1. Background
Hepatitis C virus (HCV) infection is a major global public health challenge, affecting an estimated 3% (120 to 180 million) of people worldwide (1). Approximately, 70 to 80% of HCV infected patients become chronic hepatitis C carriers and may progress to chronic liver diseases including hepatocellular carcinoma (HCC), cirrhosis of the liver and liver failure (2, 3). Although effective antiviral therapy with a combination of pegylated interferon-α 2a plus ribavirin (Peg-IFNα-2a/RBV) may prevent these complications, treatment outcomes vary. Different sustained virologic response (SVR) rates (50%) have been observed in different populations (4-6).
Viral factors, including pre-treatment HCV RNA load, HCV genotype, liver cirrhosis, and host factors, including gender, age, innate immunity, and genetic variation in Interleukin-28B (IL28b) are beneficial in predicting response to interferon (7, 8). Several viral factors may predict the outcome of Peg-IFNα-2a/RBV combination therapy; for instance, 40 amino acids in the non-structural 5A (NS5A) region (9, 10), and amino acid substitutions in the core region have been reported as predictors of hepatitis C therapy outcome (11, 12).
Numerous common polymorphisms have been found in the human genome that may be correlated with the effectiveness of interferon therapy. For instance, two single nucleotide polymorphisms (SNPs) were observed in the IL28B locus. The rs12979860 (located 3 kb upstream of the IL28B gene) and the rs8099917 (located 8.9 kb upstream of the IL28B gene) SNPs are the strongest SVR predictors in patients infected with chronic HCV (13, 14). One of the most acceptable, reliable, and fastest methods to determine the rs12979860 polymorphism is PCR-RFLP assay. This method can be used by every molecular diagnostic laboratory with PCR-RFLP assay capability. Determining this polymorphism will be clinically useful in predicting treatment response in participants with HCV infection (15, 16).
Recently, a meta-analysis indicated that the rs12979860 allele is more important than the rs8099917 allele and more accurately predicts SVR (17). Furthermore, the prevalence of SVR and these SNPs varies among ethnicities and HCV genotypes (18). Thus, more studies are warranted to identify the mechanisms. Unfortunately, there are currently limited reports in literature about the role of rs12979860 polymorphisms in Azerbaijani patients with established chronic hepatitis C. The patients taking part in this study had come from the Republic of Azerbaijan to Iran for medical treatment by Peg-IFNα-2a/RBV combination therapy.
2. Objectives
We aimed to investigate the influence of rs12979860 polymorphism on the rate of response to Peg-IFNα-2a/RBV combination therapy in Azerbaijani patients with HCV infection.
3. Methods
3.1. Study Population
This cross-sectional study was conducted on a total of 102 Azerbaijani patients with established chronic hepatitis C between January 2010 and September 2015. The patients had come from the Republic of Azerbaijan to Iran for medical treatment while all of them were anti-HCV Abs and HCV-RNA positive. They referred to hospitals affiliated to Iran University Medical Sciences and Tehran hepatitis centre. Informed consent was obtained from all of the participants. The exclusion criteria included (i) having other hepatitis infection [HBV (HBV-DNA and hepatitis B surface antigen negative), and HDV (HDV-RNA and anti-HDV antibody negative)], (ii) using immunosuppressive drugs, (iii) coexistent autoimmune liver diseases (negativity for antimitochondrial and antinuclear antibodies, etc.), (iv) diabetes mellitus, (v) genetic disorders, (vi) alcohol intake, (vii) drug toxicity, and (viii) having any other disease that suppress the immune system for instance human immunodeficiency virus infection (anti-HIV Abs negative). The study was approved by the local ethics committee of Gastrointestinal and liver disease research center (GILDRC) at Iran University of Medical Sciences, Tehran, Iran.
3.2. The Treatment Protocol
The treatment protocol with Peg-IFNα-2a/RBV combination therapy was based on standard protocols for hepatitis C treatment (19): Peg-IFNα-2a (Pegasys, Roche, Basel, Switzerland) 180 mg per week and ribavirin (RBV) (Copegus, Roche) 1,000 - 1,200 mg per day for 48 weeks. Determination of the response to anti-HCV treatment has been described previously in detail (20).
