Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China

Abstract

The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

Introduction

Chronic infection of HBV has been a major public health problem, with 240 million people chronically infected worldwide, including 93 million people in China.^1,2 So far, nine genotypes named by alphabetical capital letters (A−I) have been defined by a sequence divergence of more than 8% in the entire genome, which have distinct ethnogeographical distribution.^3,4 In China, HBV/C and HBV/B were two major ones, distributing predominantly in North and South China, respectively.^5,6

Nucleos(t)ide analogues (NAs) are commonly used in clinic. Five kinds of NAs, including lamivudine (LAM), adefovir (ADV), telbivudine (LdT), entecavir (ETV), and tenofovir (TDF), have been approved to be used in clinic. Among them, TDF was just approved in 2014 in China. In current practice of China, LAM and ADV are still widely used in single or in combination with other NAs, mostly due to financial reasons. Thus, drug resistance is still a challenge for anti-HBV treatment in China.

HBV genotypes may vary in virological features and therefore have potential impact on clinical outcome and therapeutic efficacy. Some studies suggested that HBV genotypes might affect the efficacy of NA treatment.⁷ For example, patients with genotype B achieved virological response significantly more rapidly than those with genotype C.⁷ However, the opposite results were also reported.⁸ The resistance mutations are located in the reverse transcriptase (RT) region of the HBV polymerase gene. The rtM204I and rtM204 V are classic LAM-resistant mutations, and rtM204I is also an LdT-resistant mutation; rtN236T and rtA181 V are two well-recognized ADV-resistant mutations; substitutions in rtT184 (including rtT184G/L/A/S/I/F), rtS202G/C, or rtM250V/I/L in conjunction with LAM-resistant mutations result in ETV resistance.⁹ Some mutations do not decrease drug susceptibility, but may rescue replication capacity of resistant mutant. They were termed as compensatory or secondary mutations. In addition, rtA181T was suggested to be an atypical mutation associated with LAM and ADV selection and may reduce the typical extent of virological breakthrough, but it was not well recognized as a primary resistant mutation.^10,11 Multidrug-resistant mutation was defined as concomitance of mutations resistant to both nucleoside analogues (LAM ± ETV) and nucleotide analogues (ADV) in the same sample. Multidrug-resistant HBV has mutations collocated on the same viral genome that confer resistance to multiple antiviral agents that have a favorable cross-resistant profile, that is, resistance to NAs.^12,13

Although drug resistance may influence therapeutic efficacy, only limited studies have investigated the association between HBV genotype and drug-resistant mutations. Mirandola et al.¹⁴ suggested that HBV genotypes might be relevant in the evolution and development of drug resistance and showed different mutational patterns in the YMDD motif between HBV/D and HBV/A. Zollner et al.¹⁵ reported that the incidence of LAM-resistant mutation in patients with HBV/A infection was higher than that of HBV/D infection. In our previous study, we investigated a group of HBV-infected patients, finding that HBV/C2 and HBV/B2 differed in LAM and ADV resistance-associated mutational patterns in HBV-infected Chinese patients.¹⁶ The sample size of above studies was relatively small, which restrained getting a full interpretation. So far, there is still a paucity of data on association of HBV genotypes with ETV-resistant and multidrug-resistant mutations.

In this study, we aimed to clarify whether HBV drug-resistant mutations were affected by the different genotypes through investigating in a large cohort of Chinese patients with chronic HBV infection.

Materials and Methods

Patients

A total of 13, 847 patients who visited Beijing 302 Hospital from July 2009 to June 2014 were enrolled in the study. All patients were positive for HBV DNA (≥100 IU/ml or 500 copies/ml) and HBsAg at sampling. Illness categories included chronic hepatitis B and HBV-related liver cirrhosis. Patients were from different regions of North China. All patients received treatment of NAs. The study was approved by the Ethics Committee of Beijing 302 Hospital.

