Biochem Genet DOI 10.1007/s10528-014-9655-8

Association Between IL-10 Gene Promoter Polymorphism and Hepatitis B Viral Infection in an Egyptian Population Roba M. Talaat • Mahmoud F. Dondeti • Soha Z. El-Shenawy • Omaima A. Khamiss

Received: 19 May 2013 / Accepted: 22 December 2013 Ó Springer Science+Business Media New York 2014

Abstract Cytokines play critical roles in the pathogenesis of hepatitis B virus infection (HBV). This work was designed to study the effect of IL-10 gene polymorphisms (-1082G/A and -819C/T) on susceptibility of Egyptians to HBV. Genotyping was performed using single-stranded polymorphism-polymerase chain reaction in 118 Egyptian hepatitis B patients and 119 healthy controls, and IL-10 serum levels were measured using ELISA. The frequency of IL-10 -1082G/G was significantly higher in HBV patients than in healthy controls, and G/A and A/A were not significantly different between groups. The distribution of IL-10 -819 genotypes was not significantly different between the HBV and healthy control groups. Although AT was significantly different between controls and patients, the distribution of the other haplotypes was not. IL-10 levels were significantly lower among hepatitis B patients. Our data stress the importance of IL-10 gene polymorphism in HBV infection. Depending on our preliminary work, IL-10 -1082G/G may act as a host genetic factor in the susceptibility to HBV infection in Egyptians. Keywords

IL-10  Hepatitis B  Egyptians

R. M. Talaat (&)  M. F. Dondeti Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt e-mail: [email protected] S. Z. El-Shenawy Biochemistry Department, National Liver Institute, Menofia University, Menufia, Egypt O. A. Khamiss Animal Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Sadat City, Egypt

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Introduction Despite the availability of a highly effective vaccine, hepatitis B virus (HBV) infection remains a global dilemma, and it is still a significant etiology of chronic HBV, cirrhosis, and hepatocellular carcinoma, especially in Asian and African countries (Dienstag 2008; Sonneveld et al. 2010). More than 350 million people, representing 5% of the world population, are affected by HBV infection, with more than 1.2 million deaths per year (Liang et al. 2009; Kew 2010). HBV infection may persist and progress to chronic hepatitis, fibrosis, and hepatocellular carcinoma (Fattovich 2003). The risk of HBV persistence is mainly related to two major factors, the age at which infection is contracted and the immune status of the host, as chronic hepatitis B occurs in less than 5% of adults and more than 90% of infants (Vildo´zola Gonzales and Salinas 2009). There is growing evidence of the effect of host genetic factors on the natural history of chronic liver diseases (Bataller et al. 2003). The majority of host genetic studies with HBV infection have been conducted on human leukocyte antigen (Thursz et al. 1995; Ho¨hler et al. 1997). A broad spectrum of possible clinical outcomes in chronic hepatitis (Fattovich 2003; Ganem and Prince 2004) is significantly determined by host genetic factors (Ho¨hler et al. 1998; Thursz 2001b; Yeh et al. 2002; Liu et al. 2004). Cytokines are a large family of proteins that mediate many innate and adaptive immune responses. IL-10, produced by Th2 cells and macrophages, is one of the critical modulators of the immune response through its suppressive effect on Th1 cells (Spits and Malefyt 1992; Moore et al. 1993, 2001) by suppression of macrophage-dependent antigen presentation, T cell proliferation, and downregulation of Th1 cytokines such as IFN-c, IL-2, and TNF-a (Fiorentino et al. 1989, 1991a; Pestka et al. 2004; Redpath et al. 2001; Wu et al. 2010), besides its antifibrogenic properties (Tsukamoto 1998). IL-10 has also been reported to play a vital role in the regulation of immune responses in HBV infection (Payvandi et al. 1998; Powell et al. 2000). There is a strong belief that the capacity for cytokine production has a major genetic component ascribed to polymorphisms within the regulatory regions or signal sequences of cytokines, leading to differential cytokine production (Mosmann 1994; Westendorp et al. 1997; Bidwell et al. 1999; Dorman and Holland 2000). The IL-10 gene is located on chromosome 1q31–32 with a total size of about 10 Kb. Its expression is regulated at the transcriptional, post-transcriptional, and translational levels (Hurme et al. 1998). It has been reported that 50% of the observed difference in IL-10 production capacity among individuals may be attributed to genetic factors (Eskdale et al. 1998; Reuss et al. 2002; Sua´rez et al. 2003). Several polymorphic sites within the proximal IL-10 promoter region have been described, including the most critical biallelic single nucleotide polymorphisms (SNPs) at positions -1082G/A (rs1800896), -819C/T (rs1800871), and -592C/A (rs1800872) (Turner et al. 1997; Crawley et al. 1999; Edwards-Smith et al. 1999). The T and C alleles at IL-10-819 were reported to be in complete linkage disequilibrium with the A and C alleles at IL-10-592, respectively (Eskdale et al. 1999). In addition, the IL-10-592A allele was reported to be associated with the IL-10-1082A allele (Tountas and Cominelli 1996; Turner et al. 1997; Eskdale

