【作者】 王婷婷；

【导师】 周建农；

【作者基本信息】 南京医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 背景:家族性腺瘤性息肉病（familial adenomatous polyposis,FAP）是一种常染色体显性遗传病,表现为大肠内布满了腺瘤（几百至几千）。其全球发病率为出生婴儿的1/10000-1/7000。进一步的研究发现除了经典型家族性腺瘤性息肉病（classical familial adenomatous polyposis,CFAP）,还有减弱性家族性腺瘤性息肉病（attenuated familialadenomatous polyposis,AFAP）和一些新类型的息肉病,如Gardner综合征、Peutz-Jeghers综合征（Peutz-Jeghers syndrome,P-J综合征）、MYH相关性息肉病（MYH associated polyposis,MAP）等。目前已知FAP的致病基因是位于5q21-22的结肠腺瘤性息肉病（adenomatus polyposis coli,APC）基因。迄今已报道了800多种APC基因的病理性改变（http://www.hgmd.cf.ac.uk/）,且多集中在15外显子的5’端。这种改变的结果是形成截短的APC蛋白,导致APC蛋白功能的障碍从而致病。Peutz-Jeghers综合征（Peutz-Jegherssyndrome,P-J综合征）作为息肉病的一种特殊类型,有独特的致病基因—LKB1/STK11,位于19p上。LKB1改变使丝氨酸/苏氨酸激酶失活从而致病。APC和LKB1基因都是肿瘤抑制基因,它们的改变导致肿瘤的发生。目的:FAP的发生具有较高的遗传倾向,外显率几乎达到100%。采取相应措施如相关基因的检测,有助于发现高危人群做到早发现、早诊断和早治疗。设计本实验试图找到中国人群中相关基因的改变,用于临床。方法:本实验分两部分:1、提取9个FAP家系18名成员的外周血DNA,进行多重连接依赖性探针扩增（multiplex ligation-dependent probeamplification,MLPA）检测APC基因的大片段缺失。应用聚合酶链式反应（polymerase chain reaction,PCR）扩增APC基因的15个外显子区域,经变性高效液相色谱（denaturinghigh performance liquid chromatograph,DHPLC）筛查出流出峰异常的片段,DNA测序验证小片段的改变。2、提取5名P-J综合征患者外周血和组织标本的DNA,应用PCR扩增LKB1基因的9个外显子,直接测序。甲基化处理组织标本DNA,应用甲基化特异聚合酶链式反应（Methylationspecific polymerase chain reaction,MSP）检测基因启动子区域5’-CpG岛的甲基化情况。结果:第一部分实验:1、通过实验发现9个家系中有3个家系有APC基因的胚系突变:1.1家系2为为移码突变即c.3184₃187 del CAAA。这使APC基因终止密码子提前于1123位出现,形成截短的蛋白。1.2家系4为错义突变即c.5432C＞T。这导致编码蛋白质的丝氨酸（Ser）变为亮氨酸（Leu）,这在数据库中未见报道,目前推测此突变可能使APC蛋白功能受到影响。1.3家系9为移码突变即c.3925₃929 del AAAAG,形成功能异常的截短蛋白。2、未检测出胚系突变的家系,发病可能和其他的机制有关。如:2.1存在APC基因启动子部位的高甲基化;2.2 E.Coli（Escherichia coli,E.coli）中的碱基切除修复（baseexcision repair,BER）基因同源的MYH基因双等位基因系的变异;2.3内含子的基因突变导致转录时剪接位点发生变化。3、胚系突变可能与临床表型相关。基因突变筛查和密切随访监测可以发现FAP高危人群,做到早发现、早诊断和早治疗。第二部分实验:1.没有发现LKB1基因外显子区域的胚系突变及体细胞突变;2.没有发现启动子区域的5’-CpG岛的高甲基化;3.仅发现内含子1-2区域和内含子2-3区域的点突变G→T,提示可能与剪切有关,或者仅为基因的多态性位点。由实验可知,P-J综合征的发生存在其他机制:1.基因的大片段缺失;2.与LKB1相互作用的蛋白的改变,如BRG1,STRADalpha,andMO25alpha等。结论:家族性腺瘤性息肉病患者APC基因的胚系突变可引起FAP;无APC胚系突变的FAP患者的发病可能存在其他的机制。P-J综合征的发生与LKB1基因的改变有关,但还有其他机制在起作用。检测基因的改变,可以为判断预后、指导临床治疗提供实验依据。更多还原

