Germline Mutations of the E-cadherin(CDH1) and TP53

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 patientscontribute to genetic predisposition in German

,HPP1 and RUNX3 genes, rather than of TP53 and E-cadherin(CDH1)Germline mutations of the

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ONLINE MUTATION REPORT

Germline mutations of the E-cadherin(CDH1) and TP53genes, rather than of RUNX3 and HPP1, contribute togenetic predisposition in German gastric cancer patientsG 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

Germline mutations of the cell adhesion molecule E-cadherin have been shown for the first time tounderlie a hereditary diffuse type gastric cancer

syndrome (HDGC) in Maori families,1 and subsequently havebeen reported in HDGC patients of various ethnic origins.29

Gastric cancer may also be associated with other hereditarytumour syndromes, which are mainly characterised bycarcinomas of other organs. One of these syndromes is theHNPCC syndrome (hereditary nonpolyposis colorectal cancersyndrome), which is caused by germline mutations in DNAmismatch repair genes.10 Furthermore, gastric cancer hasbeen observed in the context of the Li Fraumeni syndrome, arare cancer syndrome due to germline mutations of the TP53tumour suppressor gene,1114 as well as in association with theFAP and PeutzJeghers syndrome.15

Despite known molecular genetic causes, contributing to agenetic predisposition to gastric cancer, a considerablenumber of familial cases have been reported that were notattributable to one of this hereditary syndromes, suggestingthat unknown susceptibility genes for gastric cancer mightexist. Putative tumour suppressor genes that are commonlyinactivated in sporadic gastric cancers, such as the RUNX3 orHPP1 genes, are potential candidate susceptibility genes.16 17

In a previous study, we identified one E-cadherin germlinemutation among seven diffuse type familial gastric cancerpatients, indicating that in addition to E-cadherin, other genesmight be involved in genetic predisposition to the disease inthis patient group.4

There were three goals of the present study. First, we aimedto extend our analysis of E-cadherin germline mutations to ahigher number of patients, to evaluate the contribution ofgermline mutations in this gene to German familial and earlyonset gastric cancer patients and to characterise identifiedmissense mutations for their functional relevance. Secondly,we wished to analyse the role of TP53 germline mutations fora familial aggregation of the disease and for early onsetpatients. Our third purpose was to investigate two putativetumour suppressor genes, RUNX3 and HPP1, for theirpotential role in genetic predisposition to gastric cancer.

MATERIALS AND METHODSDefinition of familial gastric cancerCriteria for the definition of familial gastric cancer are: atleast two first or second degree relatives with gastric cancer,one diagnosed before the age of 50; or at least three first orsecond degree relatives with gastric cancer, independent ofage at diagnosis. Gastric cancer of the index patients wasconfirmed by histopathological reports.The study protocol was reviewed and approved by the local

ethics committee, and DNA samples and family history wereobtained with the informed consent of the patients.

Patients for germline mutation analysisDNA samples from 35 familial gastric cancer patients fromGermany were analysed for germline E-cadherin mutations;seven of these patients were included in a previous study.4 Ofthe index patients, 24 were affected with diffuse type, sixwith mixed type, and five with an intestinal type of gastriccancer according to the classification of Lauren.18 Four of the24 families fulfilled the strict criteria for HDGC, defined bythe International Gastric Cancer Linkage Consortium(IGCLC).19 In these four families, besides the histologicalverification of the index case, the diagnosis was confirmed inat least another family member (table 1).

Abbreviations: HDGC, hereditary diffuse type gastric cancer; HNPCC,hereditary non-polyposis colorectal cancer; CHO, Chinese hamsterovary; DHPLC, denaturing high pressure liquid chromatography

Key points

N The aim of the study was to investigate the role of E-cadherin germline mutations in a series of Germanfamilial and early onset gastric cancer patients, and toevaluate three other genes, TP53, RUNX3, and HPP1,which are involved in gastric carcinogenesis, for theirpotential role in a genetic predisposition to the disease.

