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Putative Association of ITGB1 Haplotype with the Clearance of HBV Infection. Integrins are transmembrane receptor proteins that mediate cell-cell adhesion and cell-extracellular matrix (ECM) adhesion. The deregulation of cellECM adhesion and the abnormal expression of beta1 (β1) integrins (ITGB1s) are involved in tumor development and metastasis. In the liver, the expression of integrins and ECM proteins can be a cause of hepatocellular carcinoma (HCC) development. We performed direct DNA sequencing of 24 individuals, and identified 23 sequence variants of ITGB1 polymorphisms. Among these 23 variants, 7 common variants were selected based on frequencies and linkage disequilibrium, and then genotyped in a larger-scale group of subjects (n=1,103). The genetic associations of ITGB1 polymorphisms with the clearance of HBV and HCC outcome of HBV patients were analyzed using logistic regression models and Cox relative hazard models. Although there was no significant association observed between the polymorphisms and the HCC outcome of HBV patients, the second most common haplotype (ITGB1 haplotype2 [C-C-C-C-T-C-T]) was putatively associated with HBV clearance (OR=0.75, p=0.008 and Pcorr=0.05). The minor allele frequency (MAF) of ITGB1 haplotype2 of the spontaneously recovered (SR) group was significantly higher than that of the chronic carrier group (CC) (freq. = 0.248 vs. 0.199). The information derived from this study could be valuable for understanding the genetic factors involved in the clearance of HBV. The hepatitis B virus (HBV) is a crucial factor in bringing about acute and chronic liver diseases (Lin et al., 2005). Approximately 350 million people are chronically infected with HBV all over the world. In fact, more than 1 million HBV carriers die annually because of HBVrelated diseases such as liver cancer (Chang et al., 2005). Each year, nearly 50 million cases are newly diagnosed, and out of that, 90% of the affected individuals were infants while the remaining 5∼10% were adults (Merican et al., 2000). Continuous HBV infection can cause liver cirrhosis (LC) and hepatocellular carcinoma (HCC) (Lin et al., 2005). HCC is the most widespread and severe form of malignancies that are diagnosed in adults. Patients infected with the hepatitis B or C virus are more likely to develop HCC, which in turn is accompanied by liver cirrhosis. The progression of HCC results in a stepwise series of events. Each step in the development of HCC appears to be linked to separate genetic and epigenetic aberrations. These changes are associated with alterations in the expression or formation of an oncogene or a tumor suppressor gene (Patil et al., 2009). In addition, several previous studies have also reported associations between genetic polymorphisms and the risk of HCC and/or HBV clearance, e. g., histone deacetylase10 (HDAC10) and secreted phosphoprotein1 (SPP1) polymorphisms, and interleukin10 (IL10) haplotypes were also shown to be associated with HBV clearance and/or HCC development (Lin et al., 2005; Park et al., 2007; Shin et al., 2003; Shin et al., 2007). Members of the integrin family, the beta1 (β1) integrins (ITGB1; MIM# 135630) are heterodimeric structures consisting of a common β1 subunit that is noncovalently associated with one of nine different α subunits. These molecules are widely distributed in various cells and they mediate cellcell and cellECM interactions that are related to many biological functions in the development of cell or tissues, hemostasis and immune response (Garrido et al., 2001). It has been shown that the deregulation of cell adhesion to the ECM and the abnormal expression of ITGB1s, particularly the α5β1 downregulation, are closely associated with tumor development and metastasis. Especially in the liver, the 　expression of integrins and ECM proteins has been linked to HBV infection and HCC development (Lara-Pezzi et al., 2001; Lee et al., 2009). Based on these observations in the area of tumor and cancer development, we hypothesized that the polymorphisms in the ITGB1 gene could influence the clearance of HBV and HCC progression among HBVinfected patients. We performed extensive screening of ITGB1 by direct sequencing to detect polymorphisms, and we examined their genetic associations with HBV clearance and HCC progression. Here, we report 23 genetic polymorphisms identified in ITGB1 and their genetic associations with HBV clearance and HCC progression in a Korean