Polymorphisms of α1-antitrypsin and Interleukin-6 genes and the progression of hepatic cirrhosis in patients with a hepatitis C virus infection

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.

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.

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.

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 (PCR-RFLP) analyses.

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 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].

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’.

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.

In this study, we recruited two groups of chronic HCV-infected patients; the chronic hepatic patients and the cirrhotic patients in addition to the control group. We found that the chronic hepatic patients exhibited significantly higher values of ALT, AST and ALP compared with the control group (p <0.05). In the same way, the cirrhotic patient group exhibited significantly higher activities of ALT, AST and ALP in addition to higher values of total bilirubin, prothrombin time and AFP and lower albumin compared to the control group (p <0.05). Interestingly, the cirrhotic patient group exhibited significantly higher values of AST, albumin, prothrombin time, AFP and fibrosis score compared to the chronic hepatic patient group (p <0.05). These results confirm the progression of the end stage complications associated with HCV infection in the cirrhotic patients. The demographic and biochemical parameters of all studied groups are summarized in Table 1.

Table 1

The clinical characteristics and biochemical parameters of the control group, chronic hepatitis patients and cirrhotic patients.

The SNP of IL-6 (–174 G/C, rs 1800795) was detected in the control, chronic hepatic and cirrhotic patients by the PCR-RFLP method. We found that the G allele was distributed at 100.0% in the control group, while the C allele was found only in the chronic hepatitis and cirrhotic patients at frequencies of 17.6 and 19.2%, respectively (Table 2). In more detail, the IL-6 (GG genotype) showed significantly lower frequency in the cirrhotic patients (61.5%) when compared to the control group (100.0%) at p <0.05. On the other hand, the IL-6 (CC genotype) was only found in the chronic hepatic patients (5.9% frequency), while the heterozygous IL-6 (GC genotype) was detected in both chronic hepatic and cirrhotic patients at 23.5 and 38.5% frequencies, respectively (Table 2).

Table 2

The single nucleotide polymorphism pattern of the IL-6 gene in the control group, chronic hepatitis patients and cirrhotic patients.

The S (264 Glu/Val, rs17580) and Z (342 Glu/Lys, rs28929474) mutations of the A1AT gene were detected in the control, chronic hepatic and cirrhotic patients by the PCR-RFLP method. We found that the wild type M allele was the most common allele in all groups. However, the frequency of the M allele decreased significantly in cirrhotic patients (65.4%) when compared to the control group (92.5%). Interestingly, the cirrhotic patients also showed a significantly increased frequency of the S allele (34.6%) compared to the control group (5.0%), while the Z allele was not detected in the cirrhotic patients (Table 3). The distribution of the A1AT genotypes revealed that the A1AT (SS genotype) was only detected in the cirrhotic patients (23.1% frequency) compared to control and chronic hepatic patients (both at 0.0% frequencies). In the same way, the A1AT (ZZ genotype) was only detected in the chronic hepatic patients (11.8% frequency) when compared to control and cirrhotic patients. The heterozygous MS and MZ genotypes were detected mostly in the chronic HCV patients (Table 3).

Table 3

The single nucleotide polymorphism pattern of the A1TAT gene in the control group, chronic hepatitis patients and cirrhotic patients.

As the low producer, IL-6 (CC genotype), was detected only in the chronic hepatitis patients, comparing the CC genotype with the high producer IL-6 (GG+GC) genotypes in the same group did not show significant increased values of ALT, AST, ALP, total and direct bilirubin or AFP (Table 4). These findings indicate that inheritance of the IL-6 (CC genotype) is not associated with increased risk of developing progressive liver diseases.

Table 4

The biochemical parameters within different IL-6 genotypes in the control group, chronic hepatitis patients and cirrhotic patients.

The chronic hepatitis patients with the Z allele (MZ+ZZ genotypes) showed a significantly increased AST activity when compared to the chronic hepatitis patients with either the MM or MS genotypes (p <0.05). Interestingly, the cirrhotic patients with the S allele (MS+SS genotypes) showed significantly increased levels of AST, ALT, total bilirubin, direct bilirubin and lower albumin levels when compared with the cirrhotic liver patients with the A1AT (MM genotype) at p <0.05 (Table 5). These results indicate that inheritance of A1AT (S or Z allele) increases the severity of liver disease in either cirrhotic or chronic hepatitis patients, respectively.

Table 5

The biochemical parameters within different A1AT genotypes in the control group, chronic hepatitis patients and cirrhotic patients.

Multivariate regression analyses showed that AST (p = 0.003, OR = 1.6, 95% CI = 1.3-2.0) and the S allele of A1AT represented as SS+MS genotypes (p = 0.001, OR = 7.7, 95% CI = 3.4-17.4) to be significantly independent predictors for development of liver cirrhosis. Although the IL-6 gene is known to regulate the expression of acute phase reactants such as A1AT, we did not find a statistically significant correlation between any of the genotypes of IL-6 (GG, GC, CC) and any of the A1AT (MM, MS, MZ, SS, ZZ) genotypes.