N Two E-cadherin germline mutations, one truncatingand one novel missense mutation, were found among30 (7%) familial gastric cancer patients with diffuse ormixed type gastric cancer. Functional analysis of themissense mutation demonstrated alterations of celladhesion and cell invasion, which strongly support itspathogenic role. One novel truncating mutation wasidentified among the 15 (7%) early onset gastric cancerpatients. The mother of this patient was affected withbilateral breast carcinoma, which underlines theassociation of breast carcinoma with germline muta-tions in the E-cadherin gene.

N Mutation analysis of the TP53 gene revealed onegermline mutation among 34 (3%) familial gastriccancer patients, indicating that the TP53 gene shouldbe included in the genetic screening of familial gastriccancer patients with a suspected genetic predispositionto the disease.

N No mutations were identified in the RUNX3 and HPP1genes, suggesting that these are not major gastriccancer predisposition genes.

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In addition to the 35 families showing aggregation ofgastric cancer, 15 early onset patients, with gastric cancerdiagnosed before the age of 45 years, were included in thisstudy. Of these patients, 14 had a diffuse type and one amixed type gastric carcinoma (table 1).Germline mutation analysis of the TP53, RUNX3, and HPP1

genes was performed for 34 German familial gastric cancerpatients, 20 with diffuse type, five with mixed type, and ninewith intestinal type of gastric cancer, according to theclassification of Lauren (table 2).18 For mutation analysis ofthe HPP1 gene, one Portuguese and one Canadian familywith gastric polyposis and diffuse type gastric cancer20 wereincluded in this part of the study. The 15 early onset gastriccancer patients were analysed for germline mutations in theTP53 gene, but not in the RUNX3 and HPP1 genes. Gastriccancer patients related to HNPCC due to germline mutationsin the DNA mismatch repair genes hMLH1 or hMSH2 were notincluded in this study.

Mutation analysisGenomic DNA was isolated from peripheral lymphocytes byphenol chloroform extraction or by the flexigene DNAextraction kit (Qiagen, www1.qiagen.com) according to themanufacturers instructions. DNA from paraffin embeddedtumour tissue was isolated after manual microdissection asdescribed previously.4

Mutation analysis was performed by DHPLC (WaveSystem, Transgenomic, Omaha, NE, USA) of the PCRproducts of all coding exons, including the intron-exonboundaries of the corresponding genes. Primers for theamplification of all 16 coding exons of the E-cadherin genewere according to published primer sequences.21 For TP53,exons 211 were analysed using primers, PCR, and DHPLCconditions as previously described.22 23 The five coding exonsof the RUNX3 gene were amplified according to publishedprimer 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 werebased on the DNA sequence of a chromosome 2 genomiccontig containing the HPP1 gene (NT005403, gi29794150,NCBI data bank). The primer sequences and detailed PCRconditions 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 MgCl2; 0.01% gelatine; 200 mM eachdNTP; 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 themelting behaviour of the PCR fragments in the temperaturerange corresponding to the calculation of the WavemakerTM

software. The analysis temperature for each fragment was thepoint at which at least 75% of the DNA was present as analpha helix, or 12 C higher. Fragments with more than onemelting domain were analysed at additional temperatures.DHPLC conditions for each exons are available from theauthors on request.

DNA sequence analysisPCR products with an aberrant DHPLC chromatogram weredirectly sequenced in both directions, starting from a newPCR product, as previously reported.4 Samples demonstratinga putative pathogenic mutation were confirmed by anotherindependent PCR and sequencing reaction.

Immunohistochemistryp53 immunohistochemistry of the tumour tissue of thepatient with the TP53 germline mutation was per-formed using the DO7 monoclonal antibody (Dako, www.dakocytomation.com) as described previously.22 E-cadherinimmunohistochemistry of selected cases was performed witha monoclonal E-cadherin antibody (Clone 36, TransductionLaboratories, Hamburg, Germany) as described.5 Appropriatepositive and negative controls were included with eachstaining series.

