Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients

GENE-38753; No. of pages: 5; 4C: Gene xxx (2013) xxx–xxx

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Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients Tarek Kamal Motawi a, Olfat Gamil Shaker b, Manal Fouad Ismail a,⁎, Noha Hussein Sayed a a b

Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

a r t i c l e

i n f o

Article history: Accepted 16 June 2013 Available online xxxx Keywords: HCV hepatitis Hepatocellular carcinoma HFE Hepcidin Ghrelin Genetic variants

a b s t r a c t Background: Hepatocellular carcinoma (HCC) associated to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths. Genetic variations may play an important role in the development of HCC in HCV patients. Ghrelin exerts anti-inflammatory, antifibrotic and hepatoprotective effects on chronically injured hepatic tissues. Ghrelin gene shows several single nucleotide polymorphisms (SNPs) including − 604G/A, Arg51Gln, and Leu72Met. Hemochromatosis gene (HFE) mutations namely C282Y and H63D may cause hepatic iron overload, thus increasing the risk of HCC in HCV patients. Aim: To investigate the association of progression of HCC with ghrelin and HFE gene polymorphisms in HCV Egyptian patients. Methods: Seventy-nine chronic HCV patients (thirty-nine developed HCC and forty did not), and forty healthy control subjects were included in the study. The polymorphisms were evaluated by PCR/RFLP analysis, and related protein levels were measured by either ELISA or colorimetric assays. Results: The three tested SNPs on ghrelin gene were detected in the studied groups, only one SNP (Arg51Gln) showed significantly higher GA, AA genotypes and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC and controls. Of the two mutations studied on HFE gene only H63D heterozygous allele was detected, and its frequency did not statistically differ among studied groups. Conclusion: Our results suggest that A allele at position 346 of the ghrelin gene is associated with susceptibility to HCC in hepatitis C patients. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Each year, hepatocellular carcinoma (HCC) is diagnosed in more than half a million people worldwide (SEER program, 2010). It is the fifth most common cancer in men, the seventh in women, and the third leading cause of cancer related deaths, responsible for about 692,000 deaths Abbreviation: ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; Bcl I, Restriction enzyme from Bacillus caldolyticus; Bsr I, restriction enzyme from Bacillus stearothermophilus; C/EBP-α, CCAAT/enhancer-binding protein alpha; Dra I, Restriction enzyme from Deinococcus radiophilus; EDTA, Ethylenediaminetetra acetic acid; ELISA, Enzyme-linked immunosorbent assay; Fe2-TF, Iron–transferrin complex; HCC, Hepatocellular carcinoma; HCV, Hepatitis C virus; HH, Hereditary hemochromatosis; IL-1β, Interleukin-1 beta; IL-6, Interleukin-6; IL-8, Interleukin-8; MCP-1, Monocyte chemoattractant protein-1; MHC-1, Major histocompatibility complex class 1; PCR, Polymerase chain reaction; Pro-Arg, Proline-arginine; Pro-Gln, Proline-glycine; RFLP, Restriction fragment length polymorphism; ROS, Reactive oxygen species; Rsa I, Restriction enzyme from Rhodopseudomonas sphaeroides; Sac I, Restriction enzyme from Streptomyces achromogenes; TFR1, Transferrin receptor 1; TFR2, Transferrin receptor 2; TNBS, Trinitrobenzene sulfonic acid; TNF-α, Tumor necrosis factor alpha; STAT-3, Signal transducer and activator of transcription-3. ⁎ Corresponding author at: Biochemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt. Tel.: +20 1223103229; fax: +20 02 23628426. E-mail address: [email protected] (M.F. Ismail).

annually (IARC, 2011). Most of the burden of the disease (85%) is borne in developing countries (WHO, 2008). Egypt has rising morbidity and mortality rates from HCC, where the relative frequency of HCC in Egypt increased from 4.0% in 1993 to 7.3% in 2003 (Lehman and Wilson, 2009). HCC related to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths (SEER program, 2010), as 31% of the HCC cases are thought to be a result of HCV infection (Llovet et al., 2012). Egypt has the highest prevalence of HCV worldwide reaching 22% of the population (Shepard et al., 2005). The Egyptian HCV epidemic resulted from the use of unsterile syringes during the campaigns of parenteral antischistosomal therapy extensively practiced from the 1920s to 1980s (Frank et al., 2000). Apart from classic risk factors, molecular genetic studies have revealed that genetic variations may play an important role in the pathogenesis of HCV (Bouzgarrou et al., 2008). Ghrelin, a 28 amino acid gut hormone has been identified as a potent growth hormone secretagogue. Moreover, it is demonstrated to have various metabolic functions, including regulation of energy balance, control of appetite, stimulation of gastric acid secretion, and regulation of gastrointestinal motility (Kojima and Kangawa, 2005). Other effects include cytoprotection, and vasodilatation (Van der Lely et al., 2004), in addition to anti-inflammatory effects (Dixit et al., 2004). Ghrelin

