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5 575582
DOI: 10.1093/hmg/ddg048
Received November 19, 2002; Revised and Accepted December 20, 2002
E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct
evidence of E-cadherin mutations triggering tumorigenesis has come from the nding of inactivating germline
mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young
gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an
intronic variant were identied. We then analysed the functional signicance of the two exonic missense variants
found here as well as a third germline missense variant that we previously identied in a HGDC family. cDNAs
encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese
hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation,
motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas
are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of
missense variants are associated with signicant functional consequences, suggesting that our cell model can
be used as an adjunct in deciding on the potential pathogenic role of identied E-cadherin germline alterations.
*To whom correspondence should be addressed. Tel: 0044 1223 3319 89; Fax: 0044 1223 3317 53; Email: cc234@cam.ac.uk and Tel: 351
225570764; Fax: 351 225570799; Email: rseruca@ipatimup.pt
{
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Human Molecular Genetics, Vol. 12, No. 5 # Oxford University Press 2003; all rights reserved
576 Human Molecular Genetics, 2003, Vol. 12, No. 5
Table 1. Details of patients, tumours and CDH1 putative deleterious sequence alterations in five cases of early-onset gastric cancer
Case Geographic origin Age/gender Histologic tumour type Ex/In Nucleotide changea Protein change Comments
293 USA 43/F Diffuse Ex 12 1849 G!A Ala617Thr (mis) Previously describedb
294 USA 43/F Diffuse Ex 12 1849 G!A Ala617Thr (mis) Previously describedb
J68 Portugal 30/M Diffuse Ex 12 1901 C!T Ala634Val (mis) Previously describedc
54 England 51/M Diffuse Int 4 532-18 C!T New
CE137 Portugal 42/F Mixed Int 4 532-18 C!T New
a
Nucleotide positions refer to an E-cadherin sequence deposited in the EMBL/GenBank Data Libraries, accession no. Z13009; the A of the ATG of the initiator
Met codon is denoted nt1.
b
Somatic mutation in endometrial and gastric cancer (16,17).
c
Mutation described in a colon cancer cell line (11).
Germline truncating mutations of the CDH1 gene (EMBL/ isolated cell type cancers predominate in young patients.
GenBank Data Libraries no. CDH1-Z13009) resulting in Furthermore, the diffuse type of gastric carcinoma in young
E-cadherin inactivation have been identified in hereditary patients demonstrates a nearly equal sex ratio, compared with a
diffuse gastric carcinoma (OMIM no. Gastric cancer-137215) male preponderance in the intestinal form.
(5,6). To date, 30 HDGC families harbouring CDH1 germline The mutation screening identified five cases (7.6%) with poten-
Figure 1. Germline missense mutations in the CDH1 gene. (A) CDH1 exon Figure 2. Western blot analysis of E-cadherin transfected cell lines using the
12 PCR/SSCP analysis, showing a band pattern in normal DNA (N) and specific antibody HECD1. Equal amounts of protein were loaded in each lane.
band shifts in DNA from patients with early-onset diffuse gastric cancer
(1, J68; and 2, 293). (B) Sequence chromatograms. (1) Missense mutation
in codon 634 (1901 C to T) in patient J68; (2) Missense mutation in
codon 617 (1849 G to A) in patient 293 (patient 294 had an identical Table 2. Median values of particle diameters in fast
mutation). aggregation assay
missense variant, present in two African-American individuals, with gastric cancer has now been reported, further supporting a
was a transition in codon 617 (1849 G to A) leading to an pathogenic association (17). The remaining germline sequence
amino acid substitution (Ala to Thr). It occurs in the fifth alteration was a transition in intron 4, at position 532-18 (C to
extracellular repeat (EC5) of E-cadherin, affecting a conserved T transition), present in two individuals. The results obtained in
sequence encoding one of the calcium binding motifs (12). As one of these individuals, CE 137, were consistent with
known, cadherins are functional only in the presence of disruption of gene expression as a result of inactivation of
calcium ions, which stabilize the protein active conformation. one allele by the germline alteration (first hit) and of the second
Adhesion is promoted in a process of cis-dimerization between (wild-type) allele by somatic methylation of the CDH1
neighbour E-cadherin molecules, for which a key role played promoter in tumour cells (second hit), although this could not
by repeats 1 and 2 (EC1 and EC2) was demonstrated (1315). be formally proven. One can only speculate why the germline
Although less information is available for the other repeats, a sequence variant would not be expressed, although its location
comparable activity could be predicted on the basis of their near the two splicing branch sites where the splicing lariat loop
sequence homology, suggesting a putative pathogenic effect could potentially form (A nucleotides at positions 13 and
for the mutation we found. Besides, this variant was also 27 upstream of the 30 splice acceptor site) could somehow
previously reported as a somatic mutation in an endometrial disrupt transcript stability.