Functional assaysAs previously described,24 construction of the plasmidsencoding the E-cadherin mutants corresponding to theDNA sequence variants G1774A and C2396G were obtainedby nested PCR, using wild type E-cadherin cloned intopcDNA3 vector as DNA template and primers carrying thedesired 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 ccccgg tat ctt cgc -39; Rev: 59- gcg aag ata ccg ggg gac act cat gag -39). Plasmids were stably transfected into Chinese hamsterovary (CHO); DG44 dhfr2 cells and single emerging clonesexpressing equal amounts of E-cadherin protein wereselected for the functional characterisation. According to aprevious report,24 cell-cell adhesion was evaluated in a slowaggregation assay. Briefly, 2610+4 cells were seeded on top ofa matrix of soft agar (Bacto-agar, Difko Laboratories),evaluating aggregates formation at 24 hours and 48 hourswith a phase contrast microscope. For the invasion studies,single cell suspensions were seeded on top of Matrigel

Table 1 Summary of E-cadherin germline mutationanalysis

Indexpatient

Patients(n)

Mutations(n) Mutation* Location Type

FamilialDiffuse type 24, ` 1 372delC exon 3 frameshiftMixed type 6 1 2396C.G,

P799Rexon 15 missense1

Intestinal type 5 0 Total 35 2Early onsetDiffuse type 14 1 1619insG exon 11 frameshiftMixed type 1 0 Total 15 1

There were no early onset intestinal type cancers. *Nucleotide positionrefers to the E-cadherin sequence NM_004360.2 deposited in the NCBI/GenBank. Seven patients, including carriers of the germline mutation inexon 3, were reported in a previous study.4 `Four patients fulfilled thestrict criteria for hereditary diffuse type gastric cancer.19 1Not foundamong 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 missensemixed 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.

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Invasion Chambers (BD Biosciences, www.bdbiosciences.com) and the ability of cells to invade was evaluatedaccording to the manufacturers instructions. In all experi-ments, at least two independent clones were used in order toexclude clonal dependence of the observed result.

RESULTSE-cadherin mutation analysisIn the 35 German familial gastric cancer patients analysed sofar, we found two germline mutations in the E-cadherin gene.Results in comparison with the histopathological type of theindex patient are summarised in table 1. One mutation was anovel missense variant represented by a heterozygous C.Gchange at nucleotide position 2396, leading to an amino acidexchange from prolin to arginin at codon 799 (P799R). Thiswas not found in an analysis of 50 control individuals, butwas found in a 41 year old patient with a mixedadenocarcinoma of the oesophageal-gastric junction.Immunohistochemical analysis of E-cadherin expression inthe tumour of this patient revealed a positive, membranousstaining pattern in the tumour cells. DNA sequencing of exon15 in the tumour revealed that the mutation was present in aheterozygous state, indicating that there was no LOH. Therewere two affected second degree relatives with gastric cancerin the maternal line (fig 1A); no samples were available fromthese family members for mutation testing.The other mutation found in the familial group was a

truncating mutation in exon 3, described in our previousstudy.4

One novel truncating mutation was detected among the 15early onset patients (14 with diffuse type and one with mixedtype gastric cancer). The mutation was an insertion G atnucleotide 1619 in exon 11, leading to a frameshift with apremature stop at codon 547 (table 1). The patient haddiffuse type gastric cancer at the age of 29. No family historyof gastric cancer was elicited, but the mother had bilateral

breast carcinoma at the age of 49, an abdominal tumour wasreported for the maternal grandmother, and lung cancer forthe maternal grandfather (fig 1B). Immunhistochemicalanalysis of E-cadherin expression in this case showed positivebut diffuse cytoplasmatic staining in the tumour, where thismutation 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 achange 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 patientsand in one of the 50 control individuals. A missense variantA592T (1774G.A) in exon 12 was identified in one familialgastric cancer patient and in one control. This missensevariant was found in the patient who also carried a TP53germline mutation and, in addition, several other knownpolymorphisms were found.To address the pathogenic role of the two E-cadherin

missense variants A592T and P799R, soft-agar aggregationand matrigel invasion assays were performed on CHO stablytransfected cells, in comparison with cells expressing the wildtype protein. Cells expressing the E-cadherin variant A592Twere not invasive into Matrigel and gave rise to aggregateswhen seeded on top of an agar matrix, resembling thebehaviour of cells expressing the wild type E-cadherin. Theseresults reinforce the hypothesis that A592T is non-patho-genic. Cells expressing the E-cadherin missense variantP799R failed to aggregate and invade into Matrigel, suggest-ing that P799R pathogenic effect. Results are summarised intable 3.