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.06.053

Please cite this article as: Motawi, T.K., et al., Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.053

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T.K. Motawi et al. / Gene xxx (2013) xxx–xxx

also exerts antifibrotic and hepatoprotective effects on chronically injured tissues (Moreno et al., 2010). Hemochromatosis gene (HFE) encodes a membrane protein which maintains iron homeostasis (Goswami and Andrews, 2006). Mutations in the HFE gene may lead to increased intestinal iron absorption and liver iron overload as evidenced in human hereditary hemochromatosis (HH type 1) (Vujic Spasic et al., 2008). HH patients may develop hepatic cirrhosis and HCC (Ajioka and Kushner, 2002). To our knowledge, no enough data are available concerning the association of ghrelin and HFE gene polymorphisms with HCC in HCV Egyptian patients. Therefore the current study aimed to analyze the polymorphisms -604G/A, Arg51Gln, and Leu72Met within ghrelin gene, and C282Y and H63D within HFE gene, and evaluate their contribution to susceptibility to HCC in HCV patients. 2. Subjects and methods 2.1. Study subjects The study included 79 unrelated chronic HCV patients diagnosed at least 5 years before, the patients admitted to the Internal Medicine Department of Kasr El Aini hospital. Thirty-nine of them (29 males and 10 females) developed HCC, while the remaining 40 (26 males and 14 females) did not. All patients were anti-HCV positive, HCC was diagnosed by high alpha-fetoprotein levels (2125.3 ± 845.4 ng/mL). The mean age of HCV patients with HCC was 51.8 ± 6.8 years compared to 49.8 ± 7.4 years for the HCV patients without HCC. Forty healthy subjects (24 males and 16 females) with negative anti-HCV and negligible level of alpha-fetoprotein(5.9 ± 2.0 ng/mL) with mean age 52.6 ± 8.1 years were also included in the study and served as control group. The study protocol was approved by the Research Ethics Committee, Faculty of Pharmacy- Cairo University (REC-FOPCU). A written informed consent was obtained from each participant before testing. 3. Methods

rs34911341) and Leu72Met (408C/A rs696217)) in exon 2 of the coding region of the preproghrelin gene. Amplification of the target DNA in the promoter region was carried out using PCR with forward primer: 5′-CACAGCAACAAAGCTGCACC-3′, and reverse primer: 5′-AAGTCCAGCCAGAGCATGCC-3′, the 929 bp fragment was amplified using 135 ng DNA, 1 μmol/L of each primer, 25 μL master mix (Qiagen-USA), the volume was completed to 50 μL with nuclease-free water. PCR was performed by initial denaturation step at 94 °C for 5 min, followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The product was incubated at 37 °C for 2 h with 10 IU of Dra-I restriction enzyme, then detected by electrophoresis in a 1.5% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. The G allele was digested into 664 and 265 bp fragments, while the A allele resisted digestion giving the same size band (929 bp) (Yang et al., 2012). The two polymorphisms in exon 2 of the ghrelin gene were defined using PCR with forward primer: 5′-GCTGGGCTCCTACCTGAGC-3′, and reverse primer: 5′-GGACCCTGTTCACTGCCAC-3′. The 618 bp fragment was amplified using 300 ng DNA, 0.24 μmol/L of each primer, 25 μL master mix, the volume was completed to 50 μL with nuclease-free water. PCR was performed by initial denaturation at 95 °C for 3 min, annealing at 60 °C for 1 min, extension at 72 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 75 s, followed by final extension at 72 °C for 10 min. The product was divided into two parts: the first was incubated at 37 °C for 3 h with 5 IU of Sac-I restriction enzyme for detection of Arg51Gln polymorphism, the other part was incubated at 65 °C for 3 h with 5 IU of Bsr-I restriction enzyme for Leu72Met polymorphism detection, then products were detected by electrophoresis in a 1.5% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. The Arg51Gln G allele was digested into 455 and 163 bp fragments, while the A allele resisted digestion giving 618 bp fragment. On the other hand, the Leu72Met C allele was digested into 517 and 101 bp fragments and the A allele resisted digestion giving 618 bp fragment (Ukkola et al., 2002).

