肝移植后乙型肝炎病毒再感染患者的病毒S区与P区重叠基因变异

A study on mutations of the overlapping hepatitis B virus surface and polymerase gene in patients with HBV reinfection after liver transplantations

目的 探讨乙型肝炎免疫球蛋白（HBIG）和拉米夫定对肝移植术后HBV再感染患者的HBVS基因与P基因重叠区变异的影响。方法 对2002年6月-2003年12月320例因乙型肝炎相关性终末期肝病行原位肝移植手术患者随访1．5～3．0年，选择资料完整的HBV再感染者14例为研究对象。直接基因测序法分析术前、术后HBVS基因及重叠P基因的序列改变情况；荧光定量PCR检测HBVDNA含量；微粒子捕捉酶免疫法检测血清HBIG浓度。结果14例患者术前、术后S区与P区氨基酸变异数量无明显改变，变异位点略有改变。基因型检测结果显示12例为C型，2例为B型，无其他基因型。s区“a-决定簇”基因变异发生在1126S、T131N、S143T和G145R位点，“a-决定簇”的上游和下游变异的有L110F、1113s和T160K位点。S区核昔酸突变时S区氨基酸存在同义与错义变异，可引起相应的P区氨基酸错义变异，其中包括已知的拉米夫定耐药位点变异及其以外的多位点氨基酸变异，且变异位点差异较大，大部分变异位点移植前已存在变异。术前有4例血清HBVDNA低于检测限，术后均阳性。HBIG浓度为0U／L者7例，≤100U／L5例，〉100U／L2例。结论 肝移植术后免疫抑制剂治疗对氨基酸变异位点的数目影响不大，对变异位点的位置有一定的影响；除了“a-决定簇”的基因变异点以外，“a-决定簇”的上游或下游基因位点也存在变异，可能是引起HBV再感染的另一原因；S区核昔酸位点的突变可引起S区氨基酸的同义和错义变异，并可同时引起重叠P区相应位点的错义变异，其临床意义有待进一步探讨。

Objective To investigate the influence of combined hepatitis B immune globulin （HBIG） and lamivudine （LMV） treatment on hepatitis B virus （HBV） surface antigen and polymerase overlapping gene mutations in HBV reinfected liver transplant recipients. Methods From June 2002 to December 2003, 320 patients who underwent liver transplantations due to HBV-related end-stage liver diseases were followed-up for 1.5 to 3 years postoperatively. Fourteen patients developed HBV reinfection. They had LMV before their liver transplantations and had LMV and HBIG after the transplantations to prevent HBV infections. Their serum levels of HBV DNA were measured by polymerase chain reaction. G-ene sequencing method was used to analyze HBV genotype and mutations of the S gene. Micro-particle enzyme immunoassay was used to measure the serum concentration of HBIG. Results （1） There was no obvious difference in the number of amino acid mutation sites in S and P regions before and after the transplantations. （2） The HBV genotypes were B-type （n = 2） and C-type （n = 12） in the reinfected group before the transplantations, and genotypes after the transplantations remained the same. （3） HBIG concentrations were 0 U/L in 7 patients, 〈 100 U/L in 5 patients, and 〉100 U/L in 2 patients. Mutations were detected as I126S, T131N, S143T and G145R in ‘a＇determinant and L110F, I113S, T160K in up- or down-stream of ‘a＇ determinant. （4） Mutations in S gene caused missense mutation in the surface antigen region. These mutations also caused corresponding missense mutations in the polymerase region. The missense mutation in the polymerase region involved lamivudine mutation sites and other mutation sites. Conclusion Immunosuppressant therapy has no obvious influence on the numbers of mutations, but it can influence the sites of the mutations. Besides ‘a＇ determinant mutations, there exist mutations in up- or down-streams of ‘a＇ determinant and they may cause HBV reinfection.