Association of ISG20 RS4566136 polymorphism with hepatitis B virus-related liver diseases

T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 169 ASSOCIATION OF ISG20 RS4566136 POLYMORPHISM WITH HEPATITIS B VIRUS-RELATED LIVER DISEASES Pham Van Dung1,2, Dao Thi Phuong4, Tran Thi Phuong Thao2, Ta Phuong Linh2 Nguyen Thanh Viet2, Nguyen Quang Duat1, Nguyen Linh Toan3, Hoang Van Tong2,3 SUMMARY Background: Interferon stimulated gene 20 (ISG20) plays an important role in viral infection and cancers. This study investigated the association between ISG20 rs4566136 polymorphism with HBV-related hepatocellular carcinoma (HCC). Subjects and methods: The polymorphism ISG20 rs4566136 were genotyped by Sanger sequencing in 199 patients with HCC, 100 liver cirrhosis patients (LC) and 156 healthy controls (HC). Results: Rs4566136C allele was associated with HCC as compared with healthy controls (allelic model: OR(95%CI) = 4.2 (2.7 - 6.5), p < 0.001). The rs4566136C allele was associated with HBV-related LC (allelic model: OR(95%CI) = 4.1 (2.6 - 6.6), p < 0.001). Patients with genotype TT had higher AST and ALT levels, followed by patients with genotype TC and CC. However, the difference was not significant. Conclusions: ISG20 rs4566136 polymorphism is associated with HBV-related liver diseases in the Vietnamese population. Allele rs4566136C contributes to an increased the risk of HBV-related HCC. * Keywords: SNPs; Rs4566136; Hepatitis B virus; Hepatocellular carcinoma; Mutation. INTRODUCTION Hepatocellular carcinoma is a primary tumor of the liver, accounting for over 90% of the primary liver cancers. HCC occurs from 80% to 90% of patients with cirrhosis [6] and is now the fifth most common cancer worldwide and the second leading cause of cancer-related death after lung cancer [3]. Significant risk factors for HCC include hepatitis B virus (HBV). Chronic HBV and HCV (hepatitis C virus) infections account for more than 70% of HCC cases. HBV affects more than 250 million individuals in the world and is the most common cause of chronic hepatitis globally, and Vietnam is one of the countries with high prevalence of HBV infection [5]. The replication of viruses is inhibited by type I interferon-stimulated genes (ISGs). Despite the understanding of the molecular basis of ISG restriction, the antiviral mechanisms remain unclear. One of the proteins in the ISG family is 20-kDa exonuclease interferon-stimulated gene 20 (ISG20), which has antiviral activity against viruses. ISG20 can inhibit the infection of a board range of viruses including HBV infection. ISG20 has been shown as an innate anti-HBV effect that can inhibit HBV infection through degrading HBV-RNA [4]. 1Military Hospital 103, Vietnam Military Medical University 2Institute of Biomedicine and Pharmacy, Vietnam Military Medical University 3Department of Pathophysiology, Vietnam Military Medical University 4Haiduong Medical Technical University, Haiduong Corresponding author: Hoang Van Tong (hoangvantong@vmmu.edu.vn) Date received: 30/8/2020 Date accepted: 08/11/2020 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 170 HBV-related- and induced- HCC development is a significant research area. Single nucleotide polymorphisms (SNPs) is a crucial factor in the development of the tumor. ISG20 might be a potential indicator for liver injury and the clinical outcome of HBV-related HCC [1]. However, so far, no studies have reported the association between ISG20 polymorphisms and HCC, and whether ISG20 polymorphisms could affect the development of HBV-related HCC in Vietnamese populations. Therefore, we carried out a study: To investigate the relationship between ISG20 rs4566136 polymorphisms and the risk of HBV-related HCC. The study of hereditary factors may shed more light on a better understanding of the molecular mechanisms and the pathogenicity of HBV-related HCC. SUBJECTS AND METHODS 1. Subjects A total of 199 patients with HBV-related HCC and 100 patients with HBV-related liver cirrhosis (LC) were enrolled for this study. HCC patients were diagnosed according to the AASLD 2018 recommendation, including liver tumor size > 1 cm and rapid absorption drug in the fast-release artery in the venous tenses and later on the CT scan film with contrast injection and/or histopathology confirmed HCC. The patients were positive for HBsAg and negative for anti-HCV and anti-HIV. HCC stages were classified based on Barcelona criteria. Liver cirrhosis (LC) patients were diagnosed with two symptoms including impaired liver function syndrome and portal hypertension syndrome. Demographic and clinical parameters including age, sex, AST and ALT levels, RBC, WBC, PLT, prothrombin and AFP levels were collected. The liver cirrhosis stages were classified based on Child-Pugh criteria. We also included 156 healthy individuals, which were negative for HBV, HCV and HIV infections, as control group. A sample of 5 mL of venous blood was collected from each patient or healthy individual, and sera were separated and stored at -80ºC until use. Written informed consent from patients and healthy controls were obtained. The study was approved by the Institutional Review Board of Vietnam Military Medical University (VMMU). All samples collected were anonymized after completion collection. 