3.1. Blood sampling 3.4. Detection of HFE gene polymorphisms Venous blood was obtained from patients and controls after an overnight fast of 12 h and was divided into three portions. One portion of blood (2 mL) was added to EDTA and stored at −20 °C for detection of polymorphism of ghrelin and HFE genes. A second portion of 2 mL blood was left for 10 min to clot and then centrifuged at 1000 ×g for 5 min. The serum was then separated and stored at −20 °C for determination of ALT, AST, ferritin, and hepcidin levels. The last blood portion (2 mL) was added to EDTA and centrifuged at 1000 ×g for 10 min. The plasma was then separated and stored at −20 °C for determination of fasting plasma ghrelin. Serum hepcidin and ferritin concentrations and fasting plasma ghrelin level were assessed using enzyme-linked immunosorbent assay (ELISA) kits provided from DRG-USA according to the manufacturer's protocol. Serum ALT and AST activities were measured using commercially available kits following the manufacturer's instructions. 3.2. DNA extraction Genomic DNA was isolated from peripheral WBCs using Qia-amplification extraction kit (Qiagen-USA) according to the manufacturer's instructions. 3.3. Detection of ghrelin gene polymorphisms Three common SNPs were selected for ghrelin: one SNP (−604G/A rs27647) in the promoter region, and two SNPs (Arg51Gln (346G/A

Two sites of gene polymorphism were studied: C282Y (845G/A rs1800562) and H63D (187C/G rs1799945). The sequence of primers used for detection of C282Y polymorphism was: forward primer: 5′-TGGCAAGGGTAAACAGATCC-3′, and reverse primer: 5′-CTCAGGCACTCCTCTCAACC-3′. The 400 bp fragment was amplified using 200 ng DNA, 0.6 μmol/L of each primer, 25 μL master mix, the volume was completed to 50 μL with nuclease-free water. The sequence of primers used for detection of H63D polymorphism was: forward primer: 5′ ACATGGTTAAGGCCTGTTGC-3′, and reverse primer: 5′ GCCACATCTGGCTTGAAATT-3′. The 208 bp fragment was amplified using 800 ng DNA, 0.6 μmol/L of each primer, 25 μL master mix, the volume was completed to 50 μL with nuclease-free water. The same PCR conditions were conducted for both amplifications: initial denaturation at 96 °C for 2 min, followed by 35 cycles of denaturation at 96 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min. The 400 bp product used for detection of C282Y polymorphism was incubated at 37 °C for 2 h with 10 IU of Snab-I restriction enzyme, while the 208 bp product used for detection of H63D polymorphism was incubated at 50 °C for 2 h with 10 IU of Bcl-I restriction enzyme. Each product was detected by electrophoresis in a 4% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. The C282Y G allele yielded one 400 bp fragment, while the A allele was not detected. On the other hand, the H63D C allele was digested into 138 and 70 bp fragments and the G allele resisted digestion giving 208 bp fragment (Bittencourt et al., 2002).

Please cite this article as: Motawi, T.K., et al., Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.053

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Table 1 Demographic and clinical characteristics of all subjects.

Sex (M/F) Age ALT(IU/L) AST(IU/L) Ghrelin (ng/mL) Hepcidin (ng/mL) Ferritin (ng/mL)

Control (n = 40)

HCV (n = 79)

P

HCV without HCC (n = 40)

HCV with HCC (n = 39)

P

24/16 52.6 ± 29.5 ± 28.1 ± 3.37 ± 71.5 ± 94.6 ±

55/24 50.8 ± 7.1 118.4 ± 55.2 140.2 ± 66.2 6.5 ± 1.1 30.5 ± 12.9 490.8 ± 246.1

0.3 0.2 b0.001 b0.001 b0.001 b0.001 b0.001

26/14 49.8 ± 7.4 91.2 ± 55.2 114.1 ± 75.6 6.01 ± 0.8 36.7 ± 13.1 278.9 ± 71.5

29/10 51.8 ± 6.8 146.3 ± 39.5 166.9 ± 40.9 6.987 ± 1.07 24.2 ± 9.2 708.0 ± 153.1

0.5 0.1 b0.001 b0.001 b0.001 b0.001 b0.001

8.1 5.5 6.6 0.76 25.0 48.4

HCV = Hepatitis C virus, HCC = Hepatocellular carcinoma. P value b 0.05 was considered significant.

