Hepatol Int DOI 10.1007/s12072-015-9624-2
ORIGINAL ARTICLE
HCV NS5A resistance-associated variants in a group of real-world Japanese patients chronically infected with HCV genotype 1b Yosuke Hirotsu1 • Tatsuo Kanda2 • Hiroshi Matsumura3 • Mitsuhiko Moriyama3 Osamu Yokosuka2 • Masao Omata1,4
•
Received: 12 December 2014 / Accepted: 27 February 2015 Ó Asian Pacific Association for the Study of the Liver 2015
Abstract Background Recent advances in interferon-free treatment could lead to the eradication of hepatitis C virus (HCV) from patients infected with HCV. One of the direct-acting anti-viral agents, HCV NS5A inhibitor, is available for these combination therapies. However, naturally occurring resistance-associated variants (RAVs) to HCV NS5A inhibitors in treatment-naı¨ve patients chronically infected with HCV genotype 1b are still unknown. Methods We performed ultra-deep sequencing and analysed previously reported RAVs in a total 132 HCV genotype 1b-infected Japanese patients who had never used HCV NS5A inhibitors. We also performed direct-sequencing by Sanger method in consecutively selected 50 of the total 132 samples, and the differences between the results of the two methods were compared. Results In the comparison of the variant frequencies of ultra-deep sequencing with RAVs of direct-sequencing by Electronic supplementary material The online version of this article (doi:10.1007/s12072-015-9624-2) contains supplementary material, which is available to authorized users. & Tatsuo Kanda [email protected] 1
Genome Analysis Center, Yamanashi Prefectural Central Hospital, 1-1-1 Fujimi, Kofu-shi, Yamanashi 400-8506, Japan
2
Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan
3
Division of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan
4
University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Sanger method in 50 patients, we identified 32 RAVs by direct-sequencing with the Sanger method; minimum variant frequency was shown by ultra-deep sequencing to be 9 %. A total of 110 RAVs were identified only by ultradeep sequencing. In the samples from all 132 patients, L31W (2.3 %), L31V (49.2 %), L31F (41.7 %), L31M (1.5 %), L31I (5.3 %), L31S (2.0 %), L31P (3.0 %) and L31R (0.8 %), and Y93N (2.3 %), Y93H (25 %), Y93C (0.8 %), Y93P (2.3 %) and Y93D (0.8 %) were identified. Conclusions We demonstrated naturally-occurring RAVs of HCV NS5A inhibitors by ultra-deep sequencing and that several mutations including Y93H are common in HCV NS5A inhibitor-treatment-naı¨ve patients with chronic HCV genotype 1b. Careful attention should be paid to these RAVs, and further improvement of treatment options might be needed. Keywords HCV Drug resistance NS5A RAV Ultradeep sequencing
Introduction Chronic hepatitis C virus (HCV) infection causes hepatic cirrhosis and hepatocellular carcinoma [1, 2]. It is crucial for the prevention of this disease progression to eradicate HCV from patients with chronic hepatitis C [3, 4]. Antiviral therapy including interferon therapy even for cirrhotic patients with chronic hepatitis C, especially those in whom HCV had been eradicated, could inhibit the development of hepatocellular carcinoma and improve survival [3]. HCV is a single-positive stranded RNA virus *9600 nt. in length. HCV genomes are translated into single open reading frames of *3000 amino acids. Cellular and viral
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proteases (NS2 cysteine protease and NS3 serine protease) turn this protein into structural (core, E1, E2 and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) [5–7]. HCV RNA replicates by RNA-dependent RNA polymerase (NS5B) in the cytoplasm of hepatocytes. Recent advances in interferon-free treatment could lead to the eradication of HCV from patients infected with HCV without the use of interferon [8–18]. The interferon-free combination of protease inhibitor ABT-450 with ritonavir (ABT-450/r), HCV NS5A inhibitor ombitasvir (ABT-267), non-nucleoside polymerase inhibitor dasabuvir (ABT333), and ribavirin for 12 weeks led to 96.2–99.5 and 96.3 % of sustained virological response in previously untreated and treated patients infected with HCV genotype 1, respectively [10, 12, 13]. Another interferon-free combination of HCV NS5A inhibitor ledipasvir and nucleotide polymerase inhibitor sofosbuvir with or without ribavirin for 12 weeks led to 95–97 and 94–96 % of sustained virological response in previously untreated and treated patients infected with HCV genotype 1, respectively [14–16]. HCV NS5A inhibitor daclatasvir plus protease inhibitor asunaprevir for 24 weeks led to 80.5 and 87.4 % sustained virological response in previous non-responders and interferon-ineligible/intolerant patients infected with HCV genotype 1b, respectively [18]. Thus, all these interferon-free regimens for HCV genotype 1-infected patients included HCV NS5A inhibitors. In Japan, 98–99 % of HCV genotype 1 belongs to HCV genotype 1b [19]. Several resistance-associated variants (RAVs) to HCV NS5A inhibitors have been reported [20, 21]. Resistance mutations were mapped to the N-terminal region of HCV NS5A [21]. L31 M and Y93H have been identified as resistance-associated polymorphisms of HCV NS5A in HCV genotype 1-infected patients treated with daclatasvir and asunaprevir [22]. Ledipasvir has a similar potency and resistance profile to that of daclatasvir [21]. In the present study, to reveal the current prevalence of RAVs at positions L31 and Y93 in HCV genotype 1binfected patients who were previously untreated with HCV NS5A inhibitors, we examined the procedures available for identifying these RAVs, ultra-deep sequencing with or without direct-sequencing by Sanger method.
Materials and methods Patients Sera from 132 HCV NS5A inhibitor-treatment-naı¨ve chronic hepatitis C genotype 1b patients were consecutively collected and stored at -80 °C until RNA extraction.
