IL28B Polymorphism Associated with Spontaneous Clearance of Hepatitis C Infection in a Southern Brazilian HIV Type 1 Population

Abstract

About one-third of people infected with human immunodeficiency virus-1 (HIV-1) are coinfected with hepatitis C virus (HCV) because of shared transmission routes. Studies report that HIV-1 complicates hepatitis C infection by increasing HCV viral load and reducing spontaneous clearance. Single nucleotide polymorphisms (SNPs) upstream of the IL28B gene have been associated with spontaneous and treatment-induced clearance of HCV infection. The aim of this study was to evaluate the association between the SNP rs12979860 of the IL28B gene and spontaneous clearance of HCV infection in a Brazilian HIV-1 population. The SNP was analyzed by polymerase chain reaction (PCR) followed by restriction digestion in 138 anti-HCV-positive patients. Spontaneous clearance was observed in 34 subjects (24.6%). Genotype distribution was significantly different between spontaneous clearance and HCV chronic patients. The CT/TT genotypes conferred a nearly 3-fold increased odds to chronic HCV infection relative to the CC genotype (odds ratio, 2.78; 95% confidence interval, 1.16–6.64; p=0.011). In conclusion, the rs12979860 polymorphism is associated with spontaneous clearance of HCV in HIV-1 Brazilian infected patients.

Introduction

Human immunodeficiency virus type 1 (HIV-1) infects more than 38 million people worldwide and is the etiologic agent of the current AIDS pandemic. Approximately 10 million of the HIV-1-infected subjects are also coinfected with hepatitis C virus (HCV) because of shared transmission routes.¹ The introduction of highly active antiretroviral therapy (HAART) resulted in the effective control of viral replication and reduced the majority of AIDS-related opportunistic diseases. However, hepatitis C is still a serious problem and one of the main causes of morbidity and mortality in HIV-1 subjects.

Up to 25% of HIV-1-positive patients exposed to HCV (anti-HCV positive) may show spontaneous viral clearance. Some factors have been associated with this situation, such as differences in immune response, ethnic background, and parenteral exposure.^2,3 Recently, single nucleotide polymorphisms (SNP) near the gene of interleukin 28B (IL28B), which encodes a type III interferon (IFN-lambda3), were identified as genetic determinants of spontaneous resolution of HCV infection. Specifically, one polymorphism (rs12979860) was associated with the response to standard treatment for HCV (pegylated interferon plus ribavirin) in coinfected HCV/HIV-1 individuals.^4,5 This polymorphism has varied distribution among ethnic groups.⁶ The present Brazilian population is one of the most heterogeneous populations in the world, formed mainly by the admixture between European, African, and Native American populations.⁷ It is intriguing to consider whether a previous association between IL28B polymorphism and HCV clearance is also observed in this mixed population. This study aimed to correlate the genotypes of the rs12979860 polymorphism with spontaneous clearance of HCV infection in a Southern Brazilian HIV-1 population.

Materials and Methods

Patients and samples

A cross-sectional study was conducted with HIV-1-positive patients, included in a broader research project entitled “Clinical-epidemiological study on HIV coinfections with hepatitis B and C, HTLV-I and II, in the population of Canoas, a medium-sized city of the State of Rio Grande do Sul, Brazil,” between July 2008 and January 2009.

The target population of the project was comprised of adult patients, either receiving or not receiving HAART and users of the referral services for patient care and antiretroviral drug supply in the city of Canoas, situated in the Porto Alegre metropolitan area of Rio Grande do Sul state, Brazil. A total of 580 HIV-1-positive patients were originally included in the study. Sociodemographic factors and potential risk factors for HIV infection were obtained from the interviews with patients. Ethnicity was investigated as self-reported skin color, and patients were classified as white or nonwhite. Clinical data of each patient, as well as the treatment, CD4⁺ T cell count, and viral loads were obtained from medical records.

Samples were collected by venipuncture in 5-ml tubes, using ethylenediaminetetraacetic acid (EDTA) as anticoagulant, centrifuged to separate the plasma and the buffy coat in specific tubes and stored at −20°C. The presence of antibodies to HCV (anti-HCV) was determined using third-generation enzyme-linked immunosorbent assay (ELISA). Spontaneous HCV clearance was defined as HCV seropositivity and negative HCV RNA findings (<50 IU/ml) without any hepatitis C specific treatment (interferon and ribavirin). HCV-RNA viral load and genotype in positive samples were determined by real-time PCR and restriction fragment length polymorphism (RFLP) as previously described.^8,9 The assay used to quantify the HCV-RNA has the sensitivity to detect approximately 50 IU/ml.

