e抗原陽性慢性B型肝炎患者接受干安能治療結束後，precore/basal core promoter 基因突變和e抗原持久陰轉之相關性研究

Abstract

背景：B型肝炎病毒感染是臺灣醫學和公衛上重要的課題。在台灣，約有參百萬人是B型肝炎帶原者。從病毒自然病程中發現，自發性e抗原陰轉會造成病毒量下降，肝炎情況緩解，及長期良好的預後。目前針對e抗原陽性之慢性B型肝炎患者的治療準則如下：如有持續或間歇性丙胺酸轉胺酶值上升，且大於正常值上限的2倍，可先觀察3至6個月，看是否有自發性的e抗原陰轉，若無則可考慮給予治療。治療的方法目前以干安能（lamivudine）最廣為使用，不過治療後e抗原的持久陰轉率卻不高，約38％至77％，累積三年的復發率為36％至54％，然而大部分的復發都發生在停藥後的第一年，復發率的問題成為干安能治療上的瓶頸。核前區在核苷酸1896位置發生G轉成A突變，形成一個轉譯停止的訊號，導致無法合成e抗原。核心啟動子在核苷酸1762位置發生A轉成T突變，位置1764發生G轉成A雙突變，會減少約70%的e抗原的合成，且增加病毒的複製，這是在e抗原陰性的B型肝炎病毒中最廣被研究的突變。早期病例報告指出核前區1896變異與慢性肝病的進展或猛爆性肝炎的發生有關，另外發現當核心啟動子1762/1764雙突變產生時，減少e抗原合成，反而可能增強宿主免疫反應，而加重肝組織的發炎壞死。最近報告也發現在自發性e抗原陰轉前後，核前區比核心啟動子變異種之上升比例更顯著。若在干安能使用一年下，除了使病毒數目快速下降之外，一年內也會讓核前區及核心啟動子突變數目增加，不過在停藥後常會造成殘存的野生種病毒復發。在e抗原血清陽轉的研究中發現，會造成e抗原血清陽轉的原因可能由於無法持久的產生核前區1896和核心啟動子1762/1764雙變異的突變種，一旦轉為野生種病毒，則較容易回歸e抗原陽性。過去在討論干安能治療後是否能產生持久反應的因素，如年紀、病毒基因型、e抗原陰轉後再延長干安能的治療時間及停藥時病毒濃度皆有相關，但對於系列的核前區1896和核心啟動子1762/1764雙變異的突變種的研究和持久性e抗原陰轉的相關，目前仍不清楚。 目的：針對自發性和干安能治療後產生持久性e抗原陰轉的患者，系列探討核前區1896和核心啟動子1762/1764雙變異突變種是否和其有關。假設一：自發性e抗原陰轉中，若伴隨e抗體產生，會有較高之核前區1896和核心啟動子1762/1764的突變種比例。假設二：干安能治療之B型肝炎e抗原陽性患者發生e抗原陰轉時或治療結束時之B型肝炎病毒的核前區1896和核心啟動子1762/1764，若呈現穩定的突變種（sustained mutant strain），則患者會有較高的e抗原持久陰轉率。 材料和方法：第一部份：回溯收集臺大醫院慢性B型肝炎且屬於自發性e抗原持久陰轉患者32位，符合表面抗原陽性大於6個月，e抗原陽性超過3個月且日後產生e抗原持久陰轉的患者。依照收案時及日後追蹤e抗原和e抗體的狀況，收集6個時間點的血清，分別是T1、T2、T3分別是e抗原陰轉的時間點前1年、6個月及3個月，T4是e抗原陰轉的時間點，T5、T6是e抗原陰轉的時間點後6個月及1年。分別測量其病毒基因型、病毒量和核前區1896和核心啟動子1762/1764雙變異的突變種比例，了解其對e抗原陰轉的影響。第二部份：回溯收集臺大醫院慢性B型肝炎患者45位，皆符合表面抗原陽性超過6個月，e抗原陽性超過3個月，HBV DNA陽性（≧105 copies/ml），丙胺酸轉胺酶超過正常值上限的5倍。給予每天100毫克的干安能治療，持續治療至e抗原消失後至少3個月以上，停藥標準至少達到其治療反應，排除治療中產生YMDD motif突變、C型肝炎、D型肝炎感染和HIV感染，自體免疫性肝炎，也沒有酒精或毒品濫用，並排除肝移植後和惡性腫瘤。收集三個時間點的血清，分別是治療前、e抗原陰轉和治療結束時。並至少追蹤至治療結束後6個月，以是否產生e抗原持久陰轉為結果，測量病毒基因型、病毒量和核前區1896和核心啟動子1762/1764雙變異的突變種比例，來了解和e抗原持久陰轉的相關。 結果：在自發性e抗原陰轉前一年即產生了病毒量的下降，並伴隨肝炎的發生，在核前區1896和核心啟動子1762/1764雙變異的突變種比例會隨著e抗原陰轉而緩慢上升，而病毒量和肝炎的發生會隨e抗原陰轉後而下降。中位數病毒量是基因型B比例高（p=0.03~0.61）。核前區1896突變株是基因型B比例高，而核心啟動子1762/1764雙變異的突變株則是基因型C比例高（p=0.004~0.03）。在e抗原陰轉中有無產生e抗體，對ALT值、病毒量和核前區1896和核心啟動子1762/1764雙變異的突變種比例沒有影響。另外在干安能治療後，依停藥後6個月分具有持久反應（sustained response, SR-6）者34位，無持久反應（non sustained response, NSR-6）者11位。在e抗原陰轉時和治療結束時，大多數病毒量下降至低於100 copies/ml，且核心啟動子1762/1764雙變異的測量也因病毒量太低，而無法在聚合酶連鎖反應中放大出來。依據干安能治療達到合併反應後停藥6個月時的情形，分為持久反應 （sustained response, SR-6），和無持久反應（non sustained response, NSR-6）兩組。依照卡方檢定和t檢定，兩組病患在年齡、性別、治療前最高ALT值、治療前病毒濃度、病毒基因型、干安能使用時間的長短、服藥後至e抗原陰轉的時間、以及e抗原陰轉後再延長干安能治療的治療時間均無統計上的差別，依邏輯回歸分析法也是沒有顯著差異。但在分析是否之前已經使用過干安能方面，顯然NSR-6那一組有較多曾經使用過的經驗（p=0.007）。在干安能治療後，核前區1896為G的野生種，在e抗原陰轉時和治療結束時，呈現比例上的增加。治療前的分析發現，持久反應（SR-6）那一組的核前區1896是單純G和無持久反應（NSR-6）那一組的核前區1896是單純G，兩者在統計上有差異（p=0.004）。另外從e抗原消失那一點到結束治療的病毒演變，持久反應（SR-6）那一組的兩時間點核前區1896都是單純A的有0位，和無持久反應（NSR-6）那一組的兩時間點都是單純A的有1位（6%），可能由於個數太少，統計上沒有意義。 結論：本研究結果顯示在自發性e抗原持久陰轉時，常伴隨著肝功能指數的上升和病毒量的下降。核前區1896和核心啟動子1762/1764雙變異的突變比例，會在此時段內產生波動變化，但並不是重要的決定因素。其中核前區1896突變株是基因型B比例高，而核心啟動子1762/1764雙變異的突變株則是基因型C比例高，和之前報導相似。在e抗原陰轉中，e抗原消失常合併e抗體的產生，應是在e抗原陰轉之前的病毒-宿主間的交互作用，產生了免疫反應，而非病毒本身變異造成e抗原持久陰轉的變化。在干安能使用下之e抗原陰轉過程中，病毒量的下降深度較自發性e抗原陰轉更為顯著（約4~5 log10比2~3 log10）。核前區1896野生種病毒隨時間而篩選成優勢種，甚至發生突變種病毒反轉為野生種病毒，這結果和自發性e抗原陰轉時會產生多數的突變種情形不同。而是否患者在e抗原陰轉後呈現持久穩定的突變種（sustained mutant strain），會不會有較高的e抗原持久陰轉率，由於個數太少，尚需大規模研究才能證實。關於核心啟動子1762/1764雙變異部份的研究，尚待未來使用更敏感的方法來克服。

