TERT启动子突变在胶质瘤中的分布以及预后分层研究

发布时间：2018-01-26 17:29

本文关键词： 脑胶质细胞瘤 TERT启动子突变分子标志物预后分析　出处：《南京医科大学》2016年博士论文　论文类型：学位论文

【摘要】：端粒是真核细胞染色体末端的一段序列,随着细胞的分裂而逐渐缩短。端粒酶逆转录酶(Telomerase reverse transcriptase,TERT)通过激活端粒酶来保持端粒的完整,使细胞获得无限增殖能力。TERT在胶质瘤细胞中存在高表达,并与胶质瘤的恶性程度密切相关,而正常脑组织中则没有TERT的表达。研究发现TERT启动子区域存在两个体细胞突变：C228T和C250T,并导致TERT启动子的转录活性增加了2-4倍。国外有报道发现TERT启动子突变在胶质母细胞瘤中频繁出现,并对预后产生一定的影响。本研究希望通过基于中国人群的大样本中进行TERT启动子突变的检测,明确其分布规律和对于预后的影响,希望能够找到一个具有预后判断价值的分子标志物。在本课题的第一部分中,我们对887例不同级别、不同组织病理学亚型的胶质瘤样本进行全基因组DNA的提取,通过巢式PCR扩增含有TERT启动子突变的特定区域,割胶回收纯化后进行Sanger测序,通过比对分析扩增片段中C228T和C250T突变情况,统计突变率,绘制C228T和C250T突变在不同级别,不同组织学亚型的胶质瘤中的分布谱,并揭示其富集规律。结果显示：在887例胶质瘤样本的TERT启动子区域上,274例被检测出含有C228T突变,占到总样本数的30.89%,83例含有C250T突变,占到总样本数的9.36%。仅有1例样本同时存在C228T和C250T双位点的突变。除10例为纯合子突变外,其余所有含有突变点的样本均为杂合突变。我们按照2007年WHO中枢神经系统肿瘤分级分析了TERT在不同级别胶质瘤中的分布规律。结果显示,在498例WHOⅡ级胶质瘤样本中,有197例被检测出含有TERT突变,突变率为39.56%；在139例WHO Ⅲ级胶质瘤样本中,有56例被检测出含有TERT突变,突变率为40.29%；在250例WHO Ⅳ级胶质瘤样本中,有104例被检测出含有TERT突变,突变率为41.6%。突变率随着胶质瘤级别的升高略有增加。我们随后在各组织学亚型中统计了TERT启动子的分布规律：其中少突胶质细胞瘤中TERT启动子突变的富集比例最高,占到75.71%(53/70),星型胶质细胞瘤中TERT启动子的突变频率最低,仅有10.84%(22/203)。为了更详细的描述TERT启动子突变在胶质母细胞中的分布,我们分别在原发性胶质母细胞瘤和继发性胶质母细胞瘤中分析TERT的突变频率,结果显示44.72%(89/199)的原发性胶质母细胞瘤含有TERT启动子突变,而只有29.41%(15/51)的继发性胶质母细胞瘤含有此突变。通过上述结果我们认为TERT启动子突变在少突胶质细胞瘤中高度富集,突变率随肿瘤级别的增加有递增趋势,且C228T突变和C250T突变呈明显的互斥现象。TERT启动子突变在以中国人群为基础的胶质瘤患者中稳定存在,可以作为针对中国人群的胶质瘤分子标志物进行预后分层研究。目前胶质瘤治疗所面临的问题主要有以下几个方面：胶质瘤细胞的侵袭性和浸润性较强,这在一定程度上限制了手术切除的范围；在化学治疗后很快就会出现具有抗药性的细胞克隆；肿瘤细胞中存在多种致癌信号通路的激活和基因突变体的相互作用等等。鉴于上述原因,胶质瘤,特别是胶质母细胞瘤患者的生存率一直很低,其中位生存期一般不超过15个月。然而,仍有不足10%的胶质母细胞瘤患者的寿命可以延长到三至五年。但是这部分患者生存期延长的原因和他们本身体质以及基因改变之间的原因仍未能明确。有报道指出：携带O6-Methylguanine-DNA methyltransferase (MGMT)启动子甲基化的年轻女性胶质瘤患者有机会获得比其他患者更长的生存期。因此,寻找具有诊断或者预后意义的特征性分子标志物,并进行相关的预后分层研究对胶质瘤的个体化治疗具有重要的指导意义。因此在本课题的第二部分中,我们针对不同级别,不同组织学亚新的胶质瘤进行TERT启动子突变的预后分层研究,并通过全转录组测序数据对胶质瘤样本进行TCGA分型注释,并在Proneural, Neural, Classical, Mesenchymal四个分子亚型中评估TERT启动子突变的预后判断价值。结果显示：TERT启动子突变组的患者生存期明显长于TERT启动子野生组的生存期(P0.05),且C250T突变组的患者生存期长于C228T突变组的生存期。在WHO Ⅱ级胶质瘤中,TERT启动子突变组的患者生存期的优势则更加明显。当采用TCGA分型标准对样本进行分子亚型注释后我们发现,TERT启动子突变在Proneural和Mesenchymal两个分子亚型中呈现出了明显的预后分层,而在Classical和Neural两个亚型中的分层并不明显。据此我们得出结论：TERT启动子突变作为判断胶质瘤预后指标的分子标志物在TCGA亚型和低级别胶质瘤中的预测意义较为明显,可以作为一个稳定的预后指标为这类胶质瘤患者预后进行评估。近年来的研究发现胶质瘤在其发生发展的过程中存在着不同的遗传背景,这些遗传背景始终贯穿着一个或几个特征性的基因改变。而这些特征性的基因改变也直接或间接的影响着胶质瘤的演化和预后。目前公认的是IDH突变是胶质瘤发生基因组改变的早期事件,而TP53突变和1P19Q缺失则分别出现在星形胶质细胞瘤和少突胶质细胞瘤的进化过程中。其中TP53突变更被认为是从低级别星形胶质细胞瘤转化为继发性胶质母细胞瘤的过程中携带的遗传标志物。而EGFR扩增则是原发性胶质母细胞瘤的特征性遗传标志物。在第三部分的研究中,我们引入目前已知的与胶质瘤预后密切相关的分子标志物,包括IDH突变、1P19Q缺失、TP53突变,PTEN突变、EGFR扩增和MGMT启动子甲基化等,进一步分析了TERT突变在这些特征性基因变异的情况下对胶质瘤患者的预后影响。结果显示：在1P19Q缺失和IDH1突变的环境中,TERT启动子突变的预后优势更加明显。此外,在针对PTEN突变和EGFR扩增的联合分析中我们发现,只有在不携带这两种分子标志物的前提下,TERT启动子突变才可作为预后较好的分子标志物而存在。PTEN突变和EGFR扩增对TERT启动子突变的预后均存在抑制作用,这种抑制效应在TP53突变组中更加明显。综上所述,本课题通过巢式PCR、Sanger测序、全转录子测序、mRNA芯片数据、生存预后统计等方法,对胶质瘤中TERT启动子突变,TP53突变、IDH突变、1P19Q缺失、PTEN突变和EGFR扩增等分子标志物进行检测,绘制TERT启动子突变在胶质瘤中的富集谱,综合评估了TERT启动子突变作为特征性分子标志物在胶质瘤中的预后价值。这些结果为后续深入研究TERT启动子突变影响胶质瘤预后的机制奠定了基础,也为寻找胶质瘤诊断和预后相关的分子标志物及分子治疗的靶点筛选提供了理论参考。
[Abstract]:Telomere is a sequence of end of eukaryotic chromosomes, with cell division and gradually shortened. Telomerase reverse transcriptase (Telomerase reverse, transcriptase, TERT) to keep the integrity of telomeres by activation of telomerase in glioma cells, the cells in the presence of high expression of proliferative ability of.TERT, and with the malignant degree of glioma is closely related however, in normal brain tissue is not the expression of TERT. The study found that the TERT promoter are two somatic mutations: C228T and C250T sub region, and lead to the transcriptional activity of TERT promoter was increased by 2-4 times. The foreign TERT promoter mutation appears frequently in glioblastoma and reported to have a certain impact on the the prognosis of the disease. The author hopes that through a large sample of China population in the TERT promoter mutations based on clearly affect the