中国人群胰腺癌遗传易感性研究

发布时间：2018-03-18 04:30

本文选题：胰腺癌　切入点：SWI/SNF　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：胰腺癌目前仍是预后差,死亡率极高的消化道恶性肿瘤,5年生存率小于5%。2008年,世界范围内估计新发病例为27万多例,估计死亡病例为26万多例,居全球恶性肿瘤死因的第8位。近年来,全求胰腺癌的发病率已趋平稳,而我国则呈上升趋势,从70年代相对少见到2005年居常见恶性肿瘤死因的第7位。胰腺癌高病死率的现状一直没得到改善的重要原因是由于病因尚未清楚。目前手术切除是唯一可能的治愈方法,由于缺乏有效的早期诊断方法,多数病例确诊时已是晚期,丧失手术切除机会。恶性肿瘤是一种慢性复杂性疾病,是环境和遗传因素长期共同作用的结果。胰腺癌的家族聚集性提示遗传因素在胰腺癌的发生过程中扮演重要角色。因此,开展胰腺癌的遗传易感性研究,寻找特异敏感的生物标志物应用于早期诊断,评估疗效及预后,以及应用发展新的治疗靶,对于我国胰腺癌的综合防治具有重要意义。第一部分SWI/SNF复合物的遗传变异与胰腺癌发生风险关系研究背景：基因的异常转录是癌症发生的基础,染色质高度紧密的折叠阻止了转录因子、辅因子与DNA的结合,所以凡涉及DNA的反应都要克服染色质的高度紧密性。ATP依赖的染色质重构复合物是利用水解ATP获得的能量来动员核小体,改变染色质的结构,使得转录因子与DNA结合,从而调节靶基因的转录。其中交配型转换/蔗糖不发酵复合物(mating type switch/sucrose non-fermenting, SWI/SNF)蛋白家族广泛参与细胞的分化、增殖和DNA修复,作为肿瘤抑制基因被研究得最为深入,大量的研究发现在包括胰腺癌在内的很多人类癌症中都发现SWI/SNF复合物亚基的突变或表达异常。研究目的：分析SWI/SNF复合物遗传变异与中国汉族人群胰腺癌发生的关系；评价SWI/SNF复合物关键基因的遗传变异基因-基因,基因-环境交互作用在于在胰腺癌发生过程中的效应。研究方法：采用两阶段病例-对照研究系统地分析ATP依赖的染色质重构复合物SWI/SNF基因的遗传变异与中国人群胰腺癌发生风险关系。第一阶段在武汉地区中应用中通量基因分型平台OpenArray对SWI/SNF复合物关键基因的标签SNP(tag SNP)与胰腺癌发生关系进行关联研究,筛选出关联SNPs;第二阶段在北京地区用Taqman基因分型技术验证第一阶段的关联SNPs。应用卡方检验比较基因型及人口学资料分布；利用Armtage's趋势检验进行基因型与胰腺癌发生风险的关联分析：应用logistic回归进行单位点分析计算各SNP的效应值;为控制因多重检验而引入的假阳性率,采用错误发现率(False discovery rate,FDR)方法进行多重假设检验校正；应用lOgistic回归对基因-基因和基因-环境交互作用进行分析。研究结果：1.第一阶段纳入310例胰腺癌病例与457位健康对照,共分析了编码SWI/SNF复合物的6个关键基因上的14个SNP,以隐性遗传模型计算,我们发现位于BRD7基因上的rs11644043(OR=2.04,95%CI=1.17-3.56)、 SMARCA4基因上的rs11085754(OR=1.64,95%CI=1.16-2.33)以及SMARCBl基因上的rs2073389(OR=1.93,95%CI=1.36-2.74)三个位点与胰腺癌发生风险显著相关：第二阶段纳入了429例胰腺癌病例与585位健康对照,成功验证了rs11644043(OR=1.97,95%CI=1.24-3.14)和rs11085754(OR=1.45,95%CI=1.04-2.02)两个位点。 2.两阶段合并,我们发现了三个位点rs11644043. rs11085754与rs2073389之间的对胰腺癌发生风险作用的累积效应(P for trend0.0001),个体随着携带风险基因型个数的增加,胰腺癌的发生风险也随之增加。 1.两阶段合并,我们发现rs2073389(Padd-FDR=6.00×10-4,Pmul-FDR=1.50×10-2)以及rs11085754(Padd-FDR=0.03)在修饰胰腺癌发生风险方面都与吸烟存在着交互作用。研究结论：1.位于BRD7基因上的rs11644043以及SMARCA4上rs11085754的遗传变异和中国人群的胰腺癌发生风险相关； 2. rs11644043、 rs11085754与位于SMARCB1基因上的rs2073389三者在对胰腺癌发生风险方面存在着累积效应； 3.rs2073389以及rs11085754在修饰胰腺癌发生风险方面都与吸烟存在着交互作用。创新点与不足之处：本研究采用两阶段病例对照研究设计首次在中国人群中探讨SWI/SNF复合复上的遗传变异和胰腺癌发生关系。重点分析了基因-基因、基因-环境交互作用,是以往很多研究所忽略的。本研究的样本量较小,特别是在研究基因-基因,基因-环境交互作用时,统计效能不足稍显不足。本次研究中对发现的遗传变异没有进行生物学功能实验,所以其背后的生物学机制需要进一步研究和验证。第二部分9p21.3区域的遗传变异与胰I腺癌发生风险关系研究背景：近来,9p21.3区域成为多种疾病的GWAS的热点区域,如慢性淋巴细胞白血病、黑色素瘤、胶质瘤、乳腺癌、心血管疾病、颅内动脉瘤、子宫内膜异位等。这一区段在30-40%的人类肿瘤中都发现失活,包含了人类三个重要的抑癌基因p16INK4a、 p14ARF、 P15INK4b,它们在调节细胞周期抑制、衰老、凋亡方面作用突出,广泛参与到各种肿瘤的发生发展过程中。此区域的多个抑癌基因在胰腺癌的发生发展过程中扮演重要角色,目前,尚无研究报道探索这一区域的遗传变异与中国人群胰腺癌发生风险的关系。研究目的：系统地分析9p21.3上的遗传变异与中国汉族人群胰腺癌发生风险的关系；对潜在的功能性位点进行深入的功能学实验研究,进一步明确真正的致病位点的作用。