【作者】 盛霞；

【导师】 施达仁； 周晓燕； 盛伟琪； 杜祥；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer, HNPCC)是一种常染色体显性遗传性恶性肿瘤综合征,外显率高达80%-90%,占所有结直肠癌的2%-15%。HNPCC与散发性结直肠癌(Sporadic Colerectal Cancer, SCRC)相比,无论在发病机制、临床特征,乃至治疗方案的选择和随访方案的制定上,HNPCC都具有其特殊性。目前,世界许多国家和地区都制定了一定的HNPCCI临床诊断标准,如Amsterdam标准,Bethesda指导纲要等。HNPCC发生的分子遗传学基础是错配修复基因(mismatch repair, MMR)的突变,该基因的突变能导致复制错误增加,微卫星不稳定而使多器官肿瘤发生,已定位并克隆的人MMR基因hMLHl、hMSH2、hMSH6、hMSH3、hPMSl、hPMS2等与HNPCC发生密切相关。本课题组先前已对58个符合不同临床标准的HNPCC家系先证者进行了hMSH2/hMLH1/hMSH6/hPMS2基因整个编码区胚系小突变和大片段异常及hMLH1基因启动子胚系甲基化的研究,结果发现29个家系存在MMR基因的胚系突变和2个hMLH1基因启动子胚系的完全甲基化,总的胚系异常率仅约为53.4%(31/58)。国内外研究均发现符合不同临床诊断标准的HNPCC家系中,约42%-75%的HNPCC家系能检测到hMSH2、hMLH1和hMSH6基因的胚系突变,但在那些不能完全符合AC标准的可疑HNPCC家系中,MMR基因胚系突变的检出率可能更低。临床上有一些携带MMR基因突变的HNPCC家系并不符合ACⅠ或ACⅡ标准,而将近一半临床高度怀疑HNPCC的人群不能检测到MMR基因的突变,这都表明可能还存在一部分尚未被鉴定出的基因参与HNPCC的发生过程。随着基因芯片技术的发展,人们可以从全基因组的水平研究基因表达谱的变化,从整体上认识基因表达与肿瘤发生、发展及转移的关系。不少研究者已经运用这一工具对乳腺癌、肺癌、前列腺癌、淋巴瘤及散发性结直肠癌等肿瘤相关的基因表达谱进行了研究,而HNPCC的基因表达谱研究尚未见报道。采用基因表达谱芯片筛选HNPCC差异基因,并对其功能深入研究,是阐明HNPCC发病机制、确立HNPCC特异性诊断标志物和治疗相关的分子靶点的重要手段。本研究收集50例符合不同临床标准的HNPCC先证者及7例散发性结直肠癌病例,首先对hMSH2/hMLH1胚系基因突变及肿瘤组织相应蛋白表达情况进行检测分析,将HNPCC分为具有MMR缺陷表型组(MLH1和/或MSH2基因突变和/或蛋白表达缺失)和无MMR缺陷表型组；然后应用Affymetrix HG-U133plus2.0寡核苷酸基因芯片技术对50例HNPCC及同期7例散发性结直肠癌肿瘤组织进行了人类全基因组表达谱芯片检测,通过SAM (significance Analysis of Microarray)软件和交集分析,筛选出符合不同临床标准的HNPCC与SCRC、MMR缺陷表型组与SCRC组之间的差异表达基因,分析与SCRC不同的HNPCC基因表达谱特征,确定与MMR缺陷表型密切相关的基因；对显著表达差异的基因采用real-time PCR和免疫组化进行验证,以期发现HNPCC发生可能相关的基因,找到代表MMR缺陷表型的分子标签。本研究共分为三个部分：目的：对50例符合不同临床标准的HNPCC家系先证者病理资料进行分析并完成hMSH2、hMLH1蛋白及其中26例基因突变检测。方法：符合Amsterdam标准家系7个、复旦标准家系9个和Bethesda指导纲要患者34例收入本项临床病理研究。收集家系先证者外周血样本10ml以供提取基因组DNA,以7例同期散发性结直肠癌为对照。利用PCR技术分别扩增2个基因共35个外显子,PCR产物纯化后进行DNA测序,并对测序结果进行分析。免疫组化采用Envision二步法。结果：在26个HNPCC先证者中共发现8个hMSH2/hMLH1突变,其中符合AC标准的4例(4/8),BG标准4例。50例中蛋白表达缺失17例(16/50),包括已检测到突变的8例。结论：hMSH2、hMLH1基因突变率及蛋白表达缺失率分别为30.8%和34%；免疫组化检测错配修复蛋白的表达对错配修复基因的突变有很好的初筛作用,Amsterdam标准对预示突变最敏感。仍有一部分符合临床标准的HNPCC患者不能检测到MMR基因的异常。目的：筛选HNPCC与SCRC、具有MMR缺陷表型组与SCRC组差异基因。方法：收集第一部分50例HNPCC患者及同期7例SCRC新鲜肿瘤标本,Trizol法提取组织RNA,制备靶标(双链cDNA合成、纯化,体外转录合成生物素标记cRNA、纯化、片段化),利用Affymetrix真核表达谱芯片杂交,清洗染色并扫描；应用SAM等软件对差异基因进行分析,结合MAS等分析工具对筛选基因进行初步的生物学功能分析。结果：获得HNPCC与SCRC组差异表达基因425个,符合AC组与SCRC组差异表达基因445个,符合复旦推荐标准与SCRC组差异表达基因469个,符合BG组与SCRC组差异基因153个,RB组与SCRC组差异基因613个,MMR缺陷表型组与SCRC组差异表达基因662个(q-value<5%, Fold change≥2)。经统计学分析、文献挖掘及聚类分析结果筛选出HNPCC与SCRC差异基因58个,代表MMR缺陷表型基因11个。结论：58个基因组成的表达模式代表了HNPCC与SCRC之间MMR基因之外的差异表达基因,具有MMR缺陷表型组与AC组基因表达谱相似,有11个基因可以构成这一表达模式；这些差异表达基因可能在HNPCC的发病中起重要的生物学作用。目的：收集第二部分57个病例并增加55例(包括6例HNPCC和49例SCRC)验证HNPCC与SCRC部分差异表达基因,筛选并分析与MMR缺陷表型相关的分子标签。方法：采用实时荧光定量PCR和免疫组化分别从mRNA和蛋白水平检测HNPCC和SCRC肿瘤组织中ANPEP/CD13、MTA-2、APCDD1和HEPACAM的表达,分析这四个基因在两组中的表达与基因芯片结果的符合情况,分析它们在具有MMR缺陷表型组与SCRC组之间的差异。结果：ANPEP/CD13、MTA-2、APCDD1和HEPACAM在基因和蛋白水平的表达与基因芯片结果一致。经统计学分析发现这四个基因在MMR缺陷表型组的表达与在CRC组的表达有明显差异,ANPEP、MTA-2在MMR基因异常组显著上调,APCDD1和HEPACAM在MMR基因异常组显著下调。结论：ANPEP/CD13、MTA-2、APCDD1和HEPACAM可能与HNPCC发生有关,并可望成为提示MMR缺陷表型的分子标签。更多还原

