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.

Keywords:
Hepcidins; Anemia; Renal Dialysis; Hepacivirus; Polymorphism, Genetic; Interleukin-6

RESUMO

Introdução:

A hepcidina está associada à patogênese da anemia por insuficiência renal crônica, considerada um estado inflamatório crônico e também infecção por HCV. A IL-6 estimula a liberação de hepcidina a partir do fígado, suprime a captação intestinal de ferro e libera ferro das reservas internas.

Método:

Para detectar a associação entre o polimorfismo do gene IL-6 e os marcadores de anemia, 80 pacientes em hemodiálise (HD) [40 pacientes em HD, negativos para HCV; e 40 em HD, positivos para HCV] foram avaliados por exames químicos de rotina e hemograma completo, além da avaliação da hepcidina sérica, parâmetros do ferro [ferro sérico e ferritina sérica] e marcadores de hepatite C. O polimorfismo da IL-6 -174G/C foi determinado por MS-PCR, enquanto os polimorfismos de IL-6 -597G/A e -572 G/C foram detectados por PCR-SSP.

Resultados:

A hepcidina não esteve significativamente elevada em pacientes com HCV em comparação com pacientes em hemodiálise negativos para HCV. Uma diferença estatisticamente significativa foi detectada entre os pacientes em HD HCV negativos comparados aos positivos nas frequências de IL-6 -174 G/C e -597 G/A (P≤ 0,01 e P≤ 0,001, respectivamente). Por outro lado, foi relatada uma diferença não significativa entre pacientes em HD HCV negativos e positivos nas frequências de IL-6 -572 G/C.

Conclusões:

Nosso estudo indicou que os polimorfismos de IL-6 -174 G/C e -597 G/A podem desempenhar um papel na suscetibilidade ao HCV em pacientes em HD. Ainda necessitamos de estudos prospectivos adicionais em uma população maior para confirmar nossos achados.

Descritores:
Hepcidinas; Anemia; Diálise Renal; Hepacivirus; Polimorfismo Genético; Interleucina-6

Introduction

Chronic renal failure is a slow, insidious, irreversible deterioration in renal function¹1 Becherucci F, Roperto RM, Materassi M, Romagnani P. Chronic kidney disease in children. Clinical Kidney Journal. 2016 Aug 1;9(4):583-91.. 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 agents²2 Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner B, Saran R, Wang AY, Yang CW. Chronic kidney disease: global dimension and perspectives. The Lancet. 2013 Jul 20;382(9888):260-72.. 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) levels³3 Brattich M. Comorbid diseases in patients on dialysis: the impact on anemia. Nephrology Nursing Journal. 2007 Jan 1;34(1)..

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 death⁴4 Agarwal SK, Dash SC, Gupta S, Pandey RM. Hepatitis C virus infection in haemodialysis: the 'no-isolation'policy should not be generalized. Nephron Clinical Practice. 2009;111(2):c133-40.. In developed countries, the prevalence of anti-HCV seropositivity among patients on maintenance HD ranges between 5 and 60%⁵5 Etik DO, Ocal S, Boyacioglu AS. Hepatitis C infection in hemodialysis patients: A review. World journal of hepatology. 2015 Apr 28;7(6):885. 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%⁶6 Soliman AR, Momtaz Abd Elaziz M. Evaluation of an isolation program of hepatitis C virus infected hemodialysis patients in some hemodialysis centers in egypt. ISRN nephrology. 2012 Oct 31;2013.. Patients with chronic HCV infection often have increased liver iron. However, little is known about the mechanism of iron accumulation in the liver⁷7 El Said HW, Abou Seif KH, Ahmed YS, Abou Elleil HA, El Said TW, Behairy MA, Mohamed MM, Ahmed FA. Relationship of serum haemojuvelin and hepcidin levels with iron level and erythropoietin requirement in prevalent hepatitis C virus positive haemodialysis patients. Nephrology. 2018 Apr;23(4):323-30..

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 inflammation⁸8 Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. The Journal of clinical investigation. 2004 May 1;113(9):1271-6.