Figure 1 Pedigree of the families with novel E-cadherin germline mutations. (A) Pedigree of the familial gastric cancer patient with the P377Rmutation. (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 andinvasion assays

Assayed Aggregation Invasion index (%)

Mock CHO39 2 10.81.2Wild type39 + 1.20.2A592T + 1.80.4P799R 2 15.01.5

Figure 2 Pedigree of the familial gastric cancer patient with the TP53germline mutation R283C. General symbols: squares, males; circles;females; numbers, age in years at diagnosis; arrow, index patient.

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TP53 mutation analysisIn one of 34 (3%) familial gastric cancer patients, a germlinemutation in the TP53 gene was detected (table 2). Themutation was a heterozygous C.T transition at nucleotide847 in exon 8, leading to a change from arginine to cysteineat codon 283 (R283C). This patient had a diffuse type gastriccarcinoma, diagnosed at the age of 52. In the family therewere three affected family members with gastric cancer. Inaddition, one sister of the patient died wwith leukemia at theage of 17 years, and another wth liver carcinoma at the age of34 years (fig 2). No samples from these family members wereavailable to test for the TP53 mutation. Mutation analysis ofexon 58 of the tumour DNA showed that the mutation waspresent in a heterozygous state, but a somatic missensemutation in exon 5, a T.A transversion at nucleotide 389resulting in an amino acid exchange from leucin to histidin atcodon 130 (L130H), was found. Immunohistochemicalanalysis demonstrated positive TP53 expression in thetumour cells, but not in the adjacent non-tumorous epithelialcells.No TP53 germline mutations were identified among the 15

early onset gastric cancer patients. A T.A transversion atnucleotide 1100+30 was identified in intron 10 in one familialand in one early onset gastric cancer patient, and was alsofound in one of the 50 control subjects. Several commonpolymorphisms, which have been already described, werealso found.

RUNX3 mutation analysisNo germline mutations or polymorphisms were found in theRUNX3 gene in an analysis of 34 familial gastric cancerpatients.

HPP1 mutation analysisNo germline mutations were detected in the HPP1 geneamong the 34 familial gastric cancer cases from Germany,nor in one of the two gastric cancer families with a gastricpolyposis phenotype, from Portugal and Canada. Twointronic DNA sequence variants, in intron 4 at nucleotide439+26G.C and at nucleotide 439+47T.A, were detected inone patient and in two patients, respectively.

DISCUSSIONIn this study we describe a comprehensive mutation analysisto 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 germlinemutations among the 35 familial and the 15 early onsetgastric cancer patients. The most interesting result was thefinding of a novel E-cadherin truncating germline mutation inexon 11, among the 15 (7%) early onset diffuse/mixed typegastric cancer patients. Interestingly, although no positivehistory of gastric carcinoma was reported for the family ofthis patient, the mother of the patient was affected withbilateral breast carcinoma at the age of 49 years. Anassociation of breast carcinoma, in particular of the lobulartype, with E-cadherin germline mutations was also found inour previous study,4 and a significant over-representation ofthis tumour type in families with E-cadherin germlinemutations has been shown.25 In addition, somatic mutationsof 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 wasavailable to prove that she was a mutation carrier and thehistopathological type of the breast carcinoma is unknown, itis intriguing to speculate that the bilateral breast carcinomamight have been associated with the E-cadherin germlinemutation in this family, emphasising that breast carcinoma ispart of the tumour spectrum associated with E-cadherin