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et al. 1998; Kube et al. 2001). Several disease association studies have reported the weighty importance of the IL-10 promoter in the susceptibility to infectious diseases (Bidwell et al. 1999; Haukim et al. 2002; Hollegaard and Bidwell 2006). Furthermore, the IL-10 promoter has been reported to be associated with response to hepatitis B surface antigen (HBsAg) vaccine (Ho¨hler et al. 2005), HBeAg seroconversion (Peng et al. 2006), and HBV-related hepatocellular carcinoma (Shin et al. 2003; Tseng et al. 2006). Thus, it is critical to investigate whether the polymorphism in the IL-10 promoter region is associated with HBV infection. In Egypt, no study has been conducted to investigate the association between functional SNPs in the IL-10 gene promoter and HBV infection. Thus, this work was designed to study the association between the polymorphisms at the two biallelic sites -1082 and -819 of the IL-10 gene promoter with HBV infection in an Egyptian population and to determine whether the genetic variation at these sites will have a considerable effect on IL-10 production levels.

Materials and Methods Patients and Controls The study enrolled 118 patients with HBV infection from the National Liver Institute, Menofia University, Egypt. The males outnumbered the females (98 men and 20 women), with a mean age of 34.4 ± 10.69 years (range 68–22 years). The study included 119 healthy controls with no history of previous liver disease, normal liver function tests, and negative HBV and HCV serology. Patients with HCV or other viral infections or any liver diseases were excluded from the study. All investigations were performed in accordance with the university’s Health and Human Ethical Clearance Committee guidelines for clinical research. The local ethics committee approved the study protocol, and informed consent was obtained from all subjects. Viral Assessment HBsAg was tested using commercially available kits (Sorin Biomedica, Milan, Italy), and confirmation of the presence of HBV-DNA in HBV-positive patients was tested by a standard polymerase reaction (Roche Diagnostics, Indianapolis, IN). HCV antibodies were tested using enzyme-linked immunosorbent assay (ELISA; Murex Biotech, Dartford, UK). All patients were positive for HBsAg and HBVDNA and negative for HCV antibodies. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) (BioMe´rieux SA, Marcy l’Etoile, France), direct and indirect bilirubin (Roche Diagnostics), and albumin (Human Gesellschaft fur Biochemica und Diagnostica, Wiesbaden, Germany) were all measured according to the manufacturer’s instructions for their respective kits.

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DNA Isolation Blood was collected by withdrawal of 5 ml venous blood from each individual, into sterile vacutainer tubes containing EDTA.K3 (Tri-potassium ethylene diamine tetra acetic acid). The tubes were centrifuged at 1,500 rpm for 10 min. Plasma was separated, aliquoted, and stored at -80°C for cytokine secretion analysis. Genomic DNA was extracted from whole blood-EDTA samples using a Wizard Genomic DNA Purification Kit (Promega, Madison, USA), according to the manufacturer’s instructions. Genotyping IL-10 -1082G/A and -819C/T SNPs were genotyped by single-stranded polymorphism-polymerase chain reaction (Welsh and Bunce 1999) using a fourprimer mix (Table 1). The PCR was performed in two tubes, with each tube containing a forward primer specific to one allele in addition to a generic primer and internal control primers (forward and reverse). The final total volume for each reaction was 25 ll. Reaction components were 29 DreamTaq Green Master Mix (Fermentas, Thermo Fisher Scientific), 10 pmol each primer (Metabion, Martinsried, Germany), and 0.1 lg DNA. The PCR cycling was one cycle of 94°C for 2 min; followed by 5 cycles of 96°C for 25 s, 70°C for 45 s, and 72°C for 20 s; 11 cycles of 96°C for 25 s, 65°C for 50 s, and 72°C for 45 s; and a final 15 cycles of 96°C for 25 s, 55°C for 60 s, and 72°C for 2 min. The PCR was performed in a Biometra thermal cycler (Biometra, Germany). The PCR products were visualized on 2% agarose gel and estimated in comparison with a 100 bp DNA ladder (Fermentas, Thermo Fisher Scientific). The size of the PCR product for the IL-10 -1082G/A primers was 258 bp for the A or G allele, 233 bp for the IL-10 -819C/T primers, and 429 bp for the control primers (Fig. 1).