4. Statistical analysis Data are expressed as means ± SD for quantitative variables, frequency for qualitative variables. Quantitative variables were compared using independent Student t-test. On the other hand, qualitative variables were compared using chi square (χ2) test or Fischer's exact test. The Statistical Package for the Social Sciences software (SPSS 17.0, Chicago, IL, USA) was used. P b 0.05 was considered significant. 5. Results The demographic and clinical characteristics of the HCV patients with or without HCC and control subjects are shown in Table 1. There were no significant differences among studied groups regarding age and sex distribution. HCV patients had significantly higher ghrelin (P b 0.001) and ferritin levels (P b 0.001) together with higher ALT (P b 0.001) and AST activities (P b 0.001), and had significantly lower hepcidin levels (P b 0.001) compared to controls. Moreover, compared with the HCV without HCC group, the HCV with HCC group had significantly higher ghrelin (P b 0.001) and ferritin levels (P b 0.001) together with higher ALT (P b 0.001) and AST activities (P b 0.001), and had significantly lower hepcidin levels (P b 0.001). The three SNPs under investigation on the ghrelin gene were found in all studied groups. The distribution of different genotypes and allele frequencies among cases and controls is summarized in Table 2. Only one SNP Arg51Gln (346G/A, rs34911341) showed significantly higher GA, AA genotype distributions and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC

Table 2 Distribution of the genotypes and frequencies of alleles of -604G/A, Arg51Gln, and Leu72Met polymorphisms in the ghrelin gene. Control HCV without HCC χ2 −604G/A GG 22 GA 9 AA 9 G allele 53 A allele 27

19 9 12 47 33

0.7

346G/A GG GA AA G allele A allele

32 7 1 71 9

1.5

408C/A CC CA AA C allele A allele

35 5 0 75 5

31 7 2 69 11

28 10 2 66 14

P

HCV with HCC χ2

P

0.7*

17 10 12 44 34

0.2

0.9**

21 12 6 0.4 * 54 24

7.2

0.03**

9.1

0.003 **

0.96 0.4*

1.3

0.7

0.4

0.5*

0.7*

26 9 4 0.7 * 61 17

0.09 0.9 **

0.8

0.7**

0.5

0.6 **

HCV = Hepatitis C Virus, HCC = Hepatocellular carcinoma, *P values for comparison between controls & HCV without HCC, **P values for comparison between HCV without HCC & HCV with HCC. P value b 0.05 was considered significant.

(P = 0.03, P = 0.003, respectively). However, the genotype distributions and allele frequencies of −604G/A (P = 0.9, P = 0.9 respectively) and that of Leu72Met (P = 0.7, P = 0.6, respectively) did not significantly differ among the studied groups. On the other hand, HFE gene analysis showed that the C282Y SNP was not detected among all the studied groups. Only heterozygous H63D was detected. The distribution of different genotypes and allele frequencies among cases and controls is summarized in Table 3. H63D genotype distributions and allele frequencies did not significantly differ among HCV patients with or without HCC (P = 1, P = 1 respectively). Characteristics of patients with different Arg51Gln genotypes on ghrelin gene are shown in Table 4. There was a significant association between the Arg51Gln SNP and elevated ALT and AST activities (P b 0.001, P b 0.001 respectively), but there was no significant association between Arg51Gln SNP and ghrelin level (P = 0.6). 6. Discussion Ghrelin, a gastric mucosa-derived peptide, has various metabolic functions, including regulation of energy balance, control of appetite, stimulation of gastric acid secretion, and regulation of gastrointestinal motility (Van der Lely et al., 2004). Moreover, subsequent in vitro and in vivo data clearly showed the anti-inflammatory effects of ghrelin. Ghrelin has been demonstrated to down-regulate the expressions of pro-inflammatory and anorectic cytokines, including IL-1β, IL-6 and TNF-α, from human T cells and monocytes (Dixit et al., 2004), and to inhibit TNF-α- and H2O2-induced release of chemokines (IL-8 and MCP-1) from human endothelial cells (Li et al., 2004). In animal models, administration of ghrelin attenuated endotoxin-induced cytokine production (Li et al., 2004) and ameliorated biliary obstruction-induced chronic hepatic injury (Iseri et al., 2008), pancreaticobiliary duct ligation-induced systemic inflammation (Kasımay et al., 2006), collagen-induced arthritis (Chorny et al., 2008), and trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis (Gonzalez-Rey et al., 2006). Furthermore, previous clinical studies showed increased ghrelin levels in active inflammatory diseases (Ates et al., 2008; Kümpers et al., 2008; Toussirot et al., 2007). SNPs in the ghrelin gene may potentially cause defects in, or inactivation of the ghrelin protein which may play a role in the pathology of HCV and susceptibility to HCC. Table 3 Distribution of the genotypes and frequencies of alleles of H63D polymorphism in the HFE gene.