The present study was approved by the Research Ethics Committees of the Universidade Luterana do Brasil (process 139H/2007). All participants signed an informed consent form.

Genetic analysis

Genetic analysis of the IL28B genotypes was performed in subjects with a positive result for anti-HCV. Buffy coat samples were defrosted and processed together with positive and negative controls. Total DNA of each clinical sample was purified by a standard silica-based procedure.¹⁰

IL28B genotypes were determined by polymerase chain reaction (PCR) followed by RFLP analysis. The PCR reaction system consisted of ultrapure distilled water (DNase and RNase free), 1×PCR buffer (10 mM Tris–HCl, pH 8.5, 50 mM KCl), 1.5 mM MgCl₂, 0.0625 mM of deoxynucleoside triphosphates, 0.25 μM of each primer (IL28-3F 5′-GCGCTTATCGCATACGGCTA-3′ and IL28-5R 5′-GGGGCGAGGGGCTTTGCT-3′), 2 U of Taq DNA polymerase, and 1 μl of DNA extracted in 50 μl reaction volume. Amplification was carried out according to the following protocol: 1 cycle at 94°C for 3 min and 40 cycles at 94°C for 5 s, 65°C for 20 s, and 72°C for 30 s, followed by a final extension cycle at 72°C for 5 min. The amplified fragments (188 bp) were digested with BstUI restriction enzyme and separated by electrophoresis on a 10% gel of polyacrylamide stained with silver nitrate. PCR amplified products of the three IL28B genotypes were sequenced to validate the PCR-RFLP assay.

Statistical analysis

Double entry data was performed using the EPIDATA software, version 3.1, where consistency was checked and these data were cleaned. Data analysis was conducted with SPSS software, version 18.0. Allele frequencies were determined by direct counting of alleles. Group mean values were compared using the Mann–Whitney U test. Frequencies were compared between groups using Fisher's exact test or chi-square when indicated. Associations between IL28B genotypes and HCV infection outcome (chronic/spontaneous clearance) were assessed using Upton's chi-square test as recommended previously.¹¹ All p values presented are two-tailed and a value of p<0.05 was considered statistically significant.

Results

Anti-HCV testing was performed in the 580 HIV-positive subjects included in population study. The prevalence of anti-HCV-positive samples was 23.8% (138 individuals). Sociodemographic and clinical variables of the HIV-1/HCV-coinfected subjects are shown in Table 1. Of all the 138 individuals, 62% were men, with a mean age of 41.6±9.5 years. No significant differences were observed between patients who spontaneously cleared the virus (n=34) or had persistent infection (n=104) for demographic data, risk factor for HIV-1 infection, and clinical characteristics (Table 1). HCV genotypes were determined in the chronically infected individuals. A total of 63 (60.6%) patients had genotype 1, 5 (4.8%) had genotype 2, and 36 (34.6%) had genotype 3. The mean log₁₀ HCV-RNA was 6.8±0.7, 6.9±0.3, and 6.6±0.9 IU/ml for HCV genotypes 1, 2, and 3, respectively (p=0.14). There was a trend toward a lower HCV viral load in patients infected with HCV genotype 3 compared to genotype 1 (p=0.075). No significant differences were observed in the mean plasma HCV-RNA according to IL28B genotypes (CC 6.7±0.8, CT 6.8±0.7, and TT 6.8±0.7 IU/ml; p=0.871).

Table 1.

Sociodemographic and Clinical Characteristics of the HIV/HCV Coinfected Study Population

Variable	Total ^a (n=138)	Chronic infection (n=104)	Spontaneous clearance (n=34)	p-value ^b
Male gender	86 (62.3)	65 (62.5)	21 (61.8)	1.00
Age (years)	41.7±9.5	41.4±9.5	42.7±9.5	0.49
White skin colour	82 (59.4)	65 (62.5)	17 (50.0)	0.28
CD4⁺ count (cells/mm³)	429±243	415±225	472±295	0.30
HIV viral load (log₁₀ copies/ml)	2.7±1.3	2.6±1.2	2.8±1.4	0.42
Time of HIV diagnosis (years)	6.2±3.7	6.1±3.7	6.5±3.7	0.59
HAART use^c	101 (82.1)	76 (82.6)	25 (80.6)	0.81
Injecting drug use	55 (39.9)	37 (35.6)	18 (52.9)	0.07
Blood transfusion	37 (26.8)	28 (26.9)	9 (26.5)	0.93
Presence of tattoo	57 (41.3)	44 (42.3)	13 (38.2)	0.68
Body piercing	9 (6.5)	5 (4.8)	4 (11.8)	0.15
HCV-RNA (log₁₀ IU/ml)		6.8±0.7	NA
HCV genotype			NA
1		63 (60.6)
2		5 (4.8)
3		36 (34.6)

