Interleukin 28B Polymorphisms and Therapy Response in Egyptian Hepatitis C Genotype-4 Patients

Abstract

Hepatitis C infection represents a major health problem in Egypt; only 20% of patients undergo spontaneous clearance of the virus and around 25% of all patients progress to develop cirrhosis. More than 90% of Egyptian patients have hepatitis C virus (HCV) genotype-4. Combined pegylated interferon and oral ribavirin are the current standard therapies for HCV-4. The aim of the work is to evaluate the predictive power of the rs12979860 IL28B SNP and rs12980275 IL28B SNP for treatment response in Egyptian patients infected with HCV genotype 4. One hundred eleven HCV patients receiving combined treatment were studied for rs12979860 and rs12980275 polymorphisms by the restriction fragment length polymorphism technique. The rs12979860 CC and rs12979860 AA genotypes were significantly associated with sustained virological response (p=0.001). Our results suggest that studying IL28B polymorphisms contribute to proper prediction of response to standard therapies in Egyptian patients, optimizing cost effectiveness, and minimizing unneeded adverse effect of therapy.

Introduction

Studies focusing on hepatitis C virus (HCV) genotype 4 infection in Egypt have become a national interest. Egyptians have the highest prevalence of adult HCV infection in the world constituting 9% of the overall adult population (Kamal and Nasser, 2008) and around 20% in rural communities (El-Raziky et al., 2007), 90% of cases have HCV genotype 4 (Ray et al., 2000). HCV is an important cause of acute and chronic hepatitis, with only 20% of patients having spontaneous clearance of the virus and around 25% of all patients progressing to develop cirrhosis (Hadigan and Kottilil, 2011).

Combined pegylated interferon (Peg-IFN) and ribavirin therapy are still the most effective therapies in HCV-4 (El-Zayadi et al., 2005). The rate of sustained virological response (SVR) is around 50% (Kamal, 2011). It is of major interest for both patient care and economic approach to predict failure of response to therapy (Asselah et al., 2010). Therefore, reliable and efficient treatment predictors are needed to reduce and avoid unnecessary adverse effects and economic load of therapy.

Multiple genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) near the IL28B gene (encoding IFN-λ3) to be strongly associated with spontaneous and treatment-induced clearance of HCV infections (Rauch et al., 2010). One of these SNPs, rs12979860, was pivotal in predicting the resolution of HCV-1 infections (Thomas et al., 2009). Little is known about the IFN λ family, but evidence is increasing to support a role for them in the immune response to viral infections (Chevaliez and Hezode, 2010; Pineda et al., 2011). Results of a GWAS showed a significant association between treatment responses with SNP rs12980275 located in the IL28B gene region (Venegas et al., 2011).

The aim of the present work is to evaluate the prevalence and the predictive power of the IL28B rs12979860 and rs12980275 for treatment response in Egyptian patients infected with HCV genotype 4.

Materials and Methods

Study population

This study comprised 111 Egyptian patients with HCV-4 infection. Patients were attending the outpatient clinic of gastroenterology department, Cairo University. Patients were either newly diagnosed or attending for follow-up. There were 45 females and 76 males and their ages ranged from 20 to 63 years with a mean age of 40.5 years. The research protocol was approved by the research ethics committee of the Department of Clinical Pathology, Cairo University. Patients coinfected with human immunodeficiency virus (HIV), hepatitis B virus (HBV), patients suffering from liver cirrhosis, pediatric age group, and non-Egyptians were excluded from the study. All patients enrolled in our study were subjected to a complete HCV patient work-up adopted by the Egyptian Ministry of Health, including laboratory and radiological investigations and liver biopsies before therapy. Informed consents were signed by all patients.

