Mendy M E, Kaye S, Le Roux E, Kirk G D, Jeng-Barry A, McConkey S, Cotten M, Kuniholm M H, Leligdowicz A, Hainaut P, Rowland-Jones S, Whittle H
Viral Diseases Programme, Medical Research Council, Atlantic Boulevard, Fajara, P.O. Box 273, Banjul, The Gambia.
J Clin Microbiol. 2008 Aug;46(8):2723-30. doi: 10.1128/JCM.01622-07. Epub 2008 May 28.
Hepatocellular carcinoma (HCC) and cirrhosis are important causes of mortality worldwide. Persistent hepatitis B virus (HBV) infection is a major cause of these diseases. Double mutations in the basal core promoter (BCP) (A1762T and G1764A) and precore (pre-C) (G1896A) regions of the virus are associated with progression to HCC. The current study is aimed at developing a simple method for screening and detecting BCP and pre-C mutations in HBV carriers. We have developed and validated an oligonucleotide ligation assay (OLA) to detect point mutations in the HBV core gene. We have applied OLA methods to samples from HBV-infected carriers recruited from the Gambia Liver Cancer Study (GLCS) comprising asymptomatic HBsAg carriers, patients with cirrhosis, and patients with HCC. We observed an 89.3% and 95.8% concordance between the OLA and DNA sequencing for BCP and pre-C mutations, respectively. OLA detected the mutations in single-strain infections and in infections with mixtures of wild-type and mutant viruses under conditions where sequencing detected only the single dominant strains. BCP mutations were detected in 75.7% of patients with advanced liver disease (cirrhosis/HCC) compared to 47.6% of asymptomatic carriers, while pre-C mutations were detected in 34.5% of advanced liver disease patients and in 47.6% of asymptomatic HBsAg carriers. There was a significant association between the presence of BCP mutations and advanced liver disease. In conclusion, OLA is a simple, economical, and reliable assay for detection of pre-C and BCP mutations. Its application can lead to improvement in diagnosis and clinical care in regions where HBV is endemic.
肝细胞癌（HCC）和肝硬化是全球范围内重要的死亡原因。持续性乙型肝炎病毒（HBV）感染是这些疾病的主要病因。病毒的核心启动子（BCP）（A1762T和G1764A）和前核心（pre-C）（G1896A）区域的双重突变与HCC进展相关。当前研究旨在开发一种简单方法，用于筛查和检测HBV携带者中的BCP和pre-C突变。我们已经开发并验证了一种寡核苷酸连接分析（OLA）方法，用于检测HBV核心基因中的点突变。我们已将OLA方法应用于从冈比亚肝癌研究（GLCS）招募的HBV感染携带者的样本，该研究包括无症状HBsAg携带者、肝硬化患者和HCC患者。我们观察到，OLA与DNA测序在BCP和pre-C突变方面的一致性分别为89.3%和95.8%。在测序仅检测到单一优势菌株的条件下，OLA检测到单菌株感染以及野生型和突变病毒混合感染中的突变。在晚期肝病（肝硬化/HCC）患者中，75.7%检测到BCP突变，而无症状携带者中这一比例为47.6%；在晚期肝病患者中，34.5%检测到pre-C突变，无症状HBsAg携带者中这一比例为47.6%。BCP突变的存在与晚期肝病之间存在显著关联。总之，OLA是一种用于检测pre-C和BCP突变的简单、经济且可靠的分析方法。其应用可改善HBV流行地区的诊断和临床护理。