HBV RT-region amplification and genotype classification

HBV genotype assignment was based on phylogenetic analysis of the 1225-bp-long S/Pol-gene sequence fragment (nt 54–1278) as described previously.^17,18 Phylogenetic and molecular evolutionary analyses were performed using MEGA version 5 software, and phylogenetic trees were constructed using neighbor-joining analysis with bootstrap test confirmation performed on 1,000 resampling. Standard reference sequences were acquired from the online Hepatitis Virus Database (www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) as we described elsewhere.¹⁶

Serological markers, quantitation of HBV DNA, and sequencing of HBV RT gene

Biochemical and serological markers and HBV DNA level of the patients were routinely detected in the Central Clinical Laboratory of Beijing 302 Hospital. HBV DNA level was determined using a real-time quantitative PCR kit (Fosun Pharmaceutical Co., Ltd.) with a lower detection limit of 100 IU/ml.

Resistant mutations were detected as we previously described.^19,20 In brief, HBV DNA was extracted from patient's serum by DNAout (Tianenze), and the RT-region (nt 54–1278) was subjected to an in-house PCR with a sensitivity of 20 IU/ml (Chinese patent ZL 200910092331.1) as previously described. The sense and antisense primers for the first-round PCR were 5′-AGTCAGGAAGACAGCCTACTCC-3′ (nt 3146–3167) and 5′-AGGTGAAGCGAAGTGCACAC-3′ (nt 1577–1596), respectively. The sense and antisense primers for the second-round PCR were 5′-TTCCTGCTGGT-GGCTCCAGTTC-3′ (nt 54–75) and 5′-TTCCGCAGTAT-GGATCGGCAG-3′ (nt 1258–1278), respectively. The PCR products were directly sequenced for all patients. Drug resistance-associated mutations at sites of rt80, rt173, rt180, rt181, rt184, rt202, rt204, rt229, rt233, rt236, and rt250 were analyzed.

Clonal sequencing for HBV RT gene

Clonal sequencing for interested samples was performed as we previously described.¹⁹ In brief, PCR products were purified by the QIAquick PCR Purification Kit (Qiagen), incubated with dATP and Taq DNA polymerase, and then purified by the QIAquick Gel Extraction Kit (Qiagen). The ligation and transformation were performed according to the manufacturer's instructions of pGEM-T Vector System II (Promega). Amplicons were purified by the QIAprep Spin Miniprep Kit (Qiagen). The cloned target gene was sequenced with SP6 and T7 primers (≥20 clones per sample). Clonal sequence analysis was performed in priority for the samples that contained multidrug-resistant mutations, potential novel drug-resistant mutations, or mutational patterns detected by direct sequencing.

Statistical analysis

Continuous data are presented as mean ± SD or median (quartile deviation) and were examined by Student t-test or Wilcoxon signed-rank test. Categorical data were examined by chi-square test or Fisher's exact test. Statistical analysis was carried out in SPSS 19.0 software. A p value of <0.05 was considered statistically significant.

Results

Classification of HBV genotypes and baseline clinical features

Among the 13,847 patients, 1,987 (14.3%) patients were infected with HBV/B, 11,751 (84.9%) patients were infected with HBV/C, and 109 (0.8%) patients were infected with HBV/D (Fig. 1). No other genotypes (A, E, F, G, H, and I) were detected in enrolled samples. Clinical characteristics of patients infected with HBV/B and HBV/C are shown in Table 1. No significant difference was observed in age, gender, and major laboratorial parameters at the testing samples between the two patient subsets.

FIG. 1.

Neighbor-joining phylogenetic tree based on the 52 representative HBV genetic sequences is presented. Standard reference sequences marked with gray circle.

Table 1.

Clinical Characteristics of Patients Infected with HBV/B and HBV/C

Clinical features	Genotype B (n = 1,987)	Genotype C (n = 11,751)	p-Value
Age	43 ± 15	43 ± 14	0.954
Male (%)	1,747 (87.9)	9,831 (83.7)	0.107
HBV DNA (log₁₀ IU/ml)^a	5.3 (2.6, 6.5)	5.5 (2.7, 6.5)	0.850
Alanine aminotransferase (U/L)^a	40 (25, 83)	41 (26, 78)	0.301
Total bilirubin (μM)^a	13.8 (10.0, 21.33)	14.4 (10.6, 23.1)	0.119
HBeAg positive (%)	1,154 (58.1)	7,410 (63.1)	0.212

Data are presented as median and interquartile (Q1, Q3).

