From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Analysis of Epstein-Barr Virus Gene Polymorphisms in Normal Donors and
in Virus-Associated Tumors From Different Geographic Locations
By Farhat Khanim, Qing-Yun Yao, Gerald Niedobitek, Simritpal Sihota, Alan B. Rickinson, and Lawrence S. Young
While Epstein-Barr virus (EBV) infection is associated with
the development of certain lymphoid and epithelial tumors,
the role of the virus in the pathogenesis of these malignancies remains unknown. It has been suggested that EBV
strain variation may contribute to tumor development. Two
major strains of EBV, type 1 and type 2, have been identified
on the basis of genetic polymorphismsand other minor genetic variations give rise to distinct EBV isolates. We analyzed EBV strain variation in healthy individuals and compared them with EBV isolates present in lymphoid and
epithelial neoplasmsfrom the same geographic regions. In
particular, the incidence of the 30-bp latent membrane protein (LMP1) gene deletion, recently implicated in the development of more aggressive forms of virus-positivelympho-
mas and Hodgkin’s disease (HD), was examined in the
normal population. While a preferential association of the
LMPl deletion with the type 2 strain of EBV was observed,
there was no increased incidence of virus isolates carrying
this deletion in HD, Burktt’s lymphoma, or virus-associated
carcinomas compared with the appropriate normal population. A polymorphism in the 6amHl F region of the EBV
genome, previously identified in Chinese populations, was
found at increased incidence in European HD biopsies. Overall, we found that most of the EBV gene polymorphisms
detected in EBV isolatesfrom healthy virus carriers occurred
with similar frequency in virus-associated tumors from the
same geographical region.
0 1996 by The American Society of Hematology.
E
EBNA3 genes, two major virus strains have been identified,
EBV types 1 and 2. The results of many studies looking at
the frequency of type 1 versus type 2 EBV have shown
markedly different frequencies of type 1 and 2 EBV in different geographic location^.'^ In addition to this broad distinction between EBV types 1 and 2, there is also minor heterogeneity within each virus type, which is most easily detected
as variation in the size of the EBNA proteins. These differences have been used to trace virus transmission within families and from transplant donors to recipients.I6
Further attempts to identify variability between virus isolates has led to the identification of a defined polymorphism
in the BamHI F region of the EBV genome.” In contrast to
the prototype B95.8 EBV strain (“F” type), EBV genomes
from NPC biopsies obtained from Southern Chinese patients
were shown to contain an additional BamHI restriction site
in this region (‘T’variant), whereas this variant was only
rarely detected in spontaneous LCLs generated from healthy
Southern Chinese or North American donors.’*.’’These observations suggested that the “f” variant of EBV may be
implicated in the pathogenesis of NPC. However, it has recently been reported that the “f’ EBV variant is not present
in NPC specimens from North Africa,” an area of intermediate NPC incidence, indicating that the “f‘ ’ polymorphism
is not an indispensable prerequisite for NPC development.
Cloning and sequencing of the LMPl gene from EBV
PSTEIN-BARR virus (EBV) is a ubiquitous human herpes virus that is found as a widespread infection in all
human communities. First identified in the endemic form of
Burkitt’s lymphoma (BL) in Africa, EBV is also associated
with lymphoproliferative diseases in immunosuppressed individuals‘.’ with Hodgkin’s disease (HD)3 and with certain
T-cell lymphomas! Delayed primary EBV infection is responsible for infectious mononucleosis (IM),’ a self-limiting
lymphoproliferative disorder. Recent work indicates that
while up to 50% of HD cases are EBV-associated in Western
countries, the prevalence of this association is increased in
certain parts of the world (ie, South
and in human
immunodeficiency virus (H1V)-positive patients.’ This geographic variation in the association of EBV with certain
tumors is also demonstrated by undifferentiated nasopharyngeal carcinoma (NPC),* an epithelial neoplasm that occurs
at high incidence in Southeast Asia and is consistently associated with EBV. In contrast to BL, where only around 20%
of cases occurring outside endemic areas are EBV-associated, all cases of NPC are EBV-positive regardless of geographic origin. While ethnic factors may influence the prevalence of EBV-associated tumors in different countries, it is
also possible that EBV strain variation contributes to this
geographic variation.
A characteristic feature of EBV is its ability to transform
resting human B cells into permanently growing lymphoblastoid cell lines (LCLs) in vitro.’ In LCL cells and also in
posttransplant lymphoproliferative disorders (PTLDs)?’’ a
limited set of latent viral proteins is expressed, including six
Epstein-Barr nuclear antigens (EBNA 1,2, 3A, 3B, 3C, and
LP), three latent membrane proteins (LMPl, 2A, and 2B),
and two small nuclear, noncoding RNAs (EBER 1 and 2).’
However, in other EBV-associated tumors, different forms
of EBV latency have been described in which only a subset
of viral latent genes is expressed suggesting that the contribution of EBV to the development of virus-associated tumors
is va”able.’.’’-l3While several viral latent gene products have
been implicated in the process of virus-induced cellular
transformation, as yet only LMPl has a well-established
oncogenic function, as well as diverse effects on cell phenotype. l~~
On the basis of genetic polymorphism in the EBNA2 and
Blood, Vol 88,No 9 (November I), 1996: pp 3491-3501
From the Institute for Cancer Studies and Department of Pathology, The University of Birmingham, CRC Institute for Cancer Studies, Birmingham, U.K.
Submitted January 16, 1996; accepted June 26, 1996.
Supported by grants from the Cancer Research Campaign (to
L.S.Y. and A.B.R.) and from the Medical Research Council (to G.N.,
L.S.Y., and A.B.R.).
Address reprint requests to Lawrence S. Young, PhD, Institute
for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TJ, United Kingdom.
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 I8 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
OO06-4971/96/8809-0O$3.00/0
3491
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3492
KHANIM ET AL
isolates derived from Chinese NPCs identified several mutations as compared with the wild-type B95.8 strain, including
a point mutation leading to the loss of an XhoI restriction
site in the first exon and a 30-bp deletion in the carboxy
terminal region of the LMPl gene.21322
It has been suggested
that these changes are associated with increased tumorgenicity and reduced immunogenicity of the LMPl prot e i ~ ~ . ’Recently,
~.’~
a similar deletion in the LMPl gene has
been found in a proportion of EBV-associated European and
certain virus-associated lymphoproliferation~.~~
The LMP 1
gene from different EBV isolates also contains variable numbers of a 33-bp repetitive sequence, which has also been
used as a marker of heterogeneity between EBV strains.25
The aim of this study was to analyze specific EBV polymorphisms in virus isolates obtained from healthy individuals in comparison to human lymphoid and epithelial neoplasms from the same geographic location. In particular,
polymorphisms in the BamHI F region and in the LMPl
gene were analyzed in an attempt to identify disease-associated virus strain variation.
