doi:10.1136/jmg.2003.

015594
2004;41;89- J. Med. Genet.

Siewert and H Hfler
Seruca, K Biedermann, D Huntsman, C Dring, E Holinski-Feder, A Neutzling, J R
G Keller, H Vogelsang, I Becker, S Plaschke, K Ott, G Suriano, A R Mateus, R

gastric cancer patients
contribute to genetic predisposition in German
, HPP1 and RUNX3 genes, rather than of TP53
and E-cadherin(CDH1) Germline mutations of the
http://jmg.bmjjournals.com/cgi/content/full/41/6/e89
Updated information and services can be found at:
These include:
Rapid responses
http://jmg.bmjjournals.com/cgi/eletter-submit/41/6/e89
You can respond to this article at:
service
Email alerting
top right corner of the article
Receive free email alerts when new articles cite this article - sign up in the box at the
Topic collections
(3360 articles) Genetics
(994 articles) Cancer: gastroenterological
(870 articles) Molecular Medicine
(498 articles) Stomach and duodenum
(134 articles) Online mutation reports

Articles on similar topics can be found in the following collections
Notes
http://www.bmjjournals.com/cgi/reprintform
To order reprints of this article go to:
http://www.bmjjournals.com/subscriptions/
go to: Journal of Medical Genetics To subscribe to
on 14 June 2005 jmg.bmjjournals.com Downloaded from
ONLINE MUTATION REPORT
Germline mutations of the E-cadherin(CDH1) and TP53
genes, rather than of RUNX3 and HPP1, contribute to
genetic predisposition in German gastric cancer patients
G Keller, H Vogelsang, I Becker, S Plaschke, K Ott, G Suriano, A R Mateus, R Seruca,
K Biedermann, D Huntsman, C Doring, E Holinski-Feder, A Neutzling, J R Siewert, H Hofler
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J Med Genet 2004;41:e89 (http://www.jmedgenet.com/cgi/content/full/41/6/e89). doi: 10.1136/jmg.2003.015594
G
ermline mutations of the cell adhesion molecule E-
cadherin have been shown for the first time to
underlie a hereditary diffuse type gastric cancer
syndrome (HDGC) in Maori families,
1
and subsequently have
been reported in HDGC patients of various ethnic origins.
29
Gastric cancer may also be associated with other hereditary
tumour syndromes, which are mainly characterised by
carcinomas of other organs. One of these syndromes is the
HNPCC syndrome (hereditary nonpolyposis colorectal cancer
syndrome), which is caused by germline mutations in DNA
mismatch repair genes.
10
Furthermore, gastric cancer has
been observed in the context of the Li Fraumeni syndrome, a
rare cancer syndrome due to germline mutations of the TP53
tumour suppressor gene,
1114
as well as in association with the
FAP and PeutzJeghers syndrome.
15
Despite known molecular genetic causes, contributing to a
genetic predisposition to gastric cancer, a considerable
number of familial cases have been reported that were not
attributable to one of this hereditary syndromes, suggesting
that unknown susceptibility genes for gastric cancer might
exist. Putative tumour suppressor genes that are commonly
inactivated in sporadic gastric cancers, such as the RUNX3 or
HPP1 genes, are potential candidate susceptibility genes.
16 17
In a previous study, we identified one E-cadherin germline
mutation among seven diffuse type familial gastric cancer
patients, indicating that in addition to E-cadherin, other genes
might be involved in genetic predisposition to the disease in
this patient group.
4
There were three goals of the present study. First, we aimed
to extend our analysis of E-cadherin germline mutations to a
higher number of patients, to evaluate the contribution of
germline mutations in this gene to German familial and early
onset gastric cancer patients and to characterise identified
missense mutations for their functional relevance. Secondly,
we wished to analyse the role of TP53 germline mutations for
a familial aggregation of the disease and for early onset
patients. Our third purpose was to investigate two putative
tumour suppressor genes, RUNX3 and HPP1, for their
potential role in genetic predisposition to gastric cancer.
MATERI ALS AND METHODS
Defi ni ti on of fami l i al gastri c cancer
Criteria for the definition of familial gastric cancer are: at
least two first or second degree relatives with gastric cancer,
one diagnosed before the age of 50; or at least three first or
second degree relatives with gastric cancer, independent of
age at diagnosis. Gastric cancer of the index patients was
confirmed by histopathological reports.
The study protocol was reviewed and approved by the local
ethics committee, and DNA samples and family history were
obtained with the informed consent of the patients.
Pati ents for germl i ne mut at i on anal ysi s
DNA samples from 35 familial gastric cancer patients from
Germany were analysed for germline E-cadherin mutations;
seven of these patients were included in a previous study.
4
Of
the index patients, 24 were affected with diffuse type, six
with mixed type, and five with an intestinal type of gastric
cancer according to the classification of Lauren.
18
Four of the
24 families fulfilled the strict criteria for HDGC, defined by
the International Gastric Cancer Linkage Consortium
(IGCLC).
19
In these four families, besides the histological
verification of the index case, the diagnosis was confirmed in
at least another family member (table 1).