distribution and the prognosis, hoping to find A prognostic value of molecular markers. In the first part of this paper, we studied 887 cases of different grade glioma samples, extracted from different histopathological subtypes of whole genome DNA, specific regions containing TERT promoter mutations by nested PCR amplification, recovered after purification by Sanger sequencing. Through the comparison and analysis of C228T amplification and C250T mutation fragments, statistical mutation rate, draw C228T and C250T mutation in different levels, the distribution of different histologic subtypes of gliomas in the spectrum, and reveals its enrichment regularity. The results showed that in 887 cases of glioma samples in the promoter region of TERT, 274 cases were detected with the C228T mutation, accounted for the total sample number 30.89%, 83 cases with C250T mutations, mutations accounted for the total number of samples only 1 9.36%. samples C228T and C250T double sites exist at the same time. Except for 10 cases of homozygous mutation, the More than all the sample containing mutations were heterozygous. We analyzed the distribution of TERT in different grades of glioma in 2007 in accordance with the WHO central nervous system tumor grade. The results showed that in 498 cases of WHO grade II glioma samples, 197 cases were detected with the TERT mutation, the mutation rate was 39.56%; in 139 cases of WHO grade III glioma samples, 56 cases were detected with the TERT mutation, the mutation rate was 40.29%; in 250 cases of WHO grade IV glioma samples, 104 cases were detected with the TERT mutation, the mutation rate of 41.6%. mutation rate with gliomas slightly increased. We then the statistical distribution of the TERT promoter in histologic subtypes in various tissues: the enrichment ratio of oligodendroglioma TERT promoter mutation is the highest, accounted for 75.71% (53/70), TERT promoter of glioma in mutation frequency is lowest, only 10.84% (22/203). For a more detailed description of the TERT promoter mutation distribution in glioblastoma cells, we analyze the frequency of TERT mutation in primary glioblastoma and secondary glioblastomas, the results showed that 44.72% (89/199) of primary glioblastoma cells containing TERT promoter the mutation, while only 29.41% (15/51) of the secondary glioblastomas containing this mutation. The above results we concluded that TERT promoter mutations are highly enriched in oligodendroglioma, mutation rate increased with tumor grade have an increasing trend, and the C228T mutation and C250T mutation in.TERT mutual exclusion obvious promoter mutation there are stable in patients with glioma in Chinese population based, can be used for glioma molecular marker for prognosis China population stratification research matter. At present, glioma treatment problems are the following : glioma cell invasion and infiltration is strong, which limits the scope of the surgical resection in a certain extent; in the chemical treatment appear soon after cell clones resistant; there are many oncogenic signaling pathways activated in tumor cells and the mutant gene interaction and so on. In view of the above reasons, glioma in particular, the survival rate of patients with glioblastoma is very low, the median survival period is generally not more than 15 months. However, there is still a shortage of 10% glioblastoma patients life span can be extended to three to five years. But the reason between the causes of the prolonged survival of patients and their health and the change is not clear. The gene has been reported with O6-Methylguanine-DNA methyltransferase (MGMT) promoter in glioma hypermethylation of young female patients have access than other patients Longer survival. Therefore, looking for the characteristic molecular markers with diagnostic or prognostic significance, and has important