A cross sectional study on the profile of drug resistant mutations in nucleos(t)ide analogue experienced chronic hepatitis b patients in tianjin

The study targeted to discover the profile of mutation site and mutation patterns in patients with chronic hepatitis B CHB in Tianjin, China.. Table 1: HBV genotype–specific implications

T I T L E A cross-sectional study on the profile of drug-resistant

mutations in nucleos(t)ide analogue-experienced chronic

hepatitis B patients in Tianjin

一级学科：临床医学

二级学科：内科学

传染病学

论文作者：TRIEUCONGDOANH 指导教师：侯 伟 教授

天 津 医 科 大 学 研 究 生 院

二〇一八年五月

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中文摘要

目的：乙型肝炎病毒（HBV）感染是全世界最严重和最普遍的健康问题之一。HBV 感染呈世界性流行，但不同地区 HBV 感染的流行强度差异很大。据世界卫生组织（ WHO）报道，全球约 20 亿人曾感染 HBV，其中 2.4 亿人为慢性 HBV 感染者，每年约有 65 万人死于 HBV 感染所致的肝功能衰竭、肝硬化（LC）和肝细胞癌（HCC）。HBV 属嗜肝 DNA 病毒科（ hepadnaviridae），基因组长约 3.2

HBeAg、HBcAg、病毒 DNA 多聚酶和 HBx 蛋白。P 区编码ＤＮＡ聚合酶，此酶既具有反转录酶活性，又有催化合成 DNA 的多聚酶功能。P 区包括四个编码区域，其中逆转录酶区（RT 区）包含五个功能保守区域：A 区、B 区、C 区、D 区、E 区，其中 A、C、D 区为酶与三磷酸核苷结合的区域，B、E 区为 RNA 模板和引物定位域。HBV 至少有 A-J 这几个基因型，我国以 B 型和 C 型为主。目前使用核苷(酸)类似物（NAs）对乙型肝炎病毒（HBV）感染者行直接抗病毒药物治疗。口服药物方面，在拉米夫定(LAM)、阿德福韦酯(ADV) 、恩替卡韦(ETV)、替比夫定(LDT) 、替诺福韦酯(TDF)的基础上，新增替诺福韦艾拉酚胺富马酸(TAF)这一新药。NAs 其进入机体后，形成三磷酸活性成分与机体天然的脱氧三磷酸核苷（dNTP） 竞争结合到 HBV 聚合酶上，使 HBV 的 DNA 链合成终止，这是核苷（酸）类似物抑制 HBV 复制的机制。NAs 使用核苷(酸)类似物能导致在 HBV 聚合酶逆转录酶(RT)结构域位点的保守区发生突变。HBV 常见的耐药突变位点多位于 HBV 逆转录酶区的 B、C、D 结构域内。不同抗病毒药物所致的耐药突变位点不同：如①拉米夫定(LAM)耐药：在 HBV 基因组中反转录酶区的 C 结构域内有一个高度保守的 YMDD 基序，即酪氨酸-甲硫氨酸-天门冬氨酸-天门冬氨酸。拉米夫定能够与其特异性结合，通过抑制前基因组 RNA 反转录为负链 DNA 并终止 DNA 链延伸而有效抑制 HBV DNA 复制。②阿德福韦酯(ADV)耐药：阿德福韦酯同样也能抑制逆转录酶活性，但其耐药突变概率比拉米夫定低，阿德福韦酯耐药与逆转录酶 B 区或 D 区位点有关，从而降低阿德福韦

酯对 HBV DNA 的抑制作用。③恩替卡韦(ETV)具有高耐药基因屏障，至少需要三个位点发生突变才可构成耐药。在接受抗病毒药物治疗的患者体内，HBV 突

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病毒疗效，进而发生基因型耐药（genotypic resistance）、表型耐药（phenotypic resistance）及临床耐药。检测到与 NAs 相关的 HBV 耐药基因突变，称为基因型耐药（genotypic resistance）。体外实验结果显示抗病毒药物敏感性降低，且与基因型耐药相关，称为表型耐药（phenotypic resistance）。针对一种抗病毒药物出现的耐药突变对另一种或几种抗病毒药物也出现耐药，称为交叉耐药（cross resistance ）。 至 少 对 两 种 不 同 类 别 的 NAs 耐 药 ， 称 为 多 药 耐 药 （ multidrug

的基因变异； 另一方面耐药变异株和野生株的共生生态的变化， 耐药变异株要

床表现。HBV 逆转录酶区耐药基因变异的检测方法:运用 DNA 测序原理的检测方法如(1)直接测序法,该方法对 HBV 全基因组测序，既可对已知的耐药变异位点进行检测，也可对未知的耐药变异进行检测研究。临床上以检测常见耐药变异位点为主。在直接测序基础上发展克隆测序法。(2) 焦磷酸测序法,该方法的检测通量高，可以对 HBV 基因组上的任意片段进行测序，在同时检测多个已知的

具有高灵敏、高特异性和准确性等特点，能早期检测核苷（酸）类似物治疗患者

的 HBV 逆转录酶区基因耐药突变异。还可以运用杂交原理的检测方法如(1)线性探针技术,将特异的寡核苷酸探针固定于硝酸纤维素膜条上对生物素化 PCR扩增的目标片段进行反向杂交后显色，使检测结果肉眼可见。本法对检测样本病毒载量的要求与直接测序法相近,可用于临床上早期发现耐药变异的出现。但本方法受探针的局限，只能检测常见的几种变异位点，不能用于检测和研究未知的耐药变异。(2)荧光实时定量 PCR, 该方法结合了 PCR 技术和 FRET 核酸定量检测技术。在 DNA 扩增的同时释放可供检测的荧光信号，实现对模板 DNA 的定量分析。该方法应用于单核苷酸多态性分析，检测的敏感性高，对血清样本病毒载量要求较低。此法敏感性较高，可同时检测多个耐药变异位点。但本方法只能检测常见的几种变异位点，不能用于检测和研究未知的耐药变异。(3) 基因芯片法,该方法在芯片表面有序地固定高密度的寡核苷酸或特定基因片段探针， 再与生物素标记的待检基因片段反应， 然后通过显色产生荧光信号， 软件处理信号获得待测基因序列信息。表型耐药检测是指在体外细胞培养系统中使 HBV 基因组中一个或多个位点突变，再检测病毒对药物的敏感性降低或丧失，可作为耐

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药诊断的依据。已发展多种技术如瞬时转染细胞模型、稳定转染细胞模型、酶活