Data are reported as number of patients (% in parentheses) or mean±standard deviation.

Reported p-values were obtained using Fisher's exact or Chi-square test for categorical variables and Mann–Whitney U test for continuous variables comparing chronic and spontaneous clearance HCV patients.

Percentuals do not coincide due to lack of data from certain participants in the study.

HIV, human immunodeficiency virus; HCV, hepatitis C virus; HAART, highly active antiretroviral therapy; NA, not applicable.

Allele and genotype frequencies of the rs12979860 polymorphism in the HIV-1/HCV coinfected study population are shown in Table 2. Alleles T and C frequencies were 38% and 62%, respectively. The genotype distribution was significantly different between HCV chronic infected patients and spontaneous clearance subjects (p<0.05). When a dominant model for the allele T was tested significant differences were observed, where the CT/TT genotypes conferred a nearly 3-fold increased odds to chronic HCV infection relative to the CC genotype (odds ratio, 2.78; 95% confidence interval, 1.16–6.64; p=0.011).

Table 2.

Allele and Genotype Frequencies of the rs12979860 Interleukin-28B Gene Polymorphism in the HIV/HCV Coinfected Study Population

	All patients ^a (n=138)	Chronic infection (n=104)	Spontaneous clearance (n=34)	p-value^b
Allele				0.142
T	106 (38.4)	85 (40.9)	21 (30.9)
C	170 (61.6)	123 (59.1)	47 (69.1)
Genotype				0.015
TT	16 (11.6)	11 (10.6)	5 (14.7)
TC	74 (53.6)	63 (60.6)	11 (32.4)
CC	48 (34.8)	30 (28.8)	18 (52.9)

Data are number (%) of patients.

Upton's chi-square test.

Discussion

Studies in developed countries have shown a HCV prevalence of around 30% in different HIV-1-positive populations. However, the prevalence can reach 90% in some risk groups, mainly in injecting drugs users.¹² Our results showed a prevalence of 23.6% of HIV-1/HCV coinfection, similar to previous findings in other HIV-1 Brazilian populations.^13,14

Between 20% and 30% of individuals monoinfected with HCV have spontaneous clearance of hepatitis C after the acute phase.¹⁵ The rate of spontaneous resolution appears to be lower in patients coinfected with HIV-1.² In the present study, we observed that almost 25% of the subjects had spontaneous clearance of HCV infection. It is likely that genetic variations of the patients also explain this heterogeneity between coinfected individuals as observed in other studies with different populations.^16
–18

The first studies that linked IL28B SNPs and HCV clearance came from studies of large cohorts of patients with chronic HCV infection who received standard treatment for HCV.^19
–21 These findings have been confirmed in other independent population, including HIV/HCV-coinfected subjects.^5,22 The same IL28B SNPs were also associated with spontaneous clearance in both HCV monoinfected and HIV/HCV coinfected patients. Thomas et al.²³ reported that stratification based on HIV status did not modulate the effect of the SNP rs12979860 on HCV clearance. Similarly, Rauch et al.²⁴ found that the rs8099917 polymorphism, another SNP located upstream of the IL28B gene, was associated with HCV clearance in Swiss HCV-infected patients with or without HIV infection. Intriguingly, although not discussed by the authors, the prevalence of spontaneous clearance was much higher in coinfected HIV/HCV patients than in monoinfected patients (47.3% and 14.8%, respectively). Clausen et al.²⁵ reported the association of the rs12979860 polymorphism and other two SNPs located near the IL28 gene with spontaneous clearance in HIV-positive patients.