研究方法：首先,采用两阶段病例-对照研究研究设计,分析9p2113区域的遗传变异与中国汉族人群胰腺癌发生风险的关系,第一阶段选取我们已完成的中国人群胰腺癌外显子芯片中9p21.3(19.9-29Mb)区域的标签SNP,应用卡方检验以及logistic回归分析它们与胰腺癌发生风险关系；得出与胰腺癌发生风险相关的阳性的位点后,通过VISTA Enhancer Browser数据库、Encode中chip-seq数据以及e-QTL数据库SNPexp-A等生物信息分析手段分析此位点或与之连锁的位点可能的潜在的功能：利用双荧光素酶报告基因实验以及电泳迁移率变动分析实验研究验证这些位点的生物学功能；再对生物学功能实验得出的潜在功能位点进行第二阶段的人群的关联研究的验证；研究结果：1.第一阶段我们纳入了942例胰腺癌病例与1504例健康对照,分析了9p21.3区域的39个SNP,发现位于ANRIL基因上的rs6475609位点与胰腺癌发生风险显著相关,每增加一个rs6475609-A等位基因,胰腺癌的发生风险下降19%(OR=0.81,95%CI=0.72-0.92)。经过生物信息学分析与关联研究分析,发现与之高度连锁的两个可能存在潜在功能的位点rsl537373(OR=0.84,95%CI=0.74-0.95)与rs1333042(OR=0.84,95%CI=0.74-0.95)也与胰腺癌发生风险显著相关。第二阶段我们对生物学功能实验验证出来的位点rsl537373进行人群的关联研究,纳入了来自武汉地区的624例胰腺癌病例与1244例健康对照,发现rs1537373位点的遗传变异仍和胰腺癌发生风险显著相关,个体每增加一个rs1537373-T,发生胰腺癌的风险下降16%(OR=0.84,95%CI=0.73-0.98)。 2.e-QTL分的结果显示,rsl537373位点的遗传变异与CDKN2B基因的表达水平相关,携带TG/TT基因型的个体的CDKN2B的表达水平要显著高于携带GG基因型的个体(P dominant:6.00×10-4)。 3.双荧光素酶报告基因结果显示,在胰腺癌的两个细胞系Panc-1和SW1990中,都发现了含有rsl537373-G的片段较含有rsl537373-T的片段有更强的增强子活性(Ppanc-10.01,Psw19900.01)。 4.电泳迁移率变动实验发现,含有rs1537373-G的片段与某转录因子的结合能力强于含有rs1537373-T的片段。研究结论：1.位于ANRIL基因上的rsl537373位点的遗传变异与中国人群的胰腺癌发生风险显著相关； 2.rs1537373位点上的遗传变异可能由于与某转录因子的结合活性不同,从而影响所在片段的增强子活性,进而影响胰腺癌发生风险。创新点与不足之处：本研究采用两阶段的病例对照研究结合生物学功能实验的策略首次系统全面地分析了9p21.3区域的功能性遗传变异与中国人群胰腺癌发生风险的关系。本研究未能获得胰腺癌病人的组织样本,不能明确发现的阳性位点具体影响临近的哪个基因的表达,只能根据现的的数据库的数据推测,证据级别稍显不足。本次研究第二阶段的样本量较小,统计学效能稍显不足。
[Abstract]:Pancreatic cancer is still poor prognosis and high mortality rate of malignant tumor of digestive tract, 5 years survival rate of less than 5%.2008, the worldwide estimated new cases of more than 270 thousand cases, estimates of deaths of more than 260 thousand cases of malignant tumor death worldwide eighth. In recent years, the global incidence of pancreatic cancer has become stable, while China is a rising trend, from 70s in 2005 to see relatively few common malignant tumor and the seventh leading cause of death. An important reason of high mortality rate of pancreatic cancer has not been improved because the cause is not yet clear. At present, surgical resection is the only cure for may, due to the lack of effective early diagnosis methods, the majority of cases the diagnosis is already late, lost the opportunity of surgery. Malignant tumor is a kind of chronic and complex disease, is the interaction of environmental and genetic factors. The pancreatic cancer familial aggregation suggest that genetic factors In play an important role in the process of pancreatic cancer. Therefore, to carry out genetic susceptibility to pancreatic cancer, looking for specific and sensitive biomarkers used for early diagnosis, curative effect evaluation and prognosis, and the target for the development of new applications, has important significance for the comprehensive prevention and treatment of pancreatic cancer in China.