炎的治疗和管理密切相关。NAs 不能完全清除体内病毒，目前用药患者数巨大且患者长期服用 NAs，对 NAs 耐药问题在临床上越发严重，往往导致患者病情反复甚至加重。不同 NAs 药物导致慢性乙肝患者 HBV 逆转录酶区变异位点、

和突变模式的分布特点。

方法：本研究分为三部分。第一部分：对 1320 例慢性乙型肝炎患者 HBV 基因组

RT 区扩增，直接测序后分析耐药突变相关的位点，其中包括 rt80，rt169，rt173，rt180，rt181，rt184，rt194，rt196 ，rt202，rt204，rt207，rt213，rt214，rt215，rt236，rt237，rt238 和 rt250。然后分析其基因型、性别、年龄等是否存在统计学显着性差异。在第二部分中，我们进一步研究各个突变位点的突变百分比以了解抗病毒耐药的分子机制，包括 LAM，ADV，ETV，LDT 和 TDF。第三部分，我们又进一步分析了基因型，性别和年龄组之间的突变模式。

结果：收集 2012 年 7 月至 2015 年 12 月在天津市第二人民医院，天津市肝病研究所诊治的慢性乙型肝炎感染者 1320 名，其中男性占比 73.79％，女性占比

型包括基因型 B 和基因型 C 两种，感染率分别为 8.71％和 91.29％，之后对耐药相关突变位于 HBV P 基因的 RT 区域进行测序分析。

以 1320 例天津地区慢性乙型肝炎患者为研究对象，对所有患者的临床资料进行突变分析，包括 rt80，rt169，rt173，rt180，rt181，rt184，rt194，rt196，rt202， rt204，rt207，rt213，rt214，rt215，rt236，rt237，rt238 和 rt250，其中以 rtM204突变率最多，占 24.25％，其次为 rt180（14.97％）和 rt181（12.43％）。之后，我们进一步分析了不同 HBV 基因型、性别和年龄在各个位点突变所占的比例。rtA181 位点 ADV 相关突变在基因型 C 中的百分比显着高于基因型 B（13.11％

比 4.25％，p <0.001），其他突变位（rtS213 p = 0.005，rtN236 p <0.001， rtV214

变率高于基因型 B（3.1％vs.0.47％，p = 0.019）。然而，rtM204 和 rtL180 的 LAM

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相关突变和 rtT184，rtS202 和 rtM250 的 ETV 相关突变在基因型 B 和 C 之间无显着差异（P> 0.05）。在性别分层亚组分析中，rtV207 位点的 LAM 相关突变在女性中的表现比男性高（p = 0.002），在 rtI169 位点的 ETV 相关突变也是如此（p =

和 18.03％。

（1） 我们发现了 16 个相关耐药突变位点，即 rtL80I，rtL80V，rtV173L，rtV173M，rtL180M，rtL180F，rtL180Q，rtM204V，rtM204I，rtM204K，rtM204L，rtM204Q，rtM204S，rtV207I，rtV207L 和 rtV207M，其中以 rtL180M 最为常见，占 26.34％，其次为 rtM204I，rtL80I 和 rtM204V，占比分别为 24.26％，16.33％和 12.73％。此外，耐药性位点的分布在基因型、性别、年龄等方面亦存在差异。在基因型 B中的比例明显高于基因型 C 的突变位点包括 rtV207M（5.32％vs1.04％p = 0.006）、

因型 C 之间没有显着差异（p> 0.05）。当按性别分层时，突变位点 rtV207I 在女性中的比例显着高于男性（11.46％vs7.06％，p = 0.017）。突变位点 rtV207L 显示男性比女性低得多（0.96％vs2.87％，p = 0.025）。在性别分层分析中，除 rtV207I

和 rtV207L 外，其余位点突变无显着性别差异。在按年龄分层的亚组分析中，我们在突变位点 rtL180M，rtM204V，rtV207I，rtV173L 和 rtL180V 中检测到显着的差异比例（所有 p <0.05）。其他与 LAM 相关的耐药突变位点在年龄方面没有差异。

（2）对于 ADV 相关耐药突变，检测到的突变位点，即 rtA181T，rtA181C，rtA181G，rtA181S，rtA181D，rtA181P，rtA181H，rtS213T，rtS213N，rtV214A，rtV214E，rtV214I，rtV214P，rtV214T，rtQ215H， rtN236L，rtI233V，rtI233L，

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rtN236T，rtN236H，rtP237H，rtP237T，rtP237S，rtN / H238A，rtN / H238K，rtN / H238D，rtN / H238S，rtN / H238T，rtN / H238G 和 rtN / H238Q，其中 rtA181T

占 20.85％，其次为 rtS213T，rtV214A 和 rtA181V，分别占 16.17％，15.1％和10.26％。基因 B 型患者 S213T 突变的患病率高于携带基因型 C 的患者（32.84％vs.15.01％，p <0.001），Q215H（14.93％vs 5.28％，p = 0.004）和 rtP237T （2.99％

型 B（10.77％vs2.99 ％，p = 0.038）。其他 ADV 相关的突变位点，基因型之间没有发现显着差异。当按年龄分层时，在突变位点 rtS213T（p = 0.033），rtN236T

（0.001），rtQ215H（p = 0.02）和 rtP237（p = 0.038）中检测到显着的差异。其

突变位点：rtI169T，rtI169M，rtI169L，rtI169V，rtL180M，rtT184S，rtT184A，rtT184I，rtT184L，rtT184F，rtS202I，rtS202G，rtS202G， rtS202C，rtS202N，rtM204V，rtM250I，rtM250L，rtM250R，rtM250V。其中，rtL180M 和 rtM204V

占 24.04％，其次为 rtS202G，rtT184I 和 rtT184L，分别为 11.28％，10.39％和 8.31％。突变位点 rtI169I 在女性中的表达显着高于男性（6.25％vs0.35％，p = 0.01），突变位点 rtI169L 也是如此（8.33％vs1.73％，p = 0.027）。男性和女性之间在其他突变位点之间没有差异。在按年龄分层的亚组分析中，突变位点 rtL180M，

差异。在基因型 B 和基因型 C 之间的 ETV 相关突变位点亦未发现显着差异。

（4） 在 LDT 和 TDF 关系突变位点中，我们仅检测到 LDT 的一个突变位点：rtM204V 和 TDF 的三个突变位点，即 rtA194T，rtA194I 和 rtA194V（表 9 和表

