与慢加急性肝衰竭（ACLF）相关的乙型肝炎病毒（HBV）突变位点的研究
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摘要
乙型病毒性肝炎仍是一个世界性的公共卫生问题,全球范围内乙型肝炎病毒(hepatitis B virus, HBV)的慢性感染人群高达3.5亿以上。HBV相关的慢加急性肝衰竭(acute on chronic liver failure, ACLF)是慢性肝炎患者死亡的重要原因,如果不进行肝移植,其死亡率高达60-80%,每年大约有22600的患者死于肝衰竭。长期以来宿主免疫一直被认为是乙型肝炎加剧的主要原因,但是近年的研究结果表明病毒因素在ACLF的发病中同样起重要作用。HBV是嗜肝病毒家族的一员,其DNA全长约为3200个核苷酸,呈现部分双链环状。HBV基因组包含4个相互重叠的开放性读码框,分别编码HBV表面抗原、核心抗原、聚合酶和多功能的非结构性蛋白X。由于HBV的复制过程包含前基因组RNA的逆转录,其逆转录酶缺乏校正功能,故HBV的突变率显著高于其它的DNA病毒。HBV基因组的突变率可随着病毒的复制不断增加,其中一些突变可能与慢性肝炎的急性加剧相关,可作为ACLF的预警的生物标志物和抗病毒治疗的靶标。近来有研究表明基本核心启动子区的A1762T/G1764A突变与ACLF发病相关,A1846T、G1896A和C1913A/G位点的突变在ACLF患者中发生率显著高于慢性肝炎患者。目前关于HBV突变和ACLF发病的关系主要集中于基本核心启动子区和前核心区,对HBV基因其它部位的研究尚少。ACLF是否有另外的一些HBV突变位点与ACLF发病有关尚不清楚,为此本研究用HBV全基因组测序筛选出与ACLF发病相关的HBV突变,并发现一些新的突变位点,经过扩大样本的验证,以期为ACLF的预警提供依据。
方法
1HBV全基因测序筛选与ACLF发病可能相关的HBV突变位点
1.1Illumina高通量测序。样本来自两组(12例ACLF和12例慢性轻度乙型肝炎(mildchronic hepatitis B, CHB-M))年龄配对、性别和基因型分布无显著差异患者,所有样本HBV的DNA水平均>100,000copies/mL。首先从200μl的血清中提取病毒DNA,分3段重叠扩增HBV全基因组。PCR产物在Genome Analyzer II平台上进行高通量测序,然后处理高通量测序数据。
1.2检测上述样本的肝功能指标(ALT, AST, TBIL, PTA), HBV血清标记物和HBV DNA滴度。
1.3分析GenBank数据库的HBV基因突变情况：在GenBank数据库中搜索中国地区报道的ACLF和CHB-M相关的HBV全基因序列共258条(ACLF患者序列143条,CHB-M患者序列115条),分析其突变。
2HBV突变与ACLF发病的关系的验证
2.1样本。来自80例无症状携带者(asymptomatic carriers, ASC)、152例CHB-M、102例慢性重度乙型肝炎患者(severe chronic hepatitis B, CHB-S)和104例ACLF患者,共438例。
2.2从200μ1血清中提取HBV DNA,半巢式PCR扩增HBV基因组目标序列,PCR产物行常规Sanger测序。
2.3检测上述样本肝功能指标,HBV血清标记物和HBV DNA商度。
结果
1Illumina高通量测序筛选结果
1.1作为质量控制的质粒测序结果显示：所测得的读长36bpDNA序列的不同位置发生测序错误的频率不一致,在20位碱基之后其碱基替换率呈现上升趋势,当位于序列的第30-36个碱基时,测序的错误率显著上升。为了降低错配率,我们截取每个短片段序列前面的20bp进行质粒全长的拼接。质粒序列的总体误差率为0.49%,其中每一百个核苷酸中错配碱基读取频率>4%的核苷酸平均为2.4个,将4%作为区分真实突变和技术误差的界线。
1.2T216C、G285A、A1846T、G1896A、C1913A/G、G2095A、A2159G、A2189C和G2345A为ACLF患者常见的突变位点,在12例ACLF患者中发生上述突变的病例数分别为10例、9例、10例、10例、8例、5例、10例、11例和7例。
1.3T216C、G285A、A1846T、G1896A、C1913A/G、G2095A、A2159G、A2189C和G2345A这9个位点的突变频率在ACLF患者中显著高于慢性轻度肝炎患者(P<0.05或P<0.01)。
1.4基本核心启动子区(basal core promoter, BCP)(A1762T/G1764A)突变在12例ACLF和12例CHB-M患者中的发生率均为6例。其突变频率在ACLF患者和慢性轻度肝炎患者未见显著差异。
1.5从Genbank数据库下载258条HBV全基因序列,分析上述突变的分布情况发现：T216C、G285A、A1846T、G1896A、C1913A/G、A2159G、A2189C和G1764A在ACLF中的发生率均显著高于CHB-M, G2095A、G2345A和A1762T在ACLF组和CHB-M组间未见差异。分别对HBV B基因型和C基因型患者的突变进行分析发现,在B基因型的患者中,T216C、G285A、A1846T、G1896A和C1913A/G这5个位点突变在ACLF组中显著高于CHB-M组；C基因型患者中,A1846T、C1913A/G、A2159G、A2189C和G1764A这5个突变在ACLF组中显著高于CHB-M组。A1762T/G1764A双联突变在B基因型和C基因型的ACLF患者中均未见显著升高。
2HBV点突变与ACLF发病的关系的验证结果
2.1ACLF组的B基因型比率显著高于其余三组慢性乙型肝炎患者。
2.2T216C、G285A、A1846T/G、G1896A、C1913A/G、A2159G/C和A2189T/C这7个突变的发生率在ACLF组显著高于ASC、CHB-M和CHB-S组(P<0.05或P<0.01)。
2.3T216C、G285A、A1846T/G、G1896A、C1913A/G这5个位点在HBV基因型B的感染者中显著高于HBV基因型C感染者。
2.4对ASC、CHB-M、CHB-S和ACLF患者的B基因型和C基因型毒株的突变进行比较发现：以ASCs为对照组,B基因型毒株感染者中,T216C、G285A、G1896A、 C1913A/G突变在CHB-S和ACLF组中均显著升高；C基因型毒株感染者中,A1846T/G、G1896A、C1913A/G、A2159G/C突变在ACLF组和CHB-S组中显著升高。以CHB-M为对照组,HBV B基因型毒株感染者中,T216C、G285A、G1896A、 C1913A/G和A2159G/C在ACLF患者中显著升高；C基因型毒株感染者中,G285A、A1846T/G、G1896A和C1913A/G在ACLF患者中显著升高。当以CHB-S为对照时,基因型B毒株感染者中,A2159G/C和A2189T/C在ACLF组中显著升高。
