DPB1 variants against Hepatitis B virus infection in an Indonesian population

Infection, Genetics and Evolution 41 (2016) 177–184

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Research paper

Protective effects of HLA-DPA1/DPB1 variants against Hepatitis B virus infection in an Indonesian population Widya Wasityastuti a,b,c, Yoshihiko Yano d,⁎, Neneng Ratnasari e, Teguh Triyono f, Catharina Triwikatmani e, Fahmi Indrarti e, Didik Setyo Heriyanto g, Laura Navika Yamani a,b, Yujiao Liang a,b, Takako Utsumi h,i, Yoshitake Hayashi a,b a

Division of Molecular Medicine & Medical Genetics, Department of Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan Division of Infectious Disease Pathology, Department of Microbiology and Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan c Department of Physiology, Faculty of Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia d Department of Gastroenterology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan e Subdivision of Gastroenterohepatology, Department of Internal Medicine, Dr. Sardjito Hospital, Faculty of Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia f Department of Clinical Pathology, Dr. Sardjito Hospital, Faculty of Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia g Department of Anatomical Pathology, Dr. Sardjito Hospital, Faculty of Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia h Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan i Indonesia–Japan Collaborative Research Centre for Emerging and Re-emerging Infectious Disease, Institute of Tropical Disease, Airlangga University, Surabaya 60115, Indonesia b

a r t i c l e

i n f o

Article history: Received 12 September 2015 Received in revised form 7 March 2016 Accepted 30 March 2016 Available online 2 April 2016 Keywords: Host gene HLA-DP variants HBV infection Indonesian population Protection

a b s t r a c t Human leukocyte antigen (HLA) DPA1/DPB1 variants have been reported to influence Hepatitis B virus (HBV) infection. HLA-DPA1/DPB1 plays a pivotal role in antigen presentation to CD4+ helper T cells and influences the outcome of HBV infection. To investigate the influence of HLA-DP variants on the outcome of HBV infection in an Indonesian population where it has the third-highest prevalence of HBV infection worldwide, we performed a case–control study of 686 participants, including patients with HBV-related advanced or nonadvanced liver disease, patients with spontaneously resolved HBV, and healthy controls. Single-nucleotide polymorphisms in HLADPA1 (rs3077) and HLA-DPB1 (rs3135021, rs9277535, and rs228388) were genotyped using real-time TaqMan® genotyping assays. Because rs2281388 deviated from Hardy–Weinberg equilibrium, it was excluded from subsequent analyses. The results of logistic regression analyses showed that the HLA-DPB1 rs9277535 variants were associated with a reduced risk of persistent HBV infection (odds ratio [OR] 0.70, 95% confidence interval [95% CI] 0.52–0.96, P = 0.026, additive genetic model; OR 0.60, 95% CI 0.38–0.96, P = 0.033, dominant genetic model). The HLA-DPA1 rs3077 variant was associated with a protective effect increasing the spontaneously resolved HBV infection (OR 0.64, 95% CI 0.41–0.98, P = 0.039, dominant genetic model). By contrast, the HLADPB1 rs3135021 variant was not associated with the outcome of HBV infection, including susceptibility, spontaneously resolved, or disease progression. Combinations of haplotype markers were also associated with HBV susceptibility (CA for rs3077–rs9277535, OR 0.57, 95% CI 0.36–0.92, P = 0.021; GA for rs3135021–rs9277535, OR 0.56, 95% CI 0.36–0.86, P = 0.0087). In conclusion, these findings confirm that HLA-DPA1/DPB1 variants were associated with the outcomes of HBV infection in an Indonesian population. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: HBV, Hepatitis B virus; SNP ID, single-nucleotide polymorphism identification number; HLA, Human Leucocyte Antigen; MAF, minor allele frequency; HC, healthy controls; SRH, spontaneously resolved HBV infection; ALD, HBV-related advanced liver disease; NALD, HBV-related nonadvanced liver disease; vs., versus; Additive, additive genetic model (minor allele (m) vs. major allele (M)); Dominant, dominant genetic model (heterozygote and minor homozygote (Mm + mm) vs. major homozygote (MM)); Recessive, recessive genetic model (minor homozygote (mm) vs. major homozygote and heterozygote (MM + Mm)); OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Department of Gastroenterology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail address: [email protected] (Y. Yano).

http://dx.doi.org/10.1016/j.meegid.2016.03.034 1567-1348/© 2016 Elsevier B.V. All rights reserved.

