AHR和PPAR-γ基因多态性与肿瘤遗传易感性研究

【作者】 陈丹；

【导师】 卢大儒；

【作者基本信息】 复旦大学 ， 遗传学， 2009， 博士

【摘要】 第一部分肺癌是全世界头号癌症“杀手”。吸烟是导致肺癌的主要因素,大约87%的肺癌都是由吸烟引起的。然而,只有不到20%的吸烟者最终会罹患肺癌,提示遗传因素以及基因与吸烟的交互作用在肺癌的发生、发展中可能起到重要作用。存在于烟草烟雾中的致癌物多环芳烃(PAHs)的致癌作用主要是通过芳香烃受体(AHR)所介导的,它是一个配体依赖的转录因子,可以调节受烟草烟雾诱导表达的致癌物代谢酶的转录。我们推测AHR基因上的遗传变异可能会影响个体对于肺癌易感性的差异,为了检验这一假设,我们采用了病例-对照的研究方法,选择了该基因上的8个单核苷酸多态(SNP)位点,利用Illumina和Taqman分型方法对500例中国汉族肺癌患者和517例年龄、性别、地区与之相匹配的正常对照进行了基因分型,研究了不同等位基因型、基因型、单倍型与肺癌风险的相关性,并探讨了基因-环境的交互作用与肺癌遗传易感性的关系。对于多重检验可能带来的假阳性问题,本研究采用100,000次的置换检验方法予以控制。在单位点分析中,我们发现rs2158041和rs7811989两个SNP位点的基因型分布在病例和对照中存在显著的统计学差异,P值分别为0.0038和0.008。经过置换检验后,这两个位点的基因型分布差异仍显示出显著性(P值分别为0.0193和0.0407)。非条件logistic回归分析表明,rs2158041位点的突变杂合基因型可以显著增加患肺癌的风险(与GG基因型相比,GA基因型的校正OR=1.53,95%CI=1.17-1.99),rs7811989位点的突变杂合基因型对于罹患肺癌具有相同的效应(与GG基因型相比,GA基因型的校正OR=1.48,95%CI=1.13-1.93)。此外,单倍型分析表明,AHR基因的单倍型在病例和对照组中的分布具有显著性差异(全局性检验P=1.38e-5)。我们还发现,当把累计吸烟量作为分类变量或是连续变量来计算时,非同义突变位点rs2066853(p.Arg554Lys)与累计吸烟量均存在统计学上显著的交互作用(P值分别为0.033和0.019)。突变基因型Lys/Lys在重度吸烟者中可以显著增加患肺癌的风险(校正OR=3.36,95%CI=1.07-10.55)。以上研究结果提示,AHR基因多态和该基因与吸烟的交互作用可能在肺癌的发病过程中扮演着一定角色。第二部分很多研究证据表明过氧化物酶增殖活化物受体γ(PPAR-γ)被活化后可以削弱由吸烟引起的炎症反应并且可以抑制肺部肿瘤的生长,提示PPAR-γ在肺癌的发生、发展中可能起到肿瘤抑制因子的作用。我们推测,PPAR-γ基因上的遗传变异可能对个体罹患肺癌的风险造成影响。为了验证这一假设,本文采用病例-对照的研究方法,在中国汉族人群的500例肺癌患者和517例年龄、性别、地区与之相匹配的正常对照中,通过Illumina高通量基因分型技术对该基因上11个SNP多态与肺癌易感性的关系进行了研究,并且探讨了PPAR-γ基因与吸烟的交互作用对于肺癌患病风险的影响。我们首次发现,该基因上的7个SNP的突变基因型与肺癌风险降低具有相关性,在显性模型下,rs13073869和rs1899951的P值分别为0.0004和0.0130;在加性模型下,rs4135247的P值为0.0310;在超显性模型下,rs2972162、rs709151、rs1175541和rs1175543的P值分别为0.0468、0.0175、0.0172和0.0386。其中,rs13073869和rs1899951两个位点经过100,000次的置换检验校正后仍为阳性。与单位点分析一致,单倍型和二倍型分析均显示由rs13073869,rs1899951和rs4135247位点组成的单倍型‘AGA’和‘AAA’具有保护效应。与携带有单倍域1中最常见的单倍型‘GGG’的个体相比,单倍型‘AGA’(校正OR=0.80,95%CI=0.65-0.98)和‘AAA’(校正OR=0.57,95%CI=0.37-0.87)的携带者患肺癌的风险显著降低。携带有1～2个‘AGA’拷贝的个体与非携带者相比,罹患肺癌的风险降低了28%(校正OR=0.72,95%CI=0.56-0.93);而携带有1～2个‘AAA’拷贝的个体与非携带者相比,罹患肺癌的风险降低了42%(校正OR=0.58,95%CI=0.37-0.90)。此外,我们发现rs1899951位点与吸烟之间存在显著的基因-环境交互作用。根据HapMapⅡ期中国汉族人群的基因分型数据,rs13073869与PPAR-γ3亚型启动子区的已知功能位点C-681G(rs10865710)之间存在完全的连锁不平衡(A等位基因与-681 G等位基因连锁,D’=1.00,r~2=1.00)。此外,rs1899951位点与PPAR-γ2亚型特异外显子B上的已知功能位点p.Pro12Ala(rs1801282)存在完全的连锁不平衡(A等位基因与Ala12等位基因连锁,D’=1.00,r~2=1.00)。这个基因真正的致病位点的确切位置及生物功能有待于进一步的研究和探索。第三部分神经胶质瘤(简称胶质瘤)是神经系统最常见的原发性肿瘤,占所有脑肿瘤的70%以上。其中,多形性胶质母细胞瘤(简称胶质母细胞瘤)是成年人中恶性程度最高、入侵性最强的胶质瘤。越来越多的体内外研究证实,PPAR-γ被配体活化后可以抑制胶质瘤的生长、增殖,诱导其调亡,并阻断胶质瘤细胞的迁徙和入侵性。目前,该基因上已知的功能比较明确的两个位点分别是C-681G(rs10865710)和p.Pro12Ala(rs1801282)。在本论文的第二部分的研究工作中,我们发现与肺癌关联最显著的两个位点rs13073869和rs1899951分别与上述两个已知的功能位点存在完全的连锁不平衡。鉴于脑胶质瘤和肺癌在发病机制上的相关性,以及PPAR-γ在二者中具有相似的抗肿瘤作用,我们推测C-681G和p.Pro12Ala可能会影响脑胶质瘤的遗传易感性。为此,我们在包含有241例胶质母细胞瘤,284例星形细胞瘤(胶质母细胞瘤除外),241例其他类型胶质瘤病例以及824例正常对照的中国人群病例一对照研究中,探讨了C-681G和p.Pro12Ala与脑胶质瘤的相关性。logistic回归表明,与C-681G位点的野生型等位基因C相比,突变型等位基因G与胶质母细胞瘤风险降低显著相关(校正OR=0.76;95%CI=0.61-0.96;P=0.020),并且这种保护效应随着突变等位基因数目的增加而增强(趋势检验P=0.024)。p.Pro12Ala与胶质母细胞瘤的相关性没有达到统计学意义的显著性,但表现出保护性趋势(校正OR=0.68;95%CI=0.41-1.15;P=0.151)。因此,在胶质母细胞瘤中,C-681G和p.Pro12Ala的突变等位基因可以看作是潜在的低风险等位基因。然而,在星形细胞瘤和其他胶质瘤类型中,并没有观察到这两个位点的任何效应。当我们对这两个位点进行联合效应分析时,与不携带低风险等位基因的个体相比,携带有1个低风险等位基因的个体与胶质母细胞瘤相关性的OR值和95%CI分别为0.85和0.61-1.18,而携带有两个以上低风险等位基因的个体与胶质母细胞瘤相关性的OR值和95%CI分别为0.50和0.32-0.79,显示出PPAR-γ基因功能位点的突变等位基因对胶质母细胞瘤的保护作用具有显著的等位基因剂量依赖效应(趋势检验P=0.004)。我们的研究表明PPAR-γ基因的C-681G位点可能在胶质母细胞瘤中发挥作用,这与和它存在完全LD的rs13073869位点突变等位基因在肺癌中的保护效应一致。更多还原

