Japanese Journal of Clinical Oncology 32:219-221 (2002)
© 2002 Foundation for Promotion of Cancer Research
Concurrent Mutations of K-ras Oncogene at Codons 12 and 22 in Colon Cancer
1 Department of Surgery, Jichi Medical School, Minami Kawachi, Tochigi, 2 Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, Utsunomiya and 3 Department of Surgery, Jichi Medical School Omiya Medical Center, Saitama, Japan
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ABSTRACT |
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 TOP ABSTRACT GENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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K-ras mutation is the most common oncogenic alteration in various human cancers including colorectal carcinomas. Point mutations have the potential to activate the K-ras gene if they occur in the critical coding sequences. Almost all of these mutations have been localized in codons 12, 13 and 61. We report a case of colon cancer presenting point mutations at both codons 12 and 22 of the K-ras gene. PCR-SSCP and subsequent sequencing revealed that GGT (glycine, wild-type) to AGT (serine) substitution at codon 12 and CAG (glutamine, wild-type) to CGG (arginine) substitution at codon 22 occurred in the same allele.
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GENETIC SUMMARY |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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Disorder: Colon cancer
Ethnicity of patient: Japanese
Gene: K-ras
GeneBank accession number: L00045
Type of DNA variant: Somatic point mutations
Mutation: GGT (Gly, wild-type) to AGT (Ser) substitution at codon12 and CAG (Gln, wild-type) to CGG (Arg) substitution at codon 22 of the K-ras gene
Allelic frequency: Not tested
Methods of mutation detection: PCR-SSCP and direct sequencing
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CASE REPORT AND GENETIC ANALYSIS |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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The ras mutation is the most common oncogenic alteration in human cancers. K-ras mutation is present in up to 40% of colorectal adenomas and carcinomas (1,2). K-ras gene has transforming activity after single point mutation within its coding sequence. Mostly, these mutations have been localized in codons 12, 13 and 61. Rare variants of point mutations at codons 11, 18 and 59 have been reported in a few human tumors (3–5). Recently, Tsukuda et al. (6) reported a point mutation at codon 22 in a primary colon cancer and suggested that this mutation might be advantageous for the growth of tumor cells. Here we report a case of colon cancer presenting point mutations of the K-ras oncogene at codons 12 and 22 concurrently in the same allele.
An 82-year-old female was diagnosed with ascending colon cancer and underwent surgical resection. Histological diagnosis was well differentiated adenocarcinoma. We previously reported microsatellite instability (MSI) status (MSI-negative) and methylation profile (partial methylation) of the hMLH1 promoter region in tumor tissues of this case (7). PCR-SSCP analysis for the K-ras gene revealed two significant shifted bands in the tumor sample (Fig. 1). Direct sequencing of this tumor-derived PCR product identified two point mutations which caused a substitution of serine for glycine (GGT to AGT) at codon 12 and arginine (Arg) for glutamine (CAG to CGG) at codon 22 (Fig. 2). To confirm whether these mutations occurred in the same allele or not, each of the two shifted bands were cut from the gel, reamplified and subjected to SSCP analysis and direct sequencing (Figs 1 and 2). It was shown that DNA fragments amplified from these two bands had the same mobility shift in SSCP, indicating that they are complementary single-strands. Furthermore, sequencing analysis confirmed that these two mutations were detected in the same electropherogram without contamination of the wild-type sequence. These findings indicate that mutations at codons 12 and 22 were localized in the same allele. Previously, Tsukuda et al. reported that the novel point mutation at codon 22 was C to A transversion substituting lysine (Lys) (AAG) for glutamine (CAG) (6). The tumorigenecity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants. In this study, it was not identified whether this K-ras Arg22 mutation gives rise to tumorigenesis. Further studies are needed to elucidate the biological activity of this combination of point mutations at codons 12 and 22. To our knowledge, this is the first report of K-ras point mutations occurring concurrently in the same allele.
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METHODS FOR MUTATION DETECTION |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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PCR-SSCP and PCR/direct sequencing was performed with the following conditions and parameters:
PCR primer, forward: 5'ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT3'.
PCR primer, reverse: 5'TA A TAT GTC GAC TAA AAC AAG ATT TAC CTC3'.
Size of PCR product: 135 bp.
Thermal cycle profile:
40 cycles of 96°C, 1 min/55°C, 1 min/73°C 30 s.
Final extension: 72°C, 7 min.
The 5' end of the forward primer was labeled with indodicarbocyanine (Cy5) fluorescent dye.
PCR-SSCP: non-RI SSCP analysis as described previously with minor modifications (8,9).
Electrophoresis: 15% polyacrylamide gel, 18°C.
Sequencing primer: same as the reverse PCR primer.
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Acknowledgments |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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This work was supported in part by Grants-in-Aid for Cancer Research and for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare, Japan and a Grant-in-Aid from the Vehicle Racing Commemorative Foundation.
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FOOTNOTES |
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+ For reprints and all correspondence: K. Sugano, Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, 4–9–13 Yohnan, Utsunomiya, Tochigi 320-0834, Japan
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REFERENCES |
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TOPABSTRACTGENETIC SUMMARYCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONAcknowledgmentsREFERENCES |
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7 Miyakura Y, Sugano K, Konishi F, Ichikawa A, Maekawa M, Shitoh K, et al. Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability. Gastroenterology 2001;121:1300–9.[Web of Science][Medline]
8 Sugano K, Kyogoku A, Fukayama N, Ohkura H, Shimosato Y, Sekiya T, et al. Methods in laboratory investigation. Rapid and simple detection of c-Ki-ras2 gene codon 12 mutations by nonradioisotopic single-strand conformation polymorphism analysis. Lab Invest 1993;68:361–6.[Web of Science][Medline]
9 Kondo H, Sugano K, Fukayama N, Kyogoku A, Nose H, Shimada K, et al. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer 1994;73: 1589–94.[Web of Science][Medline]
Received February 12, 2002; accepted March 13, 2002
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