| BackgroundLung cancer is a major public health concern worldwide,causing as many deaths as next four most deadly cancers combined(breast,prostate,colon,and pancreas).Non-small cell lung cancer(NSCLC)is the commonest lung cancer histology,comprised primarily of adenocarcinoma and squamous cell carcinoma.In 2014,being the leading cause of cancer-related deaths in China,lung cancer accounted for 23% of all female cancer deaths and 29% of all male cancer deaths,totally approximately 220,5200 deaths(World Health Statistics,WHO,2014).It has been established that multiple environment(mainly cigarette smoking and asbestos)and genetic factors are involved and gene-environment interactions exist in the development and process of lung cancer.In the past few years,several genetic variants and loci have been identified to be the genetic risk factors of lung cancer,to date,however,these known common loci only explain a small fraction of the familial risk of lung cancer and it is vital to investigate the remaining missing heritability to understand the development and process of lung cancer.The TOX3/LOC643714 locus on chromosome 16q12.1 was one of the first breast cancer regions identified through genome-wide association study(GWAS)in populations of European and East Asian origin.The chromosomal region spanning the 5’ end of TOX3,the intergenic region between TOX3 and LOC643714,and the entire coding part of LOC643714 are located in a 133 kb linkage disequilibrium(LD)block.LOC643714 is an uncharacterized gene of unknown function [http://www.ncbi.nlm.nih.gov/gene/643714].TOX3 gene,also termed trinucleotide repeat containing 9(TNRC9),belongs to the high-mobility group(HMG)family of nonhistone chromatin proteins,indicating that it may play important roles in banding and unbinding of DNA and alteration of chromatin structure.TOX3 is largely expressed within the brain in the frontal and occipital lobe,the central nervous system(CNS),and the ileum in normal human tissues.Through interaction with the c AMP-response-element-bindingprotein(CREB),TOX3 regulates Ca2+-dependent neuronal transcription.The overexpression of TOX3 induces transcription involving isolated estrogen responsive elements and estrogen-responsive promoters,and protects neuronal cells from cell death caused by endoplasmic reticulum stress or BCL2-Associated X Protein(BAX)overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts.In the several GWAS,a number of novel genetic variants and loci at the TOX3/LOC643714 locus were identified to be independently associated with elevated risk of breast cancer and rs3803662 was highlighted for its potential biological contribution to the development of breast cancer.The other SNPs at the TOX3/LOC643714 locus reported to be associated with risk of breast cancer or the other human diseases included rs3104746,rs8051542,rs4784227,rs12443621,rs3112612,rs3112562,rs3104793,rs8046994,and rs3104788.Though the known etiology and carcinogenesis of lung cancer are different from that of breast cancer,the patients of the two diseases could be treated by some common chemotherapeutic agents such as taxanes(paclitaxel and taxotere),vincristine(Navelbine,NVB),and platinum-containing anticancer drugs(cisplatin and carboplatin).Most importantly and interestingly,a recent meta-analysis based on four lung cancer GWAS in populations of European ancestry,the MD Anderson Cancer Center(MDACC)GWAS,the Institute of Cancer Research(ICR)GWAS,the National Cancer Institute(NCI)GWAS,and the International Agency for Research on Cancer(IARC)GWAS,identified a rare variant of BRCA2 gene,a well-known risk factor for breast,ovarian,and aggressive prostate cancers,to an increased risk of squamous cell lung cancer among cigarette smokers,suggesting that lung cancer and breast cancer are linked at a molecular and genetic level to a certain extent.Due to the findings that there is a molecular and genetic linkage between lung cancer and breast cancer and TOX3 may play important roles in banding and unbinding of DNA and alteration of chromatin structure,we deduced that the previously identified breast cancer susceptibility-associated variants and loci at the TOX3/LOC643714 locus may contribute to lung cancer risk.To test the hypothesis,16 SNPs at the TOX3/LOC643714 locus were selected and genotyped in a Han Chinese population from Southwestern China based on a case-control study.The genotyping data demonstrated that three SNPs(rs9933638,rs12443621 and rs3104746)at the TOX3/LOC643714 locus were associated with an elevated risk of lung cancer and might be potentially biologically relevant to lung carcinogenesis.Materials and methodsStudy population352 Patients with primary lung cancer,newly diagnosed,histologically confirmed and previously untreated from September 2007 to December 2008 were collected from the Institute of Human Respiratory Disease of the Second Affiliated Hospital,the Third Military Medical University.407 healthy control samples with matched age-and sex frequency were recruited at the Center of Physical Examination of the Second Affiliated Hospital between November 2007 and December 2008.Any history of cancer was the exclusion criterion for the healthy control.At least within three generations,the subjects were unrelated.All participants signed a written informed consent form,completed a detailed questionnaire regarding their smoking habits,and donated 5 ml peripheral blood after