【作者】 王福如；

【导师】 沈洪兵；

【作者基本信息】 南京医科大学 ， 流行病与卫生统计学， 2010， 硕士

【摘要】 乳腺癌是女性最常见的恶性肿瘤,约占所有女性恶性肿瘤患者的23%。近年来,我国乳腺癌的发病率呈快速增长趋势,乳腺癌的发病率已位于女性恶性肿瘤发病率的首位,成为威胁我国女性健康的重大疾病之一。目前普遍认为乳腺癌的发生发展是基因与环境共同作用的结果,而遗传背景的差异可能影响个体对乳腺癌的易感性。研究表明,在一般人群中,乳腺癌的遗传易感性主要与常见的低共显性基因的变异有关。近年来的全基因组关联(Genome-wide association study,GWAS)研究确定了大量的乳腺癌低共显性遗传易感性位点,但这些研究多集中于欧洲高加索裔人群,在亚洲人群,目前正开展一项包括中国人群的乳腺癌全基因组关联研究,初步筛选出了染色体1p31(rs12041465 C>A)、20q13.3(rs6100755 A>G)、7q33(rs1806667 T>G)、9q33.3(rs3829849 C>T)以及16q12.1(rs4784227 C>T)5个中国人群潜在乳腺癌易感区域,但尚未经过大样本的人群验证和评价。我们假设这些易感区域的基因多态可能与中国汉族人群乳腺癌遗传易感性相关。昼夜节律调控机制与肿瘤遗传易感性的关系目前广受关注。昼夜节律主要受到一系列核心生物钟基因调控。神经元PAS域蛋白2(NPAS2)是最大的生物钟基因,它位于2号染色体长臂11.2区域。NPAS2可以与BMAL1形成异二聚体,进而转录激活另外两类生物钟基因,PER和CRY。此外,该异二聚体还可以调控癌基因c-MYC并且抑制它的转录,从而抑制肿瘤的形成。此前的高加索人群研究报道,NPAS2第13外显子上的非同义性多态rs2305160 C>T与乳腺癌危险性的之间存在关联,鉴于NPAS2是调控下游肿瘤相关基因表达的重要转录因子以及其表达水平改变的生物学意义,我们在NPAS2 3’-UTR区发现了一个潜在功能性单核苷酸多态性(SNP)位点(rs3739008 C>T),其多态改变可能影响到多种miRNAs与NPAS2 3’-UTR区的结合,进而可能影响乳腺癌的遗传易感性。为了验证上述假设,本研究基于亚洲人群GWAS研究数据,采用病例-对照研究设计,探讨了染色体1p31 rs12041465 C>A、20q13.3 rs6100755 A>G、7q33 rs1806667 T>G、9q33.3 rs3829849 C>T以及16q12.1 rs4784227 C>T基因多态性与中国江苏汉族人群乳腺癌易感性的关系。此外,我们利用细胞转染、荧光素酶报告基因等分子生物学手段,研究2q11.2区域NPAS2 3’-UTR区的潜在功能性多态位点rs3739008C>T的功能学意义,并进一步在中国江苏人群中对rs3739008C>T以及此前报道的rs2305160C>T两位点与乳腺癌危险性关联进行研究。第一部分染色体部分区域遗传变异与乳腺癌易感性关联研究近年来的全基因组关联研究已经确定了大量的乳腺癌低共显性遗传易感性位点,但这些研究多集中于高加索裔人群,所发现的乳腺癌遗传易感性位点中,只有少数SNPs与乳腺癌的易感性可以在中国人群中得到验证,得到验证的乳腺癌遗传易感性位点在中国人群的效应往往也弱于高加索裔人群。因此,在亚洲人群,尤其是中国人群中寻找更多的乳腺癌遗传易感性位点显得尤为重要。最近,Zheng等开展了一项旨在发现亚洲人群乳腺癌遗传易感性位点的全基因组关联研究,该研究Ⅰ期采用Affymetrix Human SNP Assay 6.0以及Human Mapping 500K Array平台对2073例病例和2084例对照检测了684,457个SNP位点,并依据:(i)MAF≥0.05;(ii)基因分型成功且分簇明确;(iii)此前并未被证实为乳腺癌遗传易感性位点或与这些位点间存在高度连锁不平衡(r2≥0.8);(iv)在CGEMS以及SBCS GWA数据库中meta分析的P≤1×10-3,且方向一致的原则,选取了53个SNP位点在iPLEX Sequenom MassArray平台进行Ⅱ期重复验证。我们结合该研究的前期筛检标准,利用HapMap数据库中CHB的人群数据,根据染色体SNPs最小等位基因频率≥0.05的原则,最终筛选出位于染色体1p31(rs12041465 C>A)、20q13.3(rs6100755 A>G)、7q33(rs1806667 T>G)、9q33.3(rs3829849 C>T)以及16q12.1(rs4784227 C>T)5个显著与乳腺癌发病风险相关多态性位点(Ⅰ期Ⅱ期联合趋势性P≤1×10-3)在江苏汉族女性进行验证。本研究采用病例对照研究设计(1006病例和1017对照),应用TaqMan基因分型技术,在江苏汉族人群中对上述位点与乳腺癌发病风险的关联进行验证,结果显示:(1)染色体16q12.1区域rs4784227 C>T多态性改变能够显著增加乳腺癌的发病风险(P=0.008),与携带野生纯合型CC基因型的个体比较,携带变异基因型(CT+TT)的个体发生乳腺癌的风险增加了27%(调整OR=1.27,95%CI=1.06-1.52)。(2)rs3829849 C>T(调整OR=1.13,95%CI=0.90-1.41)、rs12041465 C>A (调整OR=0.97,95%CI=0.81-1.17)、rs6100755 A>G(调整OR=1.10,95%CI=0.90-1.36)和rs1806667 T>G (调整OR=0.99,95%CI=0.82-1.21)多态位点与江苏汉族人群乳腺癌的发病风险不存在显著的统计学关联(P>0.05)。本研究结果显示,染色体16q12.1区域rs4784227 C>T可能是江苏汉族人群乳腺癌的易感位点。第二部分染色体2q11.2区域NPAS2功能性遗传变异与乳腺癌易感性研究流行病学研究发现,生物钟节律的破坏会增加乳腺癌的发病风险。神经元PAS域蛋白2(NPAS2)编码基因NPAS2位于染色体2q11.2区域,是最大的生物钟基因,主要表达于哺乳动物的前脑区,编码螺旋-环-螺旋-PAS类的转录因子。NPAS2与脑肌类芳香烃受体核转位子蛋白1(BMAL1)形成异二聚体可以调控癌基因c-MYC并且抑制它的转录,从而抑制肿瘤的形成。高加索人群研究报道,NPAS2第13外显子的非同义性多态rs2305160 C>T与乳腺癌发病风险存在关联,表明NPAS2的基因变异可能参与乳腺癌发生发展。鉴于NPAS2是调控下游肿瘤相关基因表达的转录因子以及其表达水平改变的生物学意义,我们系统的筛选了位于5’-侧翼区,5’-非翻译区(5’-UTR)和3’-UTR等区域可能影响基因转录表达或活性的功能性SNPs,新发现了一个位于NPAS2基因3’-UTR区域的功能性SNP位点(rs3739008 C>T),我们通过构建荧光素酶报告基因检测,表明rs3739008 C>T的碱基改变可能影响microRNA- (miR-) 17-5p和miR-519e与NPAS2 3’-UTR区的结合,从而使得NPAS2转录水平显著增加(P<0.05)。通过病例对照研究,我们分析了NPAS2 rs3739008 C>T和rs2305160C>T两位点与江苏汉族人群乳腺癌发病风险的关系。研究结果表明,两位点与乳腺癌的发病风险均未有统计学显著性(显性模型:rs3739008 C>T:调整OR=1.13,95% CI=0.95-1.35;rs2305160 C>T:调整OR=0.99,95% CI=0.82-1.19)。尽管我们未能发现显著的统计学关联,但由于SNP位点rs3739008 C>T基因型与NPAS2表达存在显著相关性,而NPAS2表达与乳腺癌预后有关,进一步的研究将有助于评价该多态在乳腺癌预后转归中的作用。更多还原

