Association Study Of Nicotinic Acetylcholine Receptor And CYP2A6 Gene Polymorphisms With The Susceptibility Of Lung Cancer Among Chinese Southern Male Populationin

Background and objective:The incidence and the mortality of lung cancer were growing fastest in malignant tumor worldwide and continue to rise. In our country and most industrialized countries, lung cancer is the leading cause of cancer-related mortality. The occurrence of lung cancer is the result of the interaction of environmental and genetic factors. At present, the pathogenesis of it has not been fully elucidated, but the epidemiological data showed that smoking is the environmental risk factor which most closely to lung cancer. The risk of lung cancer in smokers is 20 times more than in nonsmokers. And more than 80% lung cancer patients are associated with tobacco dependence or addictive. At the same time, it takes our attention that only 10%-15% smokers have lung cancer. It suggests that susceptibility to lung cancer is different from one to another, and the cause of such differences is derive from genetic polymorphisms.To study complex diseases such as the genetic susceptibility of lung cancer, Genome-wide association studies(GWAS) is an effective means. In recent years, the human genome project and the implementation of the international genome haplotype map project and develop, genotyping detection system also in constant development and improvement at the same time, which laid a foundation for GWAS, also open a door to the study of complex diseases. GWAS is used millions of Single nucleotide polymorphisms (SNP) in the human genome cases as sign to make case-control correlation analysis, hope to found the affect of genetic characteristics to disease development. Its fundamental is setting up the case group and control group in the target population and using SNP as a genetic marker, then use genome-wide scale contrast analysis. If the frequency of some SNP loci alleles or genotype in case group has significant differences compared with control group, the site may be associated with the risk of disease.The researchers through gene location and linkage disequilibrium analysis to determine which is the susceptibility genes to the desease. At present, in the worldwide GWAS have screened a number of genetic susceptibility heteromorphosis to lung cancer, such as nicotine acetylcholine receptors and CYP2A6 function related to gene mutation loci, genetic polymorphisms of these sites can increase or decrease risk of lung cancer in the individual. The above findings suggest that by examining the associated with the risk of lung cancer genetics, molecular marker, contribute to etiology research and screening high-risk groups for lung cance.SNP is refers to the genomic DNA because a single nucleotide occur transition, upside down insertions or deletions etc result in variation of DNA sequence polymorphism. SNPS as human genome has the characteristics of numerous, widely distributed and stable genetic.It will occur in gene coding sequence, as well as in non-coding sequences. According to the location in gene, the SNP can be divided into gene regulatory SNPS(rSNP) and coding SNPS(cSNP). According to the influence on biological genetic traits cSNP can be divided into two types:one is Synonymous cSNP, namely the change of base sequence to the SNP does not cause the change of amino acid sequence of its translation protein and the mutation of the bases and mutation base make the same meaning. Another is the non-synonymous cSNP, which refers to the amino acid residues can be changed by SNP nucleotide sequence, thus affecting the structure and function of protein. CSNP in about half of the non synonymous cSNP, which often the direct cause of biological traits changed.In addition, the regulation region of rSN through impact the combination of the transcription factor or miRNA, caused a change of the stability of gene expression and translation level, there by affecting the normal cell function. Therefore, the potential function of SNP is usually the preferred choice for the relationship between the occurrence and progression of diseases, and to provide the scientific basis for prevention and treatment of malignant tumors including lung cancer, and making the clinical treatment decisions.At present, the relationship between SNP and lung cancer susceptibility has become a hot field of lung cancer research. GWAS has found multiple Susceptibility loci and pseudoautosomal region which affect the risk of lung cancer in several parts of the world. For example, members of nicotine gene family CHRNA3, CHRNB4,CHRNB2 (cholinergicreceptor, nicotinic, alpha 3 beta 4 B2andB2)can encode nicotinic acetylcholine receptors(nAChRs) which associated with smoking behavior of individual. The past researches suggested that nAChRs exists only in the nervous system, but recent studies have confirmed that pulmonary bronchial epithelial cells on the distribution of nicotine acetylcholine receptors play an important role in the process of the occurrence of lung