年龄之间未发现显着差异。

除了突变位点的不同表现之外，我们进一步分析了基因型，性别和年龄组之间的突变模式。基于 1320 例 CHB 患者，确定了 331 个突变模式。其中，ADV相关突变模式 rtS213T 最常见，占 8.33％，其次是 rtA181T，rtV214A，分别占6.67％和 5.91％。按照性别进行分层分析，采用卡方检验，观察到 ADV 相关模

式 rtA181T 在男性和女性之间的分布存在显着差异，男性比例显着高于女性（7.7％

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vs3.76％，p = 0.012） 。然而，突变位点 ADV 相关 rtV214A 突变率女性比男性高（8.67％vs.4.93％，P = 0.016），LAM 相关突变 rtV207I（5.49％vs.2.77％，p=

+ rtS202G 和 rtA181T + rtN236T 在基因型之间也没有差异。

结论: 中国天津地区慢性乙型肝炎患者存在多种突变位点和突变模式。对于应用核苷（酸）类似物抗病毒治疗的患者,应该注重耐药临床管理,加强患者依从性，提高患者对疾病的认识，避免随意停药、换药；加强对医务人员抗病毒治

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疗耐药预防和管理的教育，避免不规范治疗；治疗中应定期检测 HBV DNA 以及时发现原发性无应答或病毒学突破，及时调整治疗策略，以降低耐药风险；应长期监测治疗效果及耐药位点，及时发现耐药突变，尽早干预；初治选择强效且

序贯治疗也能够降低耐药的发生； 已经发生多重耐药突变的患者，应立即选用恰当的挽救治疗，选择有效的抗病毒药物(无交叉耐药)，可将诱导发生多药耐药的风险降至最低。针对 HBV 感染人体的各个环节作用的多靶位抗病毒药物研究开发是治疗多重耐药 HBV 感染的新途径。

年龄

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ABSTRACT

Objective: Hepatitis B virus (HBV) infection is one of the most serious and common prevalence health problems throughout the world Chronic HBV infection can bring about chronic hepatitis B (CHB) and then progress to liver cirrhosis (LC); moreover, it may eventually develop to hepatocellular carcinoma (HCC) The characteristics of drug- mutant resistance in nucleot(s)ide analogs have a strong association with treatment and management hepatitis B Mutation site and mutation patterns are more and more complicated and currently limited The study targeted to discover the profile of mutation site and mutation patterns in patients with chronic hepatitis B (CHB) in Tianjin, China

Methods: This study was stratified into three parts The first part, 1320 case patients with chronic hepatitis B were amplifier of the RT region in the HBV genome and 18AA positions related to drug resistance mutations in the RT region, including rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt196, rt202, rt204, rt207, rt213, rt214, rt215, rt236, rt237, rt238 and rt250 were analyzed according to sequencing results And then statistical analysis was used to investigate whether significant difference was detected with dividing genotypes, gender and age In the second part, we took a further step to study the proportion of mutant sites to comprehend the mechanism of molecular in antiviral resistance, including LAM, ADV, ETV, LDT and TDF In the third part, we farther analyzed the mutation patterns between genotype, gender and age groups Result: In the first part, the drug resistance was detected at eighteen positions, including rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt196, rt202, rt204, rt207, rt213, rt214, rt215, rt236, rt237, rt238 and rt250 Position at rt204 was the most of frequently occurred with the percentage of 24.25%, follow by rt180 with 14.97% and rt181 with 12.43% Genotype C isolates had higher rates of ADV-related mutation rtA181 than genotype B (13.11% vs 4.25%, p<0.001) The ADV – associated mutation at position rtS213, rtN236, rtV214, and rtI233 showed a remarkable higher percentage in genotype C than genotype B (p<0.05, all) The LAM- associated mutation site rtV173 with genotype C had a much higher mutation proportion than

In the second part, overall sixty resistant mutation sites were identified Mutation site rtM204I was the most common with the proportion 32.35% Study the percentage of mutant sites to comprehend the molecular mechanism of antiviral resistance, (1) LAM associated drug resistance, we discovered 16 mutation sites, namely rtL80I, rtL80V, rtV173L, rtV173M, rtL180M, rtL180F, rtL180Q, rtM204V, rtM204I, rtM204K, rtM204L, rtM204Q, rtM204S, rtV207I, rtV207L and rtV207M The rtL180M was the most frequently occurred with the most frequently occurred with 26.34% The mutation site rtV207M indicated an essential higher proportion in genotype B than genotype C (5.32% vs.1.04% p=0.006) Genotype B isolates had higher rates of mutation site rtL80V than genotype C (6.38% vs.2.08% p=0.021) The noteworthy difference proportion was found in mutation site rtL180M, rtM204V, rtV207I, rtV173L and rtL180V (p<0.05, all) in terms of age (2) ADV- related mutation resistance, thirsty two mutation sites was detected, namely rtA181T,rtA181C, rtA181G, rtA181S, rtA181D, rtA181P, rtA181H, rtS213T, rtS213N, rtV214A, rtV214E, rtV214I, rtV214P, rtV214T, rtQ215H, rtQ215R, rtI233V, rtI233L, rtN236T, rtN236H, rtP237H, rtP237T, rtP237S, rtN/H238A, rtN/H238K, rtN/H238D, rtN/H238S, rtN/H238T, rtN/H238G, rtN/H238Q and rtN/H238R The mutation site rtA181T was the most common with the percentage 20.85% The mutation site mutation S213T in patients with genotype B had a much higher prevalent than patients carrying genotype C (32.84% vs.15.01%, p<0.001) The mutation site Q215H, rtP237T and rtN/H238R indicated an essential higher proportion in genotype B than genotype C (14.93% vs 5.28%, p=0.004), (2.99% vs.0.1%, p=0.012) and (10.45% vs.0.1%, p<0.001) The mutation site rtA181V demonstrated a remarkable higher proportion in genotype C than genotype B (10.77% vs.2.99%, p=0.038) When

