臺灣博碩士論文加值系統

1. 前言
週產期感染B型肝炎病毒的自然病程可分為三個階段：(1)免疫耐受期(immune tolerance stage)。通常可持續至青春期，此時病人血清中e抗原(HBeAg)陽性，B型肝炎病毒(HBV DNA)濃度很高，肝功能正常或輕微升高。(2)免疫廓清期(immune clearance stage)。約發生在15至35歲之間，此時期e抗原可為陽性或陰性，肝功能有多次惡化現象，肝組織呈活動性肝炎的變化。(3)殘存期(residual stage)或嵌入期(integrated stage)。此時e抗原呈陰性，而e抗體為陽性，此即為e抗原血清轉換(HBeAg seroconversion)。病毒複製能力大為降低，B型肝炎病毒濃度很低，肝組織較少發炎的變化，有些病人已進入肝硬化的階段。因此近30年來，診斷慢性B型肝炎(Chronic hepatitis B)時，e抗原被視為B型肝炎病毒持續複製的指標。
一般認為e抗原陰性的帶原者，多數其病毒是處於失去複製活性的情況，因此病人的肝功能正常，稱為無症狀帶原者(asymptomatic carrier)。因此，e抗原陰性的帶原者，若呈現肝功能異常，則必須考慮其他的原因，如﹕C型或D型肝炎病毒(Hepatitis D virus)附加感染、酒精性肝炎(alcoholic hepatitis)、自體免疫性肝炎(autoimmune hepatitis)等。
1980年初期，開始有學者提出B型肝炎病毒仍能於e抗原陰性的情況進行複製，並且導致肝功能異常。因此主張這類病人應歸類為「e抗原陰性慢性B型肝炎」(HBeAg negative chronic hepatitis B)。文獻報告指出，B型肝炎表面抗原陽性(HBsAg positive)的病人中，e抗原陰性慢性B型肝炎所佔的比例，各個地區並不相同，其中以地中海地區最高約33%，而亞洲地區則約15%。
1989年，Carman 等發現關於e抗原陰性慢性B型肝炎分子病毒學的變化。B型肝炎病毒基因位於核前區(precore region)核苷酸1896(Nucleotide1896)的位置發生突變，guanine 為adenine所取代，形成一個轉譯(translation)的停止訊號(stop codon)，導致病毒無法合成e抗原，但病毒仍持續複製。這種突變種病毒首先被認為侷限於地中海地區，後來陸續發現分佈於全球各地，且盛行率也不斷增加中。
除了核前區發生突變，導致病毒無法合成e抗原以外，另外一個區域basal core promoter (BCP)核苷酸1762及1764 (Nucleotide1762、1764)的位置發生雙突變(double mutation)，分別是核苷酸1762的位置adenine 被thymine取代，及核苷酸1764的位置guanine被adenine取代。經由雙突變的發生，調降preC mRNA的轉錄，進一步減少e抗原的合成。
因此，目前認為核前區1896突變和basal core promoter 1762/1764雙突變是造成慢性B型肝炎e抗原血清轉換後病毒仍持續複製的主因。這些病患不但肝臟發炎反應持續進行，甚至可能發生猛爆性肝炎或肝衰竭的情形。根據文獻報告，慢性肝炎患者不論是自然產生e抗原的血清轉換，或經由藥物治療(干擾素α， interferonα) 導致e抗原的血清轉換，皆可能出現核前區1896和basal core promoter 1762/1764變異種病毒。
核前區1896和basal core promoter 1762/1764變異種病毒的盛行率依血清e抗原的表現不同有所差異。義大利曾報告其e抗原陰性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率達80%。日本曾報告e抗原陽性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率約17%，而e抗原陰性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率則為37.5%。中國大陸的研究，e抗原陰性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率達52%，亦遠高於e抗原陽性者(16% )。香港地區e抗原陰性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率約48%。亞洲地區e抗原陰性的慢性B型肝炎患者，核前區1896變異種病毒的盛行率較地中海地區低的原因，可能是B型肝炎病毒基因型分佈不同所致。
關於核前區1896和basal core promoter 1762/1764變異種病毒的致病性(pathogenicity)還未有定論。核前區1896和basal core promoter 1762/1764變異種病毒最初被認為可能引起猛爆性肝炎(fulminant hepatitis)，但隨後有報告並不同意此論點。大部份研究顯示核前區1896和basal core promoter 1762/1764變異種病毒也存在於無症狀帶原者，而且大部份病患於e抗原血清轉換前即出現basal core promoter 1762/1764變異種病毒，之後再出現核前區1896變異種病毒，因此核前區1896和basal core promoter 1762/1764變異種病毒是否是e抗原血清轉換的預測因素(predictive factor)以及核前區1896和basal core promoter 1762/1764變異種病毒是否具致病性，或只是免疫篩選(immune selection)的結果，還需進一步研究。
台灣地區核前區1896和basal core promoter 1762/1764變異種病毒的盛行率，長庚醫院朱嘉明醫師曾報告，慢性B型肝炎急性發作時，核前區1896變異種病毒的比例為58% (含e抗原陰性和e抗原陽性)； 慢性B型肝炎，核前區1896變異種病毒的比例則為70%。而e抗原陰性的猛爆性B型肝炎，高達75%為核前區1896變異種病毒。台大高嘉宏教授針對e抗原陽性慢性肝炎病患的研究，發現basal core promoter 1762/1764變異種病毒的盛行率約26%。
至於不同的B型肝炎病毒基因型其臨床表現是否不同，並無完整的報告。目前已確定的B型肝炎病毒基因型共有A、B、C、D、E、F和G七種。亞洲地區主要是基因型B和C，西方國家主要是基因型A和D，基因型F分佈於中美洲，基因型G分佈於法國和美國，而基因型E只出現於非洲。目前為止，有關核前區1896和basal core promoter 1762/1764變異種病毒與B型肝炎病毒基因型的相關性，還不十分清楚。肯定的是基因型A型幾乎不發生核前區1896突變，除非伴隨核苷酸1858 突變。台大高嘉宏教授的研究顯示基因型C型的慢性B型肝炎患者其臨床表現較基因型B型的患者嚴重。基因型C型病毒出現basal core promoter 1762/1764變異種病毒的比例較高，且對干擾素治療的反應較差。香港的研究也顯示基因型C型的e抗原陰性慢性B型肝炎患者，其肝生檢呈現較嚴重的發炎反應。但B型肝炎病毒基因型於無症狀帶原者、慢性肝炎、肝硬化及肝癌的分佈有無差異，仍待進一步研究。
2. 研究目的
