Microsatellite instability and frameshift mutations in genes involved in cell cycle progression or apoptosis in ovarian cancer

The loss of mismatch repair enzymes increases the mutation rate in microsatellites and coding regions of the genome and appears to be involved in drug resistance. The replication error (RER+) phenotype, associated with microsatellite instability, has been widely described for both familial and sporadic colon cancers and for gastric and endometrial tumors. For ovarian cancer, the incidence of RER+ cases among sporadic tumors is still uncertain. We analyzed epithelial ovarian tumors and ovarian carcinoma cell lines for microsatellite instability and for mutations in the coding regions of different genes, including the recently discovered human CHK-1 gene, which has an important role in controlling cell cycle progression and whose coding region contains a poly(A)9 tract. Microsatellite instability and frameshift mutations in coding regions of BAX, TGFbetaRII, IGFIIR, E2F-4, ICE, and CHK-1 genes were analyzed in ovarian cancer samples and cell lines by polymerase chain reaction (PCR). Approximately 26% of patients showed microsatellite instability in two or more loci. BAT-26 locus showed no alteration in primary tumors. We detected a BAX mutation in one tumor sample and a TGFbetaRII mutation in one cell line. Our findings confirm the presence of the RER+ phenotype in sporadic ovarian cancer. The low rate of mutation in genes previously reported to be altered in colon and gastric cancer suggests that other not yet identified genes might be altered and could play a role in tumor progression and response to treatment in RER+ ovarian tumors.     

Mutation analysis of transforming growth factor beta type II receptor, Smad2, Smad3 and Smad4 in esophageal squamous cell carcinoma

Mutations of the transforming growth factor beta type II receptor (TGFbetaRII) gene and Smad family genes have been observed in several human cancers. However, there has been no report on mutation analysis of the entire coding regions of these genes in esophageal cancer, and the role of these genes in the development of esophageal cancer remains unknown. We performed polymerase chain reaction-single strand conformation polymorphism analysis of TGFbetaRII, Smad2, Smad3 and Smad4 genes and microsatellite assay in 20 esophageal squamous cell carcinomas (ESCC). We detected polymorphisms at exon 2 of Smad3 and intron 6 of Smad4. No mutation was found in TGFbetaRII, Smad2, Smad3 and Smad4 genes. These results suggest that mutation of TGFbetaRII, Smad2, Smad3 and Smad4 genes is a rare event in ESCC.     

Infrequent mutations of the transforming growth factor beta-type II receptor gene at chromosome 3p22 in human lung cancers with chromosome 3p deletions

Mutations in the transforming growth factor beta-type II receptor (TGFbeta RII) gene have been detected in several types of human cancers that represent the phenotype of genomic instability. The TGFbeta RII gene has been mapped to chromosome 3p, on which loss of heterozygosity (LOH) was frequently detected in both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). To investigate whether the TGFbeta RII gene on 3p22 is inactivated in lung cancers, we examined 35 sporadic lung cancers (15 SCLC and 20 NSCLC) with LOH on 3p for mutations of the TGFbeta RII gene. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbeta RII gene. Using these primers, we screened for mutations of the TGFbeta RII gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. A mutation was detected in a case of SCLC: one base insertion in the polyadenine tract of exon 3. This tumor showed the replication error (RER) phenotype. There were no mutations in exons 1, 2, 4, 5, 6 and 7. These results indicate that the polyadenine tract is a mutational hot spot in the TGFbeta RII gene in RER positive tumors, and that TGFbeta RII mutations occur rarely in lung cancers with LOH on chromosome 3p.      

Birt-Hogg-Dubé (BHD) gene mutations in human gastric cancer with high frequency microsatellite instability

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by benign hamartomatous skin lesions and an increased risk of pneumothorax and renal tumors. Many of the patients harbor insertion/deletion mutations in the hypermutable poly(C)(8) tract in exon 11 of the BHD gene. This mutational hot spot is also reported to be a target of mutation in microsatellite instability (MSI) sporadic colorectal tumors. To test if the BHD gene is a potential mutational target in gastric cancer, we screened for mutations in all of the coding exons of the BHD gene in 30 cases of MSI gastric cancer as well as 50 cases of microsatellite stable (MSS) gastric cancer. Mutations in the poly(C)(8) tract of BHD were detected in 3 of 19 MSI-high cases (15.8%), and none of 11 MSI-low cases. All BHD mutated cases also showed mutations of both BAX and TGFbetaRII. No mutations were detected in the other exons of the BHD gene. No BHD mutations were found in MSS gastric cancer cases. Taken together, these findings show that the BHD gene is a rare target in MSI-high gastric cancer, and BHD mutation tends to occur downstream in the mutational events of other major MSI-high target genes.     