2.5与非ACLF组比较,B基因型毒株的T216C、G285A、A1846T/G、G1896A、 C1913A/G、A2159G/C和A2189T/C与ACLF密切相关,而C基因型毒株的G285A、 A1846T/G、G1896A、C1913A/G和A2159G/C与ACLF密切相关。
2.6多因素分析显示T216C、G1896A、C1913A/G和A2159G/C是ACLF发病的独立危险因素。
2.7T216C、G1896A、C1913A/G和A2159G/C的两个或以上的联合突变与ACLF发生密切相关。
2.8“含C216的联合突变”,“含A/G1913的联合突变”,“含G/C2159的联合突变”对ACLF诊断具有较高的特异性。
结论
1B基因型病毒感染的慢性乙型肝炎患者更容易发展为ACLF。
2T216C、G1896A、C1913A/G和A2159G/C突变为ACLF发生的独立危险因素。其中T216C和A2159G/C系本研究新发现的ACLF发病的独立危险因素(T216C位点已申请国际专利,国际申请号PCT/CN2012/071108),提示了HBV S区和核心区突变在ACLF发病过程中可能起着重要作用。
3HBV联合突变的分析发现,联合突变在ACLF发病中发挥更为重要的作用。“含C216的联合突变”,“含A/G1913的联合突变”,“含G/C2159的联合突变”对ACLF的诊断特异性较高。
4上述病毒突变可作为预测乙型肝炎加剧的分子标志物,为阻止ACLF发病和改善HBV感染者的预后提供依据。
Background
Infection of hepatitis B virus is one of the most serious and prevalent global health problems. More than350million people worldwide are chronically infected with HBV. HBV related acute-on-chronic liver failure (ACLF) is a main cause of death for patients with chronic hepatitis. The mortality of ACLF is60-80%, resulting in22,600deaths a year. In addition to host factors, viral factors per se could also play an important role in determining clinical outcomes of chronic hepatitis B. HBV is a small enveloped DNA virus of the Hepadnaviridae family with approximately3200nucleotides. The virus has a partially double-stranded DNA genome made of four overlapping open reading frames that encode hepatitis B surface (S) antigen, a core protein, a polymerase, and a multifunctional nonstructural protein named X. Since HBV replicates through the reverse transcription of pregenome RNA, mutations occur more frequently in HBV than in other DNA viruses. Therefore, mutations in the HBV genome may accumulate with time, and some of them might serve as viral markers for predicting the development of HBV-associated liver failure. Recently, several studies have indicated that patients with basal core promoter (BCP) mutations (A1762T/G1764A) had higher risks of ACLF. However, BCP mutations are also frequently found among patients with mild chronic hepatitis B and asymptomatic carriers. In addition to these two mutations, mutations at nt.1846, nt.1896, and nt.1913have also been found to be associated with the development of ACLF. All of these studies focused mainly on the particular portions of the HBV genome (e.g. BCP and precore (PC) regions), and rarely analyzed the other locus that might play important roles. Hence, it remains unclear whether other predictive markers might be found by comparative analysis of the complete HBV genomes between different stages of hepatitis B. The aims of present investigation were to screen new variants of HBV related to the risk of ACLF development and to find some specific biomarkers in the HBV genome for chronic hepatitis B progression.
