B型肝炎X基因對於兒童B型肝炎感染自然病史所扮演的角色

Abstract

中文摘要 研究背景及目的 慢性B型肝炎感染是一個全球的健康議題。持續性的B型肝炎感染常常會演變成為慢性肝臟發炎或是肝癌。B型肝炎感染，流行地區例如--台灣，通常開始於嬰幼兒期，並主要藉由母嬰垂直感染而使兒童成為帶原者。兒童的慢性B型肝炎感染在年幼時期通常沒有症狀，這段免疫耐受期通常會持續15年甚至更久。 B型肝炎病毒如何躲匿並對抗宿主固有的先天性免疫，並使患童不產生免疫反應的機制目前還是未明。近來發現B肝病毒功能性基因之一的HBx基因可能在B型肝炎自然病史中扮演了某種重要的角色。HBx基因的變異也曾被報導與肝臟腫瘤發生的原因相關。然而，對慢性B型肝炎兒童來說， HBx基因的變異是否跟免疫耐受期(immune tolerance phase)的維持或結束有關係，目前仍然不清楚。HBx基因的突變對於兒童慢性B型肝炎的長程自然病史之影響仍然有待研究。我們希望探討HBx基因變異與兒童慢性B型肝炎感染長程病史之間的交互作用。 研究方法 臺大醫院兒童肝膽腸胃科自兒童期開始對一群慢性B型肝炎感染的患者做成長程追蹤。自幼年開始，他們在門診規律性定期接受病史詢問及理學檢查，並且也接受抽血檢驗，其中包括肝轉胺酶、B型肝炎病毒標記(HBsAg, HBeAg, Anti-HBe, Anti-HBs, Anti-HBc)、胎兒蛋白、B型肝炎病毒去氧核醣核酸的定量、與B肝病毒基因型測定等。在這些慢性B型肝炎感染的個案中，我們追蹤未曾接受B型肝炎抗病毒藥物治療、未合併有其他C型肝炎或D型肝炎…等可能導致肝臟發炎因素的病患，取其兒童期第一次接受抽血檢查之血清，萃取其中的B型肝炎病毒去氧核醣核酸，並進行HBx基因的定序。接著與已知資訊中的HBx基因序列進行比對並分析，以找出突變點，並且與臨床資料整合做分析， 我們希望能探討B肝病童之HBx基因突變與免疫耐受期長度以及與自然病程之間的關連性。 研究結果 在這個研究中，我們追蹤了155位同意加入本研究之病患至少5年以上，其中有105位是男性。他們平均起始追蹤年紀為8.9± 4.5歲，平均追蹤時間為23.4± 6.7年。有110位病患在追蹤期間經歷了免疫耐受期的結束，並且有81位患者經過B型肝炎e抗原抗體轉換的過程。根據155位患者聚合酶連鎖反應附帶分析比對，基因型B型占所有病患的77.4%。另外， HBx基因出現機率最高的突變位點有五個，包括V44G/L, A66T, R87G, N118T, I127V。利用Kaplan-Meier存活分析，我們發現基因型B型、HBx基因的R87V、 N118T、以及I127V突變，相較於基因型C型及沒有發生突變者，有較高的比例容易結束免疫耐受期而進入到免疫廓清期(發炎期)，並且達到統計學上的顯著意義(p<0.001, p=0.017, 0.044, 0.001)﹔在多變項分析，僅有HBx基因的I127V突變發現到有較高的機會較早突破免疫耐受期而進入到免疫廓清期，其HR為2.07; p值為0.022，達統計學上顯著意義。 結論 在慢性B型肝炎感染自然病史中， 我們發現HBx基因的I127V突變和較早突破免疫耐受期有顯著相關。

Abstract Backgrounds and Aims Chronic hepatitis B virus (HBV) infection is a world-wide health problem. Persistent HBV infection is often associated with chronic liver disease and may lead to liver cirrhosis and hepatocellular carcinoma (HCC). HBV infections were mostly acquired in the infancy and early childhood through the route of mother-to-infant transmission, especially in endemic areas such as Taiwan. Children with HBV infection often remained asymptomatic at their young age, and their immune tolerance phases may last 2 decades or longer. The mechanism of HBV to evade or overwhelm the human innate immune system, and to keep a state of relative hypo-responsiveness to HBV in children still remained largely unclear. Recently, HBx gene, which was one of the four HBV functional genes, had been reported to be involved in the host immune abrogation. However, the relationship between HBx mutation and either the persistence or the ending of immune tolerance was unknown. The impacts of HBx mutation on long term natural course of chronic HBV infection remained to be elucidated, particularly in children. We aim to investigate prognostic value of mutant HBx gene on the natural history of HBV infection from childhood to adult life. Materials and Methods Patients were recruited from long term prospective chronic HBV carrier cohort, which was conducted in Pediatric department of National Taiwan University Hospital. They received evaluations periodically including history taking and physical examinations. Liver enzymes, HBV markers (HBsAg, HBeAg, Anti-HBe, Anti-HBs, Anti-HBc), AFP, DNA quantitation, and HBV genotyping were also assessed. The blood samplings collected at initial time of enrollment were examined for HBx gene. We analyze the sequences of HBx genes and tried to find the mutations. We attempt to elucidate the correlations between HBx gene mutations and the lasting or ending of immune tolerance phase. Results A total of 155 children (105 males) were followed for 23.4± 6.7 years with initial age of 8.9± 4.5 years. Genotype B accounted for 77.4% of all patients according to sequence findings. One hundred and ten patients had breakthrough immune tolerance phase and 81 patients achieved HBe seroconversion. HBx gene mutations of V44G/L, A66T, R87G, N118T, I127V had highest frequencies. In Kaplan-Meier survival analysis, genotype B and mutants of R87V, N118T, and I127V in HBx gene had significantly higher cumulative proportion of breakthrough of immune tolerance phase (log rank test, p<0.001, p=0.017, 0.044, and 0.001, respectively). In multivariate analysis, we found that mutation of I127V in HBx gene was an independent favorable factor to predict earlier breakthrough of immune tolerance phase, with a statistically significant value (hazard ratio 2.07, 95% CI 1.11-3.85, p=0.022) Conclusions In chronic HBV infection, patients with I127V mutation in HBx gene had higher potential to breakthrough the immune tolerance phase.