9 De Domenico I, Ward DM, Kaplan J. Regulation of iron acquisition and storage: consequences for iron-linked disorders. Nature reviews Molecular cell biology. 2008 Jan;9(1):72.^-1010 Costa E, Swinkels DW, Laarakkers CM, Rocha-Pereira P, Rocha S, Reis F, Teixeira F, Miranda V, do Sameiro Faria M, Loureiro A, Quintanilha A. Hepcidin serum levels and resistance to recombinant human erythropoietin therapy in haemodialysis patients. Acta haematologica. 2009;122(4):226-9.. Impaired hepcidin regulation may play a role in alterations of iron metabolism in HD patients with HCV infection⁷7 El Said HW, Abou Seif KH, Ahmed YS, Abou Elleil HA, El Said TW, Behairy MA, Mohamed MM, Ahmed FA. Relationship of serum haemojuvelin and hepcidin levels with iron level and erythropoietin requirement in prevalent hepatitis C virus positive haemodialysis patients. Nephrology. 2018 Apr;23(4):323-30.. 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 activity⁸8 Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. The Journal of clinical investigation. 2004 May 1;113(9):1271-6.^,1111 Papanikolaou G, Tzilianos M, Christakis JI, Bogdanos D, Tsimirika K, MacFarlane J, Goldberg YP, Sakellaropoulos N, Ganz T, Nemeth E. Hepcidin in iron overload disorders. Blood. 2005 May 15;105(10):4103-5.. IL-6, one of the well-studied pro-inflammatory cytokines, is a major mediator of the acute phase response¹²12 Ryu J. and Eung-Jun Kim S. Interleukin-6 -634 C/G and -174 G/C Polymorphisms in Korean Patients Undergoing Hemodialysis. Korean J Intern Med. 2012 Sep; 27(3): 327-337..

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 inter-individual variability for susceptibility and disposition to diseases¹³13 Talaat RM, Abdelkhalek MS, El-Maadawy EA, Abdel-Mageed WS, El-Shenawy SZ, Osman MA. Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians. Human immunology. 2017 Nov 1;78(11-12):739-46.. 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 CKD¹⁴14 Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nature immunology. 2015 May;16(5):448.. 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.

Materials And Methods

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) method¹⁵15 Talaat RM, Abdel-Aziz AM, El-Maadawy EA, Abdel-Bary N. CD38 and interleukin 6 gene polymorphism in Egyptians with diffuse large B-cell lymphoma (DLBCL). Immunological investigations. 2015 Apr 3;44(3):265-78.. 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/primer-blast), 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 independentt-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/cgi-bin/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 r²). The genotype, allele, and haplotype frequencies were compared between cases and controls using a chi-square test (χ²2 Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner B, Saran R, Wang AY, Yang CW. Chronic kidney disease: global dimension and perspectives. The Lancet. 2013 Jul 20;382(9888):260-72.). The odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the risk associated with a particular allele, genotype, or haplotype.

Results

Patient baseline characteristics

the baseline characteristics of the 80 chronic hemodialysis (HD) patients are given in Table 2. The sample included 49 males and 31 females with mean age of 52.26±12.28 years. The 40 negative HCV-HD patients were 27 males and 13 females with mean age 51.42±2.14 years, labeled as group 1 and the 40 positive HCV-HD patients were 22 males and 18 females with mean age 53.10±1.73 years, labeled as group 2. Iron level increased significantly (P≤0.001) in positive HCV-HD (391.65±32.43) in comparison with HCV-negative HD group (108.07±11.57) (Table 2). Similarly, Hb, hematocrit, and ferritin (9.81±0.24, 28.54±0.78, 998.62±90.19, respectively, were significantly elevated (P≤0.001, P≤0.001, P≤0.01, respectively) in HCV-positive HD patients compared with HCV-negative HD patients (8.36±0.29, 25.61±1.31, 276.77±53.66, respectively). No statistical difference was found in hepcidin level in HCV-positive HD patients (595.86±52.96) compared to HCV-negative HD patients (570.64±59.38).

