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. 2012 Sep;31(9):1492–1498. doi: 10.1089/dna.2012.1709

Association of GNLY Genetic Polymorphisms with Chronic Liver Disease in a Korean Population

Geun-Hee Park 1, Kyoung-Yeon Kim 1, Jae Youn Cheong 2, Sung Won Cho 2, KyuBum Kwack 1,✉
PMCID: PMC3429283  PMID: 22788687

Abstract

Granulysin (GNLY) is found in cytotoxic granules of cytolytic T lymphocytes and natural killer (NK) cells, which are critical for hepatitis B virus (HBV) clearance. GNLY cytotoxicity plays an important role in the defense against viruses or intracellular bacteria. We hypothesized that genetic variation in the GNLY gene could affect the resistance of hosts against HBV infection. We compared the distribution frequencies of GNLY polymorphisms between an HBV-induced chronic liver disease (CLD) group and a spontaneous recovery (SR) control group to determine whether GNLY polymorphisms play a role in HBV clearance. A total of 117 patients in the SR group and 230 patients in the CLD group were enrolled. Samples derived from complex infections, including hepatitis C and human immunodeficiency virus, and those associated with insufficient clinical information (10 samples in SR and 24 samples in CLD) were excluded from the study. The final analysis included 107 SR and 206 CLD samples. DNA was extracted from peripheral blood, and GNLY genotypes were determined by the GoldenGate® method. The genotype distribution of the single-nucleotide polymorphisms (SNPs) rs2886767 (C>T), rs1561285 (G>C), and rs11127 (T>C) were significantly different between the SR and CLD groups in a recessive model (p<0.015). These three SNPs were in a complete linkage disequilibrium (LD) block. Diplotype distributions of haplotype (HT) 1 (C-G-T) and HT2 (T-C-C) were significantly different between the SR and CLD groups in a recessive model (p=0.025) and a dominant model (p=0.008). All p-values remained significant after multiple comparisons. GNLY polymorphism genotypes and diplotypes were associated with the chronicity of HBV. These data suggested that genetic variation of GNLY may be an important factor in HBV clearance through the CD8+ T or NK cell-mediated removal of HBV-infected cells from the host.

Granulysin is one of four bioactive mediators released from the granules of cytolytic T cells and natural killer cells. Since CTLs are critical for eliminating viral infections, a polymorphism in one of these proteins might lead to altered disease. In this study, hepatitis B virus infection is modulated by granulysin polymorphisms.

Introduction

Between 350 and 400 million people worldwide are chronically infected with the hepatitis B virus (HBV) (Lee, 1997; Lok and McMahon, 2007). While most HBV-infected patients show spontaneous recovery (SR) mediated by the host immune system, 5%–10% of patients fail to recover and develop chronic liver disease (CLD), including chronic hepatitis and cirrhosis (Thio et al., 2003). HBV-induced CLD is a major risk factor for hepatocellular carcinoma (HCC) (Cha and Dematteo, 2005), which is one of the most common causes of cancer-related mortality. Although the effects of HBV infection have been studied extensively, the factors involved in the progression from acute hepatitis B to CLD have not been clearly defined. The chronicity of HBV is not only affected by environmental factors, viral strains, and gender and age at infection but also by the genetic variation of the host.

The Lin group (Thio et al., 2003) reported that age- and sex-matched monozygotic twins and dizygotic twins showed significant differences in the concordance rates for HBV infection. Moreover, the Carrilho group (Carrilho et al., 2005) measured the frequency of HBV markers in genetically related (consanguineous) and nongenetically related (nonconsanguineous) Brazilian families of Asian and Western origins and reported significantly higher (p<0.001) HBsAg levels in Asian than in Western family members. These results suggest that genetic variations can result in different outcomes of HBV infection. Single-nucleotide polymorphisms (SNPs) are important sources of genetic variation, and the pathophysiology of HBV infection has been shown to be affected by individual genetic variants, including SNPs (Frodsham, 2005). The results of an SNP association study on the chronicity of HBV infection showed a variety of innate immunity-associated genes, including human leukocyte antigens (Guo et al., 2011), NKG2D (Ma et al., 2010), tumor necrosis factor-alpha (TNF-α) (Kim et al., 2003), mannose-binding lectin (Thio et al., 2005), interleukin-18 (IL-18) (Cheong et al., 2010), and cytotoxic T lymphocyte antigen 4 (Thio et al., 2004). In Korea, which is an HBV endemic area (Cheong, 2008; Park et al., 2010), the annual mortality of HBV-induced HCC is an important problem (Lavanchy, 2004). The study of variation in innate immunity-associated genes is important to better understand the development of HBV chronicity in the Korean population.

