Germline EPHB2 Receptor Variants in Familial Colorectal
George Zogopoulos1,2, Claus Jorgensen1, Julinor Bacani1, Alexandre Montpetit3, Pierre Lepage3,
Vincent Ferretti3, Lauren Chad3, Subani Selvarajah1, Brent Zanke4,5, Thomas J. Hudson4,5, Tony
Pawson1, Steven Gallinger1,2,4*
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, 2Dr. Zane Cohen Digestive Diseases Clinical Research Centre, Mount Sinai
Hospital, Toronto, Ontario, Canada, 3McGill University and Genome Que ´bec Innovation Centre, Montreal, Quebec, Canada, 4Cancer Care Ontario, Toronto, Ontario,
Canada, 5The Ontario Institute for Cancer Research, Toronto, Ontario, Canada
Familial clustering of colorectal cancer occurs in 15–20% of cases, however recognized cancer syndromes explain only a small
fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has
recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the
contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-
based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized
the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected.Two
of these variants(A438TandG787R) result in significantresiduechanges,while the third leads to a conservative substitution in
the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant
was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and
G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain,
causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function
and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity,
with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline
EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.
Citation: Zogopoulos G, Jorgensen C, Bacani J, Montpetit A, Lepage P, et al. (2008) Germline EPHB2 Receptor Variants in Familial Colorectal Cancer. PLoS ONE 3(8):
Editor: Jo ¨rg Hoheisel, Deutsches Krebsforschungszentrum, Germany
Received December 20, 2007; Accepted July 3, 2008; Published August 6, 2008
Copyright: ? 2008 Zogopoulos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Cancer Institute, National Institutes of Health under RFA # CA-96-011 (to SG) and through cooperative
agreements with members of the Colon Cancer Family Registry and P.I.s. The content of this manuscript does not necessarily reflect the views or policies of the
National Cancer Institute or any of the collaborating institutions or investigators in the Colon CFR, nor does mention of trade names, commercial products, or
organizations imply endorsement by the US Government or the Colon CFR. Cancer Care Ontario, as the host organization to the ARCTIC Genome Project,
acknowledges that this Project was partly funded by Genome Canada through the Ontario Genomics Institute, by Ge ´nome Que ´bec, the Ministe `re du
De ´veloppement y ¨conomique et Re ´gional et de la Recherche du Que ´bec and the Ontario Institute for Cancer Research. GZ is a Scholar of the Society of University
Surgeons and a recipient of a Terry Fox Foundation Research Fellowship from the National Cancer Institute of Canada. TJH is a recipient of a Clinician-Scientist
Award in Translational Research from the Burroughs Wellcome Fund.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Colorectal cancer is a common malignancy in Western society,
with 15–20% of cases developing on the basis of apparent genetic
predisposition . However, the recognized familial colorectal
cancer syndromes–Familial Adenomatous Polyposis (FAP), He-
reditary Non-Polyposis Colorectal Cancer (HNPCC) and MYH-
Associated Polyposis (MAP)-account for much less than one-third
of inherited colorectal cancer, leaving the additional heritable
component of this disease unexplained .
In the great majority of colorectal cancers, mutational activation
of the WNT signaling pathway plays an essential role in initiating
tumorigenesis [2–4]. Hyperactivation of WNT signaling most
commonly occurs early in the adenoma to carcinoma sequence as
a consequence of inactivating mutations in the APC tumour
suppressor gene or activating oncogenic b2catenin mutations. These
genetic alterations lead to constitutive activation of b2catenin /
TCF4 complex which, in turn, drives overexpression of WNT
signaling targets. These downstream effectors of the WNT pathway
include genes with a critical role in the development of colorectal
tumors, such as the EPHB and ephrin genes [5,6].