Background：Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver disease worldwide, persistent hepatitis B virus infection is closely associated with the development of cirrhosis and hepatocellular carcinoma in Taiwan. Learning from nature history of hepatitis B infection, seroconvesion from HBeAg to anti-HBe usually indicates lower viral loads, resolved hepatitis activity and improved long-term outcome. Lamivudine is the first nucleoside analogue for the treatment of chronic hepatitis B, which has been shown to increase hepatitis B e antigen（HBeAg） seroconversion and reduce progression of hepatic fibrosis. Nevertheless, the relapse rate is high after cessation of lamivudine therapy unless HBeAg seroconversion occurs. Most of the published studies have shown that full HBeAg seroconversion response was durable in Western patients. However, it was not durable in Oriental countries, such as Korea and Taiwan. Hepatitis B e antigen (HBeAg) and an antibody（anti-HBe）are closely related to the level of hepatitis B virus replication and thus frequently used in assessing the activity of liver disease and monitoring the response to antiviral therapy.Therefore, it is important to identify factors associated with the development of HBeAg seroconversion in patients with HBeAg-positive chronic hepatitis B. The molecular basis of HBeAg is only partly clarified. From the viral replication’s point of view, when the precore region and core gene in HBV DNA are transcribed and translated, HBeAg is produced and secreted into the circulation. A point mutation from G to A at nucleotide (nt) 1896（A1896）converts codon 28 in the precore region from TGG for tryptophan to TAG for a stop codon and aborts the synthesis of HBeAg at the translation level. Likewise, a double mutation in the basal core promoter（BCP）changing nt 1762 and 1764 from A to T and G to A（T1762/A1764）,respectively, reduces the production of HBeAg by down-regulating the transcription of precore mRNAs. Convincing lines of evidence have indicated a close association of HBeAg seroconversion with the appearance of precore and BCP mutations as well as a decrease in the serum viral load. Another study revealed that precore 1896 wild vs. mutant strains ratio also played a role in the seroconversion of HBeAg and severity of disease activity. Liu et al reported that non-sustained HBeAg seroconversion might be due to the lack of sustained precore and BCP mutations after seroconversion. Lin et al reported that lamivudine therapy might result in the rapid selection of basal core promoter mutation of hepatitis B, but this mutation might revert to wild type gradually after cessation of therapy. The results of the present study have shown the genotype, age, and additional lamivudine therapy after HBeAg seroconversion are independent factors of sustained response to lamivudine therapy. However, the role of precore and BCP mutations in the development of sustained HBeAg seroconversion, either spontaneous or after lamivudine therapy remained unknown. Aim：Are the precore and/or BCP mutations of hepatitis B virus,either spontaneous or after lamivudine therapy associated with sustained HBeAg seroconversion？