Abbreviations: HDGC, hereditary diffuse type gastric cancer; HNPCC,
hereditary non-polyposis colorectal cancer; CHO, Chinese hamster
ovary; DHPLC, denaturing high pressure liquid chromatography
Key poi nts
N
The aim of the study was to investigate the role of E-
cadherin germline mutations in a series of German
familial and early onset gastric cancer patients, and to
evaluate three other genes, TP53, RUNX3, and HPP1,
which are involved in gastric carcinogenesis, for their
potential role in a genetic predisposition to the disease.
N
Two E-cadherin germline mutations, one truncating
and one novel missense mutation, were found among
30 (7%) familial gastric cancer patients with diffuse or
mixed type gastric cancer. Functional analysis of the
missense mutation demonstrated alterations of cell
adhesion and cell invasion, which strongly support its
pathogenic role. One novel truncating mutation was
identified among the 15 (7%) early onset gastric cancer
patients. The mother of this patient was affected with
bilateral breast carcinoma, which underlines the
association of breast carcinoma with germline muta-
tions in the E-cadherin gene.
N
Mutation analysis of the TP53 gene revealed one
germline mutation among 34 (3%) familial gastric
cancer patients, indicating that the TP53 gene should
be included in the genetic screening of familial gastric
cancer patients with a suspected genetic predisposition
to the disease.
N
No mutations were identified in the RUNX3 and HPP1
genes, suggesting that these are not major gastric
cancer predisposition genes.
1 of 6
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from
In addition to the 35 families showing aggregation of
gastric cancer, 15 early onset patients, with gastric cancer
diagnosed before the age of 45 years, were included in this
study. Of these patients, 14 had a diffuse type and one a
mixed type gastric carcinoma (table 1).
Germline mutation analysis of the TP53, RUNX3, and HPP1
genes was performed for 34 German familial gastric cancer
patients, 20 with diffuse type, five with mixed type, and nine
with intestinal type of gastric cancer, according to the
classification of Lauren (table 2).
18
For mutation analysis of
the HPP1 gene, one Portuguese and one Canadian family
with gastric polyposis and diffuse type gastric cancer
20
were
included in this part of the study. The 15 early onset gastric
cancer patients were analysed for germline mutations in the
TP53 gene, but not in the RUNX3 and HPP1 genes. Gastric
cancer patients related to HNPCC due to germline mutations
in the DNA mismatch repair genes hMLH1 or hMSH2 were not
included in this study.
Mutati on anal ysi s
Genomic DNA was isolated from peripheral lymphocytes by
phenol chloroform extraction or by the flexigene DNA
extraction kit (Qiagen, www1.qiagen.com) according to the
manufacturers instructions. DNA from paraffin embedded
tumour tissue was isolated after manual microdissection as
described previously.
4
Mutation analysis was performed by DHPLC (Wave
System, Transgenomic, Omaha, NE, USA) of the PCR
products of all coding exons, including the intron-exon
boundaries of the corresponding genes. Primers for the
amplification of all 16 coding exons of the E-cadherin gene
were according to published primer sequences.
21
For TP53,
exons 211 were analysed using primers, PCR, and DHPLC
conditions as previously described.
22 23
The five coding exons
of the RUNX3 gene were amplified according to published
primer sequences,
16
with the exceptions of exon 1 and exon 5,
for which modified primer sequences were used. Primer
sequences for the 10 coding exons of the HPP1 gene were
based on the DNA sequence of a chromosome 2 genomic
contig containing the HPP1 gene (NT005403, gi29794150,
NCBI data bank). The primer sequences and detailed PCR
conditions are available from the authors on request.
The PCR reactions were performed in 50 ml of reaction
mixture consisting of 10 mM Tris-HCl, pH 8.3; 50 mM KCl;
1.0, or 1.5, or 2.0 mM MgCl
2
; 0.01% gelatine; 200 mM each
dNTP; 0.4 mM of each primer; and 2.5 U Taq Polymerase.
The optimal temperatures for resolution of heteroduplex
and homoduplex DNA for the exons of the E-cadherin,
RUNX3, and HPP1 genes were established by analysing the
melting behaviour of the PCR fragments in the temperature
range corresponding to the calculation of the Wavemaker
TM
software. The analysis temperature for each fragment was the
point at which at least 75% of the DNA was present as an
alpha helix, or 12

C higher. Fragments with more than one

melting domain were analysed at additional temperatures.
DHPLC conditions for each exons are available from the
authors on request.
DNA sequence anal ysi s
PCR products with an aberrant DHPLC chromatogram were
directly sequenced in both directions, starting from a new
PCR product, as previously reported.
4
Samples demonstrating
a putative pathogenic mutation were confirmed by another
independent PCR and sequencing reaction.
I mmunohi stochemi stry
p53 immunohistochemistry of the tumour tissue of the
patient with the TP53 germline mutation was per-
formed using the DO7 monoclonal antibody (Dako, www.
dakocytomation.com) as described previously.
