【作者】 吴昊；

【导师】 吕明；

【作者基本信息】 山东大学 ， 内科学（消化病）（专业学位）， 2017， 博士

【摘要】 研究的背景和目的炎症性肠病(inflammatory bowel diseases,IBD)主要包括两种临床表现,即溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn’sdisease,CD)。调查结果显示,全世界每年新增UC、CD患者数量逐年上升。但到目前为止IBD的病因和发病机制尚未完全明确,总结现有的研究结果,IBD是遗传因素、环境因素、免疫因素等复杂因素综合作用于遗传易感者,使宿主体内发生针对自身肠道菌群的异常免疫应答,导致难以控制的炎症反应,最终造成肠组织损害。肠道菌群紊乱或许是其中的重要环节,与IBD的发病相关[1]。动物模型以及不同地区不同种族人群的研究结果显示,宿主肠道菌群的结构和IBD肠组织炎症之间存在紧密的联系[2]。人类组织血型抗原(histo-blood group antigens,HBGA)主要在肠道黏膜及其分泌液中表达,分为ABH抗原以及Lewis抗原。有学者在研究中指出,HBGA的表达在一定程度上决定了肠道菌群的构成[3-5]。而岩藻糖基转移酶(FUT)2和3基因的编码产物负责催化血型前体物质和岩藻糖结合生成ABH抗原和Lewis抗原,如在α-(1,2)-岩藻糖基转移酶(由FUT2基因表达)的催化下,血型前体物质与岩藻糖基结合后变为H抗原,而后者在A酶催化下成为A抗原、受到B酶催化则成为B抗原,从而形成ABH组织血型抗原。而Lewis抗原,则是在α-(1,3/1,4)-岩藻糖基转移酶(由FUT3基因表达)和α-(1,2)-岩藻糖基转移酶共同调节下生成的。在FUT2基因表达的酶作用下,血型前体物质盐藻糖基化后生成H抗原,该抗原和FUT3基因表达的酶催化下,变成Lewis b抗原;若FUT2基因发生突变,FUT2酶失去正常催化功能,血型前体物质将不能被催化生成H抗原,只能在α-(1,3/1,4)-岩藻糖基转移酶的催化生成Lewis a抗原。Philipp Rausch等在临床CD患者的研究中发现,FUT2(rs601338)基因多态性导致ABH以及Lewis抗原表达异常在一定程度上影响肠道菌群的构成[6]。也就是说,FUT2、FUT3基因多态性可能通过调节肠道人类组织血型抗原的表达水平,影响肠道菌群的构成,从而对IBD患病风险产生影响。并且有研究指出,FUT2基因多态性与CD[7,8]、Ⅰ型糖尿病[9]和原发性硬化性胆管炎患病风险之间存在一定的关联[10],但目前有关FUT2基因多态性和IBD易感性的研究结论存在较大的分歧[11-12]。如McGovern DP等研究显示,在欧美高加索人群中,FUT2(rs601338)基因多态性并非UC患者易感性关联因素[8]。但另一项基于芬兰人群的相似研究,结果显示存在该单核苷酸多态性(SNP)的野生基因型GG的人群UC患病率更高[13]。同样针对欧美高加索人群中研究发现,FUT2基因突变所导致的非分泌状态会增加CD患病风险[8],不过针对日本人群FUT2基因多态性的研究却得出了完全相反的结论[14]。截止目前,FUT3基因多态性与IBD的相关性方面研究报道也较少。基于此,笔者在本课题的研究中,以浙江籍汉族人群为研究对象,同时分析FUT2、FUT3基因多态性,Lewis抗原表达和IBD之间的关联,试图为诠释IBD遗传学病因机制寻找新的线索。第一部分岩藻糖基转移酶2和3基因多态性及Lewis抗原表达与溃疡性结肠炎的关系方法于2005年1月到2015年10月期间,在温州医科大学附属第二医院、温州医科大学附属第一医院、温州市中心医院、温州市人民医院接受过诊疗的485例UC患者纳入病例组,UC诊断通过结肠镜检查、联合临床、病理和影像学检查结果综合确定,按照“炎症性肠病诊断与治疗的共识意见(2012年,广州)”进行疾病确诊[14]并以同一时间段在温州医科大学附属第二医院体检中心的正常体检者580名(男女比例为319:261)为对照,对照组人员是基于年龄、性别匹配原则筛选出来的。纳入前排除心脑血管疾病、代谢性疾病、肝肾功能不全、肿瘤以及自身免疫性疾病和炎症性肠病家族史,所有研究对象均源于无血缘关系的浙江籍汉族人群。抽取外周空腹静脉血,提取全基因组DNA,通过SNaPshot技术对FUT2(rs281377、rs601338、rs1047781)和 FUT3(rs28362459、rs3894326、rs3745635)6个SNP位点的基因多态性进行测定。另随机选取7例存在乙状结肠炎症的UC患者,再选7例确诊为良性乙状结肠息肉的患者为对照组,采用免疫组织化学法检测乙状结肠中Lewis a和Lewis b抗原的表达水平,对比Lewis a和b抗原表达水平的组间差异。结果比较FUT3(rs3745635)突变等位基因(A)和基因型(GA+AA)的频率这两项指标,UC 组较对照组更高(17.0%比 13.3%,P=0.016;30.7%比 25.0%,P=0.038)。但 FUT2(rs281377、rs601338、rs1047781)三个位点和 FUT3(rs28362459、rs3894326)两个位点的等位基因和基因型频率组间对比,结果不具有显著性差异(均P>0.05)。对疾病分层分析结果显示,与远端结肠炎相比,广泛性结肠炎患者FUT3(rs28362459)突变等位基因(G)和基因型(TG+GG)的频率明显下降(17.8%比27.7%,P=0.001;31.6%比47.9%,P<0.001)。在FUT3(rs3745635)突变等位基因(A)和基因型(GA+AA)频率这两项指标上,广泛性结肠炎患者也低于远端结肠炎患者(12.9%比 19.3%,P=0.011;23.0%比 35.0%,P=0.006)。通过 Haploview4.2软件对FUT2,FUT3几个SNP位点进行连锁不平衡和单倍型分析,结果显示FUT2(rs281377)和(rs1047781)两个 SNP 位点存在连锁不平衡(D’=0.96,r2=0.10),FUT3(rs28362459)、(rs3745635)、(rs3894326)三个位点也互相之间存在连锁不平衡关系:rs3894326/rs3745635(D’=1.0,r2=0.02),rs3894326/rs28362459(D’=0.86,r2=0.3),rs3745635/rs28362459(D’=0.92,r2=0.45)。但是在单倍型频率的分布上,组间比较无显著性差异(均P>0.05)。在乙状结肠炎症的UC患者和良性乙状结肠息肉对照组的肠组织Lewis抗原的表达水平比较中发现,虽然Lewis b抗原在UC组和对照组的表达水平无统计学差异(P>0.05),但Lewis a抗原在UC患者乙状结肠隐窝上皮中的表达水平显著高于对照组(P=0.028)。结论FUT3基因多态性及肠道Lewis a抗原表达水平可能与浙江籍汉族人群UC的易感性相关。第二部分岩藻糖基转移酶2和3基因多态性与克罗恩病的关系方法于2005年3月至2014年3月期间,在温州医科大学第一附属医院、温州医科大学附属第二医院、温州市中心医院、温州市人民医院接受过诊疗的275例CD患者。