Methods
1Illumina high-throughput sequencing
1.1The samples from two groups (12ACLF patients and12mild chronic hepatitis B (CHB-M)) were analyzed with Illumina high-throughput sequencing. There were no significant differences of age, sex and genotype distribution between two groups. The plasma HBV DNA levels of these patients were>100,000copies/mL. HBV DNA was extracted from200μl serum and three primers pairs were used to amplify3overlapping HBV full-length fragments. PCR products were utilized for the high-throughput sequencing, which was carried out on a Genome Analyzer II platform. Then the high-throughput sequencing data were analyzed.
1.2The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers and the titer of HBV DNA were measured.
1.3Analysis of mutations.258full genome sequences of HBV subimitted by Chinese were found from the Genbank database and analyzed.
2HBV mutations were validated by direct sequencing in large samples
2.1The samples were from438subjects (80asymptomatic carriers (ASC),152CHB-M patients,102severe chronic hepatitis B (CHB-S) patients and104ACLF patients).
2.2HBV DNA was extracted from200μl serum, and semi-nest PCR was used to amplify the target regions of HBV genome. The nucleotide sequences of PCR products were carried out by Sanger sequencing.
2.3The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers, the titer of HBV DNA in438subjects were measured.
Results
1Result of Illumina high-throughput sequencing:
1.1An examination of the qualities revealed that lower values were more common near the end of the reads (positions20and higher in reads of length36), so we trimmed the last16nuclotieds before the alignment. The overall plasmid sequence error rate was0.49%, and the mean number of nucleotides with>4%mismatch error rate per one hundred nucleotides was2.4. According to this result,4%was empirically served as the limit to distinguish the authentic variants from technological artifacts.
1.2In ACLF group, the mutations at nine sites (T216C, G285A, A1846T, G1896A, C1913A/G, G2095A, A2159G, A2189C and G2345A) were frequently found.
1.3Mutation frequencies at nt.216, nt.285, nt.1846, nt.1896, nt.1913, nt.2095, nt.2159, nt.2189and nt.2345of twelve ACLF patients were significantly higher than those of twelve CHB-M (P<0.05or P<0.01)
1.4The cases with mutation at nt.1762/nt.1764were six in both ACLF group and CHB-M group. Mutation frequencies of BCP mutations (A1762T, G1764A) were not significantly higher in12ACLF patients than in12CHB-M (P>0.05).
1.5Based on the analysis of258HBV genomic sequences downloaded from Genbank database, we found that in genotype B virus, the prevalence of T216C, G285A, A1846T, G1896A and C1913A/G were significantly higher in ACLF as compared with CHB-M; in genotype C virus, the prevalence of A1846T, C1913A/G, A2159G, A2189C and G1764A were significantly higher in ACLF. The prevalence of A1762T/G1764A double mutations was not much higher in ACLF as compared with CHB-M.
2Result of the validation
2.1A significantly higher ratio of genotype B to C was found in patients with ACLF than in patients with non-ACLF.
2.2The prevalence of seven mutations (T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C)in ACLF group enhanced significantly, as compared with ASC, CHB-M, and CHB-S groups (P<0.05).
2.3The prevalence of five mutations (T216C, G285A, A1846T/G, G1896A and C1913A/G) was significantly higher in genotype B virus than in genotype C virus.
2.4As compared with ASCs, mutations at nt.216, nt.285, nt.1896, nt.1913in genotype B were associated significantly with an increased risk of CHB-S and ACLF. respectively. In subjects with genotype C virus, prevalence of mutations at nt.1846, nt.1896, nt.1913, and nt.2159were significantly higher in ACLF and CHB-S than those in ASC. As compared with patients with CHB-M, mutations at nt.216, nt.285, nt.1896, nt.1913and nt.2159were significantly associated with an increased risk of ACLF in genotype B, whereas mutations at nt.285, nt.1846, nt.1896, and nt.1913were significantly associated with an increased risk of ACLF in genotype C. As compared with the patients with CHB-S, mutations at nt.2159and nt.2189in genotype B were significantly associated with an increased risk of ACLF.
2.5T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C in genotype B were each associated with ACLF compared with non-ACLF, whereas G285A, A1846T/G, G1896A, C1913A/G, and A2159G/C in genotype C were each associated with ACLF compared with non-ACLF.
2.6Multivariate analysis showed that T216C, G1896A, C1913A/G and A2159G/C were independent risk factors for ACLF.
2.7Combinations with any2or more of T216C, G1896A, C1913A/G and A2159G/C were significantly associated with the development of ACLF.
2.8C216in any combination, A/G1913in any combination, and G/C2159in any combination had high specificity for ACLF.
Conclusions
1The patients with CHB infected with genotype B virus were more prone to development of ACLF than those with genotype C virus.
2T216C, G1896A, C1913A/G and A2159G/C were independent factors associated with ACLF. T216C and A2159G/C mutations were novel independent risk factors related to ACLF development, which highlighted the influence of genetic variants in the HBV S gene and core gene on the progression of hepatitis B.
3A combined examination of different viral mutations could predict more precisely the progression of liver disease. C216in any combination, A/G1913in any combination, and G/C2159in any combination were specific for ACLF.