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RNA extraction Total RNA was extracted from 140 lL of each serum sample using a QIAamp Viral RNA Mini QIAcube Kit (Qiagen, Tokyo, Japan) on the QIAcube. RNA was stored at -80 °C until analysis. cDNA synthesis and amplification by PCR for ultradeep sequencing cDNA was synthesized with random primers using a highcapacity cDNA reverse transcription kit with RNase inhibitor according to the manufacturer’s instructions (Applied BiosystemsÒ, Tokyo, Japan). Amplification was performed with 50 -GCAGTGGATGAACCGGCT-30 (sense) and 50 -GTGGACGCCTTCGCCTTCAT-30 (antisense) for 30 cycles at 98 °C for 15 s, 55 °C for 5 s, and 72 °C for 2 min using PrimeSTARÒ HS DNA Polymerase (Takara Bio Inc., Otsu, Japan). The first PCR product was further amplified with two inner primer sets, 50 -TGGGACTGGA TATGCACGGT-30 (sense) and 50 -RGTCATGCCCGT CACRTAGTG-30 (antisense), using PrimeSTARÒ HS DNA Polymerase (Takara Bio), by the same PCR conditions. Amplified products were separated by agarose gel electrophoresis and purified using a QIAquick PCR Purification Kit (QIAGEN, Tokyo, Japan). For deep sequencing, purified PCR products were amplified with 50 -TGGGACTGGA TATGCACGGT-30 (sense) and 50 -TCATGGARCCGT TYTTGACAT-30 (antisense) or 50 -ATGTCAARAACGGY TCCATGA-30 (sense) and 50 -RGTCATGCCCGTCACR TAGTG-30 (antisense) using a PlatinumÒ PCR SuperMix High Fidelity in Ion Plus Fragment Library Kit (Life Technologies, Tokyo, Japan) for 3 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s and 68 °C for 30 s, ending with a holding period at 4 °C. Amplicons were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter, Tokyo, Japan). Each amplicon was quantified using a NanoDrop Lite spectrophotometer (Thermo Scientific, Yokohama, Kanagawa, Japan), and all amplicons from a single viral genome were pooled together at equimolar ratios. The PCR amplicons were treated with 1 ll End Repair Enzyme (Life Technologies) at room temperature for 20 min. End-repaired amplicon were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter). The amplicons were ligated to adapters with diluted barcodes of the Ion XpressTM Barcode Adapters kit for 15 min at 25 °C, and then at 72 °C for 5 min. Adaptor ligated amplicon libraries were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter). The library concentration was determined using an Ion Library Quantitation Kit (Life Technologies), and then each library was diluted to 26 pM and the same amount of libraries was pooled for one sequence reaction. Next, emulsion PCR was carried out using
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the Ion OneTouchTM System and Ion OneTouchTM 200 Template Kit v2 (Life Technologies) according to the manufacturer’s instructions. Template-positive Ion SphereTM Particles were then enriched with DynabeadsÒ MyOneTM Streptavidin C1 Beads using an Ion OneTouchTM ES system (Life Technologies). Purified ion sphere particles were loaded on an Ion 314 Chip. Massively parallel sequencing was carried out on a personal genome machine (PGM) sequencer (Ion TorrentTM) using the Ion PGM Sequencing 200 Kit version 2. The sequence data were processed using standard Ion Torrent SuiteTM software running on the Torrent Server. Sequence data were visually confirmed and obtained with the Integrative Genomics Viewer (IGV). Direct-sequencing by Sanger method In 50 consecutively selected samples from a total of 132, direct-sequencing was also performed by the Sanger method. We amplified the cDNA with 50 -GCAGTGGATGAACCGG CT-30 (sense) and 50 -GTGGACGCCTTCGCCTTCAT-30 (antisense) for 30 cycles at 98 °C for 15 s, 55 °C for 5 s, and 72 °C for 2 min using PrimeSTARÒ HS DNA Polymerase (Takara Bio). Sanger sequencing was performed with BigDyeÒ Terminator v3.1. PCR products were purified and subsequently analyzed by 3500 Genetic Analyzer (Applied Biosystems). Nucleotide sequence accession number All sequence reads have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA002429. Statistical analysis We used univariate analyses, applying the Student’s t test; p \ 0.05 was considered statistically significant.
Results Sequencing of HCV NS5A regions To perform deep sequencing of HCV NS5A regions, we used two primer pairs covering 316 bp of the target sequence and amplified the NS5A regions by PCR. Target sequencing was performed using the Ion TorrentTM PGM System to generate sequence reads of approximately 200 bp. Read alignments were performed on the Torrent Server. The results were average mapped reads of 6238 (range 663–9311), mean sequencing depth of 1914 9 (range 185–3563), and uniformity of 99 % (range
48–100 %) (Table 1). HCV genotype 1b sequence (Accession Number: GU133617.1) was used for identifying the variation sequences. We divided 148 samples into seven runs of ultra-deep sequencing. In the runs, 29, 19, 38, 9, 15, 22, and 16 samples were pooled, respectively. Comparison of variant frequencies of ultra-deep sequencing with resistance-associated variants (RAVs) of direct-sequencing by Sanger method In 50 of the total 132 patients, we performed both ultradeep sequencing and direct-sequencing by Sanger method and compared the results (Fig. 1, Table S1). The threshold of measured variant frequencies between variant negative/positive by ultra-deep sequencing was set at 1 %. We identified 32 RAVs by direct-sequencing with the Sanger method; minimum variant frequency was shown by ultradeep sequencing to be 9 % (Fig. 1, left part). A total of 110 RAVs not detectable using direct-sequencing by Sanger method were identified by ultra-deep sequencing and, of interest, eight of these 110 RAVs had above 9 % variant frequency by ultra-deep sequencing (Fig. 1, right part; p \ 0.001). Resistance-associated variants (RAVs) at positions L31 and Y93 in HCV genotype 1b-infected patients Next we focused on RAVs at positions L31 and Y93, which were reported with the use of daclatasvir [22]. We performed ultra-deep sequencing of samples of the total 132 patients (Fig. 2). L31W, L31V, L31F, L31 M, L31I, L31S, L31P and L31R were identified in 2.3, 49.2, 41.7, 1.5, 5.3, 2.0, 3.0 and 0.8 % (Fig. 2, left part). We also observed Y93N (2.3 %), Y93H (25 %), Y93C (0.8 %), Y93P (2.3 %) and Y93D (0.8 %) (Fig. 2, right part). RAVs and EC50 of daclatasvir The association between the number of RAVs identified in the present study and EC50 of daclatasvir [21] is shown in Fig. 2 and Table 2. RAVs L28T and R30E with EC50 of daclatasvir above 10 pM were not observed in Japanese patients infected with HCV genotype 1b even by ultra-deep sequencing (Table 2).
