Impact of IL 28 B genetic variant ' s and viral genotype on treatment outcome of hepatitis C infected patients

Introduction: Viral genotype and variation in host genes involved in the immune response may predict the treatment response in patients infected with HCV. The present study was designed to determine the distribution pattern of HCV and host genotypes in Chronic Hepatitis C (CHC) patients and their association with virological response and other risk factors. Methodology: Two hundred and fifty (n = 250) HCV positive patients were included in the study. HCV and Interleukin 28B (IL28B) genotyping was carried out by PCR-RFLP. Results: Viral genotype 3 was the predominant genotype seen in 187 (74.8%) patients. Wild genotype predominated in rs12979860, rs12980275 and rs8099917 SNP of IL28B gene. A significant difference was found in end stage virological response (EVR) between HCV genotype 1 infected patients with wild and variant genotype for rs12980275 and rs8099917 SNPs respectively (P < 0.05). On multivariate analysis all the SNPs were found to be associated with each other (P < 0.05) with rs12980275 SNP associated with history of Jaundice (P < 0.05). Viral genotype 3 was significantly associated with age (< 50 years) and rapid virological response (RVR) while as viral genotype 1 was significantly associated with history of surgery on multivariate analysis (P < 0.05). Conclusions: The viral genotype and IL28B polymorphisms are important factors to personalize antiviral therapy of patients with CHC.


Introduction
It is now well established that HCV infection affects all countries and chronic infection with HCV is a global health problem that affects more than 180 million people worldwide, approximately 3% of the worldwide population is infected with the HCV [1,2].In Asia, the prevalence of HCV ranges between 2% and 5% [1,3].It is estimated that in India approximately 1.8-2.5% of the population is presently infected by HCV and about 20 million people are already having HCV infection [3].HCV infection leads to chronic liver disease in 60-70% of patients, 5-20% will develop cirrhosis, and 1-5% will die of complications including hepatocellular carcinoma (HCC) [4,5].Direct acting antivirals like protease inhibitors are expensive and not available at all medical centers, therefore the most effective current standard therapy for chronic HCV infection consists of long-acting pegylated interferon-α plus oral treatment with ribavirin (Peg-IFN-a/R) [6,7].
Virus-specific characteristics (viral load, genotype) and clinical parameters (age, gender, BMI, Stage of liver fibrosis and liver enzymes) are among the baseline predictors of response to HCV treatment [8].Moreover, host genetic variation may influence the response to HCV treatment and is assumed to explain the heterogeneity in HCV clearance across individuals.Several independent studies have consistently shown that single nucleotide polymorphisms (SNPs) near IL28B, which encodes the type III interferon IFN-λ3 are strongly associated with the response to treatment of chronic hepatitis C [9,10] and affects the spontaneous and induced clearance of HCV and progression to CHC [11].The mechanism by which SNPs influence the outcome of HCV infection and its treatment is completely explained.The first and largest genome wide association study (GWAS) identified a top discovery SNP, rs12979860, which is located on chromosome 19q13, 3kb upstream of the IL28B gene, which codes for IFN-λ-3 [9,12].Following this discovery, other SNP, rs8099917 has been identified and is in linkage disequilibrium with the previous SNP.In particular, the homozygous genotypes CC at marker rs12979860, AA at marker rs12980275 and TT at marker rs8099917 are all associated with favorable treatment outcomes [13].These data have been confirmed in multiple ethnic groups, populations of different ancestry and HCV genotypes, and in various clinical scenarios [14].Multiple clinical cohort studies have validated the findings of the GWA studies and regression modelling has shown that the IL28B polymorphism is the strongest baseline predictor of SVR in patients treated with Peg-IFN-a/R [15,16].IL28B genotype testing has become part of the standard of care as it can predict which patients are more likely to respond to treatment [2].
A sizeable chunk of patients with hepatitis C are being treated in our clinical setting at out tertiary care hospital where we observe that clinical response is quite good in majority of patients.With this background in mind, the present study was conceived to study the rate of response and predictors of response based on clinical, viral and genetic characteristics in patients with chronic HCV infection who were treated with a combination of Peg-IFN-a/R in a superspeciality hospital in the valley of Kashmir.

Patient Samples
The present study was a prospective nonrandomized open labeled clinical trial conducted between July 2013 and July 2016 in the Department of Gastroenterology and Clinical Biochemistry, Sher-I-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, Kashmir, India.All consecutive patients (n = 250) above age of 14 years with hepatitis C infection were enrolled.The diagnosis of HCV was established by the presence of HCV-RNA level greater than 50 IU/mL.The study was approved by the Ethical Committee of SKIMS, Soura-190011, Srinagar, India.Written informed consent was obtained from all patients before taking their blood samples.