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 [9 (22.5%), 31 (77.5%), 0 (observed) vs. 15.01 (37.5 %), 18.99 (47.5%), 6.01 (15%) (predicted) (P≤0.001)] in HCV-negative HD patients. The frequency of IL-6 -597G/A genotypes significantly deviated from the HWE for GG, GA, AA [8 (20%), 29 (72.5%), 1 (2.5%) (observed) vs. 13.68, 17.64, 5.68 (predicted) (P≤0.001)] in HCV-positive HD patients.

The distribution of IL-6 alleles and genotypes are shown in Table 3. A statistically significant difference was detected between the HCV negative and positive HD patients in frequencies of IL-6 -174 G/C (P≤0.01). There was a significant increase in the frequency of -174 GG genotype (P<0.01, OR=4.66, 95%CI=1.76-12.31) in HCV-positive HD patients compare to HCV-negative HD patients and a significant decrease in the frequency of -174 GC genotype (P<0.01, OR=0.16, 95%CI=0.06-0.45) in HCV-positive HD patients compare to negative. The frequencies of G alleles were also higher among HCV-positive HD patients than negative (77.5% vs. 61.25%, P≤0.05, OR=2.17, 95%CI=1.09-4.35). On the other hand, no significant difference in the SNP frequency at the IL-6 -572 was recorded between HCV-positive and -negative HD patients

Analysis of IL-6 -597 G/A SNP revealed a significant difference in the distribution of different genotypes between HCV-positive and -negative HD patients (P≤0.001). HCV-positive HD patients had increased frequency of IL-6 -597 G/A vs. HCV-negative HD patients (P≤0.01, OR=6.15, 95%CI=2.33-16.21). The frequencies of A alleles were also higher among HCV-positive HD patients compared to negative (39.2% vs. 15.4%, P≤ 0.01, OR=3.54, 95%CI=1.63-7.67).

We analyzed the linkage disequilibrium (LD) pattern between the SNPs. There was LD between IL-6 -174 G/C and IL-6 -572 G/C (r²= 0.00086 and D’ 0.9975), between IL-6 -174 G/C and IL-6 -597 G/A (r²= 0.0038 and D’= 0.7486), and between IL-6 -572 G/C and IL-6 -597 G/A (r²= 0.00068 and D’= 0.3699).

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).

Association between markers of anemia and il-6 gene polymorphisms in hcv-hd patients

In HCV-negative HD patients, anemia markers did not significantly differ in the genotypes IL-6 -572 G/C and IL-6 -597. Hepcidin level increased significantly (P≤0.05) in IL-6 GC genotype (640.18± 399.44) in comparison to IL-6 GG genotype (331.09±78.14). In contrast , ferritin level was significantly elevated (P≤0.01) in IL-6 GG genotype (307.54 ±372.98) than IL-6 GC genotype ( 167.34 ±93.34) (Table 5). Anemia markers did not differ significantly in any of the studied IL-6 SNPs in HCV-positive HD patients (Table 6).

Discussion

High circulating levels of IL-6 have been documented in several clinical inflammatory conditions including liver diseases such as viral chronic hepatitis¹⁶16 Radovic M1, Jelkmann W, Djukanovic L, Ostric V. Serum erythropoietin and interleukin-6 levels in hemodialysis patients with hepatitis virus infection.. J Interferon Cytokine Res. 1999 Apr;19(4):369-73., and various diseases in patients with end-stage renal disease¹⁷17 Buraczynska M, Jozwiak L, Ksiazek P, Borowicz E, et al. (2007). Interleukin-6 gene polymorphism and faster progression to end-stage renal failure in chronic glomerulonephritis. Transl. Res. 150:101-105.^,1818 Ryu JH and Kim SJ (2012). Interleukin-6 -634 C/G and -174 G/C polymorphisms in Korean patients undergoing hemodialysis. Korean J. Intern. Med. 27:327-37..

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 HCV¹⁹19 Barrett S, Collins M, Kenny C, Ryan E, Keane CO, Crowe J. Polymorphisms in tumour necrosis factor-a, transforming growth factor-ß, interleukin-10, interleukin-6, interferon-, and outcome of hepatitis C virus infection. Journal of medical virology. 2003 Oct 1;71(2):212-8.. 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 HCV-positive 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.²⁰20 Saifan C, El-Charabaty E, Kleiner M, El-Sayegh S. Effect of hepatitis C virus infection on erythropoiesis in patients on hemodialysis. International journal of nephrology and renovascular disease. 2013;6:121..