In the present study we evaluated the association of polymorphism rs12979860 located near the IL28B gene with spontaneous clearance of HCV infection in a Brazilian HIV-1 population. We observed that the allele distribution was different between HCV chronic infected subjects and patients with spontaneous clearance. Although the biological action mechanism of SNP rs12979860 has not been identified, previous studies used the allele T dominance model in the analysis of this polymorphism.^26,27 This model was also adopted in our study showing an association between the CC genotype and spontaneous clearance and suggesting that this genotype may play an important role in the resolution of HCV infection. Our data are in agreement with previous studies that have observed this association in HCV/HIV-coinfected patients.^23
–25 Together these studies indicate that the IL28 gene is also important for the control of HCV infection in HIV-infected subjects. However, the SNPs of the IL28B gene were not associated with susceptibility for HIV infection^28,29 or HIV disease progression.²⁹

SNP rs12979860 of the IL28B gene presents a varied distribution among ethnic groups, with the C allele having the lowest frequencies among black subjects (∼0.42), the highest frequencies in East Asian subjects (∼0.95), and intermediate frequencies in European-American (∼0.73) and Hispanic subjects (∼0.7) (reviewed by Balagopal et al.⁶). In association studies there are concerns about spurious associations due to the population structure secondary to ethnic variability in allele frequencies. The population of the present study included individuals of mixed ancestry, according to their self-reported background (data obtained from the interview with patients), and similar results were observed when white and nonwhite subjects were evaluated (data not shown). However, self-reported background is not a good predictor of genomic ancestry in Brazil.^30,31 Brazil has one of the most heterogeneous populations in the world, the result of five centuries of massive interethnic crosses between peoples from different continents.^7,32 Parra et al.³⁰ emphasize that large urban areas (as, for example, Porto Alegre and the respective metropolitan area) do not present significant isolated ethnical groups; thus our patients are from a single panmictic population.

The IL28B genotype provides important information about a patient's likelihood of achieving a sustained virological response and has clinical relevance. In this sense, a commercial test has been available in the United States since July 2010.⁶ The majority of the current protocols to study IL28B genotypes is based on real-time PCR using TaqMan probes (e.g., Tilmann et al.³³) or direct sequencing of PCR products (e.g., Aparicio et al.³⁴). In the present study, we used a PCR-RFLP assay to genotype the rs12979860 SNP. Samples of each genotype were direct-sequenced and confirmed the PCR-RFLP results (data not shown). This assay allowed an easy identification and interpretation of the polymorphism in polyacrylamide gels and could be an excellent alternative to laboratories with resource-limited settings, since it does not require real time PCR or DNA sequencing equipments.

It is important to mention that some limitations must be considered in the interpretation of our findings. First, the statistical power to detect differences is limited, due to the small sample size, preventing any valid multivariate analysis from being performed. Second, it is difficult to estimate the time of infection of the two viruses (HCV and HIV-1) and clarify the exact relation between HCV clearance and HIV infection. Third, this is a cross-sectional study, which is not the best method to identify the causality of factors studied, and the potential effects of reinfections in individuals who have spontaneous clearance as well as individuals analyzed as chronically infected even though they might be on their way to spontaneous clearance were not considered.

Nonetheless, the results obtained contribute to a better understanding of the genetic basis of the hepatitis C evolution in HIV patients and provide evidence that IL28B gene polymorphism has a significant impact on spontaneous HCV clearance.

Footnotes

Acknowledgments

The authors thank the personnel staff, physicians, and patients of the Serviço de Atendimento Especializado (SAE) and Centro de Testagem e Aconselhamento (CTA) of Canoas, RS, for their collaboration in making this study possible. This research project was funded by the Brazilian Ministry of Health, Secretaria de Vigilância em Saúde (Department of Health Surveillance), Programa Nacional de Doenças Sexualmente Transmissíveis e Aids (MS/SVS/PN-DST/AIDS—Brazilian Program of Sexually Transmitted Diseases and AIDS—Cooperation Term 282/07), through the International Technical Cooperation Project AD/BRA/03/H34, established between the Brazilian government and the United Nations Office on Drugs and Crime (UNODC). The procedures of molecular biology analysis of HCV were determined by the project entitled “Development of Molecular Test Kits to Detect and Quantify Viral Agents—Hepatitis B and C and HIV—Based on the Real Time PCR Technology,” sponsored by the Financiadora de Estudos e Projetos (FINEP—Financial Support Organization for Research and Projects) and the Simbios Biotecnologia (FINEP/FULBRA/LDM–Simbios Biotechnology—Ação Transversal 2006—convênio no. 0.1.07.0102.00).

Author Disclosure Statement

No competing financial interests exist.

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