Microsatellite alterations and K-ras, TGFbetaRII, IGFRII and bax mutations in sporadic cancers of the gastrointestinal tract

DNA was analyzed from 57 sporadic gastrointestinal tumors (34 pancreatic cancers, 23 colon tumors) and cognate normal tissues to verify whether mutations at coding sequences were associated with microsatellite instability (MSI). Genomic instability was present in 41% (14/34) of pancreatic samples and in 26% (6/23) of colon cancers previously tested by six microsatellite markers. The tumors included 37 cases showing no MSI; 15 cases with MSI at only 1 locus and 5 cases with MSI at 2 or more loci. All the samples were screened for mutations in genes containing repeated tracts in their coding sequences (TGFbetaRII, IGFRII and bax) and in codon 12 of the K-ras oncogene. Furthermore, loss of heterozygosity (LOH) at NM23.H1 locus was tested, 17/34 (50%) pancreatic tumors and 6/23 (26%) colon cancers showed mutations in codon 12 of K-ras; allelic loss of NM23. H1 locus was found in 6/18 (33%) informative colon tumors and in no pancreatic cancers. The TGFbetaRII, IGFRII and bax genes were altered in 3 (13%), 1 (4%) and 3 (13%) out of 23 colon tumors respectively, but no mutation was detected in pancreatic cancers. Mutations in the repeated nucleotide stretches within the coding sequences of TGFbetaRII, IGFRII and bax genes were found only in colon tumors with a high unstable phenotype (more than 3 microsatellite loci altered).     

Genetic and epigenetic analysis of the VHL gene in gastric cancers

The von Hippel-Lindau tumor suppressor gene (VHL), which is located on chromosome 3p25, plays an important role in tumorigenesis, particularly in tumor growth and vascularization. Mutations of the VHL gene have been observed in the hereditary VHL syndrome and a variety of other sporadic cancers. In this study, in order to investigate whether the VHL gene is involved in gastric carcinogenesis, we have examined the genetic alterations, including somatic mutations and allelic loss, with the two microsatellite markers, D3S1038 and D3S1110, as well as promoter hypermethylation of the VHL gene in 88 sporadic gastric adenocarcinomas. No mutation was detected in the coding region of the VHL gene. Allelic loss was found in 20 (33.9%) of 59 informative cancer cases at one or both markers. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and epigenetic alterations of the VHL gene in gastric cancers. Our results suggest that genetic and epigenetic alterations of the VHL gene may be not involved in the development or progression of gastric cancers. The findings also provide evidence for the presence of another gastric cancer specific tumor suppressor gene at the 3p25 region.     

RER phenotype and its associated mutations in familial gastric cancer

To clarify the genetic background of gastric cancer, we collected 28 familial gastric cancers (FGCs) with reference to the Amsterdam criteria in hereditary non-polyposis colorectal cancer (HNPCC) and investigated the frequency of replication error (RER) at six microsatellite loci and frameshift mutations in its related genes in these tumors. RER was detected in seven (25%) of the 28 gastric cancers. Five (18%) cases showed RER at more than two loci. The apparent increased incidence of RER in FGC was not detected compared with that reported in sporadic gastric cancers previously. Among four cases with RER at more than three loci, frameshift mutations in the (A)8 track of the hMSH3 gene were detected in all the four cases and mutations in the (A)10 track of the transforming growth factor-beta type II receptor (TGF-beta RII) gene were detected in the three of them. Histologically, three of the four cases were of the intestinal type, and the other one was the diffuse type. No mutation was detected in the (C)8 and (GT)3 tracks of the hMSH6 and TGF-beta RII genes respectively. These results indicate that the acquisition of the RER phenotype equally influences the gastric carcinogenesis of both sporadic and familial cases, and that the majority of FGC is pathogenetically distinct from HNPCC.      

Mutations in the ST7/RAY1/HELG locus rarely occur in primary colorectal,gastric, and hepatocellular carcinomas

Human cancers frequently show a loss of heterozygosity on chromosome 7q31, which indicates the existence of broad-range tumour-suppressor gene(s) at this locus. Truncating mutations in the ST7 gene at this locus are seen frequently in primary colon cancer and breast cancer cell lines. Therefore, the ST7 gene represents a novel candidate gene for the tumour suppressor at this locus. However, more recent studies have reported that ST7 mutations are infrequent or absent in primary cancer and cell lines. To ascertain the frequency of mutations of the ST7 gene in cancer cells, we examined mutations in the ST7 coding sequence in 48 colorectal, 48 gastric, and 48 hepatocellular carcinomas using polymerase chain reaction-single-strand conformational polymorphism and direct sequencing. We detected somatic mutations, which were located near the exon-intron junction in intron 8, in only three out of 144 cases. We conclude that mutations in the ST7 gene are rare in primary colorectal, gastric, and hepatocellular carcinomas.     

