利用多重荧光定量PCR协助诊断Crigler-Najjar综合征Ⅱ型及Gilbert综合征

Using multiple fluorescence PCR technique to assist in the diagnosis of Crigler-Najjar syndrome type Ⅱ and Gilbert syndrome

目的尝试建立一个多重荧光定量PCR反应体系对Crigler-Najjar综合征Ⅱ型(Crigler-Najjar syndromeⅡ,CNSⅡ)及Gilbert综合征(Gilbert syndrome,GS)患者的尿苷二磷酸葡糖醛酸基转移酶1A1(uridine diphosphate glucuronosyltransferase 1A1,UGT1A1)基因突变进行检测,以运用于协助以上疾病的临床诊断。方法选取国内外目前报道较多UGT1A1基因常见突变的4个位点[A(TA)7TAA、Gly71Arg、Pro229Gln及Try486Asp],利用多重荧光定量PCR Tapman探针法对96例临床诊断为GS或CNSⅡ患者及100名健康体检者全血基因组进行检测,利用T-A克隆法对扩增产物进行测序验证。结果多重荧光定量PCR反应体系可有效检测出实验样本中所存在的以上4种基因突变。A(TA)7TAA、Gly71Arg、Pro229Gln及Try486Asp在试验组测得的阳性率高于对照组,差异有统计学意义(P<0.05)。PCR产物电泳及测序结果与其基因检测表型完全一致。结论利用多重荧光定量PCR技术可以更简便、快捷、有效地对GS或CNSⅡ患者中的UGT1A1基因突变进行检测,为临床诊断提供有用信息。

Objective To establish a multiple fluorescence quantitative PCR reaction system for Crigler-Najjar syn- drome type Ⅱ （CNS Ⅱ） and Gilbert syndrome （GS） in patients with uridine diphosphate glucuronosyl transferase 1AI （ UGT1A1 ） gene mutation testing, and apply to assist in the clinical diagnosis of the disease. Methods We selected 4 common .mutation sites [A（TA）7TAA, Gly71Arg, Pro229Gln and Try486Asp] from UGT1A1 gene that currently repor- ted at home and abroad at present. Using multiple fluorescence PCR Tapman probe to detect the whole genome that 96 patients were clinical diagnosis of GS or CNS H and 100 healthy controls, using the method of T-A clone sequencing to test the PCR-amplified product. Results This multiple fluorescence quantitative PCR reaction system could effectively detect the test samples of the above 4 kinds of genetic mutations. The positive rate of A （TA） 7TAA, Gly71Arg, Pro229Gln and Try486Asp measured in clinical group were higher than the control group, with statistical significance. PCR products electrophoresis and T-A clone sequencing were completely conform to the gene types （P 〈 0.05 ）. Con- clusion Using multiple fluorescent quantitative PCR technology can be more convenient, fast and effectively to detect UGT1A1 gene mutations in patients of GS or CNS Ⅱ , provide information for clinical diagnosis.