MATERIALS AND METHODS
provided by Dr W. Sheng, College of Medicine, National Taiwan
University, Taiwan. Nine further snap frozen NPC biopsies were a
kind gift of Dr J. Nicholls, University of Hong Kong. All NPC cases
from Hong Kong and from Birmingham had been shown to be EBVpositive in previous st~dies.”~
NPC cases obtained from Taiwan
were diagnosed as World Health Organization (WHO) 111 or WHO
I1 NPCs, but material for in situ hybridization analysis was not
available. Paraffin sections from 1 1 Caucasian NPCs and four gastric
carcinomas were obtained from the files of the Department of Pathology, University of Birmingham. The four gastric carcinomas had
been demonstrated to be EBV-positive in a previously published
study.2x
DNA Extraction
Crude DNA for polymerase chain reaction (PCR) amplification
was extracted from LCLs and BLs by incubation of cell pellets in
Proteinase K lysis solution (Proteinase K 100 pg/mL, Tween 80 5
pUmL, IO mmoUL Tris HCL (pH 8.3), 50 mmollL KCI. 1.5 mmol/
L MgCI2, and 0.01% gelatin) for 1 hour at 55°C followed by heat
inactivation of the enzyme. DNA from frozen biopsies was extracted
by proteinase K lysis at 55°C for 1 hour, phenolkhloroform purification and ethanol precipitation. For preparation of DNA from paraffin
blocks, 30-pm sections were first deparaffinized with xylene and
ethanol and DNA extracted as described above for frozen biopsies.
Cell Culture and Biopsies
Lymphoblastoid cell lines (sp-LCLs) were established from
healthy chronic EBV carriers by spontaneous outgrowth of B lymphocytes in culture and maintained in RPMI 1640 medium (GIBCO,
Paisley, UK) supplemented with 8% heat inactivated fetal calf serum
(FCS), 2 mmollL glutamine, penicillin (120 U/mL), and streptomycin (120 pg/mL) (Sigma, Dorset, UK). Of the donors, 22 were of
European origin, 39 were from Africa (Kenya or Gambia), 11 from
New Guinea, and 10 were of Chinese extraction. In addition, four
LCLs were obtained from Caucasian patients in the acute phase of
IM. The African LCLs were generated in our labs through collaboration with Drs H. Rupani, H. Whittle, and K. Lam.’” LCLs from New
Guinea were kindly provided by Dr D. Moss (QIMR, Brisbane,
Australia). LCLs from Chinese donors were established in our laboratories. Previously established BL cell lines were maintained in
RPMI 1640 supplemented with 10%FCS, glutamine, and gentimycin
(8 pg/mL). All cultures were grown at 37°C in 5% C02 and passaged
weekly.
Formalin-fixed and paraffin-embedded tissue samples from eight
patients with IM were included in this study (see Table 2). Five
lymph nodes and four palatine tonsils were available and have been
included in previous studies (G.N. et al, submitted). Histological
changes in keeping with acute IM were seen in all cases and this was
supported by clinical andor serological information. Furthermore,
numerous EBV-infected lymphoid blasts were detected in all IM
cases, as described previously (G.N. et al, submitted).
Twenty-five formalin-fixed and paraffin-embedded biopsies of
histologically confirmed Hodgkin’s disease (HD) were obtained
from the files of the Department of Pathology, University of Birmingham, the Department of Pathology, Heartlands Hospital Birmingham, Birmingham, UK (courtesy of Dr J. Crocker) and the
Department of Pathology, Bristol Royal Infirmary, Bristol, UK
(courtesy of Dr N. Rooney). The EBV status of the cases had been
assessed previously using in situ hybridization with RNA probes
specific for the small virus-encoded nuclear RNAs (EBERs) as described in detail elsewhere.2” HD cases were selected from previously studied series on the basis of the detection of EBV in the
Hodgkin Reed-Sternberg (HRS) cells. The histological subtype of
each case I S presented (see Table 3).
Ten snap frozen NPC biopsies ( I 17- 135 in Table 5). were kindly
DNA PCR Amplijication
DNA aliquots (100 ng to 1 pg) were amplified in 100-pL reactions
containing 1 pmoVL of each primer. 200 pmol/L dATP, dCTP,
dGTP, and dTTP, IO mmoUL Tris HC1 (pH 8.8), 1.5 mmoVL MgClz,
50 mmoVL KCI, 0.1% Triton X-100, and overlaid with mineral oil.
The samples were denatured for 5 minutes at 94°C. Two units of
Taq polymerase were added and PCR amplification performed in a
Perkin Elmer-Cetus (Warrington, UK) 480 Thermal Cycler. The
primer combinations used, cycle parameters for PCR amplification,
and product sizes of the different regions of EBV are described in
Table 1.
Restriction Fragment Length Polymorphism Analysis
To perform an analysis of the BamHI F and XhoI polymorphisms,
amplified PCR DNA was purified by phenokhloroform extraction
followed by ethanol precipitation with glycogen carrier. DNA pellets
were resuspended in 50 pL distilled water and 5 pL aliquots were
digested with 20 U of either BamHI for the BamHI F restriction
fragment length polymorphism (RFLP) and XhoI for the XhoI RFLP
(Boehringer Mannheim, Germany). Restriction enzyme digests were
performed in 20 pL volumes according to manufacturer’s instructions and the results analyzed on polyacrylamide gels as described
below.
Analysis of PCR and RFLP Products
To analyze the products of the PCR amplifications, samples were
electrophoresed on 10%to 15% polyacrylamide gels and bands visualized by ethidium bromide staining. Specificity of the PCR reactions
was confirmed by hybridization with oligonucleotide probes as described. Gels were denatured (0.2 mom NaOH, 0.5 m o w NaCI),
neutralized (4 X TBE, 1 X TBE) and DNA transferred onto nylon
membranes (Hybond N+, Amersham, UK) by electroblotting at 200
V for 2 hours. Nonspecific binding was bIocked with incubation of
the membrane in prehyb solution (0.5% sodium dodecyl sulfate
[SDS], 4 x SSC, 5 X Denhardts, and 200 p g / d denatured salmon
sperm DNA) for 60 minutes at 42°C. Following hybridization with
10 pmoles of ’2P-5‘ end-labelled oligonucleotide probe in prehyb
solution for a minimum of 4 hours at 42°C. membranes were washed
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3493
ANALYSIS OF EBV GENE POLYMORPHISMS
Table 1. PCR Primer Combinations and Conditions
Primer
PCR
EBNA 2
EBNA 3C
BamHlF RFLP
LMPl Repeats
Xhol RFLP
LMPl Deletion
2A-3‘
28-3’
2C-5‘
2A-P
2B-P
5’
3‘
Type 1-P
Type 2-P
5’
3‘
P
5’
3‘
P
5’
3‘
5’ P
3‘ P
5’
3‘
P
Primer Sequence 5’-3‘
PCR Product
Size (bp)
94°C. 30 sec
52T, 60 sec x 35
72°C’ 60 sec
Type 1 = 249
Type 2 = 300
99939-958
100091-072
100002-021
94°C. 30 sec
52°C. 60 sec x 35
72“C, 60 sec
Type 1 = 153
Type 2 = 246
55611-630
55832-813
55800-781
168594-575
168374-393
168446-427
169569-550
169081-100
169490-471
169356-337
168389-370
168130-149
168219-200
94”C,
52°C.
72°C.
94°C.
50°C.
72°C.
94”C,
52T,
72°C.