Hepatitis B infection is a major threat to public health on a global scale. It is estimated that two billion people worldwide are infected with Hepatitis B virus (HBV). The clinical outcomes of HBV infection range from asymptomatic infection to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Furthermore, end-stage liver disease related to HBV infection is a major reason for liver transplantation and is responsible for more than one million deaths each year (Lavanchy, 2008; Lee, 1997; WHO, 2012). Although HBV was identified as the causative pathogen of liver disease about 50 years ago (Blumberg, 2002), the exact pathogenesis of

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HBV infection remains unclear. The development of single-nucleotide polymorphism (SNP) databases and the advances in technology have provided an opportunity to explore the issue of host susceptibility (Nishida et al., 2013). Recent studies have suggested that complex interactions among genetic variations, HBV virulence, and environmental factors determine disease development and progression (Frodsham, 2005; Thio et al., 2000; Yano et al., 2013). One noteworthy finding of genome-wide association studies is that polymorphisms in the Human Leucocyte Antigen (HLA) DPA1/DPB1 genes, which are located on the short arm of chromosome 6, influence HBV infection (Chang et al., 2014; Kamatani et al., 2009; Kim et al., 2013; Mbarek et al., 2011; Nishida et al., 2012). HLA-DP molecules are heterodimers of specialized glycoproteins that deliver foreign peptides to the surface of the cell, presenting the peptides to T cells, and enhancing the cellular responses to eliminate pathogens such as HBV. Two classes of HLAs, class I and class II, interact with specific CD8+ cytotoxic T cells and specific CD4+ helper T cells, respectively (Blackwell et al., 2009; Murphy, 2011). Consequently, the inability of HLA-DP to adequately present HBV peptide fragments will impair the host's immune response (Schmidt et al., 2013). The first genome-wide association study examined the associations between chronic HBV infection and 11 SNPs in the HLA-DP gene, a major isotype of HLA class II, made up of a 29-kDa α chain and a 34-kDa β-chain, in Japanese subjects (Kamatani et al., 2009). Different risk alleles or genetic models have been identified in different populations, and HLA-DPA1 rs3077 and HLA-DPB1 rs9277535 were important SNPs in terms of the susceptibility to HBV infection in several studies (Al-Qahtani et al., 2014; Guo et al., 2011; Li et al., 2011; Liao et al., 2014; Migita et al., 2012; Posuwan et al., 2014; Vermehren et al., 2012; Wang et al., 2011; Wong et al., 2013; Zhang et al., 2013). Associations between these SNPs and the spontaneously resolved of HBV infection were subsequently reported in Chinese (An et al., 2011; Guo et al., 2011; Li et al., 2011), Japanese, and Koreans populations (Nishida et al., 2012). However, other SNPs have yet to be widely evaluated. HLA-DPB1 rs3135021 and rs2281388 were reported to be associated with hepatocellular carcinoma progression in a Chinese population (Zhang et al., 2013). Unfortunately, the association between HLA-DPA1/DPB1 and the progression of HBV infection was not repeated in other studies (Al-Qahtani et al., 2014; An et al., 2011; Hu et al., 2012; Li et al., 2011; Liao et al., 2014; Migita et al., 2012). These conflicting findings raised our interest. Indonesia has the third-highest prevalence of HBV infection worldwide, with a moderate to high incidence that affects 242 million people. Therefore, HBV infection places a heavy burden on the healthcare system (WHO, 2013). However, to date, the influence of HLA-DP variants on HBV infection has not been fully investigated in an Indonesian population. Due to the prevalence rate of HBV infection ranges from 2.1% to 10.5% (Utsumi, 2014), this study was performed to investigate the profiles and the genetic influence of HLA-DPA1/DPB1 SNPs on the susceptibility, spontaneously resolved, and progression of HBV-related advanced liver disease in an Indonesian population. We focused on one SNP in HLA-DPA1 (rs3077) and three SNPs in HLA-DPB1 (rs3135021, rs9277535, and rs2281388).

2. Materials and methods

between populations, we also included 86 Japanese subjects who were recruited from Kobe University Hospital. The HBV status of each subject was determined based on the serological results of Hepatitis B surface antigen (HBsAg) and total antibody against HBV core antigen (anti-HBc) tests. The healthy controls were blood donors from blood donation units who were seronegative for both HBsAg and anti-HBc. HBV infection was considered to have spontaneously resolved in blood donors who were seronegative for HBsAg and seropositive for anti-HBc. Blood donors and patients with seropositive for HBsAg were considered to be HBV carriers. All blood samples were negative for Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV). The HBV carriers were divided into two groups of patients with nonadvanced liver disease (asymptomatic and chronic Hepatitis B) or advanced liver disease (cirrhosis and hepatocellular carcinoma). Subjects who were seropositive for HBsAg, had normal serum alanine aminotransferase (ALT) levels (b 35 U/L), and had no obvious clinical symptoms were considered to be asymptomatic carriers. Chronic HBV infection, cirrhosis, and hepatocellular carcinoma were classified by a gastrohepatologist at the research site using the criteria of the Asian Pacific Association for the Study of the Liver (Liaw et al., 2012; Omata et al., 2010; Shiha et al., 2009). The diagnosis of HBV infection was based on clinical, biochemical, imaging, and/or histological examinations. Each subject gave his/her written informed consent before enrolment. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committees of Kobe University, Japan, and Gadjah Mada University, Indonesia (code KE/FK/194/EC). 2.2. Serological and biochemical tests All subjects were screened for HBsAg, anti-HCV antibodies, and antiHIV antibodies using automated chemiluminescent enzyme immunoassays on an Architect analyzer (Abbott Laboratories, IL, USA). Subjects who were seronegative for HBsAg were also tested for anti-HBc antibodies using a passive hemagglutination assay (Mycell® Anti-rHBc; Institute of Immunology, Tokyo, Japan). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using standard procedures. 2.3. DNA extraction and SNP genotyping For SNP genotype determination, peripheral blood was drawn and collected into EDTA blood tubes. Genomic DNA was extracted from 200 ml of buffy coats on the day of collection using a DNA extractor kit (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions. Genotyping was performed using the Allelic Discrimination Assay on a 7500 Real-Time PCR System with TaqMan® Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA). SNPs were genotyped as previously described with specific primers (Zhang et al., 2013) and FAMand VIC-labeled probes provided by Sigma-Aldrich (Hokkaido, Japan), in accordance with the recommended protocol (Malkki and Petersdorf, 2012). The four SNPs were successfully genotyped at rates of N97.5%. Samples with known genotypes obtained by direct sequencing were used as the quality controls in each TaqMan® genotyping assay.