HCV serology and genotyping
Serum samples were screened for Hepatitis C and anti-HCV antibodies using commercially available ELISA kits (HCV: Anti HCV (ELISA): Murex, Biotech, Kyalami, South Africa).Identification of HCV genotype was carried out by PCR-RFLP using the method of Chinchai et al. [17].In HCV RNA positive samples, subtypes were determined by performing PCR using type specific primers for the core region of the HCV genome [18].
HCV RNA levels and treatment responses The HCV RNA was determined using a COBAS Amplicor HCV Monitor, v2.0 (Roche Diagnostics, Branchburg, NJ, USA) with a precision value of 50 IU/mL before, during and after the treatment.

Intervention
The patients underwent interventional treatment in accordance with the standard protocol consisting of 180µg of peg interferon α-2a or 1.5µg/kilogram of body weight of pegylated interferon α-2b administered subcutaneously once per week.In genotype 1 and 4 patients, 1000 mg and 1200 mg of ribavirin was administered orally in daily doses in patients < 75 kg and > 75 kg of weight respectively.In genotype 2 and 3 patients, ribavirin was administered orally in 800 mg daily doses.

IL28B gene polymorphism
High-molecular-weight DNA was isolated from blood samples using DNA extraction kit (Zymo Research, USA).The amplification reaction was carried out in 25 μL reaction volume in a 0. digestion products for determining the rs12979860, rs12980275 and rs8099917 SNPs.In order to perform RFLP assay for the rs12979860 genotype, 20 μL of amplicons were digested with 5U of BstUI restriction endonuclease (New England Biolabs, MA, United States) at 60°C for 2 h.For RFLP assay at rs12980275, 20 μL of amplicons were digested with 5U of BslI restriction endonuclease (New England Biolabs, MA, United States) at 55°C for 2 h.For RFLP assay at rs8099917, 20μL of amplicons were digested with 1U of MaeIII restriction endonuclease (Roche Molecular Systems) at 55°C for 2 hours.Restriction digestion products for each were separated on 3% agarose gels stained with ethidium bromide for visualization on a UV trans-illuminator.

Statistical analysis
Pearson's chi-square test for proportions was used to analyze data.Fisher's exact test was used when the sample observation in a particular group was low.A two-tailed P < 0.05 was considered significant.Statistical analysis was performed using the statistical software package SPSS (SPSS v 20 Inc., Chicago, IL, USA).

Results
The present study was conceived to assess the various genotypes of HCV, the treatment response to antiviral therapy and impact of various modifiable and non-modifiable factors on treatment outcome among patients infected with Hepatitis C. Predominance of male gender (68.0%) and HCV genotype 3 (74.8%)was seen in our set of patients.Only 4% (10 of 250) were alcoholics in our study.RVR was achieved by 74% of patients.Table 2 depicts the frequency distribution of selected demographic and risk factors and Table 3 contains demographic and baseline parameters of Hepatitis C infected patients.
Of the polymorphisms of IL28B rs12979860 SNP the wild genotype (CC) was seen in 136 patients (54.4%) while as variant genotype (CT+TT) was seen in 114 patients (45.6%).42.9% of patients (27 of 63) infected with genotype 1 virus were wild for rs12979860 SNP compared to 58.9% of patients infected with genotype 3 virus and the association was statistically significant (P < 0.05).Only 12.5% of non-SVR patients were having wild genotype compared to 55.8% of patients in SVR group with wild genotype (P < 0.05) (Table 3).In rs12980275 SNP, AA (wild) genotype was seen in 132 patients (52.8%) while AG+GG (variant) genotype in 118 patients (47.2%).In case of rs8099917 SNP 59.6% were having wild genotype (TT) compared to 40.4% with variant genotype (TG+GG).The pattern of the various IL28B polymorphisms and their significant association with various clinicopathological and risk factors of HCV infected patients are shown in Table 4.
Patients infected with viral genotype 1 and 3 were stratified with respect to virological response and host polymorphism respectively and shown in Table 5.    Variant genotypes of rs12980275 (AG+GG) and rs8099917 (TG+GG) were significantly associated with SVR in patients infected with genotype 1 as well as genotype 3 (P < 0.05).In case of rs8099917 SNP the variant genotypes were also significantly associated with SVR in patients infected with both viral genotypes (P < 0.05).All the non-EVR patients infected with only genotype 1 virus had variant genotype for rs12980275 and rs8099917 SNP (P < 0.05) (Table 5).
Most of the patients with < 50 years of age and < 70 kilograms of weight achieved SVR (P < 0.05).The patients having history of alcohol consumption were relatively poor responders (P < 0.05).Almost 99.0% of patients achieving RVR and EVR went on to achieve SVR (P < 0.05).149 of 185 (80.5%) patients with < 50 years of age were infected with genotype 3 as against 19.5% patients infected with genotype 1 virus (P < 0.05).About 32% (40 of 122) patients having history of surgery in past were infected with genotype 1 and 68% were infected with genotype 3 (P < 0.05).Most of the patients infected with genotype 3 achieved RVR and EVR as compared to the patients infected with viral genotype 1 (P < 0.05).The various parameters in relation to SVR and viral genotypes are shown in Table 6.
Table 7 depicts the multivariate analysis of various parameters with respect to viral and host genotype by logistic regression.Viral genotype was significantly associated with age, Rapid virological response and history of surgery in past (P < 0.05).All the three SNPs were significantly associated with each other (P < 0.05).rs12980275 SNP was associated with history of Jaundice (P < 0.05).