Discussion The present ultra-deep sequencing study showed that among RAVs with EC50 above 10 pM at positions L31 and Y93, L31W, L31V or L31F and Y93N or Y93H were observed in 3/132 (2.3 %), 65/132 (49.2 %) or 55/132 (41.7 %) and 3/132 (2.3 %) or 33/132 (25 %) of Japanese
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Hepatol Int Table 1 The average mapped reads, on target (%), mean depth, and uniformity (%) of ultra-deep sequencing in the present study
Parameter
Number of mapped reads
On target (%)
Mean depth
Average
Uniformity (%)
6238
93
1914
Min
663
78
185
99 48
Max
9311
97
3563
100
Mapped reads, number of reads that were mapped to the full reference genome; on target, percentage of mapped reads that were aligned over a target region; mean depth, mean average target base read depth, including non-covered target bases; uniformity, percentage of target bases covered by at least 90.2 the average base read depth
Table 2 Frequency analysis after ultra-deep sequencing of known HCV NS5A resistance-associated variants (RAVs) conferring resistance to daclatasvir [10 pM RAVs Y93N
Fig. 1 Comparison of variant frequencies (%) of ultra-deep sequencing with resistance-associated variants (RAVs) of direct-sequencing by Sanger method in 50 patients. In a total of 142 RAVs, 32 RAVs detected and 110 RAVs not detected with direct-sequencing by Sanger method, were analyzed. The definition of the term ‘‘variant frequency’’ is the allele frequency in variant calling
Fig. 2 Resistance-associated variants (RAVs) at positions L31 and Y93 in 132 patients infected with HCV genotype 1b and EC50 of daclatasvir. Results of ultra-deep sequencing. AA amino acids, EC50 half maximal (50 %) effective concentration [21]
HCV genotype 1b-infected patients previously untreated with HCV NS5A inhibitors, respectively (Table 2). These results revealed that RAVs to HCV NS5A inhibitors are
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EC50 (pM)
Number of RAVs/132 (%)
74
3 (2.3 %)
Y93H
49
33 (25.0 %)
L31W L31V
210 61
3 (2.3 %) 65 (49.2 %)
L31F
13
55 (41.7 %)
L28T
52
0 (0 %)
R30E
16
0 (0 %)
EC50, half maximal (50 %) effective concentration [21]
present in HCV NS5A inhibitor-untreated HCV genotype 1b-infected patients in Japan as well. It is known that the Ion Torrent system has a high error rate, up to nearly 2 % [23]. It was reported that the sequence generated by Ion Torrent displays nearly perfect coverage behavior on GC-rich, neutral and moderately ATrich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the Ion Torrent PGM, resulting in a lack of coverage for approximately 30 % of the genome [23]. Context-specific errors were observed in the Ion Torrent PGM [23]. Further studies will be needed at such poorcoverage regions. When examined with 200 bp from the enterohemorrhagic Escherichia coli (EHEC) strain genome, the number of substitutions per 100 bp was 0.0303 by the PGM platform [24]. In the present study, we sequenced *200-bp PCR products from the HCV genome, which has an errorprone RNA-dependent RNA polymerase, and therefore the error rates in the sequenced HCV genome might be different from those we expected. We also performed nested RT-PCR to obtain PCR products for deep sequencing, and these methods might result in PCR-induced error and bias. We also found high prevalence rates of L31V (49.2 %) and L31 F (41.7 %) in this cohort (Table 2). They were even more common than Y93H. It is possible that these observations might be
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related to PCR-induced error and bias caused by the abovementioned methods. Sequences including hyper variable regions such as HCV RNA may cause mis-assembly as well as independent assembly of haplotypes, thus resulting in poor coverage. However, we observed no regions with poor coverage, because average uniformity was 99 % in the deep sequencing of the present study (Table 1). In this study, there were no significant differences in clinical background including patients’ ages, platelet counts, AST/ ALT levels, albumin levels or the existence of cirrhosis between the patients with and without L31V and/or Y93H, although the number of patients was rather limited. There are two forms of RAVs: one form is created by the pressure from drugs, and it is difficult to predict the emergence of these kinds of RAVs, and the other is indwelling, with replication fitness, which is possible to predict by direct-sequencing or ultra-deep sequencing before the use of drugs. In general, the former might be HCV NS3 D168A/V/D or HCV NS5A L31V/M, which emerges with the use of asunaprevir and daclatasvir [22]. The present study also demonstrated that one of the latter forms of RAVs seems to be HCV NS5A Y93H polymorphism. It was reported that RAVs to both daclatasvir and asunaprevir were found in all six HCV genotype 1a-infected patients with viral breakthrough while receiving therapy with those two direct-acting antiviral agents (DAAs) [25]. It was reported that all-oral daclatasvir plus asunaprevir treatment resulted in 90, 82, and 82 % SVR in the treatment-naı¨ve cohort, in the non-responder cohort and in the ineligible and/or intolerant cohort, respectively [26]. Kumada et al. [18] reported that in a phase 3 study of daclatasvir plus asunaprevir for Japanese patients chronically infected with HCV genotype 1b, of the 34 patients with virologic failure, 29 had resistance-associated substitutions to both daclatasvir (predominantly NS5AL31 M/V-Y93H) and asunaprevir (predominantly NS3D168 variants) detected at failure, and that 22 patients with virologic failure had NS5A polymorphisms L31 M/V and/ or Y93H prior to treatment. They also reported that, of the 37 patients with L31 M/V and/or Y93H at baseline, only 11/23 interferon-ineligible/intolerant patients and 4/14 non-responder patients achieved SVR [18]. Before the use of daclatasvir plus asunaprevir, RAVs to HCV NS5A inhibitors at positions L31 and Y93 should be examined to avoid treatment failure of HCV genotype 1b patients. The ION-1 study of ledipasvir and sofosbuvir for untreated HCV genotype 1 infection [16] reported that two patients with relapse had mutations associated with RAVs both at baseline and at the time of relapse, although the ION-3 study of ledipasvir and sofosbuvir for HCV genotype 1 infection [14] reported that the presence of any given HCV NS5A RAVs at baseline was not associated with relapse. It has already been shown that with many of
