Association of Polymorphisms intrail and Chronic Hepatitis B in Chinese Han Populations from Shandong Province
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1 374 Available online at Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 Association of Polymorphisms intrail and Chronic Hepatitis B in Chinese Han Populations from Shandong Province Fang Wang 1, Xueliang Zhang 2, Yan Wang 3, and Zhaohui Sun 1 1 Department of Clinical Laboratory, The Second Hospital, Shandong University, Jinan, Shandong, 2 Jinan Blood Center, Jinan, Shandong, 3 Traditional Chinese Medical Hospital, Rongchen, Shandong, China Abstract. Background. HBV is the most common and serious infectious disease in China. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to thetnf-super family and can induce apoptosis in addition to other biological effects. It has been found that the occurrence of chronic hepatitis B is related to polymorphisms of many apoptotic factors. This study therefore aims to investigate the association of TRAIL polymorphism with chronic hepatitis B by observing the polymorphism and gene frequency of the TRAIL gene at 1525G/A and 1595C/T of the 3 -UTR among people of the Chinese Han ethnicity. Methods. This study followed a case-control design. Polymorphisms of the TRAIL gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 187 patients with chronic hepatitis B and 142 healthy controls. Results. The alleles of TRAIL at 1525G/A and 1595C/T have achieved genetic equilibrium in normal individuals (P<0.05) and patients with chronic hepatitis B(P=0.547). The genotype frequencies of TRAIL at 1525GG/1595CC in female patients were significantly higher than those in male patients. The frequencies of the AA genotype at 1525G/A and the TT genotype at 1595C/T sites were significantly lower in patients with chronic hepatitis B with HBeAg positive than in healthy controls (10.5 vs 23.2%, P<0.05). The frequency of the AA genotype at 1525G/A and TT genotype at 1595C/T sites were significantly lower in the chronic hepatitis B individuals than in normal controls (12.3 vs 23.2%, P < 0.05). Conclusions. TRAIL may be related to susceptibility to hepatitis B. Key words: chronic hepatitis B; Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL; gene polymorphism. Abbreviations: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL; Hepatitis B virus, HBV; Hepatocellular cancer, HCC. Introduction Infection with the hepatitis B virus (HBV), a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular cancer (HCC), is a major global health problem. 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection [1]. China has a high prevalence of HBV infection; according to the latest national hepatitis B epidemiological survey, the current HBV carriers in mainland China number 93 million, including million patients with CHB [2,3]. Address correspondence to Zhaohui Sun, MD. Department of Clinical Laboratory, The Second Hospital, Shandong University, 247 Beiyuan Road, Jinan, Shandong, China ; phone: ; fax: ; e mail: szhsdey@163.com TNF-related apoptosis-inducing ligand (TRAIL) is a member of thetnf receptor superfamily. TRAIL is expressed by a wide variety of human cells, including immune cells. TRAIL expression is increased by pro-inflammatory cytokines and LPS, which supports the hypothesis that TRAIL participates in the immune system [4]. Studies revealed that TRAIL and TRAIL receptors play an important role in the innate immune response to infectious diseases, particularly to viral infections [5]. It is generally accepted that most diseases associated with hepatitis viruses contribute to the immune response to viral infection, in which apoptosis plays a significant role [6]. Both serum solubletrail and membrane-bound TRAIL are up-regulated in HBV-infected patients [7-9]. Liver damage caused /16/ by the Association of Clinical Scientists, Inc.