49058-039
TTGTGACAGAGGTGACAAAA
TTGAAGAGTATGTCCTAAGG
AGGGATGCCTGGACACAAGA
TCCAGCCACATGTCCCCCCTCTACGCCCGACA
AACGTCAACCTGTCCACAACCCTCGCCAGGAG
AGAAGGGGAGCGTGTGTTGT
GGCTCGTITITGACGTCGGC
GAAGATTCATCGTCAGTGTC
CCGTGAWCTACCGGGAGT
CAACTGCCACAGACCCCATT
GGCAATGGGACGTCTGTAA
GCTAAGCCAGGATAATCAGG
GGCGCACCTGGAGGTGGTCC
WCCAGCAGAGTCGCTAGG
AGGACCCTGACAACACTGAT
GCGTTACTCTGACGTAGCCG
ACAATGCCTGTCCGTGCAAA
ACACACTGCCCTGAGGATGG
TGTACATCGTTATGAGTGAC
GCGACTCTGCTGGAAATGAT
GACATGGTAATGCCTAGAAG
GGTTCCGGTGGAGATGATGA
PCR Conditions
895.8 Coordinates
48810-829
48997-49028
30 sec
60 sec x 35
90 sec
30 sec
45 sec x 35
90 sec
30 sec
60 sec x 30
90 sec
Uncut = 222
Cut = 125
+97
Between
155-400
94”C, 30 sec
48T, 90 sec x 35
70°C, 120 sec
wt = 184
del = 154
Uncut = 4989
c u t = 345
t144
Abbreviations: 5’. 5’ PCR primer; 3’. 3’ PCR primer; P, oligonucleotide probe; wt, wild-type or ”B95.8-like“: Sequence coordinates correspond
to the published 895.8 sequence (Baer et a14’).
at room temperature with 2 X SSC, 0.1%SDS, and 1 X SSC, 0.1%
SDS and exposed to Kodak autoradiographicfilm (Heme1 Hempsted,
UK).
Cycle Sequencing
DNA for cycle sequencing was purified from low melting point
agarose gels using the Wizard PCR Prep kit (Promega, UK). Sequencing was carried out with 32P-5’
end-labelled oligonucleotide
primers and the Amplitaq Cycle Sequencing kit (Perkin Elmer-Cetus,
UK) according to manufacturer’s instructions. Sequences were analyzed by electrophoresis on 6% 6 m o m urea polyacrylamide gels
and autoradiography.
RESULTS
Validation of PCR Methodology
Each isolate reported in this study was analyzed for polymorphisms in six different regions of the EBV genome. EBV
strain type was determined over two of the latent EBV genes,
EBNA2 and EBNA3C, using well-characterized primers and
amplification conditions from Sample et aLZ9Type 2 EBV
isolates were specifically included in this study for analysis
of the various polymorphic markers, therefore the percentages of type 1 EBV to type 2 EBV in the study groups are not
representative of infection levels in the general population.
Extensive studies have already been performed by our group
and other groups looking at the incidence of the two EBV
types in various geographic populations (for review see Gratama et aii5).
The “F” and “f” RFLP was detected by PCR amplification over the BamHI F fragment of EBV, purifying the products and digesting with BamHI restriction enzyme as pre-
viousiy described.” EBV isolates carrying the wild-type
“F” yielded a single undigested product of 222-bp (Fig
1). However, in isolates carrying the “f’ variant, BamHI
digestion of the PCR product results in the generation of
two fragments of 126 bp and 96 bp (Fig 1). To verify the
mutation in “f” virus isolates, nine isolates carrying either
the wild-type “F” or the “f” variant (sp-LCLs: D5, DH,
QX, QY, FJW, JLU, L10, H32, H36) were sequenced alongside B95.8, and in all “f” cases there was an A to C transversion at position 55707 (B95.8 coordinates) creating the extra
BamHI site. A similar PCR approach was used to identify
the XhoI RFLP in the LMPl gene. XhoI digestion of the
PCR products from EBV strains, which retained the XhoI
site, yielded two products of 345 bp and 144 bp, whereas
EBV isolates with a mutated XhoI site yielded an undigested
band of 489-bp (Fig 1). PCR products of nine randomly
selected isolates with or without the XhoI site (B95.8,
AG876, SWE, IM12, IM50, D5, QX, CKL, MTL) were
sequenced over the XhoI site revealing an identical G to T
transversion in codon 17 of the LMPl gene as described
previouslyz5~30
(data not shown).
The LMPl gene was analyzed over the 33-bp repeat region
by PCR followed by polyacrylamide gel electrophoresis,
blotting, and probing with a specific oligonucleotide probe
(Fig 1). Differences were easily detectable and the numbers
of repeats calculated with the aid of a 20-bp size marker.
For nine isolates (B95.8, AG876, GA490, M192, FJW, JLU,
MTL, 124, 125) the number of repeats was confirmed by
sequencing the PCR product. In agreement with previously
published results, a single band was noticed in all virusassociated tumors (Tables 3 through 6). Several established
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
KHANIM ET AL
3494
A
F
2 2 2 b ~ C
f
126bpL
.
II
BamHl "F"
RRP
B
IOSt-489bp b
present-345bp
Numbers
of
repeats
LMP1 Xhol
m
FlRp
61,
5
c
LMPl 33bp
repeats
4c
D
Undel-260bp
del-230bp b
4
cell lines, which had been generated by infection with the
B95.8 virus isolate, were also studied (data not shown), and
in all cases, the PCR assay yielded a band representing 4.5
x 33-bp LMPl repeats as in the original B95.8 cell line,
thus confirming the stability of the number of LMPl repeats
in a virus isolate over time.
The 30-bp deletion in the LMPl gene was detected by
PCR and subsequent blotting and probing. EBV isolates with
a deleted LMPl gene yielded a smaller product of 230 bp
compared with the full-length product of 260 bp (Fig I). All
PCR products obtained from isolates with the LMPl deletion
were of identical size suggesting lack of variability in the
deleted region. This was confirmed by sequence analysis of
I O isolates, six that did not carry the LMPl deletion (B95.8,
TIE, DAV, ROG, BYR, IM50) and four that were deleted
(AG876, IM12, MOR, BEV), and this showed a consistent
location of the 30-bp deletion between amino acids 343 and
352 (data not shown) as reported by Miller et al."
Comparison of EBV 1.solate.s From Normal Donors,
Hod<qkin'sDisease Biopsies, and NPC Biopsies of
European Origin
Of the 26 European spontaneous LCLs (sp-LCLs), 21
(8 1 %) harbored EBV type I , and 5 (1 9%) carried type 2 as
determined using PCR over the EBNA2 and EBNA3c re-
LMPl 30bp
deletion
Fig 1. Validation of PCR methodology. To study
the RFLPs, purified PCR products (see Materials and
Methods) were digested with restriction enzymes,
separated by polyacrylamide gel electrophoresis
(PAGE), blotted, and probed with an oligonucleotide
probe. (A) The BamHl F RFLP yielded a single 222bp band in samples with the "F" EBV variant and a
smaller 126-bp band in samples carrying the "f" EBV
variant. 895.8 and AG876 are both "F" EBV variant
controls. (6) Using the 3' probe in this figure, the
Xhol RFLP yielded a smaller band of 345-bp where
the site was retained. However an undigested band
of 489-bp was observed when the X h d site had been
mutated. (C) The number of repeats was determined
by electrophoresis of PCR products on polyacrylamide gels followed by blotting and probing. The
number of repeats was easily discernible between 36. 895.8 has 4.5 repeats and AG876 has 4 repeats.
(D) The LMPl deletion was detected by PCR, PAGE,
and blotting. 695.8-like or full-length LMPl yielded
a 260-bp band. However EBV isolates with the 30bp deletion in the LMPl gene yielded a 230-bp band
as for AG876 prototype strain.
gions of the viral genome with previously validated primer
combinations (Table 2). Type 1 EBV was found in all eight
IM biopsies. Thus, in a total of 34 virus isolates from lymphocytes obtained from healthy virus carriers or from otherwise normal individuals during primary infection, EBV type
1 was identified in 85%. The 23 HD biopsies, as well as 1 I
NPC biopsies and four gastric carcinoma samples obtained
from European patients, all harbored type 1 EBV (Table 3).