the sofosbuvir-based regimens, most of the RAVs are of little relevance [14, 16], although we do not know whether ledipasvir and sofosbuvir could be effective for patients with Y93H. Sofosbuvir is one of the potent HCV NS5B polymerase inhibitors, and it is likely that the more effective regimens may overcome any RAVs. It appears prudent, meanwhile, that more information concerning RAVs of HCV NS5A is gathered before using any HCV NS5A inhibitors for HCV genotype 1-infected patients. In the present study, we performed direct-sequencing by Sanger method in 50 consecutively selected subjects of the total 132. In 30 of these 50 patients, all patients achieved SVR after treatments with sofosbuvir-based regimens. Although we confirmed that RAVs are of much less significance in the sofosbuvir-based regimens, the relevance of RAVs still needs further investigation with sofosbuvir-based regimens as well. Although we could detect variants that had a variant frequency around 9 % by direct-sequencing with the Sanger method (Fig. 1), we also found five variants around 17–25 %, supporting the general consensus that direct Sanger sequencing can only reliably detect variants above 20–30 %. The minimum variant frequency of ultra-deep sequencing was 9 % in mutations detected by direct-sequencing with the Sanger method. Among the 110 RAVs without detection by direct-sequencing with the Sanger method, ultra-deep sequencing revealed that 8 RAVs (7.3 %) and 102 RAVs (92.7 %) had above and below 9 % variant frequency, respectively (Fig. 2). It may be possible that direct-sequencing by Sanger method could fail to detect some of the RAVs, although at this juncture we do not yet know the meaning of the below 9 % variant frequency of ultra-deep sequencing. A previous study by Sanger-method direct-sequencing from Japan [20] reported that L31 M and/or Y93H were detected in 33 (11.2 %) of 294 patients, and Y93H (8.2 %) was predominant over L31 M (2.7 %). The prevalences of L31 M and Y93H according to a European HCV genotype 1b database are 3.8 and 8.3 %, respectively [27]. Another study from the US reported prevalences of L31 M and Y93H of 6.3 and 3.8 %, respectively, in patients infected with HCV genotype 1b [28]. The present study by ultradeep sequencing demonstrated prevalences of L31 M and Y93H of 1.5 and 25 %, respectively (Fig. 2), similar to those of another recent report from Japan [29]. Applegate et al. [30] reported that RAVs of HCV NS3, NS5A and NS5B regions were examined by ultra-deep pyrosequencing in 50 Australian patients with HCV genotype 1a co-infected with or without HIV and that 20 % of the patients harbored dominant RAVs, while 36 % demonstrated non-dominant RAVs below the level detectable by bulk sequencing (i.e., \20 %) but above a
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threshold of 1 %. In their study, RAVs (\1 %) were observed at sites associated with DAA resistance from all classes of DAAs with the exception of sofosbuvir [30]. In conclusion, we demonstrated naturally occurring RAVs of HCV NS5A inhibitors by ultra-deep sequencing, and also that several mutations including Y93H are common in HCV NS5A inhibitor-treatment-naı¨ve patients with chronic hepatitis C. Attention should be paid to these RAVs and, of course, further treatment options will be needed. HCV NS5A inhibitors should be used in combination with one or two other DAAs. Finally, more work needs to be done to validate the high frequency of variants, and also to define the relationship between variants and drug resistance. Acknowledgements We are all thankful to our colleagues at the liver units of each hospital who cared for the patients described herein. This study was supported by a Grant-in-Aid for the Genome Research Project from Yamanashi Prefecture (to Yosuke Hirotsu and Masao Omata). Compliance with ethical requirements and Conflict of interest All procedures followed were in accordance with ethical standards of the responsible committees on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for participation in the study. Yosuke Hirotsu, Hiroshi Matsumura, Mitsuhiko Moriyama and Masao Omata declare that they have no conflicts of interest. Tatsuo Kanda reports receiving lecture fees from Chugai Pharmaceutical, MSD, Tanabe-Mitsubishi, Daiichi-Sankyo, and Bristol-Myers Squibb, and Osamu Yokosuka reports receiving grant support from Chugai Pharmaceutical, Bayer, MSD, DaiichiSankyo, Tanabe-Mitsubishi, and Bristol-Myers Squibb. Written informed consent was obtained from all patients, and this study was approved by the respective Institutional Ethics Committees (Yamanashi Prefectural Central Hospital GS-US-337-0113; Chiba University School of Medicine, No.1462, No. 502; and Nihon University School of Medicine RK-100910-14).