Table 1 Primer sequences for amplification of IL-10 SNPs at positions -1082 and -819 IL-10 SNP position

Primera

Sequence

PCR product size (bp)

-1082A/G

G (sense)

50 -CTACTAAGGCTTCTTTGGGAG-30

258

A (sense)

50 -ACTACTAAGGCTTCTTTGGGAA-30

Generic (antisense)

50 -CAGTGCCAACTGAGAATTTGG-30

C (sense)

50 -CCCTTGTACAGGTGATGTAAC-30

T (sense)

50 -ACCCTTGTACAGGTGATGTAAT-30

Generic (antisense)

50 -AGGATGTGTTCCAGGCTCCT-30

Internal control (sense)

50 -GCCTTCCCAACCATTCECTTA-30

Internal control (antisense)

50 -TCACGGATTTCTGTTGTGTTTC-30

-819T/C

Control a

Adapted from Perrey et al. (1998)

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233

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Fig. 1 PCR products of primers for IL-10 SNPs at positions -1082G/A (top) and -819C/T (bottom). Top Genotypes of five IL-10 -1082A/G samples: Samples 1 and 3 (lanes 2, 3, 6, and 7) GG, samples 2 and 4 (lanes 4, 5, 8, and 9) GA, sample 5 (lanes 9 and 10) AA. Lane 1 100 bp ladder. Bottom Genotypes of four IL-10 -819T/C samples: Samples 1 and 3 (lanes 4, 5, 8, and 9) TT, sample 2 (lanes 6 and 7) CC, sample 4 (lanes 10 and 11) CT. Lane 12 100 bp ladder

Measurement of Plasma IL-10 Total concentrations of IL-10 plasma levels were measured in HBV patients and normal controls by a sandwich enzyme-linked immunosorbent assay (ELISA; R&D System, Minneapolis, USA), according to the manufacturer’s instructions. The ELISA reader-controlling software (Softmax) readily processes the digital data of raw absorbance value into a standard curve from which cytokine concentrations of unknown samples can be derived directly and expressed as pg/ml. Statistical Analysis Statistical analyses were performed using the SPSS statistical package version 19 (SPSS, IBM, USA). Comparisons among patients and control groups were performed by independent t test, and results were presented as mean ± SD. The gender data and the frequencies of alleles and genotypes of the studied SNPs in both control and patient groups were estimated by testing descriptive statistics through crosstabs. Chi-square tests were performed to examine the differences in gender, allele frequency, and genotype distribution between groups. One-way ANOVA

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Biochem Genet Table 2 Demographic and biochemical characteristics of HBV patients and healthy controls Parameter

Control group (N = 119)

HBV group (N = 115)

p

Age (years)

33.42 ± 17.23

42.66 ± 12.59

\0.001

Correlation with disease r = 0.286 p \ 0.001

Gender (male/female)

39/80

95/20

\0.001

r = 0.509 p \ 0.001

42.01 ± 23.50

\0.001

Aspartate aminotransferase (IU/l)

20.97 ± 5.86

r = 0.560

Alanine aminotransferase (IU/l)

18.91 ± 5.05

44.13 ± 34.81

\0.001

r = 0.493

Albumin (g/l)

4.26 ± 0.39

3.43 ± 0.76

\0.001

r = -0.589

Total bilirubin (mg/dl)

0.67 ± 0.19

1.09 ± 0.60

\0.001

r = 0.463

Direct bilirubin (mg/dl)

0.11 ± 0.07

0.31 ± 0.013

\0.001

r = 0.287

Creatinine (mg/dl)

0.89 ± 0.15

1.09 ± 0.33

\0.001

r = 0.379

Urea (mg/dl)

