【作者】 朱小泉；

【导师】 杨泽； 高绍荣；

【作者基本信息】 中国协和医科大学 ， 医学遗传学， 2009， 博士

【摘要】 AS是一种常见的慢性炎症性疾病。病变主要累及骶髂关节,引起脊柱强直,部分患者伴有不同程度的髋关节、眼、肺、心血管、肾等多器官损害,目前为止,AS的发病原因仍不清楚。既往研究表明AS与HLA-627强关联。全基因组连锁和关联分析结果显示在染色体1p、2q、6q、6p、9q、10q、16q以及19q存在AS连锁区段。近年来多个群体报道2q14的Interleukin 1基因簇与AS关联。这些结果为研究非B27基因提供了线索和证据。论文一采用候选基因关联研究,在中国北方人群中定位AS的易感基因,并深入研究-850T变异对TNFa启动子转录活性的影响。本论文的第一部分旨在以HLAⅢ类基因区的TNFa启动子-850T变异作为AS的遗传标记,在其侧翼各60kb的范围内定位AS的易感基因。我们挑选了11个SNPs,在中国北方吉林群体中进行病例对照关联研究。结果显示,TNF（rs1799724）在中国北方吉林人群中与AS关联（P=0.021）,携带T等位基因增强了疾病的发病风险（OR=1.95,95%CI:1.10-3.45）。通过连锁不平衡分析,将AS可能的易感基因定位在58kb的范围内。为了进一步寻找AS的易感基因,我们从58kb区段内的7个与免疫疾病相关基因中挑选出29个SNP进行精细定位。结果尚未发现与AS关联的SNP。为了进一步研究-850T变异对TNFa基因启动子转录活性的影响,我们利用定点突变技术分别构建突变型T和野生型C两种荧光素酶报告基因载体,瞬时转染人HepG2细胞系,检测突变型T对TNFa启动子活性的影响。结果显示-850突变型T等位基因较野生型C增强了TNFa启动子活性,1.05-1.33倍。独立样本t检验未见统计学意义（t=1.645,P=0.102）。提示-850T变异可能仅作为遗传标记连锁AS的易感基因。结论:我们在HLAⅢ基因区将AS的易感基因定位于58kb的范围,-850T未能增强TNFa启动子的转录活性,其周围紧密连锁有AS的易感基因。本论文的第二部分从免疫应答和免疫调节网络入手,采用候选基因关联研究策略寻找AS的易感基因。首先通过文献检索,挑选与抗原呈递,T细胞活化,细胞免疫和体液免疫应答,免疫调节等方面的节点基因,初步作为AS研究的候选基因。经PubMed和遗传关联数据库（GAD）筛选,保留14个与三个或三个以上的免疫学疾病关联基因作为候鸦颉Ｆ浯未?4个基因中挑选出19个与三个或三个以上的免疫学疾病关联,同时显著性大于0.001的SNP作为遗传标记,分别在45个中国吉林AS患者与HapMap数据库中提供的中国北京地区正常群体中进行关联研究,保留显著性小于0.01的两个SNP rs10489629和rs1024611,在整个中国吉林AS群体中复制关联结果。其中IL23R SNPrs10489629与AS关联。最后在其周围挑选7个SNP进行病例对照关联研究。结果显示SNP rs10736406和rs10889677与AS关联。结论:我们基于免疫应答和免疫调节网络,采用候选基因关联研究策略,研究AS的易感基因。结果显示IL23基因与AS关联。DA2B是一种先天性遗传病,主要表现为2个或2个以上关节的挛缩畸形,并且不包括由神经和肌肉病变所导致的关节畸形。远端关节弯曲2B型是DA中最常见的类型之一。其有明显的遗传异质性,目前发现TNNI2,TNNT3和MYH3的突变导致了疾病的发生。论文二旨在制造携带R156X和del175K两种突变的Knock-in小鼠模型,并用其来研究DA2B的发病机理。首先我们利用定点突变技术构建打靶载体,并通过基因打靶技术获得携带TNNI2^del175K突变基因的ES细胞,经囊胚注射和假孕移植产生嵌合体小鼠,筛选F1和F2代小鼠,鉴定出TNNI2^{del175K/del175K}纯合小鼠。表型分析发现:1)部分TNNI2^del175K/+和全部TNNI2^{del175K/del175K}小鼠在哺乳期死亡。2)TNNI2^del175K/+和TNNI2^{del175K/del175K}小鼠在哺乳期内发育迟缓。结论:1.TNNI2^del175K纯合突变致死。2.TNNI2^del175K变导致小鼠发育迟缓。更多还原

