利用多重实时荧光定量PCR技术检测新生儿高胆红素血症患者UGT1A1基因多态性研究

To detect UGT1A1 gene polymorphism for neonatal patients with high blood bilirubin by using multiple real-time fluorescent quantitative PCR technique

目的利用多重实时荧光定量PCR技术检测新生儿高胆红素血症患者UGT1A1基因多态性,探讨UGT1A1基因多态性与新生儿胆红素水平的关系。方法选取2015年1月至2016年1月深圳市第三人民医院新生儿高胆红素血症组165例(总胆红素≥257μmol/L)及对照组150例(总胆红素<257μmol/L),提取全血DNA,选取UGT1A1基因常见的四个突变位点,启动子的A(TA)7TAA盒以及编码区Pro229Gln、Try486Asp、Gly71Arg,合成引物及探针,对样本进行多重实时荧光定量PCR检测并采集荧光,对PCR检测阳性结果的样本回收提纯,送基因测序鉴定。结果多重实时荧光定量PCR结果与基因测序结果一致,A(TA)7TAA突变在高胆红素血症及对照组检出率分别为7.8%、23.3%,Gly71Arg位点突变在高胆红素血症组及对照组分别为52.7%、19.3%,差异有统计学意义。Try486Asp及Pro229Gln点突变在两组检出率均较低,无统计学意义。在高胆红素血症组Gly71Arg纯合子突变者胆红素水平比杂合子突变及野生型高,有统计学意义。结论利用多重实时荧光定量PCR技术检测基因突变方便高效、经济准确,新生儿高胆红素血症与Gly71Arg突变有关。

Objective To detect UGT1A1 gene polymorphism for neonatal patients with high blood bilirubin by using multiple real-time fluorescent quantitative PCR technique and to discuss the relationship between UGT1A1 gene polymorphism and neonatal bilirubin level.Methods We chose 165 patients with high neonatal bilirubin(bilirubin≥257μmol/L)as the test group and 150 general subjects(bilirubin<257μmol/L)as control group in authors'hospitals between January 2015 and January 2016.DNA was extracted firstly from the whole blood;four common mutations of UGT1A1 gene were chosen,including A(TA)7TAA box of the promoter,and the Pro229Gln,Try486Asp,Gly71Arg in the coding region;primers and probe were synthesized;and samples were detected by multiple real-time fluorescent quantitative PCR technique and the fluorescence was collected;and the samples with positive results were recovered and purified for gene sequencing.Results The results of multiple real-time fluorescent quantitative PCR were consistent with the results of gene sequencing.The detection rates of A(TA)7TAA box mutation in test group and control group were 7.8% and 23.3%respectively,while the rate of Gly71Arg mutation in the test group and control group were 52.7% and 19.3%respectively,and the difference was statistically significant.However,the detection rates of Try486Asp and Pro229Gln mutation were relatively low with no statistical significance.In the test group,the bilirubin level of patients with homozygous Gly71Arg was higher than that of wild type or heterozygous mutations,and the difference was statistically significant.Conclusion Multiple real-time fluorescent quantitative PCR technique is a convenient,accurate,efficient and economic way to detect gene mutation,and the neonatal hyperbilirubinemia is associated with Gly71Arg mutations.