Journal of Viral Hepatitis, 2014, 21, 297–304

doi:10.1111/jvh.12139

Tumour necrosis factor-alpha, interleukin-10, interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta S. C. Karatayli,1,* Z. E. Ulger,1,* A. A. Ergul,1,* O. Keskin,2 E. Karatayli,1 R. Albayrak,3 M. Ozkan,3 R. Idilman,2 K. Yalcin,4 H. Bozkaya,2 O. Uzunalimo glu,1,2 C. Yurdaydin1,2 and 1,2 1 2 Hepatology Institute, Ankara University, Ankara, Turkey; Department of Gastroenterology, Medical School, Ankara A. M. Bozdayi University, Ankara, Turkey; 3Department of Biometry Genetics, Faculty of Agriculture, Ankara University, Ankara, Turkey; and 4Department of Gastroenterology, Medical School, Dicle University, Diyarbakir, Turkey Received March 2013; accepted for publication June 2013

SUMMARY. No data exist to assess certain polymorphisms

that have a potential effect on the immune response in patients with chronic hepatitis delta (CHD). The aim of this study was to investigate polymorphisms in 6 polymorphic sites: IL-10 -1082 (rs1800896), IL-10 -627 (rs1800872), IFN-c +874 (rs62559044), TNF-a -308 (rs1800629), vitamin D receptor (VDR) FokI (rs2228570) and VDR TaqI (rs731236). The genotypes of 67 patients with CHD and 119 patients with chronic hepatitis B (CHB) were compared. In addition, 56 individuals with resolved hepatitis B virus (HBV) infection were used as a control group for patients with CHB. Polymorphisms in TNF-a, IL-10, and VDR genes were analysed using polymerase chain reaction/restriction fragment length polymorphism methods. The IFN-c gene polymorphism was detected by allele-specific polymerase chain reaction (PCR). Patients with CDH were more likely to have advanced liver disease compared

INTRODUCTION Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV) which causes acute and chronic liver disease in humans. HDV is able to replicate within cells in the absence of HBV [1] but requires hepatitis B surface antigen as its envelope protein for packaging and release of

Abbreviation: CHB, chronic hepatitis B; CHD, chronic hepatitis delta; HBV, hepatitis B virus; HDV, hepatitis delta virus; LD, linkage disequilibrium; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; SNPs, single nucleotide polymorphisms; VDR, vitamin D receptor. Correspondence: A. Mithat Bozdayi, MD, PhD, Department of Gastroenterology, Hepatology Institute, University of Ankara Medical School, Ankara University, 06110, Cebeci-Ankara, Turkey. E-mail: [email protected] *All three authors contributed equally to the study.

© 2013 John Wiley & Sons Ltd

with patients with CHB (P < 0.0001). IL-10 -1082 and VDR TaqI polymorphisms showed significant differences between patients with CHD and CHB. The high secretory IL-10 -1082 genotype GG was less frequent in CHD compared with patients with CHB and resolved HBV (17.7%, 37.4% and 47.1%, respectively (P < 0.05 for CHD vs CHB and resolved HBV). The frequency of the high secretory VDR TaqI TT genotype was 86.6% in patients with CHD, 62.7% in patients with CHB and 62.5% in resolved HBV individuals (CHD vs CHB: P < 0.05). None of the polymorphisms analysed had an effect on HBV persistence. IL-10 -1082 and VDR TaqI polymorphisms may contribute to the more severe liver disease associated with CHD compared with CHB. Keywords: chronic hepatitis B, chronic hepatitis delta, cytokine genes, polymorphism, vitamin D receptor gene.

HDV virions [2]. HDV infection can therefore occur either concurrently with HBV infection (coinfection) or in patients with chronic hepatitis B (CHB) infection as a superinfection [3,4]. Although the implementation of HBV vaccination programmes has significantly decreased the incidence of HDV infection [5], chronic hepatitis delta (CHD) infection is still a significant health problem in various parts of the world including Turkey [6]. Several studies performed in Western European countries showed that HDV infection is not a vanishing disease in Western Europe due to migration of patients from endemic areas [5,7]. Currently HDV is divided into eight major genotypes showing characteristic geographical distribution [8]. Genotype 1 is dominant in Turkey [9–11], which has been shown to be associated with a more rapid disease progression [12]. The mechanism of liver injury remains vastly unknown. Some studies described cellular immune responses against HDV, suggesting that there is an association between the

