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Comprehensive analysis of chronic hepatitis C by large-scale viral genome wide analysis

通过大规模病毒全基因组分析对慢性丙型肝炎进行综合分析

基本信息

批准号：
20390206
负责人：
ENOMOTO Nobuyuki
金额：
$ 11.98万
依托单位：
University of Yamanashi
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

The virological response to pegylated-interferon/ribavirin therapy (PEG-IFN/RBV) for chronic hepatitis C virus (HCV)-1b infection is quite diverse. However, contribution of virological factors in determining early responses as well as the final outcome in association with interleukin 28B (IL28B) single nucleotide polymorphisms (SNPs) is not understood comprehensively. We undertook a retrospective cohort analysis for 124 consecutive HCV-1b Japanese patients treated by PEG-IFN/RBV. After classification of patients by the initial responses at week 12, the pretreatment dominant complete HCV amino acid sequences in those patients were subjected to systematic sliding window analysis in association with IL28B SNPs analyses (rs8099917) to characterize response-specific viral sequences. One hundred three patients were eligible for the study. When complete HCV open reading frames (ORFs) were compared between patients with (n=10) and without (n=93) a rapid viral response (RVR), NS5A aa2224 to 224 … More 8, a part of interferon sensitivity determining region (ISDR), was extracted as the region most significantly associated with this favorable response (p=4E-04). Between non-end of treatment response (non-EVR) (n=32) and the remainder (n=71), core aa.70 was extracted (p=7.0E-08). IL28B SNP was correlated significantly with the polymorphisms of core 70 (p=3.4E-06). On the other hand, the final sustained virological response (SVR) rate was most significantly correlated with NS5A aa2340 to 2382, almost completely coinciding with the interferon ribavirin response determining region (IRRDR) (p=1.2E-07). In PEG-IFN/RBV therapy, polymorphisms in IL28B, NS5A-ISDR, core, and most importantly, NS5A-IRRDR have a tremendous influence on the treatment response in association with the viral kinetics, resulting in significantly different outcomes in chronic HCV-1b infection.The association between hepatitis C virus (HCV) sequences along with interleukin 28B (IL28B) SNP in the development of hepatocellular carcinoma (HCC) has not been well clarified. (1) Complete HCV open reading frame sequences were determined in 20 patients developing HCC and 23 non-HCC patients with HCV-1b infection in 2 distant time points. (2) An additional 230 patients were studied for core and NS5A sequences with HCC development. (3) 98 patients were investigated for changes in the viral core sequences over time. (4) IL28B SNP was investigated in 228 patients. (1) During observation period (HCC for 10.8 years, and non-HCC for 11.1 years), changes of core a.a. 70 and three amino acid positions in NS5A were characteristic of the patients developing HCC. (2) In 230 patients, the Q/H:R ratio at core a.a. 70 was significantly higher in the HCC group (HCC group 43:22 vs. non-HCC group 66:99, p=0.001). (3) A change of core R70Q was observed over time in eleven patients associated with a decrease in platelets (p=.005) and albumin over time (p=.005) or with HCC occurrence (6/11, 54.5%), whilst a Q70R change was observed in four patients without associated