population (n=1,103). A total of 1,103 Korean subjects having either present or past evidence of HBV infection were prospectively extracted from the outpatient clinic of the liver unit and from the Center for Health Promotion of Seoul National University Hospital from January 2001 to August 2003. These subjects were divided into two groups according to their serologic markers: the chronic carrier (CC) group and the spontaneously recovered (SR) group. The CC and SR groups were composed of 670 and 433 subjects, respectively (Table 1). The HBsAg-positive patients (CC group) were hepatitis B surface antigen (HBsAg) positive over a 6-month period. They were followed up for disease progression at least every 6 months. The diagnoses of the CC and SR subjects were established by repeated seropositivity of the hepatitis B surface antigen (EnzygnostⓇ HBsAg 5.0; Dade Behring, Marburg, Germany), antiHBs (EnzygnostⓇ AntiHBs II) and antiHBc (ABCorek; DiaSorin s. r. l., Saluggia, Italy) of the IgG type without HBsAg, respectively, over a 6month period. The CC group was further divided into two subgroups, i. e., those without (the CH/LC group; n=343) and those with HCC (the HCC group; n=327), according to the absence or presence of HCC, respectively. We excluded subjects who were positive for antiHBs only and not for antiHBc, as well as those who were positive for antiHCV and antiHIV (GENEDIAⓇ; Greencross Life Science Corp., Yongin, Korea, HCVⓇ3.2; DongA Pharmaceutical Co., Seoul, Korea). The patients who had other types of liver disease such as autoimmune hepatitis, toxic hepatitis, primary biliary cirrhosis, or BuddChiari syndrome were also excluded from the sample. None of the patients had a previous history of immunosuppression or antiviral treatment. Informed consent was gained from each patient, and the Institutional Review Board of Human Research at Seoul National University Hospital approved the study protocol. Liver cirrhosis was diagnosed pathologically or by the clinical evidences of portal hypertension such as visible collateral vessels on the abdominal wall, esophageal varices on the esophagogastroscopy, palpable splenomegaly, and sonographically definite findings of cirrhotic liver or ascites. HCC was diagnosed as described previously (Bruix et al., 2001). The clinical parameters are summarized in Table 1. Using the ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City, CA), we sequenced all exons, including exonintron boundaries and promoter regions (∼1.5 kb), to discover polymorphisms of the ITGB1 gene using DNA samples of 24 unrelated healthy Korean individuals. Twenty seven primer sets for amplification (Supplementary Table 1) and sequencing analysis were designed based on GenBank sequences (NT_008705.15). Sequence variants were verified by chromatograms. In order to genotype the polymorphic sites in our study, amplifying primers and probes were designed for TaqMan (Livak, 1999). Primer Express (Applied Biosystems) was used to design both the PCR primers and the MGB TaqMan probes. One allelic probe was labeled with the FAM dye and the other with the fluorescent VIC dye (Supplementary Table 2). Typically, PCR was run in the TaqMan Universal Master mix without UNG (Applied Biosystems) at a primer concentration of 900 nM and a TaqMan MGB-probe concentration of 200 nM. The reaction was performed in a 384-well format in a total reaction volume of 5 ul using 20 ng of genomic DNA. The plate was then placed in a thermal cycler (PE 9700, Applied Biosystems) and heated for 2 min at 50oC and for 10 min at 95oC, followed by 40 cycles of 95oC for 15 s and 60oC for 1 min. The TaqMan assay plate was then transferred to a Prism 7900HT instrument (Applied Biosystems) where the fluorescence intensity of each well was read. Fluorescence data files from each plate were analyzed by automated software (SDS 2.1). Primer sequences are listed in Supplementary Table 1. Linkage disequilibrium (LD) was inferred using the algorithm (Haploview) that searches for a spine of strong |D’| and LD coefficient r2 running from one marker to another (Barrett et al., 2005). Haplotypes of each individual were determined using the algorithm (PHASE, version 2.0) developed by Stephens et al (Stephens et al., 2001). Subjects with missing genotypes were omitted in the analysis of individual singlenucleotide polymorphisms (SNPs) and haplotypes. The genotyping success rate was ＞ 99%, which makes the omission of a few individuals unlikely to change the results of the analysis. For