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quantity and quality of host T-cell responses and the infection [13,14]. More recently, HDV-specific IL-2, interferongamma (IFN) and interferon-inducible protein-10 responses were observed in CHD, suggesting predominant cellular immune activation [15]. These results suggest that HDV infection is an immune-mediated disease. Considering the critical roles of cytokines in regulating immune responses, the level of cytokine expression may have a significant role in disease outcome [16]. It has been shown that single nucleotide polymorphisms (SNPs) in the regulatory region of the cytokine genes determine the variability in cytokine production [17]. In addition to cytokine genes, polymorphisms in several noncytokine genes, such as the vitamin D receptor (VDR) [18], may also have an important role in the host response. Recent studies have suggested a crucial role for the VDR and vitamin D in activating T cells [19,20]. The role of polymorphisms of genes that have a potential effect on the immune response has been investigated in patients with HBV infection but no data exist for CHD. In this study, we aimed to examine the roles of gene polymorphisms of cytokines involved in cellular immune responses such as TNF-a and IFN-c as well as humoral immune responses such as IL-10, and further polymorphisms of VDR genes in Turkish patients with CHD and to compare them with a sex- and age-matched CHB population at a 1 to 2 ratio. In addition, we used a control group consisting of individuals with resolved HBV infection. Six gene polymorphisms associated with functional consequences in the context of increased or decreased cytokine secretions were selected. These 6 polymorphic sites include 2 on the IL-10 gene at the -1082 (A/G, rs1800896) and -627 (C/A, rs1800872) positions, 1 on the IFN-c gene at the +874 (T/A, rs62559044) position, 1 on the TNF-a gene at the -308 position (G/A, rs1800629) and 2 on the VDR gene at the FokI (exon 2-rs2228570) and TaqI (exon 9-rs731236) restriction sites.

MATERIALS AND METHODS Patients The study population is shown in Table 1: Sixty-seven patients with CHD (group 1; 20 women, 47 men; mean age, 36  11); 119 patients with CHB (group 2; 34

women, 85 men; mean age, 38  12); and 56 individuals with resolved HBV infection (group 3; 39 women, 17 men; mean age, 49  11). The study population consisted of consecutive patients seen at the ambulatory Hepatology Clinic of the Gastroenterology Department of Ankara University Medical School and were selected based on the following inclusion criteria: all group 1 patients had to be HDV RNA positive by polymerase chain reaction (PCR) and had to have ALT levels higher than the upper limit of normal. All group 2 patients had to have elevated ALT and HBV DNA levels more than 2000 IU/ mL. The inactive carrier state had to be ruled out in uncertain cases either through long-term follow-up or by liver biopsy. In this group, 74 of 119 patients were HBeAg negative, 45 patients were HBeAg positive. In patients with available liver biopsies, liver fibrosis grade was assigned according to Knodell et al. [21]. The control group (group 3) comprised anti-HBs and anti-HBc positive healthy volunteers (resolved HBV). The study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of the Ankara University Medical School. Informed consent was obtained from all patients for the use of their samples for the study.

Viral load measurement and genotype determination Serum HBV DNA had been quantified by Cobas TaqMan Analyzer using the COBAS TaqMan HBV 48 test kit (Roche Diagnostics, Mannheim, Germany). Serum HDV RNA was detected using an in-house PCR method [10]. Hepatitis B virus and HDV genotypes were determined by direct sequencing as described previously [10,11].

Peripheral DNA isolation Samples of genomic DNA were extracted from the peripheral blood leucocytes using the high salt concentration method [22] and stored at +4 °C.

Analyses of TNF-a -308, IL-10 -1082, IL-10 -627, VDR FokI and VDR TaqI gene polymorphisms Gene polymorphisms in TNF-a -308 (rs1800629), IL-10 -1082 (rs1800896), IL-10 -627 (rs1800872), VDR FokI (rs2228570)

Table 1 Baseline characteristics of the study populations

CHD CHB Resolved HBV

N

Gender Female/male

Age

ALT

Platelets

Fibrosis grade

67 119 56

20/47 34/85 39/17

36  11 38  12 49  11

94  77 105  80 22  5*,***

160  38 192  42* 230  55*,***

2.14  1.53 1.01  1.10** –

*P < 0.05 and **P < 0.01 vs CHD: ***P < 0.05 vs CHB. Age, ALT, platelets, fibrosis grade are given as mean  SD. © 2013 John Wiley & Sons Ltd