3.3. Sample Preparation
A 5 ml blood specimen was collected in an EDTA-containing vacutainer tube from each patient. Following plasma separation, peripheral blood mononuclear cells (PBMCs) were isolated by using a standard method of Ficoll-Hypaque gradient centrifugation (Lymphoprep, Oslo, Norway). After washing 3 times with phosphate-buffered saline (pH = 7.3 ± 0.1), the achieved PBMCs were counted and resuspended in 250 μL RNALater (Ambion Inc., Austin, TX, USA), and frozen at -70°C until use.
3.4. Detection of HCV-RNA and HCV-Genotyping
Viral RNA was extracted from 140 μL of plasma by using QIAamp viral RNA extraction kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s instructions. For the detection of genomic HCV RNA, the extracted viral RNAs were subjected to reverse-transcription-nested polymerase chain reaction (RT-nested PCR), as described before (20). Polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) was employed to determine HCV genotypes (21).
3.5. Single Nucleotide Polymorphism (SNP) of the IL28B Genotype
Genomic DNA was extracted from a pellet of about 3 - 5 × 106 PBMC specimens using a QIAamp DNA blood mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. rs12979860 SNP genotyping was performed using PCR-RFLP assay.
The oligonucleotide primers were as follows: 5′-GCT TAT CGC ATA CGG CTA GC-3′ (sense) and 5′-AGG CTC AGG GTC AAT CAC AG-3′ (the primers were synthesized by metabion company, Germany). Polymerase chain reaction amplification was performed (6) and PCR products (252 base pairs) were digested with 1 unit of the BstU-I restriction endonuclease (New England Biolabs, Inc.) at 60°C for 16 hours. BstU-I digestion of allele CC yields the fragments of 135, 82, and 25 base pairs, and DNA containing the allele TT polymorphism yields the fragments of 160 and 82 base pairs. Restriction digestion products of specimens with undigested products and positive and negative controls and DNA size marker were visualized using 3.5% agarose gel electrophoresis (6). In 10 patients, the genomic region surrounding the IL28B rs12979860 C/T polymorphism was also confirmed by sequencing.
3.6. Statistical Analysis
Statistical analysis was performed using SPSS software version 16 (SPSS, Inc., Chicago, IL). To evaluate the normality of data, Kolmogorov-Smirnov test was used. Analysis of continuous variables was conducted by one-way ANOVA and Kruskal-Wallis tests. The chi-square test was applied to assess associations between categorical variables. A P value of ≤ 0.05 was considered statistically significant.
3.7. Ethical Approval
The ethical approval of the present research was received from the ethics committee of Iran University of Medical Sciences (No.2083 at 2015-04-28) before starting this study.
4. Results
4.1. Clinical and Demographic Features
The current prospective study was conducted on a total of 102 Azerbaijani patients chronically infected with HCV. The mean age of the patients was 36.5 ± 12.8 (ranged between 8 and 71 years). Out of 102 patients, 58 (56.9%) were male. The HCV genotypes in the study population were determined as follows: subtype 1b in 72 (70.6%) patients, subtype 3a in 17 (16.7%), genotype 2 in 7 (6.9%), subtype 1a in 5 (4.9%), and mixed infection in 1 (1.0%) patient. The patients with HCV-1b infection took part in the present study. The data on demographic, laboratory, and epidemiological characteristics of the Azerbaijani patients with HCV infection and different types of response to anti-viral therapy with RBV/PEG-IFN2α are summarised in Table 1.