Discussion
Hepatitis C infection is a leading cause of chronic hepatitis, cirrhosis and liver cancer and a primary indication for liver transplantation [4,5].Currently, standard of care (SOC) for all HCV genotypes continues to be (Peg-IFN-a/R) for 24 weeks for genotypes 2 and 3 or 48 weeks for genotypes 1 and 4 due to the scarcity and expensiveness of oral antivirals in India.[6].
Of the 250 patients infected with HCV, alcohol intake and drug abuse was extremely uncommon in our patients (10 of 250; 04%) because of social and religious prohibitions.This is in sharp contrast to all other major studies from other parts of world including India where alcohol consumption varied from 50% to 74% [19,20] (Table 2).
Recent studies have demonstrated that single nucleotide polymorphisms (SNPs) near the IL28B gene are strongly associated with HCV patient's response to Peg-IFN-a/R treatment and spontaneous elimination of the virus in different populations [21,22].A reasonable hypothesis might involve differential levels of intrahepatic interferon-stimulated gene (ISG) expression according to IL28B genotype, where the good response variant was associated with lower levels of intrahepatic ISG expression, and therefore slightly higher levels of circulating serum HCV RNA.The mechanistic basis for this association is unknown, and it remains unclear whether it is driven by the host, the virus, or both [23].
In our study, we genotyped all three IL28B polymorphisms.Determination of rs12979860 IL28B genotypes has shown that CC genotype was found in 54.4% (136 of 250) of patients and variant genotype (CT+TT) was present in 45.6% of patients.In two independent studies distribution of rs12979860 CC genotype was 26.8% and 21%, respectively while as Fattoviich et al. reported 39% CC genotype [24][25][26].In case of rs12980275 the frequency of wild and variant genotype (AA) was 52.8% which is higher than the frequency of 39% reported by Fattoviich et al. [26].The frequency of rs8099917 TT genotype was 59.6% presented in our patients, comparable to 56% reported by Fattoviich et al. [26] (Table 3).So in this study, the incidence of favourable/wild genotypes was more frequent than other studies [16,27].
In this study, the effect of IL28B polymorphisms was evaluated comparing the favourable (wild) genotypes with the unfavourable (variant) ones.A positive correlation was found between wild genotype (TT) of rs8099917 SNP and RVR (P < 0.05) while as a positive correlation was also found between wild genotypes of rs12980275 and rs8099917 and EVR (P < 0.05).A strong association was found between favourable genotype of all the three SNPs of IL28B gene and SVR (P < 0.05) (Table 3).A number of studies have validated the effect of the favourable genotypes on RVR and SVR [24][25][26][27].
On analysing the effects of IL28B polymorphisms on SVR with respect to genotype 1 and 3 separately, the effect of the genotype rs12980275 and rs8099917 on SVR was found to be statistically significant in both viral genotypes (P < 0.05).Thus the IL28B gene polymorphism closely associates with the natural course and treatment response of CHC in different populations, irrespective of HCV genotype.However, when stratified according to EVR the effect of the genotype rs12980275 and rs8099917 was found to be statistically significant in case of patients infected with genotype 1 as compared to genotype 3 virus (P < 0.05) (Table 4).
Of the 250 patients, 238 (96.8%) went on to have undetectable HCV RNA after 6 months of treatment i.e. attained SVR (Table 5).Almost all the patients with < 50 years of age were found to achieve SVR (P = 0.005) in accordance with many studies [28][29].In consistence with many studies it was observed that overweight individuals (≥ 70 kilograms) with CHC are less likely to achieve SVR (P = 0.04) [28,30].20% of patients with history of alcohol intake didn't achieve SVR compared to only 2.5% of non-alcoholics falling in non-SVR group.We found a significant impact of alcohol intake on SVR (P = 0.03) in accordance with previous reported studies [19,31].Since it has been well validated from previous studies across the globe that alcoholism is associated with a poor treatment outcome, the virtual absence of alcohol intake in our patients may be an important factor for the high SVR in our population.Secondly, almost all the non-SVR group had a variant version of alleles in all the 3 SNPs; hampering the clearance of HCV as compared to SVR group where majority of alleles were wild for all the three SNPs of IL28 gene.Lastly, the Kashmir has an ethnic population with only inter-regional marriages which gives it a conserved gene pool; different from the rest of the country which might play an important role in the HCV clearance and perhaps the genetic basis of which needs to be elucidated in future.Although 98% of patients with genotype 3 achieved SVR, genotype 1 patients also showed very high SVR of 94% (P>0.05).So interestingly, in our study treatment response was independent of viral genotype which is in contradiction with majority of studies which associate genotype 1 and 4 with lower SVR [32][33][34].
RVR was achieved by 74.0% of patients which is considerably higher than most studies [32,33].In all, 183 of 185 patients (98.9%) who achieved RVR went on to attain SVR (P = 0.05) which is in consistence with most of the studies [35,36].In patients infected with HCV, the achievement of EVR is of major importance for the overall therapy success.All guidelines recommend to perform a quantitative assessment of HCV RNA at week 12 of treatment.EVR is highly predictive of SVR and provides a valuable tool to decide on continuation and duration of treatment, as well as providing patients with an additional motivation to adhere to treatment [37][38][39].In this study, majority of the patients had attained EVR (240 out of 250; 96.0%).Such an extremely high rates of EVR has not been reported in any of the previous studies [32,33].
Viral genotype 1 was seen in 63 patients (25.2%) and genotype 3 was seen in 187 patients (74.8%) while as genotype 2, 4, 5 and 6 were absent in our cases (Table 5).Similar frequency of viral genotypes has been reported by many studies from India [40,41].There was a significantly lower percentage of patients with ≥ 50 years of age in genotype 1 than genotype 3 (41.5% vs. 58.5%)which is in contrast to majority of studies where patients with genotype 1 or 2 were significantly older [42,43].Majority of patients with genotype 1 were having past history of surgery compared to genotype 3 patients (40 of 63 vs. 82 of 187; P = 0.005).This is in agreement with other studies from different countries where majority of the cases with genotype 1 had history of hospitalization for major/minor surgery [44,45].Of the 185 patients achieving RVR (74.0%)only 19.5% (36) belonged to genotype 1 while 80.5% (149) had genotype 3 (P = 0.001) and all the patients with genotype 3 went on to achieve complete EVR (P = 0.003).In other sense 36 out of 63 patients (57.1%) with genotype 1 while 149 out of 187 (79.6%) with genotype 3 achieved RVR which is considerably higher than most studies [32][33][34][35]46,47].Although a few studies have recorded higher RVR but their overall SVR rates were lower than this study [48,49].