本研究計畫分析台灣地區不同程度的B型肝炎病毒相關性肝疾病，其核前區1896和basal core promoter 1762/1764變異種病毒的盛行率及B型肝炎病毒基因型的分佈，以推測核前區1896和basal core promoter 1762/1764變異種病毒及B型肝炎病毒基因型是否影響慢性B型肝炎的自然病程。
另外也將分析e抗原陰性慢性B型肝炎的病患，其血清B型肝炎病毒的濃度高低，以及與核前區1896和basal core promoter 1762/1764變異種病毒、B型肝炎病毒基因型的相關性。
3.研究方法
收集仁愛醫院e抗原陰性之慢性B型肝炎病患(C型肝炎病毒抗體陰性且D型肝炎病毒抗體陰性)的血清，174例，分為以下兩組：
(1).對照組：無症狀帶原者(asymptomatic carrier)62例。
(2).實驗組：e抗原陰性之慢性B型肝炎病毒相關性肝疾病病患112例，其
中含慢性肝炎(chronic hepatitis)49例、肝硬化(liver cirrhosis)31例、肝細胞癌( hepatocellular carcinoma)32例。
將血清萃取之HBV DNA以巢式聚合酶連鎖反應(nested polymerase chain reaction)放大核前區及basal core promoter的區域。然後在將巢式聚合酶連鎖反應產物以自動核酸定序儀(automatic sequencer)分析其核酸序列。
B型肝炎病毒基因型則以聚合酶連鎖反應併限制酶片段長度多形性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)的方法分析。
取慢性肝炎病人的血清，以商用試劑Roche Cobas Amplicor HBV Monitor test 來測定血清B型肝炎病毒的濃度。
分析病毒濃度與核前區1896和basal core promoter 1762/1764變異種病毒及B型肝炎病毒基因型的相關性。分析不同階段B型肝炎病毒相關性肝疾病，核前區1896和basal core promoter 1762/1764變異種病毒之盛行率，B型肝炎病毒基因型的分佈及其臨床相關性。
資料的統計分析，將使用Chi-square test、Fisher''s exact test、Student’s t test、 logistic regression 及Pearson correlation test。所有統計均為雙尾檢定(two-tailed test)，當P值小於0.05為有意義的差別。
4. 結果
174例中，平均年齡47±14歲；男性127例、女性47例；血清ALT平均值127±245 U/L。B型肝炎病毒基因型的分佈，分別為基因型A型1例(0.6%)、基因型B型111例(64%)、基因型C型44例(25%)型、基因型D型4例(2.3%)、基因型F型6例(3.4%)，有8例(4.6%)無法判讀，並沒有發現基因型E型的病毒。核前區1896變異種病毒的盛行率為79%，basal core promoter 1762/1764變異種病毒的盛行率為67%。
分析對照組和實驗組發現，對照組62例病患，平均年齡40±11歲；男性37例、女性25例；血清ALT平均值23±8 U/L。B型肝炎病毒基因型B型佔50例(81%)，基因型C型只佔5例(8%)。核前區1896變異種病毒的盛行率為79%，basal core promoter 1762/1764變異種病毒的盛行率為50%。而實驗組慢性B型肝炎病毒相關性肝疾病病患，平均年齡51±14歲；男性90例、女性22例；血清ALT平均值185±291 U/L。B型肝炎病毒基因型B型佔61例(54%)，基因型C型佔39例(35%)。核前區1896變異種病毒的盛行率為79%，basal core promoter 1762/1764變異種病毒的盛行率為72%。
慢性B型肝炎病毒相關性肝疾病病患在平均年齡、男性比例、血清ALT平均值、病毒基因型C型比例和basal core promoter 1762/1764變異種病毒的盛行率明顯高於無症狀帶原者，其P值分別為 <0.001、0.004、<0.001、0.001和0.014。
進一步分析慢性B型肝炎病毒相關性肝疾病的次族群發現肝硬化、肝癌的病患病毒基因型B和C型的比例與慢性肝炎並無差異。在核前區1896和basal core promoter 1762/1764變異種病毒方面，慢性肝炎、肝硬化和肝癌核前區1896 突變的盛行率並無差異。但basal core promoter 1762/1764變異種病毒的盛行率，則隨疾病進展而增加，慢性肝炎、肝硬化、肝癌分別為55%、83%和88% (P=0.002)。
將B型肝炎病毒基因型B和C型分層分析發現，兩者在性別比例和血清ALT平均值並無差異。但基因型C型病患年齡較基因型B型高(分別為52歲和46歲，P=0.012)；基因型C型病患B型肝炎病毒相關性肝疾病的比例，明顯高於基因型 B型(89% vs. 55%，P=0.0001)；而且基因型C型病患basal core promoter 1762/1764變異種病毒的比例也高於基因型B型(79% vs. 59%，P=0.026)。
我們用logistic regression分析不同階段B型肝炎相關性肝疾病，發現慢性肝炎、肝硬化和肝癌，basal core promoter 1762/1764變異種病毒出現的概似比(likelihood)，均明顯高於無症狀帶原者，其odds ratio分別為慢性肝炎1.13、肝硬化4.80、肝癌7.33，odds ratio 的趨勢達統計學上有意義的差異(P=0.0001)。另外，多變項 logistic regression分析也發現，經調整性別與年齡的因素之後，基因型C型病毒basal core promoter 1762/1764變異種病毒出現的比例為79%，而基因型B型為59%，odds ratio 為2.53(P=0.04)。
進一步分析慢性B型肝炎病毒相關性肝疾病的危險因子，發現男性、年齡大於50歲以及病毒基因型C型，是造成B型肝炎病毒相關性肝疾病進展的危險因子。其odds ratio分別是男性為女性的3.81倍(P=0.003)，年齡大於50歲為小於、等於50歲的3.64倍(P=0.005)，病毒基因型C型為B型的6.56倍(P小於0.001)。由於肝硬化和肝癌是造成B型肝炎病毒慢性感染死亡的主要原因，我們特別分析形成肝硬化和肝癌的危險因子，發現年齡大於50歲及basal core promoter 1762/1764變異種病毒是形成肝硬化和肝癌的危險因子，其odds ratio分別是年齡大於50歲為小於50歲的9.71倍(P小於0.001)，basal core promoter 1762/1764變異種病毒為野生型病毒的4.24倍(P=0.003)。
進一步將病患依年齡50歲分層，63位肝硬化及肝癌的病患，basal core promoter 1762/1764變異種病毒的比例， 50歲以下(含50歲)為88%，與50歲以上的病患相當(84%)。而且50歲以下肝硬化及肝癌的病患，basal core promoter 1762/1764變異種病毒的比例，也明顯較同年齡層的無症狀帶原者高(分別為88%和45%，P=0.002)。