22
E-cadherin
immunohistochemistry of selected cases was performed with
a monoclonal E-cadherin antibody (Clone 36, Transduction
Laboratories, Hamburg, Germany) as described.
5
Appropriate
positive and negative controls were included with each
staining series.
Functi onal assays
As previously described,
24
construction of the plasmids
encoding the E-cadherin mutants corresponding to the
DNA sequence variants G1774A and C2396G were obtained
by nested PCR, using wild type E-cadherin cloned into
pcDNA3 vector as DNA template and primers carrying the
desired mutations (G1774A: For 59-gac aac acc ccc ata cca g -
39; Rev: 59- at ggg ggt gtt gtc att cac -39; For: ctc atg agt gtc ccc
cgg tat ctt cgc -39; Rev: 59- gcg aag ata ccg ggg gac act cat gag -
39). Plasmids were stably transfected into Chinese hamster
ovary (CHO); DG44 dhfr
2
cells and single emerging clones
expressing equal amounts of E-cadherin protein were
selected for the functional characterisation. According to a
previous report,
24
cell-cell adhesion was evaluated in a slow
aggregation assay. Briefly, 2610
+4
cells were seeded on top of
a matrix of soft agar (Bacto-agar, Difko Laboratories),
evaluating aggregates formation at 24 hours and 48 hours
with a phase contrast microscope. For the invasion studies,
single cell suspensions were seeded on top of Matrigel
Table 1 Summary of E-cadherin germline mutation
analysis
Index
patient
Patients
(n)
Mutations
(n) Mutation* Location Type
Familial
Diffuse type 24, ` 1 372delC exon 3 frameshift
Mixed type 6 1 2396C.G,
P799R
exon 15 missense1
Intestinal type 5 0
Total 35 2
Early onset
Diffuse type 14 1 1619insG exon 11 frameshift
Mixed type 1 0
Total 15 1
There were no early onset intestinal type cancers. *Nucleotide position
refers to the E-cadherin sequence NM_004360.2 deposited in the NCBI/
GenBank. Seven patients, including carriers of the germline mutation in
exon 3, were reported in a previous study.
4
`Four patients fulfilled the
strict criteria for hereditary diffuse type gastric cancer.
19
1Not found
among 50 control individuals. n, number.
Table 2 Results on TP53 germline mutation analysis in familial gastric cancer
Index patient Patients(n) Mutations(n) Mutation* Location Type
diffuse type 20 1 847C.T, R283C exon 8 missense
mixed type 5 0
intestinal type 9 0
total 34 1
*Nucleotide position refers to the TP53 sequence NM_000546.2 deposited in the NCBI/GenBank. n, number.
2 of 6 Onl ine mutation report
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from
Invasion Chambers (BD Biosciences, www.bdbiosciences.
com) and the ability of cells to invade was evaluated
according to the manufacturers instructions. In all experi-
ments, at least two independent clones were used in order to
exclude clonal dependence of the observed result.
RESULTS
E-cadheri n mut at i on anal ysi s
In the 35 German familial gastric cancer patients analysed so
far, we found two germline mutations in the E-cadherin gene.
Results in comparison with the histopathological type of the
index patient are summarised in table 1. One mutation was a
novel missense variant represented by a heterozygous C.G
change at nucleotide position 2396, leading to an amino acid
exchange from prolin to arginin at codon 799 (P799R). This
was not found in an analysis of 50 control individuals, but
was found in a 41 year old patient with a mixed
adenocarcinoma of the oesophageal-gastric junction.
Immunohistochemical analysis of E-cadherin expression in
the tumour of this patient revealed a positive, membranous
staining pattern in the tumour cells. DNA sequencing of exon
15 in the tumour revealed that the mutation was present in a
heterozygous state, indicating that there was no LOH. There
were two affected second degree relatives with gastric cancer
in the maternal line (fig 1A); no samples were available from
these family members for mutation testing.
The other mutation found in the familial group was a
truncating mutation in exon 3, described in our previous
study.
4
One novel truncating mutation was detected among the 15
early onset patients (14 with diffuse type and one with mixed
type gastric cancer). The mutation was an insertion G at
nucleotide 1619 in exon 11, leading to a frameshift with a
premature stop at codon 547 (table 1). The patient had
diffuse type gastric cancer at the age of 29. No family history
of gastric cancer was elicited, but the mother had bilateral
breast carcinoma at the age of 49, an abdominal tumour was
reported for the maternal grandmother, and lung cancer for
the maternal grandfather (fig 1B). Immunhistochemical
analysis of E-cadherin expression in this case showed positive
but diffuse cytoplasmatic staining in the tumour, where this
mutation was also detected in an heterozygous state.
However, we found an additional somatic E-cadherin muta-
tion, an A.T transversion at nucleotide 1373 leading to a
change from asparagin to isoleucin at codon 458 (D458I).
An unclassified sequence variant in intron 4 (53218 C.T)
was found in two familial diffuse type gastric cancer patients
and in one of the 50 control individuals. A missense variant
A592T (1774G.A) in exon 12 was identified in one familial
gastric cancer patient and in one control. This missense
variant was found in the patient who also carried a TP53
germline mutation and, in addition, several other known
polymorphisms were found.