28.93 ± 7.04

33.41 ± 13.52

\0.01

r = 0.214

p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.01

All data except gender presented as mean ± SD; gender data were assessed from crosstab by chi-square test

through a Tukey test was used to compare genotypes/haplotypes and ELISA data. Correlation between variables was determined using Sperman’s correlation test. The distribution of genotypes was examined to fit Hardy–Weinberg equilibrium by DeFinetti software (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl), and it was assessed by the chi-square test with one degree of freedom. The online tool SNPstats (http://bioinfo. iconcologia.net/SNPstats; Sole´ et al. 2006) performed the haplotype analyses and calculated the LD parameters (D0 and r2). The odds ratio (OR), which reflects the likelihood of carrying a specific allele if there is a persistent HBV infection, the 95% confidence interval, and their p-values were also calculated. All p-values were twotailed, and p values less than 0.05 were considered to be statistically significant.

Results Biochemical Assessment of the Controls and the HBV Patients All subjects were examined for biochemical and viral parameters (Table 2). The HBV patients were positive for HBsAg and HBV-DNA (1,641,329.1 ± 7,786,867.1 IU/l). On the other hand, both groups were negative for HCV antibody and HCV-RNA. Levels of liver enzymes showed significant increase in the patient

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group (p \ 0.001 for ALT and p \ 0.001 for AST). A significant reduction in the level of albumin (p \ 0.001) was demonstrated. Levels of both urea (p \ 0.005) and creatinine (p \ 0.001) were significantly elevated with the disease. Furthermore, levels of total bilirubin were significantly increased in HBV patients (p \ 0.001). Association Between IL-10 Gene Promoter Polymorphisms and HBV Infection The genotype frequencies of both IL-10 alleles (-1082 and -819) were within Hardy–Weinberg equilibrium, since testing of the deviation of their genotypes from the equilibrium shows no significant change (Table 3). Genotyping of IL-10 (-1082) showed a significant increase (p \ 0.001) in the distribution of GG genotype in the HBV patients (12.6% for controls vs. 26.1% for HBV). The frequencies of GA and AA, however, were not significantly changed in the controls and HBV patients. Furthermore, allele A was significantly greater (p \ 0.05) than allele G in both groups. Allele A was more frequent in controls (61.8%) than in the HBV group (49.6%), and the G allele was more frequent in the HBV group (47.9%) than in controls (38.2%). Both the GG genotype (OR 2.4) and the G allele (OR 1.5) could be considered risk factors for HBV. Genotyping of IL-10 (-819) showed that the CC genotype is more frequent in both the control and the patient groups. In contrast, the TT genotype is hardly detected in both groups (*4.7%). A reduction in frequency of the T allele coincides with the elevation in C allele frequency and was detected in both groups. However, an insignificant change in the distribution of all genotypes between control and patients was demonstrated. Four haplotypes emerged (GC, GT, AC, and AT) from the estimates of haplotype frequencies. Both GC and AC were the most frequent haplotypes in both groups, and GT was the least frequent, although the GT haplotype might be a risk factor for HBV infection (OR 2.4). The frequency of the AT haplotype showed a significant increase (p \ 0.05) in the controls over the HBV patients. The haplotype estimates (D0 0.69, r2 -0.33; p \ 0.001) indicate the presence of a level of LD between IL-10 -1082G/A and IL-10 -819C/T in the Egyptian population. Correlation Between IL-10 Gene Promoter Polymorphisms and IL-10 Plasma Levels Mean plasma concentrations were significantly lower (p \ 0.001) in the HBV patients (80.77 ± 226.1) than in the controls (5,053.33 ± 279.65 pg/ml) (Fig. 2). Hepatitis B was significantly correlated with reduction in plasma IL-10 level (r = -0.755; p \ 0.001). The highest IL-10 level in controls coincided with the presence of the GG genotype (5,498.67 ± 1,030.97), and the lowest level coincided with the GA genotype (4,966.33 ± 320.02) (Table 4). In the HBV group, the highest level of IL10 coincided with the presence of the GA genotype (98.71 ± 38.12) and the lowest with the AA genotype (41.75 ± 16.79). Additionally, the highest level of IL-10 coincided with the TT genotype of IL-10 -819 (189.33 ± 161.89), and the lowest