A role for EPHB2, a member of the Eph receptor tyrosine kinase
family, as a tumor suppressor gene in colorectal carcinogenesis has
been suggested by several recent findings. EPHB2 maps to a
chromosomal region (1p36.1) often deleted in these tumors . In
addition, immunohistochemical studies have shown that expression
of multiple EPHB isoforms is frequently suppressed in invasive
human colorectal tumours . Moreover, Alazzouzi et al. have
recently shown a high incidence of EPHB2 frameshift mutations in
microsatellite unstable colorectal tumors and aberrant EPHB2
promoter methylation in both microsatellite stable and unstable
neoplasms . Loss of EPHB2 expression in colorectal tumors has
also been associated with worse prognosis .
Mutational inactivation of EPHB2 has been linked to prostate
carcinogenesis. Huusko et al. recently described a variety of EPHB2
mutations in prostate tumours and in cell-lines , and a
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common nonsense mutation has been associated with prostate
cancer in African American men with a family history of this
disease . These findings taken together with the established
role of EPHB receptors in colorectal cancer, prompted us to
hypothesize that germline EPHB2 mutations may account for at
least a fraction of the genetic alterations underlying the
unexplained portion of hereditary colorectal cancer, and may
represent a new cancer syndrome causing genetic predisposition to
colorectal and prostate cancer. Therefore, in this study, we
screened 116 population-based familial colorectal cancer cases for
germline EPHB2 mutations. The 116 probands tested had at least
one additional affected first degree relative. These cases had
microsatellite stable tumors, and did not meet diagnostic criteria
for FAP, HNPCC and MAP. We also enriched our series with
individuals who met the above inclusion criteria and had personal
or family histories of prostate cancer. Our findings suggest that
rare EPHB2 alleles contribute to a small fraction of familial
Materials and Methods
Study Subjects and DNA Samples
Biospecimens were obtained from the Ontario Familial
Colorectal Cancer Registry (OFCCR), a member of the National
Cancer Institute Cooperative Family Registries for Colorectal
Cancer Studies (http://epi.grants.cancer.gov/CFR/about_colon.
html) . The OFCCR includes 3,770 colorectal cancer cases
diagnosed in the province of Ontario, Canada between 1997–
2000, with an age at the time of diagnosis of 20 to 74. Age- and
sex-matched control subjects with no personal history of colorectal
cancer were recruited by telephone from a list of randomly
selected residential telephone numbers for Ontario and from
population-based Tax Assessment Rolls of the Ontario Ministry of
Finance. Study subjects donated a venous blood sample and
peripheral lymphocytes were isolated using Ficoll-Paque, accord-
ing to the manufacturer’s recommendations (Amersham Biosci-
ences, Baie d’Urfe ´, Quebec, PQ, Canada). The phenol–chloro-
form method was used to isolate genomic DNA from lymphocytes
and colorectal cancer cell-lines. The QIAamp protocol (Qiagen
Inc., Mississauga, Ontario, Canada) was employed to extract
genomic DNA from paraffin-embedded tissues. All study subjects
signed written consent to participate in a Mount Sinai Hospital
Research Ethics Board approved research study.
EPHB2 Mutation Screening
Using automated sequencing (Applied Biosystems 3730xl DNA
Analyzer, Foster City, CA, USA), we screened for germline
(Entrez Gene ID:
NM_017449) sequence variants in 116 familial colorectal cases
(average age at diagnosis 54 years, range 22 to 74 years, 59
females, 57 males). Our analysis included patients with personal
(n=6) and/or family histories (father, n=19; sibling, n=23; half-
sibling, n=4) of prostate cancer. Patients with FAP, HNPCC or
MAP were excluded. A series of colorectal cancer cell lines (Caco2,
Colo320DM, Colo320HSR, HT29, LS513, LS1034, SW837,
SW948, SW1417, T84) were also screened. We sequenced the
entire coding region and at least 50 bp of intronic sequence at the
exon/intron boundaries. PCR primer sequences and conditions
are provided in Table S1. Variants identified are numbered
relative to RefSeq NM_004442.