Comparison of NA usage

In clinic practice, anti-HBV schedules were complex. Twenty-four NA schedules were summarized in one of our previous studies.¹⁹ To simplify comparative analysis in the current study, NA usage was achieved based on if patients received a certain NA treatment rather than how patients received antiviral schedule in detail. No significant difference in NA exposure was observed in general between HBV/B- and HBV/C-infected patient subsets. Specifically, LAM-treated patients were 50.93% in HBV/B subset vs. 52.24% in HBV/C subset (p = 0.279); ADV-treated patients were 57.88% in HBV/B subset versus 57.60% in HBV/C subset (p = 0.815); and ETV-treated patients were 35.83% in HBV/B subset versus 33.90% in HBV/C subset (p = 0.094).

Analysis of individual drug resistance-associated mutations

Eighteen drug resistance-associated mutations were comparatively analyzed between HBV/B- and HBV/C-infected patients. These mutations included classical primary and secondary drug-resistant mutations, and major atypical drug resistance-associated mutations. The results showed that the detection rates of rtV173 L, rtL180 M, rtA181 V, rtA181T, rtT184G/L/A/S/I/F, rtS202G/C, rtM204I, and rtM204 V were significantly higher in HBV/C-infected patients than in HBV/B-infected patients. In contrast, the detection rates of rtN236T and rtM250V/I/L were significantly lower in HBV/C-infected patients than in HBV/B-infected patients (Table 2).

Table 2.

Comparison of Nucleos(t)ide Analogue (NA)-Resistant Mutations Between HBV/B- and HBV/C-Infected Patients

NA-resistant mutations	HBV/B-infected patients (n = 1,987)	HBV/C-infected patients (n = 11,751)	p-Value
rtL80I/V^a	182 (9.16)	1,162 (9.89)	0.327
rtV173L^a	4 (0.20)	505 (4.30)	<0.001
rtL180M^a	244 (12.28)	2482 (21.12)	<0.001
rtA181V^b	48 (2.42)	843 (7.17)	<0.001
rtA181T	41 (2.1)	725 (6.2)	<0.001
rtT184G/L/A/S/I/F^b	23 (1.18)	264 (2.25)	0.002
rtS202G/C^b	23 (1.16)	296 (2.52)	0.001
rtM204I^b	292 (14.70)	2,197 (18.70)	<0.001
rtM204V^b	226 (11.37)	1732 (14.74)	0.001
rtL229 W/V/F^a	86 (4.33)	577 (4.91)	0.263
rtI233V^a	8 (0.40)	27 (0.23)	0.157
rtN236T^b	151 (7.60)	538 (4.58)	<0.001
rtM250V/I/L^b	32 (1.61)	105 (0.9)	0.003

Represents compensatory resistant mutations.

Represents primary resistant mutations.

Analysis of primary drug-resistant mutations for different NA-resistant types and mutational patterns

Based on the property of primary resistant mutation, mutational types were categorized as LAM-resistant mutation, ADV-resistant mutation, ETV-resistant mutation, and multidrug-resistant mutation. Mutational patterns of drug resistance were further analyzed on this basis.

LAM-resistant mutations were more frequently detected in HBV/C-infected patients compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). The detection rates of ADV- and ETV-resistant mutations were similar, but mutational patterns were different between the two genotypes. For ADV-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For ETV-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184G/L/A/S/I/F or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05) (Table 3).

Table 3.

