乙型肝炎病毒基因多态性检测芯片的临床应用

Application of a Gene Chip for Detection of Hepatitis B Virus Gene Polymorphism

目的:评价临床应用乙型肝炎病毒(HBV)基因多态性芯片(中科院上海微系统和信息技术研究所等研制),检测HBV基因前C区1896、1814,基本C区启动子(BCP)1762和1764,P区的YMDD基序552位等的点突变的效能。方法:以该芯片检测49例不同临床类型乙型肝炎病人血清,并对部分血清的PCR扩增产物进行测序分析。结果:HBV前C区1896位在血清HBeAg阳性病例中,野生株、混合(野生/突变)株、突变株和未确定(无杂交信号)的检出率,分别为57.7％,38.5％,0和3.8％;血清抗HBe阳性病例中,相应为33.3％,22.2％,33.3％和11％,后者突变株显著高于前者(P<0.01)。所有病例HBV前C区的1814位均为野生,未检出突变。BCP 1762和1764双突变在慢性重型肝炎和肝炎肝硬化的检出率分别为66.7％和69.2％,显著高于慢性肝炎的19%(P<0.01)。10例拉米夫定治疗6月以上,HBV DNA仍阳性者中,7例检出P区YIDD或YVDD突变,1例兼有YIDD和YVDD突变,余2例无杂交信号。7例1 896,4例BCP,6例YMDD的测序和芯片检测结果一致,6例无信号位点的测序结果显示,这些位点附近,出现了与杂交探针错配的突变。结论:初步结果提示,该芯片适用于临床检测HBV前C区、P区一些已知热点的突变,并可用于研究这些突变与疾病严重性,及产生抗HBV药物耐药间的相互关系。

Objective: To evaluate the efficacy of a gene chip (developed by Shanghai micro ?system and information technique institute etc.) for detection of hepatitis B virus (HBV) gene polymorphism.Methods: Applying the chip to detect relevant HBV gene amplified products by PCR from sera of 49 patients with hepatitis B of various clinical types and dideoxy terminator sequencing to analy-sis some of these products. Results: For nt 1896 in HBV pre - C region in amplified products from sera with HBeAg, the rates of wild, mixed (wild and mutation), mutation type and undetermined (no hybridized signal) were 57.7% , 38.5% , 0 and 3.8% , but in that from sera with anti - HBe were 33.3 % , 22.2% , 33.3% and 11%, respectively. No mutation at nt 1814 in HBV pre - C region was found in all amplified products. The mutation rates for basai core promoter (BCP) nt 1762 and 1764 in HBV pre - C regions in patients with severe hepatitis B and with chronic hepatitis B plus cirrhosis (66.7% and 69.2%) were significantly higher than that in patients with chronic hepatitis B (19.0%; P<0.01); The mutation of motif YMDD (nt 552) in HBV P region were detected in 8 of 10(2 undetermined) hepatitis B patients treated with Lamifudine over 6 months remaining serum HBV DNA positive, and none of this mutation was detected in other 39 patients without using such drug. AU of sequencing of 7 nt l 896, 4 BCP and 6 YMDD agreed with the results of them by the chip. The sequencing of sites without hybridized signal also showed that there was other mutation around these sites. Conclusion: Preliminary results suggest that the chip is useful for clinic to find some known hot spots of mutation in HBV pre - C and P regions and to study dynamically the interrelation among HBV and host as well as anti - HBV drugs.