A chronicity of HBV study that analyzed the recovery mechanisms leading to the host innate immune responses against HBV infection demonstrated differences in innate immunity between chimpanzees and transgenic mice (Litsov, 1975; Kagi et al., 1994). A strong cytolytic T lymphocyte (CTL) response is an important factor in the chronicity and pathogenesis of HBV infection. In particular, CD8+ T cells play a significant role in the inhibition of HBV gene expression and prevention of HBV RNA-containing capsid assembly. In addition, they contribute to the induction of apoptosis in virus-affected hepatocytes via two general pathways of cytotoxicity, the TNF receptor super family, member 6 (Fas)/Fas ligand apoptotic pathway, and the granule exocytosis pathway with granzyme B and perforin (Kagi et al., 1994; Lowin et al., 1994; Stenger et al., 1999; Robek et al., 2002; Thimme et al., 2003).

Natural killer (NK) cells also play an important role in HBV infection. NK cells do not require activation by specific virus antigens, and can therefore remove the infecting virus soon after infection. Moreover, similar to antigen-specific CD8+ T cells, NK cells can kill the infected cells directly through a physical cell–cell contact and indirectly through interferon-γ and TNF-α, as well as IL-3, granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor (Guidotti and Chisari, 2001). In chronic hepatitis B patients, hepatic NK cells have enhanced cytolytic activity against target cells compared to peripheral NK cells (Zhang et al., 2010).

GNLY, which is found in cytotoxic granules in both CTL and NK cells, is a member of the saposin-like protein (SAPLIP) family. It is released together with the pore-forming protein, perforin, and granzymes upon antigen stimulation, and its expression is regulated by IL2, IL15, and chemokines (Stefanovic et al., 1977; Jongstra et al., 1987). GNLY is encoded by the GNLY gene in chromosome 2p11.2, which encodes a 15-kDa protein that is cleaved after synthesis at its amino- and carboxytermini to produce a 9-kDa protein (Jongstra et al., 1987; Donlon et al., 1990; Yabe et al., 1990). The alternative splicing of GNLY generates NKG5, which is secreted into the extracellular space, and the 15-kDa protein designated as 519. Recombinant 9-kDa GNLY is lytic to tumors and broadly antimicrobial, and it kills gram-positive and gram-negative bacteria, yeast, fungi, and parasites (Stenger et al., 1998).

The reported functions of GNLY suggest that SNPs of GNLY could be determining factors in the chronicity of hepatitis B. Therefore, the present study evaluated the association between GNLY genetic polymorphisms and the chronicity of HBV by comparing GNLY gene polymorphism frequencies between CLD patients and an SR control group in the Korean population using a genotype and diplotype analysis.

Materials and Methods

Case–control study subjects

A total of 347 Korean blood samples were obtained from the outpatient clinic of the Gastroenterology Department and from the Center for Health Promotion of the Ajou University Hospital (Suwon, South Korea) without gender or age restrictions between March 2002 and February 2006. Samples were derived from genetically unrelated Korean patients. The experimental protocol was approved by the institutional review board. The SR group consisted of 117 subjects as controls, and HBV patient samples obtained from 230 CLD patients were divided into two groups according to serologic markers.

