本文選題：家族性腺瘤性息肉病 + 家系管理�。� 參考：《昆明醫(yī)科大學(xué)》2016年博士論文

【摘要】：[目的]收集云南省內(nèi)不同地域及民族的家族性腺瘤性息肉病(FAP)家系,建立獨(dú)具特色的遺傳性大腸癌資源庫,在此基礎(chǔ)上隨機(jī)挑選20例不同家系來源的FAP先證者通過聯(lián)合多基因(APC、MYH及AXIN2)測序及生物信息學(xué)分析技術(shù)查找FAP致病基因突變位點(diǎn),對(duì)于沒有發(fā)現(xiàn)致病性突變位點(diǎn)的FAP家系,選擇其中一個(gè)少數(shù)民族家系對(duì)其全部家系成員進(jìn)行全基因組測序以檢測其致病基因,最后通過同義突變外顯子異常剪切試驗(yàn)探討APC基因的罕見突變形式(APC基因的同義突變SNP)在家族性腺瘤性息肉病發(fā)病機(jī)制的作用。[方法]1.收集云南地區(qū)不同地域及民族的FAP先證者組織樣本,進(jìn)行家系信息收集整理,全面收集完整的FAP家系信息、血液及組織樣本,進(jìn)一步擴(kuò)充云南省遺傳性大腸癌組織標(biāo)本庫規(guī)模。2.在本課題組已建立的云南省遺傳性大腸癌組織標(biāo)本庫中隨機(jī)選擇20例來自于不同家系的FAP先證者的血液樣本,分別提取DNA后,對(duì)與FAP發(fā)病密切相關(guān)的APC、MYH及AXIN2基因進(jìn)行多基因聯(lián)合檢測,同時(shí)利用生物信息學(xué)技術(shù)篩選FAP先證者的致病性胚系突變位點(diǎn)。3.對(duì)上述經(jīng)多基因聯(lián)合篩查未見致病性突變位點(diǎn)的FAP患者,選擇其中一個(gè)少數(shù)民族(白族)FAP家系對(duì)其全部7名家系成員進(jìn)行全基因組測序以檢測其致病基因,對(duì)全基因組測序數(shù)據(jù)進(jìn)行單核苷酸變異(SNV),拷貝數(shù)變異(CNV)以及結(jié)構(gòu)變異(SV)的基因組變異分析4.根據(jù)前期研究中篩選得到的且生物信息學(xué)預(yù)測可能引起APC外顯子異常剪切的APC基因同義突變SNP(c.1458TC rs2229992,位于APC基因12號(hào)外顯子邊緣區(qū)域)的基因序列分別構(gòu)建野生型及突變型的mini gene系統(tǒng)并插入pEGFP-N1 構(gòu)建重組質(zhì)粒 pEGFP-N1+wt-minigene 以及pEGFP-N1+mt-minigene,將重組質(zhì)粒分別轉(zhuǎn)染Hela細(xì)胞進(jìn)行真核表達(dá),隨后通過RT-PCR對(duì)Hela細(xì)胞真核表達(dá)產(chǎn)物進(jìn)行對(duì)比驗(yàn)證以觀察突變型APC-sSNP是否能引起APC基因異常剪切并最終導(dǎo)致APC蛋白的截短。[結(jié)果]1.通過對(duì)云南省遺傳性大腸癌標(biāo)本庫的進(jìn)一步整理、病案調(diào)查及門診新發(fā)患者的收集,篩選出符合本實(shí)驗(yàn)研究的31個(gè)典型的FAP家系,其中3個(gè)白族家系,1個(gè)彝族家系,1個(gè)回族家系和7雜交家系,19個(gè)漢族家系。2.對(duì)20例隨機(jī)入組進(jìn)入本研究的FAP先證者的APC基因的篩檢結(jié)果中,我們發(fā)現(xiàn)了 3個(gè)致病性突變,其中1個(gè)為無義突變c.3587CA(p.S1196X),該類型突變使得終止密碼子提前出現(xiàn),從而使APC蛋白呈截短改變;另外2種突變分別是雜合性重復(fù)c.1959-143__1959-140het_dupAGAA及雜合性缺失c.6123_6124het_delAT,這兩種突變均會(huì)導(dǎo)致APC基因出現(xiàn)移碼突變,從而使APC蛋白相應(yīng)表達(dá)的功能發(fā)生改變導(dǎo)致FAP的發(fā)生。在針對(duì)MYH基因的檢測結(jié)果中,我們檢出了 MYH基因2種無義突變:c.456TA(p.Y152X)和c.1564TG(p.G522X),這2種突變?yōu)橹虏⌒酝蛔?