To address the pathogenic role of the two E-cadherin
missense variants A592T and P799R, soft-agar aggregation
and matrigel invasion assays were performed on CHO stably
transfected cells, in comparison with cells expressing the wild
type protein. Cells expressing the E-cadherin variant A592T
were not invasive into Matrigel and gave rise to aggregates
when seeded on top of an agar matrix, resembling the
behaviour of cells expressing the wild type E-cadherin. These
results reinforce the hypothesis that A592T is non-patho-
genic. Cells expressing the E-cadherin missense variant
P799R failed to aggregate and invade into Matrigel, suggest-
ing that P799R pathogenic effect. Results are summarised in
table 3.
Figure 1 Pedigree of the families with novel E-cadherin germline mutations. (A) Pedigree of the familial gastric cancer patient with the P377R
mutation. (B) Pedigree of the early onset gastric cancer patient with the insertion G at nucleotide 1619. General symbols: squares, males; circles,
females; numbers, age in years at diagnosis; arrow, index patient.
Table 3 Summary of the aggregation and
invasion assays
Assayed Aggregation Invasion index (%)
Mock CHO
39
2 10.81.2
Wild type
39
+ 1.20.2
A592T + 1.80.4
P799R 2 15.01.5
Figure 2 Pedigree of the familial gastric cancer patient with the TP53
germline mutation R283C. General symbols: squares, males; circles;
females; numbers, age in years at diagnosis; arrow, index patient.
Online mutation report 3 of 6
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from
TP53 mutat i on anal ysi s
In one of 34 (3%) familial gastric cancer patients, a germline
mutation in the TP53 gene was detected (table 2). The
mutation was a heterozygous C.T transition at nucleotide
847 in exon 8, leading to a change from arginine to cysteine
at codon 283 (R283C). This patient had a diffuse type gastric
carcinoma, diagnosed at the age of 52. In the family there
were three affected family members with gastric cancer. In
addition, one sister of the patient died wwith leukemia at the
age of 17 years, and another wth liver carcinoma at the age of
34 years (fig 2). No samples from these family members were
available to test for the TP53 mutation. Mutation analysis of
exon 58 of the tumour DNA showed that the mutation was
present in a heterozygous state, but a somatic missense
mutation in exon 5, a T.A transversion at nucleotide 389
resulting in an amino acid exchange from leucin to histidin at
codon 130 (L130H), was found. Immunohistochemical
analysis demonstrated positive TP53 expression in the
tumour cells, but not in the adjacent non-tumorous epithelial
cells.
No TP53 germline mutations were identified among the 15
early onset gastric cancer patients. A T.A transversion at
nucleotide 1100+30 was identified in intron 10 in one familial
and in one early onset gastric cancer patient, and was also
found in one of the 50 control subjects. Several common
polymorphisms, which have been already described, were
also found.
RUNX3 mutat i on anal ysi s
No germline mutations or polymorphisms were found in the
RUNX3 gene in an analysis of 34 familial gastric cancer
patients.
HPP1 mutati on anal ysi s
No germline mutations were detected in the HPP1 gene
among the 34 familial gastric cancer cases from Germany,
nor in one of the two gastric cancer families with a gastric
polyposis phenotype, from Portugal and Canada. Two
intronic DNA sequence variants, in intron 4 at nucleotide
439+26G.C and at nucleotide 439+47T.A, were detected in
one patient and in two patients, respectively.
DI SCUSSI ON
In this study we describe a comprehensive mutation analysis
to elucidate the role of four different genes, E-cadherin, TP53,
RUNX3, and HPP1, in genetic predisposition to gastric cancer.
With respect to E-cadherin, we identified three germline
mutations among the 35 familial and the 15 early onset
gastric cancer patients. The most interesting result was the
finding of a novel E-cadherin truncating germline mutation in
exon 11, among the 15 (7%) early onset diffuse/mixed type
gastric cancer patients. Interestingly, although no positive
history of gastric carcinoma was reported for the family of
this patient, the mother of the patient was affected with
bilateral breast carcinoma at the age of 49 years. An
association of breast carcinoma, in particular of the lobular
type, with E-cadherin germline mutations was also found in
our previous study,
4
and a significant over-representation of
this tumour type in families with E-cadherin germline
mutations has been shown.
25
In addition, somatic mutations
of the E-cadherin gene are frequently found in sporadic,
diffuse type gastric carcinomas and lobular breast carcino-
mas.
26 27
Although no DNA of the patients mother was
available to prove that she was a mutation carrier and the
histopathological type of the breast carcinoma is unknown, it
is intriguing to speculate that the bilateral breast carcinoma
might have been associated with the E-cadherin germline
mutation in this family, emphasising that breast carcinoma is
part of the tumour spectrum associated with E-cadherin
germline mutations. The finding of a somatic missense
mutation in exon 10 in the tumour of the patient with the
truncating germline mutation, which alters the LDFE-motif
of a Ca-binding domain
21
to LIFE, would predict a complete
loss of normal cell adhesion and is consistent with the diffuse
cytoplasmic protein staining pattern, which was demon-
strated by immunohistochemistry. According to the classical
inactivation theory of tumour suppressor genes, this mis-
sense mutation may represent the second hit that results in
complete inactivation of the E-cadherin gene, although we did
not prove that the germline and the missense mutation were
located on different alleles.