All samples were infected with HBV and classified into one of the two groups by a pathologist according to their HBV infection status, clinical data, and serological profile. Every 6 months, for more than 12 months, the 347 patients were subjected to serological tests for serum levels of the hepatitis B core antibody (HBcAb) (Anti-HBc II Reagent Kit; Abbott Laboratories, South Pasadena, CA), hepatitis B surface antigen (HBsAg) (Anti-HBs; Abbott Laboratories), and hepatitis B surface antibody (HBsAb) (HBsAg; Abbott Laboratories). Liver function tests for aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and bilirubin levels were performed using commercially available assays. HBV DNA was detected using an HBV-branched DNA assay (Bayer Diagnostics, Tarrytown, NY). All samples showed elevated ALT at least once during the follow-up period and were positive for HBV DNA, irrespective of hepatitis B e antigen (HBeAg) positivity. The patients in the HBV clearance groups were HBsAg negative, HBeAg negative, anti-HBs positive, anti- HBc positive, and had recovered from HBV infection. The patients in the CLD group were HBsAg positive for more than 6 months with elevated ALT and AST (more than 2-times the normal upper limit) levels.

Samples testing positive for the presence of antibodies against complex infections such as hepatitis C (Genedia HCV ELISA 3.0; GreenCross, Gyeonggi, Korea) or anti-human immunodeficiency virus (HIV Ag/Ab combo; Abbott Laboratories), and samples with insufficient clinical information were excluded. The final analysis included 107 SR and 206 CLD samples.

Sample preparation

Blood samples were stored at −80°C for the handling of human genomic DNA. Genomic DNA was purified using G-DEX blood genomic DNA (gDNA) purification kits (Intron Biotechnology, Inc., Seungnam, Korea). The gDNA was quantified using the picogreen dsDNA quantification reagent, according to a standard protocol (Molecular Probes, Eugene, OR). The plates were read using a VICTOR™ 3 1420 Multilabel counter, and a standard curve for gDNA concentration was created using known concentrations of lambda DNA.

SNP selection and genotyping

A total of three SNPs were selected from a public SNP database (www.ncbi.nlm.nih.gov/snp/) based on their location, suitability for the GoldenGate assay, and frequency among the tagging SNPs for the genotyping assay. The SNPs were rs2886767, which is located in the 5′ flanking region, rs1561285, which is located in the 3th intron, and rs11127, which is located in the fourth exon. Genotyping was performed using the GoldenGate® kit according to a standard protocol (Illumina, Inc., San Diego, CA). Oligos were amplified by allele-specific primer extension. After hybridization to a Sentrix array matrix, signal intensities were read by a BeadArray Reader (Illumina). GenomeStudio software (Version 1.5.16; Illumina) was used for genotyping analysis.

Statistical analysis

The genetic models for the association test were divided according to the minor allele, and the genetic model was considered additive (AA vs. Aa vs. aa), dominant (AA vs. Aa plus aa), or recessive (AA plus Aa vs. aa). Linkage disequilibrium (LD) blocks were checked by the Gabriel method using Haploview software. The χ2-test was used to assess the Hardy–Weinberg Equilibrium (HWE) between the SR group and the CHB group. The difference between groups was determined by the odds ratio (OR). ORs were presented with 95% confidence intervals (95% CIs) and were adjusted for age and sex. All statistical tests were performed using SAS software (SAS 9.1; SAS Institute, Cary, NC), and the significance level was set at p<0.05. The probability values obtained were corrected for multiple testing by using Bonferroni's correction and permutation tests. The Bonferroni's p-value for reaching significance was 0.01 (0.05/5). The Plink program (http://pngu.mgh.harvard.edu/∼purcell/plink/) was used to confirm the results and permutation test (N=10,000).