【Abstract】 PartⅠLung cancer is the leading cause of cancer deaths in the world with a poor prognosis and an overall 5-year survival rate of＜15%.The epidemic of lung cancer is directly attributable to cigarette smoking that accounts for 87 percent of lung cancer cases.However,only a small fraction of smokers(usually＜20%)develop lung cancer in their lifetime.It is well established that susceptibility to lung cancer may in part be attributable to inter-individual variation in metabolic activation or detoxification of tobacco carcinogens,suggesting the importance of genetic determinants in lung cancer etiology,including the gene-environment interaction between genetic polymorphisms and environmental factors,such as smoking.Most of the carcinogenic effects of polycyclic aromatic hydrocarbons(PAHs) present in tobacco smoke are mediated by the aryl hydrocarbon receptor(AHR),a ligand-dependent transcription factor that regulates tobacco-induced expression of carcinogen metabolic enzymes.To test the hypothesis that genetic variations in AHR may confer individual susceptibility to lung cancer,we genotyped for eight selected single nucleotide polymorphisms(SNPs)in AHR in a case-control study of 500 lung cancer patients and 517 cancer-free controls in a Chinese population.We observed statistically significant differences between case patients and control subjects in genotype distributions for two SNPs(P=0.0038 for rs2158041 and P=0.008 for rs7811989)and the significance remained after applying 100,000-time permutation tests(P=0.0193 for rs2158041 and P=0.0407 for rs7811989).Further logistic regression analyses revealed that the significantly increased lung cancer risk was associated with heterozygous genotypes of rs2158041(adjusted odds ratio=1.53 and 95%confidence interval=1.17-1.99 for GA,compared with the GG genotype)and rs7811989(adjusted odds ratio=1.48 and 95%confidence interval=1.13-1.93 for GA, compared with the GG genotype).Furthermore,haplotype analysis revealed significant differences in haplotype distributions of AHR between cases and controls(Global P= 1.38e-5).We also observed statistically significant interaction between the polymorphism rs2066853(p.Arg554Lys)and cumulative cigarette smoking as a discrete or continuous variable(P=0.033 and 0.019,respectively),and the Lys/Lys genotype conferred an increased risk of lung cancer in the heavy smokers(adjusted odds ratio=3.36 and 95%confidence interval=1.07-10.55).These findings suggest that AHR polymorphisms and potential gene-smoking interaction may be involved in the etiology of lung cancer. PartⅡAccumulating evidence indicates that activation of the peroxisome