In the familial gastric cancer group, one truncating
mutation and one missense mutation were found among 30
(7%) diffuse/mixed type familial gastric cancer patients, and
none was found in the five familial gastric cancer patients
with an intestinal type of cancer. One of the germline
mutations, previously reported by our group,
4
was detected
in the four families (25%) that met the strict criteria for
HDGC. Overall, a high variability regarding the frequency of
E-cadherin germline mutations has been reported in the
literature. A low incidence (06%) was found in familial
gastric cancer patients from Japan and Finland,
7 28 29
and two
missense mutations were identified among five (40%)
patients with a familial aggregation of the disease from
Korea.
5
In contrast, E-cadherin germline mutations have been
reported in 25% to 36% of diffuse type gastric cancer families
fulfilling the strict criteria of HDGC defined by the IGCLC,
which requires histopathological confirmation not only of the
index patient, but also of the other family members.
19
The
frequency of germline mutations we found in families within
the criteria of HDGC are in line with those reported recently.
8
High detection rates of E-cadherin germline mutations in up
to 100% has been reported for the Maori families in New
Zealand and in studies including a high proportion of
families with multiple cases of very early onset gastric
cancer.
1 6
This suggests that besides histopathological con-
firmation in respect to the inclusion criteria, variations due to
different ethnic origins and due to a specific selection of
analysed families exist, which may explain at least in part the
different findings concerning the frequency of E-cadherin
germline mutations in the different studies.
Among the two mutations identified in the familial group,
a novel missense mutation in exon 15 was found. This
mutation replaces a prolin by an arginin in the cytoplasmic
domain of the protein, and represents a non-conservative
amino acid change. This replaced prolin is conserved between
human, mouse, and chicken E-cadherin,
21
and the alteration
was not found among the 50 control participants. Moreover,
in the functional analysis, this mutant was shown to disrupt
the E-cadherin abilities to mediate cell adhesion and to
suppress cell invasion. All these findings together strongly
support a pathogenic role for this missense mutation.
With respect to the analysis of the TP53 gene, we identified
one germline mutation among the 34 (3%) familial gastric
cancer patients we analysed, namely a missense mutation in
exon 8 leading to a non-conservative amino acid change from
arginin to cystein at codon 283 (R283C). Exon 8 belongs to
the highly conserved region of the TP53 gene, and R283
represents a residue involved in DNA contact.
30
The R283C
mutation has been reported as a somatic mutation in various
cancer types, including gastric carcinoma, and the R283H but
not the R283C has been reported as a germline mutation
(TP53 data bank: http:TP53.curie.fr/). Germline mutations of
the TP53 gene are known as the molecular genetic cause of
the Li-Fraumeni syndrome.
31
The Li-Fraumeni syndrome is
a complex, autosomally inherited cancer predisposition
syndrome which is mainly characterised by the occurrence
of multiple tumours in children or young adults, a
4 of 6 Onl ine mutation report
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from
predominance of soft tissue sarcomas, osteosarcomas, and
breast cancer, and an excess of brain tumours, leukaemia,
and adrenocortical carcinomas. Gastrointestinal tumours
have been shown to be only occasionally associated with
TP53 germline mutations,
32
and only two families with a
predominant occurrence of gastric carcinomas in several
family members have been reported in the literature.
11 14
No
TP53 germline mutations were found in two studies from
Japan analysing seven and 11 familial gastric cancer patients,
respectively.
33 34
In the family of the patient with the TP53
germline mutation in our study, gastric carcinoma was the
predominant tumour type, with three affected family
members. This does not point at the first glance to an
association with the Li-Fraumeni syndrome in terms of its
classical definition. Besides gastric carcinoma in this family,
one family member died due to carcinoma of the liver at the
age of 34 years, and another due to leukaemia at the age of 18
years, which represents a neoplasia originally identified as a
component of the Li-Fraumeni-syndrome.
The patient with the TP53 germline mutation in our study
was also a carrier of a rare missense variant (Ala592Thr) in
the E-cadherin gene. This was found in a similar frequency in
healthy control individuals in a previous study
35
and in our
present study, thus classifying this variant as a polymorph-
ism, not casually related to HDGC. Moreover, in our in vitro
system, cells expressing the A592T E-cadherin variant
behaved as cells expressing the wild type protein, thus
propounding its non-pathogenic nature. Interestingly, H.
pylori infection was visible in the gastric carcinoma of the
patient with the TP53 germline mutation. It has been
suggested that H. pylori infection and TP53 act synergistically
in gastric carcinogenesis
36
and may be related to the
development of gastric cancer in this patient.
No germline mutations were identified in the RUNX3 and
HPP1 genes. The RUNX3 gene has been implicated recently as
a tumour suppressor gene in gastric cancer.