Results

Clinical characterization

The fate of patients infected with HBV is determined by several factors, including host immune reactions. T cells and NK cells, which play an important role in protection against chronic hepatitis in HBV infections, express GNLY in response to the hepatitis B virus. In the present study, the effect of GNLY polymorphisms on the chronicity of hepatitis B was investigated by analyzing 3 GNLY SNPs in samples from an SR group (HBsAg-, HBcAb+, and HBsAb+), which consisted of 107 control patients who had recovered from HBV infection without treatment, and a CLD group (HBsAg+, HBcAb-, and HBsAb-), which consisted of 206 HBV-induced CLD patients who were still at risk of HBV infection. The age and sex distribution of the patients in the CLD and SR groups was statistically different (data not shown). The characteristics of the study subjects are summarized in Table 1.

Table 1.

Clinical Characteristics of Study Subjects

Variable SR CLD
No. of subjects 107 206
Sex (Male/Female) 80/27 158/48
Age (mean±SD) 46.04±8.40 42.86±9.62
Aspartate aminotransferase (U/L, mean±SD) 30.62±25.45 93.67±161.74
Alanine aminotransferase (U/L, mean±SD) 39.40±29.40 107.36±170.25
Albumin (g/dL, mean±SD) 4.40±0.26 3.96±0.61
Bilirubin (mg/dL, mean±SD) 0.90±0.35 1.71±2.88
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Age, aspartate aminotransferase, alanine aminotransferase, albumin, and bilirubin are summarized and expressed as the mean±SD (standard deviation).

SR, spontaneous recovery; CLD, chronic liver disease induced by hepatitis B virus.

Genotype distribution and genotype analysis

Genetic variants of rs2886767, rs1561285, and rs11127 did not show evidence of departure from HWE. Three genotypes had minor allele frequencies (MAFs)>1% (Table 2). The results of the genotype analysis showed that the CC, GG, and TT genotypes were the most common in the rs2886767, rs1561285, and rs11127 polymorphisms in all groups (Table 2).

Table 2.

Genotype Frequencies and Associations Between Hepatitis B Virus-Induced Disease in GNLY Single-Nucleotide Polymorphisms

 
  
  
  
 Frequency (%)
  
 SR vs. CLD
  
  
  
SNP HWE MAF Genotype SR CLD Model OR Lower CI Upper CI pa pb pc
rs2886767     CC 29 (7.27) 73 (18.3) Additive 1.05 0.75 1.49 0.763    
C>T 0.523 0.421 CT 67 (16.79) 90 (22.55) Dominant 0.66 0.39 1.11 0.117    
Flanking_5UTR     TT 11 (2.76) 43 (10.78) Recessive 2.44 1.19 5.01 0.015 0.027 0.074
rs1561285     GG 32 (8.02) 83 (20.8) Additive 0.99 0.68 1.43 0.951    
G>C 0.121 0.381 CG 70 (17.54) 92 (23.05) Dominant 0.61 0.36 1.01 0.055    
Intron     CC 5 (1.25) 31 (7.77) Recessive 3.78 1.41 10.14 0.008 0.017 0.042
rs11127     TT 29 (7.27) 77 (19.3) Additive 1.05 0.74 1.49 0.790    
T>C 0.434 0.411 CT 70 (17.54) 88 (22.05) Dominant 0.60 0.36 1.01 0.055    
Exon     CC 8 (2.01) 41 (10.28) Recessive 3.26 1.45 7.30 0.004 0.007 0.020
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The CIs and pa values were obtained from logistic regression with additive, dominant, and recessive models.

The pb values were calculated by the permutation test.

The pc values were calculated by Bonferroni's correction.

SNP, single-nucleotide polymorphisms; HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency; OR, odds ratio; CI, confidence interval.

To analyze the genetic association between GNLY polymorphisms and clearance from HBV infection, genetic distributions were statistically analyzed. The SR and CLD samples were compared by a multiple logistic regression analysis with adjustment for gender and age. The GNLY SNP rs2886767 in the promoter region was a risk type associated with the homozygous variant TT genotype in a recessive model (OR=2.44; 95% CI=1.19–5.01; p=0.015). The intronic SNP rs1561285 CC genotype was a risk type, and significant differences in this SNP were detected between the SR and CLD groups in a recessive model (OR=3.78; 95% CI=1.41–10.14; p=0.008). In the nonsynonymous SNP rs11127, the CC genotype was also a risk type and was significantly different between the SR and CLD groups in a recessive model (OR=3.26; 95% CI=1.45–7.30; p=0.004). After performing Bonferroni's correction and the permutation test, rs1561285 and rs11127 SNPs still showed significant correlations (Table 2).