本研究中127位男性中90位(71%)為慢性B型肝炎病毒相關性肝疾病，而47位女性中只有22位(47%)為慢性B型肝炎病毒相關性肝疾病，男性明顯高於女性(P=0.004)。多變項 logistic regression的分析發現男性與女性形成肝硬化和肝癌的危險因子也有所不同。在男性方面，年齡大於50歲及basal core promoter 1762/1764變異種病毒是形成肝硬化和肝癌的危險因子，其odds ratio分別是年齡大於50歲為小於、等於50歲的4.69倍(P小於0.006)，basal core promoter 1762/1764變異種病毒為野生型病毒的4.34倍(P=0.02)。至於女性則只有年齡大於50歲罹患肝硬化和肝癌危險性較高，其odds ratio為小於、等於50歲的81.96倍(P小於0.006)。
另外有46位HBeAg陰性之慢性肝炎病患，其中男性40位、女性6位，平均年齡42±11歲，ALT平均值328±391 U/L，HBV DNA平均值為1.1´108 copies/ml (間距4.9´103-1.2´109 copies/ml)，其中44位病患之HBV DNA濃度均大於105 copies/ml。有10位病患其血清ALT大於400 U/L，臨床上正處於急性發作(acute exacerbation)的階段，其HBV DNA平均值為3.32´108±1.23´108 copies/ml。而其他慢性肝炎的病患，其HBV DNA平均值為5.33´107±1.44´107 copies/ml。急性發作者血清HBV DNA的濃度明顯高於慢性肝炎的病患(P=0.0002)。進一步利用Pearson correlation分析，發現HBV DNA與血清ALT呈顯著相關性(significant correlation)，相關係數(r)為0.333，P=0.024。若以10pg/ml（相當於2.86´106 copies/ml）為HBV DNA濃度高低之分界，則有34位病患（74%）HBV DNA濃度大10pg/ml。分析血清中HBV DNA的濃度與B型肝炎病毒基因型的相關性，發現兩者並無相關性。但當核前區1896突變或basal core promoter 1762/1764雙突變單獨發生時，HBV DNA的濃度大於10pg/ml的odds ratio較高，雖然其趨勢並未達到統計學上的意義(P=0.32)。
5.討論
本研究發現，台灣地區e抗原陰性B型肝炎病毒慢性感染者，核前區1896變異種病毒的盛行率為79%，遠比美國、北歐的12-27%高出許多。這種地區分佈的差異，目前推論是因為各地區分佈的B型肝炎病毒基因型不同所致。由於主要分佈於美國、北歐的基因型A型病毒，並不常出現核前區1896的突變。本研究發現，台灣地區e抗原陰性B型肝炎病毒慢性感染者，病毒基因型的分佈，基因型A型相當少見，只佔0.6%，而基因型B、C和D型的比例達91%，因此，台灣地區核前區1896突變的盛行率達79%，也就不足為奇了。
台灣地區e抗原陰性B型肝炎病毒慢性感染者，basal core promoter 1762/1764變異種病毒的盛行率為67%，略低於核前區1896變異種病毒的盛行率。由日本、香港及中國大陸的報告指出，e抗原陽性的無症狀帶原者，basal core promoter1762/1764雙突變的盛行率約為5-38%，較核前區1896突變的盛行率高。而e抗原陽性慢性肝炎、e抗原陰性無症狀帶原者和e抗原陰性慢性肝炎，basal core promoter 1762/1764變異種病毒的盛行率則依序增加。由於basal core promoter 1762/1764變異種病毒只減少約70% e抗原的合成，而核前區1896變異種病毒則完全無法製造e抗原，因此推論B型肝炎病毒慢性感染的自然病程中，在免疫容忍期(immune tolerance stage)的階段，即可能發生basal core promoter1 762/1764雙突變，而減少e抗原的產生，再隨著核前區1896突變的出現，使病毒完全無法製造e抗原。
無症狀帶原者、慢性肝炎、肝硬化及肝癌之間，核前區1896變異種病毒的盛行率並無差異。但是basal core promoter 1762/1764變異種病毒的盛行率卻明顯有所不同，分別為50%、55%、83%和88%。這意味basal core promoter 1762/1764雙突變較核前區1896突變，更具致病性。我們的研究顯示，basal core promoter 1762/1764變異種病毒出現的概似比(likelihood)，隨無症狀帶原者、慢性肝炎、肝硬化、肝癌的進展，逐次增加，肝癌出現basal core promoter變異種病毒的odds ratio達健康帶原者的7.33倍。因此，我們推論，basal core promoter變異種病毒的確增強B型肝炎病毒的致病性，而能作為B型肝炎病毒慢性感染的自然病程中，疾病進展的有效指標。
Basal core promoter(核苷酸1742-1849)是位於X基因(核苷酸1685-1849)內的調控序列。文獻上報告，不論由肝癌患者的血清或肝組織，所分離出的B型肝炎病毒，都有basal core promoter 1762/1764雙突變發生。日本B型肝炎病毒基因型C型感染的肝癌患者，血清B型肝炎病毒出現basal core promoter 1762/1764雙突變的比例達90%。南非的肝癌患者，basal core promoter 1762/1764變異種病毒的盛行率為66%，遠高於無症狀帶原者11%。台灣也有相同的報告，台大高嘉宏教授針對250名B型肝炎病毒慢性感染者的世代研究，發現帶有basal core promoter 1762/1764變異種病毒者，發生肝癌的危險性是沒有突變者的10.6倍。以上報告顯示，basal core promoter 1762/1764變異種病毒，是發生肝癌的危險因子。然而其致癌機轉為何，仍不清楚。Basal core promoter 1762/1764變異種病毒不但影響precore mRNA的轉錄，也影響X蛋白的編碼，造成X蛋白胺基酸序列中兩個胺基酸的改變。這種變化是否改變B型肝炎病毒的生物特性，導致病毒具直接肝細胞致癌性(hepatocarcinogenesis)，需進一步動物實驗來證明。
雖然不能肯定basal core promoter 1762/1764變異種病毒具直接肝細胞致癌性，但basal core promoter 1762/1764變異種病毒卻能透過促使肝疾病惡化的方式，增加肝癌的危險性。
由多變項logistic regression 分析，我們發現basal core promoter 1762/1764變異種病毒是肝疾病進展至肝硬化和肝癌的危險因子，其odds ratio為野生型病毒的4.24倍。因此，可推論basal core promoter 1762/1764變異種病毒導致肝癌的致癌機轉，仍然是循「肝組織發炎壞死→肝硬化→肝癌」的模式進行。