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Haplotype

-819C/T

12.6

36.7 37.3 1.55 24.5

AC

GT

AT

25.0

67.3

47.9

4.2

43.7

52.1

61.8

38.2

87.4

36.1

51.3

GC

62

T

57

CT/TT 176

5

TT

C

52

CT

147 62

CC

A

104

GA/AA 91

43

AA

G

15 61

52

178

46

6

40

69

117

113

85

32

53

30

Number

Number

%

HBV group

Control group

GG

-1082G/A

Allele

GA

Genotype

IL-10 SNP position

Table 3 Genotype frequencies of IL-10 SNPs (-1082G/A and -819C/T)

17.48

5.23

33.5

43.8

25.2

72.4

40.0

5.2

34.8

60.0

50.9

49.1

73.9

27.8

46.1

26.1

%

0.6410 (0.4438–0.9258)

\0.001

2.40 (0.51–11.34) 0.58 (0.35–0.99)

\0.05

0.74 (0.48–1.15)

1.00

0.8293 (0.5430–1.2664)

1.2059 (0.7896–1.8415)

0.7251 (0.4319–1.2175)

1.2550 (0.3722–4.2321)

0.6872 (0.4054–1.1648)

NS

NS

NS

NS

NS

NS

NS

NS

1.3790 (0.8213–2.3154)

\0.05 NS

0.4087 (0.2064–0.8089) 1.5602 (1.0802–2.2534)

\0.01

0.6814 (0.3918–1.1851)

0.8128 (0.4864–1.3583)

2.4471 (1.2362–4.844)

OR (95% CI)

NS

NS

\0.001

p

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Biochem Genet

Fig. 2 Comparison of mean plasma concentrations of IL-10 in hepatitis B patients and healthy controls. Lines inside boxes represent medians, boxes represent the 25th and 75th percentiles, and lines outside the boxes represent the 10th and 90th percentiles. Mean plasma concentrations were significantly lower (p \ 0.001) in the HBV patients (80.77 ± 226.1) than the controls (5,053.33 ± 279.65 pg/ml)

level coincided with the CT genotype (51.17 ± 15.37) in HBV patients. In the controls, however, the highest level coincided with the CT genotype (5,187.02 ± 397.22) and the lowest with the TT genotype (3,242 ± 862.74). Therefore, the reduction in IL-10 secretion level observed in HBV patients compared with normal controls was irrelevant to IL-10 genotypes, either in IL-10 -1082G/A or -819C/T. Furthermore, comparing IL-10 levels with different IL-10 haplotypes showed that the appearance of the GC haplotype was congruent with the highest level of IL-10 in both groups (5,343.12 ± 1,175.44 vs. 63.12 ± 35.36 for controls and HBV patients, respectively), and the lowest level of IL-10 coincided with the AT haplotype (3,242 ± 862.74 vs. 19.33 ± 2.67 for controls and HBV patients, respectively).

Discussion Many studies have reported genetic polymorphisms that can influence susceptibility to persistent HBV infection (Thursz et al. 1995; Ho¨hler et al. 1997; Ahn et al. 2000; Ben-Ari et al. 2003). Genetic association studies may provide some keys to clear the ambiguity of the infection’s persistence and progression in chronic viral hepatitis, and they might provide novel therapeutic approaches (Miyazoe et al. 2002). Many genetic association studies have been conducted to study the major

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Biochem Genet Table 4 Mean plasma concentrations (pg/ml) of IL-10 in hepatitis B patients and healthy controls, compared by genotype IL-10 SNP position -1082G/A

-819C/T

Haplotype

Genotype

Control group (N = 119)

HBV group (N = 115)