【Abstract】 Background:Familial adenomatous polyposis（FAP）,a autosomal dominant inherited disease,characterizes thousands or hundreds of adenoma covering intestine.The morbidity is from 1 over 10000 to 1 over 7000.It is reported that the adenomatous polyposis coli（APC）is the virulence gene to date.With deeper studying,attenuated familial adenomatous polyposis（AFAP）and other special type,such as Gardner syndrome,Peutz-Jeghers syndrome（P-J syndrome）and MYH associated polyposis（MAP）,were found one after another,besides classical familial adenomatous polyposis（CFAP）.The virulence gene,APC,locates on 5q21-22.The number of it’s mutation types is up to 800 on record,which mainly focus on exon 15. The result of the mutation is the formation of truncated APC protein, which could not perform normally and causes the disease.P-J syndrome, considered as one special type of familial hereditary polyposis,is caused by the change of LKB1/STK11,locating on 19p,which makes serin/threonine kinase inactive.It is reported that gene APC and LKB1 is tumor suppressing gene,whose changes lead the tumor to develop.Objective:FAP is a disease with higher genetic predisposition.If not treat,it tends to develop carcinoma.Earlier detection,earlier diagnosis and earlier treatment are more important.The detection of the changes of related gene will help us to find the high risk group,take action to give them reasonable therapy.So we design the experiment to find the available changes in Chinese group.Methods:Our study contains two parts:one was to study Eighteen members from nine FAP pedigrees by using multiplex ligation-dependent probe amplification（MLPA）to detect large fragment deletion of APC gene,and PCR to amplify all exons of APC gene respectively for mutation screening by denaturing high performance liquid chromatograph（DHPLC）,whose abnormal elution profile will lead the mutations of related exon to be determined.The other is to study 5 patients with P-J syndrome by using PCR to amplify all exons of LKB1 gene respectively for mutation determination through DNA sequencing and Methylation specific polymerase chain reaction（MSP）to detect whether the promoter region contains 5’CpG island high methylation.Results:In first part,we found two types of mutation in three pedigrees among nine pedigrees,frame shift mutation c.3184₃187 del CAAA in pedigree 2 and c.3925₃929 del AAAAG in pedigree 9,and novel missense mutation c.5432C＞T in pedigree 4,which all lead to the disfunction of APC protein.Development of tumor in Patients of the other six pedigrees may caused by other factors,such as higher methylation of promoter region,the mutation of MYH gene,base excision repair（BER）gene,and the change of splicing site caused by the mutation in intron.We also found there may be a connection between phenotype and genotype.In the second part,germline mutation and somatic mutation in LKB1 were not detected.So did the 5’CpG island methylation of promoter region.The only change found is the mutation, G→T,in intron 1-2 and intron 2-3,which maybe connected to the splice or just gene polymorphism.So other mechanisms maybe involved in, such as large fragment deletion and related protein’s changes,as BRG1, STRADalpha,and MO25alpha.Conclusion:The germline mutation of gene APC can lead to the development of FAR Other mechanisms may explain the development of FAP without germline mutation.P-J syndrome,a special type of FAP,is caused by the changes in gene LKB1,which referred to the mutation of gene,methylation of promoter region and so on.The detection of gene can provide experimental evidence for diagnosing the disease and guiding the clinical therapy.更多还原