A438T, D679N and G787R Allele Frequencies
A random sample of cases and matched controls from the
OFCCR series were selected for studies to evaluate the A438T,
D679N and G787R allele frequencies. Lymphocyte DNA samples
from an additional series of OFCCR cases (n=364 for A438T;
n=1160 for D679N; n=182 for G787R) and population-matched
controls (n=384 for A438T; n=1133 for D679N; n=199 for
G787R) were tested to evaluate the population frequencies of the
A438T, D679N and G787R variants. Genotyping assays for the
A438T, D679N and G787R variants were developed using
Fluorescence Polarization-Single Base Extension (FP-SBE) ,
SNPstream  and RFLP, respectively (Table S2).
Pedigree and Loss of Heterozygosity (LOH) Analyses
Testing for segregation in the families of the probands carrying
the A438T and G787R variants was performed by direct
sequencing, using either lymphocyte or archival DNA from
paraffin-embedded tissue blocks. Loss of heterozygosity was
performed by sequencing paired tumor/normal archival DNA
samples and comparing sequence autoradiograms for a decrease in
the intensity of the non-mutated signal compared to the mutated
sequence (Table S2). These reactions were performed using the
Thermo Sequenase Radiolabeled Terminator Cycle Sequencing
kit, according to the manufacturer’s protocol (USB Corporation,
Cleveland, Ohio, USA).
Biochemical Characterization of the A438T & G787R
The A438T and G787R cDNA sequence variants were
generated using PCR-based site-directed mutagenesis and RefSeq
NM_004442 (OriGene Technologies Inc., Rockville, MD, USA)
as the template. The PCR products were cloned into pcDNA3
(Invitrogen Canada Inc.), and sequence verified. DU145 (a gift
from Dr. Irene Andrulis, Samuel Lunenfeld Research Institute)
were grown in MEM/10% FBS. Cells were transiently transfected
with the various EPHB2 cDNA constructs (wild-type, A438T, or
G787R) using lipofectamine 2000 (Invitrogen Canada Inc.,
Burlington, Ontario, Canada), according to manufacturers
instructions. Five hours following transfection, medium was
changed to starvation medium (MEM/0.5%FBS) for 16h. Cells
were then stimulated with 2 mg/ml preclustered ephrin b1-Fc
(ephrin b1-Fc, R&D Systems, Minneapolis, MN, USA; anti-Fc
antibody, Jackson ImmunoResearch Laboratories Inc., West
Grove, PA, USA) for 30min. Transfected EPHB2 was immuno-
precipitated using anti-EPHB2 antiserum  and immunoblotted
with anti-phosphotyrosine (4G10, Upstate Biotechnology, Lake
Placid, New York, USA) or anti-EPHB2. Expression and
immunoprecipitation of EPHB2 variants for in vitro kinase assays
was performed as above except cells were left unstimulated prior to
cell lysis and immunoprecipitation, as previously described by
Holland et al.  Gels were analyzed and quantified using a
Storm phosphoimager (Molecular Dynamics Inc., Sunnyvale,
Mutational screening of the EPHB2 gene in 116 patients with
familial colorectal cancer identified 3 novel missense nucleotide
changes and the D679N variant previously suggested by Huusko et
al.  to be pathogenic in prostate cancer (Table 1). Three
unrelated patients were found to carry the D679N allele,
diagnosed with colorectal cancer at the ages of 57, 59 and 67
years, respectively. However, subsequent analysis of additional
subjects suggests that D679N represents a rare neutral EPHB2
polymorphism, since the variant allele was observed at similar
frequencies in patients with colorectal cancer (11 out of 1133) and
population-matched controls (11 out of 1160).
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We also screened for EPHB2 mutations in 10 colorectal cancer
cell lines, and Caco2 was found to express a novel variant, R4Q (nt.
11 ARG), in addition to the wild-type EPHB2 receptor. The R4Q
amino acid substitution may cause an alteration in the signal
peptide, which maylead to decreased EPHB2 expression.However,
immunoprecipitation with anti-EPHB2 antiserum, followed by
immunoblotting using an antiphosphotyrosine antibody, demon-
strated that despite carrying the R4Q variant, the CAC02 cell line
produces a functional EPHB2 receptor (data not shown).