cancer.Type nicotinic acetylcholine receptors belong to the ligand gating ion channel superfamily which can be activated by nicotine acetylcholine and choline and participate in the regulation of nicotine and tobacco nitrosamines.Nicotine and tobacco nitrosamine is the main carcinogens that make smoking result in lung cancer. In addition, nicotine link with nAChRs receptor proteins on the surface of the cells, affect the progress of the tumor through various channels, such as cell proliferation, invasion and interstitial blood vessel formation, but also can protect the tumor cells to escape apoptosis, exert the function of inhibiting tumor cell apoptosis. AChR alpha 3 subunit protein encoded by CHRNA3 gene can also combine with tobacco carcinogens NNK, causing oncogene activation, and promotes the proliferation of tumor cells, increase the risk of lung cancer. In addition, other carcinogen such as nitrosamine can acted on nAChRs protein,result in tumor angiogenesis and tumor cell growth,which related to the tumor occurrence and development. Independent results of GWAS from Iceland, France,the United States and Britain demonstrated that genetic susceptibility was closely related to the risk of lung cancer. Four groups of researchers through the cases of lung cancer patients and healthy people-contrast analysis, have verified the nicotinic acetylcholine receptor gene family of multiple SNPs loci polymorphisms affect the risk of lung cancer. Further study found that the distribution of SNP is different in different ethnic groups. European GWAS studies have shown that, for instance, SNP loci rs8034191, rs1051730 and rs16969968 which located in chromosome 15 q25.1 area was associated with a significant risk of lung cancer. However, studies have failed to repeat the experiment results in South Korea, Japan and China, the reason may be the smallest Allele Frequency of the polymorphic loci (Minor Allele Frequency, MAF) caused by low distribution in Asian. The above findings suggest that different lung cancer susceptibility gene loci exist in the ethnic and regional. Therefore, it is necessary to research the relation between gene polymorphisms and lung cancer. To find out lung cancer susceptibility loci of the Chinese people for our country so that to provide data basis and theoretical basis for the screening of high risk people and individual prevention and treatment for lung cancer.CHRNB2 (cholinergic receptor nicotinic beta 2) is located in chromosome 1q21 region, CHRNA3 (cholinergic receptor nicotinic and alpha 3 beta 4) is located in the 15q25.1 region and CHRNA4(cholinergic receptor nicotinic alpha 4.) is located in the 20ql3.2 region. They encode alkali acetylcholine receptors (nAChRs) which may affect the individual’s smoking behavior and tobacco addiction,and participate in the regulation of tobacco carcinogens in the signal transduction pathway at the same time, through inhibiting cell apoptosis and tumor proliferation closely associated with the occurrence of lung cancer. Although the association of the mutation loci of the gene family and the the risk of lung cancer had reported in western and the Asian people, but the mechanism why they were associated has not been determined. Is it direct effect on cancer molecular pathways or indirect effect on cancer molecular pathways through smoking or the synergy of them? Based on some research, the results show that the mutation loci of nicotine receptor gene families can encourage smoking, so the researchers believe that gene polymorphism is increase the risk of lung cancer by affect smoking behavior. For example, the Japanese study found that rs4845652 T allele carriers on 1 q21 area have low nicotine dependence(Nd) which proved CHRNA4 CHRNB2 and interactions of the gene affect Nd, such as smokers who carry CHRNA4 rs 1044397 GG or GA CHRNB2 CC with higher Nd. Besides, Iceland’s GWAS results found genetic variation of rs 1051730 loci on chromosome 15 q24-25 associated with tobacco addiction, leading to the increase of the smoking behavior and the risk of lung cancer. However, European and American scholars draw the different conclusion that the polymorphism variation of the loci has nothing to do with smoking and nicotine dependence.CYP2A6 gene which located in human chromosome 19 long arm 19 q12-q13. 