CC CG GG C allele G allele

Control

HCV without HCC

χ2

P

HCV with HCC

χ2

P

32 8 0 72 8

30 10 0 70 10

0.3

0.8 *

0.004

1.0**

0.3

0.8 *

29 10 0 68 10

0.004

1.0 **

HCV = Hepatitis C Virus, HCC = Hepatocellular carcinoma, *P values for comparison between controls & HCV without HCC, **P values for comparison between HCV without HCC & HCV with HCC. P value b 0.05 was considered significant.

Please cite this article as: Motawi, T.K., et al., Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.053

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Table 4 Characteristics of patients with different Arg51Gln genotypes on ghrelin gene.

Sex (M/F) Age Ghrelin (ng/mL) ALT(IU/L) AST(IU/L)

GG (n = 53)

GA + AA (n = 26)

P

37/16 49.8 ± 6.8 6.44 ± 1.2 92.5 ± 34.2 118.6 ± 59.7

18/8 52.8 ± 7.5 6.6 ± 0.8 171.23 ± 52.6 184.1 ± 57.01

0.9 0.07 0.6 b0.001 b0.001

P value b 0.05 was considered significant.

Studies on the functional significance of Leu72Met polymorphism generated controversial results. Ukkola and Kesäniemi (2003) reported that the Met72 allele was protective against fat accumulation and associated metabolic co-morbidities, on the other hand, Vivenza et al. (2004) and Steinle et al. (2005) demonstrated a significant association between Leu72Met polymorphism and several metabolic disorders. In the present study, genotype distributions and allele frequencies of Leu72Met SNP were not significantly different among the studied groups. These results may suggest that Met72 allele may not be a susceptibility factor for HCC in HCV patients. The current results were highly supported by the recent work of Liu et al. (2012) and Dossus et al. (2008) who showed that Leu72Met polymorphism is not associated with clinical abnormalities. Regarding −604G/A polymorphism, Moreno et al. (2010) reported that chronic HCV patients with the − 604A haplotype are more susceptible to severe liver fibrosis. On the other hand, Campa et al. (2010) found that carriers of − 604G/A polymorphism had a decreased risk of colorectal cancer. However, our results failed to show any detectable changes in genotype distributions and allele frequencies in − 604G/A SNP among the studied groups, suggesting that − 604G/A SNP is not associated with susceptibility to HCC in HCV patients. In the current study, the frequency of Arg51Gln polymorphism of ghrelin gene and the distribution of A allele were significantly higher in hepatitis C patients who developed HCC than those in hepatitis C patients without HCC and controls. There was also a significant association between the Arg51Gln SNP and elevated ALT and AST levels. These results suggest that Arg51Gln polymorphism may reduce ghrelin biological activity including its anti-inflammatory properties, and hence might be a susceptibility factor for HCC in HCV patients. The Arg51Gln polymorphism results in a change in the COOH-terminal processing site of the ghrelin peptide within its precursor protein from Pro-Arg to Pro-Gln, resulting in the failure of the normal cleavage necessary to produce the 28-amino acid ghrelin. A 94-amino-acid long pro-ghrelin peptide is produced, which might be biologically inactive (Ukkola et al., 2001). These results are in harmony with those of Pöykkö et al. (2003a, 2003b) who reported that Arg51Gln polymorphism is associated with obesity and metabolic syndrome, and Hubacek et al. (2007) who observed a significant association between Arg51Gln polymorphism and elevated cholesterol level. However, there was no significant association between Arg51Gln SNP and ghrelin level in HCV patients. This may be attributed to the fact that Arg51Gln SNP results in ghrelin inactivation rather than affecting its transcription. These results are in accordance with those noted by Vivenza et al. (2004). The results of Ukkola et al. (2002) and Pöykkö et al. (2003a, 2003b), in contrary to the current results, showed that Arg51Gln SNP caused a significant decrease in ghrelin level. In accordance with previous studies done by Elbadri et al. (2011) and Tacke et al. (2003), we found that plasma ghrelin level was significantly elevated in HCV patients than that in the control group; the change in ghrelin level may be relevant to the anorexia and metabolic disturbances due to cirrhosis and hepatic dysfunction. HFE gene encodes an atypical major histocompatibility complex class I protein (MHC1), the main role of HFE protein is to maintain

