劉燦,歐啟水
(福建醫(yī)科大學(xué)附屬第一醫(yī)院檢驗(yàn)科,福州 350005)
·綜述·
HBV基因突變及其臨床意義研究進(jìn)展
劉燦,歐啟水
(福建醫(yī)科大學(xué)附屬第一醫(yī)院檢驗(yàn)科,福州 350005)
HBV以準(zhǔn)種形式存在患者體內(nèi),不同基因區(qū)的準(zhǔn)種變化具有明顯的異質(zhì)性,其對(duì)疾病的診斷、治療及轉(zhuǎn)歸均有一定影響。理解HBV突變及其改變的分子及病理特征,有助于實(shí)現(xiàn)不同乙肝感染患者的分層管理,促進(jìn)更精準(zhǔn)的個(gè)體化治療。該文就HBV基因突變及其臨床意義研究進(jìn)展作一綜述。
HBV;基因;突變;臨床意義
HBV為部分雙鏈環(huán)狀嗜肝DNA病毒,基因組含4個(gè)部分重疊的開放讀碼框(open reading frame,ORF),即前-S/S區(qū)、前-C/C區(qū)、P區(qū)和X區(qū)。HBV侵入機(jī)體后脫衣殼形成松弛環(huán)狀DNA(relaxed circular DNA,rcDNA),進(jìn)入靶細(xì)胞核內(nèi),在DNA聚合酶作用下合成共價(jià)閉合環(huán)狀DNA(covalently closed circular DNA,cccDNA),再經(jīng)過RNA聚合酶轉(zhuǎn)錄成各種大小不同的mRNA,其中前基因組RNA(pregenomic RNA,pgRNA)在HBV逆轉(zhuǎn)錄酶作用下合成完整的HBV DNA,其他mRNA則翻譯成病毒的各種蛋白質(zhì)成份(見圖1)[1]。根據(jù)HBV基因組序列差異性>8%和S基因序列差異性>4%,可將HBV區(qū)分為至少10個(gè)基因型(A~J型)及若干個(gè)基因亞型,我國(guó)主要以B、C基因型感染為主[2]。然而,由于HBV逆轉(zhuǎn)錄過程缺乏嚴(yán)格的校正機(jī)制,每104~105核苷酸就會(huì)出現(xiàn)1個(gè)突變。因此,每一個(gè)患者體內(nèi)的病毒構(gòu)成并不均一,而是由HBV基因序列存在微小差異的多種病毒株組成,并始終處于動(dòng)態(tài)變化過程中,即以準(zhǔn)種(quasispecies)形式存在[3]。HBV準(zhǔn)種的異質(zhì)性變化包括復(fù)雜性和多樣性改變[4],不同基因型HBV的準(zhǔn)種異質(zhì)性變化對(duì)疾病的診斷、治療及轉(zhuǎn)歸均有一定的影響。本文就HBV不同基因區(qū)的突變、抗病毒療效及臨床轉(zhuǎn)歸的關(guān)系等作一綜述。
注:本圖在文獻(xiàn)[1]基礎(chǔ)上修改而成;NTCP,鈉離子/牛磺膽酸共轉(zhuǎn)運(yùn)多肽;rcDNA,松弛環(huán)狀DNA;cccDNA,共價(jià)閉合環(huán)狀DNA;pgRNA,前基因組RNA。
圖1 HBV感染流程
HBV前-S/S區(qū)基因包括preS1區(qū)、preS2區(qū)和S區(qū),分別有各自的起始密碼子,但共用一個(gè)終止密碼子[5](見圖2A)。前-S/S區(qū)基因主要編碼3種包膜蛋白:大蛋白、中蛋白和主蛋白,分別又稱L、M和S蛋白。preS1因基因亞型的不同而造成編碼氨基酸長(zhǎng)度不固定,由108至126個(gè)氨基酸構(gòu)成,此區(qū)域包含與肝細(xì)胞結(jié)合受體鈉離子/牛磺膽酸共轉(zhuǎn)運(yùn)多肽(Na+/taurocholate cotransporting polypeptide, NTCP)的結(jié)構(gòu)域,在病毒感染進(jìn)入細(xì)胞中起重要作用[6]。preS2由55個(gè)氨基酸組成,與T、B淋巴細(xì)胞免疫表位識(shí)別有關(guān)[7]。S區(qū)基因由226個(gè)氨基酸組成,包括N端區(qū)(aa1~98)、主要親水區(qū)(major hydrophilic region,MHR,aa99~169)和C端區(qū)(aa170~226),負(fù)責(zé)編碼HBsAg,是病毒包膜蛋白的主要抗原區(qū)域[7]。
