Association between interleukin-6 gene polymorphism and iron regulation in hemodialysis patients infected with HCV

ABSTRACT Backgrounds: Hepcidin is related to the pathogenesis of chronic renal failure anemia, which is considered a chronic inflammatory state as well as HCV infection. IL-6 stimulates the release of hepcidin from the liver, suppresses intestinal iron uptake, and releases iron from internal stores. Method: To detect the association between IL-6 gene polymorphism and anemia markers, 80 hemodialysis (HD) patients [40 negative HCV HD patients and 40 positive HCV HD patients] were studied by routine chemistry and complete blood count, in addition to the assessment of serum hepcidin, iron parameters [serum iron and serum ferritin], and hepatitis C markers. IL-6 polymorphism -174G/C was determined by MS-PCR, while IL-6 polymorphisms -597G/A and -572 G/C were detected by PCR-SSP. Results: Hepcidin was non-significantly elevated in HCV-positive compared with HCV-negative hemodialysis patients. A statistically significant difference was detected between the negative and positive HCV HD patients in frequencies of IL-6 -174 G/C and -597 G/A (P≤ 0.01 and P≤ 0.001, respectively). On the other hand, a non-significant difference was reported between negative and positive HCV HD patients in the frequencies of IL-6 -572 G/C. Conclusions: Our study indicated that IL-6 -174 G/C and -597 G/A polymorphisms may play a role in HCV susceptibility in HD patients. Additional prospective studies on a larger population are needed to confirm our findings.


IntroductIon
Chronic renal failure is a slow, insidious, irreversible deterioration in renal function 1 . Prevalence is estimated to be 8-16% worldwide. Anemia is a common feature of chronic kidney disease (CKD) associated with poor outcomes. The current management of patients with anemia in CKD is controversial, with recent clinical trials demonstrating increased morbidity and mortality related to erythropoiesis-stimulating agents 2 . Patients on dialysis are frequently affected by multiple comorbidities that can directly or indirectly contribute to anemia. The systemic and chronic nature of these diseases leads to frequent inter-current events that can depress hemoglobin (Hb) levels 3 .
In hemodialysis (HD) patients, hepatitis C virus (HCV) infection is the most common cause of acute and chronic hepatitis, and it increases the risk for death 4 . In developed countries, the prevalence of anti-HCV seropositivity among patients on maintenance HD ranges between 5 and 60% 5 . The frequency of HCV is much higher in patients undergoing dialysis in less developed countries. In Egypt, the prevalence of HCV antibodies in hemodialysis patients ranges from 52.3 to 82.3% 6 . Patients with chronic HCV infection often have increased liver iron. However, little is known about the mechanism of iron accumulation in the liver 7 .
Hepcidin, a 25-amino acid peptide hormone, exclusively synthesized in the liver, is thought to be a key regulator of iron homeostasis. Hepcidin is induced by infection and inflammation [8][9][10] . Impaired hepcidin regulation may play a role in alterations of iron metabolism in HD patients with HCV infection 7 . Whereas both inflammation and iron loading induces hepcidin production, erythropoietic activity suppresses its production. In the case of inflammation, the primary mediator seems to increase interleukin-6 (IL-6) levels, which in turn cause the binding of signal transducer and activator of transcription 3 (STAT3) to the hepcidin promoter, increasing its activity 8,11 . IL-6, one of the well-studied pro-inflammatory cytokines, is a major mediator of the acute phase response 12 .
The number of single nucleotide polymorphisms (SNP) in the human genome is estimated to be about one million. As SNPs may alter gene expression and function, there is considerable interest in their interindividual variability for susceptibility and disposition to diseases 13 . In HD patients, gene polymorphisms modulating IL-6 synthesis may represent a fair means for testing the link between IL-6 and the development of CKD 14 . In the present investigation, we analyzed IL-6 gene polymorphism in chronic HCV seropositive Egyptian patients on regular HD to evaluate the association between IL-6 and anemia markers.

