Association Study Of Single Nucleotide Polymorphisms In Chromosome15q25.1with The Susceptibility Of Lung Adenocarcinoma Among Chinese Southern Population

Background and objective:Lung cancer is one of the malignancies, which seriously threatens human health and life. Currently, in China and most industrialized countries, lung cancer has been the first cause of death in all malignancies. The morbidity and mortality of lung cancer in the world showed a rising trend. The reason may be related to the growing of tobacco consumption and serious environmental pollution. Lung cancer is the result of gene-environment interaction. The epidemiological data showed that the risk for lung cancer among smokers is20times higher than among non-smokers, although its pathogenesis has not yet been fully known. Therefore, smoking is the most closely environmental risk factors linked to developing lung cancer than other risks. However, only10%to15%of smokers were diagnosed with lung cancer, although more than80%of lung cancer can be attributable to the exposure to tobacco smoke. This indicates that susceptibility to lung cancer is different between individuals, and the cause of this difference comes from the genetic polymorphism.With the promoting of the international project aimed at developing a haplotype map of the human genome and the completion of the human genome project, in addition the establishment and improvement of the platform for high-throughput technology, Genome-wide association studies (GWAS) provides a powerful tool for the study of genetic susceptibility in complex diseases such as lung cancer. The basic principle of GWAS is as follows: within the target population, the case and control group are defined and the difference of allele or genotype frequencies on all single nucleotide polymorphism (SNP) within the scope of whole-genome between the two groups are compared. If the allele or genotype frequency on a SNP in case group is apparently higher or lower comparing with the control group, there is a relationship between this locus and disease. Then according to the location of the locus in the genome and linkage disequilibrium relationships, the potential disease susceptibility gene is presumed. Currently, GWAS has screened a number of common genetic variants associated with genetic susceptibility to lung cancer, in which the genetic variation located in5p15,6p21and15q25chromosome sections are involved in regulating nicotine acetylcholine receptor genes and generating telomerase genes, thus they are closely related to the developing of lung cancer. These findings suggested that it might contribute to the etiology study of lung cancer and the screening high-risk populations with lung cancer by detecting genetic molecular markers associated with developing lung cancer.Single nucleotide polymorphism (SNP) is a DNA sequence polymorphism occurring when a single nucleotide variation develops on DNA strand in the genome. There are a lot of SNP. They may occur in coding or non-coding sequence of a gene. According to the SNP position in the gene, they can be divided into regulatory SNP (rSNP) and coding SNP (cSNP). Among them, depending on whether the amino acid sequence in coding regions is changed, cSNP can be divided into synonymous cSNP and non-synonymous cSNP. The synonymous cSNP means the changes in the nucleotide sequence does not cause changes in the amino acid sequence of protein; non-synonymous cSNP means that the encoded amino acid residue type is affected, thus the structure and function of the protein changes. In addition, the rSNP in the regulation region can cause changes in gene expression levels or mRNA stability and translation process by affecting the binding with transcription factor or miRNA, thereby affecting the normal cell function. Therefore, these potential functional SNPs are commonly selected preferentially for investigating the relations with the disease development, progression and prognosis, providing a scientific basis for prevention and clinical treatment strategies of malignancies, including lung cancer.Currently, it is a hot spot in the field of lung cancer study to investigate the relationship between SNP and lung cancer susceptibility. And investigators have screened some chromosomal regions associated with susceptibility to lung cancer. For example, the independent studies from Iceland, France, the United States and the United Kingdom in2008demonstrated, through the GWAS analysis in lung cancer patients and healthy population, the changes of SNPs on CHRNA3-CHRNA5-CHRNB4(cholinergic