Two of the three novel nonsynonymous variants (A438T &
G787R) we identified result in biochemically significant and
potentially pathogenic residue changes. The residue affected by
the A438T substitution is located in the extracellular fibronectin
type III domain . Since this domain may be involved in ligand
binding, we postulated that the A438T variant might have
decreased binding activity and signaling function. The G787R
variant affects a residue in the kinase domain, and this substitution
might therefore directly affect the receptor’s kinase and biological
activity . The third novel variant (V945I) was detected in two
patients, and leads to a conservative substitution at the extreme
carboxy-terminus in the SAM domain ; we have not
characterized this allele further.
Table 1. Non-synonymous germline EPHB2 missense changes identified in familial and random colorectal cancer cases.
Cancer Cases Control Subjects
nt. 1312 GRA A438T6 Extracellular Fibronectin
nt. 2035 GRA D679N11 Protein Kinase Domain3/11611/116011/1133
nt. 2359 GRA G787R13 Protein Kinase Domain1/1160/182 0/199
nt. 2833 GRA *V945I15 SAM Domain2/116 Not determined Not determined
*A conservative residue substitution (V945I).
Figure 1. Pedigrees of colorectal cancer cases carrying the A438T (Family 1, Panel A) and G787R (Family 1, Panel B) variants. +/2,
carrier; 2/2, non-carrier; LOH+, Colorectal tumour tissue was found to manifest LOH, with loss of the wild-type allele; Ca, cancer.
EPHB2 in Colorectal Cancer
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The A438T variant was found in a patient who had two primary
cancers. He was diagnosed with prostate cancer and a microsatellite
stable right-sided colon cancer at the ages of 61 and 64 years,
respectively (Figure 1A,Family 1).Theproband’sfather also carried
the A438T variant and is the only other family member with colon
cancer, he was diagnosed with a microsatellite stable sigmoid cancer
at the age of 76 years. Of the 6 unaffected family members tested for
this missense change, 3 were found to carry the A438T variant
(Figure 1A). Sequencing analyses of paired tumor-normal genomic
DNA samples revealed loss of the wild-type EPHB2 allele in the
colon cancer from the proband, but not in the colon cancer from his
father (data not shown). The father’s tumor manifested LOH of the
variant, rather than the wild-type, EPHB2 allele. This latter
observation may reflect the frequent loss of the 1p36.1 chromo-
somal region during tumorigenesis [7,8], and not the targeted
inactivation of the EPHB2 locus by LOH.
The G787R variant was detected in a patient with a diagnosis of
rectal cancer at the age of 67 years (Figure 1B, Family 2). This
tumor showed loss of the wild-type allele (Figure 2). The patient also
reported a history of follicular type thyroid cancer at age 73 years.
This proband was selected for mutation screening because of a
family history of colorectal cancer on the maternal side. However,
genotyping data revealed that the G787R variant allele either
originates on the paternal side or is the result of a de novo mutation
(Figure 1B). The proband’s daughter is the only other carrier of this
variant, and she is currently unaffected at the age of 45 years.
We estimated the allele frequencies of the A438T and G787R
variants by screening an additional series of population-based
colorectal cancer cases and age and sex matched control subjects.
The genotyping results suggest that these two alleles are rare
variants (Table 1). Neither variant was detected in control subjects
and they were not identified in any additional colorectal cancer
cases. Therefore, to further evaluate the possible pathogenic role
the A438T and G787R receptor variants, we characterized their
intrinsic tyrosine kinase activity.
Biochemical characterization of the A438T and G787R
isoforms revealed that that the G787R variant is functionally
impaired, whereas the A438T change likely represents a neutral
polymorphism. DU145 cells, which do not express an endogenous
functional EPHB2 receptor , were transiently transfected with
either the wild-type EPHB2 receptor or one of the two variants.