proliferator-activated receptor-γ(PPAR-γ)dampens the inflammation cascade caused by cigarette smoking and inhibits tumor growth of the lung,suggesting that it has tumor suppressor functions in the pathogenesis and progression of human lung cancer.We hypothesized that genetic variation in the PPAR-γgene may have an impact on individual risk of lung cancer.To test this hypothesis,we conducted a case-control study of 500 incident lung cancer cases and 517 age and sex frequency-matched cancer-free controls in a Chinese population,and genotyped 11 single nucleotide polymorphisms (SNPs)of PPAR-γusing an Illumina high-throughput genotyping platform.We also investigated potential interactions between polymorphisms of the PPAR-γgene and cigarette smoking in lung cancer risk.The issue of multiple tests was controlled by using 10,000-time permutation tests.We found,for the first time,that decreased lung cancer risk was statistically significantly associated with seven SNPs(P=0.0004 for rs13073869 and 0.0130 for rsl899951 in a dominant model;P=0.0310 for rs4135247 in a log-additive model;and P=0.0468 for rs2972162,0.0175 for rs709151,0.0172 for rs11715541 and 0.0386 for rs1175543 in an overdominant model).The difference for two SNPs(rs13073869 and rs1899951)remained significant after applying 10,000-time permutation tests. Consistent with these results of single-locus analysis,both the haplotype and diplotype analyses revealed a protective effect of the haplotype ’AGA’ and ’AAA’ of rs13073869, rs1899951 and rs4135247.The risk of lung cancer was significantly decreased among individuals carrying the haplotype ’AGA’(adjusted OR=0.80 and 95%CI=0.65-0.98), and ’AAA’(adjusted OR=0.57 and 95%CI=0.37-0.87),compared with those carrying the most common haplotype ’GGG’ in block 1.Subjects carrying 1～2 copies of the haplotye ’AGA’ had a 28%reduced lung cancer risk(adjusted OR=0.72 and 95% CI=0.56-0.93),and those carrying 1～2 copies of ’AAA’ had a 42%reduced lung cancer risk(adjusted OR=0.58 and 95%CI=0.37-0.90)compared with their respective non-carriers Furthermore,we observed a statistically significant interaction between the rs1899951 and cigarette smoking.Our results indicate that PPAR-γpolymorphisms and their interaction with smoking may contribute to the etiology of lung cancer.According to the phaseⅡHapMap data on 45 Han Chinese,the rs13073869 polymorphism was in perfect LD(D’=1.00,r~2=1.00)with a OG substitution (rs10865710)in the PPAR-γ~3 regulatory region at position-681 in PPAR-γ~3-specific exon A2(the A allele being associated with the-681 G allele).In vitro,the-681 G allele completely abolished the binding of STAT5B to the cognate promoter element as well as the transactivation of PPAR-γ3 by the growth hormone/STAT5B pathway.Furthermore, rs1899951 is in perfect LD with the non-synonymous polymorphism p.Pro12Ala (rs1801282)in PPAR-γ2-specific exon B(the A allele being associated with the Ala12 allele).This amino acid is located in a