DNA损伤修复基因多态性与肺癌的遗传易感性及药物基因组学研究

【作者】 吴文婷；

【导师】 卢大儒；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 第一部分DNA修复基因遗传变异与肺癌易感性研究第一节GTF2H1基因多态与肺癌遗传易感性研究肺癌已成为全球范围内发病率最高的癌症,80-90%肺癌的发生与吸烟有着重要的联系,但只有10-15%吸烟者会得肺癌,提示遗传因素、遗传多态性在肺癌的发生、发展中起到重要作用。DNA修复基因多态性作为个体损伤修复能力差异的分子遗传学基础,可能是肺癌的遗传易感因素。GTF2H1 (p62)蛋白在核苷酸切除修复(NER)通路中发挥重要作用,参与XPC-HR23B蛋白早期损伤识别,并招募核酸内切酶XPG到损伤部位完成酶切过程。此外,GTF2H1蛋白参与转录并调节多个基因转录激活。我们推测GTF2H1基因上的遗传变异可能单独或联合影响中国人群肺癌的遗传易感性。为了验证这一假设,我们在中国汉族人群的500例肺癌患者和517例年龄、性别与之相匹配的正常对照中,对GTF2H1基因上的6个单核苷酸多态(SNPs)位点进行了基因分型,探讨了GTF2H1基因的遗传多态对肺癌患病风险的影响。同时,为研究-79 C>T多态改变对GTF2H1基因的转录调控作用,我们构建了分别含有C和T等位基因的GTF2H1启动子区域的pGL3-Basic报告基因载体,进行了体外转录活性实验。我们发现,在显性模型下,3个SNP位点的突变基因型与野生型相比显著增加肺癌风险效应,rs3802967(校正OR=1.38；95%CI=1.04-1.82),rs4150606(校正OR=1.44；95%CI=1.08-1.92)和rs4150678(校正OR=1.37：95%CI=1.04-1.81)。rs3802967 C／T+T／T基因型在男性人群中风险性更加显著(P=0.002)。rs4150667显示出与肺癌的负相关,这一风险在隐性模型下更为显著(校正OR=0.59,95%CI=0.38-0.93)。单倍型分析显示单倍型“222212”(1代表常见等位基因,2代表罕见等位基因)显著增加肺癌患病风险性(P=0.03)。荧光素酶报告基因检测实验显示携带T-等位基因报告基因载体有更高的荧光素酶表达活性,提示-79C→T改变可能影响GTF2H1基因转录调控。因此,我们认为GTF2H1单核苷酸／单倍型多态可能与中国人群肺癌遗传易感性有关。第二节Rad52基因多态与肺癌遗传易感性和药物基因组学研究烟草等外源物质的暴露会造成双链断裂(DSBs).同源重组(HR)修复是真核生物双链断裂损伤修复的主要方式,对于维持哺乳动物细胞的基因组完整性十分重要。Rad52在同源重组早期结合到DSBs上,保护它免于外切核酸酶的攻击,并协助由Rad51介导的同源重组的发生,在肺癌发生过程中扮演重要角色。我们在中国汉族人群的500例肺癌患者和517例年龄、性别与之相匹配的正常对照中,对Rad52基因上的9个单核苷酸多态(SNPs)位点进行了基因分型,探讨了Rad52基因的遗传多态对肺癌患病风险的影响。同时,为研究3’UTR-104G>A多态改变对Rad52基因的表达水平调控作用,我们构建了分别含有G和A等位基因的Rad52 3’UTR区域的pGL3-Promoter报告基因质粒,进行了体外转录活性实验。并通过生物信息学软件预测和SPR实验进一步探讨了等位基因间表达差异的分子机理。单位点分析发现,Rad52基因3个位于调控区SNP rs10774474(P=0.002), rs11571378(P=1.647×10-5),rs1051669(P=0.002)在肺癌病例和对照组中的分布具有显著差异,经Bonferroni多重校正后,关联均仍具有显著性。全局显著性检验表明rs10774474-rs11571378构成的单倍型在病例组和对照组中的分布频率存在显著差异(P=0.007),10000次置换检验后,关联仍显著。单倍型‘12’(顺序为rs10774474-rs11571378,1代表常见等位基因,2代表罕见等位基因)具有风险效应。对rs1051669(104G>A)多态功能研究表明：rs1051669-A报告载体荧光素酶报告基因相对活性下降。运用DINAMelt软件预测发现,G等位基因自由能更低,其可能通过形成更稳定的局部二级结构来稳定mRNA增强报告基因的表达。同时,SPR实验显示,突变型A-等位基因RNA与胞质抽提物调控因子结合能力更强。提示104G→A改变可能增强Rad52基因与负调控因子的结合而降低Rad52基因表达水平。以上结果提示Rad52基因单核苷酸多态可能与中国人群肺癌遗传易感性有关,在肺癌发生过程中发挥重要作用。第二部分非小细胞肺癌铂类药物化疗药物基因组学研究第一节XPD基因与非小细胞肺癌铂类药物化疗关联分析非小细胞肺癌(non-small cell lung cancer, NSCLC)约占肺癌的80%,其中2／3的患者在确诊时已处于晚期(Ⅲ或Ⅳ期),失去了手术机会。铂类药物(顺铂、卡铂)是目前治疗晚期NSCLC的主要化学药物。铂进入细胞后,与DNA结合形成铂-DNA加合物,导致DNA链间交联或链内交联,引起细胞死亡。DNA损伤修复能力的差异可能是决定铂类药物疗效的重要因素。XPD在机体核苷酸切除修复(NER)中扮演重要角色,拥有5’-3’DNA解旋酶活性,行使NER以及基础转录过程中DNA结链作用。我们推测,XPD基因遗传多态可能影响DNA损伤的修复移除能力,而导致个体间疗效差异。鉴于此,我们观察了445例接受铂类为主化疗的NSCLC患者治疗后情况,采用MassARRAY时间飞行质谱生物芯片系统检测XPD基因三个潜在功能位点(p.Arg156Arg, p.Asp312Asn, p.Asp711Asp)遗传多态,以探讨该基因单核苷酸多态与NSCLC患者对铂类药物化疗疗效,毒副反应及预后的关系。非条件logistic回归分析发现,XPD基因p.Arg156Arg位点纯合突变A／A基因型显著增加患者发生3-4度血液毒性风险性,进一步分析发现,该遗传多态与3-4度白细胞毒性显著相关(趋势检验P=0.007),经Bonferroni多重校正后,关联均具有显著性。单倍型／二倍型分析显示,单倍型‘CG’的携带者发生白细胞毒性的风险显著降低,经过10,000次置换检验后,关联均具有显著性。在诺维本联合顺铂用药人群中,XPD p.Arg156Arg突变基因型A／A显著增加铂类药物敏感性(P=0.003),血液毒性风险性(P=0.012),白细胞毒性风险性(P=0.003)。XPD p.Asp312Asn, p.Asp711Asp多态与肺癌患者总生存期显著相关(log-rank P分别为0.006,0.006)。XPD p.Asp312Asn多态在ⅢB期人群中与预后关联更加显著(log-rank,P=7.25×10-5)。本研究首次发现,XPD基因遗传多态与晚期肺癌患者铂类药物化疗后的血液毒性,总生存期显著相关。XPD p.Arg156Arg, p.Asp312Asn, p.Asp711Asp单核苷酸多态性有可能作为检测指标来预测铂类药物的不良反应和预后风险,成为指导该类药物的个体化用药的依据。第二节中国人群晚期非小细胞肺癌铂类为主化疗药物基因组学研究铂类耐药由多种因素引起,包括药物解毒增加；DNA损伤修复能力增强；细胞耐受性增加等。核苷酸切除修复(NER)主要修复各种大片段DNA损伤造成的DNA双螺旋扭曲,如链内交联,同源重组(HR)修复链间交联(ICL),错配修复(MMR)对药物所致的加合物的识别及处理产生一种前凋亡信号。此外,叶酸代谢,炎症因子等因素均与铂类耐药相关。