同樣使得終止密碼子提前出現(xiàn),導(dǎo)致MYH蛋白呈截短改變;同時(shí)篩檢得到4種缺失性突變,包括2種會(huì)導(dǎo)致MYH基因發(fā)生移碼突變的雜合性缺失c.1435-106_1435-63het_delGCCTAGCTAGATCAGTAGAGTCGGGGAAAGG GAGAGAGGACAAG 及 c.1566+33_1566+35het_delTGT。針對(duì) AXIN2 基因的篩查結(jié)果中,我們篩檢得到4種同義突變,其中位于第8外顯子c.2062CT(p.L688L)為已報(bào)道的致病性突變,該同義突變在mRNA水平干擾剪接增強(qiáng)子的作用導(dǎo)致APC蛋白的移碼并最終導(dǎo)致APC蛋白的截短。3.針對(duì)APC(-)FAP白族家系成員全基因組測序分析結(jié)果發(fā)現(xiàn),DCC基因的結(jié)構(gòu)變異是該家系成員發(fā)生FAP的主要原因;而造成蛋白質(zhì)功能喪失的7個(gè)SNV(OR5AN1、OR5A1、LOXL、CEACAM21、C20orf201、ABCB5、PAPR15)及發(fā)生indel的3個(gè)SNV(CELA1、C18orf25和MAGI)可能與FAP發(fā)生、發(fā)展存在一定的關(guān)系。4.包含 APC 基因的同義突變 SNPc.1458TC(rs2229992)的 wt-minigene 及mt-minigene系統(tǒng)分別轉(zhuǎn)染Hela細(xì)胞后,提取Hela細(xì)胞RNA并通過RT-PCR擴(kuò)增后,將PCR產(chǎn)物進(jìn)行電泳可見由mt-minigene構(gòu)建的重組質(zhì)粒轉(zhuǎn)染Hela細(xì)胞進(jìn)行表達(dá),經(jīng)RT-PCR擴(kuò)增目的片段,PCR產(chǎn)物行瓊脂糖凝膠電泳顯示,其最終表達(dá)產(chǎn)物可見wt-minigene系統(tǒng)的PCR產(chǎn)物未引起外顯子剪切,而mt-minigene系統(tǒng)的PCR產(chǎn)物可見12號(hào)外顯子出現(xiàn)跳躍,其對(duì)應(yīng)的PCR產(chǎn)物僅見12號(hào)外顯子兩翼的內(nèi)含子片段。[結(jié)論]1.云南省FAP家系包括少數(shù)民族FAP家系的收集和保存,是對(duì)云南省遺傳性大腸癌標(biāo)本庫的擴(kuò)充,既提高了該標(biāo)本庫的應(yīng)用價(jià)值,又較好的保存了云南省寶貴的FAP家系遺傳資源。為研究不同地區(qū)基因的差異性提供優(yōu)質(zhì)樣本,有望在民族特異性上發(fā)現(xiàn)FAP相關(guān)致病基因新的突變及新的致病基因。2.結(jié)合本研究結(jié)果,在對(duì)隨機(jī)選擇的來自20個(gè)不同家系的FAP先證者進(jìn)行多基因聯(lián)合篩查結(jié)果發(fā)現(xiàn),其中8名FAP先證者明確了致病基因及其突變位點(diǎn),致病基因的檢出率為40%。3.全基因組測序分析結(jié)果發(fā)現(xiàn),抑癌基因DCC的結(jié)構(gòu)變異是本研究中選擇的APC(-)FAP白族家系成員發(fā)生FAP的主要原因,提示DCC基因是對(duì)FAP患者進(jìn)行突變基因檢測時(shí)除了 APC、MYH以及AXIN2基因以外的另外一個(gè)候選篩查基因。4.APC基因的同義突變SNPc.1458TC(rs2229992)是導(dǎo)致FAP發(fā)生的可能致病基因突變位點(diǎn),但需進(jìn)一步行外顯子剪切依賴實(shí)驗(yàn)進(jìn)行驗(yàn)證,同時(shí)進(jìn)行細(xì)胞、動(dòng)物水平功能驗(yàn)證及擴(kuò)大樣本的人群驗(yàn)證。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735

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