Japanese Journal of Clinical Oncology 33:17-20 (2003)
© 2003 Foundation for Promotion of Cancer Research
Ile–Leu Substitution (I415L) in Germline E-cadherin Gene (CDH1) in Japanese Familial Gastric Cancer
1 First Department of Pathology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, 2 Department of Surgery, Toho University School of Medicine, Tokyo and 3 Biology Division, National Cancer Center Research Institute, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Background. Germline mutation in the cell adhesion molecule E-cadherin is known to be associated with the development of undifferentiated or diffuse-type familial gastric cancers, but the prevalence of this contribution seems to be low in Japanese familial cases, so far.
Methods. We screened all exons of the E-cadherin gene for mutations in 101 Japanese patients having an intense family history of gastric cancers.
Results. An abnormal band pattern was found in exon 9 in three patients (Y6, B21, B37) from two families by PCR–SSCP. DNA sequencing analysis of these three patients revealed isoleucine–leucine substitution at codon 415 in exon 9. B21 and B37 are siblings and the other three brothers died of gastric cancer and another brother (B36) has not been affected by gastric cancer to date. This case (B36) did not have this alteration in the exon 9 of E-cadherin.
Conclusion. Although the mechanistic basis is not clear, our findings may provide a possibility that this additional missense mutation in germline E-cadherin gene may contribute to gastric cancer predisposition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Gastric cancer is a major cause of cancer death in Japan. Epidemiological studies have shown that some cases represent a strong familial history of gastric cancer (1), but the pathogenesis of these familial gastric cancers has not been clearly defined.
E-cadherin, a calcium-dependent cell to cell adhesion molecule, which is often inactivated in various human ‘non-cohesive’ cancers, has been reported to change in three germline DNA in familial gastric cancers in New Zealand Maori (2). Additional cases were reported later in three of 10 and two of seven diffuse-type gastric cancer families of European origin (3,4), suggesting that E-cadherin mutation is responsible, at least in some part, for familial gastric cancer ubiquitously. Further surveys are still ongoing (5) and additional base substitutions have also been reported (6).
Although considerable documentation is available about familial gastric cancer in Japan, there have been few studies about the E-cadherin changes in germlines of Japanese familial cancers (7,8). In this paper, we report a new base substitution in germline CDH1 which is linked to gastric cancer in one family and has never been found in sporadic gastric cancer or healthy subjects.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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We screened all the exons of the E-cadherin gene for mutations in 101 Japanese patients having an intense family history according to a previous report (8).
Recently, a single nucleotide polymorphism in the promoter region of E-cadherin gene has been identified as influencing E-cadherin transcriptional activity (9), and we also detected this site in our series.
The criteria for familial gastric cancer are the following: (a) three and more gastric cancer patients in a family; (b) at least two successive generations should be affected; and (c) in one of the relatives, gastric cancer should be diagnosed before the age of 50 years. Familial gastric cancer cases were recruited from Yamagata Central Hospital, Jichi University Hospital, Toho University Hospital, Seirei Hamamatsu Hospital, Hamamatsu Medical Center and Hamamatsu University Hospital. Material was collected from 78 Japanese gastric cancer families including 87 gastric cancer patients and 14 healthy (unaffected) individuals who have intense familial gastric cancer history. Genomic DNA from 53 operated paraffin-embedded normal gastric mucosa obtained according to standard procedures, which have been described previously (10), and 48 peripheral blood cells were isolated for genomic DNA using Qiagen kits (Qiagen, Hilden, Germany). The project including these analyses was approved by the Ethical Committee on Genetic Analysis of Hamamatsu University School of Medicine.
Polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) was performed under four sets of conditions for detecting migration abnormality. The primer sets covering E-cadherin have been described previously (8,11). We screened from exon 1 to exon 16, by dividing the whole coding region into 32 portions. Screening with the primer of the promoter region (F 5'-TCCCAGGTCTTAGTGAGCCA-3' and R 5'-GGCCACAGCCAATCAGCA-3') was also performed as described previously (9). PCR products were diluted 1:1 with 95% formamide, 4.9% EDTA, 0.05% xylene cyanol and 0.05% bromophenol blue, denatured at 95°C for 5 min and chilled on ice. We used 8% polyacrylamide gel with or without 5% glycerine running at room temperature or 4°C. Silver staining was also performed as described (12).