16
Inactivation by
promoter hypermethylation and hemizygous deletion has
been demonstrated in 40% of early and in 90% of advanced
gastric cancers, and a missense mutation in the highly
conserved RUNT domain has been found in one tumour.
Furthermore, RUNX3 knockout mice developed hyperplasia
of the epithelial cells of the stomach glands.
16
In addition,
RUNX3 is an integral component of the TGF-b induced
signalling cascade, and it is well known that the TGF-b
mediated cell signalling plays an important part in gastric
carcinogenesis.
37
Therefore, RUNX3 is an attractive possibility
as a gastric cancer susceptibility gene. However, our finding
of no germline mutations in familial diffuse and intestinal
type gastric cancer families argues against a major role for
RUNX3 in gastric cancer predisposition.
The HPP1 gene encodes for a transmembrane protein,
which may have several roles in cell growth, maturation, and
adhesion. A high frequency of inactivation of the HPP1 gene
by promoter hypermethylation has been shown for hyper-
plastic polyps of the colon, and also for colorectal adenomas
and colorectal and gastric carcinomas.
38
In our study of 34
German familial gastric cancer patients and two families (one
Canadian and one Portuguese) with a gastric polyposis
phenotype, no HPP1 germline mutations were found.
Although we may possibly have missed mutations by the
DHPLC screening or larger genomic deletions, which are not
detectable by the methods used, our results suggest that
HPP1 and RUNX3 are not important alternative gastric cancer
predisposition genes.
In conclusion, we report a frequency of 7% of E-cadherin
germline mutations in a group of familial diffuse and mixed
type gastric cancer patients from Germany. Our finding of a
truncating germline mutation in an early onset diffuse type
gastric cancer patient, whose mother was affected with
bilateral breast carcinomas, underlines the association of E-
cadherin germline mutations with this tumour type. The
identification of a germline mutation in the TP53 gene
suggests the TP53 gene should be included in genetic
screening of familial gastric cancer patients, and supports a
heterogeneous genetic predisposition to gastric cancer. In
order to elucidate such predisposition, we have ruled out the
RUNX3 and HPP1 as major gastric cancer predisposition
genes.
ACKNOWLEDGEMENTS
We thank K F Becker and J Muller for critical reading of the
manuscript.
Authors affiliations
. . . . . . . . . . . . . . . . . . . . .
G Keller, I Becker, S Plaschke, K Biedermann, H Hofler, Institute of
Pathology, Klinikum rechts der Isar, Technische Universitat Munchen,
Germany
H Vogelsang, K Ott, C Doring, A Neutzling, J R Siewert, Department of
Surgery, Klinikum rechts der Isar, Technische Universitat Munchen,
Germany
G Suriano, A R Mateus, R Seruca, Instituto de Patologia e Imunologia
Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal
D Huntsman, Department of Pathology and Laboratory Medicine,
University of British Columbia, Vancouver, Canada
E Holinski-Feder, Medizinische Genetik, Ludwig-Maximilians
Universitat, Munchen, Germany
The study was supported by the German Cancer Foundation, Mildred-
Scheel-Stiftung, grant 70-3124-Ke1.
Conflict of interest: none declared
Correspondence to: Dr G Keller, Institute of Pathology, Klinikum rechts
der Isar, Technische Universitat Munchen, Ismaningerstr. 22, D-81675
Munich, Germany; gisela.keller@lrz.tum.de
REFERENCES
1 Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P,
Taite H, Scoular R, Miller A, Reeve AE. E-cadherin germline mutations in
familial gastric cancer. Nature 1998;392:4025.
2 Gayther SA, Gorring KL, Ramus SJ, Huntsman D, Roviello F, Grehan N,
Machado JC, Pinto E, Seruca R, Halling K, MacLeod P, Powell SM, Jackson CE,
Ponder BAJ, Caldas C. Identification of germline E-cadherin mutations in
gastric cancer families of European origin. Cancer Res 1998;58:40869.
3 Richards FM, McKee SA, Rajpar MH, Cole TRP, Evans DGR, Jankowski JA,
McKeown C, Sander DSA, Maher ER. Germline E-cadherin gene (CDH1)
mutations predispose to familial gastric cancer and colorectal cancer. Hum
Mol Genet 1999;8:60710.
4 Keller G, Vogelsang H, Becker I, Hutter J, Ott K, Candidus S, Grundei T,
Becker KF, Mueller J, Siewert JR, Hofler H. Diffuse type gastric and lobular
breast carcinoma in a familial gastric cancer patient with an E-cadherin
germline mutation. Am J Pathol 1999;155:33742.
5 Yoon KA, Ku JL, Yang HK, Kim WH, Park SY, Park JG. Germline mutations of
the E-cadherin gene in Korean familial gastric cancer patients. J Hum Genet
1999;44:17780.
6 Guilford P, Hopkins JBW, Grady WM, Markowitz SD, Willis J, Lynch H,
Rajput A, Wiesner GL, Lindor NM, Burgart LJ, Toro TT, Lee D, Nimacher JM,
Shaw DW, Findlay MPN, Reeve AE. E-cadherin germline mutations define an
inherited cancer syndrome dominated by diffuse gastric cancer. Hum Mut
1999;14:24955.