CD的诊断通过结肠镜、胃镜、小肠镜、胶囊内镜检查,联合临床、病理和影像学检查结果综合判断,按照中华医学会消化病分会推出的“炎症性肠病诊断与治疗的共识意见(2012年,广州)”进行疾病确诊[14]。以同一时间段在温州医科大学附属第二医院体检中心接受体检的502名年龄,性别匹配的健康人群,排除心脑血管疾病、代谢性疾病、肝肾功能不全、肿瘤以及自身免疫性疾病和炎症性肠病家族史后作为对照组,所有研究对象均为无血缘关系的浙江籍汉族人群。抽取外周空腹静脉血,提取全基因组DNA,多重PCR反应及产物纯化,通过SNaPshot 技术识别 FUT2(rs281377、rs601338、rs1047781)和 FUT3(rs28362459、rs3894326、rs3745635)基因多态性。通过卡方检验确定上述各SNP位点的基因型分布是否与Hardy-Weinberg平衡一致,探讨各等位基因和基因型频率的组间分布差异。在对SNP位点进行连锁不平衡以及单倍型分析使用Haploview4.2软件。结果CD组与对照组相比,FUT2(rs1047781)的突变等位基因T和基因型TT的频率显著增加(分别是49.27%比 43.33%,P=0.024;27.64%比 16.53%,P<0.001)。CD组中,FUT2(rs601338)的突变等位基因A杂合子和基因型(GA+AA)亦高于对照组(分别是 1.64%比 0.50%,P= 0.023;3.27%比 1.00%,P=0.044);然而经Bonferroni校正后(即P值界限调整为0.05/6=0.0083),只有FUT2基因的(rs1047781)位点在CD组和对照组之间比较仍有统计学差异。对于FUT3基因,在CD组和对照组之间比较,无论是各位点的基因型还是等位基因频率,都没有统计学差异。进一步分层分析发现,FUT2(rs1047781),FUT3(rs28362459)和FUT3(rs3745635)基因突变与CD疾病部位显著相关:回结肠型CD患者比结肠型CD患者中FUT2(rs 1047781)突变等位基因(T)和基因型(AT+TT)频率降低(41.67%比59.41%,P=0.001;63.33%比83.17%,P=0.002);而回肠型CD患者相较于结肠CD型患者FUT2(rs1 047781)突变等位基因(T)频率和基因型(AT+TT)频率亦降低(45.24%比 59.41%,P=0.007;64.29%比 83.17%,P=0.004);回结肠型 CD 患者较结肠型CD患者FUT3(rs3745635)的突变等位基因(A)和基因型(AA +AG)更普遍,(19.44%比 9.41%,P=0.006;34.44%比 16.83%,P=0.006);回结肠型 CD 患者与结肠型CD患者FUUT3(rs28362459)的突变基因型(GG+GT)频率比较也得出同样的结论(53.33%比34.65%,P=0.010)。采用Haploview4.2软件进行连锁不平衡和单倍型分析发现,FUT2(rs281377和rs1047781)两个位点存在连锁不平衡(D’=0.90,r2=0.10),F T3(rs28362459)、(rs3745635)和(rs3894326)三个位点彼此之间也存在连锁不平衡:rs3894326/rs3745635(D’=1.0,r2=0.01),rs3894326/rs28362459(D’=0.93,r2=0.29),rs3745635/rs28362459(D’=0.96,r2=0.45)。CD组中(TT)单倍型频率高于对照组(49%比43%,P=0.020)。结论FUT3(rs1047781)基因多态性可能与CD易感性相关。FUT3(rs28362459和rs3745635)基因多态性可能影响CD疾病部位。更多还原

【Abstract】 Backgrounds and aims:Inflmmatory bowel disease(IBD),mainly comprising ulcerative colitis(UC)and Crohn’s disease(CD),has a rising global incidence and morbidity rate in recent years.Although the etiology and pathogenesis of IBD is not yet fully clarified,it has been mostly accepted that IBD is a condition involved in multiple factors such as heredity,environment,and immunity,triggering an inappropriate immune response to normal constitutions of the gut microbiota in an individual with genetic predisposition.which leads to the injury of intestinal tissue.Of these factors,the imbalance of micriobiota has been thought to be the trigger of the onset of IBD[1].Data from clinical and colitis animal model studies supported that the composition of intestinal microbiota is closely related to the inflammation of the intestinal tissue[2].Histo-blood group antigens(HBGA),including ABH and Lewis antigens,are mainly expressed in the intestine,saliva and other secretion gland.The expression of HBGA in the intestine has been shown to affect the constitution of intestinal microbiota[3-5]Fucosyltransferases(FUTs)-encoding genes FUT2 and FUT3 are in charge of the formation of ABH and Lewis antigens by adding a fucose to precursor substrate.Blood group precursor becomes H antigen after fucosylation by a-(1,2)-fucosyltransferase encoded by FUT2 In the presence of alpha-3-N-acetylgalactosamine transferase encoded by Gene