Table 1. Demographic and Laboratory parameters of the Azerbaijani Patients with Hepatitis C Virus Infection and Different Types of Response to Anti-viral Therapy with Ribavirin/PEG-IFN2αa
| Patients | Sustained Virological Responders | Non-Responders | Relapsers | Total | P Value |
|---|---|---|---|---|---|
| No. of patients | 71 (69.6) | 17 (16.7) | 14 (13.7) | 102 (100) | |
| Gender Male/Female | 38/33 | 10/7 | 10/4 | 22/29 | 0.458 |
| Age | 34.7 ± 13.2 (8 - 71) | 38.7.7 ± 11.3 (20 - 56) | 39.6 ± 11.6 (22 - 57) | 36 ± 12.8 (8 - 71) | 0.270 |
| Laboratory parameters | |||||
| Viral Load, IU/mL | 3,138,045 (10,065 - 23,800,950) | 4,420,864 (47050 - 15,584,670) | 721,119 (14100 - 2,583,880) | 801,990 (10,065 - 23,800,950) | 0.098 |
| ALT, IU/L | 70.1 (14 - 269) | 73.3 (27 - 187) | 47.8 (17 - 180) | 71.8 (14.269) | 0.480 |
| AST, IU/L | 59.8 (15 - 171) | 58.9 (18 - 148) | 71.3 (20 - 156) | 61.3 (15 - 171) | 0.702 |
| HCV genotype | 0.933 | ||||
| 1a | 3 (75) | 1 (25) | 0 | 4 (100) | |
| 1a/1b | 2 (100) | 0 | 0 | 2 (100) | |
| 1b | 48 (66.7) | 11 (15.3) | 13 (18.1) | 72 (100) | |
| 2 | 5 (71.4) | 1 (14.3) | 1 (14.3) | 7 (100) | |
| 3a | 11 (68.8) | 4 (25) | 1 (6.2) | 16 (100) | |
| ND | 1 (100) | 0 | 0 | 1 (100) |
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ND, not determined.
aValues are expressed as No. (%) or mean ± SD.
4.2. Frequency of IL28B Genotypes
The genotyping of IL28B rs12979860 showed the presence of CC, CT, and TT genotypes in 41 (40.2%), 52 (51.0%), and 9 (8.8%) out of 102 individuals, respectively. There was a significant association between IL28B genotypes and response to HCV combination therapy with peg-IFNα-2a/RBV (Table 2) (P = 0.018).
Table 2. Correlation between IL28B Polymorphisms (rs12979860) of HCV and Response to Peg-IFNα-2a/RBV Combination Therapy in Azerbaijani Patientsa
| Response | Total | P Value | ||||
|---|---|---|---|---|---|---|
| SVR (n = 70) | NR (n = 17) | Relapse (n = 15) | ||||
| IL28B | CC | 34 (48.6) | 5 (29.4) | 2 (13.3) | 41 (40.2) | 0.018 |
| CT | 30 (42.9) | 9 (52.9) | 13 (86.7) | 52 (51.0) | ||
| TT | 6 (8.6) | 3 (17.6) | 0 | 9 (8.8) | ||
| Total | 70 (100) | 17 (100) | 15 (100) | 102 (100.0) | ||
aValues are expressed as No. (%).
4.3. Clinical and Demographic Features of Patients with HCV-1b Infection
The mean age of patients with HCV-1b infection was 36.9 ± 12.4 (ranged between 27 and 57 years). Out of 72 patients, 42 (58.3%) were male. Table 1 represents the demographic characteristics, laboratory parameters, and different responses to combination therapy with peg-IFNα-2a/RBV (SVR [n = 48, 66.7%], NR [n = 11, 15.2%], and relapsers [n = 13, 18.1%]). In the current investigation, no significant associations were observed between gender (P = 0.234), age (P = 0.248), viral load (P = 0.204), ALT level (P = 0.893), and AST level (P = 0.940) of Azerbaijani patients with hepatitis C virus infection and outcome of HCV combination therapy (Table 3).