PPAR-γ2 domain that enhances ligand independent activation,and the Pro-to-Ala exchange may cause a conformational change in the protein,thus affecting its activity.The exact location and biological functions of the real causal SNPs of the gene is of great interest and warrants further investigation. PartⅢGliomas are the most common primary tumors in the central nervous system(CNS), which account for more than 70%of all brain tumors,with glioblastoma multiformes (GBMs)as the most lethal and aggressive intracranial neoplasm striking adults. Increasing evidence demonstrates that PPAR-γactivation by agonists induce apoptosis and inhibit glioma cell migration and brain invasion in vivo and in vitro.Two functional polymorphisms have been identified in the PPAR-γgene.One is a praline to alanine substitution,located at codon 12(p.Pro12Ala,rs1801282)of the PPAR-γ2-specific exon B,which may cause a conformational change in the protein thus affecting its activity.Another is a C to G variant(c.-681C＞G,rs10865710)in the promoter region for the PPARγ-3 transcript at position-681 from the beginning of exon A2,which completely abolished the binding of STAT5B to the cognate promoter element as well as the transactivation of the PPARγ-3 promoter.The polymorphisms of rs10865710 and rs1899951 which were found to be significantly associated with lung cancer in our study were in perfect LD(D’=1.00,r~2=1.00)with c.-681C＞G and p.Pro12Ala,respectively.We hypothesized that these two functional polymorphisms might be the causal loci and may confer individual susceptibility to glioma.To test this hypothesis,we investigated the association between these two SNPs and glioma risk in a case-control study of 241 glioblastoma cases,284 astrocytoma cases(except glioblastoma),241 other glioma cases,and 824 healthy controls.We found, for the first time,the variant G allele c.-681C＞G was significantly associated with reduced risk of glioblastoma(adjusted OR=0.76;95%CI=0.61-0.96;P=0.020),with the estimated effect following a trend of decreasing magnitude by number of variant alleles (P for trend=0.024).The polymorphism rs1801282 showed a similar trend as rs10865710 toward association in glioblastoma,although it did not reach statistical significance(adjusted OR=0.68;95%CI=0.41-1.15;P=0.151).When the combined effect of these two polymorphisms were examined,the ORs(95%CIs)for glioblastoma were 0.85(0.61-1.18)for subjects possessing only one low-risk allele and 0.50 (0.32-0.79)for those possessing two or three low-risk alleles,compared to subjects without any low-risk allele,suggesting a significant locus dose-response effect on glioblastoma(P for trend=0.004).However,no significant association with risk of astrocytoma and other glioma was observed for p.Pro12Ala or c.-681C＞G in our study. These findings suggest that the functional SNP c.-681C＞G appears to play a significant role in glioblastoma,the most lethal and aggressive primary brain tumor striking adults.更多还原