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ORIGINAL ARTICLE
HCV NS5A resistance-associated variants in a group of real-world Japanese patients chronically infected with HCV genotype 1b Yosuke Hirotsu1 • Tatsuo Kanda2 • Hiroshi Matsumura3 • Mitsuhiko Moriyama3 Osamu Yokosuka2 • Masao Omata1,4
•
Received: 12 December 2014 / Accepted: 27 February 2015 Ó Asian Pacific Association for the Study of the Liver 2015
Abstract Background Recent advances in interferon-free treatment could lead to the eradication of hepatitis C virus (HCV) from patients infected with HCV. One of the direct-acting anti-viral agents, HCV NS5A inhibitor, is available for these combination therapies. However, naturally occurring resistance-associated variants (RAVs) to HCV NS5A inhibitors in treatment-naı¨ve patients chronically infected with HCV genotype 1b are still unknown. Methods We performed ultra-deep sequencing and analysed previously reported RAVs in a total 132 HCV genotype 1b-infected Japanese patients who had never used HCV NS5A inhibitors. We also performed direct-sequencing by Sanger method in consecutively selected 50 of the total 132 samples, and the differences between the results of the two methods were compared. Results In the comparison of the variant frequencies of ultra-deep sequencing with RAVs of direct-sequencing by Electronic supplementary material The online version of this article (doi:10.1007/s12072-015-9624-2) contains supplementary material, which is available to authorized users. & Tatsuo Kanda [email protected] 1
Genome Analysis Center, Yamanashi Prefectural Central Hospital, 1-1-1 Fujimi, Kofu-shi, Yamanashi 400-8506, Japan
2
Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan
3
Division of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan
4
University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Sanger method in 50 patients, we identified 32 RAVs by direct-sequencing with the Sanger method; minimum variant frequency was shown by ultra-deep sequencing to be 9 %. A total of 110 RAVs were identified only by ultradeep sequencing. In the samples from all 132 patients, L31W (2.3 %), L31V (49.2 %), L31F (41.7 %), L31M (1.5 %), L31I (5.3 %), L31S (2.0 %), L31P (3.0 %) and L31R (0.8 %), and Y93N (2.3 %), Y93H (25 %), Y93C (0.8 %), Y93P (2.3 %) and Y93D (0.8 %) were identified. Conclusions We demonstrated naturally-occurring RAVs of HCV NS5A inhibitors by ultra-deep sequencing and that several mutations including Y93H are common in HCV NS5A inhibitor-treatment-naı¨ve patients with chronic HCV genotype 1b. Careful attention should be paid to these RAVs, and further improvement of treatment options might be needed. Keywords HCV Drug resistance NS5A RAV Ultradeep sequencing
Introduction Chronic hepatitis C virus (HCV) infection causes hepatic cirrhosis and hepatocellular carcinoma [1, 2]. It is crucial for the prevention of this disease progression to eradicate HCV from patients with chronic hepatitis C [3, 4]. Antiviral therapy including interferon therapy even for cirrhotic patients with chronic hepatitis C, especially those in whom HCV had been eradicated, could inhibit the development of hepatocellular carcinoma and improve survival [3]. HCV is a single-positive stranded RNA virus *9600 nt. in length. HCV genomes are translated into single open reading frames of *3000 amino acids. Cellular and viral
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proteases (NS2 cysteine protease and NS3 serine protease) turn this protein into structural (core, E1, E2 and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) [5–7]. HCV RNA replicates by RNA-dependent RNA polymerase (NS5B) in the cytoplasm of hepatocytes. Recent advances in interferon-free treatment could lead to the eradication of HCV from patients infected with HCV without the use of interferon [8–18]. The interferon-free combination of protease inhibitor ABT-450 with ritonavir (ABT-450/r), HCV NS5A inhibitor ombitasvir (ABT-267), non-nucleoside polymerase inhibitor dasabuvir (ABT333), and ribavirin for 12 weeks led to 96.2–99.5 and 96.3 % of sustained virological response in previously untreated and treated patients infected with HCV genotype 1, respectively [10, 12, 13]. Another interferon-free combination of HCV NS5A inhibitor ledipasvir and nucleotide polymerase inhibitor sofosbuvir with or without ribavirin for 12 weeks led to 95–97 and 94–96 % of sustained virological response in previously untreated and treated patients infected with HCV genotype 1, respectively [14–16]. HCV NS5A inhibitor daclatasvir plus protease inhibitor asunaprevir for 24 weeks led to 80.5 and 87.4 % sustained virological response in previous non-responders and interferon-ineligible/intolerant patients infected with HCV genotype 1b, respectively [18]. Thus, all these interferon-free regimens for HCV genotype 1-infected patients included HCV NS5A inhibitors. In Japan, 98–99 % of HCV genotype 1 belongs to HCV genotype 1b [19]. Several resistance-associated variants (RAVs) to HCV NS5A inhibitors have been reported [20, 21]. Resistance mutations were mapped to the N-terminal region of HCV NS5A [21]. L31 M and Y93H have been identified as resistance-associated polymorphisms of HCV NS5A in HCV genotype 1-infected patients treated with daclatasvir and asunaprevir [22]. Ledipasvir has a similar potency and resistance profile to that of daclatasvir [21]. In the present study, to reveal the current prevalence of RAVs at positions L31 and Y93 in HCV genotype 1binfected patients who were previously untreated with HCV NS5A inhibitors, we examined the procedures available for identifying these RAVs, ultra-deep sequencing with or without direct-sequencing by Sanger method.
Materials and methods Patients Sera from 132 HCV NS5A inhibitor-treatment-naı¨ve chronic hepatitis C genotype 1b patients were consecutively collected and stored at -80 °C until RNA extraction.
