None of the authors have a conflict of interest to disclose.
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Research Article
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Differences in the frequencies of K-ras c12–13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method†‡
Article first published online: 25 AUG 2011
DOI: 10.1002/em.20673
Copyright © 2011 Wiley Periodicals, Inc.
Additional Information
How to Cite
Chow, L., Lin, P.-C., Chang, J. S., Chu, P.-Y., Lee, P.-K., Chen, S.-N., Cheng, Y.-M., Lee, J.-C., Chang, J.-Y. and Liu, T.-W. (2012), Differences in the frequencies of K-ras c12–13 genotypes by gender and pathologic phenotypes in colorectal tumors measured using the allele discrimination method. Environmental and Molecular Mutagenesis, 53: 22–31. doi: 10.1002/em.20673
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Dr. Tsang-Wu Liu approved and supported this project as the primary investigator of the Tumor Marker Laboratory of the National Institute of Cancer Research. Dr. Jang-Yang Chang initiated this project as the director of the National Institute of Cancer Research. Dr. Jeng-Chang Lee coordinated specimen collection as the head of the department of surgery of National Cheng Kung University. Dr. Peng-Chan Lin conducted patient enrollment as a senior hemato-oncologist in the Department of Internal Medicine of National Cheng Kung University. Dr. Jeffrey S. Chang performed statistical analyses and edited this manuscript. Dr. Pei-Yi Chu is the pathologist who reviewed this manuscript. Pao-Kung Lee worked on the early method development and result comparison. Shan-Na Chen contributed experimental support. Ying-Min Cheng modified and continued the assay. Dr. Lihui Chow designed the assay, analyzed results, and drafted the manuscript.
Publication History
- Issue published online: 5 JAN 2012
- Article first published online: 25 AUG 2011
- Manuscript Accepted: 13 JUL 2011
- Manuscript Revised: 12 JUL 2011
- Manuscript Received: 4 NOV 2010
Funded by
- Department of Health of Taiwan. Grant Number: DOH99-TD-C-111-004
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Keywords:
- K-ras codon 12–13 mutations;
- GAT prevalence;
- CRC pathologic phenotypes;
- allele discrimination;
- dual-color real-time PCR
Abstract
The frequencies of different genotypes of the K-ras oncogene in colorectal cancer (CRC) reveal complex relationships among gender, age, and tumor aggression, however, differences among these studies could also be attributed to a lack of standardization of the detection methods used. We developed the allele discrimination assay, which uses dual-color real-time polymerase chain reaction (qPCR) as a fast K-ras genotyping method, and demonstrated higher sensitivity and specificity than DNA sequencing with formalin-fixed paraffin tissues. The assay detected K-ras mutations among 83 of 204 patients with CRC (40.7%); 20.6% of these mutations were G12D (GAT) mutations, 7.4% were G13D (GAC) and G12V (GTT), and 5.3% were other types. A higher proportion of females was observed overall in tumors with K-ras mutations (60.2%, P = 0.01), codon 12 mutations (63.2%, P = 0.005), and transversions (69.6%, P = 0.02), which reflected the higher prevalence of females among the well- to moderately differentiated tumors (29% in males vs. 53% in females; interaction P = 0.03). The opposite was observed for poorly differentiated tumors (47% in males vs. 35% in females). No significant influence of age was found on the prevalence of K-ras mutation. Males with pathological changes and females with poorly differentiated tumors displayed GAT as a less common genotype compared with most other prevalence studies. In conclusion, allele discrimination, with no additional amplification step, is a fast and reliable genotyping method for detecting K-ras c12–13 mutations. Using this method, we demonstrate differences in the frequencies of K-ras genotypes by gender and pathologic phenotypes of CRC. Environ. Mol. Mutagen., 2012. © 2011 Wiley-Liss,Inc.