【Abstract】 Hereditary nonpolyposis colorectal cancer(HNPCC) is one of the most common autosomal dominantly inherited cancers syndrome, accounts for 2%-15% of all colorectal cancers and the penetrance reach up to 80%-90%. Compare with sporadic colorectal cancer(SCRC), HNPCC shows its own characteristcs associated with the molecular mechanism,clinical features,the methods for treatment and management of HNPCC kindreds. Therefore, to differentiate the HNPCC from SCRC is very important and it will interest not only in the clinic but also in genetic counseling of HNPCC kindreds. Now, many countries and territories have established the clinical diagnostic criteria for HNPCC, such as Amsterdam Criteria and Bethesda Guidelines. The molecular genetics basis of HNPCC was the mutation of mismatch repair gene, which can induce the increasement of misreplications, microsatellites instability and then result in tumorigenesis of many organs.Now, human MMR gene which has been locolized and cloned as hMLH1、hMSH2、hMSH6、hMSH3、hPMS1、hPMS2 and so on were closely associated with HNPCC. The former two account for a large majority of mutations found in HNPCC families from various countries. Previously, We detected germline mutations and large genomic variantions of the entire coding regions of hMSH2lhMLH1/hMSH6 genes and the methylation of hMLHl promoter in 58 HNPCC families fulfilling different cliinical criteria, and the final germline-variation rate of Chinese HNPCC was 53.4%(31/58). It has been reported that the germline mutation of hMSH2, hMLHl and hMSH6 was about 42%-75% in HNPCC families fulfilling different clinical criteria, while the mutation rate of MMR gene in the atypical HNPCC family was likely to less. Some HNPCC families which can found the mutation of MMR gene didn’t fulfil any clinical criteria, one the other hand, more than fifty percent suspected HNPCC patients can’t found mutation of MMR gene. Therefore, the tumorigenesis of HNPCC must be associated with other disease genes which has not been verified. With the development of gene chip technic, people can study the gene expression profile on the level of genomewide, and recognize the relationship of the gene expreesion and tumorigenesis, progress and metastasis on a whole level. Many related reseach has been reported on the gene expression profile of breast cancer, lung cancer, protaste cancer, lymphoma SCRC and so on. But the study on the HNPCC gene expression has not been reported up till now.In this study, we collected 50 HNPCC families fulfilling different clinical criteria, detected the germline mutation and protein expreesion of MLH1 and MSH2, then we devided the 50 samples into two groups:MMR-proficient and MMR-deficient phenome. Secondly, we use the the Human Genome U133A GeneChip array (Affymetrix) to analysis the the gene expreesion of this 50 samples and another 7 SCRC samples. Thirdly, we screen the differential expression gene of the HNPCC and SCRC, MMR-proficient and MMR-deficient phenome, constructed the gene signature of HNPCC and MMR-deficient tumors. Finally, we demonstrated the robustness of the signature by transferring it to a real-time RT-PCR and IHC platform.This two-step classification approach can identify MMR-deficient phenome as well as HNPCC cases merits further gene expression studies to identify prognostic signatures.The current research project is comprised for the following three parts:Object:To analyse the expression of hMLH1 and hMSH2 protein in 50 HNPCC probands fulfilling defferent clinical criteria and the germline mutation of hMLHl and