2.1. Subjects 2.4. Meta-analysis study The Indonesian subjects involved in this study were recruited from Dr. Sardjito Hospital, Yogyakarta, Indonesia, between April 2013 and November 2014. A total of 686 participants, including 222 HBV carriers, 228 spontaneously resolved of HBV, and 236 healthy controls, were enrolled from the blood donation unit, inpatient wards, and outpatient clinic of the Subdivision of Gastroenterohepatology, Department of Internal Medicine. To compare the linkage disequilibrium pattern

In order to improve the accuracy of the influence of the SNPs on the outcome of HBV infection, we performed a meta-analysis of rs3077 and rs9277535. Relevant studies were screened and identified by a computerized literature search of electronic databases, including Pubmed, Elsevier, and Web of Science, with English only as the language restriction. No manual searches of books, abstracts and conference

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proceedings were made. The following index terms were used: ‘Hepatitis B’ and ‘HLA-DP’. The inclusion criteria were: (a) the study analyzed the genotype frequencies of HLA-DPA1/DPB1 in HBV carriers, HBV spontaneously resolved, HBV-related advanced liver disease, and/or healthy (HBV-uninfected) controls; (b) the study included specific criteria for enrolling the subjects; (c) the study had a case–control design; and d) the numbers of cases and controls, and the genotype frequencies were stated. Studies that did not meet these criteria were excluded. The meta-analysis was performed for three groups: (a) healthy controls (HBV-uninfected subjects) and HBV carriers; (b) HBV carriers and subjects with HBV spontaneously resolved; and (c) subjects with HBV-related nonadvanced or advanced liver disease. 2.5. Statistical analysis The Hardy–Weinberg equilibrium (HWE) of the genotype distributions and the linkage disequilibrium (LD) of the SNPs were examined using Haploview software v4.2 (http://www.broadinstitute.org/ haploview/haploview) (Barrett et al., 2005). The differences for categorical variables and continuous variables were compared using the χ2 test and Student's t test, respectively. The genotype frequency represented the frequency of the major homozygous (MM), heterozygous (Mm) and minor homozygous (mm) which corresponding to specific allele of each SNP ID (rs3077 correspond to C/T, rs3135021 correspond to G/ A, and rs9277535 correspond to G/A). Genetic associations (genotypebased, allele-based, and haplotype-based) were tested using the χ2 test or Fisher's exact test. Three different genetic association models were calculated: additive genetic model (minor allele (m) versus (vs.) major allele (M)), dominant genetic model (heterozygote and minor homozygote (Mm + mm) vs. major homozygote (MM)), and recessive genetic model (minor homozygote (mm) vs. major homozygote and heterozygote (MM + Mm)). We used a logistic regression model to calculate the age- and gender-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) between the HLA-DP variants and the risk of HBV infection. Haplotype frequency was estimated using two-stage iterative method, the Expectation Maximization algorithm. The statistical analyses were performed using Stata version 12 (StataCorp, College Station, Texas, USA), while allele-based and haplotype-based genetic models were applied using SNPStats web tools (http://bioinfo. iconcologia.net/SNPstats) (Sole et al., 2006). The examination of heterogeneity and the calculation of overall effect of meta-analysis data were analyzed using MedCalc® software version 15.4 (www.medcalc.org) (Schoonjans et al., 1995). Forest plots were used to show the results. Values of P b 0.05 were considered statistically significant. 3. Results 3.1. Subject characteristics and evaluation of the Hardy–Weinberg equilibrium The characteristics of the subjects are summarized in Table 1. The HBV carriers were older (mean ± SD; 43.48 ± 14.37 years, P = 0.000) than the subjects with HBV spontaneously resolved (30.61 ± 10.22 years) and the healthy controls (29.18 ± 9.30 years). The subjects

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with advanced liver disease (51.94 ± 11.79 years, P = 0.000) were older than the subjects with nonadvanced liver disease (37.40 ± 12.94 years). Males accounted for 76.13%, 81.57%, and 84.75% of HBV carriers, subjects with HBV spontaneously resolved, and healthy controls, respectively; these percentages were not significantly different among the three groups (χ2 = 5.767, P = 0.560). Likewise, gender in HBV-related nonadvanced and advanced liver disease groups were also not significantly different (χ2 = 0.080, P = 0.778). Significant differences were observed in AST level (P = 0.000) and ALT level (P = 0.001). Nevertheless, no significant differences were found in platelet count (P = 0.641). Four SNPs were analyzed in this case–control study, one in HLADPA1 (rs3077) and three in HLA-DPB1 (rs3135021, rs9277535, and rs2281388). The minor alleles for rs3077, rs3135021, rs9277535, and rs2281388 in this Indonesian population were T, A, A, and T, respectively. The genotype distributions of three SNPs (rs3077, rs3135021, and rs9277535) met the HWE criterion in the HBV carriers, subjects with HBV spontaneously resolved, and healthy controls. There were no significant differences between the observed and expected frequencies of each genotype in these three groups (P N 0.05). However, rs2281388 deviated from HWE because the genotype distribution was highly skewed. Therefore, rs2281388 was excluded from further statistical genetic analyses.