X

stratified with age, the mutation site rtS213T, rtN236T rtQ215H and rtP237 showed a significant divergence rate (p<0.05, all) (3) ETV associated drug resistance, nineteen mutation was identified, including rtI169T, rtI169M, rtI169L, rtI169V, rtL180M, rtT184S, rtT184A, rtT184I, rtT184L, rtT184F, rtS202I, rtS202G, rtS202C, rtS202N, rtM204V, rtM250I, rtM250L, rtM250R, rtM250V The mutation site rtL180M and rtM204V were the most predominant prevalent with 24.04% The mutation site rtI169I and rtI169L exhibited a significant higher rate in female than male (6.25% vs 0.35%, p=0.01) and (8.33% vs 1.73%, p=0.027) The mutation site rtL180M, rtM204V and rtT184I had a remark difference frequent in terms of age (p<0.05, all) (4) LDT associated drug resistance, one mutation site M204I was detected (5) TDF associated drug resistance, three mutation sites was detected, including rtA194T, rtA194I and rtA194V The mutation site rtA194T was the most common with 80% Fifteen seldom mutation sites was found, LAM-associated resistant mutation, two mutation sites were detected, including rtL180F, rtL180Q and ADV-related resistance mutation, with eleven uncommon mutation site, containing rtA181C, rtA181D, rtA181H, rtA181P, , rtS213N, rtV214T, rtQ215R, rtI233L, rtN/H238G, rtN/H238K and rtN/H238R With ETV association resistant mutation, seldom mutation site rtM250R was discovered and rtA194I for TDF relation resistant mutation

In the third part, three hundreds thirty one mutation patterns were identified The ADV –associated mutation pattern rtS213T was the most frequently occurred with 8.33% The pattern rtA181T in ADV relation mutation were observed with significant difference between male and female (7.7% vs.3.76%, p=0.012) The mutation ADV- associated pattern rtV214A generated an essential bigger rate in female than male (8.67% vs.4.93%, p=0.016) The proportion of mutation site rtV207I in female was higher than male (5.49% vs.2.77%, p=0.025) The mutation pattern rtL80I+rtM204I in LAM association mutation exhibited a substantial bigger rate in genotype B than genotype

C (10.43% vs.4.81% p=0.016) The mutation patterns rtS213T showed a bigger rate

in genotype B than genotype C (13.91% vs.7.8%, p=0.033) The distribution in mutation rtN236T was found with remarkable divergence between genotype B and genotype C (4.35% vs.0.91%, p=0.009) The pattern rtV207L in patients with

B (7.14% vs.1.74%, p=0.029) The mutation pattern rtA181T, rtV214A, rtM204I, rtV207I, rtQ215H and rtL80I+rtM204I, rtL80I+rtL180M+rtM204I were significant differed between terms of age with p all <0.05 Three hundred and one combination mutations were found with 57.52% of CHB patients The combined mutation rtL80I+rtM204I was most frequently observed with 5.3% Twenty the most common

rtL80I+rtM204I,rtL80I+rtL180M+rtM204I,rtL180M+rtM204V,rtL180M+rtM204I,rtL180M+rtM204I,rtL180M+rtM204V+rtS202G,rtA181T+rtN236T,rtA181V+rtN236T,rtA181T+rtA181V+rtN236T,rtV173L+rtL180M+rtM204V,rtA181T+rtA181V,rtL180

rtL180M+rtM204V+rtA181V,

rtL180M+rtM204V+rtT184L,rtV207I+rtA181T,rtL180M+rtM204V+rtM204I,rtV173L+rtM204I, rtV207I+rtV214A, and rtA181T+rtV214A+rtN236T.The combined mutations rtL80I+rtM204I and rtS213T + rtQ215H, which showed the higher proportion in genotype B than genotype C (p<0.05, all)

Conclusions: The characteristic of NA-resistant HBV are complicated in CHB patients in Tianjin, China, which provided molecular epidemiology data for clinical management Therefore, persistently monitor the resistance mutations of HBV play essential role in for optimizing management strategy and preventing an outbreak of drug resistance

Keywords Hepatits B Mutation Site Mutation Patterns Nucleos(t)ide

analogues Genotype Gender Age

XII

中 文 摘 要

本研究旨在探讨中国天津地区慢性乙型肝炎患者的突变位点和突变模式的分布特点。方法：对 1320 例慢性乙型肝炎患者 HBV 基因组 RT 区扩增，直接测序后分析耐药突变相关的位点。然后分析其基因型、性别、年龄等是否存在统计学显著性差异。在第二部分中，我们进一步研究各个突变位点的突变百分比以了解抗病

核苷（酸）类似物抗病毒治疗的患者,应该注重耐药临床管理,加强患者依从性，提高患者对疾病的认识，避免随意停药、换药；加强对医务人员抗病毒治疗耐药预防和管理的教育，避免不规范治疗；治疗中应定期检测 HBV DNA 以及时发现原发性无应答或病毒学突破，及时调整治疗策略，以降低耐药风险；应长期监测治疗效果及耐药位点，及时发现耐药突变，尽早干预；初治选择强效且具有高

疗也能够降低耐药的发生； 已经发生多重耐药突变的患者，应立即选用恰当的挽救治疗，选择有效的抗病毒药物(无交叉耐药)，可将诱导发生多药耐药的风险降至最低。针对 HBV 感染人体的各个环节作用的多靶位抗病毒药物研究开发是治疗多重耐药 HBV 感染的新途径。

关键词： 耐药相关突变位点 突变模式 核苷类似物

XIII

CONTENT

ABSTRACT VIII

ABBREVIATIONS 1

1 INTRODUCTION 2

2 -MATERIALS AND METHODS 16

2.1 Patients 16

2.2 Laboratory assessment 16

2.2.1 HBV DNA extraction and amplification 16

2.2.2 HBV genotyping and antiviral resistant mutation detection 17

2.3 Statistical Analysis 17

3-RESULT 18

3.1 Clinical features of study patients 18

3.2 Mutation sites subgroup analysis by genotype, gender and age 19

3.2.1 Mutation each sites subgroup analysis by genotype, gender and age 19

3.2.2 Mutation sites subgroup analysis by genotype, gender and age with drugs 20

3.3 Mutation patterns subgroup analysis by genotype, gender and age 36

4 DISCUSSION 57

5-CONCLUSION 61

6-FUTURE OUTLOOK 62

REFERENCES 64

XIV

PUBLICATION 71

REVIEW 72

ACKNOWLEDGEMENTS 110

RESUME 111

2

1 INTRODUCTION

Hepatitis B virus (HBV) infection is one of the most serious and common frequent health problems all around the world and more than 350 million people who got chronic carriers of HBV surface antigen (HBsAg) and 2 billion human have proof of past or present infection with HBV [1] One study from The Global Burden of Disease estimated that 686,000 deaths was responsible for hepatitis B in 2013 with a 5.9 per 100,000 age-standardized death rates globally,of which 317,400 deaths from cirrhosis

of the liver secondary to hepatitis B and 300,000 deaths were attributed to liver cancer [2] The distribution of infection disease is related with a large spectrum of clinical manifestations range from acute or fulminate hepatitis to vary of chronic infection, namely asymptomatic carries, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC)[3,4]