Patients and cOntrOls
This study was conducted on 80 unrelated Egyptian patients on regular chronic HD for at least one year. They were recruited from the hemodialysis unit in Desouk General Hospital, Kafr El-Sheikh, Egypt. All investigations were performed following the University of Sadat City, Health and Human Ethical Clearance Committee guidelines for Clinical Researches. The local ethics committee approved the study protocol, and oral informed consent was obtained from all patients.
HCV infection was diagnosed by the presence of HCV antibodies using enzyme-linked immunosorbent assay (ELISA) and confirmed by the presence of HCV-RNA using real-time PCR. For HBV infection, HBV surface antigen (HBsAg) was tested by ELISA. According to the presence of HCV infection, hemodialysis patients were divided into two groups. Group (1) included 40 hemodialysis patients with negative HCV infection. Group (2) included 40 hemodialysis patients with positive HCV infection. Both groups were negative for HBV infection.
Exclusion criteria were HD patients who were not compliant with hemodialysis for six months with more than three missed dialysis sessions/month, patients exposed to hospitalization, major surgeries, episodes of gastrointestinal tract bleeding, access clotting or bacteremia, or any other infections in 4 weeks before the blood draws, pregnancy, patients with polycystic kidney disease, HBV infection, hematopoietic disorders (including multiple myeloma), decompensated liver cirrhosis, hyperparathyroidism, patients treated with interferon and/or ribavirin, and/or patients with a history of blood transfusion in the last six months.
Blood samples were withdrawn, and the following biochemical parameters were measured: serum albumin level, serum total bilirubin level, serum alanine aminotransferase (ALT) level, serum alkaline phosphatase (ALP) level, serum aspartate aminotransferase (AST) level, urea, creatinine, sodium, and potassium. Complete blood count, total iron, serum ferritin levels, and erythropoietin were also estimated. The hepcidin level was measured by the quantitative sandwich enzyme immunoassay technique (Sun Red Company, China) il-6 snPs genOtyPing blood samples were collected in EDTA sterile tubes. Genomic DNA was isolated from whole blood, according to the manufacturer's instructions (Qiagen Ltd., UK).
IL-6 (-174 G/C) rs1800795 was genotyped using a mutagenic separated polymerase chain reaction (MS-PCR) method 15 . The reaction was done in one tube containing the two alleles. The PCR mixtures consisted of DreamTaq Green PCR Master Mix (2x) (Fermentas, Thermo Fisher Scientific Inc.), 10 pmol of each allele-specific primer, 10 pmol of antisense primer, and 100 ng of DNA. PCR cycling conditions consisted of 95°C for 10 min [1 cycle], followed by 94°C for 30 seconds, 66°C for 45 seconds, 72°C for 45 seconds [40 cycles], and finally 72°C for 7 min [1 cycle]. An amplicon of 136 bp (C allele) and 121 bp (G allele) was demonstrated using 4% agarose gel electrophoresis and the size of PCR products was determined relative to the migration of a 25 bp step ladder.
IL-6 -572 G/C rs1800796 and -597 G/A rs1800797 were genotyped using the sequence-specific primer method (PCR-SSP). The primers sequence for IL-6 -572 G/C and -597 G/A were designed using the tool at HTTP://www.ncbi.nlm.nih. gov/tools/primerblast), according to Talaat et al. (Table 1). PCR reaction consisted of 25 μL in two tubes, one for each allele. Thermal cycling conditions were 94°C for 2 min (1 cycle), followed by 96°C for 25s, 70°C for 45s, and 72°C for 20s (5 cycles), followed by 96°C for 25 s, 65°C for 5s, and 72°C for 45s (11cycles), and finally 96°C for 25 s, 55°C for 60 s, and 72°C for 2 min (15 cycles). IL-6 -572 G/C primers resulted in an amplicon of 325 bp and 473 bp for IL-6 -597 G/A. The size of PCR products was determined relative to the migration of a 100 bp step ladder using 2% agarose gel electrophoresis. statistical analysis all statistical analyses were performed using the Statistical Package for Social Science (SPSS) version 19 (LEAD Technology Inc.). Data are presented as means with corresponding standard deviation (SD).
Comparisons among different groups were performed by an independent t-test. Each polymorphism was examined in the control population to confirm that the distribution of the genotypes confirmed to Hardy-Weinberg expectations (http://ihg.gsf.de/cgibin/hw/hwa1.pl) (HWE). The online tool SNP stats (http://bioinfo.iconcologia.net/SNPstats) was used to perform the haplotype analyses and calculate the LD parameters (D' and r 2 ). The genotype, allele, and haplotype frequencies were compared between cases and controls using a chi-square test (χ 2 ). The odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the risk associated with a particular allele, genotype, or haplotype.  il-6 genOtyPes and allele frequencies the frequency of the -572 G/C genotypes for IL-6 did not deviate significantly from the HWE equilibrium. However, the values predicted by the assumption of the HWE were different to those observed for Il-6 -174 G/C genotype GG, GC, and CC  Table 3.   tAble 2 HCV-negative and positive HD patients. All data are presented as mean ± SD. N: number of cases, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RBCs: red blood cells count and NS: non-significant. Differences between the 2 groups were compared by independent t-test. P < 0.05 is considered significant.
As shown in Table 4, the haplotype frequencies of IL-6 -174 G/C, -572 G/C, and -597G/A in HCV-positive HD patients demonstrated a significant increase in the IL-6 GGA and CGA haplotypes (P≤0.01 and P≤0.001 respectively). At the same time, there was a significant reduction in the CGG haplotype (P≤0.01).