【Abstract】 Ankylosing Spondylitis（AS） is a common chronic inflammatory disorder,characterized by vertebral ankylosing,accompanying impairment of eye,lung,Cardiovascular system and kidney.However,the pathogenesis of AS is unknown.Lot of studies indicated that HLA-B27 is significantly associated with AS.The results from genome wide linkage and association analysis demonstrated linkage with AS on chromosome 1p,2q,6q,6p,9q,10q,16q and 19q.The association between AS and interleukin 1 gene cluster,on chromosome 2q14 were reported by several studies in past years.All of these studies suggest that non HLA-B27 genes are susceptible to AS. In this study,we tried to position the susceptible genes to AS with candidate gene -association method in Chinese north population and exploit the effect of-850T variant on transcription of TNFαgene promoter.In the first part of the study,we wanted to position possible susceptible genes to AS in a 120kb candidate area on HLAⅢregion.11 common SNPs were selected for case-control association study in China-Jilin population.The results showed that SNP rs1799724 was associated with AS in the China-Jilin population（P=0.021） and the T allele carried increased the risk of AS（OR=1.95,95%c.i:1.10-3.45）.We position the possible susceptible genes of AS in a 58kb segment on the HLAⅢgene region by linkage disequilibrium measure.In order to further seek the disposition genes to AS within the 58kb area,we re-selected 29 common SNPs from 7 genes which relate with immune diseases on this block. Nevertheless no association was observed in these 29 common SNPs.To study whether the -850T variant impacted on the transcriptional activation of TNFαgene promoter,we constructed two type of luciferase report vectors,-850T and -850C,by site-directed mutagenesis technique,transiently transfected them into HepG2 cell line, respectively and detected the activation of TNFαpromoter by dual-luciferase reporter assay system.The results indicated that -850T variant appeared to increase the transcript from 1.05 to 1.33 fold than wild type C allele,however these is no statistical difference by Independent Sample t-test（t=1.645,P=0.102）.Conclusion:1) We positioned the susceptible genes to AS in a 58kb segment on HLAⅢ gene region.2)-850 T failed to affect the transcriptional activation of TNFαpromoter and it may be closely linked with true disposition genes to AS.The second of our study is to position susceptible genes to AS by association study with a group of candidate genes which play key role in immune response and immune regulation.Firstly,we selected genes from several sides of antigen presentation,T cell activation, cell immune response,humoral immune response and immune regulation to build a database.Screening the database with a principle of association with three different immune disorders that can be searched out from PubMed and Genetic Association Database,we retained 14 genes for candidate association study.Secondly,we selected 19 common SNPs from these 14 candidate genes according to two rules.One is association with three or more immune diseases and the other is that significant level is more than 0.001.There were two SNPs,rs10489629 and rs1024611 demonstrated association with AS between 45 case from China Jilin population and 45 health control from China Beijing offered by Hapmap database.We replicated the association results in the whole case-control population of China Jilin.The SNP rs rs10489629 on IL23 gene was remained as a genetic mark which possibly linkages with susceptible genes to AS for the association result being replication.For fine-mapping whether IL23 gene is susceptible to AS,we continually did a case-control association study with 7 common SNPs selected from IL23 gene in the population of China Jilin.The results exhibited that two of 7 SNPs,rs10736406 and rs 10889677 were associated with AS.Conclusion:we researched susceptible genes to AS with candidate-gene method on the base of immune response and regulation network in the population of China Jilin.Our result indicated that IL23 gene is associated with AS and possibly contributed susceptibility to AS.Distal arthrogryposis type 2B,one of most common types of DAs,is a multiple congenital contracture disorder characterized by contractures of the distal joints of limbs without neurological and skeletal muscle defect.DA2B has obvious genetic heterogeneity.Several reports showed mutations in TNNI2,TNNT3 and MYH3 responsible to DA2B.The partⅡof my study is to generate two types of knock-in mice carrying with mutations of R156X and del175K in TNNI2 and study the pathogenesis of this disorder on these models.Firstly,we constructed targeting vectors using site-directed mutagenesis and obtained the positive ES cell clones with two mutations by gene targeting technique,produced chimeras through microinjecting ES cell into blastocysts and transferring them into uterus of pseudopregnant recipient,and got the homogenous TNNI2^{del175K/del175K} mice by detecting the mutant gene of F1 and F2 generations.We found:1) parts of TNNI2^del175K/+ and all of TNNI2^{del175K/del175K} pups died within 20 days.2) Almost TNNI2^del175K/+ and all of TNNI2^{del175K/del175K} pups were growth retardation during suckling.Conclusion:The homogenous del175K mutation of TNNI2 is lethal.The del175K mutation of TNNI2 causes growth failure of mouse.更多还原