POLYMORPHISMS OF α1-ANTITRYPSIN AND INTERLEUKIN-6 GENES AND THE PROGRESSION OF HEPATIC CIRRHOSIS IN PATIENTS WITH A HEPATITIS C VIRUS INFECTION
Motawi T, Shaker OG, Hussein RM, Houssen M
*Corresponding Author: Rasha M. Hussein, Ph.D., Department of Biochemistry, Faculty of Pharmacy, Beni-Suef University, Salah Salem Street, 62511, Beni-Suef, Egypt. Tel: +20-12-0013-6515. Fax: +20-82-2317-958. E-mail: rasha.hussein@ pharm.bsu.edu.eg
page: 35

MATERIALS AND METHODS

Patients. In this study, 150 patients were recruited from a large group of 250 Egyptian HCV carriers together with 100 sex- and age-matched controls who attended the outpatient clinic at the Kasr El-Aini Hospital, Cairo University, Cairo, Egypt. The patients were divided into two subgroups: group 1 comprised 85 patients with chronic HCV infection and group 2 comprised 65 patients with liver cirrhosis. Inclusion Criteria. All patients underwent clinical examination and routine liver function tests such as serum alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, albumin, prothrombin time and viral hepatitis markers (anti-HCV antibodies, HBsAg and HBeAg). Patients were diagnosed with liver cirrhosis based on imaging studies in the form of abdominal ultrasound with Doppler as well as upper endoscopy for functional evaluation of decompensated cases. Staging of fibrosis was assessed using the Child Pugh assessment fibrosis score [15,16]. All patients provided written informed consent before participating in the study. The study was approved by the Ethics Committee of Kasr El-Aini, Faculty of Medicine, Cairo University, Cairo, Egypt and the study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments in humans. Exclusion Criteria. Patients with inflammatory diseases, cardiovascular diseases, thyroid dysfunction, diabetes mellitus (DM), alcohol intake, active schistosomiasis, co-infection with hepatitis B virus (HBV) and previously treated with interferon therapy, were excluded from the study. Specimen Collection. A blood sample of 10 mL was drawn from each participant after overnight fasting. The blood samples were divided into two aliquots. The first aliquot was centrifuged for separation of serum to determine all routine and serological tests. The second aliquot was collected in a vacutainer containing EDTA as anticoagulant and stored at –80 °C for polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) analyses. Routine Biochemical Tests. Serum ALT and serum AST were determined based on the method of Henry et al. [17] and Amador et al. [18] respectively using kits supplied by Roche Diagnostics GmbH (Penzberg, Germany). Serum albumin was determined using kits also supplied by Roche Diagnostics GmbH. Serum alkaline phosphatase (ALP) was determined based on the method of Bretaudiere et al. [19] using kits supplied by Bio Diagnostic (Giza, Egypt). Serum bilirubin was detected based on the method of Landis et al. [20] using kits supplied by Roche Diagnostics GmbH. Serum α fetoprotein (AFP) was detected according to the enzyme-linked-immunosorbent serologic assay (ELISA) method of Hirai [21] using kits supplied by Affymetrix (Santa Clara, CA, USA). DNA Purification. DNA was purified using a generation capture column kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. DNA was released by DNA elution buffer and heat without precipitation according to method of Glasel [22]. Detection of the Single Nucleotide Polymorphisms of α1-Antitrypsin and Interleukin 6 Genes by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. The Z mutation (342 Glu/Lys, rs28929474) and S mutation (264 Glu/Val, rs17580) on the A1AT gene in addition to the IL-6 (–174 G/C, rs1800795) mutation were identified by multiplex PCR using the thermal cycler (Techne Genius, Cambridge, Cambridgeshire, UK). Twenty pmol of each primer (described below) and 0.5 U of Taq DNA polymerase (Qiagen Ltd., Crawley, West Sussex, UK) were added to 100 ng of DNA in 30 μL (final volume) of a solution containing 20 mM Tris-HCL (pH 8.4), 50 mM KCL, 1.5 mM MgCl2, and 200 mM of each dNTP. Temperature cycling conditions were adjusted as follows for all primers used: initial denaturation for 5 min. at 94 °C; 35 cycles of 1 min. at 94 °C, 1 min. at 55 °C, and 2 min. at 72 °C; final extension for 10 min. at 72 °C. The PCR products of IL-6 were digested by 5 U of NIaIII at 37 °C for 24 hours. The presence of the –174 G/C mutation destroys a NIaIII restriction site in the respective PCR products. The generated fragments were 119 + 49 bp for the C allele and a single fragment of 168 bp for the G allele. The PCR products of the A1AT gene were digested by Taq1, where the presence of either S or Z mutation destroys a Taq1 restriction site in the respective PCR products. Fragments of (157 + 22 bp) and (100 + 21bp) were detected for the wild type M allele, a 179 bp fragment for the Z allele and a 121 bp fragment for the S allele. The analysis was carried out using a 1% agarose gel stained with ethidium bromide (Figure 1). Primer sequences of the A1AT gene: S (forward) 5’- TGA GGG GAA ACT ACA GCA CCT CG-3’; S (reverse) 5’-AGG TGT GGG CAG CTT CTT GGT CA-3’; Z (forward) 5’-ATA AGG CTG TGC TGA CCA TCG TC-3’; Z (reverse) 5’-TTG GGT GGG ATT CAC CAC TTT TC-3’. Primer sequences of the IL-6 gene: IL-6 (forward) 5’-TGA CTT CAG CTT TAC TCT TTG-3’; IL-6 (reverse) 5’-CTG ATT GGA AAC CTT ATT AAG-3’. Statistical Analyses. The obtained data were analyzed using the Statistical Package for the Social Sciences version 12 software (SPSS Inc., Chicago, IL, USA). Mean ± standard error (SE) was used to describe continuous variables, while percentages and frequencies were used to describe categorical variables. One way repeated measure analysis of variance (ANOVA) followed by post Hoc test Scheffe’s method were used to compare the continuous variables of the groups. The differences in the frequency of A1AT and IL-6 genotypes were analyzed by the c2 test. The unpaired t-test was used to compare the continuous variables between different A1AT and IL6 genotypes. Multiple logistic regression analysis was performed to evaluate the independent associations between liver cirrhosis and the polymorphic variants of the studied SNPs at both A1AT and IL-6 genes in addition to the other variables that may affect liver cirrhosis such as serum level of ALT, AST, ALP, total and direct bilirubin, albumin, prothrombin time and AFP. For this analysis, patients were divided as cases with absence of cirrhosis vs. cases with presence of cirrhosis (fibrosis score F0-F3 vs. F4 respectively). Odds ratio (OR), 95% confidence interval (95% CI) and p values were calculated with the SPSS Inc. software. A p value of <0.05 was considered statistically significant.



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