articleLack of association between the GRP 78 polymorphisms in the promoter and 3 ' UTR and susceptibility to chronic HBV infection in a Chinese Han population

Background: Hepatitis B virus (HBV) infection causes large amount of unfolding or false-folding protein accumulation in the endoplasmic reticulum (ER), which in turn induces the expression of glucose-regulated protein 78 (GRP78). The aim in the present study was to analyse the potential association between GRP78 single-nucleotide polymorphisms (SNPs) and the risk of HBV infection. Methods: The associations between seven common GRP78 polymorphisms in the promoter (rs391957, rs17840762, rs17840761, rs11355458) and in the 3' untranslated region (UTR) (rs16927997, rs1140763, rs12009) and possible risk of chronic HBV infection were assessed in a case-control study. 496 cases and 539 individually matched healthy controls were genotyped. Results: Overall, no associations were observed in genotypic analyses. In addition, haplotypes and diplotypes combining those SNPs in the promoter or in the 3' UTR in high linkage disequilibrium (LD) were also not associated with HBV risk. Conclusion: These observations do not support a role for GRP78 polymorphisms in HBV infection in a predominantly Chinese Han population. Background Hepatitis B virus (HBV) infection is one of the major infectious diseases that may lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) [1]. Approximately 30% of the world's population has been infected with HBV and approximately 350 million are persistent carriers. The mechanisms underlying resolution of acute HBV infection or its progression to chronicity remain undetermined [2]. Approximately 90% of infants infected from HBV e antigen (HBeAg) positive mothers will fail to achieve clearance and develop persistent HBV infection. Whereas, for adults, the majority of HBV-infected individuals achieve clearance with just less than 10% becoming persistent carriers[3]. Glucose-regulated protein 78 (GRP78), also recognized as immunoglobulin heavy-chain binding protein (BiP) as it was found bound to immunoglobulin heavy chains in pre-B cells, is known to be induced in the endoplasmic reticulum (ER) compartment by a variety of stresses [4]. These stress signals include glucose starvation, ER Ca2+ depletion, virus infection or cancer progress which leads to accumulation of misfolded proteins in the ER. It has been shown that GRP78 functions as an antiapoptotic chaperone playing a key role in maintaining the proper functions of proteins and organelles [5]. Recent studies have emphasized that GRP78 play important roles in the progression inhibition of chronic HBV infection and HCC progression [6,7]. Overexpression of the large superficial protein of HBV which trans* Correspondence: bioxzhu@yahoo.com 1 Institute of Oncology, Affiliated Tumor Hospital, Guangzhou Medical College, Guangzhou, PR China † Contributed equally Full list of author information is available at the end of the article © 2010 Zhu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhu et al. BMC Medical Genetics 2010, 11:83 http://www.biomedcentral. com/1471-2350/11/83 Page 2 of 6 lated from transcripts specified by a preS1 promoter in Huh7 cells results in a blockage of secretion of hepatitis B surface antigen (HBsAg), which leads to an accumulation of HBsAg in the ER lumen and in turn induces expression of GRP78[8]. The presence of pre-S mutants of HBV in sera and tissues was related to a high risk of developing HCC, and subsequently induce ER stress, leading to the expression of GRP78 [9-11]. In the previous projects, we focused on the possible association between HBV infection or HCC risk/prognosis and GRP78 polymorphism involving a new mutation (-87 T>A, from the estimated translation start site of GRP78 gene), or an intronic mutation (rs430397 G>A, in the intron 5 of GRP78 gene). These studies indicated that GRP78 was associated with HCC progression, but not associated with HBV infection [12,13]. As a part of series of studies about relationship of polymorphism(s) and stress-associated diseases, we hypothesized that polymorphisms in promoter and 3' untranslated region (UTR), which were known to be involved in regulation of gene expression, may be contributing factors or markers of a stress-associated disease, such as virus infection. To test this possibility, we further compared GRP78 polymorphisms at seven common loci in the promoter region (rs391957, rs17840762, rs17840761, rs11355458) and the 3' UTR (rs16927997, rs1140763, rs12009) between chronic HBV carriers and healthy subjects, and to reveal the possible association between HBV infection and GRP78 gene. Methods Subjects 496 Han patients with chronic HBV infection as case series who had been treated as inpatients or outpatients were enrolled in several affiliated hospitals of Guangzhou Medical College between January 2001 and April 2005. The diagnosis of chronic HBV infection was based on the presence of HBsAg and HBeAg, or HBsAg and anti-HBe, together with the absence of anti-HBs, for at least 36 months (according to the corresponding inpatient and outpatient records) prior to enrolment. All these were tested to exclude hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infection, the presence of autoimmune hepatitis, and/or alcohol consumption exceeding 8 g per day and other conspicuous diseases. Patients who received antiviral therapy including interferon or nucleoside analogues within one year of enrollment were also excluded. 539 unrelated healthy HBV non-infected controls were recruited from Han volunteer blood donors in above hospitals and the Department of Cancer, Zhongshan School of Medicine in Sun Yat-sen University in the same time. The controls were negative for HBsAg, anti-HBs and anti-HBc, with normal serum alanine aminotransferase (ALT) levels. These subjects were members of the healthcare staff of the hospitals who were periodically monitored for professional exposure to hepatitis viruses. A standard informed consent, was given to the participants, after the nature of study had been fully explained. The study was performed with the approval of the ethical committee of Guangzhou Medical College and adhered to the tenets of the Declaration of Helsinki. Genotyping Genomic DNA was extracted from peripheral blood leukocytes using QIAGEN QIAamp DNA Mini Blood Kit (Hilden, Germany). The SNPs were detected by the TaqMan Assay-by-Design service (Applied Biosystems, Foster City, CA). The details of sequences and reaction conditions are available upon request https://products.appliedbiosystems. com/ab/en/US/adirect/ab. PCR was performed using the TaqMan Universal Master Mix without UNG on the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA) and heated to 95°C for 10 minutes followed by 40 cycles of 92°C for 15 seconds and 60°C for 1 minute. Haplotype and diplotype construction Haplotype frequencies and pairwise linkage disequilibrium (LD) matrices were determined using the Haploview version 3.2.0 (Whitehead Institute for Biomedical Research, USA)[14]. Magnitudes of LD were represented by the standardized LD coefficients, complete association (D') and absolute association (r2) [15-17]. The frequencies closely agreed with results from a maximum likelihood method implemented via an expectation-maximization (EM) algorithm[18]. Haplotypes and diplotypes were selected according to the corresponding occurring probabilities with a higher likelihood (> 0.95 as cut-point) [19]. Table 1: Characterization of the participants Characteristics Cases (%) Controls (%) P