We found diminished autophosphorylation of the G787R
receptor, but not the A438T variant, following ephrinB1
stimulation compared to the wild-type receptor (Figure 3A). We
confirmed that the G787R receptor has reduced catalytic activity
by using an in vitro kinase assay. The ability of the G787R
receptor to autophosphorylate or to phosphorylate the enolase
substrate was approximately 9-fold lower than that of the wild-type
receptor (Figure 3B), demonstrating that the G787R mutation
alters receptor activity and is not a rare neutral polymorphism.
The Eph receptor family is the largest known subgroup of
receptor tyrosine kinases. This family is further subdivided into
two distinct classes, EphA (A1 to A10) and EphB (B1 to B6), based
on their binding affinities for two membrane-anchored ligand
families with the corresponding names of type A (A1 to A5) and B
(B1 to B3) ephrins [19,20]. Following ligand binding, Eph
receptors activate cell repulsion pathways to modulate cell
compartmentalization and ordered cell migration in a variety of
biological processes .
Mouse animal model studies have shown that, in the small
intestine, EphB receptors mediate intestinal stem cell proliferation
 as well as epithelial cell migration and organization along the
crypt-villous axis . Since loss of mitotic activity control, epithelial
patterning and tissue architecture are hallmarks of tumorigenesis,
disruption of normal EPHB receptor expression and function likely
promotes colorectal carcinogenesis. Constitutive EPHB receptor
expression maystimulate tumorinitiation bydisturbingproliferative
stem cell homeostasis, and secondary silencing of EPHB receptor
activity may permit expansion of cancer cells, beyond the spatial
boundaries imposed by intact EPHB receptor function to populate
adjacent tissue structures [5,6,21]. In support of this hypothesis, we
and others have recently shown a causal role for EphB inactivation
in tumor progression . We found that EphB2 or EphB3 silencing
in ApcMin/+mice results in accelerated and more aggressive
In the current study, we screened 116 population-based familial
cases of colorectal cancer for mutations in a candidate tumor
suppressor gene, EPHB2, and identified three candidate variants
(A438T, D679N, G787R), which were further characterized. Even
though the A438T allele was not observed in control subjects and
was found to segregate with disease in Family 1, biochemical
characterization suggests that A438T is a rare neutral polymor-
phism; we cannot exclude the possibility that it affects more subtle
aspects of EPHB2 signaling, such as the formation of higher order
oligomers. The D679N variant has been previously reported to be
associated with prostate cancer . Although, it remains possible
that this variant modulates predisposition to prostate cancer, our
data suggest that it occurs at a population frequency of
approximately 1% and that it does not, on its own, increase
susceptibility to colorectal cancer. We observed the D679N allele
in a similar number of patients with colorectal cancer and
population-matched controls. In contrast to these latter variants,
our data suggest that the G787R variant is functionally
compromised and may be a rare cause of hereditary colorectal
cancer. The G787R variant was identified in a patient diagnosed
Figure 2. Sequencing results demonstrating loss of the wild-
type EPHB2 allele in the G787R carrier. EPHB2 gene sequencing
results of genomic DNA extracted from paired tumor and
adjacent normal colon mucosa are shown. Relative to the
intensities of the guanine bands in the sequencing reaction, the
intensity of the guanine nucleotide at position 2359 is substantially
reduced in the tumor sample, suggesting loss of the wild type allele (G)
in the tumor but not in the adjacent normal tissue (arrow shows nt.
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with rectal cancer at 67 years of age, and biochemical
characterization revealed that the G787R substitution markedly
diminishes the receptor’s intrinsic kinase activity.