本部分研究收集445例以铂类药物为主化疗NSCLC患者血样及其临床资料,采用候选通路策略,选择了以上通路XPC, XPF, XPG, MLH1, MSH2, MTHFR, XRCC3, lig4, Rad52, IL-1a, IL-1 b等11个关键基因的17个SNP位点,通过基因多态与化疗近期疗效、不良反应及远期生存期的关联分析,寻找与NSCLC患者对铂类药物敏感性相关的基因及其多态位点。单位点分析表明,MTHFR rs1801131与化疗敏感性相关。接受诺维本联合铂类用药亚组人群分层分析发现Rad52 rs1051669 A/A基因型增加化疗疗效敏感性。7个基因上的9个SNP多态与不良反应显著相关。False discovery rate多重校正后,IL-1 b rs16944 (-511 C>T), rs1143627 (-31T>C)与3-4度白细胞毒性和3-4度粒细胞毒性关联仍具有显著性。分层分析显示,rs16944与3-4度白细胞毒性相关性在非吸烟人群中更显著(校正OR=5.10,95%CI=2.31-11.19,P=5.41×10-5)；其与3-4度粒细胞减少相关性在非吸烟者(校正OR=16.38,95%CI=5.88-45.61,P=8.84x10-8),女性(校正OR=12.87,95%CI=3.70-44.76,P=5.92×10-5)亚组人群中更显著。进一步基因-环境交互分析表明,IL-1b rs16944基因型,rsl143627基因型与吸烟状态、性别因素在对白细胞减少、粒细胞减少发生的影响中呈现显著的交互作用。在粒细胞减少不良反应中,rs16944基因型-吸烟,rs16944基因型-性别交互作用P值分别为0.001,0.007。携带XPC rs222800 T/T基因型个体生存期低于携带C/C+C/T基因型个体(MST,14个月v18个月),携带Rad52 rs1051669 A/A基因型个体生存期低于携带野生型G/G+A/G个体(MST,7个月v 18个月),差别具有统计学意义。本研究首次从整体上探索了DNA损伤修复通路,叶酸代谢通路及炎症通路基因多态和肺癌化疗疗效,不良反应及总生存期的关系。发现8个基因上的10个SNP位点可能影响中国人群晚期NSCLC患者对以铂类为主化疗方案药物敏感性和安全性,在进行多重检验校正后,发现2个位点的基因型效应仍表现显著。2个SNP位点与非小细胞肺癌患者预后相关。以上结果提示它们和NSCLC含铂化疗潜在的生物学联系,为提高NSCLC患者化疗敏感性、延长生存期和减轻毒副作用,建立个体化治疗提供依据。更多还原

【Abstract】 PartⅠDNA Repair Gene Polymorphisms and Lung Caneer CharpterⅠGenetic variants in GTF2H1 and risk of lung cancerLung cancer is the most common cause of cancer deaths worldwide. Although the risk of lung cancer is associated with exposure to cigarette smoke, which accounts for about 80-90% of lung cancer incident cases, only 10-15% of smokers develop lung cancer, suggesting possible involvement of predisposing genetic factors. It has been hypothesized that these differences may be due, in part, to genetically determined variation in DNA repair capacity.GTF2H1, the p62 subunit of the multiprotein complex TFIIH, participates in both the nucleotide excision repair process and transcription control by specifically interacting with a variety of factors important in carcinogenesis. To elucidate the role of genetic variations in GTF2H1 in the etiology of lung cancer, we conducted a case-control study of 500 incident lung cancer cases and 517 controls in a Chinese population by genotyping six common single nucleotide polymorphisms (SNPs) in GTF2H1.An increased risk was associated with the variant genotypes of rs3802967 [adjusted odds ratios (OR)= 1.38,95% confidence interval (CI)= 1.04-1.82], rs4150606 (adjusted OR= 1.44,95% CI= 1.08-1.92), and rs4150678 (adjusted OR= 1.37,95% CI=1.04-1.81) in a dominant genetic model. The risk for rs3802967 C/T+T/T genotypes was more pronounced among males subjects (P= 0.002). In contrast, a decreased risk was associated with the rs4150667 T/T genotype (adjusted OR= 0.59,95% CI= 0.38-0.93) in a recessive model. Haplotype analysis showed that the haplotype "222212" (1 for common alleles and 2 for minor alleles) was associated with increased risk of lung cancer (P= 0.03). Further evaluation using luciferase reporter constructs showed that the T allele of rs3802967 had higher luciferase expression, suggesting that the-79C→T change may affect transcriptional activation of GTF2H1. Taken together, these results suggest that GTF2H1 polymorphisms/haplotypes may contribute to genetic susceptibility to lung cancer. CharpterⅡRad52 Polymorphisms in Chinese Lung Cancer Patients: Association with Lung Cancer SusceptibilityThe removal of tobacco