论文标题:早期B细胞因子相关锌指蛋白基因（OAZ）与人类系统性红斑狼疮发病的相关性研究
Study on Association between Olf1/Ebf-Associated Zinc Finger Protein Gene (OAZ) and Systemic Lupus Erythematosus in Chinese Population
论文作者 梁栋
论文导师 黄秀琴；沈南,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 华东师范大学,点击次数 176,论文页数 87页File Size10581k
2005-06-01论文网 http://www.lw23.com/lunwen_1682827/ 红斑狼疮;系统性;狼疮肾炎;早期B细胞因子相关锌指蛋白基因基因;单核苷酸多态性;凝胶电泳迁移率改变分析(EMSA);实时定量RT-PCR;等位基因分型;单倍型
lupus erythematosus; sysytemic; Lupus Nephritis; OLF1/EBF-associated zinc finger protein gene; single nucleotide polymorphism; electrophoretic mobility shift assays (EMSA); quantitative real-time polymerase chain reaction; allelic discrimination; haplotype.
系统性红斑狼疮是典型自身免疫病,被公认为自身免疫病的原型。通过对群体遗传学、双生子发病一致率和疾病家族聚集性等一系列研究均证实遗传因素在SLE发病中的起重要作用。到目前为止,已有3个独立的研究小组在不同的人群中证实染色体16q12区域SLE的发病显著连锁。实验室的预初研究也提示中国人群该区与SLE易感连锁,微卫星标记D16S517与SLE存在传递不平衡(P<0. 0001) ,而且OAZ基因可能是该区域的易感基因。本研究在此基础上,对OAZ基因与SLE的相关性做进一步分析。首先,我们采用测序等方法对OAZ基因内部阳性微卫星D16S517附近的SNPs进行筛选和验证,新发现3个SNPs,分别为rs2292155(第三内含子),rs2292156,rs3803665(第三内含子),其中rs3803665为沉默突变(第四外显子)。为进一步研究SNP的变异与疾病的相关性,我们选择OAZ基因内部阳性微卫星D16S517附近的8个SNP标记,进行Taqman等位基因分型(AllelicDiscrimination AD)。对SNP的等位基因型(allele)、基因型(genotype)及单倍型(haplotype)的分布进行病例对照研究,并应用传递不平衡检验(TDT)的方法进行连锁不平衡分析。病例对照结果显示:发现中国汉族人群中① SNPrs1344531等位基因(T)频率在SLE患者组(47. 0%)和正常对照组(38. 1%)中差异存在统计学意义(P=0. 008) ;CC基因型的频率在SLE患者组(25. 5%)与在正常对照组(38. 1%)中相比显著下降(P=0. 005) 。 ② SNP rs1344531等位基因(T)频率在狼疮肾炎患者组(53. 0%)和正常对照组(38. 1%)中差异存在统计学意义(P=0. 001) ;CC基因型的频率在狼疮肾炎患者组(22. 9%)与在正常对照组(38. 1%)中相比显著下降(P=0. 013) ,TT基因型的频率在狼疮肾炎患者组(28. 9%)与在正常对照组(14. 3%)中相比显著增高(P=0. 003) 。③ rs1420676-rs1344531(C-T)(P=0. 001) , rs3803665-rs1420676-rs1344531(C-C-T)(P=0. 004) ,rs2292155-rs3803665-rs1420676-rs1344531(C-C-C-T)(P=0. 002) 等单倍型频率在狼疮肾炎患者组中显著增高。而rs1344531-rs2080353(C-A)(P=0. 005) ,rs1344531-rs2080353-rs933564(C-A-G)(P=0. 006) 等单倍型的频率在狼疮肾炎患者组中显著下降。此外,传递不平衡检验(TDT)未发现阳性结果,但rs1344531位点显示优势传递的趋势(T等位基因,传递:不传递=107:97,P=0. 4839) 。以上结果提示,阳性SNP位点(rs1344531) 可能参与SLE的发病,也可能与邻近的某个致病位点存在连锁不平衡。有研究发现疾病候选基因的SNPs往往决定基因的表达情况,因此在实验的第二部分我们应用AB17700进行荧光标记实时定量PCR(real time PCR)的方
Systemic lupus erythematosus (SLE) is a generally accepted prototype autoimmune disease. The importance of genetic influences on SLE has been consistently supported by studies of populations, twin concordance rates, and aggregation of disease in families.Up to now, linkage of 16q12 to Systemic lupus erythematosus (SLE) has been confirmed by 3 independent research groups. Our previous study also established linkage of chromosome 16q12 to SLE and suggested that OLF1/EBF-associated zinc finger protein (OAZ) might be a novel candidate susceptibility gene within the 16q interval.In the first phase, SNPs located around positive microsatellite marker D16S517 within OAZ gene were selected and screened for verification by sequencing. 