3.2. Associations between HLA-DP variants and susceptibility to HBV infection in healthy controls and HBV carriers To investigate the associations between the HLA-DP variants and susceptibility to HBV infection, we compared the alleles of HBV carriers and healthy controls. The minor alleles of rs3077 (T), rs3135021 (A), and rs9277535 (A) were observed at frequencies of 32.2%, 29.8%, and 39.2%, respectively, in the HBV carriers and at frequencies of 36.7%, 27.2%, and 48.6%, respectively, in healthy controls (Table 2). The frequency of the minor allele of rs9277535 was significantly greater in healthy controls than in HBV carriers. Moreover, the A allele in rs9277535 was associated with a reduced risk of HBV infection (additive genetic model, odds ratio [OR] 0.70, 95% confidence interval [95% CI] 0.52–0.96, P = 0.023). By contrast, the distributions of the minor alleles of rs3077 and rs3135021 were not significantly different between the HBV carriers and healthy controls (additive genetic model, rs3077: OR 0.92, 95% CI 0.67–1.26, P = 0.120; rs3135021: OR 1.19, 95% CI 0.85– 1.68, P = 0.310). We further examined the association between the HLA-DP genotypes and HBV susceptibility in HBV carriers and healthy controls. Among the genotype-based genetic models used, the association between HLA-DPB1 rs9277535 and HBV susceptibility was significant in the dominant genetic model (OR 0.62, 95% CI 0.39–0.99, P = 0.033). We observed a trend that CT and TT genotypes of HLA-DPA1 rs3077 was greater in healthy controls than in HBV carriers (dominant genetic model; OR 0.78, 95% CI 0.50–1.22, P = 0.228), but statistically insignificant after adjusting to age and gender. While HLA-DPB1 rs3135021 were not significantly associated with HBV susceptibility in any of the genetic models tested (all P N 0.05) (Table 2).

Table 1 Subject characteristics. Group

N

Sex (M/F)

Age (years, mean ± SD)

AST (U/ml)

ALT (U/ml)

Platelet (103/ml3)

Healthy controls HBV spontaneously resolved HBV carriers HBV-related nonadvanced liver disease HBV-related advanced liver disease

236 228 222 132 90

200/36 186/42 169/53 100/32 69/21

29.18 ± 9.30 30.61 ± 10.22 43.63 ± 14.46 37.40 ± 12.94 51.94 ± 11.79

– – – 36.31 ± 43.25 139.30 ± 179.97

– – – 37.28 ± 38.62 68.55 ± 95.56

– – – 194.58 ± 112.91 179.59 ± 97.39

Abbreviations: N, number of samples; M, male; F, female; SD, standard deviation; U, unit; ALT, alanine transaminase; AST, aspartate aminotransferase.

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Table 2 Associations of HLA-DPA1/BPB1 variants with persistent HBV infection.* Gene, chromosome position,a allele

SNPs IDb

HC

CC/GG HLA-DPA1, 33033022, C/T HLA-DPB1, 33045558, G/A HLA-DPB1, 33054861, G/A

MAF (%)

Genotype frequency (%)

rs3077

CT/GA

TT/AA

HBV

MAF (%)

Genotype frequency (%) CC/GG

CT/GA

Testing of mode HBV vs. HC of inheritance P-valuec OR (95% CI)d

TT/AA

91 (0.386) 115 (0.487) 30 (0.127) 0.371 107 (0.493)

83 (0.376) 29 (0.131) 0.322 Additive Dominant Recessive rs3135021 127 (0.557) 81 (0.355) 20 (0.088) 0.265 107 (0.495) 92 (0.418) 19 (0.086) 0.298 Additive Dominant Recessive rs9277535 63 (0.269) 116 (0.496) 55 (0.235) 0.486 82 (0.379) 106 (0.469) 34 (0.152) 0.392 Additive Dominant Recessive

0.590 0.228 0.785 0.310 0.319 0.639 0.023⁎ 0.033⁎ 0.092

0.92 (0.67–1.26) 0.76 (0.49–1.19) 1.09 (0.58–2.07) 1.19 (0.85–1.68) 1.25 (0.80–1.95) 1.21 (0.55–2.69) 0.70 (0.51–0.95) 0.60 (0.38–0.96) 0.62 (0.36–1.08)

a,b : SNP identification numbers and positions (http://www.ncbi.nlm.nih.gov/sites/) based on Human Genome Assembly build GRCh37.p17;.c,d: Logistic regression analysis were adjusted for gender and age. Bold indicates statistically significant. ⁎ Significantly different between the two groups.