No significant difference in the hepcidin level was found between groups. This result is in accordance with the data of Fujita et al.²¹21 Fujita N, Sugimoto R, Motonishi S, Tomosugi N, Tanaka H, Takeo M, Iwasa M, Kobayashi Y, Hayashi H, Kaito M, Takei Y. Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion. Journal of hepatology. 2008 Nov 1;49(5):702-10. who stated that there is no relation between HCV RNA load and serum hepcidin and supported by data of Ibrahim et al.²²22 Ibrahim M, Gadalla H, Raslan H, Abou Ellel H, William E. Effect of hepatitis-C virus infection on serum hepcidin in hemodialysis patients. [Ain Shams University, Egypt]. Anemia [M614]. World Congress of Nephrology, Milano, Italy. 2009 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 HCV²²22 Ibrahim M, Gadalla H, Raslan H, Abou Ellel H, William E. Effect of hepatitis-C virus infection on serum hepcidin in hemodialysis patients. [Ain Shams University, Egypt]. Anemia [M614]. World Congress of Nephrology, Milano, Italy. 2009.

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.²³23 Shan Y, Lambrecht RW, Bonkovsky HL. Association of hepatitis C virus infection with serum iron status: analysis of data from the third National Health and Nutrition Examination Survey. Clinical infectious diseases. 2005 Mar 15;40(6):834-41. 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.²⁴24 Sabry A, El-Dahshan K, Mahmoud K, El-Husseini A, Sheashaa H, Abo-Zenah H. Effect of hepatitis C virus infection on haematocrit and haemoglobin levels in Egyptian hemodialysis patients. Eur J Gen Med. 2007;4(1):9-15. stated that patients with end-stage renal disease and HCV-positive 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.²⁵25 Bennermo M, Held C, Green F, Strandberg LE, Ericsson CG, Hansson LO, Watkins H, Hamsten A, Tornvall P. Prognostic value of plasma interleukin-6 concentrations and the- 174 G> C and- 572 G> C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. Atherosclerosis. 2004 May 1;174(1):157-63., who concluded that IL-6 gene was associated with a variety of major diseases as HCV. Also, our results match with Nasr et al.²⁶26 Nasr MY, Deeb AS, Badra G, El Sayed IH. Lack of Any Relationship Between Circulating Autoantibodies and Interleukin-6 Levels in Egyptian Patients Infected with the Hepatitis C Virus. Asian Pacific journal of cancer prevention: APJCP. 2016;17(11):4977, who observed that IL-6 genotype and polymorphism was related to the presence and outcome of HCV. While Giannitrapani et al.²⁷27 Giannitrapani L, Soresi M, Giacalone A, Campagna ME, Marasa M, Cervello M, Marasa S, Montalto G. IL-6- 174G/C polymorphism and IL-6 serum levels in patients with liver cirrhosis and hepatocellular carcinoma. Omics: a journal of integrative biology. 2011 Mar 1;15(3):183-6. found no significant difference regarding neither genotype nor allelic frequencies of the polymorphism studied among their three groups. Moreover, Giannitrapani et al.²⁸28 Giannitrapani L, Soresi M, Balasus D, Licata A, Montalto G. Genetic association of interleukin-6 polymorphism (-174 G/C) with chronic liver diseases and hepatocellular carcinoma. World journal of gastroenterology: WJG. 2013 Apr 28;19(16):2449. proved that there is the possibility of a genetic association between IL-6 -174 G/C polymorphism 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 expression²⁹29 Morozumi T, Sharma A, De Nardin E. The Functional Effects of the- 455G/A Polymorphism on the IL-6-Induced Expression of the ß-fibrinogen Gene may be due to Linkage Disequilibrium with Other Functional Polymorphisms. Immunological investigations. 2009 Jan 1;38(3-4):311-23.