前-S序列是整個(gè)HBV基因組中異質(zhì)性最高的區(qū)域,突變類型包括替換、缺失和插入等多種形式,見表1。相對(duì)于B基因型,preS1/preS2區(qū)突變更常發(fā)生于HBV C型感染患者[5]。S區(qū)基因突變主要集中在MHR,發(fā)生在“a”決定區(qū)(aa124~147,能誘導(dǎo)機(jī)體針對(duì)所有HBV亞型產(chǎn)生保護(hù)性應(yīng)答的區(qū)域)的突變,可改變HBsAg的空間構(gòu)象引起抗原性質(zhì)改變,無法被抗HBs抗體中和,成為免疫逃逸突變;發(fā)生在“a”決定區(qū)以外的突變,除了發(fā)生免疫逃逸外,還常常影響肝臟疾病進(jìn)展及核苷(酸)類似物(NAs)耐藥的發(fā)生,見表1。前-S/S區(qū)基因突變也會(huì)對(duì)臨床HBsAg檢測(cè)造成影響[8]。如產(chǎn)生的HBsAg無法被商品化試劑檢出,容易出現(xiàn)HBsAg/抗HBs抗體雙陽(yáng)性的結(jié)果,甚至造成隱匿性肝炎(occult hepatitis B virus infection,OBI)的發(fā)生[9]。另一方面,不同的檢測(cè)系統(tǒng)對(duì)突變株的檢測(cè)能力不一[10]。Servant-Delmas等[11]比較了13種商品化試劑檢測(cè)HBsAg的結(jié)果,發(fā)現(xiàn)對(duì)不同類型突變株不同試劑檢測(cè)結(jié)果間存在差異,其總體檢出率從62.9%~97.9%不等。因此,當(dāng)臨床對(duì)HBsAg檢測(cè)結(jié)果持有疑議時(shí),應(yīng)盡可能采用另一種方法進(jìn)行復(fù)檢。
S區(qū)基因與HBV逆轉(zhuǎn)錄酶區(qū)基因重疊,有研究利用二代測(cè)序平臺(tái)(next generation sequencing,NGS)如超深焦磷酸測(cè)序(ultra-deep pyrosequencing, UDPS)等在NAs治療患者中發(fā)現(xiàn)了一些新的低比例(0.13%~0.17%)突變位點(diǎn),如sW156stop、sW163stop、sW165stop和sW191stop等[12-13],會(huì)導(dǎo)致HBsAg合成缺陷。然而,這些低比例突變的確定臨床意義目前尚不明了。
注:A,前-S/S區(qū)基因結(jié)構(gòu); B,前-C/C區(qū)基因結(jié)構(gòu)及編碼產(chǎn)物; C,HBV P區(qū)基因結(jié)構(gòu);D,HBV X區(qū)基因結(jié)構(gòu);nt,核苷酸;rt,逆轉(zhuǎn)錄酶;aa,氨基酸;MHR區(qū),主要親水區(qū);NRD負(fù)向調(diào)控原件。
圖2 HBV各基因區(qū)結(jié)構(gòu)
注:*,終止密碼;突變堿基前未加“nt”均代表氨基酸位點(diǎn)(全文同)。
HBV前-C/C區(qū)基因依據(jù)各自的起始密碼子,在基本核心啟動(dòng)子(basal core promoter,BCP)引導(dǎo)下,轉(zhuǎn)錄成preC mRNA和pgRNA,前者翻譯成前核心蛋白和核心蛋白,進(jìn)而剪切成HBeAg和HBcAg[5](圖2B)。文獻(xiàn)報(bào)道HBV前-C/C區(qū)基因的異質(zhì)性常發(fā)生在BCP和前-C區(qū),見表2。BCP和前-C區(qū)突變會(huì)促進(jìn)HBeAg血清學(xué)轉(zhuǎn)換,因此早期檢出BCP和前-C區(qū)突變可以預(yù)測(cè)HBeAg的血清學(xué)轉(zhuǎn)換[24]。與HBeAg陽(yáng)性患者相比,HBeAg陰性患者更易發(fā)生前-C區(qū)突變,而且這些突變并不引起DNA拷貝數(shù)的變化,但與肝臟疾病進(jìn)展密切相關(guān)[25]。有研究顯示,BCP/前-C區(qū)突變可能對(duì)抗病毒藥物具有更高的敏感性[26-27],但如果BCP/前-C突變聯(lián)合前-S1/S2缺失,往往會(huì)導(dǎo)致更嚴(yán)重的肝臟疾病如暴發(fā)性肝炎和HCC的發(fā)生[28]。而且,BCP/前-C區(qū)突變與基因型相關(guān),好發(fā)于HBV基因型B和D感染的患者[29]。