Gene polymorphisms in chronic hepatitis delta and VDR TaqI (rs731236) were analysed using polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) methods. The primer sequences used for PCR amplification are listed in Table 2. The restriction enzyme cleavage analysis of IL-10 -627, IL-10 -1082, TNF-a -308 and the VDR gene has been previously described by Aithal et al. [23], Zheng et al. [24], Reynard et al. [25] and Hutchinson et al. [26], respectively (Table 2). Every reaction contained 100 ng template DNA, 0.2 mM dNTP, 1 U Taq polymerase enzyme, 20 pmol of each primer and 1 mM MgCl2 (0.5 mM for IL-10-1082 polymorphism) in a 50 lL of total reaction volume. The PCR conditions were as follows: initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for IL-10 -627, 58 °C for IL-10 -1082, 63 °C for TNF-a -308 and 65 °C for both FokI and TaqI polymorphisms of the VDR gene for 1 min and extension at 72 °C for 1 min, followed by a final extension step at 72 °C for 7 min. All PCR reactions were performed in an Eppendorf Mastercycler Personal thermocycler (Eppendorf, Hamburg, Germany). Amplifications were confirmed using 1% agarose gels stained with ethidium bromide.

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Restriction endonuclease digestions were carried out according to the manufacturer’s recommendations (MBI Fermentas, LT). All restriction enzyme digestions were performed by providing convenient conditions for each region for a total of 15 h at the appropriate temperature. The digested products were visualized on 2–4% agarose gels stained with ethidium bromide.

Analyses of the IFN-c +874 gene polymorphism The IFN-c+874 gene polymorphism (rs62559044) was detected by allele-specific PCR as described by Lio et al. [27]. The PCR product size was 262 bp for IFN-c+874. The sequences of the allele-specific primers are shown in Table 2, and the lengths of PCR products are shown in Table 2. The reactions contained 100 ng template DNA, 0.2 mM dNTP, 1 U Taq polymerase enzyme, 20 pmol of each primer and 1 mM MgCl2 in a 50 lL of total reaction volume. The PCR conditions were as follows: initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension step at 72 °C for 7 min.

Table 2 Primers used for PCR and the genotyping procedures for the variants used for PCR-RFLP

Polymorphism Primer Sequence IL-10 -627

IL-10 -1082

TNF-a -308

VDR FokI

VDR TaqI

IFN-c +874T

IFN-c +874A

F: 5′-CTT AGG TCA CAG TGA CGT GG-3′ R: 5′-GTG AGC ACT ACC TGA CTA GC-3′ F: 5′-AAC TGG CTC CCC TTA CCT TC-3′ R: 5′-AGG AGG TCC CTT ACT TTC CGC-3′ F: 5′-AGG CAA TAG GTT TTG AGG GCC AT-3′ R: 5′-TCC TCC CTG CTC CGA TTC CG-3′ F: 5′-AGC TGG CCC TGG CAC TGA CTC TGC TCT-3′ R: 5′-ATG GAA ACA CCT TGC TTC TTC TCC CTC-3′ F: 5′-CAG AGC ATG GAC AGG GAG CAA G-3′ R: 5′-CGG CAG CGG ATG TAC GTC TGC AG-3′ F: 5′-TCA ACA AAG CTG ATA CTC CA-3′ R: 5′-TTC TTA CAA CAC AAA ATC AAA TCT-3′ F: 5′-TCA ACA AAG CTG ATA CTC CA-3′ R: 5′-TTC TTA CAA CAC AAA ATC AAA TCA-3′

© 2013 John Wiley & Sons Ltd

PCR product Digested fragment length Restriction length (bp) endonuclease (bp)

Reference Genotype no.

Rsa I (Fermentas, Lithuania) Mnl I (Fermentas, Lithuania) Nco I (Fermentas, Lithuania) Fok I (Fermentas, Lithuania)

412

412 412, 237, 175 237, 175 141 141, 92, 49 92,49 87, 20 107, 87, 20 107 265 265, 196, 69 196, 69

CC CA AA GG GA AA GG GA AA FF Ff ff

[23]

Taq I (Fermentas, Lithuania)

345

345 345, 260, 85 260, 85

TT Tt tt

[26]



262





[27]



262





[27]

141

107

265

[24]

[25]

[26]

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Statistical analysis The data were tested by the chi-squared and Fisher’s exact tests. P < 0.05 was considered to be statistically significant. Genotype frequencies were calculated as the percentages of variant alleles. Allele frequencies were calculated as the percentages of variant alleles. The Hardy–Weinberg equilibrium was tested by comparing expected and observed genotype frequencies by x2 test. Pairwise linkage disequilibrium (LD) was analysed between the loci by evaluating both measures of D΄ and LD correlation coefficient (R2).