2, contains more than 6000 base pairs,9 exons,and encodes 494 amino acids. Coumarin 7-hydroxylase encoded by it play an important role in physiological processs both in the metabolism of drugs and poisons metabolism, especially in the metabolism of exogenous chemicals, carcinogens before involved in many metabolic activation, such as aflatoxin B1, nitrosamines,1,3-butadiene, etc.CYP2A6 is a P450 enzyme expressed in the human respiratory tract which changed nitrosamines such as NNK, NNAL, NNN in tobacco carcinogens into the strong. NNK is the strongest carcinogen of the TSNA which is the most abundant lung carcinogens in tobacco, has high affinity to the lung tissue and is considered to be one of the important components in tobacco lead to lung cancer. CYP2A6 and CYP2A7, CYP2A13, pseudogenes CYP2A7 (CYP2A7PC and CYP2A7PT), CYP2B, CYP2F, CYP2G and CYP2S, located within a 350 KB gene cluster, CYP2A6 may exist as a loss and duplicated gene.In addiction, it contains gene transformation, nucleotide, insert, and single nucleotide polymorphisms (SNPS). Gene polymorphism is an important factor that influence the activity of CYP2A6 which has nearly 30 kinds of mutations. The homozygous wild-type of CYP2A6 was CYP2A6*1/CYP2A6 *.CYP2A6*2 is the mutation of L160H,mutation frequency is 1% 3%, which is considered to the leading cause of PMs in Europe.CYP2A6*3 is the result of the genetic inversion mutation, detection method and the research of the mutation frequency and function of which still exists a lot of controversy. CYP2A6*4 is the result of 3 non-coding region gene loss, frequency of up to 15% 20%,is considered to be the leading mutation type cause of PMs in Asian populations. The researchers of CYP2A6* 4 in our country found frequency of PMs was as high as 13.3% consistent with high frequency mutation in the phenotype of CYP2A6 in Asian.CYP2A6 allele frequency distribution between different ethnic groups with differences that most of the allele frequency is higher in Asian populations. In addition, according to the genotypes,CYP2A6 can be divided into normal metabolism (normal,100%), intermediate (intermediate,75%), slow metabolism (missile,50%-25%), low metabolism (poor,< 25%) four groups to nicotine metabolism ability. At present, in different ethnic study of CYP2A6 and lung cancer susceptibility, appear inconsistent and even contradictory results.Based on the inconsistency in the above research results, it is necessary to prove the associate between genetic polymorphisms and lung cancer in different RACES and smoking status, and to investigate environmental and genetic risk factors which affected the function of the susceptibility genes. At present, although the research of susceptibility genes of lung cancer has made great progress, most of the data is based on western population. Data of Chinese is still lack, so study of lung adenocarcinoma in Chinese patients is particularly important. Through cases-contrast analysis, this paper discusses associate beween the chromosome nAChR and CYP2A6 polymorphism and lung cancer in the southern China,provide heoretical basis for the screen of the high risk population for lung cancer, early diagnosis and individualized treatment. The study will limited in the southern China patients with lung cancer, pick out the five nicotine acetylcholine receptors SNP loci and choose five polymorphism loci detection of CYP2A6 at the same time. Using iMLDR to type of nAChR in southern China in lung cancer patients and healthy controls and using multiple LONG-PCR electrophoresis combine with SNaPshot of CYP2A6 gene polymorphism to test gene polymorphism and sort out the test results,related statistical analysis using SPSS 13.0 software. Using Codominant model, the Dominant model, the implicit model, Overdominant model and the Log-i.e. the model respectively to explore the loci polymorphism distribution and the relationship between lung cancer, verify the lung cancer susceptibility SNP loci in southern China. Comprehensive data for southern China determine the high-risk group of patients with lung adenocarcinoma and individual prevention and treatment of preliminary data basis and theoretical basis, but also for the next step to establish suitable for China southern lung adenocarcinoma susceptibility genes to prepare risk assessment model.Method1.The research object Cases come from 204 cases of lung cancer patients was confirmed by pathology in the first affiliated hospital of guangzhou medical university from May 2013 to October. Control group come from chronic disease epidemiology investigation in guangzhou and zhuhai doumen district from July 2012 to June 2013. Research object derived from southern China, peripheral blood samples of all the sample was collected.2. Choosing SNP loci. Five SNP loci of nicotine acetylcholine receptors that lung cancer may be susceptible to were chose from NCBI SNP information database, genome-wide association analysis, and other positive loci from large sample research at home and abroad, they are rs6495308 from gene CHRNA3; rs11072768 from gene CHRNA4; rs3787140 from gene CHRNA4 and rs378140 and rs2072660 from gene CHRNA2; and YP2A6*4、CYP2A6*5、CYP2A6*7、CYP2A6*9和 CYP2A6* 10 five polymorphism loci at the same time.3.Genotyping and data analysis We did nAChR gene polymorphism in lung cancer patients and healthy controls in southern China with iMLDR technology; meanwhile we use multiple LONG-PCR electrophoresis and the SNaPshot together for CYP2A6 gene polymorphism and organizing test results, using SPSS 13.0 software for related statistical analysis. Respectively discuss each locus polymorphism distribution and the relationship between the risk of lung cancer in codominant model (Codominant model), the Dominant model (Dominant model), the implicit model (Recessive