforming growth
factor
b
receptor (T
b
RII), (G)
8
tracts of Bax, and insulin-
like growth factor type II receptor (IGFIIR) genes was also
analyzed in the microsatellite-unstable cases. The primer
sequences were as described previously.
36–38
hMSH2 and hMLH1 Mutational Analysis
Mutational analysis for hMSH2 and hMLH1 was per-
formed for the high-level MSI cases using the following
methods.
In Vitro Synthesized Protein Assay
In vitro-synthesized protein assays to screen for trunca-
tion mutations in the MMR genes hMSH2 and hMLH1
were performed with primer sequences as described.
5,13
In brief, 3
m
g of total RNA was reverse transcribed using
20 to 200 ng of random hexamers or oligo(dT), 20 units of
RNAsin, 20 pmol of deoxynucleotide triphosphates, and
200 units of Superscript II reverse transcriptase (Life
Technologies, Inc., Grand Island, NY) in a 20-
m
l reaction
volume, using the manufacturers’ suggested reaction
conditions. Forty cycles of PCR were performed in 50
m
l
and included 2 to 4
m
l of first-strand cDNA mix, 10
mmol/L of Tris-HCl (pH 8.3), 50 mmol/L of KCl, 3 to 5
mmol/L of MgCl
2
, 5 pmol of each primer, 200
m
mol/L of
each nucleotide, and 2.5 units Taq polymerase (Life
Technologies). Both hMSH2 and hMLH1 were amplified
in two overlapping segments ranging between 1.2 and
2.0 kb. The left-hand primers of each segment were
tagged with a T7 promoter sequence and a translation
initiation site. The products were then subjected to in vitro
transcription/translation using the linked T7 transcription-
translation system (Amersham Corp., Little Chalfont, UK).
DNA Sequencing Analysis
Individual exons of hMSH2 and hMLH1 genes, including
intron-exon boundaries, were PCR amplified. The prim-
ers’ sequences are available on request. The PCR prod-
ucts were then purified by High Pure PCR Product Puri-
fication Kit (Boehringer Mannheim, Mannheim, Germany)
1182 Leung et al
AJP October 1998, Vol. 153, No. 4
and directly sequenced by Sequenase V2.0 (Amersham)
using both forward and reverse primers following the
manufacturer’s protocols. The sequencing products were
denatured at 80°C for 5 minutes and electrophoresed
through a 6% polyacrylamide/urea gel at 70 W for 2 to 3
hours. The gels were then fixed, dried, and exposed to
autoradiographic films.
Immunohistochemistry
Immunostaining for hMSH2 and hMLH1 was performed in
the cases showing allelic shift in one or more loci, using
the standard streptavidin-biotin-peroxidase complex
method with 3,39-diaminobenzidine as chromogen. Sec-
tions 6
m
m thick of 10% neutral buffered formalin-fixed,
paraffin-embedded tumor tissue were incubated for 1
hour at 37°C with monoclonal antibodies against the ami-
no-terminal fragment (clone GB12; dilution 1:20; Onco-
gene Research Products, Cambridge, MA) and carboxy-
terminal fragment of hMSH2 (clone FE11; dilution 1:200;
Oncogene Research Products, Cambridge, MA). For
hMLH1, sections were incubated at 37°C for 1 hour using
a monoclonal antibody (clone G168-15; dilution 1:10;
PharMingen, San Diego, CA). Microwave pretreatment at
95°C for 30 minutes in citrate buffer, pH 6.0, was per-
formed after deparaffinization. For negative control, the
primary antibodies were replaced by mouse immuno-
globulin G (Dakopatts, Glostrup, Denmark).
Results
MSI
Four of the 22 high-grade gliomas from patients ages 45
years or less (18%) showed high-level MSI (Table 1).
These included three glioblastomas and one malignant
mixed glioma. In all four cases, there was gross MSI with
75 to 100% loci involved. One additional tumor showed
MSI in one locus only, and this case was thus considered
low-level MSI. None of the tumors showed mutation in the
mononucleotide tracts in the T
b
RII and Bax genes. One
tumor showed frameshift mutation in the (G)
8
tract of the
IGFIIR gene. Representative results of the microsatellite
analysis are shown in Figure 1 and 2.