All European sp-LCLs displayed the"F" wild-type configuration as in the B95.8 prototype EBV strain (Table 2).
Of the seven IM biopsies that were available for BamHI F
RFLP analysis, six displayed the wild-type "F" configuration, whereas one showed the "f' form (Table 2). Unexpectedly, I O of the 22 HD cases (45%) harbored EBV with the
"f" variant (Table 3, Fig 2A). By contrast, the "f" variant
was detected in only one of I O (10%) Caucasian NPC biopsies and was not present in the two gastric carcinomas that
were available for analysis (Table 3).
Polymorphisms in the LMPI gene. In analyzing the
LMPl gene from the European EBV isolates, it was noted
that variation in the number of LMPl repeats was quite
limited in the type 2 EBV isolates from this group; however,
a much greater degree of variation was noted in the type 1
EBV isolates (Fig 2B). In all cases, a single band was observed, with the exception of one IM biopsy in which two
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3495
ANALYSIS OF EBV GENE POLYMORPHISMS
Table 2. Analysis of European Spontaneous LCLs and IM
Biopsies for EBV Polymorphisms
LMPl Polymorphisms
Virus Isolate
European sp-LCLs
TIE
CAR
BYR
FIN
RIC
WIL
ROG
WRI
MCC
MUR
LIF
DAV
IM50
IM52
ULA
SWE
IM53
woo
BEV
BEL
HOP
WAI
MOR
IM12
EBH9
EBHZl
I M biopsies
IM-1
IM-2
IM-3
IM-4
IM-5
IM-6
IM-7
IM-8
Virus
Type
BamHl
F/f
33-bp
Repeat
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
F
F
F
F
F
F
F
F
4
4.5
5
5
2
5
6
5
4.5
5
3
4.5
5.5
5
6
4
5
8
5
6
4
5
6
6
5
4.5
1
1
1
1
1
1
1
1
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
f
-
4.5
4.5
4
4.518
4.5
4.5
4.5
4.5
Xhol
Site
wt
wt
30-bp
Deletion
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
lost
lost
wt
wt
wt
wt
del
del
del
del
del
del
del
del
del
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
del
del
wt
del
wt
del
wt
Abbreviations: sp-LCL, Spontaneous LCL; wt, similar to that seen
in the 695.8 EBV prototype virus; F, absence of a 6amHl site in the
8amHl F fragment of the EBV genome; f, presence of an extra 8amHl
site in the BamHl F fragment of the EBV genome; lost, loss of the
Xhol site in LMPl as a result of a G to C base transition; del, presence
of a 30-bp deletion in the carboxy terminus of the LMPl gene; -,
result not obtained.
major bands representing 4.5 and 8 to 9 repeats, respectively,
were found, and these may have resulted from recombination
events associated with virus replication. Twenty-four of 26
European sp-LCLs (92%) and all IM biopsies carried EBV
with wild-type configuration of the XhoI site, whereas two
LCL isolates (8%)had lost this restriction enzyme site (Table
2). The XhoI site was preserved in all virus isolates derived
from European HD biopsies, NPC biopsies, and gastric adenocarcinomas (Table 3).
In European sp-LCLs, nine of 22 (41%) virus isolates
displayed the 30-bp LMPl deletion (Table 2). Interestingly,
four of five (80%) type 2 EBV isolates in this group dis-
played the 30-bp deletion, whereas only five of 17 (29.4%)
type 1 isolates demonstrated this polymorphism. The LMPl
deletion was also observed in four of eight (50%)IM tissues.
Thus, of all European virus isolates obtained from patients
without EBV-associated malignancies, 13 of 30 (43%) carried the LMPl deletion. EBV isolates with a deletion in the
LMPl gene were identified in only two of 23 (9%) HD
Table 3. Analysis of European HD, Gastric Adenocarcinoma.
and NPC Biopsies for EBV Polymorphisms
LMPl Polymorphisms
Virus Isolate
European HD biopsies
HD1 (mc)
HD2 (nsll)
HD3 (mc)*
HD4 (mc)
HD5 (nsl)
HD6 (nsll)
HD7 (nsll)
HD8 (mc)
HD9 (mc)
HDlO (mc)
HD11 (mc)*
HD12 (mc)
HD13 (mc)
HD14 (ns)
HD15 (nsll)'
HD16 (nsl)
HD17 (mc)
HD18 (nsll)
HD19 (nsl)
HD20 (mc)
HD21 (nsl)
HD22 (mc)*
HD23 (mc)
Gastric adenocarcinoma
biopsies
GA1
GA2
GA3
GA4
European NPC biopsies
E-NPC1
E-NPC2
E-NPC3
E-NPC4
E-NPC5
E-NPC6
E-NPC7
E-NPC8
E-NPC9
E-NPC10
E-NPCl1
Virus
Type
BamHl
F/f
33-bp
Repeats
Xhol
Site
30-bp
Deletion
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
f
F
f
F
f
f
f
F
f
F
F
f
F
f
5
4
5
4.5
5.5
5
4
4
5
4.5
5
4
5
5
4.5
4.5
4.5
4.515
4.5
wt
del
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
F
f
F
f
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
del
wt
wt
wt
wt
wt
wt
-
-
wt
F
F
F
4.515
wt
wt
wt
-
-
wt
4.5
wt
-
F
5.5
5
5
6
wt
wt
del
del
wt
wt
wt
5
5
5
5
4
4.5
4
4.5
4.5
5
5
wt
wt
del
wt
del
wt
wt
wt
del
F
F
F
F
F
F
F
F
F
f
F
wt
-
wt
wt
wt
wt
wt
wt
wt
wt
del
del
Histological subtype of HD: ns, nodular sclerosis with no further
subtype; nsl, nodular sclerosing subtype I; nsll, nodular sclerosing
subtype II; and mc, mixed cellularity.
* Histological features suggesting aggressive behavior, such as necrosis and/or the presence of bizarre forms of HRS cells.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
KHANIM ET AL
3496
A
HD biopsies
\ m P
Fig 2. Analysis of European EBV isolates. (A) EBV isolates from HD biopsies were analyzed over the B a d 1 "F" RFLP by PCR followed by
restriction enzyme digestion and PAGE. Bands were detected by Southern blotting. HD biopsies harboring EBV isolates with "F" variant
yielded an undigested band of 222-bp, whereas "f" variants gave a smaller digestion product of 126-bp. (B) Analysis of the number of 33-bp
LMPl repeats by PCR and PAGE showed a greater degree of variation in type 1 EBV isolates than in type 2 EBV isolates from European normal
donors.
biopsies, five of 10 (50%)European NPC biopsies, and two
of four gastric adenocarcinomas (Table 3).
Comparison of EBV Isolates From Normal Donors and
Burkitt Lymphoma Cell Lines of African and New Guinean
Origin
Thirty-three (85%) sp-LCLs from African individuals carried type 1 EBV and six (15%) contained type 2 virus (Table
4). Of the 1 1 African BL lines selected for this study, seven
carried EBV type I and four harbored type 2 virus (Table
4). For comparison, we also included LCLs and BL lines
from New Guinea (Table 5). All LCLs and one BL cell line
from this location contained EBV type I and 2 BL lines
harbored type 2 EBV (Table 5). All African sp-LCLs carried
EBV strains with the "F" wild-type configuration, as did
all African BL lines (Table 4). One New Guinean LCL
showed the "f" variant, whereas the remaining 10 LCLs
and all three BL lines exhibited the "F" wild-type virus
(Table 5).