p

GG

5,498.67 ± 1030.97

94.7 ± 43.10

\0.001

GA

4,966.33 ± 320.02

98.71 ± 38.12

\0.001

AA

5,021.39 ± 498.2

41.75 ± 16.79

\0.001

CC

5,087.27 ± 413.74

88.42 ± 31.70

\0.001

CT

5,187.02 ± 397.22

54.17 ± 15.37

\0.001

TT

3,242 ± 862.74

189.33 ± 161.89

\0.001

GC

5,343.08 ± 1,175.44

63.12 ± 35.36

\0.001

23.64 ± 3.64

GT

4,744.56 ± 934.79

AC

5,135.03 ± 510.31

AT

3,242 ± 862.74

\0.001

45.7 ± 13.67

\0.001

19 ± 2.67

\0.001

histocompatibility complex (MHC) gene polymorphisms. Therefore, MHC class II alleles DRB1*1302, DRB1*02, and DRB1*04 were found to be associated with HBV clearance, and DRB1*07 was found to be associated with HBV persistence (Almarri and Batchelor 1994; Thio et al. 1999, 2000). Previous studies have reported the inhibitory effect of IFN-c and TNF-a on HBV replication through noncytolytic pathways (Guidotti et al. 1994, 1996; Guidotti and Chisari 2001), but it was found that IL-10 acts against their actions (Fiorentino et al. 1989, 1991; Mosmann 1994). Therefore, IL-10 may compromise the host immune response to acute viral infection (Moore et al. 1993; Cacciarelli et al. 1996; Koziel 1999). Genetic polymorphisms are thought to have a great effect on the production of cytokines, especially SNPs in the promoter region, which are highly critical in the regulation of gene expression (Westendorp et al. 1997a; Van Deventer 2000; Mullikin et al. 2000). Thus, such studies maximized the role of IL-10 gene promoter polymorphisms in the outcome of HBV, and these are likely to be used as biomarkers to determine the disease phenotypes. Therefore, we aim to identify the role of functional IL-10 polymorphism on HBV susceptibility. In the present study, two biallelic polymorphisms in the IL-10 gene promoter were studied (-1082G/A and -819C/T). These polymorphisms have been reported to alter gene expression (Turner et al. 1997; Crawley et al. 1999; Edwards-Smith et al. 1999), and they are believed to be associated with the progression of chronic HBV infection (Miyazoe et al. 2002). According to our findings, the GG genotype was more frequent in HBV patients than in healthy controls; on the other hand, GA and AA genotypes were less frequent in HBV patients. Both genotypes GA/AA were significantly elevated in controls, compared with the HBV group. The genotype GG may be a risk factor for HBV infection, and it might have a role in susceptibility to the infection. In addition, the G allele was significantly elevated in HBV patients, and allele A was elevated in controls. About 97% of controls carry the A allele, which may indicate its protective role. These findings are similar to the study of Truelove et al. (2008) in an American population, where GA and AA were more frequent in controls than in HBV patients

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without showing any statistical significance in the two groups. In contrast to our results, the GG genotype did not show a difference between the two groups in that study. Furthermore, Gao et al. (2009) found that GA was more frequent in controls than in HBV and HCV in a Chinese population, although not significantly, and no difference was observed in the frequency of the GG genotype. They reported that IL-10 -1082A/A was associated with an increased risk, but IL-10 -1082A/G with a reduced risk of persistent HBV infection. The study of Miyazoe et al. (2002) in a Japanese population found that GA and AA genotypes were more frequent in HBV patients (inconsistent with the current results), but the frequency of the GG genotype was greater in the HBV group than in controls (consistent with the current results). In addition, the frequency of the A allele was not different between the groups, which is contradictory to our findings. In another study in a Chinese population, Yan et al. (2009) reported that GA was more frequent in the HBV group than in controls, the AA genotype was more frequent in controls, and GG was not detected at all. Also, the study found that the A allele was more frequent in controls than in HBV groups, which is consistent with our results. A study conducted by Wu et al. (2011) reported that the IL-10 -1082G/ G genotype polymorphism was associated with earlier HBeAg seroconversion and lower HBV viral load, compared with A allele carriers, and the GA genotype did not differ significantly between cases and controls. On the other hand, the study of Wang et al. (2011) in a Chinese Han population reported no statistical difference between HBV patients and controls. In a meta-analysis of an Asian population with HBV or HCV infection, it was reported that the G and A alleles in IL-10 -1082 were not associated with HBV infection in the Asian population (Lu et al. 2010). The allelic frequencies of IL-10 -819C/T did not show any significant differences between the HBV patients and healthy controls. Thus, there is no evidence to demonstrate the association between IL-10 -819C/T and HBV in Egyptians. These findings are consistent with various studies in Chinese Han populations (Yan et al. 2009; Wang et al. 2011, 2012). This is contrary to Miyazoe et al. (2002), who reported an increase in the frequencies in IL-10 -819 genotypes and both T and C alleles in Japanese HBV patients over those in controls. In another Chinese study, Zhang et al. (2006) reported no significant difference among normal controls, recovered HBV individuals, and chronic HBV patients in both IL-10 -1082G/A and IL-10 -819C/T. These controversial findings for both IL-10 -819 and IL-10 -1082 can be attributed to ethnic variations in different populations. The plasma levels of IL-10 were also found to be sharply and significantly reduced in HBV patients, compared with healthy controls, consistent with the previous study that reported the reduction of IL-10 mRNA expression in liver patients (Napoli et al. 1996). In agreement with the current data, a significant decrease of IL-10 in nonresponder HBV-infected patients was previously observed (Velu et al. 2008). In contrast to our results, Bozkaya et al. (2000) and Wu et al. (2010) reported that patients who contracted chronic HBV infection had serum IL-10 levels higher than healthy controls. Furthermore, other studies reported that IL-10 levels did not show differences between patients and healthy controls in other diseases (Cookson et al. 1999). The Th2 response predominates by producing IL-10, which in turn hinders the Th1 response and inhibits the action of IFN-c, leading to

