Association Study Of Common Genetic Variants At Chromosome 15q25 And Lung Cancer Risk

Lung cancer is one of the most common malignant tumors which forming a threat to human health worldwide.Recently,owing to the sustained growth of tobacco consumption,environment contamination and changes in lifestyle,the epidemic situation of lung cancer has become increasingly serious in our country.The occurrence of lung cancer is the result of the combination of environmental and genetic factors.Although the pathogenesis has not been fully elucidated,the epidemiological data showed that smoking is the environmental risk factor which most related to lung cancer risk.The risk of lung cancer in smokers is 20 times more than in nonsmokers.And over 90%of lung cancer cases are associated with tobacco or addiction.Meanwhile,it is worth noting that less than 20%of smokers develop lung cancer under the similar exposure,suggesting that there is an inter-individual variation in genetic susceptibility to lung cancer.Since 2008,genome-wide association studies(GWAS)have achieved great success in researching the genetic basis of lung cancer in multi-ethnic populations,identifying several lung cancer susceptibility loci,such as chromosomes 5p15,6p21 and 15q25.Independent results of GWAS from France and America showed that genetic susceptibility at chromosome 15q25 locus(CHRNA3-CHRNA5-CHRNB4)was closely related to lung cancer risk.However,studies in China have failed to repeat the results of the variants at 15q25 locus(rs8034191,rs 16969968 and rs1051730).The reason may be the genetic heterogeneity in different populations with the rarity of minor allelic frequencies(MAF)in China(all MAF,<0.05).To provide a comprehensive association map of this locus,our previous work identified four common genetic variants(all MAF,>0.05),such as rs2036534,rs667282,rs12910984 and rs6495309,the same coded allele frequency of which varied widely.Together,these findings underscored the difference of lung cancer susceptibility loci in the ethnic and regional.Besides,members of nicotine gene family CHRNA3-CHRNA5-CHRNB4 can encode nicotinc acetylcholine receptors(nAChRs),which is associated with smoking behavior and nicotine addiction of individual.And receptors located in the lung epithelial cells participated in signal transduction of tobacco carcinogens,to inhibit apoptosis and promote tumor cell proliferation.Although it has been reported that the mutation loci of the gene family was associated with lung cancer risk in western and Asian people,but the mechanism has not been determined.Is it the indirect effect mediated by smoking,or direct effect on lung cancer molecular pathways,or both,that there has been no unified conclusion at present.Previous work mainly used haplotype-tagging strategy to select representative SNPs based on linkage disequilibrium,which may screen the susceptibility polymorphisms effectively.Though further population association and functional variation,true positive variants could be found.However,it may miss vast false negative variants and also be different to test whether these variants identified are causal SNPs.Based on the deficiencies in the above research results,it is necessary to provide a comprehensive association map of 15q25 locus in different populations,and to further investigate genetic factors and smoking behavior which affected the potential function of the susceptibility genes.At present,the rapid development of next-generation sequencing(NGS)requires a separate step for capture the specific genomic regions of interest before sequencing,making up for the blind spots of previous researches based on chip.We conducted a two-stage case-control study.In the discovery phase,the 200cases defined as newly diagnosed lung cancer patients and 300 controls selected from routine physical examinations in local hospitals or a community-based screening program for noninfectious disease,which were frequently-matched to the cases for age and sex were recruited from Nanjing since 2003 to 2009.In the validation phase,samples obtained from our published NJMU GWAS(Nanjing Medical University GWAS from Nanjing and Beijing:2331 cases and 3077 controls)as internal validation and NGI GWASs(the National Cancer Institute GWASs:5716 cases and 5821 controls)as external validation.In the discovery stage,we explored the LD block spanning from chr15:78800300 to chr15:78983700 based on the combination of Agilent SureSelect Target Enrichment System and next-generation sequencing.We identified 1385 variants in total through latter multi-step analysis and screening based on bioinformatics and biostatistics.To ensure the power of results,following analysis mainly focused on the 369 common variants(MAF?0.05).Specific analysis included that:we got point mutations(SNPs)by processing the raw data with a series of software such as FASTQ,BWA,Picard Tools and GATK;we calculated the P values,odds ratio(OR)and 95%confidence intervals(CI)in the additive model by logistic regression;an approximate conditional and joint analysis approach using genome-wide complex trait analysis(GCTA)was performed to select index variants in a specific region;multiple public database resources were used to evaluate the function and expression quantitative trait locus(eQTL)of the variants;and finally,we implemented mediation analysis to assess whether smoking behavior mediated the effect of SNP on lung cancer risk.We found several significant signals at 15q25 in the discovery data and GCTA analysis revealed two novel SNPs with independent effects(rs6495304:OR=1.79,P=9.37×10-04;rs7473352:OR=1.68,P=8.05×10-03).These results were supported by in internal validation(rs6495304:P=5.16×10-10;rs74733525:P=4.29×10-04);however,external validation showed that the significance reduced(rs6495304:P=6.27×10-3),suggesting that the heterogeneity among different populations.Besides,rs6495304 demonstrated significant effects only in men or ever-smokers(P= 0.004 for interaction analysis with smoking).Analysis of eQTLs showed the risk allele(A)of rs6495304 was significantly associated with lower mRNA expression of CHRNA3(P=0.029)in 81 hypothalamic tissue samples.In addition,mediation analysis suggested that smoking behavior may mediate the effect of rs6495304 on lung cancer risk partially.In this study based on targeted resequencing technology,we systematically investigated the association between genetic architecture of common variants in this region and lung cancer risk.We identified two novel SNPs previously undetected in Chinese population through a two-stage case-control study with a large sample populations,and further mediation analysis and functional annotation suggested that rs6495304 may mediate the occurrence of lung cancer related to smoking behavior through activation of acetylcholine receptor genes.This finding provides new insights into the association between lung cancer susceptibility and the 15q25 locus.It provides theoretical basis for the screen of the high risk population for lung cancer,early diagnosis and individualized treatment.