Abstract
Background/Aim: This study examined whether the polymorphisms at the promoter region of RET gene are associated with the risk of lung cancer in the Korean population. Patients and Methods: A total of 409 Korean lung cancer patients and 409 normal subjects were recruited. PCR-RFLP, SNaP Shot assay and logistic regression analyses were performed to characterize the association between polymorphisms of RET and lung cancer risk. Results: Four SNPs (-1697 C>G, -1260 C>T, -719 C>T, -527 C>A) of RET were selected for large-scale genotyping. Statistical analyses revealed that novel -1260 C > T polymorphism and haplotype 3 (-1697G, -1260T, -719C, and -719C) were associated with the risk of lung cancer; specifically, there were significant associations within subgroups of males and smokers. Conclusion: These results demonstrated that this novel polymorphism of the RET gene is associated with an increased risk of lung cancer in the Korean population.
Lung cancer is a major cause of cancer deaths worldwide (1) and has been the most common cause of cancer deaths in Korea (2). Despite apparent resection of the primary tumour, recurrence rates remain high and the overall 5-year survival remains poor, with <15% of patients surviving 5 years from diagnosis (1). Lung cancer is a multicellular and multistage process, which involves a number of genetic changes in oncogenes and tumour suppressor genes (3) Furthermore, the genetic alteration of the RET proto-oncogene is related to lung cancer pathogenesis (4).
The RET proto-oncogene is located on chromosome 10q11.2, comprises 21 exons and encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family (5-6). It plays a crucial role in the regulation of cell proliferation, migration, differentiation and survival during embryogenesis. RET mutations are responsible for the development of several human diseases including multiple endocrine neoplasia type 2 (MEN2), familial medullary thyroid carcinoma (FMTC), papillary thyroid carcinoma (PTC) and Hirschsprung disease (HSCR) (7-10). Several groups reported that single-nucleotide polymorphisms (SNPs) of RET have been presented in endocrine tumours (11), papillary thyroid carcinoma (12), familial and sporadic MTC (13-14) and HSCR (15). In addition, a deletion of the chromosomal region including RET is involved in the pathogenesis of small cell lung cancer (SCLC) (16). Mulligan et al. (17) suggested that RET is expressed in 57% of SCLC cell lines, although RET mutation appears unlikely to be an important step in the tumourigenesis of SCLC. However, Takahashi et al. (4) reported the genetic alteration of the RET proto-oncogene in lung cancer pathogenesis. Although several studies have analysed the relationship between the genetic alterations of RET and lung cancer, these results remain contradictory and unclear. This study attempted to characterise the association between RET polymorphisms and the risk of lung cancer development in the Korean population.
Materials and Methods
Study subjects. Between August 2001 and August 2008, blood samples were collected from 818 subjects, comprising 409 lung cancer patients and 409 normal controls without cancer. Lung cancer patients were recruited from the patient pool at the Genomic Research Center for Lung and Breast/Ovarian Cancer and the Inha University Medical Center, while 409 control subjects were randomly selected from a pool of healthy volunteers who had visited the Cardiovascular Genome Center, the Genomic Research Center for Allergy and Respiratory Diseases and the Keimyung University Dongsan Medical Center. Using a structured questionnaire, detailed information about diet, smoking status, drinking status, lifestyle, and medical history were collected by a trained interviewer. Out of the 409 patients, gender and smoking status information was available for 408 and 397 cases, respectively, while out of the 409 controls, gender and smoking status information was available for 409 and 308 cases, respectively. All subjects completed a brief questionnaire and provided written consent, and the Institutional Review Board approved the study.
Map of polymorphisms, haplotypes, and linkage disequilibrium (LD) coefficients in the RET proto-oncogene. A: The location of 11 polymorphisms in the RET gene on chromosome 10q11.2 (reference sequence of RET: NM_020975). Asterisks indicate polymorphisms that were genotyped in a larger population. The frequencies of polymorphisms are based on sequencing data (n=24). The first base of the translation site is denoted as nucleotide +1. B: Haplotypes of RET. Haplotypes with frequency >0.08 are presented. C: LD analysis (|D'|) of RET. Four SNPs, namely -1697 C>G; rs3026727, -1260 C>T; Novel, -719 C>T; rs2435366 and -527 C>A; rs3097561, were used for the construction of haplotype. The LD between the polymorphisms was quantified using Haploview version 3.2.