A.or D-galactosyl transferase encoded by Gene B.H antigen would further become A antigen or B antigen,which together compose the ABH blood group antigen.FUT3.which encodes α-(1,3/1,4)-fucosyltransferase,together with FUT2,determines the formation of Lewis antigens.If the loss-of-function mutation occurred in FUT2 gene,the precursor of blood groups will not be catalyzed to H antigen,but be directly transformed to Lewis a antigen by the help of α-(1,3/1,4)-fucosyltransferase encoded by FUT3.From a study on the CD patients and healthy controls,Philipp Rausch and the colleagues revealed that FUT2(rs601338)polymorphism is strongly correlated with the constitution of the intestinal microbiota,while the expression of ABH and Lewis antigens also affect the composition of the intestinal microbiota[6].Therefore,FUT2 and FUT3 genes might influence the composition of intestinal microbiota through determining the intestinal expression of HBGA,thus potentially play a key role in the pathogenesis of IBD.Studies has showed that polymorphims of FUT2 are associated with a group of autoimmune disease such as CD[7,8],type Ⅰ diabetes[9]and primary sclerotic cholangitis[10].However,the conclusions drawn from these studies seem to be inconsistent[11-12].For example,McGovern DP and the colleages found that the single nucleotide polymorphism(SNP)in FUT2(rs601338)was not associated with the predisposition of UC in Caucasians[8],while another study from Finland suggested that the carrier with wildtype GG of this SNP in FUT2 had an increased risk of UC[31].Moreover,the conclusions of the association between FUT2 and CD drawn from different ethic background were also inconsistency.For example,one study showed that non-secretor status in Caucasian polulation increased the risk of CD[8]while in Japanese population the FUT2 non-secretor status played a protective role in patients with CD[14].As far as we know,the investigation of the association between FUT3 polymorphisms and IBD is limited.Therefore,it is necessary to investigate the relationship between IBD and the SNPs of FUT2 and FUT3 in a cohort of Han patients from Zhejiang province of China.Part I:Association of Ulcerative Colitis with FUT2 and FUT3 Polymorphisms and their expressions in colonic tissuesMethods During January 2005 and October 2015,a total of 485 UC patients were recruited from the Second Affiliated Hospital and the first Affiliated Hospital of Wenzhou Medical University and Wenzhou People’s Hospital.Five hundred and eighty age-and sex-matched healthy controls,who underwent routine health check-up,were also collected from the medical examination center of the Second Affiliated Hospital of Wenzhou Medical University.Peripheral blood was collected from each study individual.Genomic DNA was extracted from the peripheral blood.Then the polymorphisms of FUT2(rs281377,rs1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)was genotyped using SNaPshot assays.By an immunohistochemistry method,seven of these UC patients presenting inflammatory lesions in the sigmoid colon