ANALYSIS OF POLYMORPHISMS IN THE INTERLEUKIN 18 GENE PROMOTOR (-137 G/C AND -607 C/A) IN PATIENTS INFECTED WITH HEPATITIS C VIRUS FROM THE BRAZILIAN AMAZON

ARTIGO ORIGINAL / ORIGINAL ARTICLE ARQGA/1796 DOI: 10.1590/S0004-28032015000300013 ANALYSIS OF POLYMORPHISMS IN THE INTERLEUKIN 18 GENE PROMOTOR (-137 G/C AND -607 C/A) IN PATIENTS INFECTED WITH HEPATITIS C VIRUS FROM THE BRAZILIAN AMAZON Kemper Nunes dos SANTOS, Marcella Kelly Costa de ALMEIDA, Amanda Alves FECURY, Carlos Araújo da COSTA and Luísa Caricio MARTINS Received 20/2/2015 Accepted 27/4/2015 ABSTRACT - Background - The hepatitis C virus has been recognized as the leading cause of chronic liver disease in the world. Host genetic factors have been implicated in the persistence of hepatitis C virus infection. Single nucleotide polymorphisms at positions -607 C/A (rs1946518) and -137 G/C (rs187238) in the IL-18 gene promoter have been suggested to be associated with delayed hepatitis C virus clearance and persistence of the disease. Objective - Identify these polymorphisms in a population infected with hepatitis C virus from the Brazilian Amazon region. Methods - In a cross-sectional analytical study conducted in Belém, Pará, Brazil, 304 patients infected with hepatitis C virus were divided into two groups: group A, patients with persistent infection; group B, patients with spontaneous clearance. The control group consisted of 376 volunteers not infected with hepatitis C virus. Samples were analyzed by RT-PCR for the detection of viral RNA and by RFLP-PCR to evaluate the presence of the -137 G/C and -607 C/A IL-18 gene promoter polymorphisms. Results - Comparison of polymorphism allele frequencies between the patient and control groups showed a higher frequency of allele C at position -607 among patients (P=0.02). When the association between the polymorphisms and viral infection was analyzed, patients carrying genotype C/A at position -607 were found to be at higher risk of persistent hepatitis C virus infection (P=0.03). Conclusion - the present results suggest a possible role of the -607 IL-18 gene promoter polymorphism in the pathogenesis of hepatitis C virus infection. HEADINGS - Single nucleotide polymorphism. Interleukin-18. Hepatitis C. Hepacivirus. INTRODUCTION Since its discovery in 1989, hepatitis C virus (HCV) has been recognized as the leading cause of chronic liver disease in the world. Approximately 3% of the world population suffers from chronic hepatitis C(3, 22). Viral replication in the liver for a prolonged period of time can have severe clinical consequences such as fibrosis, cirrhosis, and hepatocellular carcinoma(5, 13). Host genetic factors have been implicated in the persistence of HCV infection. In this respect, two polymorphisms in the promoter region of the interleukin 18 (IL-18) gene have been shown to influence the expression of this gene(1). IL-18, also called interferon gamma (IFN-γ) inducing factor, is a proinflammatory cytokine that plays an important role in both Th1 and Th2 responses(13, 15). Immune responses and genetic factors are known to influence the prognosis of HCV infection(8). Cytokines play a critical role in the pathogenesis of HCV infection, with different clinical outcomes. Evidence indicates that genetic variants of IL-18 can influence host susceptibility, viral persistence or clearance, tissue injury, and the response to antiviral treatment(23). Two single nucleotide polymorphisms (SNP) identified at positions -607 C/A (rs1946518) and -137 G/C (rs187238) in the IL-18 promoter region seem to influence the promoter transcription activity of IL-18 and, potentially, of IFN-γ, and have been associated with delayed clearance of HCV and persistence of the disease(1). To our knowledge, no Brazilian studies have Declared conflict of interest of all authors: none Disclosure of funding: no funding received Laboratório de Patologia Clínica das Doenças Tropicais, do Núcleo de Medicina Tropical, Universidade Federal do Pará, Belém, PA, Brasil. Correspondence: Luísa Caricio Martins. Núcleo de Medicina Tropical. Av. Generalissimo Deodoro, 92 - Umarizal - CEP: 66000-000 - Belém, PA, Brasil. E-mail: 222 Arq Gastroenterol v. 52 no. 3 - jul./set. 2015 Santos KN, Almeida MKC, Fecury AA, Costa CA, Martins LC. Analysis of polymorphisms in the interleukin 18 gene promotor (-137 G/C and -607 C/A) in patients infected with hepatitis C virus from the Brazilian Amazon investigated the association between the -607 C/A (rs1946518) and -137 G/C (rs187238) SNPs in the IL-18 gene promoter and the outcomes of HCV infection. The objective of the present study was to identify these polymorphisms in a population infected with HCV from the Brazilian Amazon region. METHODS Sample characterization A total of 304 patients of Tropical Medicine Center of the viral hepatitis program, were selected and divided into 2 groups: Group A - 174 patients with persistent infection, with anti-HCV and HCV polymerase chain reaction (PCR) positive for more than 6 months. Group B - 130 patients with spontaneous clearance, characterized by positive anti-HCV and HCV PCR negative. Viral clearance was defined when anti-HCV serology was positive and HCV RNA was undetectable in serum over a period of 6 months or less in the absence of specific HCV treatment (Figure 1). For the control group, 376 anti-HCV-negative volunteers with negative HCV PCR were selected at random. None of the patients was co-infected with hepatitis B virus (HBV surface antigen negative) or Human Immunodeficiency Virus (HIV), none of the patients consumed alcoholic beverages, and all were treatment naive. All patients received detailed information about the study and signed a consent form. All results of serology confirmed by at least these parated serological tests within consecutive 6 months during the follow-up. All subjects were diagnosed by experienced physicians on the basis of clinical and laboratory findings and internationally accepted criteria(18). All subjects included (patients and control) were of the same socioeconomic status and had similar cultural habits. In addition, all subjects were born in Pará state and had the same ethnic origin, approximately 50% Portuguese, 40% Amerindian, and 10% African(20). The study was approved by the Ethics Committee of the Tropical Medicine Center, Federal University of Para (Núcleo de Medicina Tropical, Universidade Federal do Pará - NMT/ UFPA), Belém, PA, Brazil (Permit No. 042/2011). All patients gave their informed consent to participate in the study. Serological testing and HCV genotyping Peripheral venous blood (5 mL) was collected from each participant into EDTA tubes. Plasma was isolated by centrifugation and stored at -80oC until assayed. Serological markers of HCV infection (anti-HCV antibodies) were investigated using the ETI-ABHCVK-4 kit from Diasorin (Saluggia, Italy). RNA was extracted from all samples using the QIAamp Viral RNA kit (Hilden, Germany). HCV RNA was investigated by nested PCR using primers that target the 5’-UTR region. The first reaction consisted of the synthesis and amplification of cDNA in a single step us (...truncated)