Treatment regimen

Patients were treated either with Peg-IFN-α-2a and ribavirin at doses of 180 μg/week and 1000–1200 mg/day, respectively, or Peg-IFN-α-2b and ribavirin at doses of 80–120 μg/week and 800–1200 mg/day, respectively. The course of therapy was continued for 48 weeks. SVR was defined as undetectable HCV RNA 24 weeks after the end of treatment (i.e., at week 72). No-response was defined as HCV viral load decline less than 2 logs at week 12 during therapy or detectable HCV RNA in serum at any other time during the 48-week therapy. Relapse was defined as undetectable HCV RNA in serum at the end of therapy, followed by detectable HCV RNA after the completion of therapy (Domagalski et al., 2013). In this study, patients were divided into two groups: the SVR group and No SVR (nonresponders and relapsers).

DNA extraction and genotyping

Genomic DNA extraction from peripheral blood leucocytes was done using the Gene JET Genomic DNA purification kit (Fermentas, Lithuania) following the manufacturer's instructions. Genotypic analysis of the candidate SNPs was performed by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. All PCRs were performed in a total volume of 25 μL containing 5 μL genomic DNA, 12.5 μL Dream Taq Green PCR Master Mix, 1 μL of each forward and reverse primers (Bioneer, Korea), and 5.5 μL distilled water. The primer sequences, the thermocycler program, and the restriction enzymes used are presented in Tables 1 and 2, respectively. The PCR products were visualized by 2% agarose gel electrophoresis under UV light.

Table 1.

The Primer Sequences Applied for Amplification of IL28B Gene

Genetic polymorphism	Primer sequence	PCR product	Reference
IL28B	F: 5′- AGG GCC CCT AAC CTC TGC ACA GTC T-3′	403	Kurbanov et al. (2011)
rs12979860	R: 5′- GCT GAG GGA CCG CTA CGT AAG TCA CC-3′
IL28B	F: 5′-GAG AGC AAG AGG AGG GAA GGA A-3′	441
rs12980275	R: 5′- GTG TGC CAT TAG CCA GTC AGA T-3′

Table 2.

Genotyping of IL28B Single-Nucleotide Polymorphisms by Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Assay

IL28B	Thermocycler program	Restriction enzyme	Genotypes	Restriction fragments/bp
IL28B rs12979860	Initial heating at 96°C for 10 min. Forty cycles of 94°C for 1 min, 58°C for 40 s, 72°C for 1 min.	BstUI	CC genotype	25+89+105+184
			CT genotype	25+89+105+130+184
			TT genotype	89+130+184
IL28B rs12980275	Final extension at 72°C for 2 min.	BslI	AA genotype	320+121
			AG genotype	320+290+121+30
			GG genotype	290+121+30

Statistical analysis

Data are statistically described in terms of range, mean±standard deviation (±SD), median, frequencies (number of cases), and percentages, when appropriate. Comparison of quantitative variables between the study groups was done using the Kruskal–Wallis analysis of variance (ANOVA) test. For comparing categorical data, the chi square (χ ²) test was performed. The exact test was used instead when the expected frequency was less than 5. The relative frequency of gene mutation was described by OR and 95% CI. Univariate and multivariate analysis models were used to test for the preferential effect of the independent variable(s) on the dependent variable(s). A probability value (p-value) less than 0.05 was considered statistically significant. All statistical calculations were done using computer programs Microsoft Excel 2003 (Microsoft Corporation) and SPSS (Statistical Package for the Social Science; SPSS, Inc.) version 15 for Microsoft Windows.

Results

Among the 111 patients enrolled in our study, 77/111 (69.4%) patients achieved SVR and the remaining 34/111 (30.6%) showed No SVR (29/111 patients were nonresponders and 5/111 were relapsers).

The clinical and laboratory characteristics of our patients are presented in Tables 3 and 4, respectively.

Table 3.

Clinical Data of Hepatitis C Virus Patients

	HCV Patients (n=111)
Item	Number	Percentage
Male	72/111	64.9%
Female	39/111	35.1%
Splenomegaly	31/111	27.9%
Hepatomegaly	63/111	56.8%
Treatment type
PEGASYS	48/111	43.2%
PEG-INTRON	63/111	56.8%
Dose interferon
100 μg/week	49/111	44.1%
180 μg/week	62/111	55.9%
Dose ribavirin
1000 mg/day	49/111	44.1%
1200 mg/day	62/111	55.9%

HCV, hepatitis C virus.

