The Candidate Gene-Association Study Of Ankylosing Spondylitis In Chinese North Population The Study Of Pathogenesis Of DA2B

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.