guiding significance to study the relevant individual prognostic stratification for glioma treatment. In the second part of this paper, we aimed at different levels, different histological sub new glioma research prognostic stratification mutations start TERT, and glioma samples for TCGA type annotation by whole transcriptome sequencing data, and in Proneural, Neural, Classical, TERT promoter mutation prognosis sub judgment to assess the value of Mesenchymal four molecular subtypes. The results showed that TERT promoter survival in patients with mutation group significantly at the TERT promoter wild survival group (P0.05), and the survival of patients with C250T mutation group than the C228T group's survival. Mutations in WHO grade II gliomas, TERT promoter mutation group The survival advantage is more obvious. When using the TCGA classification of molecular subtypes of notes after the samples we found that TERT promoter mutations in Proneural and Mesenchymal two molecular subtypes showed significant prognostic stratification, and stratification in the Classical and Neural two subtypes in which we are not obvious. Conclusion: the TERT promoter mutation as a molecular diagnosis of glioma prognosis predicting markers in TCGA subtypes and low grade gliomas are more obvious, can be used as a prognostic indicator for stability for this kind of patients with glioma prognosis. Recent studies have found that there are different genetic background of glioma in the developing process, the genetic background was always change one or several characteristic genes. Gene change these characteristic also directly or indirectly affect glioma The evolution and prognosis of tumors. The currently accepted IDH mutation is an early event of genomic changes of glioma, and TP53 mutation and 1P19Q deletion were appeared in the process of evolution of astrocytoma and oligodendroglioma. The TP53 mutation is from low-grade astrocytes into tumor genetic markers carrying secondary glioblastomas in. EGFR amplification is a sign of genetic characteristics of primary glioblastoma. In the third part of the study, we introduce the molecule known and glioma is closely related to the prognosis of the markers, including IDH mutation and 1P19Q deletion. TP53 mutation, PTEN mutation, EGFR amplification and MGMT promoter methylation, further analysis of the effect of TERT mutation on the prognosis of patients with glioma in these characteristic gene variations. The results indicated that the deletion of 1P19Q and ID Mutations in the H1 environment, TERT promoter mutations are more obvious advantages of prognosis. In addition, the combined analysis for PTEN mutation and amplification of EGFR we found that only in the premise of not carrying the two kinds of molecular markers, TERT promoter mutations can be used as a molecular marker of good prognosis and.PTEN mutation EGFR amplification of TERT promoter mutations in the prognosis of inhibition are present, the inhibitory effect is more obvious in TP53 mutation group. In summary, this topic by nested PCR, Sanger sequencing, whole transcriptome sequencing, mRNA chip data, after the pre survival statistics of glioma TERT promoter mutation, TP53 mutation. IDH mutation, PTEN mutation and 1P19Q deletion, EGFR amplification and other molecular markers were detected, rendering the mutations of TERT promoter in glioma enrichment spectrum, a comprehensive evaluation of TERT promoter mutations as characteristic molecular markers in Prognostic value of glioma. These results provide a theoretical basis for further research on the mechanism of TERT promoter mutation affecting the prognosis of glioma, and also provide a theoretical reference for finding molecular markers and molecular therapeutic targets of glioma.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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