Clinicopathological Data and Family History
The clinical data of the four patients with high-level MSI
gliomas are shown in Table 2. The patients were all
relatively young; two of them were below 30 when they
developed the glioblastoma. Histologically, the three gli-
oblastomas showed primitive anaplastic cells in the
background and the presence of many multinucleated
Table 1. Results of Microsatellite Analysis and Immunohistochemistry for Cases Showing MSI
Patients
ABCDE
Sex/age F/27 M/23 M/35 M/37 F/38
Diagnosis GBM Mixed glioma (grade III)* GBM GBM Astro III
Tp53 11 111
D18S58 21 112
D2S123 11 112
Bat40 11 112
Bat26/A26
†
11 112
% loci with MSI 75% 100% 100% 100% 20%
T
b
RII (A)
10
22 222
Bax (G)
8
22 222
IGFIIR (G)
8
21 222
hMSH2 protein (IHC) 12 221
hMLH1 protein (IHC) 21 111
GBM, glioblastoma multiforme; Astro III, anaplastic astrocytoma; IHC, immunohistochemistry.
†
Both Bat26 and A26 amplify a polyadenine tract in intron 5 of the hMSH2 gene.
*According to World Health Organization system.
Figure 1. MSI at various loci in gliomas: A, Tp53; B, D2S123; C, D18S58; D,
Bat26; E, A26; and F, Bat40. N, normal; T, tumor.
MSI and DNA Mismatch Repair Gene Mutation in Gliomas 1183
AJP October 1998, Vol. 153, No. 4
tumor giant cells. Patient B had a malignant mixed gli-
oma, with a prominent oligodendroglial element, although
ependymal and astrocytic elements were noted in some
areas. None except patient D had a family history of
cancer. Patient D had a positive family history of colorec-
tal carcinoma, but that did not satisfy the Amsterdam
criteria for HNPCC syndrome. Interestingly, three of the
patients had metachronous colorectal adenocarcinomas,
thus satisfying the criteria of Turcot’s syndrome.
39
Patient
A was only 27 years old when she developed a glioblas-
toma, and there was no previous tumor nor any family
history of cancer.
Germline and Somatic Mutation of the
MMR Genes
All four patients with high-level MSI gliomas showed
germline mutation of the MMR genes, three of them in the
hMSH2 and one in hMLH1 (Table 2). Three of the muta-
tions resulted in truncated protein products. One case
(patient D) showed a missense mutation resulting in
amino acid substitution in an evolutionary conserved res-
idue. The wild-type allele was lost in the tumor in this
patient.
In three cases, a second hit could be identified in the
gliomas. Patient A showed two truncated protein prod-
ucts in the in vitro-synthesized protein assay of the tumor
RNA (Figure 3). The germline mutation was found in exon
8 of the hMLH1 gene, which resulted in skipping of the
exon (Figure 4A and 5). A second mutation, found only in
the tumor DNA, resulted in a stop codon (Figure 4B). For
patients C and D, the normal allele was absent when
tumor tissue was sequenced (Figure 6). For patient B, the
wild-type allele was retained in sequencing of exon 11 in
the brain tumor. We did not screen for other somatic
mutation in the hMSH2, because only paraffin blocks of
the tumor were available.
Expression of the hMSH2 and hMLH1 Protein
Immunohistochemical staining revealed complete loss of
hMSH2 protein when both antibodies on the tumor cells in
patients B, C, and D were used, whereas the normal
neurons and glial cells at the tumor borders were posi-
Figure 2. Frameshift mutation in the mononucleotide repeats of IGFIIR gene
(C) but not in the T
b
RII (A) and Bax (B) genes in microsatellite-unstable
gliomas. Lanes N, normal; lanes T, tumor.