2 Polymorphisms in TRAIL in Hepatitis B Populations 375 Figure 1. TRAIL PCR product and digestion fragments as revealed by agarose gel electrophoresis. (A) and (B) showed phenotype of TRAIL genes at loci 1525 and M, Marker (DNA standards); lanes 1-3, -1525G/A. Lane 1 is GA type, lane 2 is GG type and lane 3 is AA type; lanes 4-6, C/T. Lane 4 is CT type, lane 5 is CC type and lane 6 is TT type. in part by the host immune reaction was also demonstrated [9]. It has been found that the occurrence of chronic hepatitis B is related to polymorphisms of many apoptotic factors, including TNF-alpha promoter and Tumor necrosis factor-alpha-308a [10,11]. Therefore, in this study we sought to broaden our understanding of TRAIL polymorphism as a risk factor for HBV infection. Materials and Methods Study population. One hundred and eighty-seven consecutively hospitalized Chinese patients (30 women, 157 men, mean ± SD age of 31.23±8.84 years) with chronic HBV infection were recruited from the Department of Liver Diseases at the Second Hospital of Shandong University. These patients tested positive for hepatitis B surface antigen (HBsAg, RIA, Abbott, USA) for over 6 months and negative for hepatitis C virus antibody (anti-hcv, ELISA, LiZhu, China). These patients were diagnosed according to the Strategy for Prevention and Therapy of Viral Hepatitis published in The control group consisted of 142 age- and gender-matched healthy individuals. Patients and controls were residents of the same area and came from the same human subpopulation. Informed consent was obtained from all patients and control subjects. The study protocol was approved by the Medical Ethics Committee of the Second Hospital of Shandong University. Samples collection. EDTA-whole blood samples of all objects were collected before breakfast. Plasma was extracted by 3000 rpm centrifugation for 10 min and frozen at 20 C for further use. DNA extraction and polymorphism detection. Total DNA in the blood was extracted using a genome DNA extraction kit according to the manufacturer's instructions. The quality of DNA was further examined by measuring the optical density (OD) at wavelengths of 260 and 280 nm. The ratio of OD260/OD280 was between 1.70 and 1.80, and the final DNA concentration was 10 ng/µl. Specific primers of TRAIL (Forward, 5 -aacatcttctgtctttataatc-3 ; Reverse, 5 -aaataacacgtacttactgaag-3 ) were employed in addition to Taq PCR Master Mix and genomic DNA templates for PCR amplification. A total of 30 cycles (94 C denaturing for 30 s, 48 C annealing for 90 s, and 72 C elongation for 45 s) of PCR amplification were performed. PCR amplification products were digested by Rsa I or TSPE1 restriction enzyme, resolved on a 2% agarose gel, and then visualized by UV following ethidium bromide staining to reveal genetic polymorphisms. Statistical analysis. Data are reported as mean values ± SD, and statistical analyses were performed using the SPSS Statistical package, version 13.0 for Windows (SPSS. Inc., USA). The verification of the Hardy- Weinberg equilibrium of genotypes was performed using the χ2 test. The χ2 test was also used to evaluate differences in frequency distributions of genotypes and alleles of the TRAIL polymorphisms. All statistical tests were two-sided, and a P<0.05 was considered statistically significant. Results The PCR products of the TRAIL gene were 485 bp. Following differential restriction enzyme digestion, we found gene polymorphisms in loci 1525 and
3 376 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 Table 1. Relation of sex and TRAIL genotype in normal control group and in chronic hepatitis B group. Normal Control Genotype # (%) Chronic Hepatitis B Genotype*(%) n(100) a 1525GG 1525GA 1525AA n(100) a 1525GG 1525GA 1525AA 1595CC 1595CT 1595TT 1595CC 1595CT 1595TT male* 117(100) 40(34.2) 48(41.0) 29(24.8) 153(100) 60(39.2) 78(51.0) 15(9.8) female* 25(100) 6(24.0) 15(60.0) 4(16.0) 34(100) 5(14.7) 21(61.8) 8(23.5) Abbreviations: n, absolute number. RCC, renal cell carcinoma. CI, confidence interval. a Number of patients (number/% of total); # χ2 test: χ2=3.016, P=0.221, *χ2 test: χ2=9.680, P= Table 2. Comparison of TRAIL