were selected to evaluate the expression of Lewis a and b antigens in the sigmoid colon.In seven patients with confirmed benign colonic polyps,specimens of normal sigmoid colon mucosa were also obtained as controls.Then the expression of Lewis a and b antigens in the UC patients and the controls were compared.Results The frequencies of mutant allele(A)and genotype(GA+AA)in FUT3(rs3745635)were higher in UC patients than in controls(17.0%vs 13.3%,P=0.016,;30.7%vs 25.0%,P=0.038,respectively).There is no statistical significance of mutant allele and genotype frequencies of FUT2(rs281377,rs1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)between the two groups(all P>0.05).Stratified analyses revealed that mutant allele(G)and genotype(TG+GG)of FUT3(rs28362459)were less dominant in patients with extensive colitis than in those with distal colitis(17.8%vs 27.7%,P=0.001;31.6%vs 47.9%,P<0.001,respectively).Same conclusions could be applied for the mutant allele(A)and genotype(GA+AA)of FUT3(rs3745635)in patients with extensive colitis compared to those with distal colitis(12.9%vs 19.3%,P=0.011;23.0%vs 35.0%,P=0.006,respectively).Haploview 4.2 software was employed to analyze the linkage disequilibrium(LD)and haplotype reconstruction.Two LD blocks were presented,including block rs281377-rs 1047781 in FUT2(D’ = 0.96,r2 = 0.10)and block rs3894326-rs3745635-rs28362459 in FUT3[rs3894326/rs3745635(D’= 1,r2=0.02),rs3894326/rs28362459(D’= 0.86,r2= 0.30),rs3745635/rs28362459(D’=0.92,r’=0.45)].Furthermore,no haplotype frequencies differed significantly between UC patients and the controls(all P>0.05).Although expression of Lewis b antigen in the sigmoid colon did not differ between UC patients and controls,Lewis a antigen expression was higher in the cryptic epithelium of both inflammatory and non-inflammatory sigmoid colon of UC patients than controls(P=0.028).Conclusions Our findings indicated that the polymorphism in FUT3 and its intestinal expression of Lewis a antigen might be associated with the susceptibility of UC in Zhejiang Han population.Part Ⅱ Associations of FUT2 and FUT3 gene polymorphisms with Crohn’s diseaseMethods:During March 2005 and March 2014,a total of 275 CD patients were recruited from the Second Affiliated Hospital and the first Affiliated Hospital of Wenzhou Medical University,Wenzhou Central Hospital and Wenzhou People’s Hospital.The diagnosis of CD were based on colonoscope,gastroscope,small intestinal endoscope,and capsule endoscope,in collaboration with clinical,histopathological,and radiologic findings according to "consensus of the diagnosis and management of inflammatory bowel disease" released by the Digestive Diseases Branch of Chinese Medical Association(2012,Guangzhou).Five hundred and two age-matched and sex-matched healthy controls were also collected from the medical examination center of the Second Affiliated Hospital of Wenzhou Medical University.Diseases including cardiovascular and cerebrovascular diseases,metabolic diseases,liver and kidney dysfunction,tumors,and autoimmune