The relationship between the genetic variation of SWI/SNF complex and the risk of pancreatic cancer
Background: abnormal transcription of genes is the basis of cancer, the highly condensed chromatin prevents the transcription factor binding cofactor and DNA, so the reaction involving DNA to overcome chromatin close.ATP dependent chromatin remodeling complex is the use of ATP hydrolysis energy mobilization nucleosome, alter the structure of chromatin, the transcription factor and DNA binding, thereby regulating the transcription of target genes. The mating type switching / sucrose non fermenting compound (mating type switch/sucrose non-fermenting SWI/SNF) protein family are widely involved in cell differentiation, proliferation and repair of DNA, as a tumor suppressor gene is the best studied. A large number of studies have found that SWI/SNF complex subunit found in many human cancers including pancreatic cancer, have the mutation or abnormal expression.
Objective: to analyze the relationship between the genetic variation of SWI/SNF complex and the occurrence of pancreatic cancer in Chinese Han population, and to evaluate the genetic variation of SWI/SNF complex gene gene gene interaction in the process of pancreatic cancer.
Methods: a case-control study of two stages of systematic analysis of genetic variation of chromatin remodeling complex SWI/SNF gene dependent ATP and pancreatic cancer risk Chinese population relationship. The first stage flux application in Wuhan area in genotyping platform OpenArray of SWI/SNF complex key gene tag SNP (tag SNP). With pancreatic cancer association studies, screening Association SNPs; the second stage is divided into the first stage of technology validation with Taqman gene in the Beijing area of the association of SNPs. chi square test was used to compare genotype distribution and demographic data; using the Armtage's trend test for correlation analysis of genotype and risk of pancreatic cancer: application of logistic regression unit analysis of the effects of calculated SNP value; false positive rate control for multiple testing are introduced, the false discovery rate (False discovery rate, FDR The method was corrected by multiple hypothesis test, and the interaction between gene gene and gene environment was analyzed by lOgistic regression.
Results: 1. the first stage included 310 cases of pancreatic cancer and 457 healthy controls, a total of 14 SNP were analyzed 6 key genes encoding SWI/SNF complexes on the in recessive genetic model, we found that the BRD7 gene located on the rs11644043 (OR=2.04,95%CI=1.17-3.56), SMARCA4 gene rs11085754 (OR=1.64,95%CI=1.16-2.33) and rs2073389 the SMARCBl gene (OR=1.93,95%CI=1.36-2.74) of the three loci with pancreatic cancer were significantly related to risk: the second stage included 429 cases of pancreatic cancer and 585 healthy controls, the successful validation of the rs11644043 (OR=1.97,95%CI=1.24-3.14) and rs11085754 (OR=1.45,95%CI=1.04-2.02) two loci.
2., in the two stage of consolidation, we found the cumulative effect of three loci rs11644043. rs11085754 and rs2073389 on the risk of pancreatic cancer (P for trend0.0001), and the risk of pancreatic cancer increased with the increase of risk genotype.
1., in the two stage of merger, we found that rs2073389 (Padd-FDR=6.00 * 10-4, Pmul-FDR=1.50 * 10-2) and rs11085754 (Padd-FDR=0.03) interact with cigarette smoking in modifying the risk of pancreatic cancer.
Conclusions: 1. the genetic variation of rs11644043 on the BRD7 gene and the genetic variation of rs11085754 on SMARCA4 is associated with the risk of pancreatic cancer in Chinese people.
2. rs11644043, rs11085754 and rs2073389 three on the SMARCB1 gene have cumulative effects on the risk of pancreatic cancer.
3.rs2073389 and rs11085754 interact with smoking in modifying the risk of pancreatic cancer.