China was characteristic as an area with a high pandemic of hepatitis B virus infection and once a nationwide HBV investigation indicated that hepatitis B surface antigen carrier percentage in general population was 9.8% [5] According to survey nationwide in 2006 exhibited that the proportion of HBsAg carrier was decreased to less than 8.0% and the HBsAg rate among children with age < 5 years was only 1.0% Based on the prevalence of HBsAg and HBV infection gained from the two national sero-epidemiological surveys, the numbers of HBV-infected patients and HBsAg carriers have downfall by an measured 80 million and 20 million, respectively, from 1992 to 2006 [6] Up to now, hepatitis B is still a main disease that impacts human health, often causing chronic poverty because persistence, high prevalence, and difficult treatment

HBV has an incomplete circular double-stranded DNA genome which is fundamentally encapsidated as a pregenomic RNA which undergoes reverse transcription after encapsidation With 4%–8% divergence in the S gene or more than 8% genetic variability in the full-length nucleotide sequence, at least 10 HBV genotypes (A to J) and their subtypes have been identified [7,8] While genotype A is predominant proportion in sub-Saharan Africa, Northern Europe and Western Africa,

3

genotype B and C are common in Asia and genotype D prevails in the Mediterranean

region, Africa, Europe, and India Genotype E was frequent in West and Central

Africa previously and now might be sought in uncommon cases of immigrants or

tourists in Latin America, Turkey, Northern India and Europe and genotypes F to J are

less epidemic and usually have their own specific distributions [9] Whereas HBV

Genotypes A-H is accepted genotypes and their demonstrations have been well

identified, genotypes I and J are tentative genotypes characteristic follow by their

genome divergences [10]

Table 1: HBV genotype–specific implications in patients with chronic hepatitis B

Frequency of precore A1896 mutation Higher Lower Lower Higher

Frequency of basal core promoter

A1762T/G1764A mutation

Intracellular expression of HBV DNA Lower Higher Lower Higher

4

Modes of transmission Perinatal/vertical Perinatal/vertical Horizontal Horizontal

Incidence of progression to cirrhosis

and hepatocellular carcinoma (HCC)

Response to interferon-based therapy Higher Lower Higher Lower

ND: not detected

In China, a difference genotype distribution was showed between North and

South; genotype C was more prevalent in northern China, while genotype B was

predominant in southern provinces [11] And genotype D is more common in Xinjiang

Uygurs [12] With different HBV genotypes also lead to the biological characteristics

and varying clinical of chronic hepatitis B (CHB) patients (Table 1) Acute infection

with genotypes B and C results in lower prevalent of chronic natural than genotypes A

and D Compared to genotypes C and D, patients with genotype A and B cases have

higher percentage of spontaneous hepatitis B e antigen (HBeAg) seroconversion;

when this occurs, it may lead to delayed There was a higher prevalent of basal core

promoter (BCP) A1762T/G1764A mutation, pre-S deletion in HBV genotype B and

genotype C had a lower viral load when compare with genotype C Genotype D also

generate a higher rate of BCP A1762T/G1764A mutation than genotype A These

demonstrations proposed essential pathogenic divergence in terms of HBV genotypes

It may create more severe liver disease, containing cirrhosis and HCC in HBV

infection with genotypes C and D Moreover, patients with genotype A and B have

better responses to interferon-based treatment than genotypes C and D, but these has

not much coherent divergences for direct HBV antivirals [9]

The replication of HBV has a high proportion, with 1012 virions was produced per

day and a high mutational prevalent of approximately 10-5 substitutions/base/cycle

[13]

5

Figure 1: The replication cycle of HBV (a) with a focus on the steps of intra-capsid

reverse transcription (b)

This phenomenon leads to approximately from 1010- 1011 point mutations created per

day in individuals with active replication The genome of HBV is only with

approximately 3,200 base pairs, so all ability single base changes can be altered per

day Because HBV rt has not a proofreading function to fix incorrectly incorporated

nucleotides, point mutations can increase very rapidly as do A–G hyper-mutations

Before treating, there is a different swarm of viruses quasi-species, including mutants

with single and double mutations possibly related with the resistance drug The

mutation probability being identified during treatment is contingent on the ability

suppression of a drug to viral replication Therefore, the low antiviral activity drug

cannot exercise potential chose pressure on the virus, and the drug resistant

opportunity is not high Nevertheless, the replication of virus in suppression accepts

small chance for resistance to come out because mutagenesis is contingent on the

replication of viral genome Nucleos(t)ide analogues (NAs) drug can inhibit viral

replication, but cannot eradicate the existence of virus or affect the intrahepatic

6

cccDNA reservoir in any major way The consistent regimen of treatment should have antiviral activities aimed to divert sites and lead to decline the risk of selecting out drug-resistant species The drug resistance appears by pressuring the appearance of drug selection in replication occurs Hence, if totally complete suppression of replication could be achieved, resistance could not be an issue Some elements suggest to the existence of drug resistance, including genetic barriers to the expanding

of mutations, mechanism of drug resistance, viral replication space, and various host factors contained in suppressing viral replication [14]

To date, five nucleos(t)ide analogues have been accepted in the therapy of chronic hepatitis B (CHB), namely lamivudine (LAM), adefovir (ADV), entecavir (ETV), telbivudine (LDT) and tenofovir (TDF) [1,3] Drug NAs can suppress the replication of HBV mainly by restraining pregenomic RNA in the reverse transcription into DNA The substitution amino acids in HBV reverse transcriptase (RT) will cause NAs resistance, which is a tremendous challenge in the management of CHB The resistance in treating hepatitis B virus by nucleos(t)ide analogue therapy is remarkable structure specific, within dividing to three kinds of structural groups The resistance in each of these is determined by specific mutations in the domains with reverse transcriptase of the HBV polymerase It may relate with compensatory mutations which can cause replication The HBV polymerase is reputed to have a traditional conformation with ‘right-handed’ structure, and the resistance in each of mutations is predicted to create a specific structural change of the polymerase, consequently preventing in-corporation of NA into DNA replication The resistant selection appears at divergence proportion for each NA and affect by the high mutational proportion of HBV and the capacity of the drug to inhibit viral replication Some mutations or combinations of mutations may be related with multidrug resistance, limiting treatment options In the other way with most of other viruses, HBV resistance is confounded by the overlapping surface in gene, the major NA mutation resistance also changing the surface proteins in most cases, essentially altering virus neutralization and secretion, which may threat to world public health in the future