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers

Background

Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways.

Methods

We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene.

Results

Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected.

Conclusion

We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.     

抑癌基因PTEN在胃癌及癌前病变中的缺失和突变

目的:探讨胃癌及癌前病变中抑癌基因PTEN的缺失和突变.方法:收集中国医科大学2001～2002年胃镜中心活检标本,萎缩性胃炎、肠上皮化生、不典型增生及对应的正常胃粘膜各30例,收集同期肿瘤外科术后大体标本,早期胃癌及进展期胃癌各30例,饱和氯化钠法提取组织DNA,PCR-SSCP变性聚丙烯酰氨凝胶电泳银染法检测PTEN杂合性缺失(LOH),PCR-SSCP测序法检测PTEN基因突变.结果:在胃癌癌前期病变萎缩性胃炎、肠上皮化生、不典型增生中,PTEN的杂合性缺失率分别为10%、10%、13.3%,在早期胃癌中,PTEN的杂合性缺失率为20%,进展期胃癌中PTEN的杂合性缺失率为33.3%,而PTEN基因突变在癌前期病变及早期胃癌中无1例出现,在进展期胃癌中出现比率为10%,而且所有发生PTEN基因突变的病例均为LOH阳性病例.结论:PTEN基因缺失或失活与胃癌的浸润和转移的关系更密切.     

p73 mutations are not detected in sporadic and hereditary breast cancer

Recently, a novel tumor suppressor gene, p73, was isolated and mapped to chromosome 1p36, a region commonly associated with loss of heterozygosity in neuroblastoma and other human malignancies, including breast cancer. The p73 gene shares considerable homology with the common tumor suppressor gene p53, both in composition and function. This study examines the potential participation of p73 in the pathogenesis of sporadic and hereditary breast cancers. Mutation analysis of 29 hereditary breast cancer cases revealed five independent silent mutations in the hereditary cases that are unlikely to play a role in tumor development. Mutation analysis of 48 sporadic breast tumors did not identify any unique variants. Eleven common polymorphisms scattered throughout the gene were also detected. Thus, mutations in the p73 gene appear to play little if any role in hereditary or sporadic breast cancer.     

Lack of frameshift mutations at coding mononucleotide repeats in hepatocellular carcinoma in Japanese patients

BACKGROUND: Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage-related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor-beta receptor type II (TGFbetaRII), mannose 6-phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl-2-associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan. METHODS: Fifty HCC samples were analyzed in this study. The authors used polymerase chain reactions to screen for frameshift mutation at the TGFbetaRII (A)(10) tract, the M6P/IGFIIR (G)(8) tract, the hMSH3 (A)(8) tract, the hMSH6 (C)(8) tract, and the BAX (G)(8) tract. For MI analysis, matched tumor and nontumor liver DNA were investigated with respect to 10 microsatellite loci. RESULTS: No frameshift mutation was detected in any case, and only 4% of these cancers exhibited MI in comparisons between tumor and nontumor liver specimens. CONCLUSIONS: This study suggests that frameshift mutation at coding mononucleotide repeats within TGFbetaRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes did not seem to be involved in hepatocarcinogenesis in the Japanese population studied.     

Microsatellite instability and gene mutations in transforming growth factor-beta type II receptor are absent in small bowel carcinoid tumors

