NLR基因多態(tài)性與Behcet病和VKH綜合征的相關性研究
發(fā)布時間：2019-02-24 10:40
【摘要】：背景:Nod樣受體(Nod-like receptor,NLR)是胞漿型模式識別受體,在機體識別抗原等多種免疫應答中發(fā)揮重要作用。其中多個NLR成員(NOD1、NOD2、NLRP1、NLRP3、NLRP5和CIITA)基因多態(tài)性被證明與多種免疫性疾病相關。Vogt-小柳-原田綜合征(Vogt-Koyanagi-Harada syndrome,VKH)和白塞氏病(Behcet's disease,BD)是在亞洲人群中常見的兩種葡萄膜炎,雖然發(fā)病機制尚不清楚,但多項研究表明免疫反應和遺傳因素共同影響兩種疾病的發(fā)生發(fā)展。近期研究已發(fā)現(xiàn)多個基因與BD和VKH綜合征相關聯(lián)。目的:此項研究旨在探討在中國漢族人群中,6個NLR基因(NOD1,NOD2,NLRP1,NLRP3,NLRP5和CIITA)的多態(tài)性與BD和VKH綜合征的相關性。方法:根據(jù)文獻報道和本實驗室關于VKH綜合征的全基因組關聯(lián)研究(genome-wide association study,GWAS)結果,我們選擇了位于NLR基因上的20個單核苷酸多態(tài)性(Single nucleotide polymorphisms,SNP)位點,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)。本課題為病例對照研究,分為兩個階段共納入正常對照1440個,bd患者950個和vkh綜合征患者384個。通過taqman和聚合酶鏈式反應-限制性片段長度多態(tài)性(polymerasechainreaction-restrictionfragmentlengthpolymorphism,pcr-rflp)兩種方法進行基因分型。本研究運用計數(shù)方法統(tǒng)計snp位點各基因型和等位基因的個數(shù),運用卡方檢驗計算等位基因頻率和基因型頻率的差異p值,bonferroni校正后記為pc值,校正后的pc值0.05被認為有統(tǒng)計學差異�；騧rna和下游細胞因子的表達水平分別通過熒光定量聚合酶鏈反應(real-timepcr,rt-pcr)和酶聯(lián)免疫吸附實驗(enzyme-linkedimmunosorbentassay,elisa)進行測定。結果:bd病的研究分為三個階段,第一階段研究發(fā)現(xiàn):在bd患者中位點ciita//rs12932187的c等位基因頻率(pc=1.668e-02,or=0.713,95%ci=0.591-0.861)和位點nod1//rs2075818的g等位基因頻率(pc=4.694e-02,or=0.698,95%ci=0.562-0.868)均明顯低于正常對照組。第二階段擴大樣本數(shù)后,得到的結果與第一階段相符合,在進行數(shù)據(jù)分析后顯示:bd患者中位點ciita//rs12932187的cc基因型頻率(pc=3.331e-06,or=0.617,95%ci=0.519-0.735)和c等位基因頻率(pc=6.004e-07,or=0.709,95%ci=0.629-0.799)明顯低于正常對照組。BD患者中NOD1//rs2075818的GG基因型頻率(Pc=1.022E-02,OR=0.536,95%CI=0.386-0.745)和G等位基因頻率(Pc=6.811E-05,OR=0.720,95%CI=0.629-0.824)也明顯低于正常對照組。第三階段為功能性研究,結果顯示:在給予外周血單個核細胞(Peripheral blood monouclear cell,PBMC)LPS刺激后,CIITA//rs12932187 CC基因型攜帶者的CIITA mRNA表達被抑制而IL-10的表達水平則明顯升高。本實驗選擇的20個位于NLR基因上的SNP位點與VKH綜合征無明顯關聯(lián)。結論:研究表明CIITA//rs12932187和NOD1//rs2075818兩個位點均與Behcet病的遺傳易感性相關。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R773.9
本文編號：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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R773.9
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1 李琳;NLR基因多態(tài)性與Behcet病和VKH綜合征的相關性研究[D];重慶醫(yī)科大學;2016年
，本文編號：2429472
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