The Association of Substitutions in the Hepatitis C Virus Subtype 1b Core Gene and IL28B Polymorphisms With the Response to Peg-IFNα-2a/RBV Combination Therapy in Azerbaijani Patients

3.1. Study Population

Fifty-one chronically HCV-1b infected Azerbaijani patients were enrolled in the current cross-sectional study between March 2010 and June 2015. These patients arrived in the Islamic Republic of Iran for medical treatments from the Republic of Azerbaijan and were visited in hospitals affiliated with the Iran University of Medical Sciences.

Patients aged over thirteen years, and with HCV RNA levels in their plasma ≥ 450 IU/mL for a period of six months prior to the present study, were considered eligible. Patients co-infected with HIV or hepatitis B virus (HBV) were excluded, as were subjects affected by HCC, liver dysfunction, liver transplants, decompensated liver disease, poorly-controlled psychiatric diseases, creatinine clearance rate of 50 mL/minute, malignant neoplastic disease, active substance abuse, severe cardiac or chronic pulmonary disease, or poorly-controlled diabetes (18). This study was approved by the ethical committee of the Iran University of Medical Sciences (Tehran, Iran). All patients provided written informed consent prior to study participation and were treated with a combinational therapy of RBV (1,000 - 1,200 mg/day) (Copeguss, Roche, Basel, Switzerland) and Peg-IFNα-2a (180 µg/week) (Pegasyss, Roche, Basel, Switzerland) for 48 weeks.

3.2. Established Responses to the Treatment

Patients with sustained virological response (SVR), such as those displaying undetectable HCV RNA levels at the end of the therapy and during the six months follow-up period, were defined as responders. Patients whose HCV-RNA levels were still detected at the end of the antiviral therapy were considered non-responders (NR), while those who had undetectable HCV-RNA levels at the end of treatment, followed by a reappearance of viremia over the six months follow-up period, were designated as relapsers. Both non-responders and relapsers were considered non-viral response (NVR) patients. Finally, patients with undetectable HCV RNA levels at 12 and 4 weeks of treatment were defined as early viral response (EVR) and rapid viral response (RVR), respectively (19, 20).

3.3. Specimens Collection and Processing

At the beginning of the treatment, approximately 5 mL samples of whole peripheral blood were collected from each patient and kept in sterile vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). Following plasma separation, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll-density gradient centrifugation (Lympholyte H, Cedarlane, Hornby, Canada). After three washes with phosphate-buffered saline (pH = 7.3 ± 0.1), PBMCs were counted, resuspended in 200 μL RNAlater solution (Ambion, Inc, Austin, Texas, USA), and stored at -80°C until use.

3.4. RNA Isolation and Complementary DNA (cDNA) Synthesis

Viral RNA was extracted from 200 μL plasma samples using the high pure viral nucleic acid Kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. The quantity and quality of the isolated RNA were measured using the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, USA), and 0.5 mg of the total RNA, with an A260/A280 ratio ≥ 1.90, was used for cDNA synthesis, as previously described (4, 19, 21)

3.5. Hepatitis C Virus RNA Quantitation and Genotyping

HCV viral load was assessed using the COBAS TaqMan HCV Test v2.0 (Roche Molecular Diagnostics, Mannheim, Germany) real-time PCR. HCV genotyping was carried out using restriction fragment length polymorphism (RFLP) assay of the 5′ noncoding region (5′-NCR) of the viral genome, according to a previously described protocol (22).

3.6. Amplification of the HCV Core Region Using Reverse Transcriptase-Nested Polymerase Chain Reaction (RT-Nested PCR)

The HCV core region (nucleotides 340 - 682) was subjected to nested PCR using the following primers: outer sense 274F (5′-AAGGCCTTGTGGTACTGCCTG-3′), outer antisense oka186R (5′-ATGTACCCCATGAGGTCGGC-3′), inner sense 318F (5′-GGAGGTCTCGTAGACCGTGCA-3′) and inner antisense 709R (5′-GACCTTACCCAARTTMCGCGACCTA-3′) (23).

The first and second rounds of nested PCR were carried out in a 25 µL mixture reaction containing 1.0 U of Ex Taq DNA polymerase [TaKaRa Biotechnology (Dalian) Co., Ltd., Shiga, Japan], 2.5 µL of 10X Ex Taq buffer (Mg²⁺ free), 2.0 µL of MgCl₂ (25 mM), 2.0 µL dNTPs Mixture (25 mM each) and 10 pmol of each outer primer. In addition, 2.5 µL of DNA was added to the mixture for the first round of nested PCR while, for the second one, 2.5 µL of the resulting PCR product was used as a template. For the first round, PCR conditions were as follows: initial denaturation at 95°C for 5 minutes, 35 cycles of 30 s at 95°C, 45 s at 60°C, and 60 s at 72°C, followed by a final extension of 7 minutes at 72°C. The second round of PCR was conducted as follows: initial denaturation at 95°C for 5 minutes, 35 cycles of 30 s at 95°C, 30 s at 60°C, and 50 s at 72°C, followed by a final extension of 7 minutes at 72°C.