Diplotype analysis

LD blocks of all SNPs in GNLY were constructed by the Gabriel method using Haploview software, which set the cutoff values at MAF>0.1 and HWE p-value>0.05. A single haplotype block of the pairwise D′>0.984 and r2>0.826 is identified in the Table 3. Haplotype (HT) 1 (C-G-T) and HT2 (T-C-C) were the most common (frequency>0.05) and accounted for 98.0% of all haplotypes (Table 4).

Table 3.

Linkage Disequilibrium Coefficient Between Single-Nucleotide Polymorphisms in GNLY

 
 |D′|
  SNPs rs2886767 rs1561285 rs11127
  rs2886767 - 0.988 0.984
r2 rs1561285 0.826 - 1
  rs11127 0.929 0.882 -
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Table 4.

Diplotype Frequencies and Associations Between Spontaneous Recovery and Chronic Liver Disease in GNLY Haplotypes

 
  
 Frequency (%)
  
 SR vs CLD
  
  
  
SNP Diplotype SR CLD Model OR Lower CI Upper CI pa pb pc
HT1 HT1/HT1 28 (8.95) 73 (23.32) Additive 1.03 0.73 1.46 0.854    
C-G-T HT1/- 67 (21.41) 90 (28.75) Dominant 0.65 0.38 1.10 0.109    
  -/- 12 (3.83) 43 (13.74) Recessive 2.23 1.11 4.48 0.025 0.035 0.123
HT2 HT2/HT2 5 (1.6) 31 (9.9) Additive 0.99 0.68 1.43 0.953    
T-C-C HT2/- 69 (22.04) 92 (29.39) Dominant 0.26 0.10 0.71 0.008 0.011 0.042
  -/- 33 (10.54) 83 (26.52) Recessive 1.57 0.95 2.61 0.080    
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The CI and pa values were obtained from logistic regression with additive, dominant, and recessive models.

The pb values were calculated by the permutation test.

The pc values were calculated by Bonferroni's correction.

We noted that in this haplotype characterization. Based on three genetic models, the estimated HTs were used for a diplotype analysis by using logistic regression followed by adjustments for age and sex. The HT1 frequency reached a significant divergence in the recessive model between the SR and CLD groups (OR=2.23; 95% CI=1.11–4.48; p=0.025). Analysis of the HT2 diplotype showed a significant difference between the SR and CLD groups in the dominant model (OR=0.26; 95% CI=0.10–0.71; p=0.008) (Table 4). All diplotype p-values remained significant after the permutation test (HT1; p=0.035, HT2: p=0.011), and the HT2 of the diplotype p-values remained significant after Bonferroni's correction (p=0.042).

Discussion

GNLY has been associated with a variety of infectious diseases, and it is a critical effector molecule for a broad spectrum of pathogenic microorganisms, including Listeria monocytogenes, Trypanosoma cruzi, Salmonella typhimurium, Escherichia coli, and Mycobacterium tuberculosis (Pena et al., 1997; Stenger et al., 1998; Krensky, 2000; Deng et al., 2005). The cell-killing effect of GNLY has been demonstrated using the 9-kDa recombinant GNLY in vitro. GNLY is involved in the removal of viruses such as varicella zoster and Epstein-Barr. Expression of GNLY was associated with the recovery from infection by the herpes simplex virus and HBV (Hata et al., 2001; Sun et al., 2002; Morizane et al., 2005; Li et al., 2010). However, an association study between infectious diseases and genetic variation of GNLY has not been performed to date.