Table 4.

Laboratory Data of Hepatitis C Virus Patients

Item	Range	Mean±SD
Hemogram
Hb gm/dL	10.7–17.2	13.5±1.5
TLC×10³/cm³	2.9–16.2	6.7±2.0
Plts×10³/cm³	126–442	227.4±74.3
PC%	57–100	87.8±8.8
Albumin g/dL	3.4–5.9	4.2±0.4
ALT U/L	4.0–161	53.0±24.8
AST U/L	9.0–134	44.3±21.8
Total bilirubin mg/dL	0.18–2.3	0.76±0.3
AFP μg/L	0.3–22	5.8±3.6
HCV–RNA IU/ML	1300–8,190,361	301,557 (Median)

IL28 polymorphism and the treatment response

The frequency of IL28B SNPs and the comparison between genotypes for rs129798860 and rs1212980275 regarding response to pegylated interferon and ribavirin therapy are shown in Table 5.

Table 5.

Analysis of IL28B Genetic Polymorphisms in Hepatitis C Virus Patients

	IL28B rs12979860 CC genotype		IL28B rs12979860 CT/TT genotypes
Item	n=43	(%)	n=68	(%)	p-Value
No SVR	3	6.9%	31	45.5%	0.001
SVR	40	93.1%	37	55.5%

	IL28B rs12980275 AA genotype		IL28B rs12980275 AG/GG genotypes
Item	n=54	(%)	n=57	(%)	p-Value
No SVR	1	1.8%	33	57.8%	0.001
SVR	53	98.2%	24	42.2%

SVR, sustained virological response.

Regarding clinical data, splenomegaly was statistically prominent in patients carrying the CT/TT and AG/GG genotypes (p=0.009 and 0.003, respectively), while hepatomegaly was statistically prominent in patients carrying the AG/GG genotype. No observed statistical difference was found regarding other clinical data (data not shown).

As regards laboratory data, the albumin level was statistically lower in patients carrying the CT/TT genotype (p=0.043), and HCV RNA was also significantly lower in patients carrying the AG/GG genotype (p=0.021) (data not shown).

The frequency of SVR differed significantly in patients with rs12979860 genotype CC (93.1%; 40 of 43) and CT/TT (55.5%; 37of 68).

With respect to the genetic variation of rs12980275, the SVR frequency for genotype AA was significantly higher compared to AG/GG (98.2%; 53 of 54 and 42.4%; 24 of 57, respectively).

Thus, a significantly higher proportion of patients with the common genotypes showed SVR as compared with patients who had the variant ones.

Combined genotype profiling was studied for patients having both common genotypes [CC-AA], one common genotype [CC with AG/GG or AA with CT/TT], and both variant genotypes [CT/TT with AG/GG] (Table 6).

Table 6.

Combined Genotypes Are Shown for rs 129897860 and rs 12980275 of HCV Patients in View of Their Response to Therapy

	Combined genotypes for both rs12979860 and rs12980275
Response to treatment	Both common genotypes	One common genotype	Both variant genotypes homo/hetero	Total	p-Value
SVR	31 (40.3%)	31 (40.3%)	15 (19.5%)	77 (100%)	<0.001
No SVR	0 (0%)	4 (11.8%)	30 (88.2%)	34 (100%)
Total	31	35	45	111

Discussion

Several predictive factors have been studied for SVR rates in HCV-infected patients. They include viral factors as viral load and core amino acid substitution or host genetic variations. SNPs within a number of host genes were found to be associated with different outcomes to therapy (El-Awady et al., 2012). The rs12979860 C allele has been reported to be linked to higher serum levels of IL28B, IL28A, and IL29 levels, all of which are involved in the induction of expression of IFN responsive genes. The CC genotype has also been reported to be associated with lower levels of intrahepatic interferon-stimulated gene expression, which is linked to better IFN-based treatment response (Urban et al., 2010). The rs12980275 genotype strongly enhances the spontaneous and natural resolution of acute HCV infection, especially in Europeans and Africans (Ge et al., 2009).