Polymorphisms in the LMPI gene. PCR amplification
of the LMPl repeat region showed limited variation in the
numbers of the 33-bp repeat sequences in EBV isolates from
African and New Guinean normal donors sp-LCLs and also
in EBV isolates from BL cell lines (Fig 3A). Two of 39
African sp-LCLs (5%) carried virus with loss of the XhoI
site in the LMPl gene (Table 4). In the 1 1 New Guinean
LCLs, three virus isolates lacking the XhoI site were identified, while the remaining eight lines carried virus with an
intact XhoI site (Table 5 ) . All 1 1 African BL lines and the
three BL lines from New Guinea displayed EBV isolates
with a preserved XhoI site.
The 30-bp LMPl deletion was found in 10 of the 39 (26%)
African spontaneous LCLs (Table 4, Fig 3B). As in the
European isolates, there was an association of the LMPl
deletion with type 2 virus. Five of six (83%) African LCLs
with type 2 virus carried the LMPl deletion, whereas only
five of 33 (15.2%) type 1 isolates displayed this polymorphism (Table 4, Fig 3B). Also, there seemed to be some
variability among normal isolates from Kenya and from
Gambia. All Gambian isolates were of type I, but four of
14 (28.6%) displayed the LMPI deletion. By contrast, only
one of 19 (5.3%) type 1 EBV isolates from Kenya demonstrated the 30-bp deletion in the LMPl gene (Table 4). A
similar distribution was noted in the African BL isolates
where the 30-bp deletion was found in one of seven (14.3%)
BLs carrying type 1 EBV and in 2 of 4 (50%) BLs carrying
type 2 EBV (Table 4). None of the spontaneous LCLs or
BL cell lines from New Guinea carried EBV isolates with
the LMPl deletion.
Comparison of EBV Isolntes From Normal Donors and
From NPC Biopsies of Southeast Asian Origin
Nine of 10 Chinese sp-LCLs harbored type 1 EBV and
one contained type 2 virus (Table 6). Similarly, all NPC
biopsies except one carried type 1 EBV (Table 6). Of the
Chinese sp-LCLs. three lines (30%) displayed the "f" EBV
variant (Table 6, Fig I). The "f' polymorphism was demonstrated in 13 of 19 (68.4%) NPC biopsies from Chinese
individuals. There were no significant differences between
biopsies obtained from Hong Kong and from Taiwan (Table
6).
Polymorphisms in the LMPI gene. Little variation was
observed in the number of 33-bp repeats in EBV isolates
from normal Chinese donors and from NPC biopsies (between three and six repeats, Table 6). In contrast to the
spontaneous LCLs from Europe, Africa, and New Guinea,
the sp-LCLs from donors of Chinese extraction demonstrated
loss of the XhoI site in nine of 10 cases (90%. Table 6, Fig
4A). The one LCL carrying a preserved XhoI site was unusual in that it harbored type 2 EBV and also differed from
all other lines with respect to the 30-bp LMPl deletion (Fig
4B). All virus isolates from Chinese NPC biopsies displayed
loss of the XhoI site in the LMPl gene (Table 6, Fig 4A).
All nine Chinese sp-LCL type 1 EBV isolates displayed the
30-bp LMPl deletion, and one LCL contained type 2 virus
with a full-length LMPl gene (Fig 4B). Accordingly, all 19
Chinese NPC isolates were of type 1 and displayed the LMPl
deletion (Table 6, Fig 4B).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3497
ANALYSIS OF EBV GENE POLYMORPHISMS
DISCUSSION
Table 4. Analysis of African Spontaneous LCLs and BLs
for EBV Polymorphisms
LMPl Polymorphisms
Virus Isolate
African sp-LCLs
GA433
GA451
GA452
GA477
GA483
GA485
GA490
EBV9
87/76
a7187
M192
M214
M220
M240
Wan
Oga
Igu
Che
Mut
Nek
Ser
Waj
Mug
Wai
Muv
Mak
Hes
Pod
Mur
Ood
Wag
NVa
Huk
Alv
Nzi
Oja
Bab
And
Mun
African BL lines
Daudi
Mutu
Raji
Rae1
BL36
BL72
Makau
Chep
JC5
Elijah
AG876
Xhol
30-bp
Deletion
Virus
Type
BamHl
F/f
33-bp
Repeats
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
F
F
5
4.5
4
5
4.5
4
5
4
5
5
3
3.5
5
5
4
4
3
4
4
4
4
4
3
4
5
4
4.5
4
4
4
4
4
5
5
4
4
4
5
4
wt
wt
wt
del
wt
wt
1
1
1
1
1
1
1
2
2
2
2
F
F
4
4
4.5
5.5
5
5
5
4.5
4.5
5.5
4
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Site
wt
del
wt
wt
wt
lost
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
del
wt
del
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
lost
wt
wt
wt
del
wt
wt
wt
wt
del
del
del
del
del
wt
wt
wt
wt
wt
wt
wt
del
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
del
del
wt
wt
wt
wt
While EBV is associated with a wide range of human
malignancies, the contribution of EBV strain variation to the
pathogenesis of such tumors is unknown. Several groups
have recently proposed that certain EBV gene polymorphisms may be specifically associated with some virus-positive tumors and may confer a more aggressive malignant
phen~type.’~.~‘
To test this hypothesis, we have undertaken
an analysis of EBV isolates from different lymphoid and
epithelial malignancies and compared these with virus isolates present in the normal population. We have focused
particularly on specific polymorphisms in the LMPl gene
and in the BamHI F fragment of the EBV genome.