流行病學研究顯示男性罹患肝癌的比例較女性高，本研究發現慢性B型肝炎病毒感染者中，男性進展至慢性肝疾病的危險性較女性高(P=0.003)。若特別針對肝硬化和肝癌的病人分析，男女並無差異，這與以往文獻報告不同，由於本研究的病患均來自同一醫院，可能有取樣的偏差。經由多變項logistic regression分析男性與女性進展至肝硬化及肝癌的危險因子發現，不論男性和女性，年齡都是導致疾病進展至肝硬化和肝癌的危險因子，隨著年齡增長，肝組織長期慢性發炎壞死的反應，以致肝組織纖維化的程度日益嚴重，發生細胞轉化(cell transformation)的機會當然較高。令人意外的是男性感染basal core promoter 1762/1764變異種病毒者，進展至肝硬化和肝癌的危險性明顯高於感染野生型病毒者(P=0.02)。但basal core promoter 1762/1764變異種病毒卻不會增加女性進展至肝硬化和肝癌的危險性。目前的研究顯示X蛋白在病毒與宿主之間的交互作用中扮演多功能的調節者(multifunctional regulator)的角色，basal core promoter1762/1764變異種病毒造成X蛋白胺基酸序列中兩個胺基酸的改變，此變異的X蛋白對肝細胞癌化(carcinogenesis)的影響，在男性與女性可能有所不同。因此，未來需進行世代研究，觀察basal core promoter 1762/1764變異種病毒及其造成變異的X蛋白功能上的變化，對男性與女性的影響是否不同。另外，基因轉殖鼠的研究顯示，X蛋白是否直接導致肝細胞癌化仍無定論。人類肝細胞模式(human primary cell model)的建立，將有助於研究X蛋白是否能促使細胞自然衰老(senescence)的機制喪失，導致細胞不死(immortalization)的機會提高。
目前已確立的B型肝炎病毒基因型共有A、B、C、D、E、F和G七種。關於病毒基因型之臨床相關性，初期只知道基因型的分佈有地區性，基因型A和D盛行於西方國家及印度，基因型B和C主要分部於亞洲地區，基因型E目前只侷限於非洲，基因型F則出現於中南美洲，基因型G是最近發現分佈於法國及北美地區。在本研究中，基因型的分佈主要以基因型B型(64%)和C型(25%)為主。亞洲地區的B型肝炎病毒基因型的分佈，雖然亦以基因型B型和C型為主，但日本、香港均以基因型C型為主，台灣則以基因型B型為主。
分析無症狀帶原者和慢性B型肝炎病毒相關性肝疾病病患，發現慢性B型肝炎病毒相關性肝疾病病患，基因型C型的比例明顯高於無症狀帶原者。再將病毒基因型B和C型分層分析發現，兩者在性別比例和血清ALT平均值並無差異。但基因型C型病患年齡較基因型B型高(分別為52歲和46歲，P=0.012)。香港的研究發現，B型肝炎病毒慢性感染的自然病程中，基因型B型比基因型C型提早出現e抗原的血清轉換達10年之久。在我們的研究中無症狀帶原者，基因型B型佔81%，而且平均年齡較慢性B型肝炎病毒相關性肝疾病病患年輕10歲。基因型C型病患B型肝炎病毒相關性肝疾病的比例，也明顯高於基因型B型(89% vs.55%，P=0.0001)。我們的發現與香港的研究相符。我們推論可能與e抗原血清轉換的時間有關，基因型C型的病患，由於e抗原血清轉換的時間較晚，持續發炎的時間較久，因此肝組織的傷害較嚴重。
為了釐清基因型B型與C型，在慢性肝疾病的進展上所扮演的角色。我們用多變項 logistic regression分析造成B型肝炎病毒相關性肝疾病的危險因子，發現男性、年齡大於50歲以及病毒基因型C型，是造成B型肝炎病毒相關性肝疾病進展的危險因子。如果特別著重在造成肝硬化及肝癌的危險因子，則基因型C型的odds ratio為B型的1.69倍(P=0.13)，兩者之間並無差異。因此我們認為基因型B型與C型對e抗原陰性慢性B型肝炎病毒相關性肝疾病的影響，可分為兩階段。在無症狀帶原、慢性肝炎的階段，由於基因型C型病毒e抗原血清轉換較晚，持續發炎的時間較久，因此肝組織的傷害較嚴重。但若肝臟發炎持續，一但進入肝硬化、肝癌的階段，基因型B型和C型病毒的預後就沒有什麼不同了。
要了解不同基因型的致病性之所以不同，必須由病毒本身著手。由多變項logistic regression 分析，我們發現basal core promoter 1762/1764變異種病毒是肝疾病進展至肝硬化和肝癌的危險因子。進一步分析發現，基因型C型病患basal core promoter 1762/1764變異種病毒的比例高於基因型B型(79% vs.59%，P=0.026)。顯示basal core promoter 1762/1764變異種病毒，可能是造成基因型B型和C型致病性不同的原因之一。
本研究中46位慢性肝炎的病患，有10位其血清ALT大於400 U/L，臨床上正處於急性發作，其HBV DNA的濃度明顯高於其他慢性肝炎者。顯示HBV DNA濃度確實是造成肝臟發炎加劇的原因之一。我們進一步將HBV DNA與ALT做線性相關的分析，兩者有顯著相關性。這個結果也印證了e抗原陰性慢性B型肝炎的致病機轉，與血清病毒濃度有關。
雖然在慢性肝炎的階段，血清中HBV DNA的濃度普遍較高，但這些病患HBV DNA的濃度卻有很大的差異(4.9´103-1.2´109 copies/ml)。我們發現基因型B型和C型的HBV DNA濃度並無明顯差異。但當核前區1896突變或basal core promoter 1762/1764雙突變單獨發生時，HBV DNA的濃度大於10pg/ml的odds ratio較高，但其趨勢並未達到統計學上的意義(P=0.32)。
核前區1896突變或basal core promoter 1762/1764雙突變，對病毒複製能力的影響，已經體外實驗證實。核前區1896的突變似乎是無害的旁觀者(innocent bystander)，而basal core promoter 1762/1764變異種病毒確能增強病毒的複製能力，目前尚不能排除基因體其他位置發生突變，對HBV DNA的濃度產生影響。但是因病毒基因體突變而增強病毒複製能力的影響，通常小於20倍，臨床上HBV DNA的濃度卻有很大的差異，不能完全以基因序列的突變來解釋。人體對病毒的免疫反應，以及外在因素，如其他抗病毒藥物或免疫抑制劑的使用，也是影響病毒複製的主要因素。
6. 結論
根據本研究的發現，對於B型肝炎病毒慢性感染的病患，測定病毒基因型以及核前區1896、 basal core promoter 1762/1764是否突變是有必要的。尤其台灣地區病毒基因型C型約佔40%，basal core promoter 1762/1764雙突變的盛行率高達67%，肝疾病惡化的可能性相對較高，必須積極治療或密切追蹤其病情變化，以期能減少肝硬化和肝癌的發生。而在介入治療之時，先了解病毒基因型以及核前區1896、 basal core promoter 1762/1764是否突變、血清HBV DNA的濃度，謹慎選擇可能比較有效的藥物及治療的療程，才能提高治療的效果，進一步阻斷肝疾病的進展。但由於本研究方法為橫斷式研究(cross sectional analysis)，無法得知basal core promoter 1762/1764雙突變的發生是在疾病進展之前或之後，因此未來必須進行大型的世代研究(cohort study)，以了解不同的病毒基因型在演化過程中發生basal core promoter 1762/1764雙突變的時間點及機率，以及basal core promoter 1762/1764雙突變的發生對疾病進展的影響。

Introduction