Screening and genotyping of RET promoter polymorphisms. Eleven SNPs were identified in the RET gene by direct sequencing for the region spanning for ~2 kb upstream from the translation initiation site (NM_020975) of RET in a small set of 24 lung cancer cases. Seven SNPs (-1782 A>G; rs2505998, -1697 C>G; rs3026727, -1260 C>T; novel, -719 C>T; rs2435366, -527 C>A; rs3097561, -200 A>G; rs10900296, -196 C>A; rs10900297) out of the 11 candidate polymorphisms showed more than 5% of minor allele frequency (MAF). Four SNPs were then selected as haplotype tagging SNP (Ht SNP) (Figure 1A). Genomic DNA was isolated from peripheral blood samples using a Puregene blood DNA kit (Gentra, Minneapolis, MN, USA), following the manufacturer's protocol. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and SNap Shot assay.
Demographic characteristics of study subjects.
Genotyping of -1260 C>T and -527 C>A SNPs was carried out by PCR-RFLP. The PCR primers for amplifying DNA fragment containing the -1260 C>T SNP or -527 C>A SNP were 1260 F5′-CTGCCTGTGAGGGTGACTTCA-3′/1260 R5′-TCCTTTCAGAGA CCTGGTGC-3′ or 527 F5′-GTAGCATCCCTGCCCTCTCT-3′/527 R5′-TCCAGTGCTTG CTCTGGCTG-3′, respectively. The PCR products were digested with the restriction enzyme AatII (for -1260 C>T) or SAPI (for -527 C>A) to determine the genotypes and they were resolved by gel electrophoresis.
-1697 C>G and -719 C>T SNP genotyping were performed by the SNaP Shot assay ddNTP primer extension method as previously described (18).
Statistical analysis. Allele frequencies, genotype frequencies, and departures of the genotype distribution from the Hardy-Weinberg equilibrium for each SNP were analysed using the chi-square test or Fisher's exact test. The linkage disequilibrium (LD) between the polymorphisms was quantified using Haploview version 3.2 (http://www.broad.mit.edu/mpg/haploview). The haplotypes and their frequencies were estimated by Haploview. Genotype-specific risks were estimated as odds ratio (OR) with associated 95% confidence intervals (CI) by unconditional logistic regression (SAS Institute, Cary, NC, USA) and adjusted for age and gender. A p-value <0.05 was used as the criterion for statistical significance. Bonferroni correction was performed for multiple comparisons. All the analysis was performed using Statistical Analysis Software for Windows, version 9.1 (SAS Institute).
Results
This study population consisted of 409 lung cancer patients and 409 controls. The demographics of the patients and the controls enrolled in this study are shown in Table I. By direct sequencing of the RET promoter region (~2 kb), 11 polymorphisms of the RET promoter region were discovered among 24 lung cancer patients samples. Seven SNPs were selected, namely -1782 A>G; rs2505998, -1697 C>G; rs3026727, -1260 C>T; novel, -719 C>T; rs2435366, -527 C>A; rs3097561, -200 A>G; rs10900296, -196 C>A; rs10900297, with MAP exceeding 5% out of the 11 candidate polymorphisms. Among the 7 SNPs, 4 SNPs (-1697 C>G; rs3026727, -1260 C>T; novel, -719 C>T; rs2435366, -527 C>A; rs3097561) were selected for large-scale genotyping, based on their frequencies, their LD and their haplotype tagging status. The minor allele frequencies of the study population were 0.271, 0.375, 0.458 and 0.152. The genotype frequencies for patients and controls were in the Hardy-Weinberg equilibrium. LD coefficients (|D'|) between the polymorphisms were calculated using Haploview. The four polymorphisms were in LD (|D'|>0.95) (Figure 1). The allelic frequencies of each polymorphism and haplotype were compared between patients and controls, using logistic regression models (Table II). Logistic regression analyses revealed that the novel -1260 C>T polymorphism is associated with the risk of lung cancer in the overall population. However, the -1697 C>G, -719 C>T and -527 C>A polymorphisms were not significantly associated with the risk of lung cancer. The subsequent analysis showed that haplotype 3 (-1697G, -1260T, -719C, and -527C) was associated with an increased risk of lung cancer in the overall population. However, three haplotypes, namely haplotype 1 (-1697C, -1260C, -719T, and -527C), haplotype 2 (-1697C, -1260C, -719C, and -527C) and haplotype 4 (-1697C -1260C -719T and -527A) were not associated with the risk of lung cancer. In the stratified analyses by gender and smoking status, the novel -1260 C>T polymorphism and haplotype 3 (-1697G, -1260T, -719C, and -527C) remarkably increased the risk of lung cancer in males and smokers in this Korean population (Table III).