RESULTS General characteristics of the patient population Baseline characteristics of the patient population are summarized in Table 1. There was no difference between CHD and CHB patients with regard to age and sex. Individuals with resolved hepatitis B infection were older than patients with CHD and CHB (P < 0.05). Patients with CHD when compared with patients with CHB had lower platelet counts (P < 0.05) and had a higher Knodell fibrosis score (2.14 + 1.53 [x  SD] vs CHB 1.01  1.10, P < 0.01). Likewise, the proportion of patients with advanced liver disease (Knodell Fibosis score ≥3) was greater in patients with CDH compared with patients with CHB (17/28 vs 12/68, P < 0.0001). All patients with CHB and CHD were infected with HBV genotype D and HDV type I, respectively.

Comparisons of gene variants between patients with CHD, CHB, and resolved HBV Genotypic distribution of all alleles was in accordance with Hardy–Weinberg equilibrium (P > 0.05). Linkage disequilibrium was not observed between the loci.

IL-10 gene variants A total of 67 patients with CHD, 116 patients with CHB and 53 patients with resolved HBV were genotyped for the IL-10 -627 polymorphism (Table 3). At position -627, the frequency of the genotype AA, which has been shown to reduce the production of IL-10 [28], was similar, being 7.5% in patients with CHD, 10.3% in patients with CHB and 7.6% in resolved HBV individuals. Likewise, frequency of CC and CA genotypes was also similar between groups (Table 3). The -1082 position of the IL-10 gene was genotyped in 62 of the 67 patients with CHD, 99 of the 119 patients with CHB and 51 of the 56 patients with resolved HBV (Table 3). The frequency of AA genotype was 22.6% in patients with CHD, 13.1% in patients with CHB and 3.9% in patients with resolved HBV infection (CHD vs CHB: P > 0.05; CHD vs resolved HBV: P < 0.05; CHB vs resolved HBV: P > 0.05; Table 3). In addition, the frequency of

genotype GG was 17.7% in patients with CHD, 37.4% in patients with CHB and 47.1% in patients with resolved HBV (Table 3, P 0.05) (Table 3).

VDR gene variants Sixty-six patients with CHD, 117 patients with CHB and 56 patients with resolved HBV were genotyped for the exon 2 of the VDR gene (FokI) among the total of 67 patients with CHD, 119 patients with CHB and 56 patients with resolved HBV. No difference between groups was observed (P > 0.05). For exon 9 of the VDR gene (TaqI) polymorphism, a total of 67 patients with CHD, 118 patients with CHB and 56 patients with resolved HBV were genotyped. The results showed that the frequency of the TT genotype was 86.6% in patients with CHD, 62.7% in patients with CHB and 62.5% in resolved HBV individuals (CHD vs CHB, P: 0.001) (Table 3). As can be seen in Table 3 and mentioned above, genotyping of gene polymorphisms was not possible in every single patient where genomic DNA was available. The main reason was that amplification of respective gene areas was not always possible. Thus, to have a fairer estimate of risk factors affecting disease phenotype, that is, severe vs mild disease, we have redone the statistics after including only those patients in each group for whom the whole dataset was available. This led to a decrease in the number of patients analysed (CHD group, n = 59, CHB group, n = 95 and resolved HBV infection, n = 47), but it did not lead to a meaningful change in results in terms of significance: IL-10 -627, P = 0.89; IL-10-1082, P = 0.005; TNF-a-308: P = 0.07; IFN-c-874, P = 0.22, VDR Fok I, P = 0.71; VDR Taq I, P = 0.004.