receptor, nicotinic, α3, α5and β4) candidate gene clusters of the nicotinic acetylcholine receptor (nAChRs) coding region in15q24-25of the long arm of chromosome15are associated with the developing of lung cancer. The results of four studies showed that genetic factors also play a key role in the pathogenesis of lung cancer except smoking. In studies from U.S. MD Anderson Cancer Center, Amos et al. detected315,450SNPs in1154 smoking non-small cell lung cancer (NSCLC) patients and1,137healthy people which were matched with respect to smoking status. The results showed that the incidence of lung cancer is closely related with10SNPs, rs2808630, rs7626795, rs2202507, rsl1099666, rs1481847, rs855974, rs8034191, rs1051730, rs12956651and rs6069045, respectively. Afterwards, further studies were conducted and found that the distribution of SNPs varies in different ethnic populations. For example, European GWAS demonstrated that the SNPs of rs8034191,rs1051730and rs16969968located on chromosome15q25.1region were significantly associated with lung cancer in smokers and non-smokers. However, studies in South Korean, Japanese and Chinese population have failed to repeat the experiment findings. The reason may be attributed to the lower frequencies of these polymorphic loci in Asian populations, in which Minor Allele Frequency (MAF) were0.022,0.026and0.026in turn. These studies suggested that lung cancer susceptibility loci may be various in different ethnic populations. Therefore, studies of cancer gene polymorphism are currently needed to conduct in Chinese population to detect the lung cancer susceptibility loci in Chinese population, providing data base for screening, early diagnosis and individualized treatment in high-risk lung cancer population.The gene located on chromosome15q25.1region include AGPHD1(aminoglycoside phosphotransferase domain containing1), CHRNA5and CHRNA3(cholinergic receptor nicotinic a5and3). This gene cluster is located on nicotinic acetylcholine receptor subunits (nAChRs) in the core region of the brain. It is closely related to the nicotine addiction behavior. And receptors distributed in the lung epithelial cells are involved in signal transduction of nicotine and tobacco carcinogens,4-(methylnitrosoamino)-3-pyridinyl-1-butanone (NNK), to promote tumor cell proliferation, migration and metastasis. Although it has been reported in European, American and Japanese population, that the genetic variation of this gene family was associated with lung cancer susceptibility, it has not been known that the mechanism of this association is established by affecting the smoking behavior of individuals or directly regulating oncogenic signal transduction, or a combination of the two effects. Based on the findings of some studies, it is indicated that genetic variations in nicotine receptor gene family have a significant impact on the individual cigarettes consumption daily. Therefore, the investigators speculate that the relationship between these genetic variations and lung cancer is attributed to tobacco addiction behavior. For example, studies from Iceland deCODE Genetics Institute showed that investigator had conducted GWAS in15,771smokers and32,244control subjects in populations of European. The findings demonstrated that changes of rs1051730polymorphism in15q24-25locus are associated with nicotine dependence, increased cigarettes consumption and incidence of lung cancer. The present of each T allele at rs1051730locus increased daily cigarettes consumption9.5%(OR=0.095,95%CI:0.075-0.115, P=6.0×10-20), and raise the risk of lung cancer31%(OR=1.31,95%CI:1.19-1.44, P=1.5×10-8). Studies suggested that the changes of SNPs in15q24-25region and smoking had a synergistic effect on lung cancer. However, the GWASs from French and American have come to a different conclusion. In these studies, smoking patients with lung cancer are analyzed, and the conclusion showed that the changes of rs1051730and rs8034191polymorphism in15q25were not associated to the both of cigarettes consumption per years (packyear)(P=0.6851and0.8460) and daily cigarettes consumption (cig/day)(P=0.2145and0.4538). Therefore, investigator considered that SNPs in chromosome15q25associated with lung cancer risk were not related to smoking behavior. Meanwhile, studies showed that the changes of SNPs rs8034191and rs16969968loci were only specifically associated with lung cancer, and not associated with other smoking-related cancer (including oral cancer, throat cancer, esophageal cancer, etc.). Therefore, the relationship between gene polymorphism and incidence