There have been two other investigations examining the
contribution of germline EPHB2 mutations to colorectal cancer
susceptibility. Oba et al. screened for EPHB2 mutations in colon
Figure 3. Biochemical characterization of EPHB2 variants. Panel A: Diminished autophosphorylation of EPHB2 G787R variant in response to
ephrinB1 stimulation. DU145 cells were transiently transfected with cDNA constructs (empty vector; wild-type, wt; A438T; G787R) and either left
unstimulated (2) or stimulated (+) with preclustered ephrinB1-Fc for 30 min. EPHB2 was immunoprecipitated (IP) and immunoblotted (IB) with
antiphosphotyrosine (4G10) to evaluate receptor autophosphorylation. The cell lysate was immunoblotted with antiEPHB2 to ascertain that there was
equal transfection efficiency. Panel B: Abolished kinase activity of EPHB2 G787R variant. In vitro kinase assays were performed using wild-type EPHB2
or G787R immunoprecipitates and enolase as the exogenous substrate. Prior to imaging or immonoblotting against EPHB2, phosphorylated proteins
were separated by gel electrophoresis and stained with coomassie. Autoradiogram showing32PcATP incorporation in EPHB2 and enolase (upper
panel), anti-EPHB2 immunoblot (middle panel) and equal loading of enolase is shown (lower panel). The table shows the relative kinase activity of the
wild-type EPHB2 receptor (set to 100%) vs. the G787R variant.
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tumors and respective normal colon tissues from 50 patients with Download full-text
colorectal cancer, and identified an intron 8 alteration in a single
tumor sample, which results in a nonsense mutation. However, it is
unclear if this is a somatic mutation, as there is no indication
whether this genetic change was also observed in the paired
normal colon sample . This investigation also identified 15
cases with LOH involving the EPHB2 gene and screened for
mutations in the remaining EPHB2 allele. Since mutations in the
remaining allele were not identified, Oba et al. suggested that
EPHB2 is not a classical tumor suppressor gene. However, since
only 50 samples of likely sporadic cases of colorectal cancer were
analyzed, a tumor suppressor role for EPHB2 cannot be excluded.
In a more recent study, Kokko et al. reported an association of
three novel variants with colorectal cancer . Germline
missense changes resulting in I361V, R568W, and D861N were
observed in colorectal cancer patients, but not in healthy controls.
However, it is possible that these three variants are rare neutral
polymorphisms since the biological significance of the variants was
not evaluated using direct functional assays. The patients screened
in these latter two studies did not necessarily have significant
family histories of colorectal cancer. In contrast to these two earlier
reports, our study was designed to specifically evaluate the role of
germline EPHB2 mutations in patients with familial colorectal
cancer, and not in sporadic cases. Despite study design differences,
together these three investigations suggest that EPHB2 germline
mutations are not common occurrences in colorectal cancer.
Further investigations of larger sample sizes are needed to confirm
In summary, we identified a germline EPHB2 variant (G787R)
with diminished biological activity in a colorectal cancer patient,
and suggest that EPHB2 mutations contribute to a small fraction of
hereditary colorectal cancer. The rarity of germline EPHB2
mutations supports a more significant role for EPHB2 in colorectal
tumor progression rather than in tumor initiation. Since the EPHB
receptors (EPHB2, EPHB3 and EPHB4) follow a similar pattern of
transcriptional silencing in colorectal cancers, all EPHB receptor
family members probably play a similar role in this disease.
Therefore, the EPHB family likely accounts for a minor
proportion of genetic predisposition to colorectal cancer but has
an important role in tumor progression. Although our findings
suggest that the EPHB gene family should not be routinely
screened for germline mutations in familial cases, the EPHB genes
are candidate tumor suppressors, likely accounting for rare cases of
familial colorectal cancer.
Found at: doi:10.1371/journal.pone.0002885.s001 (0.06 MB
Found at: doi:10.1371/journal.pone.0002885.s002 (0.05 MB
The authors thank Dr. D. Daftary, Ms. S. Holter, Ms. A. Janson for their
assistance with data collection, and Ms. Joelle Fontaine, Ms. T. Selander
and the Mount Sinai Hospital Biospecimen Repository for technical
Conceived and designed the experiments: GZ BZ TJH TP SG. Performed
the experiments: GZ CJ JB AM PL VF LC SS. Analyzed the data: GZ CJ
JB. Contributed reagents/materials/analysis tools: GZ SG. Wrote the
paper: GZ SG.
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PLoS ONE | www.plosone.org6 August 2008 | Volume 3 | Issue 8 | e2885