表2 常見BCP、前-C/C區(qū)突變及臨床影響
最近有研究利用高靈敏UDPS技術(shù),分析BCP、前-C/C區(qū)基因的異質(zhì)性,發(fā)現(xiàn)在基線水平即存有少量G1896A、G1899A和前-C區(qū)起始密碼子突變,并隨疾病進(jìn)展不斷變化[26];Bayliss等[32]利用NGS檢測(cè)HBeAg陽(yáng)性患者發(fā)現(xiàn),基線水平存在少量可檢測(cè)的BCP/前-C突變,會(huì)顯著降低NAs治療后HBsAg的清除率。
P區(qū)是最長(zhǎng)的HBV基因區(qū),包含4個(gè)功能結(jié)構(gòu)域:末端蛋白(terminal protain,TP)、間隔區(qū)(spacer region, Spc)、逆轉(zhuǎn)錄酶(RT)區(qū)和RNA酶H區(qū)(RNH),其中RT區(qū)又可分為7個(gè)結(jié)構(gòu)域(圖2C)。P區(qū)編碼DNA聚合酶,參與HBV DNA的合成及pgRNA的逆轉(zhuǎn)錄。TP位于P區(qū)N端,作為DNA負(fù)鏈合成的初始引物;Spc區(qū)作為聚合酶功能非必要的補(bǔ)充,更能耐受突變,由于間隔區(qū)與前-S區(qū)部分交叉,因此該區(qū)域變異亦會(huì)引起相應(yīng)的HBsAg氨基酸位點(diǎn)的改變;RT區(qū)具備DNA聚合酶活性和逆轉(zhuǎn)錄活性,其催化中心位于nt736~747(即YMDD區(qū)),該區(qū)在所有基因型中高度保守,在HBV復(fù)制中發(fā)揮著重要作用;RNA酶H區(qū)位于P區(qū)C端,參與RNA的衣殼包裝和負(fù)鏈DNA的合成[34]。
抗病毒治療藥物NAs主要通過競(jìng)爭(zhēng)結(jié)合HBV DNA聚合酶尤其是RT區(qū),發(fā)揮強(qiáng)效抑制病毒復(fù)制的作用,一旦該區(qū)基因發(fā)生變異,就會(huì)導(dǎo)致NAs結(jié)合力下降,從而失去對(duì)突變株的抑制能力,產(chǎn)生耐藥[35]。因此,以往研究最多的HBV P區(qū)異質(zhì)性主要集中在RT區(qū),見表3。與干擾素治療相比,A、B基因型較C、D基因型具有更好的應(yīng)答效果,然而基因型差異對(duì)NAs的療效應(yīng)答情況目前仍知之甚少[2]。
表3 常見RT區(qū)突變及臨床影響
注:LMV,拉米夫定;ADF,阿德福韋酯;ETV,恩替卡韋;TDF,富馬酸替諾福韋酯;LdT,替比夫定;ETV耐藥相關(guān)變異是在rtM204V/I+rtLl80M變異基礎(chǔ)上,再聯(lián)合其他至少一個(gè)位點(diǎn)的氨基酸替代變異;*,僅在體外實(shí)驗(yàn)發(fā)現(xiàn),在臨床實(shí)踐中并未完全確認(rèn)TDF的耐藥位點(diǎn)。
本課題組曾構(gòu)建高靈敏RT-AS-LNA-qPCR[44]和RT-ARMS-qPCR[45]技術(shù),分別用于HBV YIDD(rtM204I)和YVDD(rtM204V)突變株的定量檢測(cè),初步揭示了早期檢出低比例突變DNA對(duì)NAs的療效預(yù)測(cè)價(jià)值。近來有研究利用NGS平臺(tái)顯示,NAs治療的CHB患者其HBV RT區(qū)準(zhǔn)種異質(zhì)性呈現(xiàn)多樣性及低比例變化的趨勢(shì)[5,46-48]。