4These virus mutations, as molecular biomarkers, may be useful for preventing ACLF development and predicting prognosis of patients with hepatitis B.
方法
1HBV全基因测序筛选与ACLF发病可能相关的HBV突变位点
1.1Illumina高通量测序。样本来自两组(12例ACLF和12例慢性轻度乙型肝炎(mildchronic hepatitis B, CHB-M))年龄配对、性别和基因型分布无显著差异患者,所有样本HBV的DNA水平均>100,000copies/mL。首先从200μl的血清中提取病毒DNA,分3段重叠扩增HBV全基因组。PCR产物在Genome Analyzer II平台上进行高通量测序,然后处理高通量测序数据。
1.2检测上述样本的肝功能指标(ALT, AST, TBIL, PTA), HBV血清标记物和HBV DNA滴度。
1.3分析GenBank数据库的HBV基因突变情况：在GenBank数据库中搜索中国地区报道的ACLF和CHB-M相关的HBV全基因序列共258条(ACLF患者序列143条,CHB-M患者序列115条),分析其突变。
2HBV突变与ACLF发病的关系的验证
2.1样本。来自80例无症状携带者(asymptomatic carriers, ASC)、152例CHB-M、102例慢性重度乙型肝炎患者(severe chronic hepatitis B, CHB-S)和104例ACLF患者,共438例。
2.2从200μ1血清中提取HBV DNA,半巢式PCR扩增HBV基因组目标序列,PCR产物行常规Sanger测序。
2.3检测上述样本肝功能指标,HBV血清标记物和HBV DNA商度。
结果
1Illumina高通量测序筛选结果
1.1作为质量控制的质粒测序结果显示：所测得的读长36bpDNA序列的不同位置发生测序错误的频率不一致,在20位碱基之后其碱基替换率呈现上升趋势,当位于序列的第30-36个碱基时,测序的错误率显著上升。为了降低错配率,我们截取每个短片段序列前面的20bp进行质粒全长的拼接。质粒序列的总体误差率为0.49%,其中每一百个核苷酸中错配碱基读取频率>4%的核苷酸平均为2.4个,将4%作为区分真实突变和技术误差的界线。
1.2T216C、G285A、A1846T、G1896A、C1913A/G、G2095A、A2159G、A2189C和G2345A为ACLF患者常见的突变位点,在12例ACLF患者中发生上述突变的病例数分别为10例、9例、10例、10例、8例、5例、10例、11例和7例。
1.3T216C、G285A、A1846T、G1896A、C1913A/G、G2095A、A2159G、A2189C和G2345A这9个位点的突变频率在ACLF患者中显著高于慢性轻度肝炎患者(P<0.05或P<0.01)。
1.4基本核心启动子区(basal core promoter, BCP)(A1762T/G1764A)突变在12例ACLF和12例CHB-M患者中的发生率均为6例。其突变频率在ACLF患者和慢性轻度肝炎患者未见显著差异。
1.5从Genbank数据库下载258条HBV全基因序列,分析上述突变的分布情况发现：T216C、G285A、A1846T、G1896A、C1913A/G、A2159G、A2189C和G1764A在ACLF中的发生率均显著高于CHB-M, G2095A、G2345A和A1762T在ACLF组和CHB-M组间未见差异。分别对HBV B基因型和C基因型患者的突变进行分析发现,在B基因型的患者中,T216C、G285A、A1846T、G1896A和C1913A/G这5个位点突变在ACLF组中显著高于CHB-M组；C基因型患者中,A1846T、C1913A/G、A2159G、A2189C和G1764A这5个突变在ACLF组中显著高于CHB-M组。A1762T/G1764A双联突变在B基因型和C基因型的ACLF患者中均未见显著升高。
2HBV点突变与ACLF发病的关系的验证结果
2.1ACLF组的B基因型比率显著高于其余三组慢性乙型肝炎患者。
2.2T216C、G285A、A1846T/G、G1896A、C1913A/G、A2159G/C和A2189T/C这7个突变的发生率在ACLF组显著高于ASC、CHB-M和CHB-S组(P<0.05或P<0.01)。
2.3T216C、G285A、A1846T/G、G1896A、C1913A/G这5个位点在HBV基因型B的感染者中显著高于HBV基因型C感染者。
2.4对ASC、CHB-M、CHB-S和ACLF患者的B基因型和C基因型毒株的突变进行比较发现：以ASCs为对照组,B基因型毒株感染者中,T216C、G285A、G1896A、 C1913A/G突变在CHB-S和ACLF组中均显著升高；C基因型毒株感染者中,A1846T/G、G1896A、C1913A/G、A2159G/C突变在ACLF组和CHB-S组中显著升高。以CHB-M为对照组,HBV B基因型毒株感染者中,T216C、G285A、G1896A、 C1913A/G和A2159G/C在ACLF患者中显著升高；C基因型毒株感染者中,G285A、A1846T/G、G1896A和C1913A/G在ACLF患者中显著升高。当以CHB-S为对照时,基因型B毒株感染者中,A2159G/C和A2189T/C在ACLF组中显著升高。
2.5与非ACLF组比较,B基因型毒株的T216C、G285A、A1846T/G、G1896A、 C1913A/G、A2159G/C和A2189T/C与ACLF密切相关,而C基因型毒株的G285A、 A1846T/G、G1896A、C1913A/G和A2159G/C与ACLF密切相关。
2.6多因素分析显示T216C、G1896A、C1913A/G和A2159G/C是ACLF发病的独立危险因素。
2.7T216C、G1896A、C1913A/G和A2159G/C的两个或以上的联合突变与ACLF发生密切相关。
2.8“含C216的联合突变”,“含A/G1913的联合突变”,“含G/C2159的联合突变”对ACLF诊断具有较高的特异性。
结论
1B基因型病毒感染的慢性乙型肝炎患者更容易发展为ACLF。
2T216C、G1896A、C1913A/G和A2159G/C突变为ACLF发生的独立危险因素。其中T216C和A2159G/C系本研究新发现的ACLF发病的独立危险因素(T216C位点已申请国际专利,国际申请号PCT/CN2012/071108),提示了HBV S区和核心区突变在ACLF发病过程中可能起着重要作用。
3HBV联合突变的分析发现,联合突变在ACLF发病中发挥更为重要的作用。“含C216的联合突变”,“含A/G1913的联合突变”,“含G/C2159的联合突变”对ACLF的诊断特异性较高。
4上述病毒突变可作为预测乙型肝炎加剧的分子标志物,为阻止ACLF发病和改善HBV感染者的预后提供依据。
Background
Infection of hepatitis B virus is one of the most serious and prevalent global health problems. More than350million people worldwide are chronically infected with HBV. HBV related acute-on-chronic liver failure (ACLF) is a main cause of death for patients with chronic hepatitis. The mortality of ACLF is60-80%, resulting in22,600deaths a year. In addition to host factors, viral factors per se could also play an important role in determining clinical outcomes of chronic hepatitis B. HBV is a small enveloped DNA virus of the Hepadnaviridae family with approximately3200nucleotides. The