induced adducts is carried out by homologous recombination repairing DNA double strand breaks. Rad52 is one of the key enzymes of homologus recombination repair, and has been shown to plays an important role in the maintenance of genome stability, as well as the progression of carcinogenesis.Here, we investigated 9 common polymorphisms in the Rad52 gene in a case-control study of 500 incident 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 three SNPs (P= 0.002 for rs10774474 and P= 1.647×10-5 for rs11571378, P= 0.002 for rs1051669) and the significance remained after applying Bonferroni correction. Haplotype analysis revealed significant differences in haplotype (rs10774474-rs11571378) distributions between cases and controls (Global P=0.007), and the significance remained after applying 100,000-time permutation tests. Haplotype’12’(in the order of rs10774474-rs11571378,1 for common alleles and 2 for minor alleles) was associated with increased risk of lung cancer. The 104A allele for rs1051669 3’UTR construct exhibited decreased luciferase reporter gene expression.104G→A was predicted with reduced mRNA stability, which is consistent with the luciferase results.Further SPR experiments showed another explanation in that the A variant may enhance the affinity of cytoplasmic posttranslational repression binding protein or miRNA, resulting in reduced Rad52 expression. Our results suggest that the 104G>A polymorphism of rs1051669 may be functional thus influencing the DNA repair efficacy.These data indicate that the Rad52 polymorphisms may play a role in mediating susceptibility to lung cancer. It can help us construct genetic profiles directing the prediction for cancer susceptibility. Part II Pharmacogenetic Analysis of Platinum-based Chemotherapy in Non-Small Cell Lung Cancer PatientsCharpter I Effect of XPD Polymorphisms on Outcome and Prognosis in Non-Small Cell Lung Cancer PatientsNon-small cell lung cancer (NSCLC) accounts for approximately 80% of such deaths. Most NSCLC patients are diagnosed in the advanced (stage III or IV) stages. Five-year survival rates for NSCLC remain less than 15%. Standard treatment for NSCLC involves chemotherapy with a platinum agent and another cytotoxic agent.Platinum agents cause DNA cross-linking and adducts. Xeroderma pigmentosum group D (XPD) plays a key role in the nucleotide excision repair pathway of DNA repair. Genetic polymoiphisms of XPD may affect the capacity to remove the deleterious DNA lesions and lead to individual variability. This study aimed to investigate the association of three polymorphisms of XPD at codons 156, 312, and 711 with the outcomes in advanced NSCLC patients.We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to genotype the three polymorphisms in 445 stage III and IV NSCLC patients treated with platinum combination chemotherapy. The variant homozygotes of XPD p. Arg156Arg (rs238406) polymorphism were associated with a significantly increased risk of grade 3 or 4 hematologic toxicity, and more specifically, severe leukopenia toxicity (P for trend=0.007). Consistent with these results of single-locus analysis, both the haplotype and the diplotype analyses revealed a protective effect of the haplotype ’CG’ on the risk of grade 3 or 4 leukopenia toxicity.In