BACKGROUND: Microsatellite instability (MSI) with concomitant mutations in the coding region of transforming growth factor-beta type II receptor (TGFbetaRII) results in an aberrant growth-regulatory phenotype in colorectal carcinomas. The authors postulated that a similar mechanism occurred during the malignant evolution of small bowel carcinoid tumors. METHODS: Mutational analysis of two coding regions in the TGFbetaRII gene associated with MSI and BAT-26 within intron 5 of the mismatch repair gene, hMSH2, was undertaken in small bowel carcinoids (n = 14), lymph node metasasis (n = 1) and liver metastases (n = 5). Quantitative PCR analysis [TAQMAN, Applied Biosystems, Foster City, CA] was then undertaken to examine gene alterations in mismatch repair genes (hMLH1 and hMSH2) in small bowel carcinoids (n = 7) and matched normal mucosa (n = 5). Staining was then analyzed using quantitative tissue array profiling (AQUA analysis) in a small bowel EC carcinoid tissue microarray (n = 55 tumors) with immunostaining against TGFbetaRII and MSH2. RESULTS: Mutational examination of the TFGbetaRII gene and BAT-26 demonstrated that MSI was not present in any carcinoid material. Q RT-PCR analysis demonstrated statistically significant increased message levels of hMSH2 but not hMLH1 in carcinoid tumors. Quantitative analysis of membrane TGFbetaRII immunostaining using AQUA demonstrated that TGFbetaRII expression was down-regulated (P < 0.0002) in thirty-three primary small bowel carcinoids that exhibited lymph node and liver metastases compared to normal mucosa. AQUA analysis of nuclear MSH2 immunostaining demonstrated no differences for MSH2 between normal tissue and carcinoid tumor metastasis. Small bowel carcinoids characterized by variable expression of TGFbetaRII, did not exhibit MSI and had no differences in MSH2 expression. CONCLUSIONS: The molecular events leading to the formation of carcinoid tumors in the small bowel were different from those resulting in epithelial carcinomas. The usually slow-growing and relatively nonaggressive carcinoid tumors had variable expression of TGFbetaRII but were associated with the retention of mismatch repair protein function and a microsatellite-stable phenotype.     

Von Hippel-Lindau tumour suppressor gene is not involved in sporadic human breast cancer.

OBJECTIVE: Mutations of the von Hippel-Lindau (vhl) gene, as well as allelic loss at the gene region (3p25-26) have been described in sporadic cases of the tumour types participating in VHL disease, but also in cancers not associated with the syndrome. In this study, we attempted mutation analysis of the vhl gene, as well as detection of allelic loss at 3p25-26 in sporadic human breast cancer. METHODS: Eighty-two tumour specimens were screened for loss of heterozygosity (LOH) at the vhl region, and compared to the adjacent, histologically normal tissue. Furthermore, mutations within the three exons of vhl in the same panel of tumours were detected using SSCP and heteroduplex analysis and direct sequencing. RESULTS: To our knowledge this is the first mutational analysis reported for the vhl gene in breast cancer, however we failed to reveal any mutations in the specimens examined. All the cases were informative for at least one of the microsatellite markers tested, 24 (29.2%) exhibited LOH at 3p25-26. Clinical and pathological data were available for all tumours examined, however no significant correlations were encountered. CONCLUSION: These results strongly indicate against a critical involvement of the tumour suppressor vhl in breast carcinogenesis.     

LOSS OF HETEROZYGOSITY INVOLVING THE APC TUMOR SUPPRESSOR GENE IN HUMAN COLORECTAL CARCINOMA

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Somatic Mutations of the PTEN/MMAC1 Gene in Fifteen Japanese Endometrial Cancers: Evidence for Inactivation of Both Alleles

Loss of heterozygosity (LOH) of chromosome 10q is observed in approximately 40% of endometrial cancers. Mutations in PTEN/MMAC1 , a gene recently isolated from the 10q23 region, are responsible for two dominantly inherited neoplastic syndromes, Cowden disease and Bannayan-Zonana syndrome. Somatic mutations of this gene have also been detected in sporadic cancers of the brain, prostate and breast. To investigate the potential role of this putative tumor suppressor gene in endometrial carcinogenesis as well, we examined 46 primary endometrial cancers for LOH at the 10q23 region, and for mutations in the entire coding region and exon-intron boundaries of the PTEN/MMAC1 gene. LOH was identified in half of the 38 informative cases, and subtle somatic mutations were detected in 15 tumors (33%). Our results suggest that of the genes studied so far in endometrial carcinomas, PTEN/MMAC1 is the most commonly mutated one, and that inactivation of both copies by allelic loss and/or mutation, a pattern that defines genes as "tumor suppressors,'contributes to tumorigenesis in endometrial cancers.     

Genetic alterations of the KLF6 gene in gastric cancer

The KLF6 is a zinc-finger tumor suppressor that is frequently mutated in several human cancers and broadly involved in differentiation and development, growth-related signal transduction, cell proliferation, apoptosis, and angiogenesis. To determine whether genetic alterations of KLF6 gene are involved in the development and/or progression of gastric cancer, we have screened a set of 80 sporadic gastric cancers for mutations and allele loss of the KLF6 gene. Four missense mutations, S155R, P172 T, S180L, and R198 K, were detected in transactivation domain of the KLF6 gene and one of them had biallelic mutations with somatic mutation of one allele and loss of the remaining allele. All of the cases with mutation were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 16 (43.2%) of 37 informative cases showed allelic loss at KLF6 locus. Interestingly, allelic loss was also frequent in intestinal-type gastric cancer. Therefore, our data suggest that genetic alterations of KLF6 gene might play an important role in the development or progression of sporadic gastric cancers.     