3.3. Associations between HLA-DP variants and the spontaneously resolved of HBV infection

of the gene variants were associated with the development of severe HBV-related liver disease (all P N 0.05) (Table 4).

To identify the genetic variants that were associated with the spontaneously resolved of the HBV infection, the allele and genotype frequencies of HLA-DPA1/DPB1 were compared between HBV carriers and subjects with HBV spontaneously resolved. As shown at Table 3, the HLA-DPA1 rs3077 polymorphism was associated with a protective effect increasing the spontaneous resolved of HBV infection (dominant genetic model, OR 0.64, 95% CI 0.41–0.98, P = 0.035); the frequencies of the CT and TT genotypes were significantly greater in subjects with HBV spontaneously resolved than in persistent HBV carriers (SRH vs. HBV, 0.611 vs.0.507). The other two HLA-DPB1 SNPs examined in this study seemed not to correlate with the spontaneously resolved of HBV infection (all P N 0.05) (Table 3).

3.5. Linkage disequilibrium and haplotype analysis of multiple SNPs

3.4. Associations between HLA-DP variants and progression of HBV infection In the next step, the genetic variances were also compared between subjects with HBV-related advanced or nonadvanced liver disease to identify the HLA-DPA1/DPB1 variants that were associated with the progression of HBV infection. Interestingly, the frequency of the TT genotype of the HLA-DPA1 rs3077 variant was greater in patients with HBV-related nonadvanced liver disease than in patients with HBVrelated advanced liver disease (NALD vs. ALD, 0.171 vs. 0.077). However the difference did not reach statistical significance after adjusting to gender and age (recessive genetic model, OR 2.08, 95% CI 0.77–5.59, P = 0.140) which are the important confounding factors in clinical feature of HBV infection. Thus, the multivariate analysis showed that none

The pairwise LD data for the SNPs examined in this Indonesian population are presented in Fig. 1. The physical distance separating these SNPs was 27 kb and the D′ values ranged from 0.07 to 0.94. The three SNPs in HWE (rs3077, rs3135021, and rs9277535) were in weak LD with each other. The D′ values were lower in this Indonesian population (0.07–0.60) than in a Japanese population (0.76–0.93). The associations between combinations of two or three SNPs and the susceptibility to HBV, the spontaneously resolved of HBV, and the severe outcome of HBV infection were assessed. Combinations of two or three SNPs revealed significant associations between the HLA-DP genes and a susceptibility to HBV infection. Two haplotypes (CA for rs3077– rs9277535: OR 0.57, 95% CI 0.36–0.92, P = 0.021; GA for rs3135021– rs9277535: aOR 0.56, 95% CI 0.36–0.86, P = 0.0087) were significantly associated with a reduced risk of HBV infection. Two other haplotypes (TA for rs3077–rs9277535: OR 0.77, 95% CI 0.55–1.11, P = 0.16; TGA for rs3077–rs3135021–rs9277535: OR 0.64, 95% CI 0.37–1.09, P = 0.098) also tended to be greater in healthy controls than in patients with persistent HBV infection (Table 5). However, none of the combinations of haplotypes was significantly associated with spontaneously resolved or severe outcomes of HBV infection (data not shown). 3.6. Results of the meta-analysis We extracted data from 22 studies of HLA-DPA1 rs3077 and/or HLADPB1 rs927753533 (Al-Qahtani et al., 2014; An et al., 2011; Chang et al.,

Table 3 Associations of HLA-DPA1/BPB1 variants with spontaneously resolved of HBV infection. SNPs ID

SRH

MAF (%)

Genotype frequency (%) CC/GG

HBV

MAF (%)

Genotype frequency (%)

CT/GA

TT/AA

88 (0.389)

110 (0.487)

28 (0.124)

0.367

107 (0.493)

83 (0.376)

29 (0.131)

0.322

rs3135021

118 (0.524)

92 (0.409)

15 (0.067)

0.271

107 (0.495)

92 (0.418)

19 (0.086)

0.298

rs9277535

79 (0.351)

99 (0.440)

47 (0.209)

0.429

82 (0.379)

106 (0.469)

34 (0.152)

0.392

rs3077

a,b

CC/GG

: Logistic regression analysis were adjusted for gender and age. Bold indicated statistically significant. ⁎ Significantly different between the two groups.

CT/GA

Testing of mode of inheritance

SRH vs. HBV P-valuea

OR (95% CI)b

Additive Dominant Recessive Additive Dominant Recessive Additive Dominant Recessive

0.160 0.035* 0.997 0.380 0.583 0.141 0.240 0.557 0.131

0.80 (0.59–1.09) 0.63 (0.41–0.97) 1.00 (0.53–1.88) 1.17 (0.83–1.64) 1.12 (0.73–1.73) 1.84 (0.82–4.15) 0.84 (0.63–1.13) 0.88 (0.57–1.36) 0.65 (0.38–1.13)

TT/AA

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Table 4 Associations of HLA-DP variants with progression of HBV infection. SNPs ID

NALD

MAF (%)

Genotype frequency (%)

ALD

MAF (%)

