NLR基因多态性与Behcet病和VKH综合征的相关性研究
发布时间：2019-02-24 10:40
【摘要】：背景:Nod样受体(Nod-like receptor,NLR)是胞浆型模式识别受体,在机体识别抗原等多种免疫应答中发挥重要作用。其中多个NLR成员(NOD1、NOD2、NLRP1、NLRP3、NLRP5和CIITA)基因多态性被证明与多种免疫性疾病相关。Vogt-小柳-原田综合征(Vogt-Koyanagi-Harada syndrome,VKH)和白塞氏病(Behcet's disease,BD)是在亚洲人群中常见的两种葡萄膜炎,虽然发病机制尚不清楚,但多项研究表明免疫反应和遗传因素共同影响两种疾病的发生发展。近期研究已发现多个基因与BD和VKH综合征相关联。目的:此项研究旨在探讨在中国汉族人群中,6个NLR基因(NOD1,NOD2,NLRP1,NLRP3,NLRP5和CIITA)的多态性与BD和VKH综合征的相关性。方法:根据文献报道和本实验室关于VKH综合征的全基因组关联研究(genome-wide association study,GWAS)结果,我们选择了位于NLR基因上的20个单核苷酸多态性(Single nucleotide polymorphisms,SNP)位点,即NOD1//(rs2075818,rs2907748,rs2907749),nod2//(rs8057431,rs3135499),nlrp1//(rs6502867,rs878329,rs12150220,rs8079034),nlrp3//(rs10754558,rs10925019,rs4925648,rs3806265,rs2027432),nlrp5//rs7255920和ciita//(rs12932187,rs1107438,rs8048002,rs6498122,rs4774)。本课题为病例对照研究,分为两个阶段共纳入正常对照1440个,bd患者950个和vkh综合征患者384个。通过taqman和聚合酶链式反应-限制性片段长度多态性(polymerasechainreaction-restrictionfragmentlengthpolymorphism,pcr-rflp)两种方法进行基因分型。本研究运用计数方法统计snp位点各基因型和等位基因的个数,运用卡方检验计算等位基因频率和基因型频率的差异p值,bonferroni校正后记为pc值,校正后的pc值0.05被认为有统计学差异。基因mrna和下游细胞因子的表达水平分别通过荧光定量聚合酶链反应(real-timepcr,rt-pcr)和酶联免疫吸附实验(enzyme-linkedimmunosorbentassay,elisa)进行测定。结果:bd病的研究分为三个阶段,第一阶段研究发现:在bd患者中位点ciita//rs12932187的c等位基因频率(pc=1.668e-02,or=0.713,95%ci=0.591-0.861)和位点nod1//rs2075818的g等位基因频率(pc=4.694e-02,or=0.698,95%ci=0.562-0.868)均明显低于正常对照组。第二阶段扩大样本数后,得到的结果与第一阶段相符合,在进行数据分析后显示:bd患者中位点ciita//rs12932187的cc基因型频率(pc=3.331e-06,or=0.617,95%ci=0.519-0.735)和c等位基因频率(pc=6.004e-07,or=0.709,95%ci=0.629-0.799)明显低于正常对照组。BD患者中NOD1//rs2075818的GG基因型频率(Pc=1.022E-02,OR=0.536,95%CI=0.386-0.745)和G等位基因频率(Pc=6.811E-05,OR=0.720,95%CI=0.629-0.824)也明显低于正常对照组。第三阶段为功能性研究,结果显示:在给予外周血单个核细胞(Peripheral blood monouclear cell,PBMC)LPS刺激后,CIITA//rs12932187 CC基因型携带者的CIITA mRNA表达被抑制而IL-10的表达水平则明显升高。本实验选择的20个位于NLR基因上的SNP位点与VKH综合征无明显关联。结论:研究表明CIITA//rs12932187和NOD1//rs2075818两个位点均与Behcet病的遗传易感性相关。
[Abstract]:Background: Nod like receptor (Nod-like receptor,NLR) is a cytosolic pattern recognition receptor and plays an important role in many immune responses such as recognition of antigen. The polymorphisms of multiple NLR members (NOD1,NOD2,NLRP1,NLRP3,NLRP5 and CIITA) have been shown to be associated with a variety of immunological diseases. Vogt- Xiaoliu-Harada Syndrome (Vogt-Koyanagi-Harada syndrome,VKH) and (Behcet's disease, in Behcet's Disease BD) is a common type of uveitis in Asian population. Although the pathogenesis of uveitis is not clear, many studies have shown that immune response and genetic factors affect the occurrence and development of the two diseases. Recent studies have linked multiple genes to BD and VKH syndrome. Objective: to investigate the association between the polymorphisms of six NLR genes (NOD1,NOD2,NLRP1,NLRP3,NLRP5 and CIITA) and BD and VKH syndrome in Chinese Han population. Methods: according to the results of the whole genome association study (genome-wide association study,GWAS) on VKH syndrome in our laboratory, we selected 20 single nucleotide polymorphism (Single nucleotide polymorphisms,SNP (SNP) sites located on NLR gene. NOD1// (rs2075818,rs2907748,rs2907749), nod2// (rs8057431,rs3135499), nlrp1// (rs6502867,rs878329,rs12150220,rs8079034), nlrp3// (rs10754558,rs10925019,rs4925648,rs3806265,rs2027432), nlrp5//rs7255920 and ciita// (rs12932187,rs1107438,rs8048002,rs6498122,rs4774). This