^{, 30}30 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. Journal of Biological Chemistry. 2000 Jun 16;275(24):18138-44. 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 HCV-positive HD patients was demonstrated. This result is contradictory to the results of Hirschhorn et al.³¹31 Hirschhorn JN, Lohmueller K, Byrne E, Hirschhorn K. A comprehensive review of genetic association studies. Genetics in Medicine. 2002 Mar;4(2):45. and Rothman et al.³²32 Rothman N, Skibola CF, Wang SS, Morgan G, Lan Q, Smith MT, Spinelli JJ, Willett E, De Sanjose S, Cocco P, Berndt SI. Genetic variation in TNF and IL10 and risk of non-Hodgkin lymphoma: a report from the InterLymph Consortium. The lancet oncology. 2006 Jan 1;7(1):27-38.. The study of Liu et al.³³33 Liu S, Qiu XQ, Zeng XY, Bai H, Bei CH, Yang Y. Relationship between IL6-572G/C polymorphism and hepatocellular carcinoma in men. Zhonghua gan zang bing za zhi= Zhonghua ganzangbing zazhi= Chinese journal of hepatology. 2012 Jun;20(6):463-7. 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.³⁴34 Park BL, Lee HS, Kim YJ, Kim JY, Jung JH, Kim LH, Shin HD. Association between interleukin 6 promoter variants and chronic hepatitis B progression. Experimental & molecular medicine. 2003 Apr;35(2):76. who studied the association of IL-6 among the Korean population with hepatitis and Falleti et al.³⁵35 Falleti E, Fabris C, Vandelli C, Colletta C, Cussigh A, Smirne C, Fontanini E, Cmet S, Minisini R, Bitetto D, Toniutto P. Genetic polymorphisms of interleukin-6 modulate fibrosis progression in mild chronic hepatitis C. Human immunology. 2010 Oct 1;71(10):999-1004. who observed that IL-6 -597 G/A polymorphism was related to the presence and outcome of HCV infection. Also, Cussigh et al.³⁶36 Cussigh A, Falleti E, Fabris C, Bitetto D, Cmet S, Fontanini E, Bignulin S, Fornasiere E, Fumolo E, Minisini R, Pirisi M. Interleukin 6 promoter polymorphisms influence the outcome of chronic hepatitis C. Immunogenetics. 2011 Jan 1;63(1):33-41., who reported the IL-6 -597 G/A, appear to favor a progressive HCV disease. In contrast to our study, Lu et al.³⁷37 Lu Y, Wu Z, Peng Q, Ma L, Zhang X, Zhao J, Qin X, Li S. Role of IL-4 gene polymorphisms in HBV-related hepatocellular carcinoma in a Chinese population. PloS one. 2014 Oct 8;9(10):e110061. 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-6³⁸38 Zhang X, Rovin BH. Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines. Biochimica et Biophysica Acta (BBA)-General Subjects. 2010 Dec 1;1800(12):1262-7.. Genetic polymorphisms of proinflammatory cytokine genes such as IL-6 might play a crucial role in anemia caused by chronic renal diseases³⁹39 Girndt M1, Stenvinkel P, Ulrich C, Axelsson J, Nordfors L, Barany P, Carrero JJ, Heine GH, Kaul H, Köhler H. Influence of cytokine gene polymorphisms on erythropoetin dose requirements in chronic haemodialysis patients. Nephrol Dial Transplant. 2007 Dec;22(12):3586-92..

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 genotype⁴⁰40 Balakrishnan VS, Guo D, Rao M, Jaber BL, Tighiouart H, Freeman RL, Huang C, King AJ, Pereira BJ, HEMO Study Group. Cytokine gene polymorphisms in hemodialysis patients: association with comorbidity, functionality, and serum albumin. Kidney international. 2004 Apr 1;65(4):1449-60.. 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.²⁴24 Sabry A, El-Dahshan K, Mahmoud K, El-Husseini A, Sheashaa H, Abo-Zenah H. Effect of hepatitis C virus infection on haematocrit and haemoglobin levels in Egyptian hemodialysis patients. Eur J Gen Med. 2007;4(1):9-15. 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.

References

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