they understood the purpose and procedures of the study.All of the blood samples were drawn into Na-EDTA tubes and stored at-70?C for genomic DNA extraction.The study was approved by the Ethical Committee of the Second Affiliated Hospital,the Third Military Medical University.Selection of SNPsTotally,16 SNPs at the TOX3/LOC643714 locus were selected in the study.Of the 16 SNPs,except six SNPs(rs8051542,rs12443621,rs3803662,rs3104746,rs3112562,and rs4784227)were selected based on the published references in which these SNPs were suggested to be susceptible to breast cancer or the other human diseases,the other 10 SNPs within TOX3 were selected from the genetic variation data for TOX3 gene obtained from the Hap Map project for 45 healthy Chinese Han Beijing(CHB)adults(www.hapmap.org).Haplotype blocks,representing regions inherited without substantial recombination in the ancestors of the current population,were constructed throughout the entire TOX3 gene using Haploview(version 4.0,Broad Institute of MIT and Harvard,Cambridge,MA).The history of recombination between a pair of SNPs can be estimated with the use of the normalized measure of allelic association D’(value of D prime between the two loci).The criterion for the selected SNPs to construct a haplotype block is that all SNPs in one region must be in strong LD with D’ > 0.98 for the upper 95% confidence bound and > 0.7 for the lower bound.A maximally informative ht SNP was then selected from each block using the software Tagger program(http://www.broad.mit.edu/mpg/haploview).This algorithm selects a subset of variants that capture all known common genetic variations in the TOX3 gene based on a LD threshold of r2 ≥ 0.8.The inverse of r2 represents the ratio of sample size needed to detect an indirect association with an un-analyzed SNP to direct association at the same power.Genotyping analysisGenomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit according to the manufacturer’s instructions(QIAGEN,Maryland,USA).The SNP genotyping was performed using an improved multiplexligation detection reaction(i MLDR)technique(Genesky Biotechnologies Inc.,Shanghai,China).In brief,the selected SNP loci were genotyped in one ligation reaction.Two multiplex PCR reactions were designed to amplify fragments covering all SNP loci.The primer information of the two reaction mixtures is described in Supplementary Table 1 and 2,respectively.The PCR for both reactions was 95?C,2 min;11 cycles(94?C,20s;65?C-0.5?C/cycle,40s;72?C,1 min 30s);24 cycles(94?C,20s;59?C,30s;72?C,1 min 30s);72?C,2 min;hold at 4?C.The ligation cycling programme was 95?C,2 min;38 cycles(94?C,1 min;56?C,4 min);hold at 4?C.Half a microlitre of ligation product was loaded into the ABI 3730 XL and the raw data were analysed by Gene Mapper 4.1.Data analysesCigarette smoking was stratified by the median number of pack-years of combined cases and controls(1 pack-year = 20 cigarettes per day for 1 year).Cases and controls were compared by Student’s t-test for continuous variables and Pearson’s chi-square test or Fisher’s exact test for categorical variables.The Hardy-Weinberg equilibrium of each SNP was tested by SNPStats(http://bioinfo.iconcologia.net/snpstats/start.htm).Each component of the model was: codominant model(major allele homozygotes vs.heterozygotes vs.minor allele homozygotes),dominant model(major allele homozygotes vs.heterozygotes + minor allele homozygotes),recessive model(major allele homozygotes + heterozygotes vs.minor allele homozygotes),and overdominant model(major allele homozygotes + minor allele homozygotes vs.heterozygotes).To assess the independent effect of each SNP,the multivariate logistic regression analyses with adjustments for possible confounding factors(age,gender,and smoking habits)were performed to estimate the association between the SNPs and cancer risk as well as the possible gene-environment interactions.All associations were presented as odds ratios(ORs)with the corresponding 95% confidence intervals(95%CI).All statistical analyses were performed using the Statistical Package for Social Science 15 for Windows(SPSS Inc,Chicago,IL,USA).In the statistical analysis,all statistical tests were two-sided and P < 0.05 was considered significant.ResultsSubject characteristicsTotally,352 unrelated patients and 407 unrelated controls were recruited from Southwestern China for the case-control study.No female cigarette smokers were gathered.The general descriptive characteristics of the study population were given in Table 1.The median number of pack-years of combined cases and controls was utilized as the cut-point to stratify the cigarette smoking subjects.As shown in Table 1,there were no significant difference in gender and age between the controls and cases.As expected,cases smoked more cigarettes(P < 0.001).The distribution of tumour types among the patients was as follows: adenocarcinoma,42.05%;squamous cell carcinoma,28.13%;other non-small cell carcinoma,16.19%;and small cell carcinoma,13.64%.Association of the alleles of the 16 SNPs with lung cancer riskThe basic information regarding the 16 SNPs at the TOX3/LOC643714 locus was demonstrated in Table 2.The 16 SNPs were genotyped in all of the lung cancer patients and healthy controls and qualified according to Hardy-Weinberg equilibrium(HWE)in the study population(Supplementary Table 3).As shown in Table 2,the Pearson’s chi-square test or Fisher’s exact