The hepatitis B virus (HBV), discovered in 1965, infects more than 350 million people worldwide. HBV is the major cause of acute and chronic liver diseases, and persistent HBV infection is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC), accounting for 1 million deaths annually.
The natural history of chronic HBV infection could be divided into three sequential stages: (1) the immune tolerance stage, characterized by HBeAg reactivity in serum and only minor histological activity, active viral replication continues despite little or no elevation in the aminotransferase level and no symptoms of illness; (2) the immune clearance stage, during which serum is positive for HBeAg or anti-HBe, but serum HBV DNA levels drop as the number of infected cells declines and histological signs of chronic active hepatitis are prominent, the stage may persist for 10 or more years, leading to cirrhosis; and (3)the integrated stage, when the patient is anti-HBe positive and a marked decrease in viral DNA is observed, amintransferase levels become normal. However, patients remain positive for HBsAg, presumably because of the integration of the S gene into host’s hepatocyte genome.
Hepatitis B e antigen (HBeAg) is a reliable and sensitive marker of hepatitis B virus replication. Individuals negative for HBeAg were considered to have nonreplicative HBV infection, and their serum aminotransferase levels were normal or nearly normal.
In the early 1980s it became apparent that HBV could replicate in the absence of HBeAg. It is known that precore stop codon mutation (G1896A) that abolishes the synthesis of hepatitis B e antigen (HBeAg) plays a major role in patients with HBeAg-negative chronic hepatitis B. In addition, the double mutations in the basal core promoter (A1762T and G1764A) have been demonstrated to reduce the synthesis of HBeAg by suppressing the transcription of precore mRNA. Both mutations in the precore and basal core promoter regions have been reported to be associated with the occurrence of fulminant hepatitis, and are commonly observed in chronic hepatitis B. However, the clinical significance of precore and basal core promoter mutations remains controversial and needs to be established.
Although serological and genotypic classifications of HBV have been well recognized, the clinical significance of HBV genotypes in terms of clinical outcomes and therapeutic response to antiviral therapy in patients with chronic HBV infection remains largely unknown until recently. Previous studies have indicated that, genotype B and C were the predominant HBV strains in Taiwan and Japan, and genotype C is associated with more severe liver disease. In addition, HBV genotype C is associated with a higher frequency of basal core promoter mutation and a lower response rate to interferon alfa therapy as compared to genotype B in patients with HBeAg-positive chronic hepatitis B. However, whether such an association also holds true in those with HBeAg-negative chronic hepatitis B remains largely unknown. We thus investigated the association of HBV genotypes as well as precore/basal core promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative HBV related liver disease in Taiwan.