The aberrant PCR products as revealed by SSCP were purified using a Qiagen PCR purification kit and directly sequenced using an ABI Ready Reaction Dye Terminator Cycle Sequencing Kit and ABI 373 Prism Sequencer and confirmed by cloning as described (10).
The prevalence of the variants was studied in the following background populations: random control DNAs were from 50 Japanese and 50 Chinese sporadic gastrointestinal tract cancers, 50 non-cancer Caucasians and 50 Pacific Islanders.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In screening all 16 exons by PCR–SSCP in the 87 patients from 78 gastric cancer kindreds, we identified an extra band at exon 9 in three cases (Y6, B21, B37) of two families by SSCP (Fig. 1a and c; Fig. 2a and b). The heterozygous state was clear based on the SSCP pattern. DNA sequencing analysis of E-cadherin of these three patients revealed isoleucine (ATA)–leucine (CTA) substitution at codon 415 in exon 9 (Fig. 1b). We also examined tumor tissues from the three patients; the same abnormal pattern was maintained and in tumor–normal comparisons, the wild-type band was not significantly decreased even after estimation of the quantity of tumor cells in the tumor portion (Fig. 1c). Histopathological examination showed that the three patients had diffuse-type gastric cancers. B21 and B37 are siblings and the other three brothers died of gastric cancer and another younger brother (B36) has not been affected by gastric cancer to date (Fig. 2a). This case (B36) did not have this alteration in the exon 9 of E-cadherin. Precise information about the other members of the Y6 family (Fig. 2b) was not available. All the unaffected 14 members in the cancer families had no extra band in PCR–SSCP screening.
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There was no migration abnormality of exon 9 of E-cadherin in the control samples described above (data not shown).
About the proximal promoter region and part of exon 1 of the E-cadherin gene in our series, we confirmed a C–A polymorphism site at position –160 which has recently been demonstrated to be related to the alteration of the transcriptional activity. At this polymorphic site, the PCR–SSCP patterns showed C–C and A–A homozygosity and C–A heterozygosity at nucleotide position –160, respectively. Another C–T polymorphism was detected at codon 692 of exon 13 as reported previously (2). The frequency of these polymorphisms did not differ between the familial gastric cancer patients and randomly sampled Japanese (Table 1).
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Germline mutation of E-cadherin in familial gastric cancer is not prevalent in Japanese familial cases as far as we know. In this study, we found a germline E-cadherin mutation at codon 415 in three of 87 patients analyzed. Two (B21 and B37) of the three patients are siblings and had a strong familial history of gastric cancers in this family, but no such mutation was identified in another healthy brother (B36), suggesting that this variation segregated with gastric cancer status in this family. The mechanistic basis of the possible relationship of this substitution to gastric cancer predisposition, like other reported missense mutations (13), is not clear. However, the extreme rarity or absence of this variation in the control population and the linkage with the affected status suggested to us that Ile415Leu may contribute to some of the familial clustering of gastric cancer in Japan.
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Acknowledgements |
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This work was supported by Grants-in-Aid from the MEXT of Japan, from the Ministry of Health, Labor and Welfare for the Comprehensive 10-Year Strategy for Cancer Control and from the Smoking Research Foundation. We also acknowledge all the clinicians and pathologists who enthusiastically cooperated with us.
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FOOTNOTES |
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+ For reprints and all correspondence: Haruhiko Sugimura, First Department of Pathology, Hamamatsu University School of Medicine, 1–20–1 Handayama, Hamamatsu 431-3192, Japan. E-mail: hsugimur@hama-med.ac.jp
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REFERENCES |
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Received June 25, 2002; accepted October 7, 2002
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