The present study investigated the possible association between GNLY SNPs and the chronicity of HBV. The GNLY polymorphism frequency in the Korean population was compared between 107 controls in the SR group and 206 patients in the CLD group using statistical analyses. According to the SNP database, the rs2886767, rs1561285, and rs11127 SNPs are located in the 5′ flanking region, intron, and the 4th exon of GNLY, respectively, and their genotype frequencies were significantly different between the SR and CLD groups. The risk genotype of rs2886767 was TT, and it was associated with a 2.44-fold increased susceptibility to CLD compared to the CC and CT types. The CC risk genotype of rs1561285 was associated with a 3.78-fold increased OR compared to the CC and CG genotypes. The CC risk type of rs11127 increased OR by 3.26-times compared to the TT and CT genotypes.

Li et al. (2010) reported that the expression of the 9-kDa form of GNLY is significantly increased in response to high levels of HBV DNA in a normal hepatic function group. In the present study, significant differences in the rs2886767 (C>T) genotype in the promoter region of GNLY between the SR and CLD groups led us to screen transcription factors that bind to rs2886767. The binding of several transcription factors, including E1A-binding protein p300 (EP300), IKARPS family zinc-finger (IKZF)-2, GATA-binding protein factor 1 (GATA-1), and GATA-2, to the protective allele (C) of rs2886767 was predicted by TFSEARCH (version 3.1; www.cbrc.jp/research/db/TFSEARCH.html). Interestingly, the binding of EP300 to the region containing the rs2886767 risk allele (T) was not predicted. EP300 is a histone acetyltransferase that regulates the transcriptional machinery through chromatin remodeling and functions as an adaptor molecule. EP300 interacts with several transcription factors and enhances the expression of numerous gene regulatory elements (Chan and La Thangue, 2001; Asahara et al., 2002). Our results suggest the possibility that the genotype-dependent interaction of EP300 with rs2886767 might affect the expression of GNLY.

GNLY is expressed via a toll-like receptor 2-dependent signaling pathway, and GNLY expression is regulated by specific transcription factors, including CCAAT-/enhancer-binding protein beta (C/EBPβ), and activator protein 1 in Acholeplasma laidlawii stimulates THP-1 cells (Kida et al., 2001; Kida et al., 2002). C/EBPβ is a member of the basic region/leucine zipper class of transcription factors that initiates the opening of the chromatin structure and recruits the basal transcriptional machinery. C/EBPβ can recruit so-called coactivators such as the CREB (cAMP-responsive-element-binding)-binding protein (CBP) (Ramji and Foka, 2002). EP300 binds to the rs2886767 protective allele (C) through 5 protein interaction domains, the structures of which are similar to that of CBP. The function of C/EBPβ is strongly inhibited by the E1A protein. After the coactivation of EP300 and C/EBPβ, the inhibitory effect of E1A on C/EBPβ is blocked by EP300 binding. Moreover, C/EBPβ and EP300 enhance the expression of target genes up to 18.4-fold in the presence of E1A deficiency in vivo (Janknecht and Hunter, 1996; Mink et al., 1997). The interaction between C/EBPβ and EP300 mediated by the rs2886767 protective allele may enhance GNLY expression. A genetic model derived from the current statistical analysis suggests that the combination of the protective genotypes CC and CT may efficiently eliminate HBV-infected cells by increasing GNLY expression. Therefore, polymorphisms of rs2886767 may help SR from HBV infection.

GNLY rs11127, which is located in the 4th exon, changes the amino acid sequence of the protein product as a nonsynonymous SNP through allelic exchange. To determine the effect of amino acid changes, we used the SIFT (http://sift.jcvi.org) and PolyPhen (http://genetics.bwh.harvard.edu/pph/) software programs, which were reported to have a high rate of correct predictions by functional testing. These programs showed that rs11127 amino acid changes did not affect the structure of GNLY (Luoma et al., 2010). We therefore examined the SNP database for possible protein structural changes in GNLY. In silico assay results showed that changes in the genotype of rs35146240 may have a damaging effect. GNLY rs10181739 and rs71806663 are a predicted stop codon and essential splice site, respectively, according to a genetic database (http://asia.ensembl.org). Amino acid changes caused by genetic variants, including nonsynonymous SNPs, stop codons, and frame shifts, can induce structural changes.