DISCUSSION The study represents the first study assessing certain gene polymorphisms in patients with CHD. The main findings of © 2013 John Wiley & Sons Ltd

Gene polymorphisms in chronic hepatitis delta

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Table 3 Genotype counts (%) and allele frequencies of the polymorphisms in CHD, CHB and resolved HBV groups n (%) Polymorphism

Genotype

IL-10 -627

CC CA AA C A

IL-10 -1082

AA AG GG A G

TNF-a -308

GG GA AA G A

IFN-c +874

TT TA AA T A

VDR FokI (exon 2)

FF Ff ff F f

VDR TaqI (exon 9)

TT Tt tt T t

CHD N = 67

CHB N = 119

Resolved HBV N = 56

n = 67 39 (58.2) 23 (34.3) 5 (7.5) 75.4 24.6 n = 62 14 (22.6) 37 (59.7) 11 (17.7) 52 48 n = 67 51 (76.1) 10 (14.9) 6 (9.0) 83.6 16.4 n = 64 22 (34.4) 33 (51.7) 9 (14.0) 60.2 39.8 n = 66 41 (62.1) 23 (34.9) 2 (3.0) 79.5 20.5 n = 67 58 (86.6) 8 (11.9) 1 (1.5) 92.5 7.5

n = 116 63 (54.4) 41 (35.3) 12 (10.3) 72 28 n = 99 13 (13.1) 49 (49.5)37 (37.4) 38 62 n = 118 99 (83.9) 16 (13.6) 3 (2.5) 90.7 9.3 n = 118 34 (28.8) 47 (39.8) 37 (31.4) 48.7 51.3 n = 117 82 (70.1) 32 (27.3) 3 (2.6) 83.8 16.2 n = 118 74 (62.7) 35 (29.7) 9 (7.6) 77.5 22.5

n = 53 29 (54.7) 20 (37.7) 4 (7.6) 73.6 26.4 n = 51 2 (3.9) 25 (49.0) 24 (47.1) 28 72 n = 56 41 (73.2) 14 (25) 1 (1.8) 85.7 14.3 n = 54 15 (27.8) 28 (51.8) 11 (20.4) 53.7 46.3 n = 56 38 (67.9) 18 (32.1) 0 84 16 n = 56 35 (62.5) 20 (35.7) 1 (1.8) 80.4 19.6

the study are that IL-10-1082 and VDR TaqI polymorphisms were observed more frequently in CHD compared with patients with CHB. Patients with CHD were more likely to have advanced liver disease compared with patients with CHB as reflected by higher fibrosis scores and lower platelet counts. We suggest that the observed differences in the gene polymorphisms may contribute to the more severe liver disease associated with CHD compared with CHB. No difference was observed in the studied gene polymorphisms between patients with CHB and recovered HBV individuals. The resolved HBV group was used as the control group for patients with CHB. For the CHD group, the © 2013 John Wiley & Sons Ltd

v2

P

0.76

0.94

15.8

0.003

8.1

0.09

8.2

0.09

3.9

0.41

17.7

0.001

ideal control group in the context of the outcome of an acute delta hepatitis episode, recovered HBV-HDV coinfected patients, is almost impossible to establish. Instead, patients with CHB were used as a control group for patients with CHD. One other limitation of this study is that patients with CHD with mild liver disease on liver biopsy are not compared with patients with more advanced disease within the CHD cohort because of the low number of patients. The level of IL-10 production has an important effect on immune regulation. The -1082 GG genotype was found to be associated with high IL-10 production, the -1082 GA with intermediate production and the -1082 AA with low