Table 2. Clinical Data and DNA MMR Gene Mutations in Four Patients with Microsatellite-Unstable Gliomas
Patient
Sex/
age Histology Family history
Other cancers
(age, years)
Survival after
craniotomy
MMR germline
mutation
MMR somatic mutation
in glioma
A F/27 GBM None None Died of disease at 8
months
hMLH1 last nucleotide
of exon 8
(CGgt3CAgt),
resulting in splicing
defect, skipping of
exon 8, deletion of
codon 197–226, and
frameshift
hMLH1 exon 13, codon
487 CGA3TGA(stop)
B M/23 Malignant
mixed
glioma
None Colon (25) Died of disease
(brain) at 4 years
hMSH2 exon 11 codon
580
GAA3TAA(stop)
Not determined;
(absence of hMSH2
protein by
immunostaining)
C M/35 GBM Not available
(adopted son)
Colon (29) Alive with disease
(brain) at 6
months
hMSH2 first nucleotide
of exon 12 codon
587, deletion of G
(agGCT3agCT),
generating a stop
codon 6 bp
downstream
Wild-type allele loss in
tumor by sequencing
D M/37 GBM Two sisters
had colorectal
adenocar-
cinomas
Rectum (28) Died of disease
(brain) at 0.5
months
hMSH2 exon 3 codon
199 (TGT3CGT)
resulting in change
of an evolutionary
conserved cysteine
to arginine
Wild-type allele loss in
tumor by sequencing
GBM, glioblastoma multiforme.
1184 Leung et al
AJP October 1998, Vol. 153, No. 4
tive. Staining for hMLH1 was retained in the tumors in
these three patients. The tumor cells in patient A were
negative for hMLH1 but positive for hMSH2 proteins,
whereas the normal cells were positive for both.
Discussion
Three important pieces of information resulted from this
study. 1) A proportion of young patients (18%) with high-
grade gliomas showed gross replication error, character-
istic of defects in the DNA MMR system. 2) In all of these
patients with gross replication errors, germline mutation
in one of the MMR genes could be detected. 3) In three
of the four cases, inactivation of the second allele was
found in the tumor tissue. Although the second hit could
not be detected in patient B, the tumor cells were immu-
nohistochemically negative for hMSH2, supporting the
presence of a second inactivating event. This double-hit
phenomenon for MMR genes, identical to Knudson’s the-
ory for a tumor suppressor gene, was also previously
suggested in HNPCC-related and sporadic colorectal
carcinomas.
4,6,7,40
To our knowledge, this is the first study documenting
the presence of a germline MMR gene mutation in young
patients with sporadic gliomas. A similar study in young
patients with sporadic colorectal carcinoma revealed MSI
in 58%, with germline MMR mutation detected in 42% of
the MSI-positive cases.
32
Although germline MMR gene
mutation has previously been found in four patients with
Turcot’s syndrome,
21,23,24
patient A developed and died
Figure 4. A: Sequencing result of hMLH1 exon 8 from the blood leukocytes
(lanes N) and glioblastoma (lanes T) of patient A. Both tumor and blood
leukocytes demonstrate a mutation in the last nucleotide of exon 8, CGgt to
CAgt (arrow), indicating that it is a germline mutation. B: Sequencing result
of hMLH1 exon 13 from the blood leukocytes (lanes N) and glioblastoma
(lanes T) of patient A. Somatic mutation in codon 487, CGA to TGA (arrow),
generating a stop codon, is found in the tumor but not in blood leukocytes.
Figure 5. Reverse transcription-PCR analysis using a pair of primers ampli-
fying nucleotides 471 to 813 of hMLH1 cDNA. A wild-type band of 342 bp
and an abnormal band of 253 bp (arrow), resulting from skipping of exon 8,
are noted in the RNA extracted from the leukocytes of patient A (lane A)
compared with a normal individual (lane C). Lane M: Bluescript-MSP1
marker.
Figure 3. In vitro synthesized protein assay of 59(A) and 39(B) segments of
the hMLH1 gene from the glioblastoma of patient A. The bands with highest
molecular weight and strongest intensity correspond to the normal products.
Truncated products of 21 and 18 kd (arrowheads) are noted in the 59and 39
segments. Lanes C, blood leukocytes from a normal individual as control;
lanes T, glioblastoma.