Thomas et al. (2009) reported that the allele related to HCV clearance, namely rs12979860 CC, was the predominant allele in the majority of Asian and European countries varying from 89.9% in Taiwanese to 37.2% in Caucasians (Thompson et al., 2010). Conversely, patients of African ancestry had a lower CC allele frequency of 14% (Thompson et al., 2010).

Our results revealed that the rs12979860 CC genotype was found in 38.7% of patients; these results were comparable to other studies comprising Egyptian patients, where the CC genotype varied from 23% in the De Nicola et al. (2012) study to 55% in the Asselah et al. (2012) study, while in the in sub-Saharan patients, the TT genotype was the most predominant form (Domagalski et al., 2013).

Among overall HCV patients, 77 (69.4%) achieved SVR (responders) and the remaining 34 (30.6%) patients failed to respond or relapsed (No SVR). The percent CC genotype of rs12979860 who achieved SVR was 93.1%, which is significantly higher compared with both CT and TT genotypes (55.5%). This finding was supported by previous studies (Suppiah et al., 2009; Tanaka et al., 2009; Magnia et al., 2010; Kurbanov et al., 2011; Par et al., 2011; Asselah et al., 2012; De Nicola et al., 2012).

Current data indicate that the Egyptian population infected mainly with genotype 4 has an intermediate response between that of the populations of African and European ancestries, that is, the CC allele frequency in our patients was 38.7% compared to >50% in Europeans and levels below 35% in Africans, while SVR rates in the currently studied HCV patients were 69.4% compared to 80% in Europeans and 50% in Africans (Thompson et al., 2010).

El awady et al. (2012) reported that among his 24 chronic HCV patients, only 14% had the rs12979860 CC genotype compared with 48% in controls, hypothesizing that IL28B CC in those 24 CHC patients was not sufficient to provide substantial tendency toward viral clearance.

Analysis of the influence of the IL28B rs12979860 genetic polymorphism on the clinical and laboratory characteristics of the disease revealed that splenomegaly was significantly more prominent in HCV patients harboring the CT or TT genotypes; this may be attributed to the presence of 41% of our patients having positive anti-schistosoma antibodies. Hypoalbuminemia was statistically significantly more prominent in HCV patients harboring the CT/TT genotypes. Abdo et al. (2013) reported that a significant difference was found in albumin levels between the two groups—patients with SVR and nonresponder patients.

As regards the frequency of rs12980275, the frequency of the AA genotype was identified in 48.6%, the AG genotype was detected in 33.3%, while the GG genotype in 18.1%.

Our results are in accordance with the studies of Abdo et al. (2013) that recorded the frequencies of the IL28B genotypes as follows: AA, 36.4%; AG, 51.2%; and GG, 12.4%. The AA genotype of rs12980275 that achieved SVR was 98%, which is significantly higher compared with both AG and GG genotypes (42.2%). Our results were supported by the results of Abdo et al. (2013) and Domagalski et al. (2013), which reported that 72% of patients with the AA genotype achieved an SVR compared with 46.9% with the AG and 43.7% with the GG genotypes. While Domagalski et al. (2013) reported that 62.5% of patients with the AA genotype achieved an SVR compared with 37.8% with the AG and 33.3% with the GG genotypes.

While little is known about the impact of combined genotypes on therapy response, our study revealed that harboring the common variants in both SNPs rs12979860 and rs12980275 will easily discriminate a group of patients who respond to pegylated interferon and oral ribavirin or even spontaneously clear the HCV virus after primary infections significantly better than patients harboring the variant genotypes.

Conclusion

The significant genetic results on common IL28B polymorphisms with respect to treatment response in patients with hepatitis C infection may open the possibility of personalized medicine for the treatment of this progressive disease. Further studies are urgently required to determine if patients infected with genotype 4 of HCV, and harboring a favorable SNP, will benefit from a shorter treatment duration with the current therapy protocol. Thus, optimizing the cost effectiveness and minimizing the adverse effects associated with a longer term treatment.

Footnotes

Disclosure Statement

No competing financial interests exist.

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