changes in platelets (n.s.) and albumin (n.s.), or HCC occurrence (0/4, 0%). (4) The IL28B SNP showed significant correlation with the core 70 residue. HCV core a.a.70 residue and its changes over time were associated with HCC development in genotype-1b infection in close correlation with the IL28B SNP.Protease inhibitor (PI)-resistant hepatitis C virus (HCV) variants may be present in substantial numbers in PI-untreated patients according to recent reports. However, influence of these viruses in the clinical course of chronic hepatitis C has not been well characterized. The dominant HCV nonstructural 3 (NS3) amino acid sequences were determined in 261 HCV genotype 1b-infected Japanese patients before pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy, and investigated the patients' clinical characteristics as well as treatment responses including sustained virological response (SVR) rate. HCV-NS3 sequences were also determined in 39 non-SVR patients after completion of the therapy. Four single mutations (T54S, Q80K, I153V and D168E) known to confer PI resistance were found in 35 of 261 patients (13.4%), and double mutations (I153V+T54S/D168E) were found in 6 patients (2.3%). Responses to PEG-IFN/RBV therapy did not differ between patients with and without PI-resistance mutations (mutation group, SVR 48% ; wild-type group, SVR 40% ; P=0.38). On the other hand, 2 mutations appeared in 2 non-SVR patients after PEG-IFN/RBV therapy (I153V and E168D, 5.1%). PI resistance-associated NS3 mutations exist in a substantial proportion of untreated HCV-1b-infected patients. The impact of these mutations in the treatment of PIs is unclear, but clinicians should pay attention to avoid further development of PI resistance.A proportion of patients infected with genotype 2a hepatitis C virus (HCV) cannot achieve a sustained virological response (SVR) to pegylated-interferon plus ribavirin therapy (PEG-IFN /RBV) but the reason remains unclear. The present study aimed to clarify the possible correlation between viral sequence variations and final outcome. The pretreatment complete open reading frame (ORF) sequences of genotype 2a HCV were determined by direct sequencing for two independent groups of patients (43 patients as test ; group 1 and 35 as validation ; group 2), and the correlation with the final outcome was explored. Patients with SVR (n=58) and with non-SVR (n=20) differed significantly in pretreatment HCV RNA level (p=0.002), fibrosis score (p=0.047), and cumulative ribavirin dosage (p=0.003). By comparison of all amino acid positions in the complete HCV ORFs, threonine at amino acid (aa) 110 in the core region was remarkably frequent in SVR (p=0.01 for group 1, p=0.004 for group 2, and p=5E-05 for combined). A sliding window analysis revealed that the total numbers of amino acid variations within the NS5A aa 2258 to 2306 region were significantly high in SVR compared to non-SVR patients (p=0.01 for group 1, p=0.006 for group 2, and p=0.0006 for combined). Multivariate analyses revealed that core aa 110 (p=0.02), NS5A aa 2258-2306 (p=0.03), and cumulative ribavirin dosage (p=0.02) were identified as independent variables associated with the final outcome. The outcome of PEG-IFN/RBV therapy is significantly influenced by variation in the core and NS5A regions in genotype 2a HCV infection. Less