Discussion
Based on the hypothesis that RET polymorphisms are associated with lung cancer risk, and that these polymorphisms may play a role as predictors of lung cancer, this preliminary study on RET polymorphisms was performed by sequencing of 24 samples and, for a further larger-scale study, 4 RET polymorphisms were selected via genotyping of a total of 818 samples, including of 409 lung patients and 409 controls. The findings suggested an association between the polymorphisms of the RET gene and lung cancer risk in the Korean population, especially for the male and smoking groups.
RET proto-oncogene encodes a receptor tyrosine kinase which plays an important role in the regulation of cell proliferation, migration and differentiation (6). Several groups have reported that abnormalities of RET, such as mutations and polymorphisms, are associated with an increased relative risk for the development of human diseases, including papillary thyroid carcinoma, MEN 2A and 2B, familial MTC, HSCR (7, 19-21) and SCLC (13-14, 22-25). There has been no report, however, on the association between the polymorphisms of the RET proto-oncogene and lung cancer risk. The present data indicated that the polymorphism and haplotype of the RET gene, including the novel -1697 C>G, showed remarkable association with the lung cancer risk for males and smokers.
Association of RET polymorphisms and lung cancer risk in the overall population.
Several reports described the results that provide a direct link between specific genetic alterations and exposure to tobacco carcinogens (26-27). Previous studies showed that polymorphisms of RASSF1A and PIM-1 genes are related to an increase of lung cancer risk in males and smokers (28-29). It is highly possible that smoking plays an additional role in genetic alterations for lung cancer patients (29). In the present study, polymorphisms and haplotypes of the RET gene also increased the susceptibility to lung carcinogenesis on smokers and males. This suggested that genetic constitution of individuals is important in determining the individual's susceptibility to lung cancer (30).
In summary, 11 sequence variants of RET were identified in 24 Korean individuals through direct sequencing. Four of these polymorphisms were selected for a larger-scale genotype (n=818) and significant differences in allele genotype and haploytpe were observed between the lung cancer patients and the controls. Thus, this was the first study to delineate the association between RET polymorphisms and the risk of lung cancer, and particularly to show that the novel -1260 C>T polymorphism and the haplotype 3 (-1697G, -1260T, -719C, and -527C) were associated with an increased risk of lung cancer in the overall population and in the male and smoker groups of the Korean population. These findings suggested that the polymorphisms of RET, including the novel -1260 C>T may play an important role as predictors of lung cancer. Large-scale studies are necessary in order to further verify the role of RET polymorphisms and haplotypes in determining the risk of lung cancer.
Association analysis of RET promoter polymorphisms and haplotypes and lung cancer patients.
Acknowledgements
We thank Young Lim, Dong-Hoon Shin, Choon-sik Park and Yangsoo Jang for sample collection and provision. This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea (A010250) and the Seoul Research and Business Development Program (10574).
- Received June 17, 2010.
- Revision received June 29, 2010.
- Accepted July 6, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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