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IL-10 production [28]. Patients with CHD more often had the low secretory genotype AA and less often the high secretory GG genotype compared with both patients with recovered HBV and patients with active HBV mono-infection. The frequent occurrence of the low IL-10 secretory genotype and the infrequent occurrence of the high secretory GG genotype is expected to be associated with better expression of pro-inflammatory cytokines which could potentially contribute to the more severe disease associated with CHD infection compared with CHB. Overall, no significant difference existed between patients with CHB and individuals who had cleared HBV with regard to the IL-10 -1082 polymorphism. This may have been due to the low number of patients investigated in this study but may point out to the complexity of the immune reaction in humans where single cytokine genotyping assessments could be misleading. In general, studies on the IL-10 polymorphism in HBV infection have shown inconsistent results [29–32]. Despite TNF-a being an important cytokine involved in the immune pathogenesis of HBV infection [33], we found no difference between the studied groups with regard to the TNF-a 308 polymorphism. The -308 G allele was reported to be associated with significantly higher TNF-a mRNA expression compared with the A allele [34]. However, studies performed on the association between TNF-a -308 genotype and the severity of the HBV infection have shown conflicting results. Two meta-analyses were performed on the effect of TNF-a -308 SNP and persistence of HBV infection [35,36]. In these two meta-analyses, no association was found for the -308 A allele in Caucasian patients with CHB in line with the results of the current study. In the recent meta-analysis performed by Xia et al. [35], the TNF-a -308 GG polymorphism showed significantly increased risk of HBV persistence, although here again the possibility of ethnic difference was mentioned. TNF-a has important pro-inflammatory activities affecting duration and strength of inflammatory reactions. However, other SNPs of TNF-a such as those at nucleotides -863, -538, -202 of its promoter need also to be considered. IFN-c has a pivotal role in resolution of an acute hepatitis B episode and may be the main mediator in the noncytolytic control of HBV infection [37]. High IFN-c production has been associated with the IFN-c +874 TT genotype, whereas +874 AA genotype has been associated with low IFN-c production [38]. The IFN-c +874A allele has been linked with outcome of some infectious diseases such as parvovirus, tuberculosis and intrauterine HBV infection [39–41] in the context that low IFN-gamma expression may impair host immune responses to virus infection. However, in the current study, an effect of IFN-c polymorphism was not observed either on persistence of HBV infection or when CHD patients were compared with patients with CHB. These results are in line with a Korean study [31]. As pointed out, IFN-c is a multifunctional cytokine

crucial for host defence against viral pathogens. It is possible that other SNPs of the IFN-c gene may have more functional importance such as the -746G SNP located in the proximal IFN-c promoter region and reported to be linked with HCV persistence [42]. Further, it needs to be remembered that IFN-c exerts its effects by binding to the IFN-c receptors IFN-cR-1 and IFN-cR-2 resulting in activation of the JAK-STAT intracellular signalling pathway [43,44], and studies suggest that IFN-cR SNPs may have functional significance. In one recent study, of 40 SNPs along 10 IFN-c related genes, 2 SNPs on the IFN-cR2 were found to be strongly associated with HBV viremia. Similarly, in another previous study, SNPs of IFN-AR2 had been associated with persistent HBV infection in the Gambia [45]. Finally, our somewhat unexpected results may have been due to the relatively low number of patients involved in the study. The involvement of Vitamin D in immune regulation has been reported in several studies. Vitamin D activates monocytes, stimulates cell-mediated immunity and regulates antiviral defence mechanisms which include modulating IL-12 and IFN-c at the transcriptional level [18,46]. In the current study, 2 polymorphisms of the VDR gene (FokI and TaqI) were investigated. FokI polymorphism in exon 2 (rs2228570) has an effect on creating an alternative start codon resulting in VDR proteins with different structures, a long f-VDR or a shorter F-VDR. [47]. This structural difference was shown to have an effect on immune cell behaviour, with a more active immune system for the short F-VDR [48]. However, in our study, frequency of this polymorphism did not show any significant difference between groups. This result is in line with a recent meta-analysis, performed for multiple sclerosis, which is considered to be an immune-mediated disease [49]. The other VDR gene polymorphism (TaqI) we investigated is located on exon 9 (rs7311236) and correlates with increased transcriptional activity, mRNA stability and high serum level of 1.25D3 [50]. Suneetha et al. [46] did not find any association between liver disease severity and the TaqI polymorphism in patients with CHB where this polymorphism had been compared in mild vs severe CHB. These data may be considered to be at variance with the results of the current study although the former study was confined to patients with CHB. Our study represents the first assessment of this polymorphism in CHD, and in this study, the TT genotype of VDR TaqI was more often found in patients with CHD (86.6%) when compared with patients with CHB (62.7%) (P = 0.001). It is reasonable to suggest that this difference may be linked with the more severe liver disease associated with CHD compared with CHB and that this vitamin D receptor polymorphism may have contributed to more severe liver disease through activation of the cellular immune system. In conclusion, in the current study none of the SNPs tested appeared to have an effect on HBV persistence. © 2013 John Wiley & Sons Ltd

Gene polymorphisms in chronic hepatitis delta However, the IL-10 -1082 and VDR TaqI polymorphisms showed significant differences between patients with CHD

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and CHB. These SNPs may contribute to the more severe clinical presentation of CHD.

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Tumour necrosis factor-alpha, interleukin-10, interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta.

No data exist to assess certain polymorphisms that have a potential effect on the immune response in patients with chronic hepatitis delta (CHD). The ...
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