germline mutations. The finding of a somatic missensemutation in exon 10 in the tumour of the patient with thetruncating germline mutation, which alters the LDFE-motifof a Ca-binding domain21 to LIFE, would predict a completeloss of normal cell adhesion and is consistent with the diffusecytoplasmic protein staining pattern, which was demon-strated by immunohistochemistry. According to the classicalinactivation theory of tumour suppressor genes, this mis-sense mutation may represent the second hit that results incomplete inactivation of the E-cadherin gene, although we didnot prove that the germline and the missense mutation werelocated 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, andnone was found in the five familial gastric cancer patientswith an intestinal type of cancer. One of the germlinemutations, previously reported by our group,4 was detectedin the four families (25%) that met the strict criteria forHDGC. Overall, a high variability regarding the frequency ofE-cadherin germline mutations has been reported in theliterature. A low incidence (06%) was found in familialgastric cancer patients from Japan and Finland,7 28 29 and twomissense mutations were identified among five (40%)patients with a familial aggregation of the disease fromKorea.5 In contrast, E-cadherin germline mutations have beenreported in 25% to 36% of diffuse type gastric cancer familiesfulfilling the strict criteria of HDGC defined by the IGCLC,which requires histopathological confirmation not only of theindex patient, but also of the other family members.19 Thefrequency of germline mutations we found in families withinthe criteria of HDGC are in line with those reported recently.8

High detection rates of E-cadherin germline mutations in upto 100% has been reported for the Maori families in NewZealand and in studies including a high proportion offamilies with multiple cases of very early onset gastriccancer.1 6 This suggests that besides histopathological con-firmation in respect to the inclusion criteria, variations due todifferent ethnic origins and due to a specific selection ofanalysed families exist, which may explain at least in part thedifferent findings concerning the frequency of E-cadheringermline mutations in the different studies.Among the two mutations identified in the familial group,

a novel missense mutation in exon 15 was found. Thismutation replaces a prolin by an arginin in the cytoplasmicdomain of the protein, and represents a non-conservativeamino acid change. This replaced prolin is conserved betweenhuman, mouse, and chicken E-cadherin,21 and the alterationwas not found among the 50 control participants. Moreover,in the functional analysis, this mutant was shown to disruptthe E-cadherin abilities to mediate cell adhesion and tosuppress cell invasion. All these findings together stronglysupport 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 gastriccancer patients we analysed, namely a missense mutation inexon 8 leading to a non-conservative amino acid change fromarginin to cystein at codon 283 (R283C). Exon 8 belongs tothe highly conserved region of the TP53 gene, and R283represents a residue involved in DNA contact.30 The R283Cmutation has been reported as a somatic mutation in variouscancer types, including gastric carcinoma, and the R283H butnot the R283C has been reported as a germline mutation(TP53 data bank: http:TP53.curie.fr/). Germline mutations ofthe TP53 gene are known as the molecular genetic cause ofthe Li-Fraumeni syndrome.31 The Li-Fraumeni syndrome isa complex, autosomally inherited cancer predispositionsyndrome which is mainly characterised by the occurrenceof multiple tumours in children or young adults, a


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predominance of soft tissue sarcomas, osteosarcomas, andbreast cancer, and an excess of brain tumours, leukaemia,and adrenocortical carcinomas. Gastrointestinal tumourshave been shown to be only occasionally associated withTP53 germline mutations,32 and only two families with apredominant occurrence of gastric carcinomas in severalfamily members have been reported in the literature.11 14 NoTP53 germline mutations were found in two studies fromJapan analysing seven and 11 familial gastric cancer patients,respectively.33 34 In the family of the patient with the TP53germline mutation in our study, gastric carcinoma was thepredominant tumour type, with three affected familymembers. This does not point at the first glance to anassociation with the Li-Fraumeni syndrome in terms of itsclassical definition. Besides gastric carcinoma in this family,one family member died due to carcinoma of the liver at theage of 34 years, and another due to leukaemia at the age of 18years, which represents a neoplasia originally identified as acomponent 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) inthe E-cadherin gene. This was found in a similar frequency inhealthy control individuals in a previous study35 and in ourpresent study, thus classifying this variant as a polymorph-ism, not casually related to HDGC. Moreover, in our in vitrosystem, cells expressing the A592T E-cadherin variantbehaved as cells expressing the wild type protein, thuspropounding its non-pathogenic nature. Interestingly, H.pylori infection was visible in the gastric carcinoma of thepatient with the TP53 germline mutation. It has beensuggested that H. pylori infection and TP53 act synergisticallyin gastric carcinogenesis36 and may be related to thedevelopment of gastric cancer in this patient.No germline mutations were identified in the RUNX3 and