Genotype frequency (%)

CC/GG

CT/GA

TT/AA

CC/GG

CT/GA

rs3077

60 (0.465)

47 (0.364)

22 (0.171)

0.353

47 (0.527)

36 (0.396)

7 (0.077)

0.208

rs3135021

60 (0.465)

56 (0.434)

13 (0.101)

0.318

47 (0.533)

36 (0.400)

6 (0.067)

0.270

rs9277535

44 (0.333)

66 (0.500)

22 (0.167)

0.417

38 (0.429)

40 (0.440)

12 (0.132)

0.356

Testing of mode of inheritance

ALD vs. NALD P-valuea

OR (95% CI)b

Additive Dominant Recessive Additive Dominant Recessive Additive Dominant Recessive

0.160⁎ 0.320 0.140 0.200 0.150 0.810 0.230 0.220 0.490

1.38 (0.88–2.18) 1.37 (0.73–2.59) 2.08 (0.77–5.59) 1.40 (0.83–2.37) 1.61 (0.84–3.06) 1.17 (0.33–4.17) 1.32 (0.84–2.07) 1.49 (0.78–2.85) 1.36 (0.57–3.24)

TT/AA

a,b

: Logistic regression analysis were adjusted for gender and age. We did not adjust the result for those insignificant results in bivariate analysis. ⁎ Significantly different between the two groups.

2014; Cheng et al., 2013; Guo et al., 2011; Hu et al., 2012; Hu et al., 2013; Kamatani et al., 2009; Kim et al., 2013; Lau et al., 2011; Li et al., 2011; Liao et al., 2014; Liao et al., 2015; Mbarek et al., 2011; Migita et al., 2012; Nishida et al., 2012; Posuwan et al., 2014; Vermehren et al., 2012; Wang et al., 2011; Wong et al., 2013; Yan et al., 2012; Zhang et al., 2013). Our meta-analysis of the associations between rs3077 and rs9277535 and the susceptibility of HBV, the spontaneously resolved of HBV, and the progression of HBV were analyzed using the dominant genetic model. Significant heterogeneity was observed between HBV carriers and healthy controls for rs3077 (Phet = 0.0046, I2 = 51.93%) and rs9277535 (Phet b 0.0001, I2 = 85.70%), and between HBV carriers and subjects with HBV spontaneously resolved for rs3077 (Phet b 0.0001, I2 = 69.39%) and rs9277535 (Phet b 0.0001, I2 = 80.36%). Therefore, we used a random effects model in the meta-analyses for both SNPs. The pooled results comparing HBV carriers and healthy controls or subjects with spontaneously resolved HBV in the dominant genetic model indicated that genotypes CT/GA and TT/AA for rs3077 and rs9277535 were associated with a reduced risk of HBV susceptibility (rs3077: OR 0.497, 95% CI 0.454–0.544, P b 0.001; rs9277535: OR 0.539, 95% CI 0.463–0.629, P b 0.001) and HBV clearance (rs3077: OR 0.592, 95% CI 0.525–0.668, P b 0.001; rs9277535: OR 0.626, 95% CI 0.548–0.715, P b 0.001). The OR and 95% CI for the previous studies, our study, and the overall values are shown as forest plot in Fig. 2 and Supplementary data (Tables S1–S4). The studies that compared HBV-related advanced and nonadvanced liver disease was limited. Three studies examined HLA-DPA1 rs3077

Fig. 1. Linkage disequilibrium map of the three HLA-DP SNPs. LD maps were drawn using all subjects in both populations. The LD values represent the D′ values in the Indonesian population (a) and Japanese population (b).

(Al-Qahtani et al., 2014; An et al., 2011; Migita et al., 2012) and six examined HLA-DPB1 rs9277535 (Al-Qahtani et al., 2014; An et al., 2011; Hu et al., 2012; Li et al., 2011; Liao et al., 2014; Migita et al., 2012). Because of the low heterogeneity (rs3077: Phet = 0.7586, I2 = 0,00%; rs9277535: Phet = 0.1503, I2 = 38,84%), we used a fixed effects model for both SNPs. The pooled data from 926 patients with advanced liver disease and 1542 patients with nonadvanced liver disease for rs3077 revealed that rs3077 variant was associated with the development of severe HBV-related liver disease (dominant genetic model, OR 0.812, 95% CI 0.675–0.978, P = 0.028). However, we did not observe significant association between rs9277535 variant and the development of severe HBV-related liver disease in the pooled data from 3362 patients with advanced liver disease and 5173 patients with nonadvanced liver disease (dominant genetic model, OR 0.954, 95% CI 0.870–1.046, P = 0.316) (Table S5 and Table S6). 4. Discussion HBV is the commonest liver infection with unique features. Its clinical outcome ranges from an asymptomatic carrier state to end-stage liver disease and death. These are caused by a virus with indirect cytopathic properties, indicating that the host's immune response is involved in the progression of HBV infection (Guidotti and Chisari, 2006; Lee, 1997). The first study in this field among Japanese and Thai populations showed an association between HLA-DP SNPs and chronic HBV infection (Kamatani et al., 2009). To our knowledge, this is the first study to confirm that HLA-DP SNPs are associated with HBV infection in an Indonesian population. HLA-DP is a major isotype of HLA class II, and consists of two chains, the α (DPA) and β (DPB) chains. It has vital roles in adaptive immunity. Several HBV-specific HLA class II restricted epitopes, including HLA-DP, have been identified (Desmond et al., 2008). The recognition of HBV antigen, which binds to HLA-DP on specific CD4+ T cells, activates specific effector immune cells (B lymphocytes, dendritic cells, and macrophages) (Murphy, 2011). CD4+ T cells also stimulate and preserve CD8+ cytotoxic T cells, which directly clear HBV-infected cells. Consequently, a reduction in HLA-DP expression interferes with antigen presentation and impairs cellular immunity. Inadequate HBV-specific CD4+ T-cell responses affect viral elimination and the clinical outcome of HBV infection (Boonstra et al., 2008; Guidotti and Chisari, 2006; Schmidt et al., 2013). In this study, we focused on four HLA-DP SNPs (rs3077, rs3135021, rs9277535, and rs2281388) to determine their potential associations with HBV susceptibility, the spontaneously resolved of HBV, and HBV progression. The SNP rs3077 and rs9277535 are located within the 3′untranslated regions of HLA-DPA1 and HLA-DPB1, respectively, whereas rs3135021 is located within intron 1 of HLA-DPB1 and rs2281388 is located at downstream of HLA-DPB1. The cellular activity and structural properties of proteins might be affected by SNPs in