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progression of the infection, besides its role in the control of suppression of fibrogenesis by stellate cells mediated by TGF-b1 (Zhang et al. 2006). It has been reported that there is a concomitant increase of Treg cells (T-cell CD25?), which increase the production of IL-10 and suppress Th1 response stimulated by HBcAg (Kondo et al. 2006). Additionally, it has been reported that a higher IL-10 level is associated with accelerated progression toward chronic hepatitis B (Shin et al. 2003). On the other hand, it was reported that IL-10 may increase viral replication in chronic HBV infection, which enhances the immune response (Miyazoe et al. 2002). Therefore, the current results suggest that IL-10 has a critical role in HBV infection, but that role is still enigmatic due to conflicting results. Previous studies report an increase in IL-10 levels, but other studies report a decrease. IL-10 levels thus may be associated with other factors that need to be investigated further. It has been reported that the GC haplotype is associated with an increase of IL-10 production capacity, in contrast to the AC and AT haplotypes, which are associated with a reduction of IL-10 levels. Therefore, IL-10 haplotypes might have a correlation with IL-10 expression levels, since GC has the highest levels, AC has intermediate levels, and AT has the lowest production levels (Turner et al. 1997; Edwards-Smith et al. 1999). These data agree with our findings that HBV patients who have the GC haplotype have the highest IL-10 levels, and the AT haplotype is associated with the lowest levels of IL-10 in both groups. A study by Peng et al. (2006) reports that intermediate producer haplotype of IL-10 is associated with HBeAg seroconversion, so that patients have the ability to produce anti-HBeAbs. Also, Yan et al. (2009) reported that the AC haplotype was associated with increased susceptibility to acute liver failure. Although our results suggest that there is an association between IL-10 -1082GG and susceptibility to HBV (besides the predominance of the GC haplotype concomitant with higher IL-10 production), our results cannot determine whether that IL-10 promoter polymorphism is associated with the progression of HBV infection. There are conflicting data on the role of IL-10 in the pathogenesis of HBV, as studies have reported that high levels of IL-10 may be associated with progression of the disease or enhancement of the immune response and HBeAg seroconversion. The controversial findings for both IL-10 -819C/T and IL-10 -1082G/A were reported to be due to ethnic variations in different populations (Miyazoe et al. 2002), since in some studies in which European and Asian populations were compared, the frequency of GC relevant to high IL-10 production capacity differed from approximately 50% to under 5% (Turner et al. 1997; Sua´rez et al. 2003). In addition, there are genes that affect HBV susceptibility and progression other than IL-10, besides differences in the study circumstances regarding number of patients and controls. In conclusion, the current findings stress that the IL-10 -1082G/G genotype and IL-10 -1082G allele may be genetic components of susceptibility to HBV infection. In addition, the findings suggest that IL-10 -819C/T does not have an evident role in HBV infection. Plasma IL-10 levels were significantly decreased in HBV patients compared with controls, and their levels are irrelevant to the genotypes of IL-10 (-1082G/A and -819C/T). The GC haplotype, however, may be associated with higher IL-10 levels, and AT may be associated with lower levels.

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Biochem Genet Acknowledgments This work is part of a grant from the Egyptian Academy of Scientific Research and Technology (ASRT), Cairo, Egypt. The sponsors did not participate in the study design, in the collection, analysis, and interpretation of data, in the manuscript drafting, and in the decision to submit the article for publication.

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