As a member of the SAPLIP family, GNLY is positively charged and is targeted to negative charges on the surface of cells. The crystal structure of GNLY shows 5 helix bundles, and it has been reported to lyze the membrane by using helices 1, 2, and 3 in a scissoring motion, and by twisting and tearing as it enters deeper into the membrane. The formation of a pore in the target via perforin is followed by an increase in intracellular calcium levels as a result of an influx of extracellular calcium and the release of calcium from intracellular stores (Kaspar et al., 2001; Anderson et al., 2003). Nonsynonymous genetic variation of GNLY possibly induces the formation of an abnormal structure that does not recognize HBV-infected cells or results in the loss of the lytic function mediated by the five-helix bundle. This may alter the formation of the pore in HBV-infected cells, thereby enabling the interaction with apoptosis initiation molecules and preventing the early elimination of HBV-infected cells. Therefore, modifications in the structure of GNLY may affect the chronicity of HBV.

The association of SNPs rs2886767, rs1561285, and rs11127 with the chronicity of HBV was characterized by a strong LD (r2=0.826). These SNPs formed C-G-T, T-C-C, T-G-C, and T-G-T HTs in the LD block, and the C-G-T and T-C-C HTs had more than a 95% distribution in all HTs. The LD block contained almost every region in GNLY, including the promoter region to the 4th exon. Diplotype analysis of HT1, which consists of a protective allele combination (C-G-T), showed that HT1 inclusion was associated with a 2.23-time higher chance of SR than HT1 exclusion. HT2 consisted of a risk allele combination, and diplotype analysis of HT2 (T-C-C) showed that the OR toward HBV clearance was 0.26-fold that of the HT2 inclusion type in the HT2 exclusion type. Moreover, according to the Korea association resource dataset (Cho et al., 2009) and the IMPUTE program (Marchini et al., 2007), 24 SNPs, including rs2886767, rs1561285, and rs11127 in the GNLY gene, showed strong LD (D′>0.904). These data suggest that causative variants responsible for HBV clearance may be present in rs2886767, rs1561285, and rs11127 that form an LD block. Therefore, rs2886767, rs1561285, and rs11127 are markers of causative variants associated with the chronicity of HBV.

Our study was limited by a small sample size and few genetic variants. However, this is the first study to investigate the association between GNLY polymorphisms and HBV-induced CLD. In addition, SR patients were used as a control group instead of normal healthy controls to show the effect of genomic background on the chronicity of HBV infection. Distribution of genotypes and diplotypes from both groups remained significant after multiple testing using Bonferroni's correction and permutation tests. The current results are in support of the conjecture that genetic variation in the IFI6 gene affects the clearance of HBV. Further studies should focus on investigating the effect of GNLY expression on HBV clearance. Our results suggest that the genetic variation of GNLY in HBV may play a role in the induction of CLD. In future studies, causal variants that affect GNLY molecular function should be identified through next-generation sequencing of the GNLY gene.

Conclusion

The C allele-containing genotype of rs2886767, the G allele-containing genotype of rs1561285, and the T allele-containing genotype of rs11127 were associated with HBV clearance. These data suggested that genetic variation of GNLY may be an important factor in HBV clearance through the removal of HBV-infected cells from the host by CD8+ T cells or NK cells.

Acknowledgment

We are thankful to every individual who has given us informed consent for this study and to Ju-Young Lee, Ph.D., for critical checking of statistical analysis. Informed consent was acquired from each patient, and the sampling protocols and informed consent forms used were approved by the IRB of Ajou University. Protocols for handling the human genomic materials and all the experimental procedures used were approved by the IRB of CHA University. This work was funded by grants from the Ministry of Health and Welfare, Republic of Korea (A010383 and A080734). Also, this work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093821).

Disclosure Statement

No competing financial interests exist.

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