Materials and Methods
Patients. A total of 174 patients with chronic HBV infection who were regularly followed at the Taipei municipal Jen-Ai hospital were consecutively enrolled. They included 62 asymptomatic carriers with persistently normal serum alanine aminotransferase (ALT) levels at least for 2 years in periodic biochemical examination and 112 HBV related liver disease. Among them 49 had chronic hepatitis, 31 had cirrhosis and 32 had HCC. All of them were HBsAg-positive, HBeAg-negative and anti-HBe positive. They were negative for antibodies to hepatitis C virus (HCV), hepatitis D virus (HDV) or human immunodeficiency virus (HIV), and none had received antiviral treatment during the follow-up period. Serum samples were collected and stored at -70°C until use.
Serological testing. The biochemical tests were measured by using routine automated methods. The HBsAg, HBeAg, antibodies to HCV and HDV were assayed by commercially available kits (General Biological HBsAg RIA and HBeAg/Anti-HBe RIA, General Biological Cooperation, Taiwan. HCV EIA II and Anti-Delta. Abbot Laboratories, North Chicago, IL, USA).
Genotyping of HBV. The identification of HBV genotypes was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the surface gene of HBV as previously described. Briefly, DNA was extracted from 200 μL of serum samples by using the QIAamp blood kit (Qiagen, Chatsworth, CA, USA), and the fragment of the HBV genome between nucleotide positions 256 and 796 was then amplified. The PCR products were subsequently treated with restriction enzymes. After incubation, the samples were run on a 3% agarose gel and stained by ethidium bromide. Six genotypes (A-F) of HBV could be identified by the restriction patterns of DNA fragments. Unclassified genotype was defined as an unpredicted or atypical restriction pattern. To avoid false-positive results, instructions to prevent cross contaminations were strictly followed, and results were considered valid only when they were obtained in duplicate.
Amplification and sequencing of precore/basal core promoter gene. For the first stage PCR, 25μl of reaction mixture containing 2μl of the DNA sample, 1X PCR buffer (10 mM tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin and 0.1% triton X-100), 10mM of each dNTP, 100 ng of each outer primer and 1unit of Taq DNA polymerase was amplified in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) for 35 cycles. Each cycle entailed denaturation at 95°C for 60s, primer annealing at 52°C for 30s and extension at 72°C for 60s with final extension step at 72°C for 10 min. After the first amplification, 1μl of the PCR products was reamplified for another 35 cycles with 100 ng of each inner primer. For the precore region, the outer sense primer was 5''-CTGGGAGGAGTTGGGGGA-3'', nucleotide positions 1730-1747; the outer antisense primer was 5''-CAATGCTCAGGAGACTCTAAGGC -3'', nucleotide positions 2043-2021; the inner sense primer was 5''-GGTCTTTGTACTCGGAGGCTG -3'', nucleotide positions 1763-1783; the inner antisense primer was 5''-GTCAGAAGGCAAAAAAGAGAG-3'', nucleotide positions 1966-1946. For the basal core promoter region, the outer sense primer was 5''-CTAGCCGCTTGTTTTGCTCG-3'', nucleotide positions 1282-1301; the outer antisense primer was 5''-CACAGCTTGGAGGCTTGAAC-3'', nucleotide positions 1881-1862; the inner sense primer was 5''-CTCATCTGCCGGACCGTGTG-3'', nucleotide positions 1562-1581; the inner antisense primer was 5''-TAGGACATGAACAAGAGATG-3'', nucleotide positions 1859-1840. Nucleotide sequences of the amplified products were directly determined by using fluorescence labeled primers with a 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing conditions were specified in the protocol for Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The inner primers were used as sequencing primers for both directions of each region.
Quantitation of serum HBV DNA level. Serum HBV DNA level was quantitated by a polymerase chain reaction assay with a lower limit of detection of 200 copies/ml (Cobas Amplicor HBV Monitor, Roche Diagnostic Systems Inc.)
Statistical analysis. Data were analyzed by chi-square test, Fisher''s exact test, logistic regression, Student’s t test or Pearson correlation where appropriate. All of the tests of significance were two-tailed and a p value of less than 0.05 was considered statistically significant.
Results
Patient Characteristics. The baseline clinical and virological characteristics of the 174 HBeAg negative patients with chronic HBV infection are shown in Table 2. There was a male predominance and the mean age of the patients was 47 years (range, 17 to 81 years). The genotype distribution in the 174 patients as follows: A, 1(0.6%); B, 111(63.8%); C, 44(25.3%); D, 4(2.3%); E, 0; F, 6(3.4%) and unclassified, 8(4.6%). The overall prevalence of precore stop codon mutant and basal core promoter 1762/1764 mutants was 79% and 64%, respectively.
Comparison Between Asymptomatic Carriers and HBV Related Liver Disease. The male-to-female ratio and mean age were significantly higher in HBV related liver disease patients (P=0.004 and P<0.001). Genotype B was more prevalent in asymptomatic carriers (50 in 62, 81%), and genotype C significantly increased in patients with HBV related liver disease (39 in 112, 35%) (P=0.001). The prevalence of precore stop codon mutant was comparable between asymptomatic carriers and HBV related liver disease (79% vs. 79%). Patients with HBV related liver disease had significantly higher prevalence of basal core promoter 1762/1764 mutants than asymptomatic carriers (72% vs. 50%, P=0.014).
Comparison Between Genotype B and C Patients. The baseline clinical features in patients with genotype B and C infection were comparable in terms of mean age, sex ratio, mean serum ALT level, and prevalence of precore stop codon mutant (Table 3). Patients with genotype C infection had a significantly higher mean age (52 years vs. 46 years, P=0.012), prevalence of basal core promoter 1762/1764 mutants (79% vs.59%, P=0.002) and ratio of HBV related liver disease (89% vs.55%, P=0.0001).