cancer in association with loss of heterozygosity (16), fulfilling The pathogenic significance of the two distinct missense
the classic two-hit model of tumour suppressor gene inactiva- variants (Ala634Val and Ala617Thr) identified in these
tion. E-cadherin immunohistochemistry in these two cases apparently sporadic diffuse gastric cancer cases was assessed
revealed a pattern similar to what is commonly seen in sporadic by analysing the functional consequences in a cell model
tumours with somatic missense mutations. The Ala617Thr system. We also tested a missense variant previously identified
variant has an allele frequency of 1% in Africans, suggesting by our group in a HDGC family (Thr340Ala) (8). Interestingly
it may be an uncommon polymorphism occurring in popula- this variant was shown by computer modelling to be located in
tions of African origin. Nevertheless, it would be a very a newly characterized E-cadherin sequence motif, likely to be
improbable occurrence (less than 1:10 000 chance) that both involved in the stabilization of the active protein conformation.
cases of diffuse gastric carcinoma in young African-Americans Two of the E-cadherin missense variants (Thr340Ala and
would also be carriers of the same rare polymorphism, unless Ala634Val) studied resulted in dramatic functional conse-
such polymorphism is associated with predisposition to quences: cell adhesion was disrupted, and the cells acquired a
stomach cancer. We note with interest that a third case of the highly invasive potential associated with enhanced cell motility
identical germline sequence variant in an African-American and loss of epithelial structure. One of these, Ala634Val, is the
Human Molecular Genetics, 2003, Vol. 12, No. 5 579
Patients
Constitutional DNA was isolated from 66 gastric carcinoma
patients with no familial history of stomach cancer diagnosed
before the age of 51 (65 patients were 50 or younger). Cases
originated from the UK (n 26), Portugal (n 22), Italy
(n 11), USA (n 6) and Greece (n 1). Genomic DNA was
isolated, using standard methods, either from peripheral blood
Figure 4. Collagen invasion assay. Bars represent the percentage of invasion for or from fresh frozen normal gastric mucosa. The gastric
each of the experimental conditions. carcinomas were classified according to Laure n and Carneiro
(25,26).
PCRSSCP
result of a nucleotide substitution that was shown in a colon All 16 exons including intronexon boundaries and the
carcinoma cell line to result in activation of a cryptic splicing promoter region of the CDH1 gene were amplified by PCR.
site leading to a truncated protein (11). Our results showed that
San Francisco, CA, USA). A modification of the avidin for 1 min, annealing at 58 C for 1 min and extension at 72 C for
biotinperoxidase complex method was used. 1 min and 30 s per cycle. Sequencing was performed as
aforementioned. The cDNA was inserted into the mammalian
Methylation assay expression vector pcDNA3 (Invitrogen) as a HindIII
XhoI cassette, leading to the plasmid pECAD1. Mutant
CDH1 promoter methylation analysis was performed using plasmids were obtained by nested PCR, by using specific
methylation-specific PCR (MSPCR) and primers described by primers carrying the desired mutations (Thr340Ala: Ecad1012F,
Graff et al. (28) for CpG island 3 of the CDH1 promoter. This 50 -TTC CCT GCG TAT ACC CTG G-30 and Ecad1031R,
assay entails initial modification of DNA by sodium bisulphite, 50 -ACC AGG GTA TAC GCA GGG AA-30 ; Ala634Val:
converting all unmethylated, but not methylated, cytosines to Ecad1893F, 50 -ACA CGG GGT GAG TGC CAA C-30 and
uracil, and subsequent amplification with primers specific for Ecad1911R, 50 -GTT GGC ACT CAC CCC GTG T-30 ; Ala
methylated versus unmethylated DNA. 617Thr: Ecad1841F, 50 -TCA TTG ATA CAG ACC TTC CTC-30
and Ecad1859R, 50 -GGA AGG TCT GTA TCA ATG ATG-30 )
Statistical analysis and pECAD1 as DNA template.
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