of lung cancer is still needed to be validated in different ethnic and smoking status populations, and other risk factors and biological mechanisms possibly have effects on the gene function should be further studied.Histological type of lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The most common type in NSCLC is squamous cell carcinoma and adenocarcinoma. Currently, the incidence of lung adenocarcinoma showed a rising trend worldwide, but the specific cause is not known. Studies suggested that it may be associated with passive smoking, environmental pollution, diet, hormone levels and genetic susceptibility. Lung cancer occurs mainly in female non-smoking patients. Most studies showed that female non-smoking lung cancer patients had a particular gene phenotype, suggesting that the mechanism may overlap with that in lung cancer of smokers, as well as have its unique molecular pathway. In new standards of International multidisciplinary classification of lung adenocarcinoma issued by2011International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS), lung adenocarcinoma has been divided into adenocarcinoma in situ, small invasive adenocarcinoma and invasive adenocarcinoma. This classification not only defines the development of lung adenocarcinoma histology pedigree, but also pays more attention to the characteristics of gene or molecular phenotype in the progression of neoplasm. Therefore, are there any SNPs distribution differences in the occurrence and development of lung adenocarcinoma?Currently, although the studies about genetic susceptibility to lung cancer have made great progress, the most of the data is based on Western populations. Research data about the Chinese Han population is still very limiting. In addition, Due to the rising lung cancer incidence and unique molecular phenotype, etiology studies for Chinese population of lung adenocarcinoma are particularly important. This study aims to discuss the relationship between gene polymorphism in chromosome15q25.1and the incidence of lung cancer in southern China population through the case-control analysis, providing theoretical basis and data base for screening, early diagnosis and individualized treatment in high-risk lung cancer population. The subjects of study are limited in China Guangdong Han population of lung adenocarcinoma patients. And the pathological type of cases will be re-classified in accordance with international multidisciplinary classification of lung adenocarcinoma. Six SNPs in chromosome15q25.1region were selected, which may be lung cancer susceptibility loci. Sequenom MassARRAY (?)-IPLEX was used to determine genotyping in the southern Chinese lung adenocarcinoma patients and healthy control subjects. On the one hand, the statistical analysis of each locus polymorphism and its relationship with lung adenocarcinoma susceptibility was conducted to identify lung cancer susceptibility SNPs suitable for China Guangdong population, on the other hand, the Stratified analysis of relationship between each locus polymorphism and gender, smoking status, histological type and TNM clinical stage in patients with lung adenocarcinoma was also conducted to explore the association between genotype distribution and clinicopathological factors. Meanwhile, immunohistochemistry was used to detect the expression level of protein on positive loci in lung adenocarcinoma tissues, and to further validate the role of this gene in development of lung adenocarcinoma. Based on these data, the preliminary data base and theoretical basis would be established for identifying and individualizing prevention and treatment of lung adenocarcinoma in high-risk populations in southern China, and it would also be prepared for the next step to establish a risk assessment model of lung adenocarcinoma susceptibility gene for southern Chinese populations.Methods:1. Subjects:In this study,301pathologically confirmed lung adenocarcinoma patients were recruited from Department of Thoracic Surgery of The First Affiliated Hospital of Guangzhou Medical University from September2011to September2012. During the same period,318healthy subjects from medical examination center were enrolled into control group. All of study subjects were Guangdong Han population. Peripheral blood samples were collected from all subjects.2. Selection of SNP loci: six SNPs in chromosome15q25.1region with lung cancer susceptibility were selected from the NCBI SNP Information Database, or positive loci obtained in Genome-wide association analysis and other large sample studies and abroad: AGPHD1gene rs8034191; CHRNA5gene rs16969968, and CHRNA3gene rs1051730, rs938682, rs12914385and rs8042374.3. Genotyping and data analysis: Sequenom MassARRAY (?)