云南地区家族性腺瘤性息肉病致病基因筛查及APC基因同义突变SNP在其发病机制中的研究

发布时间：2018-04-29 03:17

本文选题：家族性腺瘤性息肉病 + 家系管理　；参考：《昆明医科大学》2016年博士论文

【摘要】：[目的]收集云南省内不同地域及民族的家族性腺瘤性息肉病(FAP)家系,建立独具特色的遗传性大肠癌资源库,在此基础上随机挑选20例不同家系来源的FAP先证者通过联合多基因(APC、MYH及AXIN2)测序及生物信息学分析技术查找FAP致病基因突变位点,对于没有发现致病性突变位点的FAP家系,选择其中一个少数民族家系对其全部家系成员进行全基因组测序以检测其致病基因,最后通过同义突变外显子异常剪切试验探讨APC基因的罕见突变形式(APC基因的同义突变SNP)在家族性腺瘤性息肉病发病机制的作用。[方法]1.收集云南地区不同地域及民族的FAP先证者组织样本,进行家系信息收集整理,全面收集完整的FAP家系信息、血液及组织样本,进一步扩充云南省遗传性大肠癌组织标本库规模。2.在本课题组已建立的云南省遗传性大肠癌组织标本库中随机选择20例来自于不同家系的FAP先证者的血液样本,分别提取DNA后,对与FAP发病密切相关的APC、MYH及AXIN2基因进行多基因联合检测,同时利用生物信息学技术筛选FAP先证者的致病性胚系突变位点。3.对上述经多基因联合筛查未见致病性突变位点的FAP患者,选择其中一个少数民族(白族)FAP家系对其全部7名家系成员进行全基因组测序以检测其致病基因,对全基因组测序数据进行单核苷酸变异(SNV),拷贝数变异(CNV)以及结构变异(SV)的基因组变异分析4.根据前期研究中筛选得到的且生物信息学预测可能引起APC外显子异常剪切的APC基因同义突变SNP(c.1458TC rs2229992,位于APC基因12号外显子边缘区域)的基因序列分别构建野生型及突变型的mini gene系统并插入pEGFP-N1 构建重组质粒 pEGFP-N1+wt-minigene 以及pEGFP-N1+mt-minigene,将重组质粒分别转染Hela细胞进行真核表达,随后通过RT-PCR对Hela细胞真核表达产物进行对比验证以观察突变型APC-sSNP是否能引起APC基因异常剪切并最终导致APC蛋白的截短。[结果]1.通过对云南省遗传性大肠癌标本库的进一步整理、病案调查及门诊新发患者的收集,筛选出符合本实验研究的31个典型的FAP家系,其中3个白族家系,1个彝族家系,1个回族家系和7杂交家系,19个汉族家系。2.对20例随机入组进入本研究的FAP先证者的APC基因的筛检结果中,我们发现了 3个致病性突变,其中1个为无义突变c.3587CA(p.S1196X),该类型突变使得终止密码子提前出现,从而使APC蛋白呈截短改变;另外2种突变分别是杂合性重复c.1959-143__1959-140het_dupAGAA及杂合性缺失c.6123_6124het_delAT,这两种突变均会导致APC基因出现移码突变,从而使APC蛋白相应表达的功能发生改变导致FAP的发生。在针对MYH基因的检测结果中,我们检出了 MYH基因2种无义突变:c.456TA(p.Y152X)和c.1564TG(p.G522X),这2种突变为致病性突变,同样使得终止密码子提前出现,导致MYH蛋白呈截短改变;同时筛检得到4种缺失性突变,包括2种会导致MYH基因发生移码突变的杂合性缺失c.1435-106_1435-63het_delGCCTAGCTAGATCAGTAGAGTCGGGGAAAGG GAGAGAGGACAAG 及 c.1566+33_1566+35het_delTGT。针对 AXIN2 基因的筛查结果中,我们筛检得到4种同义突变,其中位于第8外显子c.2062CT(p.L688L)为已报道的致病性突变,该同义突变在mRNA水平干扰剪接增强子的作用导致APC蛋白的移码并最终导致APC蛋白的截短。3.针对APC(-)FAP白族家系成员全基因组测序分析结果发现,DCC基因的结构变异是该家系成员发生FAP的主要原因;而造成蛋白质功能丧失的7个SNV(OR5AN1、OR5A1、LOXL、CEACAM21、C20orf201、ABCB5、PAPR15)及发生indel的3个SNV(CELA1、C18orf25和MAGI)可能与FAP发生、发展存在一定的关系。4.包含 APC 基因的同义突变 SNPc.1458TC(rs2229992)的 wt-minigene 及mt-minigene系统分别转染Hela细胞后,提取Hela细胞RNA并通过RT-PCR扩增后,将PCR产物进行电泳可见由mt-minigene构建的重组质粒转染Hela细胞进行表达,经RT-PCR扩增目的片段,PCR产物行琼脂糖凝胶电泳显示,其最终表达产物可见wt-minigene系统的PCR产物未引起外显子剪切,而mt-minigene系统的PCR产物可见12号外显子出现跳跃,其对应的PCR产物仅见12号外显子两翼的内含子片段。[结论]1.云南省FAP家系包括少数民族FAP家系的收集和保存,是对云南省遗传性大肠癌标本库的扩充,既提高了该标本库的应用价值,又较好的保存了云南省宝贵的FAP家系遗传资源。为研究不同地区基因的差异性提供优质样本,有望在民族特异性上发现FAP相关致病基因新的突变及新的致病基因。2.结合本研究结果,在对随机选择的来自20个不同家系的FAP先证者进行多基因联合筛查结果发现,其中8名FAP先证者明确了致病基因及其突变位点,致病基因的检出率为40%。3.全基因组测序分析结果发现,抑癌基因DCC的结构变异是本研究中选择的APC(-)FAP白族家系成员发生FAP的主要原因,提示DCC基因是对FAP患者进行突变基因检测时除了 APC、MYH以及AXIN2基因以外的另外一个候选筛查基因。4.APC基因的同义突变SNPc.1458TC(rs2229992)是导致FAP发生的可能致病基因突变位点,但需进一步行外显子剪切依赖实验进行验证,同时进行细胞、动物水平功能验证及扩大样本的人群验证。
[Abstract]:[Objective] to collect familial adenomatous polyposis (FAP) families of different regions and nationalities in Yunnan province and establish a unique genetic colorectal cancer resource base. On this basis, 20 FAP precursor of different families from different families were randomly selected to search for FAP pathogeny by sequencing of combined polygenes (APC, MYH and AXIN2) and bioinformatics analysis. The mutation site, for the FAP family, which did not find the pathogenicity site, selected one minority family to complete genome sequencing of all its family members to detect its pathogenic genes. Finally, the rare mutation of the APC gene (the synonymous mutation SNP of the APC