進(jìn)一步研究發(fā)現(xiàn),選擇LAM或LdT治療4周時(shí),應(yīng)答組RT區(qū)準(zhǔn)種復(fù)雜性和多樣性顯著低于無應(yīng)答組[46],采用ETV時(shí),完全應(yīng)答組較部分應(yīng)答組準(zhǔn)種復(fù)雜性降低,而準(zhǔn)種多樣性增加[5,48],提示不同準(zhǔn)種異質(zhì)性對(duì)藥物的動(dòng)力學(xué)和藥效學(xué)變化有影響;而且高基線水平RT 2區(qū)(與S區(qū)基因重疊的B、C結(jié)構(gòu)域)的準(zhǔn)種異質(zhì)性可預(yù)測(cè)病毒學(xué)應(yīng)答[47]。盡管如此,這些在基線水平及NAs治療過程中出現(xiàn)的低比例耐藥突變,其確切的臨床意義仍未完全闡明。
X區(qū)是HBV最小的基因區(qū),N端含負(fù)向調(diào)控域(negative regulatory domain,NRD),具有抗凋亡基因;C端為轉(zhuǎn)錄因子結(jié)合區(qū)(transcription factor binding),含促凋亡基因[49]。其編碼產(chǎn)物HBx蛋白不與宿主及病毒DNA直接結(jié)合,主要通過激活或抑制轉(zhuǎn)錄因子對(duì)HBV DNA病毒轉(zhuǎn)錄、復(fù)制及宿主細(xì)胞基因起調(diào)控作用,是導(dǎo)致肝癌發(fā)生的關(guān)鍵分子[50]。X區(qū)基因突變主要發(fā)生在肝癌患者,有研究顯示,當(dāng)存在K130M+V131I雙聯(lián)突變及V5M/L+K130M+V131I三聯(lián)突變時(shí),HCC的風(fēng)險(xiǎn)分別提高3.75倍和5.34倍[51]。鑒于X區(qū)基因突變與HCC密切相關(guān),有研究利用測(cè)序技術(shù),發(fā)現(xiàn)當(dāng)8個(gè)X區(qū)基因位點(diǎn)(包括ntG1613A,ntC1653T,ntT1753V,ntA1762T,ntG1764A,ntA1846T,ntG1896A和ntG1899A)出現(xiàn)≥6個(gè)突變位點(diǎn)陽(yáng)性作為預(yù)測(cè)HCC的指標(biāo),其敏感性為44.0%,特異性可達(dá)99.7%[52]。
目前,單獨(dú)針對(duì)X區(qū)基因的二代測(cè)序研究較少。研究顯示與前-C區(qū)重疊的X區(qū)基因(nt 1 814~1 838)中,當(dāng)存有少量(0.16%)ntT1836C突變時(shí),會(huì)導(dǎo)致原有的終止密碼子TAA突變?yōu)榫幋a谷氨酰胺的密碼子CAA,從而產(chǎn)生的HBx蛋白的羧基末端比正常HBx多51個(gè)氨基酸,致使HBx蛋白發(fā)生潛在的功能性改變[26]。
HBV基因組具有明顯的準(zhǔn)種異質(zhì)性,有些與病毒治療反應(yīng)有關(guān),有些與免疫逃逸相關(guān),有些可加速肝臟疾病進(jìn)展,有些會(huì)引起檢測(cè)結(jié)果不一致。因此,借助于分子診斷技術(shù)的發(fā)展及臨床研究的深入,能進(jìn)一步了解不同HBV基因突變的臨床意義,尤其是證實(shí)一些低比例突變的時(shí)空特性,更好地針對(duì)不同的乙肝感染個(gè)體做出精準(zhǔn)醫(yī)療決策。
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(本文編輯:周萬青,劉群)
10.13602/j.cnki.jcls.2017.07.11
國(guó)家自然科學(xué)基金(81572067,81672101);福建省自然科學(xué)基金(2015J01385)。
劉燦,1979 年生,男,博士研究生,主要事乙型肝炎病毒診斷與療效監(jiān)測(cè)研究。
歐啟水,主任技師,教授,博士研究生導(dǎo)師,E-mail:ouqishui@163.com。
R446.6
A
2016-12-20)