LMPl genes carrying point mutations leading to the loss
of an XhoI restriction site in the first exon and a 30-bp
deletion in the carboxy terminal region were first identified
in virus isolates derived from Chinese NPC biopsies.”.” The
deletion overlaps with one of the putative functional domains
in the carboxy terminus of LMPl, and it has been suggested
that this variant LMPl may be more oncogenic, but less
immunogenic, than the prototype B95.8 LMPl .‘4,23 In the
present study, we confirm the presence of LMPl genes carrying both the XhoI polymorphism and the LMPl deletion
in all NPC biopsies from Hong Kong and Taiwan. These
polymorphisms were also detected in the majority of EBV
isolates from healthy Chinese donors suggesting that the
presence of variant LMPl in NPC simply reflects the overall
prevalence of this polymorphism in EBV strains infecting
the general Chinese population. This assertion is supported
by our analysis of EBV-positive NPCs and gastric adenocarcinomas from European individuals where the incidence of
EBV strains carrying the 30-bp LMPl deletion is identical
to that observed in normal European donors. However, an
interesting observation is the absence of the XhoI polymorphism from all carcinoma isolates and from virtually all virus
isolates from healthy European and African donors carrying
Table 5. Analysis of New Guinean Spontaneous LCLs
and BL Biopsies for EBV Polymorphisms
LMPl Polymorphisms
Virus Isolate
New Guinean sp-LCLs
L2
L10
L16
L24
L42
L43
H19
H21
H23
H32
H36
New Guinean BL lines
WW1
ww2
Gor
Virus
TvDe
BamHl
1
1
1
1
1
1
1
1
1
1
1
F
1
2
2
F/f
F
F
F
F
F
F
F
F
F
f
F
F
F
33-bp
ReDeatS
3
3
4
4
4
3
3
3
3
3
4
4
4
4
Xhol
Site
30-bp
Deletion
lost
wt
wt
wt
wt
wt
wt
wt
wt
wt
lost
wt
wt
wt
wt
wt
wt
wt
wt
wt
lost
wt
wt
wt
wt
wt
wt
wt
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
KHANIM ET AL
3498
6
African LCLs
Type
Tvw 2
1
1
Type 1
Type 2
D
-(-
:%
m qu F 8 . g : p a
m
8 2
5 3
4
3
Undel)
del b
Table 6. Analysis of Chinese Spontaneous LCLs
and NPC Biopsies for EBV Polvmorphisms
LMPl Polymorphisms
Virus Isolate
Chinese sp-LCLs
05
DH
QX
QY
CKL
YKO
FJW
JLU
YFK
MTL
Taiwanese NPCs
biopsies
117
118
121
124
125
127
128
129
135
126
HKU NPCs biopsies
NPC532
NPC572
NPC875
NPC876
NPC877
NPC442
NPC608
NPC934
NPC0372
Virus
Type
BamHl
F/f
33-bp
Repeat
Xhol
1
1
1
1
1
1
1
1
1
2
F
f
f
F
F
F
F
f
F
F
5
5
4
5
4
5
5
5
6
4
lost
lost
lost
lost
del
del
del
del
del
del
del
del
del
wt
wt
1
1
1
1
1
1
1
1
1
2
f
F
f
F
f
f
f
F
f
4
3
3
3
5
4
4
4
3
4
lost
lost
lost
lost
lost
lost
lost
lost
lost
lost
del
del
del
del
del
del
del
del
del
del
1
1
1
1
1
1
1
1
1
F
f
f
f
f
F
f
f
f
lost
lost
lost
lost
lost
lost
lost
lost
lost
del
del
del
del
del
del
del
del
del
F
-
African BLs
,
r
Site
lost
lost
lost
lost
lost
30-bp
Deletion
8
c
6 2 E
v)
I“$
3
Fig 3. Analysis of African EBV isolates. (A) Analysis of the 33-bp LMPl repeats in African EBV isolates
showed less variation in repeat number in both type
1 and type 2 strains. (B) The 30-bp LMPl deletion
wasdetectedbyPCRfollowedbyPAGEandblotting.
EBV isolates with a deleted LMPl produced a 230bp PCR product, and a 260-bp PCR product was generated with undeleted LMPl. An association between the 30-bp deletion and type 2 EBV isolates
was noted in both normal carriers and BLs.
the LMPl deletion. Thus, our studies demonstrate that there
is no apparent association between the 30-bp LMPl deletion
and the XhoI polymorphism in European isolates of EBV.
Similar findings have recently been published by Miller et
al.’s A recent report by Chen et al” demonstrates a high
incidence of EBV isolates carrying the 30-bp LMPl deletion
in BLs and in the general population in Brazil. This supports
our contention that there is geographic variation in the prevalence of certain EBV isolates and that this is reflected in the
virus isolates found in EBV-associated tumors common to
these locations. This would also help to explain the high
incidence of EBV isolates carrying the LMPl deletion in Tcell lymphomas in Chinese populations.33
The Chinese isolates were also different from all other
groups with respect to the relationship between virus type
and presence or absence of the LMPl deletion. Thus, almost
all isolates from Chinese individuals were of type 1 and
displayed the LMPl deletion. By contrast, isolates obtained
from all other geographic areas analyzed showed a preferential association of the LMPl deletion with type 2 virus, such
that 11 of 16 (69%) type 2 isolates carried the deletion
compared with 23 of 1 18 (19%) type 1 isolates. This association was particularly evident in Africa where infection with
type 2 EBV is more common. The analysis of EBV strains
in Africa and New Guinea confirms and extends our previous
study’“ and demonstrates that neither type 2 virus nor the
LMPl deletion is preferentially associated with BL.
Previous studies reported the presence of LMPl variants
carrying the 30-bp deletion in 10% to 30% of EBV-positive
HD cases and in various lymphoproliferative disease^.^^.^'.^^
In agreement with these studies, we identified 9% of HD
cases that carried the deletion. In their original study, Knecht
et a13’ suggested that EBV isolates with the LMPl deletion
are preferentially found in histologically aggressive forms
of HD. and this contention is supported by a more recent
study.” In our series, the two HD cases harboring LMPIdeleted EBV were of the mixed cellularity histotype, which
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ANALYSIS OF EBV GENE POLYMORPHISMS
3499
489bp)
345bpb
144bpb
Fig 4. Analysis of Chinese EBV isolates. (A) The
Xhol RFLP in the LMPl gene was detected by PCR,
restriction enzyme digestion followed by PAGE and
blotting using both the 5' and 3' "P-labelled-oligonucleotide probes (Table 1). The Xhol site was found
to be mutated and, therefore, lost in EBV isolates
from both Chinese normal donors and in NPC biopsies. (6) The 30-bp LMPl deletion was detected in
EBV isolates from both normal donor-derived spLCLs and in NPC biopsies of Chinese origin.
I
4 lost
3
B r
,
c h i i LCb
Tvpe 1
~
~
' I
~
~
a
NPC bopsles - Tvpe 1
Tvpe 2
: E t
~
P
3
b
present
m
~
r
w
P
U
)
Q
~
C
m
=
a
=
)
L
O
N
"
undel'
del'
is more frequently associated with EBV infection. All four
HD cases displaying histological signs of particular aggressiveness (ie, necrosis and/or pleomorphic HRS cells) harbored virus with the full-length LMPl gene. Thus, our findings do not support the association of LMPI-deleted EBV
with aggressive HD, but further studies of a larger number of
cases are required and the criteria for determining aggressive
tumors (ie, histiotype, stage at presentation, treatment responsiveness) needs to be more thoroughly examined.
The incidence of the LMPl deletion in normal populations
has not been extensively examined previously. In the current
study, this deletion was found in 41% of LCLs derived from
normal healthy European donors and in 50% of IM tissues.
Interestingly, results from another study identified the 30-bp
deletion in three of nine IM (30%) tissues from a Danish
population.34These results, together with the data from the
series of HD biopsies studied, suggest that EBV isolates
carrying the LMPl deletion do not significantly increase the
risk of developing HD. However, the recent demonstration
that the 30-bp deleted LMPl is always found in HIV-associated HD3' endorses the possible increased oncogenic capacity of this variant and also raises questions about the prevalence of this virus in the HIV-infected population. Our results
support the contention that the occurrence of EBV isolates
carrying the 30-bp LMPl deletion in HD reflects the overall
prevalence of this polymorphism in EBV strains infecting
the general population. The increased incidence of type 2
EBV in HIV-infected individuals"~."" may explain the prevalence of the LMPl deletion variant in HIV-associated HD,
as our data suggest a preferential association of the deletion
with type 2 virus. Clearly, the possibility that HIV-infected
individuals are infected with more than one strain of EBV
and that some of these strains may predispose to certain
EBV-associated tumors needs to be examined. In this context, it is interesting that the B-cell lymphomas arising in
HIV-positive patients are frequently found to carry type 2
EBV and thus may also harbor deleted LMP1.37Recently
published work demonstrating that patients with multifocal
HD can harbor both wild-type and deleted LMPl carrying
EBV isolates suggests that either infection with multiple
EBV strains is possible or that the LMPl deletion occurs as
a late event in the oncogenic process." Other mutational
hotspots have been identified in the carboxy terminus of the
LMPl gene including several point mutations," and these
may also contribute to the tumorigenicity of EBV. However,
the frequency of these point mutations in EBV isolates is
not known, and thus future studies examining the prevalence
of these hotspots are warranted.