Factor Associated With HBV Related Liver Disease. The prevalence of basal core promoter 1762/1764 mutants in different clinical stage of chronic HBV infection was shown in Table 4. The frequency of basal core promoter 1762/1764 mutants increased with advanced clinical stages, from 50% in asymptomatic carriers to 88% in HCC patients. To determine whether the likelihood of basal core promoter 1762/1764 mutants differed by different clinical stages of liver disease, logistic regression analysis was used. Liver cirrhosis and HCC had a significantly greater likelihood of basal core promoter 1762/1764 mutants than asymptomatic carriers (odds ratio, 4.8; 95% confidence interval [CI] 1.83-12.58 for liver cirrhosis and odds ratio, 7.33; 95% confidence interval [CI] 2.19-24.5 for HCC, score test for the trend of odds P=0.0001).
We further analyzed the risk factor associated with HBV related liver disease. The risk factors including sex, age, precore stop mutant, basal core promoter 1762/1764 mutant and genotype for HBV related liver disease in chronic HBV infection were determined by multiple logistic regression analysis (Table 5). The independent factors associated with the progression of HBV related liver disease included male (odds ratio, 3.81; 95% CI, 1.58-9.12, P=0.003), old age (>50 years) (odds ratio, 3.64; 95% CI, 1.48-8.93, P=0.005) and genotype C infection (odds ratio, 6.56; 95% CI, 2.41-17.86, P<0.001).
Liver cirrhosis and HCC are the leading causes of death for chronic HBV infection, thus we emphasized the risk factors associated with the development of liver cirrhosis and HCC. Individuals with old age (>50 years) (odds ratio, 9.71; 95% CI, 4.22-22.22, P<0.001) and basal core promoter 1762/1764 mutants (odds ratio, 4.24; 95% CI, 1.63-11.04, P=0.003) were significantly associated with the development of liver cirrhosis and HCC (Table 6). When stratified by age, the prevalence of basal core promoter 1762/1764 mutants was comparable between younger and older liver cirrhosis and HCC patients (88% vs. 84%). Nevertheless, the difference was significant between younger liver cirrhosis and HCC patients and age-matched asymptomatic carriers (88% vs. 45%, P=0.002) (Figure 3).
Comparison Between male and female patients. The baseline clinical features between male and female patients were comparable in terms of mean age, mean serum ALT level, the prevalence of precore stop codon/basal core promoter 1762/1764 mutant and the prevalence of genotype B and C. Male patients had a significantly higher ratio of HBV related liver disease (71% vs.47%, P=0.004) than female patients (Table 7).
We found the different risk factors associated with the development of liver cirrhosis and HCC in male and female. Old age (>50 years) was significantly associated with the development of liver cirrhosis and HCC in both male and female patients (odds ratio, 4.69; 95% CI, 1.55-14.28, P=0.006 and odds ratio, 81.96; 95% CI, 5.58-1203, P<0.001, respectively). But basal core promoter 1762/1764 mutants (odds ratio, 4.34; 95% CI, 1.30-14.52, P=0.02) was significantly associated with the development of liver cirrhosis and HCC only in male patients (Table 8).
The Serum HBV DNA Levels of Chronic Hepatitis. Of the 46 patients with HBeAg-negative chronic hepatitis (Table 9), all of them had detectable serum HBV DNA (mean, 1.1×108 copies/ml; range, 4.9×103-1.2×109 copies/ml). Ten of the 46 patients had acute exacerbation, the mean serum HBV DNA levels was significantly higher than the patients without acute exacerbation (3.32×108 copies/ml vs. 5.33×107 copies/ml, P=0.0002). The serum HBV DNA level was significant correlation with serum ALT level by the Pearson correlation test (r = 0.333, P=0.024). We further analyzed the association of serum HBV DNA with HBV genotype and precore/basal core promoter mutants. There was no difference between patients with genotype B and genotype C infection (Table 10). In patients with both precore and basal core promoter 1762/1764 mutations, there was a lower risk of high detectable serum HBV DNA level (>10 pg/ml) than in patients with either precore stop codon mutation or the basal core promoter mutation alone, although the difference was not significant (P=0.32)(Table 11).
Discussion
HBV mutant strains selected during HBeAg seroconversion, which allow the persistence of viral replication after loss of HBeAg have been reported. Among these mutants, precore stop codon mutation (G1896A) that abolishes the synthesis of HBeAg has been extensively studied and may play a role in HBeAg-negative chronic hepatitis B. In addition, double mutations in the basal core promoter (A1762T and G1764A) are also demonstrated to reduce the synthesis of HBeAg by suppressing the transcription of precore mRNA in both HBeAg —positive and —negative patients.
It is known that the prevalence of the precore stop codon mutation differs with geographic location and is genotype-dependent, that is less frequent in genotype A (which prevails in North America and Europe). The prevalence of such a mutation in HBeAg-negative chronic hepatitis was 12%-27% in the United States and Northern Europe, but up to 90% in the Mediterranean region and in some Asian regions. In the present study, our result consistently indicated that HBV genotype A was rare in Taiwan, and we found that the prevalence of the precore stop codon mutation in Taiwanese patients with HBeAg-negative chronic HBV infection was 79%. In addition, we firstly found that the prevalence of double mutations in the basal core promoter was 67% in patients with HBeAg-negative patients in Taiwan, and this may contribute to the hepatitis activity in them. However, the influence of precore stop codon and basal core promoter mutations in the virulence of HBV infection as well as the pathogenesis of liver cell damage remains controversial.
In this study, there was no different prevalence of precore stop codon mutation between the different clinical stages of HBeAg-negative chronic HBV infection. Thus the precore stop codon mutation alone appears to have no direct pathogenic role in chronic HBV infection. In contrast, the frequency as well as likelihood of basal core promoter 1762/1764 mutants increased with advanced clinical stages. It seems that the mutant is more pathogenic than wild-type virus and may serve as a marker for progression of liver disease.