Innovation and deficiency: This study used two stage case-control study design to investigate the relationship between SWI/SNF compound on genetic variation and pancreatic cancer in China population for the first time. The key point is the analysis of gene gene and gene environment interaction, is ignored by many previous studies.
In this study, the sample size is small, especially in the study of gene gene and gene environment interaction, lack of statistical power slightly insufficient. The study of genetic variation found no biological function experiment, so the biological mechanisms behind the need for further research and verification. The risk of relationship between genetic variation and pancreatic second part of the 9p21.3 region of I adenocarcinoma
Background: Recently, 9p21.3 has become a hot area of a variety of diseases such as GWAS, chronic lymphocytic leukemia, melanoma, glioma, breast cancer, cardiovascular disease, intracranial aneurysm, endometriosis. This segment is inactivated in 30-40% of human tumors, including three important human the tumor suppressor gene p16INK4a, p14ARF, P15INK4b, their aging in the regulation of cell cycle inhibition, apoptosis plays a prominent role, widely involved in the occurrence and development of tumors. Multiple tumor suppressor genes in this region in the development of pancreatic cancer plays an important role, at present, there is no report on the exploration of the relationship between risk the genetic variation in this region and Chinese pancreatic cancer population.
The purpose of this study is to systematically analyze the relationship between genetic variation on 9p21.3 and the risk of pancreatic cancer in Chinese Han population, and conduct in-depth functional experiments on potential functional sites, to further clarify the role of real pathogenic sites.
Research methods: firstly, the two stages of a case-control study design, analysis of the relationship between the risk of genetic variation in 9p2113 region and pancreatic cancer China Han population, the first stage of selection of pancreatic cancer China population we have completed the explicit 9p21.3 chip (19.9-29Mb) region of the SNP tag, using chi square test and logistic regression analysis of their relationship with the risk of pancreatic cancer; the risk associated with pancreatic cancer positive sites, through the VISTA Enhancer Browser database, Encode ChIP-seq database and e-QTL SNPexp-A and other biological information analysis method to analyze the site and the chain site may potentially function: using dual luciferase assay and electrophoresis mobility shift analysis Experimental study verified the biological function of these sites; and the biological function of the experiment The potential functional loci are verified by the association study of the second stage population.
Results: 1. the first phase we included 942 cases of pancreatic cancer and 1504 healthy controls, analyzed 39 SNP 9p21.3 region, found at risk was significantly related to ANRIL gene rs6475609 locus and pancreatic cancer, every increase of one rs6475609-A allele and the risk of pancreatic cancer decreased 19% (OR=0.81,95%CI=0.72-0.92) after analysis and association analysis of bioinformatics, found with highly linked two potentially functional sites of rsl537373 (OR=0.84,95%CI=0.74-0.95) and rs1333042 (OR=0.84,95%CI=0.74-0.95) also occurred and the risk of pancreatic cancer was significantly correlated. Second stage of our association study sites of rsl537373 biological function experimental verification of the crowd, included in the Wuhan area of 624 cases of pancreatic cancer and 1244 healthy controls, found that the genetic variation of rs1537373 locus and pancreas The risk of cancer was significantly correlated, and the risk of pancreatic cancer decreased by 16% (OR=0.84,95%CI=0.73-0.98) per individual increase of one rs1537373-T.
The results of 2.e-QTL analysis showed that the genetic variation of rsl537373 locus was related to the expression level of CDKN2B gene. The expression level of CDKN2B in individuals carrying TG/TT genotype was significantly higher than that in individuals carrying GG genotype (P dominant:6.00 * 10-4).
3. double luciferase reporter gene results showed that rsl537373-G and Panc-1 fragments were found in two cell lines of pancreatic cancer, including rsl537373-G and SW1990, which had stronger enhancer activity than those containing rsl537373-T (Ppanc-10.01, Psw19900.01).
4. electrophoretic mobility change experiments showed that the binding ability of rs1537373-G containing fragments to a transcription factor was better than that of rs1537373-T containing fragments.
Conclusions: 1. the genetic variation of the rsl537373 locus on the ANRIL gene is significantly associated with the risk of pancreatic cancer in the Chinese population.
Genetic variation on the 2.rs1537373 locus may be due to the different binding activity with a transcription factor, which affects the activity of the enhancer in the fragment and further affects the risk of pancreatic cancer.
Innovations and shortcomings: in this study, the two stage case-control study combined with the strategy of biological function experiment was used for the first time to systematically analyze the relationship between functional genetic variation in 9p21.3 region and the risk of pancreatic cancer in Chinese population.
This study failed to obtain tissue samples from patients with pancreatic cancer, the expression of which genes near the positive loci found the specific effect is not clear, only according to the database of data speculation, slightly less than the level of evidence. The second phase of the study sample size, statistical performance slightly less.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9

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