virus has a partially double-stranded DNA genome made of four overlapping open reading frames that encode hepatitis B surface (S) antigen, a core protein, a polymerase, and a multifunctional nonstructural protein named X. Since HBV replicates through the reverse transcription of pregenome RNA, mutations occur more frequently in HBV than in other DNA viruses. Therefore, mutations in the HBV genome may accumulate with time, and some of them might serve as viral markers for predicting the development of HBV-associated liver failure. Recently, several studies have indicated that patients with basal core promoter (BCP) mutations (A1762T/G1764A) had higher risks of ACLF. However, BCP mutations are also frequently found among patients with mild chronic hepatitis B and asymptomatic carriers. In addition to these two mutations, mutations at nt.1846, nt.1896, and nt.1913have also been found to be associated with the development of ACLF. All of these studies focused mainly on the particular portions of the HBV genome (e.g. BCP and precore (PC) regions), and rarely analyzed the other locus that might play important roles. Hence, it remains unclear whether other predictive markers might be found by comparative analysis of the complete HBV genomes between different stages of hepatitis B. The aims of present investigation were to screen new variants of HBV related to the risk of ACLF development and to find some specific biomarkers in the HBV genome for chronic hepatitis B progression.
Methods
1Illumina high-throughput sequencing
1.1The samples from two groups (12ACLF patients and12mild chronic hepatitis B (CHB-M)) were analyzed with Illumina high-throughput sequencing. There were no significant differences of age, sex and genotype distribution between two groups. The plasma HBV DNA levels of these patients were>100,000copies/mL. HBV DNA was extracted from200μl serum and three primers pairs were used to amplify3overlapping HBV full-length fragments. PCR products were utilized for the high-throughput sequencing, which was carried out on a Genome Analyzer II platform. Then the high-throughput sequencing data were analyzed.
1.2The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers and the titer of HBV DNA were measured.
1.3Analysis of mutations.258full genome sequences of HBV subimitted by Chinese were found from the Genbank database and analyzed.
2HBV mutations were validated by direct sequencing in large samples
2.1The samples were from438subjects (80asymptomatic carriers (ASC),152CHB-M patients,102severe chronic hepatitis B (CHB-S) patients and104ACLF patients).
2.2HBV DNA was extracted from200μl serum, and semi-nest PCR was used to amplify the target regions of HBV genome. The nucleotide sequences of PCR products were carried out by Sanger sequencing.
2.3The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers, the titer of HBV DNA in438subjects were measured.
Results
1Result of Illumina high-throughput sequencing:
1.1An examination of the qualities revealed that lower values were more common near the end of the reads (positions20and higher in reads of length36), so we trimmed the last16nuclotieds before the alignment. The overall plasmid sequence error rate was0.49%, and the mean number of nucleotides with>4%mismatch error rate per one hundred nucleotides was2.4. According to this result,4%was empirically served as the limit to distinguish the authentic variants from technological artifacts.
1.2In ACLF group, the mutations at nine sites (T216C, G285A, A1846T, G1896A, C1913A/G, G2095A, A2159G, A2189C and G2345A) were frequently found.