dIscussIon
High circulating levels of IL-6 have been documented in several clinical inflammatory conditions including liver diseases such as viral chronic hepatitis 16 , and various diseases in patients with end-stage renal disease 17,18. Studies on the cytokine gene polymorphisms suggest that inheritance of some genotypes related to polymorphisms of cytokine genes, such as the IL-6 gene, which clearly affect cytokine production and maybe host genetic factors associated with the progression of HCV 19 . With this overview, the present study was planned to identify the relationship between IL-6 and iron regulation in chronic hepatitis C seropositive Egyptian patients on regular HD.
HD duration was significantly longer in HCVpositive HD patients than in HCV-negative HD patients, as patients with HCV infection spent a significantly longer time (P≤0.001) on hemodialysis than those without HCV infection. These findings were supported by Saifan et al. 20 .
No significant difference in the hepcidin level was found between groups. This result is in accordance with the data of Fujita et al. 21 who stated that there is no relation between HCV RNA load and serum hepcidin and supported by data of Ibrahim et al. 22 who concluded that there is no significant difference in hepcidin level between HCV-negative and -positive HD patients. This may be explained by a multiplicity of factors that influence hepcidin level. While inflammation, as well as HD treatment may increase its level, both anemia and iron deficiency may decrease its level. Moreover, infection with HCV may impair liver ability to secrete hepcidin, which might have important implications in the treatment of anemia in HD patients infected with HCV 22 .
There was a highly significant (P≤0.001) elevation of iron level among HCV-positive HD patients. At the same time, ferritin was also highly elevated (P≤0.01) in HCV-positive than -negative HD patients. These results agree with the study of Shan et al. 23 in the US population, who stated that there is an association between HCV infection and higher levels of serum iron and ferritin. Besides, Sabry et al. 24 stated that patients with end-stage renal disease and HCVpositive appear to have higher serum iron and TS compared to HCV-negative patients; moreover, there was a significant difference in serum iron and ferritin between groups.
Our study showed an increase in the frequency of IL-6 -174 G/C G/G, C/G, and GCCC genotypes in the HCV-positive HD patients than -negative, suggesting that susceptibility to HCV-positive HD patients may influence the frequency of IL-6. Concerning allele frequency, a non-significant change was demonstrated in G and C alleles in both groups, although the G allele was severely decreased compared to the C allele. These results are in agreement with Bennermo et al. 25 , who concluded that IL-6 gene was associated with a variety of major diseases as HCV. Also, our results match with Nasr et al. 26 , who observed that IL-6 genotype and polymorphism was related to the presence and outcome of HCV. While Giannitrapani et al. 27 found no significant difference regarding neither genotype nor allelic frequencies of the polymorphism studied among their three groups. Moreover, Giannitrapani et al. 28 proved that there is the possibility of a genetic association between IL-6 -174 G/C polymorphism il-6 (-174g/c, -572g/c, and-597g/a) haPlOtyPes in hd Patients  tAble 4 OR, odds ratio; 95%CI, 95% confidence interval. NS, non-significant. The genotype, allele, and frequencies were compared using a chi-square test (χ 2 ). P < 0.05 is considered significant.     All data are presented as mean ± SD. N: number of cases, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, RBCs: Red blood cells count and NS: non-significant. Differences among groups were compared by ANOVA. P < 0.05 is considered significant.