7

The resistance to nucleos(t)ide analogues and cross-resistance is potentially

structure specific, and the five NAs were approved can be stratified under three

main kinds of structural groups ( Table ) 2: (1) L –nucleoside analogues,

containing lamivudine , its 5-fluoro derivative 7 odeli-itabine and telbivudine , (2)

acyclic nucleoside phosphonates, generated by the dAMP analogues adefovir and

tenofovir , and (3) deoxyguanosine analogues in which the deoxyribose moiety is

changed by a cyclopentane sugar derivative, including entecavir

Table 2: Patterns and pathways of antiviral drug resistance in chronic hepatitis B in

the context of cross-resistance

Pathway Aminoacid substitutions

in the rt domain

LAM ADV ETV LDT TDF

D-cyclopentane (ETV) L180M+M204V/I

± I169 ± T184

± S202 ± M250

I = Intermediate sensitivity; R = resistant; S = sensitive I, R, and S are based on cell

culture and clinical reponses Modified from [14, 46]

(a) - Resistance to LMV and Other L –Nucleosides

LAM is the first approved antiviral drug with the highest resistance rate and the

resistance with drug LAM appeared at a cumulative percentage from 14–24% per

year getting levels of over 70% by 5–6 years in treating monotherapy [15, 16] Mutation

is the result which replacing of methionine in the motif tyrosine-methionine-aspartate

(YMDD) catalytic site by isoleucine or valine The change from this replacing are

8

invented rtM204V/I, utilizing the genotype independence nomenclature system which proposed previously [17] The mutation rtM204I substitution in isolation has been detected, but the rtM204V alter in the HBV DNA in those patients who failing LAM therapy is identified in association with other changes, which are remarkably compensatory; containing rtL80V/I, rtV173L and rtL180M [18,19] These compensatory replace typically result in the promoted replication of viral with setting

of the primary resistance These compensatory changes in genotypes have showed that they only grow replication and do not direct effect on antiviral drug resistance [20]

9

(b) - Resistance to Acyclic Nucleoside Phosphonates

Resistance in ADV occurs slower than LAM, appearing at a cumulative annual prevalent of approximately more than 25% over 4 years [16], though long-term (5-year) studies in chronic hepatitis B indicated higher proportion [23]; when utilized ADV as rescue treatment after LAM failure it is also more common in individuals [24] The changes in single nucleotide that bring about substitutions in rtN236T and/or rtA181V/T are sufficient to produce ADV clinical failure [24, 25]

Regarding to TDF resistance, to date, no typical TDF-resistant mutations have been described, even antiviral therapy which treated after 6 years and linked to the rtA194T substitution, but only in relation with changes that lead to LAM resistance and typically in the setting of co-infection with HIV-1 [26] The results from recent clinical studies offer that TDF is less influence in the creating of ADV resistance, but

no singular single substitution has been characteristic that might be unclear in association to primary TDF resistance [27]

ETV resistance has been mainly indicated in patients who had previous LAM failure Sequencing in HBV isolates from these patients usually reveals mutations which create additional substitution amino acid in relation with the classical rtL180M and rtM204V LMV resistance changes [28] These changes conclude at least one of rtI169T, rtT184G, rtS202I, or rtM250V The overall resistance percentage for ETV in nạve patients is <1% at 5–6 years and lead to 1.2% after treating 5-6 years [16, 29]

( c ) - Resistance to More than One NA

Regarding to 8 codons had a relation with primary resistance drug (rtI169T, rtT184G, rtL180M, rtA181T/V, rtS202I, rtM204V/I, rtN236LT, and rtM250V), only mutation site rtA181T and combined mutation rtL180M+rtM204V with substitutions have been definitely related with multidrug resistance

(d) - Molecular Mechanisms of Resistance to NAs

10

Figure 3: Structure of HBV polymerase

HBV polymerase is a multifunctional protein containing four domains: a priming region, a spacer region, a catalytic region as a RNA dependence RNA polymerase/DNA polymerase, and carboxyl terminal region that includes ribonuclease H activity [30] Although the structure crystal of HBV polymerase which has not been completely bring out, much of its structure has been received from structure homology with HIV-1 reverse transcriptase [31] which may lead to 3-dimensional models of the reverse transcriptase (rt) region in the HBV polymerase Therefore, the substitution resulting amino acid from mutations that confer drug antiviral resistance can be mapped to functional regions to give a new insight in understanding the mechanisms in resistance [32] To the highest grade of polymerases can be considered to get a common right-handed configuration containing a thumb, palm, and finger domain Whereas the domain palm contains catalyzes the phosphoryl transfer reaction and the active site, the domain finger facilitates interactions with the incoming dNTPs as well as the template base to which

it is paired and the thumb domain which is essential role in positioning the duplex DNA facilitating grew up processivity and translocation, like acting as a primer domain [33] dNTPs and NAs bind at the site which is location in the palm sub-domain adjacent to the 3 terminus in the primer strand Differing with HIV, HBV rt indicates

a preference the L –configuration for nucleotides in contrast to other polymerases

11

which prefer nucleotides with the D – configuration [34]

The mechanism molecular of resistance for LAM has been suggested a basic on the HIV rt crystal structure [31] LAM adheres at a pocket in the surface of HBV rt which belongs palm region formed in the part by residue rtM204 The primary substitutions rtM204V/I in LMV are predicted to drop the accessible surface area between LMV and the HBV rt because the β-branched side chain in the methyl group

of valine/isoleucine now encroach on the sulfur atom in the unnatural L – oxathiolane ring of LAM [ 35] From structural alterations lead to have two kind main effects: (1) steric hindrance to decline binding of LAM to the viral polymerase and (2) decrease catalytic activity to incorporate LAM-triphosphate into the replication viral DNA [35, 36] These substitutions do not impact the binding of the nature substrate, cytosine, to the viral polymerase, and the polymerase still gets action in viral replication