Table 3. Demographic and Laboratory Parameters of the Azerbaijani Patients with Hepatitis C Virus 1b Infection and Different Types of Response to Anti-Viral Therapy with Peg-IFNα-2a/RBVa
| Patients | Sustained Virological Responders | Non-Responders | Relapsers | Total | P Value | |
|---|---|---|---|---|---|---|
| No. of patients | 48 (66.7) | 11 (15.2) | 13 (18.1) | 72 (100) | ||
| Gender | Female | 19 (39.6) | 7 (63.6) | 4 (30.8) | 30 (41.7) | 0.234 |
| Male | 29 (60.4) | 4 (36.4) | 9 (69.2) | 42 (58.3) | ||
| Age, Mean | 35.2 ± 12.8 (8 - 60) | 39.6 ± 11.7 (20 - 56) | 40.9 ± 10.8 (27 - 57) | 36.9 ± 12.4 (27 - 57) | 0.248 | |
| Laboratory parameters | ||||||
| Viral Load, IU/mLb | 672447.5 (10065 - 23800950) | 1032875 (47050 - 11015400) | 391180 (14100 - 2853880) | 659865 (10065 - 23800950) | 0.204 | |
| ALT, IU/L | 63.5 (21 - 269) | 56 (27 - 157) | 49 (17 -180) | 60.5 (17 - 269) | 0.893 | |
| AST, IU/L | 52.2 (17 - 171) | 50 (50 - 148) | 45 (20 - 156) | 50.5 (17 - 171) | 0.940 |
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;
aValues are expressed as No. (%) or mean ± SD.
bThese parameters had not a normal distribution.
4.4. Frequency of IL28B Genotypes Among Patients With HCV-1b Infection
The genotyping of IL28B rs12979860 showed the presence of CC, CT, and TT genotypes in 24 (33.3%), 42 (58.3%) and 6 (8.3%) out of 72 patients, respectively. There was a significant correlation between IL28B rs12979860 genotypes and response to HCV combination therapy with peg-IFNα-2a/RBV (Table 4) (P = 0.001).
Table 4. Genetic Variation in Interleukin-28B and Response to Peg-IFNα-2a/RBV Combination Therapy in Azerbaijani Patients with Hepatitis C Virus Subtype-1b Infectiona
| Parameters | Response to Peg-IFNα-2a/RBV Combination Therapy | P Value | ||||
|---|---|---|---|---|---|---|
| SVR (n = 48) | NR (n = 11) | Relapse (n = 13) | Total (n = 72) | |||
| IL28B | CC | 23 (47.9) | 1 (9.1) | 0 | 24 (33.3) | 0.001 |
| CT | 21 (43.8) | 8 (72.7) | 13 (100) | 42 (58.3) | ||
| TT | 4 (8.3) | 2 (18.2) | 0 | 6 (8.3) | ||
| Total | 48 (100) | 11 (100) | 13 (100) | 72 (100) | ||
aValues are expressed as No. (%).
4.5. Association of IL28B Genotypes with HCV Genotype and Viral Load
The association between IL28B rs12979860 with HCV genotype and viral load was investigated using linear regression analysis. No significant correlation was observed between IL28B rs12979860 polymorphism and HCV genotype (P = 0.263), while there was a significant association between IL28B rs12979860 genotypes and viral load, as the mean viral load was significantly lower in patients with CT and TT genotypes than those with CC genotype (P = 0.001).
4.6. Factors Associated with SVR
A multivariate logistic regression analysis was performed to determine the correlated factors with SVR. After adjusting for potential confounder variables, the results showed that age and IL28B polymorphism could be significant predictors of SVR. The results showed that by a unit increase in the age, the chance of SVR occurrence decreases 0.949 times (95% CI 0.909 - 0.992, P = 0.021). Also, the chance of SVR occurrence in IL28B polymorphism of CT and TT genotypes compared to CC genotype (as a reference group) was 0.111 (95% CI: 0.026 - 0.465, P = 0.003) and 0.110 (95% CI: 0.014 - 0.843, P = 0.034) times, respectively, which means that the chance of SVR occurrence was significantly more in CC type of IL28B polymorphism than two others types (Table 5).