analysis of viral clearance as an outcome, logistic regression models were used for calculating odds ratios (95% confidential interval) and corresponding pvalues controlling for age (continuous value) and sex (male=0, female=1) as covariates. Cox models were used for calculating relative hazards and Pvalues controlling for sex and status of liver cirrhosis among the CC group. The effective number of independent marker loci in ITGB1 was calculated to correct for multiple testing, using the software SNPSpD (http://genepi. qimr. edu. au/ general/daleN/SNPSpD/), which is based on spectral decomposition (SpD) of matrices of pairwise LD between SNPs (Nyholt, 2004). The resulting number of independent marker loci was applied to check for multiple testing. Statistical powers were calculated using the software, “Power for Genetic Association Analyses” (PGA) (Menashe et al., 2008). PGA is an application specifically designed to calculate statistical power and other values in case-control association studies. A co-dominant (1df) model with relative risk 1.3, disease prevalence value of HBV 7.1% (Lee et al., 1998), effective degree of freedom (EDF) 2, and an alpha error level of 5% was used to calculate the statistical power. Through direct sequencing of 24 individuals, we identified 23 sequence variants in the ITGB1 gene: 4 in the promoter region, 7 in coding regions of exons, 11 in introns, and 1 in the 3’-untranslated region (3’UTR) (Fig. 1A). Pairwise comparisons of all 23 polymorphisms revealed two sets of markers in absolute linkage disequilibrium (LD) (|D’|=1 and r2=1, Fig. 1A). Among these 23 variants, 7 common polymorphisms (−401T＞C, −170C＞A, ＋5479A＞C, ＋7377C＞T, ＋9685T＞C, ＋33920G＞T, and ＋34701C＞T) were selected for larger-scale genotyping (n=1,103) based on location, minor allele frequency (MAF＞0.05), and LD. No significant deviations from Hardy-Weinberg equilibrium (HWE) were observed (p＞0.05, Table 2). Five major haplotypes showed frequencies greater than 0.05 and accounted for over 89.6% of the distribution (Fig. 1B). Statistical powers of each polymorphism are shown in Table 3. Association analyses of HBV clearance (CC vs. SR) and HCC occurrence (HCC vs. CH/LC) for each polymorphism and haplotype of the ITGB1 gene were performed using logistic regression models, controlling for age and sex as covariates (Table 3). The Pvalues and odds ratios of each polymorphism and haplotype are shown in Table 3. Among polymorphisms, the second most common haplotype of the ITGB1 gene, ITGB1 haplotype-2 [C-C-C-C-T-C-T], was found to be putatively associated with HBV clearance, i. e., the frequency of individuals bearing the ITGB1 haplotype2 allele among the SR group was significantly higher than those in the CC group (freq. =0.248 vs. 0.199, OR=0.75, p=0.008, Pcorr=0.05). Although the significances were not retained after correction for multiple testing, similar associations were also observed in −401T＞C (rs2504001) and ＋33920G＞T (rs2153875). There was no association observed between ITGB1 polymorphisms or haplotypes and HCC occurrence (Table 3). To analyze the role of ITGB1 polymorphisms in the onset age of HCC, Cox relative hazards analysis for age of HCC occurrence was performed for the CC group. No significant association was observed (Table 3). Integrins play a major role in cell-cell and cellECM adhesion. They mediate signaling cascades that are involved in many cell functions. Upregulated β integrins in the vascular endothelium function as angiogenesis inhibitors in blood vessels of some human tumors (Bridger et al., 2008; Carlson et al., 2008). The abnormal expression of β integrins and the deregulation of cell ECM adhesion can be causes of tumor cell development, growth, and metastasis. Previous studies have suggested that the downregulation of integrin α5β1 is associated with the growth of tumor cells (LaraPezzi et al., 2001; Bridger et al., 2008), and higher expression of β1 integrin (ITGB1) mRNA is significantly associated with docetaxel resistance, which inhibits the effect of chemotherapy for esophageal squamous cell carcinoma (Mori et al., 2008). In addition, β integrins have key roles in the primary tumor formation, metastatic dissemination and inhibition of tumor cell senescence in mouse models which have breast and pancreatic cancer (Streuli and Akhtar, 2009). In this study, we demonstrated that the second most common haplotype, ITGB1 haplotype2 [CCCCT CT], was putatively associated with HBV clearance. In addition, there were