Hypothesis one is that the precore and/or BCP mutations of hepatitis B virus could determine the appearance of anti-HBe in the spontaneous HBeAg seroconversion. Hypothesis two is that the precore and/or BCP mutations of hepatitis B at the time of HBeAg loss or end of therapy in patient serum could determine the sustained e seroconversion after lamivudine treatment. Methods：We studied the factors in 25 patients with sustained HBeAg seroconversion as well as appearance of anti-HBe and 7 patients with sustained loss of HBeAg in the first part. We determined viral factors and status of precore and BCP mutations in the serum obtained 1 year before, 6 months before, 3 months before, at the time of , 6 months after and 1 year after HBeAg seroconversion or HBeAg loss. Secondly, both of host and viral factors as well as the drug factors were compared between 34 patients with sustained HBeAg seroconversion and 11 patients whose response was no sustained. All of patients with HBeAg positive and ALT level more than 5 times of normal upper limit without hepatitis C; hepatitis D and HIV were enrolled before therapy. All of them received a mean period 15 months（range, 6-35months）lamivudine therapy and had achieved complete responses（HBeAg seroconversion plus HBV DNA seroclearance by hybrid capture assay and normal alanine aminotransferase）and were followed-up for 6 months after end point of therapy. Stepwise logistic regression model was used to estimate the sustained response on the presence of the following variables: age; gender; genotype; pretherapy maximal ALT; the status of cirrhosis; time to HBeAg seroconversion; additional lamivudine treatment after HBeAg seroconversion; total duration of treatment; as well as HBV DNA level and status of precore mutation; basal core promoter mutation at the different time points which are of pretherapy, e antigen loss and end of therapy. The precore and basal core promoter regions were amplified and directly sequenced by polymerase chain reaction（PCR）-based assays. Besides, viral titer was checked by LightCycler real time PCR amplification machine. Results：We found that decline of serum viral load, frequently accompanied by hepatitis exacerbation, occurred since 1 year before HBeAg seroconversion. The proportions of precore and BCP mutations also increased gradually throughout the process of HBeAg seroconversion. The virologic features and proportions of precore and BCP mutations were similar between HBeAg seroconversion with anti-HBe group and HBeAg loss group. Before HBeAg seroconversion or loss, genotype B patients had higher serum viral loads（p=0.03~0.61）and lower proportions of BCP mutation（p=0.004~0.03）compared with genotype C patients. Secondly, no significant determinant was noted by step logistic regression analysis before lamivudine therapy between sustained and non-sustained HBeAg seroconversion groups. The mixed type and mutant type of precore region were replaced by wild type at the end of therapy and HBeAg seroconversion. The wild type（G1896）became dominant strain after lamivudine therapy, that was different from status of spontaneous HBeAg seroconversion.

Conclusion: our data showed that to clarify the role of the sustained precore mutant strain in the sustained HBeAg seroconversion, it was possible to increase case number for improving the statistic power in the future. Besides, there is little PCR product amplified from the BCP region due to lower viral load after lamivudine therapy. We need a more sensitive method to detect the BCP mutation in the future.