Hemoglobin
and some specific liver diseases as they observed a correlation between the presence of the high producer genotype (GG) and a worse evolution of the HCV. The polymorphism at position IL-6 -174 G/C is one of several IL-6 polymorphisms that have been suggested to affect the IL-6 expression 29,30 . According to our findings, there is evidence for a genetic association between the genotype of IL-6 -174 G/C except C/C, and the susceptibility to the HCVpositive HD patients was demonstrated. This result is contradictory to the results of Hirschhorn et al. 31 and Rothman et al. 32 . The study of Liu et al. 33 showed null associations between IL-6 -174 G/C and several common types of cancer, including breast, colorectal, prostate, lung, gastric cancer, lymphoma, multiple myeloma, and melanoma.
When studying IL-6 polymorphism at position -597 G/A there was a highly significant difference observed in carriage rate of 'A' allele in HCV-positive HD patients and highly significant in 'G' allele in HCV-negative HD patients (P<0.01) for both groups. These results agree with Park et al. 34 who studied the association of IL-6 among the Korean population with hepatitis and Falleti et al. 35 who observed that IL-6 -597 G/A polymorphism was related to the presence and outcome of HCV infection. Also, Cussigh et al. 36 , who reported the IL-6 -597 G/A, appear to favor a progressive HCV disease. In contrast to our study, Lu et al. 37 found no significant difference in the IL-6 -597 G/A allele or genotype frequencies between the HCV patients and the control group.
Pro-inflammatory cytokines interact with hematopoiesis at various stages. Synthesis of hepcidin and ferritin are induced by IL-6 38 . Genetic polymorphisms of proinflammatory cytokine genes such as IL-6 might play a crucial role in anemia caused by chronic renal diseases 39 .
There was a significant correlation (P<0.05) between IL-6 -174 G/C GC and hepcidin levels in the HCV-negative HD population. In HD patients, the higher production for IL-6 has been associated with IL-6 -174 G/C GC genotype 40 . Thus, hepcidin level may be increased in IL-6 (-174 G/C) GC genotype patients, and this supports our result. On the other hand, ferritin level showed a significant correlation (P<0.01) with IL-6 (-174 G/C) CC genotype. Sabry et al. 24 proved that serum ferritin was significantly increased in HCV-positive HD patients than in HCV-negative HD patients. Genetic polymorphism of IL-6 did not show any correlation with anemia in HCV-positive HD patients.

conclusIons
In conclusion, our preliminary study indicated that IL-6 (-174 G/C and -597 G/A) polymorphisms may play a role in HCV susceptibility in HD patients, and IL-6 -174 G/C polymorphisms may play a role in the regulation of hepcidin and iron levels in HD patients. Finally, more extensive prospective studies are needed to confirm our findings.

dAtA AvAIlAbIlIty
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.