1.3Mutation frequencies at nt.216, nt.285, nt.1846, nt.1896, nt.1913, nt.2095, nt.2159, nt.2189and nt.2345of twelve ACLF patients were significantly higher than those of twelve CHB-M (P<0.05or P<0.01)
1.4The cases with mutation at nt.1762/nt.1764were six in both ACLF group and CHB-M group. Mutation frequencies of BCP mutations (A1762T, G1764A) were not significantly higher in12ACLF patients than in12CHB-M (P>0.05).
1.5Based on the analysis of258HBV genomic sequences downloaded from Genbank database, we found that in genotype B virus, the prevalence of T216C, G285A, A1846T, G1896A and C1913A/G were significantly higher in ACLF as compared with CHB-M; in genotype C virus, the prevalence of A1846T, C1913A/G, A2159G, A2189C and G1764A were significantly higher in ACLF. The prevalence of A1762T/G1764A double mutations was not much higher in ACLF as compared with CHB-M.
2Result of the validation
2.1A significantly higher ratio of genotype B to C was found in patients with ACLF than in patients with non-ACLF.
2.2The prevalence of seven mutations (T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C)in ACLF group enhanced significantly, as compared with ASC, CHB-M, and CHB-S groups (P<0.05).
2.3The prevalence of five mutations (T216C, G285A, A1846T/G, G1896A and C1913A/G) was significantly higher in genotype B virus than in genotype C virus.
2.4As compared with ASCs, mutations at nt.216, nt.285, nt.1896, nt.1913in genotype B were associated significantly with an increased risk of CHB-S and ACLF. respectively. In subjects with genotype C virus, prevalence of mutations at nt.1846, nt.1896, nt.1913, and nt.2159were significantly higher in ACLF and CHB-S than those in ASC. As compared with patients with CHB-M, mutations at nt.216, nt.285, nt.1896, nt.1913and nt.2159were significantly associated with an increased risk of ACLF in genotype B, whereas mutations at nt.285, nt.1846, nt.1896, and nt.1913were significantly associated with an increased risk of ACLF in genotype C. As compared with the patients with CHB-S, mutations at nt.2159and nt.2189in genotype B were significantly associated with an increased risk of ACLF.
2.5T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C in genotype B were each associated with ACLF compared with non-ACLF, whereas G285A, A1846T/G, G1896A, C1913A/G, and A2159G/C in genotype C were each associated with ACLF compared with non-ACLF.
2.6Multivariate analysis showed that T216C, G1896A, C1913A/G and A2159G/C were independent risk factors for ACLF.
2.7Combinations with any2or more of T216C, G1896A, C1913A/G and A2159G/C were significantly associated with the development of ACLF.
2.8C216in any combination, A/G1913in any combination, and G/C2159in any combination had high specificity for ACLF.
Conclusions
1The patients with CHB infected with genotype B virus were more prone to development of ACLF than those with genotype C virus.
2T216C, G1896A, C1913A/G and A2159G/C were independent factors associated with ACLF. T216C and A2159G/C mutations were novel independent risk factors related to ACLF development, which highlighted the influence of genetic variants in the HBV S gene and core gene on the progression of hepatitis B.
3A combined examination of different viral mutations could predict more precisely the progression of liver disease. C216in any combination, A/G1913in any combination, and G/C2159in any combination were specific for ACLF.
4These virus mutations, as molecular biomarkers, may be useful for preventing ACLF development and predicting prognosis of patients with hepatitis B.
引文
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[1]Inoue J. Factors involved in the development of fulminant hepatitis B:Are the mutations of hepatitis B virus implicated?. Hepatol Res 2009; 39:1053-5.
[2]Sugiyama M, Tanaka Y, Kurbanov F et al. Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology 2007 1; 365:285-91.
[3]Jammeh S, Tavner F, Watson R, et al. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 2008; 89:901-9
[4]Ozasa A, Tanaka Y, Orito E, et al. Influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis B virus infection. Hepatology 2006; 44:326-334.
[5]Nagasaki F, Ueno Y, Niitsuma H,et al. Analysis of the Entire Nucleotide Sequence of Hepatitis B Causing Consecutive Cases of Fatal Fulminant Hepatitis in Miyagi Prefecture Japan. J Med Virol 2008; 80:967-73.
[6]Xu Z, Ren X, Liu Y, et al. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease:an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure. J Gastroenterol 2011; 46:391-400.
[7]Sainokami S, Abe K, Sato A, et al. Initial load of hepatitis B virus(HBV), its changing profile, and precore/core promoter mutations correlate with the severity and outcome of acute HBV infection. J Gastroenterol 2007; 42:241-9.
[8]Hou J, Lin Y, Waters J, et al. Detection and significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis. J Gen Virol 2002; 83:2291-8.
[9]任晓强,许智慧,刘志国等.958例乙型肝炎患者HBV前C/BCP区变异检测及其意义分析.解放军医学杂志2009；34：663-665.