the subgroup analysis for patients receiving Navelbine in combination with cisplatin treatment, XPD p.Arg156Arg A/A genotype was associate with increased platinum sensitivity (P=0.003), grade 3-4 hematologic toxicity risk (P=0.012) and severe leukopenia toxicity (P=0.003).We observed significant associations between XPDp.Asp312Asn, p.Asp711Asp polymorphisms and overall survival(log-rank P=0.006,0.006 respectively).The survival of patients in stageⅢB with XPD p.Asp312Asn A/G+A/A genotypes was significant shorter than that of G/G genotype (log-rank P=7.25×10-5).This investigation, for the first time, provides suggestive evidence of XPD p.Arg156Arg, p.Asp312Asn, p.Asp711Asp polymorphisms effects on toxicity and prognosis variability among platinum-treated non-small cell lung cancer patients.Charpter II Pharmacogenetic Assessment of Outcome and Survival after Platinum Chemotherapy in Non-Small Cell Lung Cancer PatientsThe large individual variability for NSCLC in both outcome and survival from chemotherapy makes the identification of pharmacogenetic markers that can be used to screen patients before therapy selection an attractive prospect.We assessed 17 selected polymorphisms based on previously described associations or putative functional effects in 11 key genes from pathways that may influence cellular sensitivity to platinum (XPC, XPF, XPG, MLH1, MSH2, MTHFR, XRCC3, lig4, Rad52, IL-1a and IL-1b) using matrix-assisted laser desorption /ionization time-of-flight mass spectrometry in 445 NSCLC patients.MTHFR rs1801131 was associated with risk of response rate. Rad52 rs1051669 A/A genotype increased response rate in subgroup analysis for patients receiving Navelbine plus platinum treatment. We observed significant associations between 9 SNPs in 7 genes and toxicity risk. Statistically significant associations for IL-1 b rs16944 (-511 C>T), rs1143627 (-31T>C) and severe leukocytopenia, agranulocytosis toxicities remained after False discovery rate correction. The risk for leukopenia was increased for rs16944 among nonsmokers (adjusted OR=5.10, 95%CI= 2.31-11.19, P=5.41×10-5); and the association between this SNP and agranulocytosis was more significant in nonsmokers (adjusted OR=16.38, 95%CI=5.88-45.61, P=8.84×10-8), female (adjusted OR=12.87,95%CI=3.70-44.76, P=5.92×10-5) patients. Further gene-environment interaction analysis suggests interactive effects between IL-1 b rs16944, rs1143627 with smoking status and gender. The interaction P value for rs16944-smoking status, rs16944-gender in agranulocytosis toxicity was 0.001,0.007 respectively.The survival of patients with XPC rs222800 T/T genotype was shorter than that of C/C+C/T genotypes(MST,14 months v 18 months), and survival of patients with Rad52 rs1051669 A/A genotypes was shorter than that of G/G+A/G genotypes (MST, 7months v 18 months), the difference was statistically significant.This exploratory study suggests that polymorphisms in specific genes encoding for DNA repair, methylenetetrahydrofolate reductase enzyme and anti-inflammatory cytokines are associated with platinum-related response, toxicities and overall survival. It can help us understand the functional consequences of chemotherapy and construct genetic profiles directing the choice of optimal therapy.更多还原