model), superdominance model (Overdominant model) and the additional model (Log-i.e. the model) five genetic model, Verify the lung cancer susceptibility SNP loci in southern China.4. Correlation study in each point polymorphism and lung adenocarcinoma patients gender, smoking status, pathological types, discuss genotype distribution and smoking behavior interaction in lung cancer.5. Smoking behavior and acetylcholine receptor and CYP2A6 gene polymorphism have interaction in the occurrence of lung cancer.ResultPart 1:relationship between nAChR, CYP2A6 gene polymorphism and lung cancer. In this study, we selected five SNPs in chromosome 15 q25.1 area and did genotypin g and statistical analysis in 204 lung adenocarcinoma patients in Guangdong provinc e and 821 healthy controls. The results show that except rsl 1072768, The other four site distribution of gene frequency were accord with Hardy Weinberg equilibrium (p >0,05). Case-control analysis find two polymorphism loci is associated with lung ad enocarcinoma risk. The gene loci rs2072660 is located in CHRNA2. Comparing with C/C genotype, its C/T genotype carriers are of higher risk, OR rate(95%Cl) is 1.708 (1.169-2.496), P= 0.006.This research did statistical analysis in parting of metabolism in 204 cases of lu ng cancer patients and 821 healthy controls group in Guangdong. The result show th at comparing with the healthy controls group, the intermediary metabolism and slow metabolism group show a reduced risk, the OR rate (95%CI) is 0.142 (0.041-0.485) P<0.001 and 0.561 (0.316-0.996), P<0.048 receptively. The low metabolism group has a risen risk, OR rate (95%CI) is 2.334 (1.552-3.581), P<0.001.The results show that gene polymorphism are not associated with the pathologic types of lung cancer.But for the anatomical position, CHRNB2 rs2072659 site G/G genotype may increase the risk of peripheral lung cancer, OR rate (95% CI) is 8.360(1.153-60.623), low metabolism CYP2A6 may decrease the risk of peripheral lung cancer,OR rate (95% CI) is 0.453(0.223-0.921)。 Part 2 Interaction of nAChR and CYP2A6 gene polymorphisms and genotype genotypes on smoking behaviorThe study analysis results show that CHRNB2rs2072660 C/T genotype reduced the smoking risk compared C/C genotype (OR=0.710,95%CI=0.530-0.953); The intermediary metabolism type (OR=5.809,95%CI=0.2611-12.924)and slow metabolism type (or=1.713,95%CI=1.107-2.649) CYP2A6 increased the risk of smoking, and low metabolic type (OR=0.466,95%CI=0.338-0.643) decreased the risk of smoking.At the same time, the interaction analysis results show CHRNA3rs6495308C/T genotype combine with low metabolic type CYP2A6 have positive interaction for smoking behavior (OR= 2.00,95% CI=1.021～3.919). Among the other genotypes interaction on smoking behavior does not exist. Part 3 The intersection between smoking nAChR and CYP2A6 polymorphism on lung cancer.This study analysis the counterpart between nAChR polymorphism and lung ca ncer; smoking and nAChR gene polymorphism; smoking and CYP2A6 gene polymo rphism. The results of multiple factors logistic regression analysis show that after fac tors like age, occupation, education, smoking and marriage was controlled, CHRNA 3rs6495308C/T genotype combine with CHRNB2rs2072659G/C genotype patients, CHRNA3rs6495308C/T genotype combine with CHRNA4rs3787140C/T genotype p atients, CHRNA3rs6495308C/T combine with CHRNB2rs2072660C/T genotype pat ients all show a vice interaction with lung cancer, the OR rate respectively is 0.378 (95%CI:0.170～0.841)、0.268 (95%CI:0.107-0.670)、0.277 (95%CI: 0.124-0.620), the other genotype doesn’t show interaction with smoking behavior. When age, occupation, education and marriage are combined, CYP2A6 metabolism parting with 0-15 cigarettes per day(OR=13.194,95%CI: 2.394～72.716), CYP2A6 metabolism parting with more than 15 cigarettes per day (OR=4.348,95%CI:1.672～11.308) show interaction with lung cancer, the others don’t show interaction.Conclusion1.This study shows that rs2072660 loci located in gene CHRNB2 is associated with population risk of lung cancer in southern China, in which rs8042374 site C/T genot ype may increase the risk of the individual. CHRNB2 rs2072659 site G/G genotype may increase the risk of peripheral lung cancer, low metabolism CYP2A6 may decrease the risk of peripheral lung cancer. CHRNB2 rs2072660 C/T genotype reduced the smoking risk; The intermediary metabolism type and slow metabolism type CYP2A6 increased the risk of smoking, and low metabolic type decreased the risk of smoking; CHRNA3 rs6495308C/T genotype combine with low metabolic type CYP2A6 have positive interaction for smoking behavior.2. Interaction analysis show that CHRNB2rs2072660 gene polymorphism and CHR NA3rs6495308 gene polymorphism show a negative interactions in lung cancer, CH RNA3rs6495308 and CHRNB2rs2072659 gene polymorphism show a negative inter actions in lung cancer, CHRNA3rs6495308 and CHRNA4rs3787140 gene polymorp hism show a negative interactions in lung cancer. CYP2A6 gene polymorphism and smoking behavior has a positive interaction on lung cancer.