iron homeostasis through the following mechanism: HFE binds to transferrin receptor 1(TFR1) during low or basal serum iron levels. Serum Fe2-TF competes with HFE for binding to TFR1. Increased serum transferrin saturation results in the dissociation of HFE from TFR1. HFE, acting as an iron sensor, then binds transferrin receptor 2 (TFR2) thus generating a signal transduction effector complex. Downstream induction of hepcidin production contributes to whole-body iron homeostasis (Goswami and Andrews, 2006). Hepcidin is a peptide hormone produced predominantly by the liver. Its major role is binding to and triggering the internalization and degradation of the iron exporter, ferroportin (Nemeth et al., 2004). Thus, negatively regulating iron efflux from enterocytes, liver macrophages, and hepatocytes into the blood. Mutation of HFE prevents formation of a functional iron sensor and signal transduction effector complex leading to decreased hepcidin production and increased intestinal iron absorption and liver iron overload as evidenced in human hereditary hemochromatosis (HH type 1) (Vujic Spasic et al., 2008). Hereditary hemochromatosis is characterized by excessive intestinal iron absorption from the diet, leading to accumulation of iron and eventually to the saturation of transferrin and the deposition of iron in vital organs. Free iron is toxic, probably due to its ability to catalyze the production of reactive oxygen species (ROS). If untreated, hemochromatosis may progress to hepatic cirrhosis and hepatocellular carcinoma, cardiomyopathy, destruction of endocrine glands and damage to joints (Ajioka and Kushner, 2002). Studies in the middle east for HFE gene C282Y and H63D allele frequencies showed the presence of C282Y mutation in Tunisians and north African Israelis, but in a very low frequency (0.17% and 1.02% respectively), and the absence of C282Y mutation in Algerians and Jordanians, supporting the Celtic origin of the disease, but H63D mutation was found in about 9% of the chromosomes genotyped among Algerians, 15.7% among Tunisians and 12.5% among oriental Jews (Alkhateeb et al., 2009; Reish et al., 2010; Roth et al., 1997; Sassi et al., 2004). In agreement with these results, our study revealed the absence C282Y mutation among our cases and controls while H63D mutation has been detected in the heterozygous state among 23.3 % of all participants. However, our study revealed that there was no significant difference in H63D mutation among studied groups. These results may suggest that heterozygous H63D mutation is not associated with HCC in HCV patients. Our results are in accordance with those of Jin et al. (2010), Settin et al. (2006) and Valenti et al. (2010). However, Gharib et al. (2011) suggested that carriers of the D allele of H63D mutation were significantly more likely to develop HCC, and Geier et al. (2004) reported that heterozygous H63D mutation might be a risk factor for hepatic inflammation and fibrosis in chronic hepatitis C. The present study showed that the mean serum level of hepcidin was significantly lower in HCV patients than in control group, this might be attributed to HCV protein-induced ROS which increases histone deacetylase activity with subsequent inhibition of the binding of the transcription factor CCAAT/enhancer-binding protein α (C/EBP-α) and phospho-STAT3 to hepcidin promoter, leading to decreased hepcidin transcription (Miura et al., 2008). This finding was in agreement with Girelli et al. (2009) who reported a significantly lower hepcidin level in HCV patients. Hepcidin level was further reduced in HCV patients who developed HCC, as in advanced stages, hepcidin may be further decreased by impaired protein synthesis due to markedly reduced functional hepatic mass, contributing to additional iron overload (Ludwig et al., 1997). In conclusion, our results suggested that Arg51Gln carriage in ghrelin is associated with increased risk of hepatocellular carcinoma in Egyptian HCV patients. However, association of -604G/A, Leu72Met in ghrelin or HFE polymorphisms with incidence of HCC was not demonstrated. Further studies are still needed to replicate the results in larger populations and to investigate whether the reported polymorphism affect either acylated or des-acylated ghrelin where only the total ghrelin level was assessed.

Please cite this article as: Motawi, T.K., et al., Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.053

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Please cite this article as: Motawi, T.K., et al., Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.053