3 new SNPs were discovered, which are rs2292155 (in intron 3), rs2292156 (in intron 3) and rs3803665 (in exon 4, silent mutation).To observe whether single nucleotide polymorphisms (SNPs) within OAZ gene are associated with SLE in Chinese population, eight SNPs located around D16S517 were genotyped by allelic discrimination-PCR (AD-PCR) with the TaqMan universal PCR master mix and ABI Prism 7900 sequence detector (PE Applied Biosystems). TaqMan MGB probes were used for AD. SNP, haplotype and linkage disequilibrium studies were performed by using the Case-control and Transmission Disquilibrium Test (TDT) approach.Results of Case-control association analysis showed: In Chinese population (1) SNP rs1344531 T allele was significantly associated with SLE susceptibility(T allele: SLE patients(47.0%) versus normal controls(38.1%) ;X2=7.300 ,P=0.008,OR(95%CI)=1.441 (1.105-1.878));Genotypic distribution for SNP rsl 344531(CC/CT/TT) differed significantly between SLE patients (25.5%/54.9%/19.6%) and normal controls (38.1 %/47.6%/14.3%) (x2=8.394, P=0.015 ) , CC genotype frequencies decreased significantly in SLE patients ( 25.5% ) ,as compared with normal controls(3 8.1%) (x2=7.976, P=0.005,OR(95%CI)= 0.557(0.370-0.838)) (2) SNP rs1344531 T allele was significantly associated with LN susceptibility (T allele: SLE patients(53.0%) versus normal controls(38.1%); X2=11.769, P=0.001,OR(95%CI)= 1.832 (1.293-2.596)); Genotypic distribution for SNP rs1344531(CC/CT/TT) differed significantly between LN patients (22.9%/48.2%/28.9%) and normal controls ( 38.1 %/47.6%/14.3%)(x2=12.065, P=0.002) .CC genotype frequencies decreased significantly in LN patients (22.9%) ,as compared with normal controls(38.1%) (x2=6.578, P=0.013, OR(95%C1)= 0.481(0.274-0.848) ), TT genotype frequencies increased significantly in LN patients (28.9%) ,as compared with normal controls(14.3%) (x2=9.423, P=0.003, OR(95%CI)= 2.431(1.363-4.334)). No statistical significance was observed between non-LN patients group and normal controls group.(3)Haplotype analysis produced additional support for association. In LN patients, frequencies for some haplotypes containing rsl344531 (rsl420676-rsl344531(C-T) (x2=11.731,P=0.001, OR(95%CI)=1.867(1.302~2.676)),rs3803665-rsl420676-rsl344531(C-C-T) (X2=8.876,P=0.004,OR(95%CI)=1.772(1.213-2.589)),rs2292155-rs3803665-rsl420676-rsl344531(C-C-C-T) (x2=9.962,P=0.002, OR (95%CI)= 1.915(1.274-2.880)) were detected to increase significantly(41.0% versus 27.1% ; 33.3% versus 21.8% ; 27.0% versus 16.2%);while others (rsl344531-rs2080353(C-A) (x2=8.06,P=0.005, OR (95% CI)=0.603(0.424~0.856)), rsl 34453 l-rs2080353-rs933564(C-A-G) (x2=7.929,P=0.006,OR(95%CI)=0.602(0.422~0.859)) decreased significantly(39.5% versus 52.2%; 36.6% versus 49.2%).Although TDT analysis results didn"t show correlation between SNPs and SLE, the trend was observed for the preferential transmission of T polymorphism of rsl344531 from parents to offspring. Our results indicate that SNPs within OAZ gene may either confer susceptibility to Lupus Nephritis or have a linkage disequilibrium with the susceptibility gene in Chinese population.SNPs of disease susceptibility gene may have influence on the expression of gene. In the second phase, we detected the mRNA expression of OAZ gene by fluorescence-based TaqMan real time quantitative PCR with
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