A notable feature of EBV strains from the Chinese population was the high prevalence of isolates carrying the BamHI
F fragment polymorphism or so-called 'f variant. Thus, in
agreement with previous
the majority of NPC
biopsies (68%)from Chinese patients carried the "f' polymorphism, but this variant was only observed in 30% of virus
isolates from healthy Chinese donors suggesting a possible
association of this variant with the development of NPC.
However, the "f' polymorphism was identified in only one
of I I (9%) European NPC biopsies and was not found in
any European LCLs. but was detected in one of seven IM
biopsies, and in one of 50 LCLs from Africa and New
Guinea. Interestingly, we found an increased incidence of
virus isolates containing the "f' polymorphism in HD biopsies from European patients. This was unexpected. as to date,
this variant has only been described in Chinese populations.
The implication of this finding with regard to the role of
EBV in the pathogenesis of HD remains obscure. Although
several open reading frames (ORF) have been identified in
the BamHI F fragment of EBV, no specific viral function
has yet been attributed to this region, and it may be that this
variation is a surrogate marker of a particular strain of EBV
with polymorphisms at other sites of the viral genome.
Previous studies have suggested that variations in the
number of 33-bp repeats in the LMPl gene between different
virus isolates may be useful for the determination of the
clonality of the EBV-carrying cell population>" Thus, it has
been argued that polyclonal EBV-carrying cell populations
"
"
"
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3500
KHANIM ET AL
should display LMPl repeat sequences of variable lengths,
while monoclonal proliferations would harbor only one virus
isolate with a defined number of repeats. Here we confirm
that PCR analysis of this region yields a single band in all
virus-associated malignancies. However, a single band was
also identified in all but one biopsy from individuals with
the polyclonal EBV-associated lymphoproliferative disease,
IM, suggesting that this polymorphism is not a suitable
marker for the determination of the cionality of virus-carrying cell populations.
In summary, although this is not a comprehensive epidemiological study, our results suggest that the specific EBV
gene polymorphisms detected in virus-associated tumors occur with similar frequencies in EBV isolates from healthy
virus carriers from the same geographic region. The only
exception to this is the “f” variant, which was detected with
increased frequency in both Chinese NPC biopsies and in
European HD tissues. While EBV isolates carrying the
LMPl deletion are found in a similar proportion of healthy
EBV carriers and virus-associated tumors from the same
geographic area, it remains possible that this LMPl variant
may influence the efficiency of malignant transformation,
thereby contributing to the increased incidence of certain
EBV-associated tumors in regions where this polymorphism
is common.
ACKNOWLEDGMENT
We are grateful to Drs N. Rooney, J. Crocker, D. Wright, D.
Moss, H. Rupani, K. Lam, J. Nicholls, W. Sheng and to P. Murray
for providing biopsy and cell line material for this study. We would
like to thank Sue Williams for her excellent help with photography.
REFERENCES
I . Rickinson AB, Kieff E: Epstein-Barr virus, in Fields BN, Knipe
DM, Howley PM (eds): Fields Virology, (ed 3 ) , Vol 2. New York,
NY, Lippincott-Raven, 1996, p 2397
2. Hamilton-Dutoit SJ, Pallesen G, Franzmann MB, Karkov J,
Black F, Skinhoj P, Pedersen C: AIDS-related lymphoma: Histopathology, immunophenotype, and association with Epstein-Barr virus
as demonstrated by in situ nucleic acid hybridization. Am J Pathol
138:149, 1991
3 . Weiss LM, Strickler JG, Wamke RA, Purtillo DT, Sklar J:
Epstein-Barr viral DNA in tissues of Hodgkin’s disease. Am J Pathol
12936, 1987
4. Pallesen G, Hamilton-Dutoit SJ, Zhou XJ: The association of
Epstein-Barr virus (EBV) with T cell proliferation and Hodgkin’s
disease: Two new developments in the EBV field. Adv Cancer Res
62:179, 1993
5. Ambinder RF, Browning PJ, Lorenzana I, Leventhal BG, Cosenza H, Mann RB, MacMohan EME, Medina R, Cardona V, Gruffennan S, Olshan A, Levin A, Petersen EA, Blattner W, Levine PH:
Epstein-Barr virus and childhood Hodgkin’s disease in Honduras
and the United States. Blood 81:462, 1993
6. Armstrong AA, Alexander FE, Pinto Paes R, Morad NA, Gallagher A, Krajewski AS, Jones DB, Angus B, Adams J, Cartwright
RA, Onions DE, Jarrett RF: Association of Epstein-Barr virus with
paediatrics Hodgkin’s disease. Am J Pathol 142: 1683, 1993
7. Uccini S, Monardo F, Stoppacciaro A, Gradilone A, Agliano
AM, Faggioni A, Manzari V, Vago L, Costanzi G, Ruco LP, Baroni
CD: High frequency of Epstein-Barr virus detection in Hodgkin’s
disease of HIV-positive patients. Int J Cancer 467:581, 1990
8. Raab-Traub N: Epstein-Barr virus and nasopharyngeal carcinoma. Semin Cancer Biol 3:297, 1992
9. Young L, Alfieri C, Hennessey K, Evans H, O’Hara C, Anderson KC, Ritz J, Shapiro RS, Rickinson A, Kieff E, Cohen J1: Expression of Epstein-Barr virus transformation-associated genes in tissues
of patients with EBV lymphoproliferative disease. N Engl J Med
321 :1080, 1989
10. Gratama JW, Zutter MM, Minarovits J, Oosterveer MAP,
Thomas ED, Klein G, Emberg I: Expression of Epstein-Barr virusencoded growth-transformation-associated proteins in lymphoproliferations of bone-marrow transplant recipients. Int J Cancer 47: 188.
1991
I I . Deacon EM, Pallesen G, Niedobitek G, Crocker J, Brooks L,
Rickinson AB, Young LS: Epstein-Barr virus and Hodgkin’s disease:
Transcriptional analysis of virus latency in the malignant cells. J
Exp Med 177:339, 1993
12. Brooks L, Yao QY. Rickinson AB, Young LS: Epstein-Barr
virus latent gene transcription in nasopharyngeal carcinoma cells:
Coexpression of EBNAl, LMPI, and LMP2 transcripts. J Virol
66:2689, 1992
13. Rowe M, Rowe DT, Gregory CD, Young LS, Farrell PJ,
Rupani H, Rickinson AB: Differences in B cell growth phenotype
reflect novel pattems of Epstein-Barr virus latent gene expression
in Burkitt’s lymphoma. EMBO J 6:2743, 1987
14. Rowe M: The EBV latent membrane protein-I (LMPI): A
tale of two functions. Epstein-Barr Virus Report 2:99, 1995
15. Gratama JW, Ernberg I: Molecular epidemiology of EpsteinBarr virus infection, in Woude GFV, Klein G (eds): Advances in
Cancer Research, vol 67. San Diego, CA, Academic, 1995, p 197
16. Gratama JW, Oosterveer MAP, Klein G, Emberg I: EBNA
size polymorphism can be used to trace Epstein-Barr virus spread
within families. J Virol 64:4703, 1990
17. Lung ML, Chang RS, Huang ML, Guo HY, Choy D, Sham
J, Tsa SY, Cheng P, Ng MH: Epstein-Barr Virus phenotypes associated with nasopharyngeal carcinoma in Southem China. Virology
185:67, 1990
18. Lung ML, Lam WP, Chan KH. Li S, Sham J , Choy D: Direct
detection of Epstein-Barr virus in peripheral blood and comparison
of EBV genotype present in direct specimens and lymphoblastoid
cell lines established from nasopharyngeal carcinoma patients and
healthy camers in Hong Kong. Int J Cancer 52: 174, I992
19. Lung ML, Chang GC, Miller TR, Wara WM, Phillips TL:
Genotypic analysis of Epstein-Barr virus isolates associated with
nasopharyngeal carcinoma in Chinese immigrants to the United
States. Int J Cancer 59:743, 1994
20. Bouzid M, Djennaoui D, Dubreuil J. Bouguermouh A, Ellouz
D, Abdelwahab J, Decaussin 0,Ooka T: Epstein-Barr virus genotypes in NPC biopsies from North Africa. Int J Cancer 56:468, 1994
2 I . Hu L-F, Zabarovsky ER, Chen F, Cao S-L, Ernberg I, Klein
G, Winberg G: Isolation and sequencing of the Epstein-Barr virus
BNLF-I gene (LMPl) from a Chinese nasopharyngeal carcinoma.