7 Yabuta T, Shinmura K, Tani M, Yamaguchi S, Yoshimura K, Katai H,
Nakajima T, Mochiki E, Tsujinaka T, Takami M, Hirose K, Yamaguchi A,
Takenshita S, Yokota J. E-cadherin gene variants in gastric cancer families
whose probands are diagnosed with diffuse gastric cancer. Int J Cancer
2002;101:43441.
8 Oliveira C, Bordin MC, Grehan N, Huntsman D, Suriano G, Machado JC,
Kiviluoto T, Aaltonen l, Jackson CE, Seruca R, Caldas C. Screening E-cadherin
in gastric cancer families reveals germline mutations only in hereditary diffuse
gastric cancer kindred. Hum Mutat 2002;19:51017.
9 Humar B, Toro T, Graziano F, Muller HJ, Dobbie Z, Kwang-Yang H, Eng C,
Hampel H, Gilbert D, Winship I, Parry S, Ward R, Findlay M, Christian A,
Tucker M, Tucker K, Merriman T, Guilford P. Novel germline CDH1 mutations
in hereditary diffuse gastric cancer families. Hum Mutat 2002;19:51825.
10 Lynch HT, Smyrk T. Hereditary nonpolyposis colorectal cancer (Lynch
syndrome). Cancer 1996;78:114967.
11 Toguchida J, Yamaguchi T, Dayton SH, Beauchamp RL, Herrera GE,
Ishizaki K, Yamamuro T, Meyers PA, Little JB, Saski MS, Weichselbaum RR,
Yandell DW. Prevalence and spectrum of germline mutations of the TP53 gene
among patients with sarcoma. N Engl J Med 1992;326:13018.
Online mutation report 5 of 6
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from
12 Sameshima Y, Tsunematsu Y, Watanabe S, Tsukamato T, Kawa-ha K,
Hirata Y, Mizoguchi H, Sugimura T, Terada M, Yokota J. Detection of novel
germline p53 mutations in diverse cancer prone families identified by selecting
patients with childhood adrenocortical carcinoma. Journal of the National
Cancer Institute 1992;84:7037.
13 Varley JM, McGown G, Thorncroft M, Tricker KJ, Teare MD, Santibanez-
Koref MF, Martin J, Birch JM, Evans DG. An extended Li-Fraumeni kindred
with gastric carcinoma and a codon 175 mutation in TP53. J Med Genet
1995;32:94650.
14 Horio Y, Suzuki H, Ueda R, Koshikawa T, Sugiura T, Ariyoshi Y, Shimokata K,
Takahashi T, Takahashi T. . Predominantly tumor-limited expression of a
mutant allele in a Japanese family carrying a germline TP53 mutation.
Oncogene 1994;9:12315.
15 Lindor NM, Greene MH. The concise handbook of family cancer syndromes.
Mayo familial cancer program. Journal of the National Cancer Institute
1998;90:103971.
16 Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ, Lee KY, Nomura S,
Lee CW, Han SB, Kim HM, Kim WJ, Yamamoto H, Yamashita N, Yano T,
Ikeda T, Itohara S, Inazawa J, Abe T, Hagiwara A, Yamagishi H, Ooe A,
Kaneda A, Sugimura T, Ushijima T, Bae SC, Ito Y. Causal relationship
between the loss of RUNX3 expression and gastric cancer. Cell
2002;109:11324.
17 Shibata DM, Sato F, Mori Y, Perry K, Yin J, Wang S, Xu Y, Olaru A, Selaru F,
Spring K, Youing J, Abraham JM, Meltzer SJ. Hypermethylation of HPP1 is
associated with hMLH1 hypermethylation in gastric adenocarcinomas. Cancer
Res 2002;62:563740.
18 Lauren P. The two histological main types of gastric carcinoma: diffuse and so-
called intestinal type of carcinoma. Acta Pathol Microbiol Scand
1965;64:3149.
19 Caldas C, Carneiro F, Lynch HT, Yokota J, Wiesner GL, Powell SM, Lewis FR,
Huntsman G, Pharoah PDP, Jankowski JA, MacLeod P, Vogelsang H, Keller G,
Park KGM, Richards F, Maher ER, Gayther SA, Oliviera C, Grehan N,
Wight D, Seruca S, Roviello R, Ponder BAJ, Jackson CE. Familial gastric
cancer: overview and guidelines for management. J Med Genet
1999;36:87380.
20 Seruca R, Carneiro F, Castedo S, David L, Lopes C, Sobrino-Simoes M.
Familial gastric polyposis revisited: autosomal dominant inheritance
confirmed. Cancer Genet Cytogenet 1991;53:97100.
21 Berx G, Staes K, van Hengel J, Molemans F, Bussemakers MJG, van
Bokhoven A, van Roy F. Cloning and characterization of the human invasion
suppressor gene E-cadherin (CDH1). Genomics 1995;26:2819.
