METHODS

Twenty-one Korean families affected with familial gastric cancer were investigated for CDH1 and MET germline mutations. Criteria for family inclusion were at least two first or second degree relatives affected with gastric cancer, at least one of whom was diagnosed with cancer before the age of 50.⁵ Blood samples from probands of each of these families were collected from Seoul National University Hospital, and four additional family members of pedigree SNU-G7 were sampled following identification of a mutation in this family. Informed consent was obtained from all participants before testing. Out of 21 probands, nine represented families suffering from diffuse types of gastric cancer, four represented families suffering from intestinal types, and histological data for the type of the remaining eight were not available. The classification of HDGC (hereditary diffuse gastric cancer) or HIGC (hereditary intestinal gastric cancer) suggested by Caldas et al¹⁵ was not possible in these families owing to the lack of histological information.¹⁵

Peripheral blood lymphocytes were isolated from each family member using Ficoll-Paque according to the manufacturer’s instructions (Pharmacia Biotech, Uppsala, Sweden). Total genomic DNA was extracted using the TRI reagent (Molecular Research Center, Cincinnati, OH, USA) or the automatic magnetic bead based system (KingFisher, ThermoLabsystems, Finland) according to the manufacturers’ instructions. CDH1 mutational screenings were performed with reported primer sets⁵ using DHPLC (denaturing high performance liquid chromatography; WAVE, Transgenomic, Omaha, USA) as previously described.^16,17 Coding sequences of exon 2 and exons 4–21 of the MET gene were screened by DHPLC using PCR primer pairs published by Duh et al.⁸ Primer sequences were not available for exon 3, which was excluded from screening.

Four general positive controls (WAVE DNA sizing control, WAVE low range mutation control standard, WAVE mid range mutation control standard, and WAVE high range mutation control standard; Transgenomic, Omaha, USA) for DHPLC were used to check the reliability of the experiments. The melting temperatures of each exon were optimised by analysing melting curves using WAVEMAKER software (Transgenomic, Omaha, USA). All samples showing abnormal DHPLC patterns were subsequently cloned using the TA cloning system (Invitrogen, Carlsbad, CA, USA) and bi-directionally sequenced using the Taq dideoxy terminator cycle sequencing kit on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

RESULTS AND DISCUSSION

No CDH1 germline mutations were found in 21 probands from Korean pedigrees afflicted with familial gastric cancers. These 21 probands without CDH1 germline mutations were then screened for MET germline mutations by DHPLC analysis. One subject (SNU-G7) out of 21 harboured a MET missense mutation (C to T) at exon 10 (codon 791), which produced a non-conservative amino acid substitution of proline to leucine (P791L, fig 1A). Residue 791 is conserved in mice, rats, and humans, suggesting that mutation of this residue could be pathologically relevant. To examine whether this sequence change is a naturally occurring polymorphism, we screened 181 unrelated, healthy, Koreans by DHPLC. None harboured the variation. Segregation analysis was not possible in this pedigree because there was only one living affected family member (SNU-G7, fig 1B). This person’s grandfather and father both died from gastric cancer without operations.

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Figure 1

MET germline mutation in exon 10 of patient SNU-G7. (A) Automatic sequencing showing the nucleotide substitution that results in an amino acid change (P791L). (B) Pedigree of SNU-G7 familial gastric cancer family. Solid symbols, affected with gastric cancer; arrows, mutation carriers.

Subject SNU-G7 initially presented with indigestion and intermittent epigastric pain of seven months’ duration. Gastrofibroscopic examination showed a gastric cancer lesion on the high body of the stomach. Tumour metastasis to the perigastric lymph node was not detected. SNU-G7 is the oldest in her generation and shows no symptoms of papillary renal carcinoma. Out of her four younger sibs, two inherited the MET mutation and two did not. The MET mutation carriers are 43 and 38 years old at the time of this report, raising the possibility that these patients will develop gastric cancer in the years to come. Therefore, these people should be carefully monitored so that the disease may be diagnosed in its early stages.