test demonstrated that of the 16 SNPs,three SNPs(rs9933638,rs12443621,and rs3104746)were found to be significantly associated with lung cancer risk(P < 0.001,P < 0.001,and P = 0.005,respectively)and rs3095661 displayed a marginally significance(P = 0.041,Table 2).Among the four SNPs,rs9933638,representing a haplotype block covering rs12443621,is located at intron 2 of TOX3.Rs3095661 and rs3104746 are located at intron 4 of TOX3 and intron 2 of LOC643714,respectively.Association of the genotypes of the four SNPs(rs9933638,rs12443621,rs3104746,and rs3095661)with lung cancer riskAs shown in Table 3,consistent with the association between the alleles of the three SNPs(rs9933638,rs12443621,and rs3104746)and lung cancer risk,there was a significantly different distribution of the genotypes of the three SNPs between lung cancer cases and controls in the Southwestern Han Chinese population(P < 0.001,P < 0.001,and P = 0.003,respectively).The multivariate logistic regression analyses with adjustment of age,gender,and smoking revealed that individuals with rs9933638 GG had an elevated risk of lung cancer compared with rs9933638 GA and rs9933638 AA carriers(codominant model,OR = 2.571,95%CI = 1.710-3.867,P < 0.001,and OR = 1.509,95%CI = 1.022-2.229,P = 0.038,respectively).The dichotomic analysis further demonstrated that individuals with rs9933638 GG showed an increased risk of lung cancer compared with rs9933638(GA+AA)carriers(dominant model,OR = 1.877,95%CI = 1.423-2.476,P < 0.001).Additionally,individuals with rs9933638(GG+GA)also displayed an increased risk of lung cancer compared with rs9933638 AA carriers(recessive model,OR = 1.893,95%CI = 1.308-2.741,P = 0.001),suggesting that individuals with the allele G of rs9933638 were susceptible to lung cancer in a dose-dependent manner.Similar findings were discovered for rs12443621,indicating that subjects with the allele G of rs12443621 were susceptible to lung cancer in a dose-dependent manner.For rs3104746,the dichotomic analysis demonstrated that compared with individuals with rs3104746(TA+AA),rs3104746 TT carriers had an increased risk of lung cancer(dominant model,OR = 1.665,95%CI = 1.243-2.230,P = 0.001),suggesting that individuals with rs3104746 TT were susceptible to lung cancer compared with rs3104746(TA+AA)carriers.Moreover,though a marginally significant difference of rs3095661 alleles was found between controls and cases(P = 0.041,Table 2),no significant difference of rs3095661 genotypes was found in the four models between controls and cases(P = 0.362,P = 0.066,P = 0.999,P = 0.197,respectively,Table 3).Notably,the detection of rs3095661 CC carriers only in lung cancer patients(6/352,1.70%)but not in controls strongly suggested that rs3095661 CC might be risk factor of lung cancer.Distribution of rs9933638,rs12443621,and rs3104746 among lung cancer patients stratified by cigarette smokingBecause rs9933638,rs12443621,and rs3104746 were found to be associated with lung cancer risk,the distribution of the three SNPs was analysed among lung cancer patients stratified by median number of pack-years of cigarette smoking to investigate whether the gene-environment interaction exists.The linear-by-linear association test revealed that except rs3104746(P = 0.638),the risk allele G of both rs9933638 and rs12443621 showed an increasing trend from light smoking to heavy smoking groups(P = 0.004 and P < 0.001,respectively,p trend < 0.001,Table 4).Multivariate logistic regression analyses adjusted for age and gender revealed that there was a significantly different distribution of genotypes of both rs9933638 and rs12443621 between light smoking patients and heavy smoking patients.Compared with patients with rs9933638 GA and 9933638 AA,patients with rs9933638 GG were enriched and demonstrated an increased risk of lung cancer in heavy smoking subjects(codominant model,OR = 1.714,95%CI = 1.155-2.542,P = 0.007;OR = 2.811,95%CI = 1.405-5.625,P = 0.003,respectively,Table 4).In the dominant model,patients with rs9933638 GG carriers were enriched and showed an elevated risk of lung cancer in heavy-smoking patients compared with rs9933638(GA+AA)carriers(OR = 1.878,95%CI = 1.290-2.736,P = 0.001).In the recessive model,lung cancer risk of individuals with rs9933638(GG+GA)in heavy-smoking patients was 2.87 times higher than that of rs9933638 AA carriers(OR = 2.868,95%CI = 1.573-5.227,P = 0.001).Similarly,patients with rs12443621 GG were also enriched in heavy smoking patients and demonstrated an increase risk of lung cancer compared with rs12443621 GA and rs12443621 AA carriers(codominant model,OR = 1.821,95%CI = 1.222-2.713,P = 0.003,and OR = 3.067,95%CI = 1.541-6.104,P = 0.001,respectively,Table 4).In the dominant model,individuals with rs12443621 GG carriers were enriched in heavy-smoking patients and showed an elevated risk of lung cancer compared with rs12443621(GA+AA)carriers(OR = 2.018,95%CI = 1.381-2.947,P < 0.001).In the recessive model,lung cancer risk of individuals with rs12443621(GG+GA)in heavy-smoking patients was 2.87 times higher than that of rs12443621 AA carriers(OR = 2.868,95%CI = 1.599-5.143,P < 0.001).The findings demonstrated that the individuals carrying risk genotypes and with heavy cigarette smoking may have a higher risk of lung cancer,suggesting that the gene-environment interaction exists in the development and process of lung cancer. |
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