22 Ott K, Vogelsang H, Mueller J, Becker K, Muller M, Fink U, Siewert JR,
Hofler H, Keller G. Chromosomal instability rather than TP53 mutation is
associated with response to neoadjuvant cisplatin-based chemotherapy in
gastric carcinoma. Clin Cancer Res 2003;9:230715.
23 Keller G, Hartmann A, Mueller J, Hofler H. Denaturing high pressure liquid
chromatography (DHPLC) for the analysis of somatic TP53 mutations. Lab
Invest 2001;12:17357.
24 Suriano G, Oliveira C, Ferreira P, Machado JC, Bordin MC, De Wever O,
Bruyneel EA, Moguilevsky N, Grehan N, Porter TR, Richards FM, Hruban RH,
Roviello F, Huntsman D, Mareel M, Carneiro F, Caldas C, Seruca R.
Identification of CDH1 germline missense mutations associated with functional
inactivation of the E-cadherin protein in young gastric cancer probands. Hum
Mol Genet 2003;12:57582.
25 Pharoah PD, Guilford P, Caldas C. The IGCLC. Incidence of gastric cancer
and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary
diffuse gastric cancer families. Gastroenterology 2001;121:134853.
26 Becker KF, Atkinson MJ, Reich U, Becker I, Nekarda H, Siewert JR, Hofler H.
E-cadherin gene mutations provide clues to diffuse type gastric carcinomas.
Cancer Res 1994;54:384552.
27 Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human E-cadherin
(CDH1) gene. Hum Mutat 1998;12:22637.
28 Avizienyte E, Launonen V, Salovaara R, Kivikluoto T, Aaltonen L. E-cadherin
is not frequently mutated in hereditary gastric cancer. J Med Genet
2001;38:4952.
29 Iida S, Akiyama Y, Ichikawa W, Yamahita T, Nomizu T, Nihei Z, Sugihara K,
Yuasa Y. Infrequent germline mutation of the E-cadherin gene in Japanese
familial gastric cancer kindreds. Clin Cancer Res 1999;5:14457.
30 May P, May E. Twenty years of p53 research: structural and functional aspects
of the p53 protein. Oncogene 1999;18:762136.
31 Malkin D, Li FP, Strong LC, Fraumeni JF, Nelson CE, Kim DH, Kassel J,
Gryka A, Bischoff FZ, Tainsky MA, Friend SH. Germline TP53 mutations in a
familial syndrome of breast cancer, sarcomas, and other neoplasms. Science
1990;250:12338.
32 Kleihues P, Schaubele B, zur Hausen A, Esteve J, Ohgaki H. Tumors
associated with TP53 germline mutations: a synopsis of 91 families.
Am J Pathol 1997;150:113.
33 Kusano M, Kakiuchi H, Mihara M, Itoh F, Adachi Y, Ohara M, Hosokawa M,
Imai K. Absence of microsatellite instability and germline mutations of E-
cadherin, APC and p53 genes in Japanese familial gastric cancer. Tumour
Biol 2001;22:2628.
34 Shinmura K, Kohno T, Takahashi M, Sasaki A, Ochiai A, Guilford P, Hunter A,
Reeve AE, Sugimura H, Yamaguchi N, Yokota J. Familial gastric cancer:
clinicopathological characteristics, RER phenotype and germline TP53 and E-
cadherin mutations. Carcinogen 1999;20:112731.
35 Salasher S, Haixin L, Huo H, Kristensen VN, Loman N, Sjoberg-Margolin S,
Borg A, Boressen-Dale AL, Vorechovsky I, Lindblom A. Low frequency of E-
cadherin alterations in familial breast cancer. Breast Cancer Res
2001;3:199207.
36 Fenoglio-Preiser CM, Wang J, Stemmermann GN, Noffsinger A. TP53 and
gastric carcinoma. A review. Hum Mutat 2003;21:25870.
37 Park K, Kim SJ, Bang YJ, Park JG, Kim NK, Roberts AB, Sporn MB. Genetic
changes in the transforming growth factor beta (TGF-beta) type II receptor
gene in human gastric cancer cells: correlation with sensitivity to growth
inhibition by TGF-beta. Proc Natl Acad Sci U S A 1994;91:87726.
38 Young J, Biden KG, Simms LA, Huggard P, Karamatic R, Eyre HJ,
Sutherland GR, Herath N, Barker M, Anderson GJ, Fitzpatrick DR, Ramm GA,
Jass JR, Leggett BA. HPP1: a transmembrane protein-encoding gene
commonly methylated in colorectal polyps and cancer. Proc Natl Acad
Sci U S A 2001;98:26570.
39 Suriano G, Mulholland D, De Wever O, Ferreira P, Mateus AR, Bruyneel E,
Mareel MM, Yokota J, Huntsman D, Seruca R. The intracellular E-cadherin
germline mutation V832M lacks the ability to mediate cell-cell adhesion and to
suppress invasion. Oncogene 2003;22:571619.
6 of 6 Onl ine mutation report
www.jmedgenet.com
on 14 June 2005 jmg.bmjjournals.com Downloaded from