genotype at 1525G/A and 1595C/T sites between chronic hepatitis B with HBeAg negative/hbeag positive and normal control. genotype (%) n(100) a 1525GG 525GA 1525AA 1595CC 1595CT 1595TT HBeAg negative* 62(100) 15(24.2) 37(59.7) 10(16.1) HBeAg positive # 95(100) 37(39.0) 48(50.5) 10(10.5) normal control* 142(100) 46(32.4) 63(44.4) 33(23.2) a Number of patients (number/% of total); *χ2 test: χ2=4.070, P=0.131, # χ2 test: χ2=6.230, P= Table 3. Comparison of TRAIL genotype and allele at 1525G/A and 1595C/T sites between chronic hepatitis B and normal control group. genotype* (%) Allele # (%) n(100) a 1525GG 1525GA 1525AA 1525G 1525A 1595CC 1595CT 1595TT 1595C 1595T chronic hepatitis B 187(100) 65(34.8) 99(52.9) 23(12.3) 229(61.2) 145(38.8) normal contro 142(100) 46(32.4) 63(44.4) 33(23.2) 155(54.6) 129(45.4) a Number of patients (number/% of total); χ2 test: *genotype, χ2=7.014, P=0.03. #Allele, χ2=2.940, P= loci 1595 of the TRAIL gene. The products of the TRAIL gene at 1525G/A included one band at 473 bp (GG type), two bands at 286 and 187 bp (AA type) and three bands at 473, 286, and 187 bp (GA type) (Figure 1A). The products of the TRAIL gene at 1595C/T included two bands at 291 and 193 bp (CC type), three bands at 291, 131, and 62 bp (TT type), and four bands at 291, 193, 131, and 62 bp (CT type) (Figure 1B). The alleles of TRAIL at 1525G/A and 1595C/T have achieved genetic equilibrium in normal individuals (χ2=0.917, P=0.632) and patients with chronic hepatitis B (χ2=1.207, P=0.547), indicating that study subjects included in the present study were representative of the target population. The genotype frequencies of TRAIL at loci 1525 and 1595 in all subjects showed the same genetic variation, indicating that TRAIL at sites 1525 and 1595 appeared to be in complete linkage disequilibrium in the Chinese population. There was no significant difference between the male and female subjects in genotype frequencies of TRAIL at two sites in normal controls (χ2=3.016, P=0.221). However, the SNPs of TRAIL gene at
4 Polymorphisms in TRAIL in Hepatitis B Populations G/A and 1595C/T sites have significant difference between the male and female subjects in chronic hepatitis B group. The genotype frequencies of TRAIL at 1525GG/1595CC in males were significantly higher than those in females (χ2=9.680, P=0.008) (Table 1). The frequency of the AA genotype at 1525G/A and of the TT genotype at 1595C/T sites were significantly lower in the chronic hepatitis B individuals with HBeAg positive than in normal controls (10.5 vs 23.2%, P<0.05). There was no significant difference in genotype frequencies for the two sites between the normal controls and chronic hepatitis B subjects with HBeAg negative (χ2=4.070, P=0.131) (Table 2). The frequency of the AA genotype at 1525G/A and of the TT genotype at 1595C/T sites were significantly lower in the chronic hepatitis B individuals than in normal controls (12.3 vs 23.2%, P<0.05). Between controls and patients with chronic hepatitis B, the difference in allele frequencies of TRAIL at 1525G/A and 1595C/T sites was not significant (χ2=2.940, P=0.086) (Table 3). Discussion Because of the country s HBV vaccination program, the incidence of HBV infection in China decreased from 9.75% in 1992 to 7.18% in However, 93 million people remain chronically infected with HBV, and 20 million people are symptomatic of chronic hepatitis B [12]. Chronic HBV carriers manifest variable stages of liver inflammation and fibrosis, both clinically and pathologically. The most serious outcome in chronic HBV carriers is cirrhosis, which is not only a major cause of liverrelated death but also a primary risk factor for hepatocellular carcinoma (HCC) [13].The natural history of chronic HBV infection is generally divided into four phases: immune tolerant phase; immune clearance phase; low replicative or inactive carrier stage; and reactivation phase [14]. It is clear that in the immune clearance phase, NK cell activation induces TRAIL-mediated death of hepatocytes in CHB, leading to liver injury [9]. It was also found that individuals with HBV infection had higher serum-soluble TRAIL and membrane-bound TRAIL [7,8]. The