The second round PCR products were purified using the high pure PCR product purification kit (Roche Diagnostic, Mannheim, Germany), according to the manufacturer’s instruction. Next, the samples were sequenced bi-directionally, according to the dye termination method, using the ABI 3730 XL sequencer. The sequences of the HCV Core region were determined from the pre-treatment plasma of the fifty-one Azerbaijani patients with chronic HCV-1b infection.

3.7. Genotyping of the IL28B rs12979860 C/T Polymorphism

Genomic DNA was extracted from PBMC samples using the QIAamp DNA blood mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. Genotyping of the IL28B rs12979860 C/T polymorphism was conducted using PCR-based RFLP assay as described previously (24).

3.8. Sequence Analysis of the HCV Core Region

A consensus sequence of the core region was generated by aligning all of the nucleotides encoding the core amino acid residues obtained from the current study to the HCV-1b reference genome (GenBank accession number D90208), using MEGA software (version 6.0). The phylogenetic tree of the core amino acid residues (codons 1 - 122) was inferred by aligning the sequences obtained from the pre-treatment plasma samples of responders, non-responders (NR) and relapsers with the HCV-1b reference genome according to the Neighbor-Joining method, using MEGA software (version 6.0) (Figure 1). Statistical significance of the phylogenetic trees was assessed using the bootstrap method (one thousand replicates).

Figure 1.

Sequences of the Core Amino Acid Residues (codons 1 - 122) obtained From the Pre-Treatment Plasma of Responders (Indicated as SVR), Non-Responders (NR) and Relapsers Aligned to the HCV-1b Reference Genome (GenBank Accession Number D90208)

3.9. The Accession Numbers of Nucleotide Sequences

The nucleotide sequences of the HCV-1b core region presented in this study were submitted to GenBank, and will be available with the accession numbers KT329291 to KT329341.

3.10. Statistical Analysis

All calculations were performed using the SPSS software package (SPSS Inc., Chicago, IL, USA). The normality of the data was assessed using the Kolmogorov-Smirnov test. The analysis of continuous variables was carried out using the One-way ANOVA and the Kruskal-Wallis tests. The χ²-test was employed to evaluate the associations between categorical variables. A P ≤ 0.05 was considered statistically significant.

4.1. Baseline Characteristics

Fifty-one Azerbaijani patients, chronically infected with HCV-1b, were enrolled in this prospective study. The mean age of participants was 39.6 ± 10.6 (range 20 - 58 years). Twenty-two (43.1%) out of fifty-one patients were male. The demographic features, laboratory parameters, and different responses to the Peg-IFNα-2a/RBV combination therapy (responders [n = 32, 62.7%], NR [n = 7, 13.3%], relapsers [n = 12, 23.5%]) are summarized in Table 1.

Significant differences were observed in terms of gender (P = 0.018), viral load titer (P = 0.020) and aspartate aminotransferase (AST) level (P = 0.024) but not for age (P = 0.547) or alanine aminotransferase (ALT) level (P = 0.992) (Table 1).

4.2. HCV Core Substitutions and Treatment Response

The core region of HCV-1b was amplified, sequenced and aligned with the HCV-1b reference genome (GenBank accession number D90208) (Figure 1). In the present study, a significant association was observed between the outcome of the antiviral therapy and amino acid substitutions in the HCV-1b core region at positions 43 [arginine (R) to lysine (K), P = 0.047], 70 [R to glutamine (Q), P < 0.001], 91 [methionine (M) to leucine (L), P = 0.037], and 106 [Serine (S) to Asparagine (N), P = 0.018]. In contrast, no association was observed between the outcome of the Peg-IFNα-2a/RBV treatment and amino acid substitutions at residues 20 [Q to leucine (L)], 30 [isoleucine (I) to T], and 101 [R to Histidine (H)] (Table 2).

A phylogenetic tree was labelled with patient numbers and responses to the Peg-IFNα-2a/RBV treatment. It is noteworthy that most of relapsers are clustered in one of the clades (Figure 2). No clustering was observed in other sequences, according to treatment response.

Figure 2.

Phylogenetic Tree Based on the Core Amino Acid Sequences (Codons 1 - 122) Obtained From Pre-Treatment Plasma of Responders (Indicated as SVR), Non-Responders (NR) and Relapsers, Aligned With the HCV-1b Reference Genome (GenBank Accession Number D90208)

4.3. Single Nucleotide Polymorphisms (SNPs) of the IL28B Genotype

Twelve (23.5%), thirty-six (70.6%) and three (5.9%) out of fifty-one patients possessed, respectively, the CC, CT and TT genotypes of IL28B rs12979860. Interestingly, all of the homozygous CC patients achieved SVR, whereas TT homozygous patients were found exclusively in relapsers. Twenty out of thirty-six CT heterozygous patients (62.5%) showed SVR. It is noteworthy that the CC genotype was associated with SVR (P = 0.009), while the TT genotype was associated with a relapse response (P = 0.006) (Table 3).