-IPLEX was used to detect polymorphism genotyping in China Southern lung adenocarcinoma patients and healthy controls. The test results were analyzed by SPSS13.0. The relationship between the various SNPs distribution and the risk of lung adenocarcinoma was explored under Codominant, Dominant, Recessive, Overdominant and Log-additive genetic model to identify lung cancer susceptibility SNP in China Southern population.4. Stratified analysis by gender, smoking status, histological type and tumor, node, metastasis (TNM) classification was performed to explore the relationship between genotype distribution and clinicopathological factors.5. Immunohistochemistry method was used to detect the expression level of the protein of susceptible gene in lung adenocarcinoma tissues.Results:Part Ⅰ: the relationship between genetic polymorphisms and the occurrence of lung adenocarcinomaSix SNPs in chromosome15q25.1region were selected in this study. Genotyping and statistical analysis were performed in301lung cancer patients and318healthy control subjects in Guangdong population. The results showed that except for rs12914385, the gene frequency distribution of other five loci were in compliance with Hardy-Weinberg equilibrium (P>0.05). The case-control analysis found two polymorphism loci associated with lung adenocarcinoma risk. CHRNA3gene rs8042374meets the co-dominant and over-dominant model. Compared with the G/G and A/A genotype, the risk increased in patients carrying G/A genotype, with OR (95%CI)1.76(1.17-2.65), P=0.024and1.71(1.15-2.54), P=0.008. Another one, CHRNA3gene rs938682, meets the dominant model. Compared with the T/T genotype, the risk may reduce in individual patient carrying T/C or C/C genotype, with OR (95%CI)0.68(0.45-1.02), P=0.063. The other three SNPs(rs8034191, rs16969968and rs1051730) showed no polymorphism and no significant association with ADC risk.The stratified analysis showed that the rs8042374genotype (G/A) was significantly associated with the stratifications in female, non-smoking, TNM stage I Ⅱ, female non-smoking and TNM stage Ⅰ+Ⅱ in female, with OR (95%CI)1.79 (1.04-3.07),1.80(1.18-2.75),1.81(1.14-2.87),1.81(1.05-3.12) and1.92(1.03-3.57), respectively. In addition, rs938682genotype (T/C or C/C) showed a protective effect for patients stratified by smoking, TNM stage Ⅲ+Ⅳ and TNM stage Ⅲ+Ⅳ in male. It may reduce the risk of individuals, with OR (95%CI)0.50(0.26-0.97),0.54(0.33-0.89) and0.40(0.20-0.82), respectively.Part Ⅱ: the expression of CHRNA3protein in lung adenocarcinoma tissueBased on the study finding that both of two positive SNPs were in CHRNA3gene, we further used immunohistochemical method to detect the expression of CHRNA3protein in the lung adenocarcinoma and tumor adjacent lung tissue of81patients. The results showed CHRNA3protein were expressed in both of lung adenocarcinoma and tumor adjacent lung tissue. Varying degrees of positive staining were showed in both of the alveolar epithelium and bronchial epithelial in normal lung tissue. However, in the tumor tissue, positive staining was only in cancer cells, but not in stromal cells. The statistics showed significantly different expression level of CHRNA3protein between lung adenocarcinoma tissues and tumor adjacent normal lung tissues (P=0.001). But there was no relationship between the SNP genotype and protein expression.Conclusion:1. The results of this study showed that two SNPs on chromosome15q25.1were associated with the risk of lung adenocarcinoma in China Southern population. The risk of cancer increased in patients carrying rs8042374genotype (G/A), and reduced in patients carrying rs938682genotype (T/C or C/C).2. The stratified analysis in lung adenocarcinoma group showed that rs8042374 genotype (G/A) was significantly associated with the stratifications in female, non-smoking, TNM stage Ⅰ+Ⅱ, female non-smoking and TNM stage Ⅰ+Ⅱ in female, suggesting that the relationship between rs8042374genotype (G/A) and lung cancer risk was not associated with smoking behavior; on the contrary, rs938682genotype (T/C or C/C) showed a protective effect for patients stratified by smoking, TNM stage Ⅲ+Ⅳ and TNM stage Ⅲ+Ⅳ in male.3. The statistics showed significantly different expression level of CHRNA3protein between lung adenocarcinoma tissues and tumor adjacent normal lung tissues, suggesting that the transcripts of this gene may be involved in the development of lung adenocarcinoma.