capacity for cytokine production in individuals largely depends on promoter genetic polymorphisms [15]. In our study, the difference in genotype frequencies of TRAIL at 1525G/A and 1595C/T sites between the normal control and chronic hepatitis B group was significant. Therefore, the occurrence of chronic hepatitis B may be related to polymorphisms of TRAIL. The clinical outcome of HBV infection is variable, from spontaneous recovery to persistent infection. The mechanisms of different clinical outcomes of HBV infection have not been fully elucidated. Environmental factors such as viral strain, gender, infection age, and the immune status of the host were suspected to affect the outcomes [16]. Genetic factors that determine a host s immune mechanisms may also play an important role in HBV infection [17,18]. Research on genetic polymorphisms( SNPs) indicates that human leukocyte antigen (HLA) genes [19,20], tumor necrosis factor (TNF)- alpha genes [10,11], vitamin D receptor genes [21,22], HLA-DR1 genes [23,24], and Paraoxonase 1 genes [25] may modulate immune response to HBV infection or response for antioxidant defense against reactive oxygen species and anti-inflammatory were hot. In our study, the difference in genotype frequencies at 1525G/A and 1595C/T sites between the normal controls and chronic hepatitis B individuals with HBeAg positive was significant, while the difference between the normal controls and chronic hepatitis B subjects with HBeAg negative was not. Whether TRAIL polymorphism is associated with the outcome of HBV infection still requires further study focusing on screening more patients with cirrhosis and hepatocellular carcinoma patients. In control subjects, the gene polymorphisms of 1525G/A and 1595C/T were not related to sex, but in chronic hepatitis B patients the gene polymorphism was related to sex. The sex disparity of HBVrelated liver diseases has long been apparent. In terms of the sex distribution of HBV infection, our study included many more male than female patients [26-29]. The clinical data show that the cure rate of female patients with acute hepatitis B was higher than that of male patients. In addition, the chronic liver disease progresses more rapidly to cirrhosis in males than in females, and HBV-related HCC occurs much more often in men than in
5 378 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 women, with a ratio of approximately 5-7:1 [30]. Higher HBV infection in males may be due to their risky social activities, unhygienic living habits, or other factors. These factors may increase the risk of contact and rate of infection [29]. In addition, human immune response was characterized by marked gender differences. Females have a stronger cellular and humoral immune response than males. Sex hormones have important regulatory effects on the immune system. Androgen and estrogen pathways have been shown to play oppositely regulate HBV transcription by targeting viral enhancer I at the molecular level [31]. This may be the cause of the significant HBV chronicity in males. In our study, the gene polymorphism of 1525G/A and 1595C/T in control subjects was not related to sex, but in chronic hepatitis B patients the gene polymorphism was related to sex. Whether gender differences in infection course of hepatitis B virus have any relationship to the SNPs of TRAIL requires further investigation. Conclusion. In conclusion, genotype frequencies of TRAIL at 1525G/A and 1595C/T sites showed significant differences between the male and female patients with chronic hepatitis B. Significant differences were also found in genotype frequencies of TRAIL at the same sites between the controls and the chronic hepatitis B cases with HBeAg positive. There was a significant difference in SNPs of TRAIL at 1525G/A and 1595C/T sites between the controls and the patients with chronic hepatitis B, indicating that TRAIL correlated with susceptibility to hepatitis B. Acknowledgements This work was supported by the Youth Fund of the 2nd Hospital of Shandong University. References 1. Lok A S and McMahon B J. Chronic hepatitis B. 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