HPP1 genes. The RUNX3 gene has been implicated recently asa tumour suppressor gene in gastric cancer.16 Inactivation bypromoter hypermethylation and hemizygous deletion hasbeen demonstrated in 40% of early and in 90% of advancedgastric cancers, and a missense mutation in the highlyconserved RUNT domain has been found in one tumour.Furthermore, RUNX3 knockout mice developed hyperplasiaof the epithelial cells of the stomach glands.16 In addition,RUNX3 is an integral component of the TGF-b inducedsignalling cascade, and it is well known that the TGF-bmediated cell signalling plays an important part in gastriccarcinogenesis.37 Therefore, RUNX3 is an attractive possibilityas a gastric cancer susceptibility gene. However, our findingof no germline mutations in familial diffuse and intestinaltype gastric cancer families argues against a major role forRUNX3 in gastric cancer predisposition.The HPP1 gene encodes for a transmembrane protein,

which may have several roles in cell growth, maturation, andadhesion. A high frequency of inactivation of the HPP1 geneby promoter hypermethylation has been shown for hyper-plastic polyps of the colon, and also for colorectal adenomasand colorectal and gastric carcinomas.38 In our study of 34German familial gastric cancer patients and two families (oneCanadian and one Portuguese) with a gastric polyposisphenotype, no HPP1 germline mutations were found.Although we may possibly have missed mutations by theDHPLC screening or larger genomic deletions, which are notdetectable by the methods used, our results suggest thatHPP1 and RUNX3 are not important alternative gastric cancerpredisposition genes.In conclusion, we report a frequency of 7% of E-cadherin

germline mutations in a group of familial diffuse and mixedtype gastric cancer patients from Germany. Our finding of atruncating germline mutation in an early onset diffuse typegastric cancer patient, whose mother was affected with

bilateral breast carcinomas, underlines the association of E-cadherin germline mutations with this tumour type. Theidentification of a germline mutation in the TP53 genesuggests the TP53 gene should be included in geneticscreening of familial gastric cancer patients, and supports aheterogeneous genetic predisposition to gastric cancer. Inorder to elucidate such predisposition, we have ruled out theRUNX3 and HPP1 as major gastric cancer predispositiongenes.

ACKNOWLEDGEMENTSWe thank K F Becker and J Muller for critical reading of themanuscript.

Authors affiliations. . . . . . . . . . . . . . . . . . . . .

G Keller, I Becker, S Plaschke, K Biedermann, H Hofler, Institute ofPathology, Klinikum rechts der Isar, Technische Universitat Munchen,GermanyH Vogelsang, K Ott, C Doring, A Neutzling, J R Siewert, Department ofSurgery, Klinikum rechts der Isar, Technische Universitat Munchen,GermanyG Suriano, A R Mateus, R Seruca, Instituto de Patologia e ImunologiaMolecular da Universidade do Porto (IPATIMUP), Porto, PortugalD Huntsman, Department of Pathology and Laboratory Medicine,University of British Columbia, Vancouver, CanadaE Holinski-Feder, Medizinische Genetik, Ludwig-MaximiliansUniversitat, 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 rechtsder Isar, Technische Universitat Munchen, Ismaningerstr. 22, D-81675Munich, Germany; [email protected]

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Germline Mutations of the E-cadherin(CDH1) and TP53