In addition to the single seemingly pathological mutation, we also identified four polymorphisms in exons 2, 7, 20, and 21 in both familial gastric cancer patients and normal controls. Out of the four, two (D1304D and A1357A) had previously been reported⁷ and two (N375S and Q648Q) were novel. The D1304D polymorphism was found in 12 of 21 (57%) gastric cancer patents and in 23 of 80 (29%) cancer free controls. A1357A was found in three of 21 (14%) gastric cancer patients and 19 of 80 (24%) normal controls. N375S and Q648Q were found in 5% (1/21) and 14% (3/21) of gastric cancer patients, respectively, and 9% (7/80) and 21% (17/80) of normal controls, respectively.

Gastric cancer is the most common cancer in Korea, yet studies have suggested that truncating mutations in CDH1 do not significantly contribute to the development of diffuse gastric cancers in Asians. Recently, MET has been proposed as a candidate gene for gastric cancer.¹¹ Germline mutations in this gene, found in the tyrosine kinase domain, have been associated with HPRC. These tyrosine kinase domain mutations may constitutively activate the protein by altering its substrate specificity or catalytic activity.^7,13 In contrast, an oncogenic MET mutation (P1009S) reported in gastric cancer patients was localised to the juxtamembrane domain.¹¹ In this study, we identified a novel germline MET mutation in a Korean gastric cancer patient in the extracellular domain (exon 10), an area which has not been extensively studied to date.^7,11 This is one of two MET mutations that have been identified in gastric cancer patients, both of which (P1009S and P791L) were identified in Korean gastric cancer patients and localise to upstream sites (the juxtamembrane and extracellular domains, respectively). The observation that MET mutations associated with HPRC or sporadic papillary renal carcinomas localise to the tyrosine kinase domain suggests that there may be different pathological mechanisms responsible for MET induced gastric cancer and papillary renal carcinomas. MET mutations in gastric cancers may affect dimerisation/oligomerisation of MET molecules on the cell surface while missense mutations in MET in HPRC alter the conformation leading to constitutive activation of the MET protein in the absence of ligand. Germline MET mutations in papillary renal carcinomas and gastric cancers are summarised in fig 2.

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Figure 2

A spectrum of MET germline mutations in papillary renal carcinomas and gastric cancers. Numbered boxes indicate the exons of MET. (1) MET germline mutations reported by Schmidt et al⁷; (2) reported by Schmidt et al¹⁸; (3) reported by Schmidt et al¹⁰; (4) reported by Olivero et al¹⁹; (5) reported by Lee et al.¹¹*Identified in this work.

Because many previous studies have not screened MET extracellular domain sequences,^11,14 it is possible that other patients harbour MET mutations in this domain. Alternatively, it was suggested that MET mutations might be specific to people of Asian or Korean ancestry.¹⁴ In support of this, no MET germline mutations were found in 18 white or Indian gastric cancer kindreds.¹⁴ However, although all MET germline mutations found in gastric cancer patients to date have been associated with Koreans, further investigation will be required to determine the ethnic specificity of these mutations.

In addition to ethnic specificity, it is important to examine the type specificity of various mutations. CDH1 germline mutations were found in diffuse gastric cancer patients, while the previously identified MET mutation (P1009S) was found in an intestinal gastric cancer patient. This led researchers to suggest a correlation between MET mutations and intestinal gastric cancer.¹⁴ However, patient SNU-G7 suffered from the diffuse type of gastric cancer, suggesting that further research is needed to address the association between MET mutations and the gastric cancer types.

Family history was not available for the patient described by Lee et al¹¹ (harbouring the P1009S MET mutation),¹⁴ making this the first report of a MET germline mutation in a patient with familial gastric cancer. However, as the frequency of both MET and CDH1 mutations is quite low in Korean gastric cancer patients, it seems that neither gene plays a major role in familial gastric cancer in this population. Further studies on the mechanism of MET and identification of other genes involved in gastric cancer development will be required.