【Abstract】 Breast cancer is the most common cancer and the leading cause of cancer related death among women worldwide, accounting for 23%. Although the incidence rate of breast cancer in China is low, it shows a significantly increasing trend in the last two decades. Breast cancer has already been one of main causes contributing to disease related deaths and a serious one to be resolved in public health. It is well accepted that the etiology of breast cancer appears to entail a complex combination of genetic and environmental. Studies indicate that most of the genetic susceptibility to breast cancer was related to the common low-penetrance genes in general population. Recent genome-wide association (GWAS) studies have identified multiple low-penetrance genetic susceptibility loci for breast cancer. However, most of these GWA studies are conducted among women of European ancestry, it is particular important to find additional genetic risk variants for breast cancer in the Asian population, especially in Chinese population. For this reason, we follwed a genome-wide association study, which aims to the genetic susceptibility to breast cancer locus in Asian population, and selected five promising SNPs that including rs12041465 C>A, rs6100755 A>G, rs1806667 T>G, rs3829849 C>T and rs4784227 C>T, which located in 1p31, 20q13.3, 7q33, 9q33.3and 16q12.1, respectively. We hypothesized that these polymorphisms may be associated with susceptibility to breast cancer risk in Chinese population. In addition, the relationship between the circadian rhythm regulatory mechanisms and genetic susceptibility to cancer received wide attention now. Circadian rhythm is mainly driven by a set of core circadian clock genes. Neuronal PAS domain protein 2 (NPAS2), the largest circadian gene, maps on chromosome 2q11.2. NPAS2 forms heterodimers with BMAL1 and then transcriptional activates expression of 2 other circadian genes, PER and CRY. Moreover, NPAS2 has been suggested to be involved in tumorigenesis, by regulating the oncogene c-MYC to suppress its transcription. A previous study in Caucasian population reported that the non-synonymous polymorphism rs2305160 C>T in exon 13 of NPAS2 was associated with susceptibility to breast cancer risk, as NPAS2 is a trans-activator to control the expression of down stream tumor related genes, we searched and found a putative functional SNP (rs3739008 C>T) located at 3’UTR of NPAS2, and hypothesized the polymorphism of this SNP may disrupt the binding of several microRNAs (miRNAs) to the 3’UTR of NPAS2, and may affect the genetic susceptibility