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Table 5 Haplotype association model in HBV carriers and healthy controls.** Haplotypes

2 markers

3 markers

Frequency (%)

rs3077-rs3135021 C-G T-G C-A T-A rs3077-rs9277535 C-G T-A C-A T-G rs3135021-rs9277535 G-G G-A A-G A-A rs3077-rs3135021-rs9277535 C-G-G T-G-A C-A-G C-G-A T-A-A T-G-G C-A-A

HBV vs. HC

HC

HBV

P-valuea

OR (95%CI)b

0.485 0.253 0.148 0.118

0.489 0.215 0.191 0.106

– 0.42 0.67 0.64

1.00 0.84 (0.55–1.29) 1.33 (0.65–1.94) 1.14 (0.66–1.94)

0.4447 0.2989 0.1846 0.0719

0.537 0.249 0.142 0.073

– 0.16 0.021⁎ 0.71

1.00 0.77 (0.55–1.11) 0.57 (0.36–0.92) 0.88 (0.45–1.72)

0.383 0.352 0.135 0.131

0.451 0.252 0.159 0.138

– 0.0087⁎ 0.68 0.93

1.00 0.56 (0.36–0.86) 0.88 (0.49–1.59) 1.02 (0.61–1.71)

0.322 0.193 0.123 0.163 0.107 0.057 0.020

0.369 0.140 0.168 0.120 0.108 0.073 0.021

– 0.098 0.75 0.057 0.93 0.65 0.94

1.00 0.64 (0.37–1.09) 1.10 (0.60–2.04) 0.56 (0.31–1.02) 1.03 (0.59–1.80) 1.20 (0.55–2.63) 0.93 (0.14–6.01)

a,b

: Logistic regression analysis were adjusted for gender and age. Bold indicated statistically significant. ⁎ Significantly different between the HBV carriers and healthy control groups.

noncoding or intergenic region as the result of an allelic imbalance, RNA instability, or protein dysfunction (Olivier, 2004). Surprisingly, HLADPB1 rs2281388 deviated from HWE, which might be attributable to laboratory or genotyping factors, sample stratification, or evolution random changes, and could lead to false-positive or unreliable results (Lewis, 2002; Zintzaras and Lau, 2008). Therefore, rs2281388 was not included in the subsequent genetic analyses. The results of this study support previous findings of the impact of rs3077 variants on the spontaneous resolved of HBV under the dominant genetic model (An et al., 2011; Guo et al., 2011; Hu et al., 2012; Liao et al., 2014; Liao et al., 2015; Wang et al., 2011; Yan et al., 2012; Zhang et al., 2013). There was also a marked that rs3077 variant tended to reduce the probability of persistent HBV infection and the risk of progression to advanced liver disease in HBV infection. Accordingly, a single copy of the minor T allele (TX) of HLA-DPA1 rs3077 represented the prominent allele that was protective against HBV infection in this Indonesian population (Lewis, 2002). Furthermore, our meta-analysis studies using the dominant genetic model also revealed that a non-CC genotype of HLA-DPA1 rs3077 reduced the probability of persistent HBV infection, increased the spontaneously resolved of HBV infection and reduced the risk of progression to advanced liver disease in HBV infection. By combining data from several independent studies and multiethnic populations, this meta-analysis could provide more reliable insight into the association between HLA-DP variants and HBV infection than individual studies (Haidich, 2010). SNP rs9277535 was not significantly associated with the spontaneously resolved of HBV infection in this Indonesian population, but associated with persistent HBV infection. Other studies involving Japanese, Korean (Nishida et al., 2012), Chinese (An et al., 2011; Hu et al., 2012; Hu et al., 2013; Li et al., 2011; Liao et al., 2014; Wang et al., 2011; Wong et al., 2013), Taiwanese (Chang et al., 2014), and Thai (Posuwan et al., 2014) populations showed that the A allele might protect against persistent HBV infection, but opposite findings were reported for Han Chinese (Guo et al., 2011) and Saudi Arabian (Al-Qahtani et al., 2014) populations. This discrepancy could be attributed to the variability of participants, the evaluated clinical factors, and the population heterogeneity among studies (Blackwell et al., 2009). Nevertheless, the overall effects of our meta-analysis showed that rs9277535 variant associated