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RNA extraction Total RNA was extracted from 140 lL of each serum sample using a QIAamp Viral RNA Mini QIAcube Kit (Qiagen, Tokyo, Japan) on the QIAcube. RNA was stored at -80 °C until analysis. cDNA synthesis and amplification by PCR for ultradeep sequencing cDNA was synthesized with random primers using a highcapacity cDNA reverse transcription kit with RNase inhibitor according to the manufacturer’s instructions (Applied BiosystemsÒ, Tokyo, Japan). Amplification was performed with 50 -GCAGTGGATGAACCGGCT-30 (sense) and 50 -GTGGACGCCTTCGCCTTCAT-30 (antisense) for 30 cycles at 98 °C for 15 s, 55 °C for 5 s, and 72 °C for 2 min using PrimeSTARÒ HS DNA Polymerase (Takara Bio Inc., Otsu, Japan). The first PCR product was further amplified with two inner primer sets, 50 -TGGGACTGGA TATGCACGGT-30 (sense) and 50 -RGTCATGCCCGT CACRTAGTG-30 (antisense), using PrimeSTARÒ HS DNA Polymerase (Takara Bio), by the same PCR conditions. Amplified products were separated by agarose gel electrophoresis and purified using a QIAquick PCR Purification Kit (QIAGEN, Tokyo, Japan). For deep sequencing, purified PCR products were amplified with 50 -TGGGACTGGA TATGCACGGT-30 (sense) and 50 -TCATGGARCCGT TYTTGACAT-30 (antisense) or 50 -ATGTCAARAACGGY TCCATGA-30 (sense) and 50 -RGTCATGCCCGTCACR TAGTG-30 (antisense) using a PlatinumÒ PCR SuperMix High Fidelity in Ion Plus Fragment Library Kit (Life Technologies, Tokyo, Japan) for 3 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s and 68 °C for 30 s, ending with a holding period at 4 °C. Amplicons were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter, Tokyo, Japan). Each amplicon was quantified using a NanoDrop Lite spectrophotometer (Thermo Scientific, Yokohama, Kanagawa, Japan), and all amplicons from a single viral genome were pooled together at equimolar ratios. The PCR amplicons were treated with 1 ll End Repair Enzyme (Life Technologies) at room temperature for 20 min. End-repaired amplicon were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter). The amplicons were ligated to adapters with diluted barcodes of the Ion XpressTM Barcode Adapters kit for 15 min at 25 °C, and then at 72 °C for 5 min. Adaptor ligated amplicon libraries were purified using AgencourtÒ AMPureÒ XP reagents (Beckman Coulter). The library concentration was determined using an Ion Library Quantitation Kit (Life Technologies), and then each library was diluted to 26 pM and the same amount of libraries was pooled for one sequence reaction. Next, emulsion PCR was carried out using
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the Ion OneTouchTM System and Ion OneTouchTM 200 Template Kit v2 (Life Technologies) according to the manufacturer’s instructions. Template-positive Ion SphereTM Particles were then enriched with DynabeadsÒ MyOneTM Streptavidin C1 Beads using an Ion OneTouchTM ES system (Life Technologies). Purified ion sphere particles were loaded on an Ion 314 Chip. Massively parallel sequencing was carried out on a personal genome machine (PGM) sequencer (Ion TorrentTM) using the Ion PGM Sequencing 200 Kit version 2. The sequence data were processed using standard Ion Torrent SuiteTM software running on the Torrent Server. Sequence data were visually confirmed and obtained with the Integrative Genomics Viewer (IGV). Direct-sequencing by Sanger method In 50 consecutively selected samples from a total of 132, direct-sequencing was also performed by the Sanger method. We amplified the cDNA with 50 -GCAGTGGATGAACCGG CT-30 (sense) and 50 -GTGGACGCCTTCGCCTTCAT-30 (antisense) for 30 cycles at 98 °C for 15 s, 55 °C for 5 s, and 72 °C for 2 min using PrimeSTARÒ HS DNA Polymerase (Takara Bio). Sanger sequencing was performed with BigDyeÒ Terminator v3.1. PCR products were purified and subsequently analyzed by 3500 Genetic Analyzer (Applied Biosystems). Nucleotide sequence accession number All sequence reads have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA002429. Statistical analysis We used univariate analyses, applying the Student’s t test; p \ 0.05 was considered statistically significant.
Results Sequencing of HCV NS5A regions To perform deep sequencing of HCV NS5A regions, we used two primer pairs covering 316 bp of the target sequence and amplified the NS5A regions by PCR. Target sequencing was performed using the Ion TorrentTM PGM System to generate sequence reads of approximately 200 bp. Read alignments were performed on the Torrent Server. The results were average mapped reads of 6238 (range 663–9311), mean sequencing depth of 1914 9 (range 185–3563), and uniformity of 99 % (range
48–100 %) (Table 1). HCV genotype 1b sequence (Accession Number: GU133617.1) was used for identifying the variation sequences. We divided 148 samples into seven runs of ultra-deep sequencing. In the runs, 29, 19, 38, 9, 15, 22, and 16 samples were pooled, respectively. Comparison of variant frequencies of ultra-deep sequencing with resistance-associated variants (RAVs) of direct-sequencing by Sanger method In 50 of the total 132 patients, we performed both ultradeep sequencing and direct-sequencing by Sanger method and compared the results (Fig. 1, Table S1). The threshold of measured variant frequencies between variant negative/positive by ultra-deep sequencing was set at 1 %. We identified 32 RAVs by direct-sequencing with the Sanger method; minimum variant frequency was shown by ultradeep sequencing to be 9 % (Fig. 1, left part). A total of 110 RAVs not detectable using direct-sequencing by Sanger method were identified by ultra-deep sequencing and, of interest, eight of these 110 RAVs had above 9 % variant frequency by ultra-deep sequencing (Fig. 1, right part; p \ 0.001). Resistance-associated variants (RAVs) at positions L31 and Y93 in HCV genotype 1b-infected patients Next we focused on RAVs at positions L31 and Y93, which were reported with the use of daclatasvir [22]. We performed ultra-deep sequencing of samples of the total 132 patients (Fig. 2). L31W, L31V, L31F, L31 M, L31I, L31S, L31P and L31R were identified in 2.3, 49.2, 41.7, 1.5, 5.3, 2.0, 3.0 and 0.8 % (Fig. 2, left part). We also observed Y93N (2.3 %), Y93H (25 %), Y93C (0.8 %), Y93P (2.3 %) and Y93D (0.8 %) (Fig. 2, right part). RAVs and EC50 of daclatasvir The association between the number of RAVs identified in the present study and EC50 of daclatasvir [21] is shown in Fig. 2 and Table 2. RAVs L28T and R30E with EC50 of daclatasvir above 10 pM were not observed in Japanese patients infected with HCV genotype 1b even by ultra-deep sequencing (Table 2).