Table 5. Factors Predictive of Sustained Virologic Response Obtained by Multivariate Logistic Regression Analysis
| Variables | SVR | |||
|---|---|---|---|---|
| Odds Ratio | 95% CI | P Value | ||
| Age | 0.949 | 0.909 - 0.992 | 0.021 | |
| Gender | Female | 1 | - | - |
| Male | 0.417 | 0.150 - 1.158 | 0.093 | |
| Genotype | 1a | 1 | - | - |
| 1b | 0.505 | 0.039 - 6.465 | 0.600 | |
| 2 | 0.738 | 0.025 - 21.15 | 0.860 | |
| 3a | 0.265 | 0.014 - 4.764 | 0.368 | |
| Viral Load, IU/mL | 0.999 | 0.999 - 1 | 0.236 | |
| IL28 | CC | 1 | - | - |
| CT | 0.111 | 0.026 - 0.465 | 0.003 | |
| TT | 0.110 | 0.014 - 0.843 | 0.034 | |
| ALT, IU/L | 1.003 | 0.979 - 1.028 | 0.771 | |
| AST, IU/L | 0.998 | 0.969 - 1.028 | 0.940 | |
5. Discussion
Combination therapy with Peg-IFNα-2a/RBV is still the standard treatment protocol in Azerbaijani patients with chronic HCV infection. New, direct-acting antiviral agents are unavailable. The most frequent HCV genotype in Azerbaijani patients is subtype 1b, which is one of the most difficult-to-treat genotypes (22). Therefore, study of effective pre-treatment predictors of therapeutic outcome is unnecessary. Recent studies have suggested that polymorphisms near the IL28B locus are associated with the response to Peg-IFNα-2a/RBV combination therapy (13, 14, 17).
Identification of SNPs including rs12979860 and rs8099917 in the IL28B locus provides an extremely effective pre-treatment predictor of response to interferon therapy. Although both SNPs are independent pre-treatment predictors of response to anti-HCV therapy, recent investigations have suggested that rs12979860 contributes directly to therapeutic responses (13, 17). The rs12979860 CC genotype was associated with higher chances of SVR. Hence, the facts mentioned above motivated us to investigate the prevalence of rs12979860 genotypes in Azerbaijani patients who were chronically infected with HCV subtype 1b and evaluate the influence of rs12979860 genotypes in anti-HCV treatment response.
In the present study, the PCR-RFLP assay was used to determine the rs12979860 polymorphism. It is noteworthy that PCR-RFLP assay has been utilized to determine this polymorphism in several studies such as those conducted by Sharafi et al. (23), Irman et al. (24), Fateh et al. (16), and Nakamoto et al. (25). These studies have reported that this assay is simple, fast, inexpensive, and valid. This method is applicable in every molecular diagnostic laboratory with PCR-RFLP assay capability (16).
Among 102 Azerbaijani patients with chronic HCV infection, CC, CT, and TT genotypes were present in 41 (40.2%), 52 (51.2%), and 9 (8.8%) cases, respectively (Table 2). On the other hand, because the most studied patients were infected with HCV genotype 1b, this polymorphism was analyzed separately in these participants. The genotype frequencies of rs12979860 polymorphisms in these patients with CC, CT, and TT genotypes were 33.3%, 58.3%, and 8.3%, respectively, which are close to the corresponding frequencies in Iranian and Turkish patients (26-28).
The results of the current study demonstrated a significant association between rs12979860 CC genotype and SVR rate. The CC genotype of rs12979860 occurred in 48.6% of patients who achieved SVR, and 47.9% of patients who were infected with HCV subtype 1b, which were significantly higher than the corresponding values of CT and TT genotypes. These findings are consistent with the results of other studies in different ethnic groups (29). Based on these findings, the rs12979860 CC genotype could constitute an effective pre-treatment predictor in Azerbaijani chronic hepatitis C patients, especially when associated with HCV subtype 1b. In contrast, a significant association between rs12979860 CT genotype and non-response or relapse was observed. Frequencies of CT genotype in NR group and relapsers were 72.7% and 100%, respectively. This finding is consistent with those of previous studies (16, 28, 30).
In this study, the rs12979860 CC genotype of IL28B was associated with response to Peg-IFNα-2a/RBV combination therapy in Azerbaijani patients with chronic HCV infection. The results of this study may help identify the patients for whom the treatment may be successful. Further studies with larger sample sizes seem necessary to evaluate the present results.