Comparative Analysis of Resistant Mutation Profile for HBV/B- and HBV/C-Infected Patients

Resistant type	Mutational pattern		HBV/B n (%)	HBV/C n (%)	p-Value
Lamivudine	Overall	rtM204 V/I	502 (25.26)	3,721 (31.67)	<0.001
	Subsets	rtM204I	276 (13.89)	1,989 (16.93)	0.001
		rtM204V	210 (10.57)	1,524 (13.0)	0.003
		rtM204I+M204V	16 (0.81)	208 (1.77)	0.002
Adefovir	Overall	rtA181 V/N236T	178 (8.96)	1,160 (9.87)	0.204
	Subset	rtA181V	27 (1.36)	622 (5.29)	<0.001
		rtN236T	130 (6.54)	317 (2.70)	<0.001
		rtA181V+N236T	21 (1.06)	221 (1.88)	0.010
Entecavir	Overall	rtM204 V/I+T184/S202/M250 substitution	72 (3.62)	508 (4.32)	0.152
	Subset	rtM204 V/I+T184/S202 substitution	43 (2.16)	430 (3.66)	<0.001
		rtM204 V/I+M250 substitution	29 (1.46)	78 (0.67)	<0.001
Multidrug	Overall	rtA181 V/N236T+M204 V/I	7 (0.35)	97 (0.83)	0.035
	Subset	rtM204 V/I+A181V	6 (0.30)	85 (0.72)	0.032
		rtM204 V/I+N236T	1 (0.05)	12 (0.10)	0.487

rtT184 substitutions include rtT184G, L, A, S, I, and F; rtS202 substitutions include rtS202G and C; rtM250 substitutions include rtM250V, I, and L.

The analytic results of multidrug-resistant mutational patterns are shown in Table 4. A total of six patterns of multidrug-resistant mutation were detected. The dominant mutational pattern was rtL180M+A181V+M204 V. Twelve patients were detected with mutations triply resistant to LAM, ADV, and ETV, of which the mutational patterns included rtL180M+S202G+M204V+N236T and rtL180M+T184A+M204V+N236T. These mutants were confirmed by clonal sequencing.

Table 4.

Analysis of Multidrug-Resistant Mutational Patterns in HBV/B- and HBV/C-Infected Patients

Mutational pattern		HBV/B n (%)	HBV/C n (%)	p-Value
Overall	rtA181V/N236T+M204V/I (±T184/S202/M250 substitution)	7 (0.352)	97 (0.825)	<0.05
Subsets	rtA181V+M204I	0	14 (0.119)	0.245
	rtL180M+A181V+M204V	5 (0.252)	65 (0.553)	0.049
	rtL180M+S202G+M204V+N236T	1 (0.050)	6 (0.051)	0.732
	rtL180M+T184A+M204V+N236T	0	5 (0.043)	0.458
	rtL80I+A181V+M204I	1 (0.050)	6 (0.051)	0.732
	rtL80I+L180M+A181V+M204V+N236T	0	1 (0.009)	0.855

rtT184 substitutions include rtT184G, L, A, S, I, and F; rtS202 substitutions include rtS202G and C; rtM250 substitutions include rtM250V, I, and L.

Discussion

In China, NA resistance remains a big problem because of the following: (1) a large number of patients are still using the first generation of NAs largely due to economic reason; (2) a large number of LAM-experienced/LAM-resistant patients have been accumulated, which provides the basis for ETV resistance; and (3) TDF was newly approved with a higher price over other four drugs and has not been taken into medical insurance. Therefore, study for HBV drug resistance has its particular significance in China and other countries that have similar situation.

A few studies have verified that HBV genotype is associated with virus replication, disease prognosis, and the efficacy of antiviral therapy.^7,21−23 However, data on the association of HBV/B and HBV/C, which are dominant genotypes in China, with drug-resistant mutation have been very limited and not shown the whole picture because of relatively small size of samples. In the current study, we investigated a large number of patients, which enabled us to make a full interpretation from the analysis. In real-life clinical practice, patients received a variety of antiviral schedules, which may influence the occurrence of drug-resistant mutations. The influence by antiviral schedules was relatively minor in our study because a large size of samples may largely balance the bias overall between the HBV/B- and HBV/C-infected patients.