gene) in the family was investigated by the abnormal exon shear test of the synonymous mutation (the SNP of the APC gene) in the family. The role of the pathogenesis of sexual adenomatous polyposis. [method]1. collect FAP precursor tissue samples from different regions and nationalities in Yunnan, collect and collate the family information, collect complete FAP family information, blood and tissue samples, and further expand the scale of the specimen bank of hereditary colorectal carcinoma in Yunnan Province, which has been built in this group. The blood samples of 20 FAP precursor from different families were randomly selected from the Yunnan hereditary colorectal carcinoma tissue specimen bank. After extracting DNA, the APC, MYH and AXIN2 genes, which were closely related to the pathogenesis of FAP, were detected by multiple genes, and the pathogenicity site of the FAP precursor was screened by bioinformatics. .3. for the FAP patients who had no pathogenic mutation site by multi gene screening, one of the ethnic minorities (Bai nationality) FAP family was selected to complete genome sequencing to detect the genome of all 7 family members. Single nucleotide variation (SNV), copy number variation (CNV) and structural variation (SV) were carried out on the whole genome sequencing data. Genomic variation analysis 4. based on the prediction of the APC gene synonymous mutation SNP (c.1458TC rs2229992, located in the marginal region of exon 12 of the APC gene) that may cause abnormal shear of the APC exon, the gene sequence is screened by bioinformatics, and the mini gene system of wild type and mutant type is constructed and inserted into the pEGFP-N1 structure, respectively. Recombinant plasmid pEGFP-N1+wt-minigene and pEGFP-N1+mt-minigene were constructed to transfect the recombinant plasmid to Hela cells for eukaryotic expression. Then the eukaryotic expression products of Hela cells were compared and verified by RT-PCR to observe whether the mutant APC-sSNP could cause abnormal APC gene shear and eventually lead to the truncation of APC protein. 31 typical FAP families, including 3 Bai families, 1 Yi families, 1 Hui families and 7 hybrid families, 19 Han families, and 19 Han families, were selected to enter the FAP first evidence of this study, including the further sorting out of the specimen bank of the hereditary colorectal carcinoma in Yunnan province and the collection of new outpatients in the outpatient clinic. In the screening results of the APC gene, we found 3 pathogenic mutations, 1 of which were nonsense mutations c.3587CA (p.S1196X), which made the termination codon appear early, so that the APC protein was truncated and the other 2 mutations were heterozygous repeat c.1959-143__1959-140het_dupAGAA and heterozygosity c.6123_6124he, respectively. T_delAT, these two mutations all lead to the mutation of the APC gene, which leads to the change of the function of the APC protein to cause the occurrence of FAP. In the results of the detection of the MYH gene, we detected 2 nonsense mutations of the MYH gene, c.456TA (p.Y152X) and c.1564TG (p.G522X), and the 2 mutations were pathogenicity mutations that also made the termination of the gene. The codon appears in advance, leading to a truncated change