J Gen Virol 72:2399. 1991
22. Chen ML, Tsai CN, Liang CL. Shu CH, Huang CR, Sulitzeanu D. Liu ST, Chang YS: Cloning and characterization of the
latent membrane protein (LMP) of a specific Epstein-Barr virus
variant derived from nasopharyngeal carcinoma in the Taiwanese
population. Oncogene 7:2 131, 1992
23. Trivedi P, Hu LF, Chen F, Christensson B, Masucci M, Klein
G, Winberg G: Epstein-Barr virus (EBV)-encoded membrane protein
LMPl from a nasopharyngeal carcinoma nonimmunogenic in a murine model system, in contrast to a B-cell derived homolog. Eur J
Cancer 30A:84, 1994
24. Knecht H, Bachmann E, Brousset P, Sandvej K, Nadal D,
Bachmann F, Odermatt BF, Delsol G, Pallesen G: Deletions within
the LMPl oncogene of Epstein-Barr virus are clustered in Hodgkin’s
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ANALYSIS OF EBV GENE POLYMORPHISMS
disease and identical to those observed in nasopharyngeal carcinoma.
Blood 82:2937, 1993
25. Miller WE, Edwards RE, Walling DM, Raab-Traub N: Sequence variation in the Epstein-Barr virus latent membrane protein
1. J Gen Virol 75:2729, 1994
26. Young LS, Yao QY, Rooney CM, Sculley TB, Moss DJ,
Rupani H, Laux G, Bornkamm GW, Rickinson AB: New type B
isolates of Epstein-Barr virus from Burkitt’s lymphoma and from
normal individuals in Burkitt-endemic areas. J Gen Virol 68:2853,
1987
27a. Niedobitek GN, Rowlands DC, Young LS, Herbst H, Williams A, Hall P, Padfield I, Rooney N, Jones EL: Overexpression
of p53 in Hodgkin’s disease: Lack of correlation with Epstein-Barr
virus infection. J Pathol 169:207, 1993
27b. Niedobitek GN, Aganthanggelou A, Barber P, Smallman
LA, Jones EL, Young LS: p53 overexpression and Epstein-Barr
virus infection in undifferentiated nasopharyngeal and squamous cell
carcinoma. J Pathol 170:457, 1993
28. Rowlands DC, Ito M, Mangham DC, Reynolds G, Herbsty
H, Hallissey MT, Fielding JWL, Newbold KM, Jones EL, Young
LS, Niedobitek GN: Epstein-Barr virus and carcinomas: Rare association of the virus with gastric adenocarcinoma. Br J Cancer 68: 1014,
1993
29. Sample J, Young L, Martin B, Chatman T, Kieff E, Rickinson
A, Kieff E: Epstein-Barr virus type-1 (EBV-I) and 2 (EBV-2) differ
in their EBNA 3A, EBNA 3B, and EBNA 3C genes. J Virol64:4084,
1990
30. Abdel-Hamid M, Chen JJ, Constantine N, Massoud M, RaabTraub N: EBV strain variation: Geographical distribution and relation to disease state. Virology 190:168, 1992
31. Knecht H, Bachmann E, Brousset P, Rothenberger S, Einsele
H, Lestou VS, Delsol G, Bachmann F, Ambros PF, Odermatt BF:
Mutational botspots within the carboxy terminal region of the LMPl
oncogene of Epstein-Barr virus are frequent in lymphoproliferative
disorders. Oncogene 10523, 1995
32. Chen WG, Cben YY, Bacchi MM, Bacchi CE, Alvarenga M,
Weiss LM: Genotyping of Epstein-Barr-virus in Brazilian Burkitts-
3501
lymphoma and reactive lymphoid-tissue-type-A with a high prevalence of deletions within the latent membrane-protein gene. Am J
Pathol 148:17, 1996
33. Chang Y-S, Su I-J, Chung P-J, Shu C-H, Ng C-K, Wu S-J, Liu
S-H: Detection of an Epstein-Barr virus variant in T-cell lymphoma
tissues identical to the distinct strain observed in nasopharyngeal
carcinoma in the Taiwanese population. Int J Cancer 62:673, I995
34. Sandvej K, Peh SC, Storstein Andreson B, Pallesen G: Identification of potential hotspots in the carboxy-terminal part of the
Epstein-Barr virus (EBV) BNLF-I gene in both malignant and benign EBV-associated diseases: High frequency of a 30-bp deletion in
Malaysian and Danish peripheral T-cell lymphomas. Blood 84:4053,
1994
35. Santon A, Manzanal AI, Camlo E, Bellas C: Deletions in the
Epstein-Barr virus latent membrane protein-I oncogene in Hodgkin’s disease. J Clin Pathol 48:M184, 1995
36. Sixbey JW, Shirley P, Chesney PJ, Buntin DM, Resnick L:
Detection of a second widespread strain of Epstein-Barr virus. Lancet
2:761, 1989
37. Boyle MJ, Sewell WA, Sculley TB, Apolloni A, Turner JJ,
Swanson CE, Penny R, Cooper DA: Subtypes of Epstein-Barr virus
in human immunodeficiency virus-associated non-Hodgkin
lymphoma. Blood 78:3004, 1991
38. Borisch B, Finke J, Hennig I, Delacretaz F, Schneider J, Heitz
PU, Laissue JA: Distribution and localisation of Epstein-Barr virus
subtypes A and B in AIDS-related lymphomas and lymphatic tissue
of HIV-positive patients. J Path01 168:229, 1992
39. Vasef MA, Kame1 OW, Chen YY, Medeiros U and Weiss
LM: Detection of Epstein-Barr virus in multiple sites involved by
Hodgkin’s disease. Am J Pathol 147:1408, 1995
40. Takano Y, Kat0 Y: Epstein-Barr virus association with early
cancers found together with gastric medullary carcinomas demonstrating lymphoid infiltration. J Pathol 175:39, 1995
41. Baer R, Bankier AT, Biggin MD, Deininger BL, Farrell PJ,
Gobson TJ, Hatful G, Hudson GS, Satchwell SC, Seguin C, Tuffnell
PS, Barrel1 BG: DNA sequence and expression of B95-8 EpsteinBarr virus genome. Nature (London) 310:207, 1984
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1996 88: 3491-3501
Analysis of Epstein-Barr virus gene polymorphisms in normal donors
and in virus-associated tumors from different geographic locations
F Khanim, QY Yao, G Niedobitek, S Sihota, AB Rickinson and LS Young
Updated information and services can be found at:
http://www.bloodjournal.org/content/88/9/3491.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.