with reduced risk of persistent HBV infection and increased the spontaneously resolved of HBV infection. Consistent with earlier studies (Al-Qahtani et al., 2014; An et al., 2011; Hu et al., 2012; Li et al., 2011; Liao et al., 2014; Migita et al., 2012), we found no association between HLA-DPB1 rs9277535 and HBV progression. One possible explanation is the highly complex interactions between multiple factors underlying the progression of HBV infection, including host factors, the route of HBV infection, HBV genotype and mutations, the environment, food toxins, and history of treatment (Chisari et al., 2010; Guidotti and Chisari, 2006; Mutimer and Lok, 2012). Further studies should focus on the multifactorial effects on HBV infection. We found that the C/G variants of rs3077 and rs9277535, but not rs3135021, were associated with chronic HBV infection in an Indonesian population. These differences might be attributable to the sample size and the genetic diversity of the populations. A phylogenetic tree of 54.794 autosomal SNPs of 75 populations showed that the Indonesian clade clustered separately from that of Japanese, Korean, Chinese, Taiwanese, and Thai, with a high bootstrap value. Indonesia, which is located near the equator, has a higher haplotype diversity than other populations at northern latitudes (Hugo Pan-ASIAN SNP Consortium, 2009). Moreover, the number of ethnic groups in Indonesia is highly more heterogeneous than Japan. Japan is dominated by Mainland Japanese (98,5%) (Jinam et al., 2015). Whereas Indonesia is composed by 3 dominant ethnics (Javanese, 40.06%; Sundanese, 15.51%; and Malay, 3.7%) and more than 10 smaller ethnic groups (Ananta et al., 2015). The multi-ethnic provides higher opportunity of population admixture and gene flow which reducing LD (Pfaff et al., 2001). Therefore, the LD values differed between the Japanese and Indonesian populations. The rs3077 and rs9277535 SNPs, which influence the host's immunity to chronic HBV infection, are associated with the reduced expression of complementary DNA (cDNA) in the liver and monocytes of patients, as well as the reduced expression of HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277535) in human liver (O'Brien et al., 2011). These effects were thought to be attributable to cis-acting variants (Cheung and Spielman, 2009; Zhang et al., 2014), miRNA binding site variations (http://snpinfo.niehs.nih.gov/) and occurred independently (O'Brien et al., 2011).

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Fig. 2. Forest plot for the association between the HLA-DP variants and HBV outcome in dominant genetic model. (a) HBV susceptibility and HLA-DPA1 rs3077. (b) HBV spontaneously resolved and HLA-DPA1 rs3077. (c) HBV progression and HLA-DPA1 rs3077. (d) HBV susceptibility and HLA-DPB1 rs9277535. (e) HBV spontaneously resolved and HLA-DPB1 rs9277535. (f) HBV progression and HLA-DPB1 rs9277535.

We also found differences in the distributions of the four haplotypes between HBV carriers and healthy controls, but not among the other groups of subjects. A combination of multiple haplotype markers on the same chromosome could provide more information than a single marker (Liu et al., 2008). Based on the computer-estimated frequencies, two haplotypes (CA for rs3077–rs9277535 and GA for rs3135021– rs9277535) might exert a protective effect against chronic HBV infection. Both haplotypes carried the minor A allele of HLA-DPB1 rs9277535. The rs3077 and rs3135021 corroborated the effect of rs9277535. This finding reinforces the protective role of A allele with lower susceptibility to chronic HBV infection. Recently, it was reported that rs9277535 variant was also associated with antibody titers after two doses of Hepatitis B vaccination in Indonesian children in the Riau Archipelago (Png et al., 2011). This concordant result suggests that a response mechanism shared by HBV vaccination and chronic infection is related to the role of HLA-DP in HBV antigen presentation, T-cell priming, and adaptive immunity (Murphy, 2011). In summary, this study has demonstrated that HLA-DP variants were protective against HBV infection in an Indonesian population. The heterozygote and minor homozygote genotypes of HLA-DPB1 rs9277535 and HLA-DPA1 rs3077 exerted a protective effect by reducing the susceptibility to HBV and increasing the spontaneously resolved of HBV infection, respectively. Larger-scale studies and functional analyses of the molecules involved are required to confirm and extend our findings. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.03.034. Acknowledgments The authors are thankful for all willing subjects who participated in this research. We cannot thank those medical staffs enough for being helpful throughout the entire process of this study. We also thank Adi Utarini, Bambang S. Riyanto, and Ginus Partadiredja for their encouragement; Woraphat Ratta-apha, Dewiyani I. Widasari, Hanggoro T. Rinonce, and Bayu S. Wiratama for their invaluable advise of technical

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