聚乙二醇干扰素/利巴韦林治疗（PEG-IFN/RBV）治疗慢性丙型肝炎病毒(HCV)-1b感染的病毒学反应是相当多样化的。然而，病毒学因素在决定早期反应以及与白细胞介素28B （IL28B）单核苷酸多态性（snp）相关的最终结果中的作用尚未得到全面了解。我们对124名连续接受PEG-IFN/RBV治疗的日本HCV-1b患者进行了回顾性队列分析。根据第12周的初始反应对患者进行分类后，对这些患者的预处理优势完整HCV氨基酸序列进行系统滑动窗口分析，并与IL28B snp分析（rs8099917）相关联，以表征反应特异性病毒序列。103名患者符合这项研究的条件。当比较有（n=10）和没有（n=93）快速病毒反应（RVR）的患者的完整HCV开放阅读框（orf）时，NS5A aa2224至224…More 8，干扰素敏感性决定区（ISDR）的一部分，被提取为与这种有利反应最显著相关的区域（p=4E-04）。在治疗未结束反应（non-EVR）（n=32）和剩余（n=71）之间，核心aa。提取70例（p=7.0E-08）。IL28B SNP与core70多态性显著相关（p=3.4E-06）。另一方面，最终持续病毒学应答（SVR）率与NS5A aa2340 ~ 2382相关性最显著，与干扰素利巴韦林应答决定区（IRRDR）几乎完全吻合（p=1.2E-07）。在PEG-IFN/RBV治疗中，IL28B、NS5A-ISDR、core以及最重要的NS5A-IRRDR的多态性与病毒动力学相关，对治疗反应有巨大影响，导致慢性HCV-1b感染的结局有显著差异。丙型肝炎病毒（HCV）序列与白细胞介素28B (IL28B) SNP在肝细胞癌（HCC）发展中的关系尚未得到很好的阐明。(1)对20例HCC患者和23例HCV-1b感染的非HCC患者进行2个时间点的完整HCV开放阅读框序列测定。(2)另外研究了230例与HCC发展相关的核心和NS5A序列。(3)研究了98例患者病毒核心序列随时间的变化。(4)对228例患者进行IL28B SNP检测。(1)观察期间（肝癌10.8年，非肝癌11.1年），NS5A核心a.a. 70和3个氨基酸位置的变化是HCC患者的特征。(2) 230例患者中，HCC组在核心a.a. 70处的Q/H:R比（43:22 vs.非HCC组66:99，p=0.001）显著高于HCC组。(3)在11例血小板（p= 0.005）和白蛋白随时间减少（p= 0.005）或HCC发生（6/11,54.5%）的患者中观察到核心R70Q随时间的变化，而在4例血小板（n / 5）和白蛋白（n /11）无相关变化的患者中观察到Q70R变化，或HCC发生（0/4,0%）。(4) IL28B SNP与core 70残基显著相关。HCV核心a.a.70残基及其随时间的变化与基因型1b感染的HCC发展相关，与IL28B SNP密切相关。根据最近的报道，蛋白酶抑制剂（PI）耐药丙型肝炎病毒（HCV）变体可能大量存在于未经PI治疗的患者中。然而，这些病毒在慢性丙型肝炎临床病程中的影响尚未得到很好的表征。对261例HCV基因型1b感染的日本患者进行聚乙二醇化干扰素加利巴韦林（PEG-IFN/RBV）治疗前的HCV非结构3 （NS3）优势氨基酸序列测定，并研究患者的临床特征和治疗反应，包括持续病毒学反应（SVR）率。39例非svr患者在完成治疗后也测定了HCV-NS3序列。261例患者中发现4个已知导致PI耐药的单突变（T54S、Q80K、I153V和D168E） 35例（13.4%），6例（2.3%）发现双突变（I153V+T54S/D168E）。有和没有pi耐药突变的患者对PEG-IFN/RBV治疗的反应没有差异（突变组，SVR 48%；野生型组，SVR 40%； P=0.38）。另一方面，2例非svr患者在PEG-IFN/RBV治疗后出现2个突变（I153V和E168D， 5.1%）。在未经治疗的hcv -1b感染患者中存在相当大比例的PI耐药相关NS3突变。这些突变对PI治疗的影响尚不清楚，但临床医生应注意避免PI耐药性的进一步发展。一部分感染基因2a型丙型肝炎病毒（HCV）的患者对聚乙二醇干扰素加利巴韦林治疗（PEG-IFN /RBV）不能实现持续的病毒学应答（SVR），但原因尚不清楚。本研究旨在阐明病毒序列变异与最终结果之间可能存在的相关性。采用直接测序法测定两组独立患者（43例为试验组，1组为验证组，35例为验证组，2组）的基因2a型HCV预处理完整开放阅读框（ORF）序列，并探讨其与最终结局的相关性。SVR患者（n=58）和非SVR患者（n=20）在预处理HCV RNA水平（p=0.002）、纤维化评分（p=0.047）和利巴韦林累积剂量（p=0.003）方面存在显著差异。通过比较完整的HCV orf中所有氨基酸位置，核心区氨基酸（aa） 110处苏氨酸在SVR中出现频率显著（组1 p=0.01，组2 p=0.004，组合p=5E-05）。滑动窗口分析显示，与非SVR患者相比，SVR患者NS5A 2258 ~ 2306区域内的氨基酸变异总数显著高于非SVR患者（组1 p=0.01，组2 p=0.006，组合p=0.0006）。多因素分析显示，core aa 110 （p=0.02）、NS5A aa 2258-2306 （p=0.03）和利巴韦林累积剂量（p=0.02）是影响最终结果的独立变量。PEG-IFN/RBV治疗的结果受基因2a型HCV感染核心区和NS5A区变异的显著影响。少