hMSH2 gene in 26 probands.Methods:The peripheral blood was collected from the probands of 26 HNPCC families fulfilling different clinical criterial, of which 7 families fulfilled AC,19 kindreds fit BG. Genomic DNA was extracted following the manafacturer’s protocol and used as the template to amplify 35 exons of the 2 genes by PCR. PCR products were purified and used as the template for direct DNA sequencing. The results of sequencing were analysed by different bioanalysis software. To further investigate the pathological effects of detected missense mutations, we analyse the same exon of hPMS2 gene using PCR-based sequencing in 130 healthy persons without family history. IHC envision two step method was performed.Results:8 germline mutation of hMLHl and hMSH2 was found in 26 HNPCC probands families.4 fulfilled AC and 4 fulfilled BG.17 probands was found to be hMLHl and hMSH2 protein deficient.Conclusions:The rate of germline mutation and protein expression was 30.8% and 34%. AC are the most sensitve clinical creteria to predict mutations. Immunohistochemical staining are reliable screening method with high predictive value for the detection of mutation. Some suspected HNPCC patients can’t found mutation of MMR gene.Object:To screen the differential genes between HNPCC and SCRC, MMR-proficient and MMR-deficient phenotype.Methods:Collected the 50 HNPCC probands and 7 SCRC tumor specimen. Labelling of RNA, hybridisation and scanning. Biotin-labelled cRNA was prepared from 10μg of total RNA and hybridised to the Human Genome U133A GeneChip array (Affymetrix). The readings from the quantitative scanning were analysed by SAM and the CapitalBio(?) Molecule Annotation System V4.0.Results:Results in 425 differential expression genes btween HNPCC and SCRC,445 genes between AC and SCRC,469 between FD and SCRC,153 between BG1 and SCRC,613 between RB and SCRC,662 between RB and SCRC for further analysis.Conclusions:58 gene was found for further analysis and to accompany the differential genes between HNPCC and SCRC besides MMR gene. A eleven-gene signature was constructed to seperating MMR-proficient and MMR-deficient phenotype.Object:To demonstrate the differential expression genes between HNPCC and SCRC, screen and preliminary identify molecular signature of MMR-deficient phenotype in 112 cases(included the 57 cases in part 2).Methods:Tissue section from 28 HNPCC and 28 SCRC patients were examined using real-time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR) technique for ANPEP, MTA2, APCDD1 and HEPACAM, and immunohistochemistry for the expression of the former three genes. Another 56 cases of HNPCC and SCRC were also used to perform the former three genes immunostaining. Demonstrate the exression of the four genes in different groups.Results:The expression of ANPEP, MTA2 in HNPCC was significantly higher than in SCRC, while APCDD1, HEPACAM was lower in HNPCC. The results was coincidence with the gene chip. The expression of these four genes was also different between the group of MMR-proficient and MMR-deficient phenotype.Conclusions:ANPEP, MTA2, APCDD1 and HEPACAM may be a signature capable of separating HNPCC and SCRC as well as MMR-proficient and MMR-deficient phenotype.更多还原