In other way, based on these homology models focusing on the A and D domains a molecular mechanism of the resistance to ADV has also been proposed for how the most frequent ADV mutation, rtN236T, which is outside the catalytic site, create ADV resistance [25] Domain A and domain D are thought with closely associated in dNTP binding and the modeling has suggested that D domain with rtN236 may be hydrogen bonded to physically adjacent rtS85 [37] from the A domain, direct interaction with the γ phosphate of ADV diphosphate The hydrogen bond between rtS85 and rtN236 were may be interrupted by the rtN236T mutation which may also disrupt between ADV diphosphate and rtN236, thereby declining the binding affinity for ADV In contrast, the mutations that affect the α-helix of the B domain appear to alter the positioning of the template strand of nucleic acid relative to the catalytic region The changes in rtA181T/V could bend the helices through hydrogen bond formation, and the drug resistance may easily result from an allosteric change in the catalytic site [32, 38]

Regarding to TDF, residue rtA194 which is located in a loop at the end of B domain, includes the α-helix that interacts with the template of nucleic acid The substitution from A to T in rt194 may create polymerization efficiency by causing allosteric alters

12

which result in misalignment between the template and dNTP-binding site The modeling in molecular of the rt194 codon has observed that the residue is located in the hinge of an α-helix which forms the B domain α-helix (DNA template-binding region) with the β-sheet of the C domain And the rtA194T may change the position

of the DNA template strand with association to the dNTP-binding site and thereby impact DNA polymerization [26]

With ETV resistance, the modeling molecular has proposed two possible pathways with rather a divert mechanisms ETV resistance In the first group, the modeling of altering at the positions rt169 and rt250 indicated that these codons alternations have

an influence on the primer-binding region of the RT, which interacts with the

3 ′terminus of the template of DNA The appearance of rt250 mutation changes the binding interaction between the primer strand and the template of DNA While residue rt169 interacts with dNTP, may have strongly related with the mutation at position rt250 The elongation of the DNA strand may be changed by the net impact

of these mutations These predictions have prolonged by in vitro studies; such as, the rtM250V in the absence of other changes confers low-level resistance to ETV, but cause a >250-fold growth in resistance to ETV in the appearance of the LMV with rtM204V mutation Moreover, with the third mutation, rtI169T, can cause further increases resistance to ETV >750-fold, but no affection by itself [28] Interestingly, the combination mutation between rtM250V and the rtM204V essentially impairs the HBV replication, but addition of the rtI169T mutation appears to promote the replication of HBV and functions as an adaptive compensatory change as well [28] Regarding the second group, the modeling of substitutions shows an altered geometry

of the binding pocket in polymerase nucleotide near YMDD site Single mutations rtS202I and rtT184G could interact and thereby impact the hydrophobic of core region between the β-sheet and the α-helix of the DNA template-binding region, which encodes two catalytic aspartate residues in the C domain The mutation substitutions at position rt184 and rt202 can predict to enlarge the affect of the LMV resistance M204V in the YMDD loop, impacting substrate discrimination upon nucleotide loading and addition to the primer terminus These altering would have to

13

interact cooperatively with the rtM204V to support a structural basis for the appeared

cross-resistance between the two drugs The recombinant constructs which including

the LMV-resistant mutations rtL180M and rtM204V in addition to either rtT184G or

rtS202I exhibited only a modest grow up in resistance to ETV with approximately

40-fold [28] Nevertheless, if all four substitutions are appeared, the resistant-ETV can

increased >750-fold [28] Interestingly, with the LAM-resistant mutations was

absence, the rtT184G and rtS202I mutations do not impact the susceptibility of HBV

to ETV in vitro [28]

Figure 4: Flow chart describes the major pathway to resistance, and other

mechanisms that may contribute to viral rebound

The mainly mutations resistance related with LAM, ADV, ETV, and LDT also have

the potential of changing the HBsAg with C-terminal region Such as, changes have

association with LAM and ETV resistance, for example with rtM204V, result in a

change at sI195M in the HBsAg, while the rtM204I altering which is related with

both LAM and LDT, is linked to three possible HBsAg changes at sW196S and

sW196L, or a termination codon sW196* The effect of the major resistance

14

mutations in LAM on the altered anti-genicity of HBsAg have been checked in vitro and been indicated to affect virus secretion and neutralization in animal models [39] Among binding assays, HBsAg expressing this NA-resistant with association residues have decreased anti-HBs binding to a level which was the same with classical vaccine escape mutant, sG145R, and so get a high public health potential [40] While the mutation rtA181T selected by LAM and ADV results in a stop codon mutation at sW172*, ADV-associated mutation rtA181V results in a modification at sL173F The HBV mutations which result in a stop codon in the envelope of gene for example those to LAM and ADV need to be rescued in trans with a low proportion of wild-type HBV in order to be capacity of viral assembly and release [41] The substitution rtN236T with ADV resistance does not impact the envelope of gene and overlaps with the stop codon at the end of the region encoding the surface antigen

The ETV resistant relation substitutions at rtI169T, rtS184G, rtS202I and also impact HBsAg and outcome in alter at sF161L, sL/V176G, and sV194F The resistant mutation rtM250V is located after at the terminal HBsAg The location sF161L within the region which was identified as the ‘a’ determinant or major hydrophilic region, that contains amino acid 90–170 of the HBsAg [42] The various possible effects from the pathways of NAs resistance are indicated in Figure 2

With multidrug resistance, several reports of complex patient histories including the sequential use of different antiviral, such as patients are at the risk of developing

“multi-drug resistant” HBV [43]

The appearance of such the complex genomic variants in HBV might be exceptionally high in the suppress immunity for example following liver transplantation [44] Specially, mutations in the HBV polymerase which has an association with resistant drug might affect on the structure of the HBV envelope (S protein) as well, due to open reading frames of these genes partially overlap For example, the mutation polymerase rtV191I which was revealed in a co-infected HBV/HIV patient during TDF-including treatment produce a stop-codon

in the overlapping surface antigen (sW182stop) These results were determined in the deletion of the terminal 44 amino acids of the HBsAg, which then lead to escape detection (“HBsAg-negativity”) in routine diagnostic serum tests [45]

15

(e) - Practical management of drug resistance

Regarding patients who were detected virological breakthrough and exclusion of non-adherence, they should be either switched to another antiviral monotherapy which has high genetic barrier to resistance (ETV or TDF) In this case, patients can also utilize second antiviral drug that contains complementary resistance profile should be added [47] Though it occurs reasonable from a virological point to decrease resistance by combination therapy, there are unclear long-term data favoring the

“add-on” over the “switch” concept Table 3 indicates the current recommendations for the management in treatment failure

Table 3: Practical management of treatment failure

Resistance to Rescue therapy

Typical treatment options for the management of HBV antiviral resistance In most cases, the switch to a monotherapy with a noncross-resistance profile is sufficient HBV: hepatitis B virus