study was a case-control study, which was divided into two stages: 1440 normal controls, 950 bd patients and 384 vkh syndrome patients. Genotyping was carried out by taqman and polymerase chain reaction-restriction fragment length polymorphism (polymerasechainreaction-restrictionfragmentlengthpolymorphism,pcr-rflp). In this study, the number of genotypes and alleles in snp loci was counted by counting method. The difference p value of allele frequency and genotype frequency was calculated by chi-square test. The bonferroni correction was recorded as pc value. The corrected pc value was considered to be statistically different. The expression levels of gene mrna and downstream cytokines were measured by fluorescence quantitative polymerase chain reaction (real-timepcr,rt-pcr) and enzyme linked immunosorbent assay (enzyme-linkedimmunosorbentassay,elisa) respectively. Results: the study of bd's disease was divided into three stages. In the first stage, it was found that the c allele frequency (pc=1.668e-02,) of locus ciita//rs12932187 was found in bd patients. The g allele frequency (pc=4.694e-02,or=0.698,95%ci=0.562-0.868) of or=0.713,95%ci=0.591-0.861 and nod1//rs2075818 were significantly lower than that of normal controls. After expanding the sample size in the second stage, the result was consistent with that of the first stage. The data analysis showed that the cc genotype frequency (pc=3.331e-06,) of ciita//rs12932187 in the middle site of bd patients. Or=0.617,95%ci=0.519-0.735) and c allele frequency (pc=6.004e-07,or=0.709,95%ci=0.629-0.799) were significantly lower than those in normal controls. GG genotype frequency (Pc=1.022E-02,) of NOD1//rs2075818 in BD patients was significantly lower than that in normal controls. OR=0.536,95%CI=0.386-0.745 and G allele frequency (Pc=6.811E-05,OR=0.720,95%CI=0.629-0.824) were also significantly lower than those in normal controls. The third stage was functional study. The results showed that the CIITA mRNA expression of CIITA//rs12932187 CC genotype carriers was inhibited and the expression of IL-10 was significantly increased after LPS stimulation of peripheral blood mononuclear cells (Peripheral blood monouclear cell,PBMC). Twenty SNP loci located on the NLR gene were not associated with VKH syndrome. Conclusion: both CIITA//rs12932187 and NOD1//rs2075818 loci are associated with the genetic susceptibility of Behcet disease.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R773.9
本文编号：2429472
[Abstract]:Background: Nod like receptor (Nod-like receptor,NLR) is a cytosolic pattern recognition receptor and plays an important role in many immune responses such as recognition of antigen. The polymorphisms of multiple NLR members (NOD1,NOD2,NLRP1,NLRP3,NLRP5 and CIITA) have been shown to be associated with a variety of immunological diseases. Vogt- Xiaoliu-Harada Syndrome (Vogt-Koyanagi-Harada syndrome,VKH) and (Behcet's disease, in Behcet's Disease BD) is a common type of uveitis in Asian population. Although the pathogenesis of uveitis is not clear, many studies have shown that immune response and genetic factors affect the occurrence and development of the two diseases. Recent studies have linked multiple genes to BD and VKH syndrome. Objective: to investigate the