The distribution of HBV genotypes vary geographically. In Europe, HBV/D is the most frequently encountered viral genotype (63%), followed by HBV/A (26%).²⁴ In East Asia, HBV/B and HBV/C are dominant HBV genotypes, while in North China, the prevalence of HBV/C was significantly higher than HBV/B.¹⁶ In a largest-to-date European survey that involved 1,568 NA-experienced chronic hepatitis B patients, drug resistance was observed in half of the cases,²⁴ similar with that in our current study (45.1%). This European study showed that the incidence of LAM-resistant mutations rtM204 V/I (48.7%) was much higher than the incidence of ADV-resistant mutations rtA181T/V (3.8%) and rtN236T (2.6%), while the difference between the incidences of LAM- and ADV-resistant mutations was not so great in our current study. This and other European studies showed that there was a mutational pattern bias of rtM204 V for HBV/A and rtM204I for HBV/D.^24,25 By contrast, no such bias was observed between HBV/B- and HBV/C-infected patients in our current study. In NA-untreated patients, drug-resistant mutations were detected in 2/140 (1.4%) of European patients with HBV/D.²⁶ This figure was only slightly lower than 2.01% (17/845) in NA-untreated Chinese patients infected with HBV/C or HBV/B in our recent investigation.²⁷

As shown in Table 2, the detection rates of drug resistance-associated mutations at nine sites were found to be significantly different between HBV/C- and HBV/B-infected patients, including all seven primary resistant mutations. The detection rate of LAM-resistant mutation was significantly higher in HBV/C-infected patients than in HBV/B-infected patients, both in overall and in three subsets. The higher resistance occurrence could be one reason for poorer response to NAs in HBV/C-infected patients than in HBV/B-infected patients as reported previously.⁷ The detection rates of ADV- and ETV-resistant mutations were similar between HBV/C-infected patients and HBV/B-infected patients in overall, while each genotype had a different dominant mutational pattern individually. These findings suggested that HBV/C and HBV/B had different inclinations to develop certain drug resistance-associated mutations.

HBV/C-infected patients had higher occurrence of multidrug-resistant mutations compared with HBV/B-infected patients. This could be mainly due to higher LAM-resistant mutation occurrence in HBV/C-infected patients compared with HBV/B-infected patients, because multidrug-resistant mutations usually occurred in patients who received sequential LAM and ADV treatments and successively developed resistant virus to both drugs.

Multidrug-resistant mutations may come from two sources: (1) coexistence of mutants that harbored mutations resistant either to nucleoside analogue (LAM, LdT, ETV) or to nucleotide analogue (ADV) and (2) existence of mutant that harbored both nucleoside and nucleotide analogue-resistant mutations in the same viral genome. Previous studies showed that development of multidrug-resistant strain was a stepwise process and coexistence of mutant strains resistant to individual drugs was its basis.²⁸⁻³⁰ In one of our previous studies, we found that mutant strains resistant to individual drugs (rtN236T and rtL180M+M204V+S202G, respectively) cocirculated with multidrug-resistant strains (rtL180M+M204V+S202G+N236T and rtL180M+A181V+M204V+S202G+N236T).¹³ In this study, we detected multidrug-resistant mutations in 104 patients' samples. Using clonal sequencing, we verified the existence of multidrug-resistant mutant strains in 87 samples (data not shown in detail). It could be deduced that ultradeep pyrosequencing would increase the detection rate of multidrug-resistant mutations across the study samples.

Conclusion

In this study, for the first time, we clarified that HBV/C-infected patients had a higher risk to develop multidrug resistance. In addition, for the first time, we clarified that HBV/C had an inclination to develop rtT184/rtS202 substitution-containing patterns, while HBV/B had an inclination to develop rtM250 substitution-containing pattern for ETV-resistant mutations. The study provided new insight on clinical implications of HBV genotype, and the information is valuable for the management of HBV drug resistance.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation (81171616, 81171617, 81371852) and the capital health research and development of special (2014-2-5034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank all individuals who participated in this study.

Authors' Contributions

Designed and organized the experiments: D.X. and X.Z. Performed the experiments and collected patients' samples: X.L., Y.L., S.X., D.J., S.Y., J.H., J.Z., and J.W. Analyzed the data: X.L., Y.L., and H.L. Wrote the article: D.X. and X.L.

Disclosure Statement

The authors have declared that no competing interests exist.

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