diseases and those having inflammatory bowel disease family history have been ruled out of these controls.All the subjects were unrelated to each other in terms of heredity.Around 2ml peripheral blood from each study individual was collected.Genomic DNA was extracted from the peripheral blood,followed by multiplex PCR reaction and.purification steps according to the manufacturer’s instructions.Then the polymorphisms of FUT2(rs281377.rs 1047781 and rs601338)and FUT3(rs28362459,rs3745635 and rs3894326)was genotyped using SNaPshot assays.Hardy-Weinberg equilibrium for each of the studied polymorphisms and the distributions of genotypes in the compared groups were evaluated by chi-squared test.Haploview 4.2 was employed to analyze the linkage disequilibrium(LD)and haplotype reconstruction.Results:Compared with controls,mutant allele and homozygote TT frequencies of FUT2(rs1047781)was significantly increased in CD patients(49.27%vs 43.33%,P=0.024;27.64%vs 16.53%,P<0.001,respectively).Same conclusions were drawn from the mutant allele A of FUT2(rs601338)in CD patients as compared with the controls(1.64%vs 0.50%,P=0.023;3.27%vs 1%,P=0.044,respectively).However,after Bonferroni correction(P value threshold was adjusted to 0.05/6=0.0083),only mutant homozygote TT frequencies of FUT2(rs 1047781)was still significantly increased in CD patients.Nevertheless,the allelic and genotypic distributions of FUT3 were not statistically different between CD patients and controls.Stratified analysis revealed that the polymorphisms of FUT2(rs1047781),FUT3(rs28362459)and FUT3(rs3745635)were significantly associated with the disease location of CD.The mutant allele(T)and genotype(AT+TT)of FUT2(rs1047781)were less prevalent in patients with ileocolonic CD than in colonic CD(41.67%vs 59.41%,P=0.001;63.33%vs 83.17%,P=0.002,respectively).The frequencies of mutant allele and genotype in FUT2(rs1047781)were also lower in patients with ileal CD compared to colonic CD(45.24%vs 59.41%,P=0.007;64.29%vs 83.17%,P=0.004,respectively).The mutant allele(A)and genotype(AA+AG)of FUT3(rs3745635)were more prevalent in patients with ileocolonic CD than in colonic CD(19.44%vs 9.41%,P=0.006;34.44%vs 16.83%,P=0.006,respectively).The frequencies of mutant genotype in FUT3(rs28362459)were also higher in patients with ileal CD compared to colonic CD(53.33%vs 34.65%,P=0.010).Haploview 4.2 software was employed to analyze the linkage disequilibrium and haplotype reconstruction,showing that LD were presented in block rs281377-rs 1047781 in FUT2(D’=0.90,r2=0.10)and in block rs3894326-rs3745635-rs28362459 in FUT3[rs3894326/rs3745635(D’=1.0,r2=0.01),rs3894326/rs28362459(D’=0.93,r2=0.29),rs3745635/rs28362459(D’=0.96,r2= 0.45)].Furthermore,the haplotype TT formed with FUT2(rs281377 and rs1047781)was more prevalent in CD patients than in controls(49%vs 43%,P=0.020).Conclusions:The polymorphic locus of FUT2(rs 1047781)might be associated with the susceptibility of CD.Mutations of FUT3(rs28362459 and rs3745635)might influence the lesion locations in CD patients.更多还原