The region of basal core promoter (nucleotides 1742 to 1849), overlaps with the X gene (nucleotide 1685-1849), controls the transcription of both precore messenger RNA and pregenomic RNA. The region also encodes the carboxyl-terminus of the X protein, which has transactivating function. Mutation in this region could influence HBeAg production and viral replication as well as the amino acid sequence of the X protein. The diminishes production of HBeAg and increases viral replication result in increased host immune response and enhanced liver cell damage. In present study, the basal core promoter 1762/1764 mutants was significantly associated with the development of liver cirrhosis and HCC (odds ratio, 4.24, P=0.003). The data indicated an association of basal core promoter 1762/1764 mutant with progression of chronic hepatitis, and may be caused liver cirrhosis and malignant transformation of liver cell after a long period of chronic hepatitis. In addition, the alternation of the amino acid sequences of the X protein may play a role in hepatocarcinogenesis. The effect of basal core promoter 1762/1764 mutant was remarkable in male. Reasons for the difference between male and female patients remain unknown. Many lines of evidence have demonstrated the modulatory effect of X protein in cell cycle progression and in the inactivation of tumor suppressors. Therefore, further cohort study to observe the effect of basal core promoter 1762/1764 mutant and the functional consequences of mutated X protein in male and females patients are needed to clarify this important issue.
The clinical, virological and therapeutic implications of HBV genotypes have been partially clarified. Previous reports shown genotype C is associated with more severe liver disease and genotype B is associated with the development of HCC in young noncirrhotic patients in Taiwan. By contrast, genotype B has a relatively good prognosis in Japan and Hong Kong. Similar to other Asian region, genotype B and C were the predominant strains in Taiwan. But we found the prevalence of genotype B is higher than genotype C in Taiwan. It is different from Japan and Hong Kong that the majority of HBeAg-negative chronic hepatitis infected with genotype C.
Several studies implied that the patients with genotype B infection experience earlier HBeAg seroconversion and genotype C seems to stay longer in the immune clearance phase and shifts to severe liver inflammation. In consistency with our study, the mean age of patients with genotype C infection was significantly higher than genotype B infection (52 years vs. 46 years, respectively, P=0.012). In addition, the proportion of HBV related liver disease including chronic hepatitis, liver cirrhosis and HCC was also significantly higher than patients with genotype B infection (89% vs. 55%, respectively, P=0.0001). Nevertheless, the pathogenic-link between the genotypes and the progression of liver disease remains largely unknown. In our study, the patients with genotype C had greater prevalence of basal core promoter 1762/1764 mutants than the patients with genotype B (79% vs.59%, P=0.002). This may contribute to the different clinical outcomes between genotype B and genotype C infection.
Of particular note is the role of genotype B and C in the progression of chronic liver disease. Genotype C was associated with progression of HBV related liver disease (odds ratio, 6.56, P<0.001). But genotype B and genotype C were at similar risk for the development of liver cirrhosis and HCC. Our result suggests that, although the patients with genotype B infection had earlier HBeAg seroconversion, but a proportion of patients progressed slowly and slower development of liver cirrhosis and HCC. The life-long risk of progression to advanced liver disease is similar among genotype B and C related liver disease.
Currently it is accepted that pathogenetic mechanisms of liver cell damage in HBeAg-negative chronic hepatitis B stem from immune response against replicating HBV. Ongoing HBV replication triggers strong immune responses against the virus. These mechanisms are most likely similar to those operating in HBeAg-positive chronic hepatitis B. So it is not unanticipated that the serum HBV DNA level was significant correlation with serum ALT level (P=0.024).
The potential impact of precore stop codon mutation and basal core promoter mutation on the replication of HBV remains controversial. In the treatment of HBeAg-positive chronic hepatitis B, low serum HBV DNA level (< 10 pg/ml) is one of the predictors for better response to interferon therapy. Thus we defined serum HBV DNA concentration > 10 pg/ml as a high HBV DNA level in the present study. Our data showed that patients with both mutations had a lower proportion of high detectable serum HBV DNA level than those with either precore stop codon mutation or basal core promoter mutation alone. These findings suggested that the impact of basal core promoter mutation on the replication of HBV is influenced by the emergence of precore stop codon mutation.
In conclusions, our results indicated that HBV genotype B and C were predominant in Taiwan. The risk of progression of HBV related liver disease is greater in patients infected with HBV genotype C and basal core promoter 1762/1764 mutants. In addition, basal core promoter 1762/1764 mutants may contribute to the different pathogenicity of different HBV genotype. The impact of mutations in the precore/basal core promoter on viral replication should also be further analyzed.

目 錄
一.中文摘要
1. 前言 3
2. 研究目的 5
3. 研究方法 5
4. 結果 6
5. 討論 9
6. 結論 12
二. 緒論
1. 前言 14
2. B型肝炎病毒基因體及其蛋白功能 14
3. 分子流行病學 16
4. B型肝炎病毒慢性感染的自然病程 16
5. e抗原陰性慢性B型肝炎
5.1 定義 17
5.2 核前區及核心區的突變 18
5.3 盛行率 18
5.4 臨床表現 20
5.5 核前區變異種病毒和basal core promoter 1762/1764
變異種病毒的免疫篩選 21
5.6 致病機轉 22
5.7 基因型的臨床相關性 24
5.8台灣地區B型肝炎病毒感染現況 25
5.9目前的重要課題 26
6. 研究假說 26
7. 研究目的 27
三. 研究方法與材料
1. 研究材料 28
2. 研究方法 28
四. 結果 31
五. 討論
1. 台灣地區核前區1896和basal core promoter 1762/1764
變異種病毒的盛行率 34
2. 核前區1896和basal core promoter 1762/1764變異種病
毒的致病性 35
3. B型肝炎病毒的致癌機轉 37
4. B型肝炎病毒基因型的影響 41
5. B型肝炎病毒基因型及precore/basal core promoter
1762/1764 變異與HBV DNA濃度的相關性 44
六. 展望
1. B型肝炎流行病學的變化 46
2. 現階段慢性B型肝炎的療
2.1. e抗原的狀態 46
2.2. B型肝炎病毒基因型 48
2.3. 變異種B型肝炎病毒 48
3. 治療的新觀點 49
4. 未來前瞻性之研究 50
七. 論文英文簡述 51
八. 參考文獻 60
九. 圖表 73

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