[10]Kusakabe A, Tanaka Y, Mochida S,et al. Case-control study for the identification of virological factors associated with fulminant hepatitis B. Hepatol Res 2009; 39:648-56.
[11]Ikegami T, Matsuki Y, Tanaka Y, et al. Impact of determination of hepatitis B virus subgenotype and pre-core/core-promoter mutation for the prediction of acute exacerbation of asymptomatic carriers. Hepatol Res 2009; 39:341-5.
[12]Ren X, Xu Z, Liu Y, et al. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis. J Viral Hepat 2010; 17:887-95
[13]Chen CY, Crowther C, Kew MC.et al. A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen. Hepatol Res 2008; 38:580-92.
[14]Guarnieri M, Kim KH, Bang G, et al. Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression. J Virol 2006; 80:587-95.
[15]Inoue J, Ueno Y, Nagasaki F, et al. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 2009; 395:202-9.
[16]Parekh S, Zoulim F, Ahn SH, et al. Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants. J Virol 2003; 77:6601-12.
[17]Liu CJ, Kao JH, Lai MY, et al. Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis. J Med Virol 2004; 72:545-550.
[18]Farci P, Diaz G, Chen Z, et al. B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure. Proc Natl Acad Sci U S A 2010; 107:8766-71.
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[20]Sugiyama M, Tanaka Y, Kurbanov F, et al. Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology 2007; 365:285-291.
[21]Seyec JL, Chouteau P, Cannie I, et al. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol 1999; 73:2052-7.
[22]Bock CT, Kubicka S, Manns MP,et al. Two control elements in the hepatitis B virus S-promoter are important for full promoter activity mediated by CCAAT-binding factor. Hepatology 1999; 29:1236-47.
[23]Chen Y, Michitaka K, Matsubara H, et al. Complete genome sequence of hepatitis B virus (HBV) from a patient with fuhninant hepatitis without precore and core promoter mutations:comparison with HBV from a patient with acute hepatitis infected from the same infectious source. J hepatol 2003; 38:84-90
[24]Pollicino T, Zanetti AR, Cacciola I, et al.Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis. Hepatology 1997; 26:495-499.
[25]Chua PK, Wang RY, Lin MH, et al. Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus. J Virol 2005; 79:13483-96.
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[1]1 Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003; 23:47-58.
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[4]Gu X, Qi P, Zhou F, Ji Q, et al.+49G/A polymorphism in the cytotoxic T-lymphocyte antigen-4 gene increases susceptibility to hepatitis B-related hepatocellular carcinoma in a male Chinese population. Hum Immunol 2010; 71: 83-87.
[5]Sojka DK, Hughson A, Fowell DJ, et al. CTLA-4 is required by CD4+CD25+ Treg to control CD4+ T-cell lymphopeniainduced proliferation. Eur J Immunol 2009; 39:1544-1551.
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[1]Inoue J. Factors involved in the development of fulminant hepatitis B:Are the mutations of hepatitis B virus implicated?. Hepatol Res 2009; 39:1053-5.
[2]Sugiyama M, Tanaka Y, Kurbanov F et al. Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology 2007 1; 365:285-91.
[3]Jammeh S, Tavner F, Watson R, et al. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 2008; 89:901-9
[4]Ozasa A, Tanaka Y, Orito E, et al. Influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis B virus infection. Hepatology 2006; 44:326-334.
[5]Nagasaki F, Ueno Y, Niitsuma H,et al. Analysis of the Entire Nucleotide Sequence of Hepatitis B Causing Consecutive Cases of Fatal Fulminant Hepatitis in Miyagi Prefecture Japan. J Med Virol 2008; 80:967-73.
[6]Xu Z, Ren X, Liu Y, et al. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease:an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure. J Gastroenterol 2011; 46:391-400.
[7]Sainokami S, Abe K, Sato A, et al. Initial load of hepatitis B virus(HBV), its changing profile, and precore/core promoter mutations correlate with the severity and outcome of acute HBV infection. J Gastroenterol 2007; 42:241-9.
[8]Hou J, Lin Y, Waters J, et al. Detection and significance of a G1862T variant of hepatitis B virus in Chinese patients with fulminant hepatitis. J Gen Virol 2002; 83:2291-8.
[9]任晓强,许智慧,刘志国等.958例乙型肝炎患者HBV前C/BCP区变异检测及其意义分析.解放军医学杂志2009；34：663-665.
[10]Kusakabe A, Tanaka Y, Mochida S,et al. Case-control study for the identification of virological factors associated with fulminant hepatitis B. Hepatol Res 2009; 39:648-56.
[11]Ikegami T, Matsuki Y, Tanaka Y, et al. Impact of determination of hepatitis B virus subgenotype and pre-core/core-promoter mutation for the prediction of acute exacerbation of asymptomatic carriers. Hepatol Res 2009; 39:341-5.
[12]Ren X, Xu Z, Liu Y, et al. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis. J Viral Hepat 2010; 17:887-95
[13]Chen CY, Crowther C, Kew MC.et al. A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen. Hepatol Res 2008; 38:580-92.
[14]Guarnieri M, Kim KH, Bang G, et al. Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression. J Virol 2006; 80:587-95.