in the MYH protein; at the same time, 4 kinds of deletion mutations are screened, including 2 heterozygosity missing c.1435-106_1435-63het_delGCCTAGCTAGATCAGTAGAGTCGGGGAAAGG GAGAGAGGACAAG and c.1566+33_1566+ 35het_delTGT. for the screening of the AXIN2 gene, which may lead to the mutation of the MYH gene. We screened 4 synonymous mutations, in which the eighth exon c.2062CT (p.L688L) was reported as the reported pathogenic mutation. The synonymous mutation at mRNA level interfered with the splicing enhancer, resulting in the APC protein truncation and the final cause of the APC protein's truncated.3. for the whole genome sequencing analysis of the APC (-) FAP white family members. The DCC base was found. The 7 SNV (OR5AN1, OR5A1, LOXL, CEACAM21, C20orf201, ABCB5, PAPR15) and the 3 SNV (CELA1, ABCB5, and PAPR15) causing the loss of protein function may occur in the family, and the development in a certain relationship contains the synonymous mutation of the gene. 992) after transfection of wt-minigene and mt-minigene system to Hela cells respectively, the Hela cell RNA was extracted and amplified by RT-PCR, and the recombinant plasmid constructed by mt-minigene was transfected to Hela cells by RT-PCR, and the target fragment was amplified by RT-PCR. The PCR product was shown by agarose gel electrophoresis, and the final expression product was visible. The PCR product of the wt-minigene system did not cause exon shear, and the PCR product of the mt-minigene system showed that exon 12 appeared jumping, and the corresponding PCR product was only seen in the intron fragments of the two wings of exon 12. [conclusion]1. in Yunnan Province, including the collection and preservation of minority FAP family, was a specimen of hereditary colorectal cancer in Yunnan province. The expansion of the library not only improves the application value of the specimen bank, but also preserves the precious FAP family genetic resources in Yunnan province. It provides high quality samples for studying the difference of genes in different regions. It is hopeful that the new mutation of FAP related genes and the new pathogenic gene.2. combined with the results of this study are expected to be selected in the random selection. The results of multiple gene screening by FAP precursor from 20 different families found that 8 of the FAP precursor identified the pathogenic gene and its mutation site. The detection rate of the pathogenic gene was found in the whole genome sequencing analysis of 40%.3., and the structural variation of the tumor suppressor gene DCC was a member of the APC (-) FAP Bai family selected in this study. The main reason for the occurrence of FAP suggests that the DCC gene is the synonymous mutation of the.4.APC gene of the other candidate screening gene other than APC, MYH and AXIN2 gene, except APC, MYH and AXIN2 gene, which is a possible mutation site for the pathogenesis of FAP, but the exon shear dependence experiment should be further carried out. Validation was carried out at the same time, cell and animal level functional verification and population verification of expanded samples were conducted.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735

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