Conclusion
Viral Genotype 3 and IL28B wild genotype predominates in the HCV infected patients of this closed population.Although SVR was achieved by majority of patients and SVR was independent of viral genotype, IL28B polymorphisms might serve as important factor to personalize antiviral therapy of patients with CHC.However, reproduction of genetic results in various clinical settings is a prerequisite for establishing such therapy regimens.

Table 1 .
Primers and restriction enzymes used for screening the various SNPs in the upstream of IL28B gene.

Table 2 .
Frequency distribution analysis of selected demographic and risk factors in Hepatitis C infected patients.

Table 3 .
Levels of various demographic and baseline parameters.

Table 4 .
Association between rs12979860 (C to T) rs12980275 (A to G) and rs8099917 (T to G) Genotypes and Clinico-pathological Characteristics of Hepatitis C (HCV) infected patients.

Table 5 .
Association between rs8099917 (T to G), rs12980275 (A to G) and rs8099917 (T to G) genotypes and sustained virological response (SVR), rapid virological response (RVR), end stage virological response (EVR) with respect to viral genotype 1 and 3. Sustained virological response, RVR: Rapid virological response, EVR: End stage virological response, ETR: End of treatment response.

Table 6 .
Association of various parameters of Hepatitis C (HCV) Patients with Sustained Virological Response (SVR) and Viral Genotype.

Table 7 .
Multivariate analysis of viral genotype and SNPs with various parameters by logistic regression.