Up to now, the incomplete information about the incidence and patterns of genotypic resistance mutations have not been extensively investigated in various parts of China Therefore, we conducted a this study to investigate the characteristics of resistant- mutations drug in nucleot(s)ide analogs treated patients with chronic hepatitis B in Tianjin, one of the biggest coast city in north China with population more than 14 million

2.2 Laboratory assessment

2.2.1 HBV DNA extraction and amplification

The HBV nucleic acid was extracted from 200 μ l patient serum samples following the operators manual The RT coding region was amplified by designed primers as follows: HBV-NY-F4, HBV-NY-R1 A typical amplification was performed in a 50 μ l reaction volume containing 24.6 μ H2O (HPLC level), 5 μ 10*PCR Buffer contain 2.0 mM MgCl2, 4 μ dNTP mix (2.5 mM each), 3 μ HBV-NY-F4, 3 μ HBV-NY-R1, 0.4 μ Taq®(5U/ μ) and 10 μ temple DNA at 94 °C for 5 min, followed by 40 cycles at

94 °C for 45 sec, 58 °C for 45 sec, and 72 °C for 90 sec, 72 °C for 5 min and final extension at 12 °C 3 ul PCR products were identified using agarose gel electrophoresis with agarose gel concentration: 1.0% and marker sample volume: 3ul at Voltage: 250V to eliminate the redundant single-strand DNA

Sample processing, PCR, products identification and digestion were done in separate laboratory rooms which were certified for molecular diagnostics using standard

17

precautions to prevent contamination

2.2.2 HBV genotyping and antiviral resistant mutation detection

The genomic sequences obtained for the HBV P gene via a direct sequencing method were compared with all HBV reference sequences used on the NCBI website

to analyze mutations in the reverse transcription (RT) region and to genotype HBV (www.ncbi.nlm.nih.gov/projects/genotyping/), as follows (accession numbers are given): subtype A, X02763, X51970, and AF090842; subtype B, D00329, AF073846, and AB602818; subtype C, X04615, AY123041, and AB014381; subtype D, X65259, M32138 and X85254; subtype E, X75657 and AB032431; subtype F, X69798, AB036910, and AF223965; subtype G, AF160501, AB064310, and AF405706; and subtype H, AY090454, AY090457, and AY090460

2.3 Statistical Analysis

All statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL,

USA) Categorical variables, like the percentages of HBV genotypes, gender and age were compared by Chi-square test (χ 2 test) A two-tailed P < 0.05 was considered to

be statistically significant.

18

3-RESULT 3.1 Clinical features of study patients

A total of 1320 NA – experienced CHB patients who visited Tianjin Second People’s Hospital and Tianjin Institute of Hepatology from July 2012 to December 2015 with Drug resistance- associated mutations located in the RT domain of the P gene was enrolled in the study The media age of these patients was 46.94 (interquartile range: 10- 80) years, and 73.79 % of them was male Two genotypes (B and C) were identified

in the 1320 patients Among them 8.71% patients were infected with genotype B, 91.29% with genotype C

Table 4: Characteristic of chronic hepatitis B patients

19

3.2 Mutation sites subgroup analysis by genotype, gender and age

3.2.1 Mutation each sites subgroup analysis by genotype, gender and age

Based on CHB patients in Tianjin, we promoted to analysis mutation each site in

1320 patients with completely clinical data and the drug resistance was detected at seventeen positions, including rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt196, rt202, rt204, rt207, rt213, rt214, rt215, rt236, rt237, rt238 and rt250 Of them, rtM204 was the most of frequently occurred with the percentage of 24.25%, follow by rt180 (14.97%) and rt181 (12.43%) Next, we further analyzed the proportion of mutation each site in different HBV genotype strains, gender and age, were all shown in Table

5 ADV – associated mutation at position rtA181 showed a remarkable higher percentage in genotype C than genotype B (13.11% vs 4.25%, p<0.001), and so did other mutations (rtS213 p=0.005, rtN236 p<0.001 rtV214 p< 0.001 and rtI233 p=0.044) And LAM- associated mutation site rtV173 with genotype C had a much higher mutation rate than genotype B (3.1% vs.0.47%, p=0.019) Nevertheless, LAM-associated mutation at rtM204 and rtL180, and ETV-associated mutations at rtT184, rtS202 and rtM250 did not differ significantly between genotype B and C (P > 0.05) When stratified with gender, LAM associated mutation at position rtV207 displayed a bigger rate in female than male (p=0.002) and so did with ETV associated mutation at rtI169 (p=0.04) However, mutation at position rtS202 in male had higher mutation proportion than female (p=0.003) With others, no difference of mutation rates between male and female were observed In the subgroup analysis stratified by age, significant differences were identified in rtL80 and rtT184 with all p<0.001, and

so did others position (rtV214 p=0.024, rtN236 p=0.001, rtQ215 p=0.005 and rtP237 p=0.007) Nonetheless, no divergences were determined in other positions

20

Table 5: The proportion mutation each site in genotype, gender and age in CHB patient population from Tianjin

(1)-With LAM associated drug resistance, we discovered 16 mutation sites, namely rtL80I, rtL80V, rtV173L, rtV173M, rtL180M, rtL180F, rtL180Q, rtM204V, rtM204I, rtM204K, rtM204L, rtM204Q, rtM204S, rtV207I, rtV207L and rtV207M In totally, rtL180M was the most frequently occurred with the most frequently occurred with 26.34%, follow by rtM204I, rtL80I and rtM204V with 24.26%, 16.33% and 12.73%, respectively Nevertheless, the distribution of resistance site had divergence in dividing terms of genotype, gender, age As shown

in Table 6, mutation site rtV207M indicated an essential higher proportion in genotype B than genotype C (5.32% vs.1.04% p=0.006), and the same to rtL80V (6.38% vs.2.08% p=0.021) Among other LAM related mutation sites did not differ significantly between genotype B and genotype C (p>0.05, all) When stratified with gender, mutation site rtV207I demonstrated a significant bigger percentage in female than male (11.46% vs 7.06% p=0.017) And the mutation site rtV207L indicated a much lower rate in male than female (0.96% vs 2.87% p=0.025) Among gender, except rtV207I and rtV207L, others showed no difference rate of mutation between male and female In the subgroup analysis stratified by age, we detected a noteworthy difference proportion in mutation site rtL180M, rtM204V, rtV207I, rtV173L and rtL180V (p<0.05, all) With other LAM -associated resistant mutation did not differ between terms of age as well

>60 (n=196)