association between the polymorphisms of six NLR genes (NOD1,NOD2,NLRP1,NLRP3,NLRP5 and CIITA) and BD and VKH syndrome in Chinese Han population. Methods: according to the results of the whole genome association study (genome-wide association study,GWAS) on VKH syndrome in our laboratory, we selected 20 single nucleotide polymorphism (Single nucleotide polymorphisms,SNP (SNP) sites located on NLR gene. NOD1// (rs2075818,rs2907748,rs2907749), nod2// (rs8057431,rs3135499), nlrp1// (rs6502867,rs878329,rs12150220,rs8079034), nlrp3// (rs10754558,rs10925019,rs4925648,rs3806265,rs2027432), nlrp5//rs7255920 and ciita// (rs12932187,rs1107438,rs8048002,rs6498122,rs4774). This study was a case-control study, which was divided into two stages: 1440 normal controls, 950 bd patients and 384 vkh syndrome patients. Genotyping was carried out by taqman and polymerase chain reaction-restriction fragment length polymorphism (polymerasechainreaction-restrictionfragmentlengthpolymorphism,pcr-rflp). In this study, the number of genotypes and alleles in snp loci was counted by counting method. The difference p value of allele frequency and genotype frequency was calculated by chi-square test. The bonferroni correction was recorded as pc value. The corrected pc value was considered to be statistically different. The expression levels of gene mrna and downstream cytokines were measured by fluorescence quantitative polymerase chain reaction (real-timepcr,rt-pcr) and enzyme linked immunosorbent assay (enzyme-linkedimmunosorbentassay,elisa) respectively. Results: the study of bd's disease was divided into three stages. In the first stage, it was found that the c allele frequency (pc=1.668e-02,) of locus ciita//rs12932187 was found in bd patients. The g allele frequency (pc=4.694e-02,or=0.698,95%ci=0.562-0.868) of or=0.713,95%ci=0.591-0.861 and nod1//rs2075818 were significantly lower than that of normal controls. After expanding the sample size in the second stage, the result was consistent with that of the first stage. The data analysis showed that the cc genotype frequency (pc=3.331e-06,) of ciita//rs12932187 in the middle site of bd patients. Or=0.617,95%ci=0.519-0.735) and c allele frequency (pc=6.004e-07,or=0.709,95%ci=0.629-0.799) were significantly lower than those in normal controls. GG genotype frequency (Pc=1.022E-02,) of NOD1//rs2075818 in BD patients was significantly lower than that in normal controls. OR=0.536,95%CI=0.386-0.745 and G allele frequency (Pc=6.811E-05,OR=0.720,95%CI=0.629-0.824) were also significantly lower than those in normal controls. The third stage was functional study. The results showed that the CIITA mRNA expression of CIITA//rs12932187 CC genotype carriers was inhibited and the expression of IL-10 was significantly increased after LPS stimulation of peripheral blood mononuclear cells (Peripheral blood monouclear cell,PBMC). Twenty SNP loci located on the NLR gene were not associated with VKH syndrome. Conclusion: both CIITA//rs12932187 and NOD1//rs2075818 loci are associated with the genetic susceptibility of Behcet disease.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R773.9
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1 李琳;NLR基因多态性与Behcet病和VKH综合征的相关性研究[D];重庆医科大学;2016年
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