[15]Inoue J, Ueno Y, Nagasaki F, et al. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 2009; 395:202-9.
[16]Parekh S, Zoulim F, Ahn SH, et al. Genome Replication, Virion Secretion, and e Antigen Expression of Naturally Occurring Hepatitis B Virus Core Promoter Mutants. J Virol 2003; 77:6601-12.
[17]Liu CJ, Kao JH, Lai MY, et al. Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis. J Med Virol 2004; 72:545-550.
[18]Farci P, Diaz G, Chen Z, et al. B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure. Proc Natl Acad Sci U S A 2010; 107:8766-71.
[19]Nakayama J, Nakanishi T, Obatake T, et al. Fulminant hepatitis caused by a hepatitis B virus core region variant strain. J Hepatol 1995; 23:199-203.
[20]Sugiyama M, Tanaka Y, Kurbanov F, et al. Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology 2007; 365:285-291.
[21]Seyec JL, Chouteau P, Cannie I, et al. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol 1999; 73:2052-7.
[22]Bock CT, Kubicka S, Manns MP,et al. Two control elements in the hepatitis B virus S-promoter are important for full promoter activity mediated by CCAAT-binding factor. Hepatology 1999; 29:1236-47.
[23]Chen Y, Michitaka K, Matsubara H, et al. Complete genome sequence of hepatitis B virus (HBV) from a patient with fuhninant hepatitis without precore and core promoter mutations:comparison with HBV from a patient with acute hepatitis infected from the same infectious source. J hepatol 2003; 38:84-90
[24]Pollicino T, Zanetti AR, Cacciola I, et al.Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis. Hepatology 1997; 26:495-499.
[25]Chua PK, Wang RY, Lin MH, et al. Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus. J Virol 2005; 79:13483-96.
[26]Khan N, Guarnieri M, Ahn SH, et al. Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene. J Virol 2004; 78:3262-70.
[27]Kalinina T, Riu A, Fischer L, et al. A dominant hepatitis B virus population defective in virus secretion because of several S- gene mutations from a patient with fulminant hepatitis. Hepatology 2001; 34:385-94.
[28]Ohkawa K, Takehara T, Ishida H, et al. Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion. Biochem Biophys Res Commun 2010; 394:87-93.
[29]Ayres A, Bartholomeusz A, Lau G, et al. Lamivudine and Famciclovir resistant hepatitis B virus associated with fatal hepatic failure. J Clin Virol 2003; 27:111-116.
[30]Baumert TF, Thimme R, von Weizsacker F. Pathogenesis of hepatitis B virus infection. World J Gastroenterol 2007; 13:82-90.
[1]1 Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003; 23:47-58.
[2]Thio CL, Mosbruger TL, Kaslow RA, et al. Cytotoxic T-lymphocyte antigen 4 gene and recovery from hepatitis B virus infection. J Virol 2004; 78: 11258-11262.
[3]Mohammad Alizadeh AH, Hajilooi M, Ranjbar M, et al. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B. World J Gastroenterol 2006; 12:630-635.
[4]Gu X, Qi P, Zhou F, Ji Q, et al.+49G/A polymorphism in the cytotoxic T-lymphocyte antigen-4 gene increases susceptibility to hepatitis B-related hepatocellular carcinoma in a male Chinese population. Hum Immunol 2010; 71: 83-87.
[5]Sojka DK, Hughson A, Fowell DJ, et al. CTLA-4 is required by CD4+CD25+ Treg to control CD4+ T-cell lymphopeniainduced proliferation. Eur J Immunol 2009; 39:1544-1551.
[6]Masteller EL, Chuang E, Mullen AC, et al. Structural analysis of CTLA-4 function in vivo. J Immunol 2000; 164:5319-5327.
[7]Kirman J, McCoy K, Hook S, et al. CTLA-4 blockade enhances the immune response induced by mycobacterial infection but does not lead to increased protection. Infect. Immun 1999; 67:3786-3792.
[8]Pedicord VA, Montalvo W, Leiner IM, et al. Single dose of anti-CTLA-4 enhances CD8+ T-cell memory formation, function, and maintenance. Proc Natl Acad Sci USA 2011; 108:266-2671.
[9]Karandikar NJ, Vanderlugt CL, Walunas TL, et al. CTLA-4:a negative regulator of autoimmune disease. J Exp Med 1996; 184:783-788.
[10]Kavvoura FK, Ioannidis JP, et al. CTLA-4 gene polymorphisms and susceptibility to type 1 diabetes mellitus:a HuGE Review and meta-analysis. Am J Epidemiol 162:3-16,2005.
[11]Zhao SX, Pan CM, Cao HM, et al. Association of the CTLA4 gene with Graves' disease in the Chinese Han population. PLoS One 2010; 5:e9821.
[12]Lee YH, Harley JB, Nath SK, et al. CTLA-4 polymorphisms and systemic lupus erythematosus (SLE):a meta-analysis. Hum Genet 2005; 116:361-367.
[13]Balic I, Angel B, Codner E, et al. Association of CTLA-4 polymorphisms and clinical-i
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