to breast cancer in turn. .To test these hypotheses, case-control study was performed to detect the association of polymorphisms on chromosomes 1p31, 20q13.3, 7q33, 9q33.3 and 16q12.1 with breast cancer risk in Chinese Jiangsu population, which based on a GWAS research projects. In addition, based on current clock gene research, we used cell transfection, luciferase reporter gene and other molecular biology methods to verify the NPAS2 3’-UTR region of potential functional polymorphism rs3739008 C>T functional significance and further test the association of rs3739008 C>T and rs2305160 C>T polymorphisms on chromosomes 2q11.2 with breast cancer risk in Chinese Jiangsu population. PartⅠ: Association study of some regions of chromosomes polymorphisms with the susceptibility of breast cancerRecent genome-wide association (GWAS) studies have identified multiple low-penetrance genetic susceptibility loci for breast cancer. However, most of these GWA studies are conducted among women of European ancestry and only few of the SNPs initially identified in European descents can be directly replicated in Chinese. Furthermore, the associations of breast cancer with the replicated SNPs are often weaker in Asian women than those reported initially in studies conducted among European descendants. Therefore, it is particular important to find additional genetic risk variants for breast cancer in the Asian population, especially in Chinese population. Zheng et al, have reported a novel genetic susceptibility locus at 6q25.1 for breast cancer risk in Chinese population in a previous genome-wide association study. Recently, A genome-wide association study, which aims to the genetic susceptibility to breast cancer locus in Asian population was conducted. This study analyzed 684,457 SNPs in 2,073 cases and 2,084 controls in Stage I by using Affymetrix Human SNP Assay 6.0 and Human Mapping 500K Array, and then selected 53 promising SNPs for a fast-track replication in Stage II on iPLEX Sequenom MassArray. Selection criteria for these SNPs were (i) MAF≥5%; (ii) very clear genotyping clusters; (iii) not previously confirmed as breast cancer genetic risk variant or in strong LD (r2≥0.8) with these variants; and (iv) P≤1×10-3 in the meta analyses of CGEMS and SBCS GWA data, having the same direction of the association in both studies. We combined the study of pre-screening criteria, using HapMap database CHB populations of data, according to the chromosome SNPs minior allele frequency≥0.05 in principle, finally selected five promising SNPs that including rs12041465 C>A, rs6100755 A>G, rs1806667 T>G, rs3829849 C>T and rs4784227 C>T, which located in 1p31, 20q13.3, 7q33, 9q33.3and 16q12.1, respectively. We then typed these SNPs in a case-control study, by using TaqMan allelic discrimination techniques to test the putative associations with breast cancer risk in Chinese population. The results showed that: (1) the variant genotype (CT + TT) of the rs4784227 C>T polymorphism on chromosome 16q12.1 was associated with a significantly increased risk of breast cancer (adjusted OR = 1.27, 95% CI = 1.06-1.52, P=0.008), compared with the wild-type CC genotype. (2) we failed to find the rs3829849 C>T (adjusted OR=1.13, 95%CI=0.90-1.41), rs12041465 C>A (adjusted OR=0.97, 95%CI=0.81-1.17), rs6100755 A>G (adjusted OR=1.10, 95%CI=0.90-1.36) and rs1806667 T>G (adjusted OR=0.99, 95%CI=0.82-1.21) polymorphisms significantly associated with breast cancer risk (P>0.05). These results suggest that, the rs4784227 C>T polymorphism on chromosome 16q12.1 may be related to the pathogenesis of breast cancer in Chinese women.PartⅡ: Association study of 2q11.2 region NPAS2 functional genetic variation with the susceptibility of breast cancerDisruption of the circadian rhythm has been reported to increase the risk of breast cancer. Neuronal PAS domain protein 2 (NPAS2) is the largest circadian gene, maps on chromosome 2q11.2, which expressed primarily in the mammalian forebrain and encodes for a member of the basic helix-loop-helix-PAS class of transcription factors. NPAS2 forms heterodimers with brain and muscle ARNT-like-1(BMAL1) and then regulates the oncogene c-MYC to suppress its transcription, which has been suggested to be involved in tumorigenesis. Previous study in Caucasian population reported that the non-synonymous polymorphism rs2305160 C>T in exon 13 of NPAS2 was associated with susceptibility to breast cancer risk, which has shown that the functional genetic variations in NPAS2 might be candidate biomarkers for breast cancer risk, where as NPAS2 expression is a significant biomarker for breast cancer survival. As NPAS2 is a trans-activator to control the expression of down stream tumor related genes, in the current study, we searched potentially functional SNPs in the promoter region, 5’-untranslated region (UTR) and 3’UTR, which may influence gene expression, and found a putative functional SNP (rs3739008 C>T) located at 3’UTR of NPAS2 and the C to T changing of the SNP may disrupt the binding of microRNA- (miR-) 17-5p and miR-519e to the 3’UTR of NPAS2 by constructing luciferase reporter gene assay test, enabling a significant increase in NPAS2 transcription (P<0.05). We then typed NPAS2 rs3739008 C>T and rs2305160 C>T polymorphisms in case-control studies of in Chinese Jiangsu Han population to test their putative associations with breast cancer risk. However, we failed to find any significant associations by different genetic models in both two sites (dominant genetic model: rs3739008 C>T: adjusted OR = 1.13, 95% CI = 0.95-1.35; rs2305160 C>T: adjusted OR = 0.99, 95% CI = 0.82-1.19). Although we did not find any significant associations, due to the functional relevance of rs3739008 C>T on NASP2 expression, it will be promising to investigate the influence of this variant on clinical characteristics of breast cancer and breast cancer survival.更多还原