Discussion The present ultra-deep sequencing study showed that among RAVs with EC50 above 10 pM at positions L31 and Y93, L31W, L31V or L31F and Y93N or Y93H were observed in 3/132 (2.3 %), 65/132 (49.2 %) or 55/132 (41.7 %) and 3/132 (2.3 %) or 33/132 (25 %) of Japanese
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Hepatol Int Table 1 The average mapped reads, on target (%), mean depth, and uniformity (%) of ultra-deep sequencing in the present study
Parameter
Number of mapped reads
On target (%)
Mean depth
Average
Uniformity (%)
6238
93
1914
Min
663
78
185
99 48
Max
9311
97
3563
100
Mapped reads, number of reads that were mapped to the full reference genome; on target, percentage of mapped reads that were aligned over a target region; mean depth, mean average target base read depth, including non-covered target bases; uniformity, percentage of target bases covered by at least 90.2 the average base read depth
Table 2 Frequency analysis after ultra-deep sequencing of known HCV NS5A resistance-associated variants (RAVs) conferring resistance to daclatasvir [10 pM RAVs Y93N
Fig. 1 Comparison of variant frequencies (%) of ultra-deep sequencing with resistance-associated variants (RAVs) of direct-sequencing by Sanger method in 50 patients. In a total of 142 RAVs, 32 RAVs detected and 110 RAVs not detected with direct-sequencing by Sanger method, were analyzed. The definition of the term ‘‘variant frequency’’ is the allele frequency in variant calling
Fig. 2 Resistance-associated variants (RAVs) at positions L31 and Y93 in 132 patients infected with HCV genotype 1b and EC50 of daclatasvir. Results of ultra-deep sequencing. AA amino acids, EC50 half maximal (50 %) effective concentration [21]
HCV genotype 1b-infected patients previously untreated with HCV NS5A inhibitors, respectively (Table 2). These results revealed that RAVs to HCV NS5A inhibitors are
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EC50 (pM)
Number of RAVs/132 (%)
74
3 (2.3 %)
Y93H
49
33 (25.0 %)
L31W L31V
210 61
3 (2.3 %) 65 (49.2 %)
L31F
13
55 (41.7 %)
L28T
52
0 (0 %)
R30E
16
0 (0 %)
EC50, half maximal (50 %) effective concentration [21]
present in HCV NS5A inhibitor-untreated HCV genotype 1b-infected patients in Japan as well. It is known that the Ion Torrent system has a high error rate, up to nearly 2 % [23]. It was reported that the sequence generated by Ion Torrent displays nearly perfect coverage behavior on GC-rich, neutral and moderately ATrich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the Ion Torrent PGM, resulting in a lack of coverage for approximately 30 % of the genome [23]. Context-specific errors were observed in the Ion Torrent PGM [23]. Further studies will be needed at such poorcoverage regions. When examined with 200 bp from the enterohemorrhagic Escherichia coli (EHEC) strain genome, the number of substitutions per 100 bp was 0.0303 by the PGM platform [24]. In the present study, we sequenced *200-bp PCR products from the HCV genome, which has an errorprone RNA-dependent RNA polymerase, and therefore the error rates in the sequenced HCV genome might be different from those we expected. We also performed nested RT-PCR to obtain PCR products for deep sequencing, and these methods might result in PCR-induced error and bias. We also found high prevalence rates of L31V (49.2 %) and L31 F (41.7 %) in this cohort (Table 2). They were even more common than Y93H. It is possible that these observations might be
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related to PCR-induced error and bias caused by the abovementioned methods. Sequences including hyper variable regions such as HCV RNA may cause mis-assembly as well as independent assembly of haplotypes, thus resulting in poor coverage. However, we observed no regions with poor coverage, because average uniformity was 99 % in the deep sequencing of the present study (Table 1). In this study, there were no significant differences in clinical background including patients’ ages, platelet counts, AST/ ALT levels, albumin levels or the existence of cirrhosis between the patients with and without L31V and/or Y93H, although the number of patients was rather limited. There are two forms of RAVs: one form is created by the pressure from drugs, and it is difficult to predict the emergence of these kinds of RAVs, and the other is indwelling, with replication fitness, which is possible to predict by direct-sequencing or ultra-deep sequencing before the use of drugs. In general, the former might be HCV NS3 D168A/V/D or HCV NS5A L31V/M, which emerges with the use of asunaprevir and daclatasvir [22]. The present study also demonstrated that one of the latter forms of RAVs seems to be HCV NS5A Y93H polymorphism. It was reported that RAVs to both daclatasvir and asunaprevir were found in all six HCV genotype 1a-infected patients with viral breakthrough while receiving therapy with those two direct-acting antiviral agents (DAAs) [25]. It was reported that all-oral daclatasvir plus asunaprevir treatment resulted in 90, 82, and 82 % SVR in the treatment-naı¨ve cohort, in the non-responder cohort and in the ineligible and/or intolerant cohort, respectively [26]. Kumada et al. [18] reported that in a phase 3 study of daclatasvir plus asunaprevir for Japanese patients chronically infected with HCV genotype 1b, of the 34 patients with virologic failure, 29 had resistance-associated substitutions to both daclatasvir (predominantly NS5AL31 M/V-Y93H) and asunaprevir (predominantly NS3D168 variants) detected at failure, and that 22 patients with virologic failure had NS5A polymorphisms L31 M/V and/ or Y93H prior to treatment. They also reported that, of the 37 patients with L31 M/V and/or Y93H at baseline, only 11/23 interferon-ineligible/intolerant patients and 4/14 non-responder patients achieved SVR [18]. Before the use of daclatasvir plus asunaprevir, RAVs to HCV NS5A inhibitors at positions L31 and Y93 should be examined to avoid treatment failure of HCV genotype 1b patients. The ION-1 study of ledipasvir and sofosbuvir for untreated HCV genotype 1 infection [16] reported that two patients with relapse had mutations associated with RAVs both at baseline and at the time of relapse, although the ION-3 study of ledipasvir and sofosbuvir for HCV genotype 1 infection [14] reported that the presence of any given HCV NS5A RAVs at baseline was not associated with relapse. It has already been shown that with many of