Role of MYH Polymorphisms in Sporadic Colorectal Cancer in China: A Case-control,Population-based Study

Purpose: Biallelic germline variants of the 8-hydroxyguanine (8-OG) repair gene MYH have been associatedwith colorectal neoplasms that display somatic G:CgT:A transversions. However, the effect of single germlinevariants has not been widely studied, prompting the present investigation of monoallelic MYH variants andsusceptibility to sporadic colorectal cancer (CRC) in a Chinese population. Patients and Methods: BetweenJanuary 2006 and December 2012, 400 cases of sporadic CRC and 600 age- and sex-matched normal blood donorswere screened randomly for 7 potentially pathogenic germline MYH exons using genetic testing technology.Variants of heterozygosity at the MYH locus were assessed in both sporadic cancer patients and healthy controls.Univariate and multivariate analyses were performed to determine risk factors for cancer onset. Results: Fivemonoallelic single nucleotide polymorphisms (SNPs) were identified in the 7 exon regions of MYH, which weredetected in 75 (18.75%) of 400 CRC patients as well as 42 (7%) of 600 normal controls. The region of exon 1proved to be a linked polymorphic region for the first time, a triple linked variant including exon 1-316 GgA,exon 1-292 GgA and intron 1+11 CgT, being identified in 13 CRC patients and 2 normal blood donors. Avariant of base replacement, intron 10-2 AgG, was identified in the exon 10 region in 21 cases and 7 controls,while a similar type of variant in the exon 13 region, intron 13+12 CgT, was identified in 8 cases and 6 controls.Not the only but a newly missense variant in the present study, p. V463E (Exon 14+74 TgA), was identified inexon 14 in 6 patients and 1 normal control. In exon 16, nt. 1678-80 del GTT with loss of heterozygosity (LOH)was identified in 27 CRC cases and 26 controls. There was no Y165C in exon 7 or G382D in exon 14, the hotspotvariants which have been reported most frequently in Caucasian studies. After univariate analysis andmultivariate analysis, the linked variant in exon 1 region (p=0.002), intron 10-2 AgG (p=0.004) and p. V463E(p=0.036) in the MYH gene were selected as 3 independent risk factors for CRC. Conclusions: According to theseresults, the linked variant in Exon 1 region, Intron 10-2 AgG of base replacement and p. V463E of missensevariant, the 3 heterozygosity variants of MYH gene in a Chinese population, may relate to the susceptibility tosporadic CRC. Lack of the hot-spot variants of Caucasians in the present study may due to the ethnic differencein MYH gene.     

Germline mutations in E-cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer

Somatic mutations in the E-cadherin (CDH1) gene have frequently been reported in cases with diffuse gastric and lobular breast cancers. Recently, germline mutations have been identified in families with diffuse gastric cancers. In families with hereditary prostate cancer (HPC), a significant association of prostate cancer, gastric and/or breast cancer has been observed in epidemiological studies. The aim of this study was to investigate if germline mutations in CDH1 could explain the risk for cancer in HPC families with an excess of gastric and breast cancer. In total, 17 members from 13 HPC families and 3 members from 3 families with hereditary gastric cancer (HGC) were screened for germline CDH1 sequence alterations using PCR/Denaturing HPLC for initial screening of nucleotide variants followed by confirmatory direct sequencing analysis. The frequency of identified novel germline mutations were tested for in 136 cases with hereditary prostate cancer and 215 cases of sporadic prostate cancer with 422 age matched controls in an allelic discrimination assay. In total, 8 sequence variants were detected in 20 samples tested. In the HPC families, we found 2 missense mutations, A592T in exon 12 and a novel D777N in exon 15 and a mutation in intron 5, 687+92T>A. A previously known polymorphism in exon 13 and 3 sequence variations in introns and untranslated regions were also found, of which the significance is unknown. In HGC-023 with early onset diffuse gastric cancer a truncating mutation, R335X, was identified in exon 7. None of the missense mutations or 687+92T>A were found in the extended HPC material or in the sporadic prostate cancer cases with age-matched controls in the allelic discrimination assay. We found several germline mutations of unknown clinical significance in the CDH1 gene that probably do not explain the association of prostate, gastric and/or breast cancers in the HPC-families. Two missense mutations and a mutation in intron 5 were identified that do not influence the risk of hereditary or sporadic prostate cancer in general and are considered to be pedigree specific. In a family with hereditary gastric cancer of the diffuse type, we identified the first truncating germline mutation in a Scandinavian family.