MSI and DNA Mismatch Repair Gene Mutation in Gliomas 1185
AJP October 1998, Vol. 153, No. 4
of the glioma without antecedent cancer. Patient B pre-
sented with the glioma first and only subsequently devel-
oped the colorectal carcinoma. Neither A nor B had a
family history of cancer. Two other patients have ante-
cedent colorectal carcinoma. In patient D, a family history
of colorectal carcinomas was also obtained. This raises
an important point concerning the management of young
patients with microsatellite-unstable gliomas and their
family members. From the information in our study, we
conclude that screening for replication error is useful in
young patients with high-grade gliomas. For high-level
MSI patients, germline mutation of the MMR gene should
be sought. Regular colonoscopic screening for colorectal
carcinoma should be offered to the patient and the family
members with demonstrable MMR gene mutation. Also,
we should be alert to and check for the possibility of brain
tumor by regular neurological examination. It is of note
that, whereas the colorectal carcinoma could be suc-
cessfully treated, three of the patients succumbed to the
gliomas 0.5 months to 4 years after the craniotomy. This
was in contrast to the prolonged survival noted in three
patients with MSI-positive glioblastoma in a previous
series.
23
Concerning the histological type of high-level MSI gli-
omas, three were glioblastomas. Interestingly, one case
was a malignant mixed glioma with a prominent oligoden-
droglial component and also focal ependymal differenti-
ation. In HNPCC kindred, the possible histological type of
brain tumor includes not only astrocytomas but also oli-
godendrogliomas and rarely ependymomas.
21
Thus, mu-
tation of the MMR gene may lead not only to glioblas-
tomas, but to high-grade gliomas of oligodendroglial or
even ependymal differentiation.
Mononucleotide tracts of various growth-regulatory
genes are frequently the target of mutational inactivation
in microsatellite-unstable tumors. The (A)
10
tract in the
T
b
RII gene is mutated in 70 to 90% of microsatellite-
unstable colorectal and gastric cancers.
36,41
Frameshift
mutation of the (G)
8
tract in Bax is also reported in more
than 50% of these cancers.
37,42,43
Apart from frameshift
mutation in the (G)
8
tract, somatic mutation of Bax genes
is frequent in MSI-positive gastric and colorectal carci-
nomas,
43
but not in gliomas.
44
Interestingly, none of the
MSI-positive gliomas in this study showed mutation in the
T
b
RII and Bax genes. This may be the result of selection
pressure, in which mutation of genes caused by MMR
defects are selected for if they confer growth advantage
in that organ.
We identified a frameshift mutation in the IGFIIR gene
in the malignant mixed glioma from patient B, the first
reported mutation in this gene in a glioma, although IG-
FIIR mutation has been reported in MSI colorectal, gas-
tric, and endometrial carcinomas.
38,45
IGFIIR plays a role
in activation of transforming growth factor
b
,
46
which is a
potent growth inhibitor. Also, it antagonizes the growth-
stimulatory effect of IGFII by internalizing and degrading
the protein.
47
Given that enhanced expression of IGFII
mRNA has been reported in gliomas,
48
inactivating mu-
tation of IGFIIR may remove the growth-inhibitory signal
and confer growth advantage.
The molecular genetic pathways of different subsets of
glioblastoma have been increasingly clarified in recent
years.
29,49
Those arising de novo are referred to as pri-
mary glioblastomas, and those developed from a pre-
existing astrocytoma are referred to as secondary glio-
blastomas. Most primary glioblastomas develop in older
patients (mean, 55 years) with epidermal growth factor
receptor amplification or overexpression, loss of het-
erozygosity in chromosome 10, and p16 deletion. Sec-
ondary glioblastomas tend to occur in younger patients
(mean, 39 years), and most of them harbor p53 muta-
tions.
50–54
We have demonstrated that a proportion of
primary glioblastomas in young patients can be caused
by germline MMR gene mutations, and these patients
and their family members are at risk of developing other
HNPCC-related tumors, in particular colorectal carcino-
mas. Screening for MSI and MMR gene mutation is thus
of importance in the management of these patients.
Acknowledgments
We thank Mr. Samson W. C. Shum for the technical
assistance, Miss Kedo Kwan for collecting the family
history, and Dr. R. J. Collins for his assistance with the
manuscript.
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