the sofosbuvir-based regimens, most of the RAVs are of little relevance [14, 16], although we do not know whether ledipasvir and sofosbuvir could be effective for patients with Y93H. Sofosbuvir is one of the potent HCV NS5B polymerase inhibitors, and it is likely that the more effective regimens may overcome any RAVs. It appears prudent, meanwhile, that more information concerning RAVs of HCV NS5A is gathered before using any HCV NS5A inhibitors for HCV genotype 1-infected patients. In the present study, we performed direct-sequencing by Sanger method in 50 consecutively selected subjects of the total 132. In 30 of these 50 patients, all patients achieved SVR after treatments with sofosbuvir-based regimens. Although we confirmed that RAVs are of much less significance in the sofosbuvir-based regimens, the relevance of RAVs still needs further investigation with sofosbuvir-based regimens as well. Although we could detect variants that had a variant frequency around 9 % by direct-sequencing with the Sanger method (Fig. 1), we also found five variants around 17–25 %, supporting the general consensus that direct Sanger sequencing can only reliably detect variants above 20–30 %. The minimum variant frequency of ultra-deep sequencing was 9 % in mutations detected by direct-sequencing with the Sanger method. Among the 110 RAVs without detection by direct-sequencing with the Sanger method, ultra-deep sequencing revealed that 8 RAVs (7.3 %) and 102 RAVs (92.7 %) had above and below 9 % variant frequency, respectively (Fig. 2). It may be possible that direct-sequencing by Sanger method could fail to detect some of the RAVs, although at this juncture we do not yet know the meaning of the below 9 % variant frequency of ultra-deep sequencing. A previous study by Sanger-method direct-sequencing from Japan [20] reported that L31 M and/or Y93H were detected in 33 (11.2 %) of 294 patients, and Y93H (8.2 %) was predominant over L31 M (2.7 %). The prevalences of L31 M and Y93H according to a European HCV genotype 1b database are 3.8 and 8.3 %, respectively [27]. Another study from the US reported prevalences of L31 M and Y93H of 6.3 and 3.8 %, respectively, in patients infected with HCV genotype 1b [28]. The present study by ultradeep sequencing demonstrated prevalences of L31 M and Y93H of 1.5 and 25 %, respectively (Fig. 2), similar to those of another recent report from Japan [29]. Applegate et al. [30] reported that RAVs of HCV NS3, NS5A and NS5B regions were examined by ultra-deep pyrosequencing in 50 Australian patients with HCV genotype 1a co-infected with or without HIV and that 20 % of the patients harbored dominant RAVs, while 36 % demonstrated non-dominant RAVs below the level detectable by bulk sequencing (i.e., \20 %) but above a
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threshold of 1 %. In their study, RAVs (\1 %) were observed at sites associated with DAA resistance from all classes of DAAs with the exception of sofosbuvir [30]. In conclusion, we demonstrated naturally occurring RAVs of HCV NS5A inhibitors by ultra-deep sequencing, and also that several mutations including Y93H are common in HCV NS5A inhibitor-treatment-naı¨ve patients with chronic hepatitis C. Attention should be paid to these RAVs and, of course, further treatment options will be needed. HCV NS5A inhibitors should be used in combination with one or two other DAAs. Finally, more work needs to be done to validate the high frequency of variants, and also to define the relationship between variants and drug resistance. Acknowledgements We are all thankful to our colleagues at the liver units of each hospital who cared for the patients described herein. This study was supported by a Grant-in-Aid for the Genome Research Project from Yamanashi Prefecture (to Yosuke Hirotsu and Masao Omata). Compliance with ethical requirements and Conflict of interest All procedures followed were in accordance with ethical standards of the responsible committees on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for participation in the study. Yosuke Hirotsu, Hiroshi Matsumura, Mitsuhiko Moriyama and Masao Omata declare that they have no conflicts of interest. Tatsuo Kanda reports receiving lecture fees from Chugai Pharmaceutical, MSD, Tanabe-Mitsubishi, Daiichi-Sankyo, and Bristol-Myers Squibb, and Osamu Yokosuka reports receiving grant support from Chugai Pharmaceutical, Bayer, MSD, DaiichiSankyo, Tanabe-Mitsubishi, and Bristol-Myers Squibb. Written informed consent was obtained from all patients, and this study was approved by the respective Institutional Ethics Committees (Yamanashi Prefectural Central Hospital GS-US-337-0113; Chiba University School of Medicine, No.1462, No. 502; and Nihon University School of Medicine RK-100910-14).
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