association signals in −401T＞C (rs2504001) located in the promoter region and ＋33920G＞T (rs2153875) in intron 14, although significances were not retained after correction for multiple testing. If the promoter polymorphism is located in transcription binding sites, it can induce the alteration of the transcription factor binding and affects transcriptional regulation. Previous studies demonstrated the associations of SNPs in promoter regions with HBV infection and/or HCC occurrence. First of all, histone deacetylase 10 (HDAC10) polymorphism (HDAC10589C>T) was associated with the clearance of HBV infection and onset age of HCC. The promoter activity of this SNP was measured by luciferase activity reporter assay. The functional assay showed that luciferase activity of “T” allele was significantly higher than that of “C” allele of HDAC10589C＞T (Park et al., 2007). Second, interleukin18 (IL18) polymorphism (IL18148G＞C) in promoter is associated with the risk of HCC. The promoter activity of IL18148G＞C measured by luciferase assay revealed that −148C allele represses transcriptional activity compared with the −148G allele (Kim et al., 2009). In addition, the promoter SNPs of tumor necrosis factorα (TNFα) (TNFα863 and TNFα308) (Kim et al., 2003b) and transforming growth factor β1 (TGF β1) (TGFβ1509) were associated with HBV clearance and/or HCC occurrence (Kim et al., 2003a). The involvements of β integrin polymorphisms in HBV infection and HCC development have not been reported in previous studies. However, SNPs in other family member of integrin genes showed associations with 　　　　　　　HBV and HCC. Integrin αv (ITGAV) SNPs, which are located in introns (rs9333289, rs11685758, and rs2290083) and 3’-UTR (rs1839123), and a haplotype (haplotype1 [C-C-G]) were found to be significantly associated with chronic hepatitis and HBV-infected HCC in a Korean population (Lee et al., 2009). In that study, rs9333289, rs11685758 and rs1839123 were associated with susceptibility to HBVinfected HCC, and rs2290083 was associated with susceptibility to both chronic infection of HBV and HBVinfected HCC. In addition, the major hap lotype, haplotype-1 [C-C-G] for rs11685758rs2290083 rs1839123, was associated with decreased susceptibility to chronic infection of HBV (OR=0.59, 95% CI=0.36∼0.97, p=0.04) and HBV-infected HCC (OR=0.58, 95% CI=0.34∼0.98, p=0.04). In comparison with our study, the association between haplotype-1 [CCG] and the decreased susceptibility to chronic HBV infection was similar to our results with ITGB1 haplotype2 [CCCC T-C-T]. The previous study may be less reliable than our study because of relatively small subjects (n=304) and p-values which did not undergo multiple testing corrections. However, in spite of the relatively less reliability, this study demonstrated the association between ITGAV polymorphisms and susceptibility to HBV infection (Lee et al., 2009). Therefore, the alpha subunit of integrin, ITGAV may be a candidate partner for the beta subunit of integrin, ITGB1 in the case of HBV infection. Additional studies have suggested the associations between integrin polymorphisms in introns and human diseases. In particular, α4 and α9 integrin polymorphisms (ITGA4 and ITGA9, respectively) were shown to be associated with autism and nasopharyngeal carcinoma (NPC), respectively. One ITGA4 polymorphism in intron (rs155100) showed association with autism in a Portuguese population, and eight ITGA9 intron polymorphisms (rs169188, rs197721, rs149816, rs169111, rs197770, rs2212020, rs189897, and rs197757) were associated with NPC in a Malaysian Chinese population (Correia et al., 2009; Ng et al., 2009). Similar associations between integrin polymorphisms and human diseases in various European populations were proposed by another study, i. e., α4 integrin polymorphisms (ITGA4) (rs1449263 in the promoter and rs3770138 in an intron) were significantly associated with multiple sclerosis (MS) in Basque and Nordic populations (The rs1449263 polymorphism was associated with MS only in the Basque population, whereas the rs3770138 was associated only in the Nordic population. ) (O'Doherty et al., 2007). In summary, we identified 23 genetic variants in the human ITGB1 gene. Seven common polymorphic sites were selected for genotyping in our HBV cohort, and statistical analyses showed that ITGB1 haplotype2 [C-C-C-C-T-C-T] was putatively associated with HBV clearance. Our findings will provide useful information for further genetic studies of this important gene.