2. Methods * Genotyping of ISG20 rs4566136 polymorphism: Total DNA was extracted from venous blood samples by a Gene JET Whole Blood Genomic DNA Purification Mini Kit (Thermo, USA). Primers were designed to amplify a fragment covering exon 2 and intron 2-3 using the Primer3 and NCBI primer BLAST software. PCR primers sequence were ISG20F:5’-GAG GGG CTT ACC TTT GTA GC-3’ and ISG20R:5’TCA GAA CAC ATC CCA CTC CT-3’. The temperature cycling was as follows: 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 25 sec and extension at 72°C for 30 sec, and final extension for 5 min at 72°C. The total reaction mixture (25 µL) contained 2 µL T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 171 DNA template, 12.5 µL Master Mix (2X), (2X) Dream Taq, 0.625 µl of each primer. PCR products were purified using the GeneJET Genomic DNA Purification Kit (Thermo, USA). Sanger sequencing was carried out on all the purified PCR products on the ABI 3130 automated genetic analyzer (Applied Biosystems, USA). Sequencing data were analyzed using the Biodit software version 7.0 to determine ISG20 rs4566136 SNP. * Statistical analysis: All statistical analysis was conducted by SPSS 22.0. Continuous variables were presented as means or median where appropriate. Categorical variables are expressed as frequencies and percentage. Comparisons of continuous data between groups were performed with Mann-Whitney U test or Kruskal-Wallis test. The comparison of the differences in categorical variables between groups was performed by Pearson’s Chi-square or Fisher’s exact test. The logistic regression model was used to analyse the association of ISG20 rs4566136 SNP with HBV-related HCC adjusted for confounding factors such as age and gender. OR and OR 95%CI were also calculated and p-value < 0.05 was considered statistical significance. RESULTS 1. Baseline characteristics of the study groups Table 1: Baseline characteristics of patient group. HCC group LC group Characteristics Median (min - max) Median (min - max) p Ages (years) 59.6 ± 11.4 58.1 ± 10.2 Gender (male) 182 (91.5) 74 (74) AST (U/L) 54 (17 - 596) 77 (18 - 2,461) 0.01 ALT (U/L) 45 (11 - 469) 47.6 (12 - 1,327) 0.04 GGT (U/L) 151 (13 - 1.184) 133 (17 - 939) 0.86 Bilirubin total (µmol/L) 16.9 (7.5 - 470) 45 (8.2 - 400) < 0.001 Bilirubin direct (µmol/L) 4.9 (0.6 - 249.7) 16.5 (1.6 - 252.7) < 0.001 Protein total (G/L) 78.9 (56 - 98) 69.8 (35 - 91) < 0.001 Albumin (G/L) 39.6 (18 - 50) 28 (17 - 49) < 0.001 RBC (T/L) 4.7 (1.6 - 6.5) 3.6 (2 - 6.3) < 0.001 WBC (G/L) 6.5 (1.6 - 16.9) 6.2 (1.6 - 60.9) 0.31 PLT (G/L) 177 (27 - 841) 100 (28 - 499) < 0.001 Prothrombin (%) 87 (33 - 123) 58 (9 - 110) < 0.001 The demographic and clinical parameters such as age, sex, AST and ALT levels, RBC, WBC, PLT, prothrombin and AFP levels of the patients were presented in table 1. Significant differences in AST, ALT, bilirubin, protein, albumin, prothrombin levels and T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 172 RBC, PLT were observed while GGT levels and RBC were not significantly different between HCC and LC patients. Importantly, AFP levels were significantly increased in HCC compared to LC patients (p = 0.001). Table 2: Characteristics of liver function and stage of disease. BCLC (HCC) (n = 199) Child Pugh (HCC) (n = 199) Child Pugh (LC) (n = 100) Stage n (%) A 7 (3.5) 154 (77.4) 0.0 B 119 (59.9) 26 (13.1) 43 (43) C 54 (27.1) 19 (9.5) 56 (56) D 19 (9.5) HCC patients made up 77.4% in Child A stage and 59.9% in the intermediate stage. LC patients in Child C stage were present in 56%. 2. ISG20 rs4566136 polymorphism analysis Figure 1: PCR products amplified DNA fragment of ISG20 gene. The polymorphism rs4566136 was located on intron 2 region of ISG20 gene. A DNA fragment covering exon 2 and intron 2 - 3 was amplified by the specific primers. PCR products were observed by agarose 1.5% gel electrophoresis with standard maker 100 bp. Figure 2: Sequence by Sanger method showing rs4566136 genotype. PCR products were purified and subjected to Sanger sequencing and the DNA sequences and genotype of the ISG20 rs4566136 SNP were analysed by Bioedit software aligning with reference sequence. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 173 3. Association of ISG20 rs4566136 polymorphism with hepatocellular carcinoma Table 3: Genotype and allele distribution of rs4566136 SNP in study groups. ISG20 SNP HCC (n = 189) (%) LC (n = 100) (%) HC (n = 128) (%) OR (95%CI) HCC vs. LC p OR (95%CI) HCC vs. HC p OR (95%CI) LC vs. HC p Genotype (n) TT 61 (32.3) 29 (29) 48 (37.5) Reference Reference Reference CT 92 (48.7) 48 (48) 53 (41.4) 1.1 (0.6 - 2) 0.75 6.5 (3.1 - 13.9) < 0.001 5.7 (2.6 - 12.4) < 0.001 CC 36 (19) 23 (23) 27 (21.1) 1.5 (0.7 - 3.1) 0.27 3.3 (1.5 - 7.4) 0.003 3.3 (1.4 - 8) 0.007 Allelic (2n) T 214 (56.6) 106 (53) 149 (58.2) Reference Reference Reference C 164 (43.4) 94 (47) 107 (41.8) 1.2 (0.8 - 1.7) 0.32 4.2 (2.7 - 6.5) < 0.001 4.1 (2.6 - 6.6) < 0.001 Dominant (n) TT 61 (32.3) 29 (29) 48 (37.5) Reference Reference Reference TC + CC 128 (67.7) 71 (71) 80 (62.5) 1.2 (0.7 - 2.5) 0.54 10.3 (5.1 - 20.9) < 0.001 9.6 (4.5 - 20.3) < 0.001 Recessive (n) TT + TC 153 (81) 77 (77) 101 (87.9) Reference Reference Reference CC 36 (19) 23 (23) 27 (21.1) 1.4 (0.8 - 2.5) 0.31 2.7 (1.4 - 5.3) 0.005 2.9 (1.4 - 6.1) 0.006 The frequencies of genotype and allele of ISG20 rs4566136 polymorphism in patients and healthy controls were presented in table 3. We analysed the association of ISG20 rs4566136 polymorphism with HBV-related HCC in different genetic models including genotypic, allelic, dominant and recessive models. The results showed that the rs4566136C allele was associated with HCC when compared with healthy controls (allelic model: OR(95%CI) = 4.2 (2.7 - 6.5), p < 0.001; dominant model: OR(95%CI) = 10.3 (5.1 - 20.9), p < 0.001 and recessive model: OR(95%CI) = 2.7 (1.4 - 5.3), p = 0.005. In addition, we also compare allele and genotype frequencies between HCC and LC groups. However, no significant difference was observed. * Association of ISG20 rs4566136 polymorphism with liver cirrhosis: To analyse the association of ISG20 rs4566136 polymorphism with HBV-related liver cirrhosis, we compared genotype and allele frequencies of ISG20 rs4566136 SNP between patients with HBV-related LC and healthy controls in genotypic, allelic, dominant and recessive models. The results showed that the rs4566136C allele was associated with HBV-related LC (allelic model: OR(95%CI) = 4.1 (2.6 - 6.6), p < 0.001; dominant model: OR(95%CI) = 9.6 (4.5 - 20.3), p < 0.001 and recessive model: OR(95%CI) = 2.9 (1.4 - 6.1), p = 0.005) . OR and p values were calculated by logistic regression model adjusted for age and gender. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 174 4. Association of rs4566136 SNP with clinical parameters of HCC patients Figure 3: Association of rs4566136 SNP with clinical characteristics of HCC patients. We analysed the association of rs4566136 polymorphism genotype with clinical parameters of HCC patients by comparing the clinical parameters such as AST, ALT, bilirubin, protein, albumin, prothrombin levels, RBC and PLT among patients with different genotypes. The results showed that patients with genotype TT had higher AST and ALT levels, followed by patients with genotype TC and CC. However, the difference was not significant (figure 3A and figure 3B). In contrast, patients with genotype TT had significantly lower albumin levels, followed by patients with genotype TC and CC (figure 3C). Table 4: Association of rs4566136 SNP with development of HCC. rs4566136 genotypes Characteristics TT TC CC p A 43 (22.8) 73 (38.6) 28 (14.8) B 6 (3.2) 14 (7.4) 6 (3.2) Child-Pugh C 12 (6.3) 5 (2.6) 2 (1.1) 0.045 A 3 (1.6) 3 (1.6) 1 (0.5) B 30 (15.9) 61 (32.3) 23 (12.2) C 16 (8.5) 23 (12.2) 11 (5.8) Barcelona D 12 (6.3) 5 (2.6) 2 (1.1) 0.095 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 175 We also classified HCC patients into subgroups with different development stages according to Child-Pugh and Barcelona criteria, compared genotypes of ISG rs4566136 SNP among classified groups. The results showed that ISG rs4566136 SNP was significantly related to Child-Pugh with p = 0.045 but it was not significantly related to BCLC with p = 0.095. DISCUSSION Human genetic polymorphism regulates the susceptibility to a certain disease of individuals and a number of studies showed the association of SNPs with cancers. ISG20 rs4932196 SNP was proven to play an important role in hearing loss syndrome in elderly individuals. However, to the best of our knowledge, there is no study that determine the role of rs4566136 polymorphism in HCC disease. This is the first study showing an association of rs4566136 polymorphism with the risk of HCC and liver cirrhosis in the Vietnamese population. ISG20 has been shown to promote metastasis and angiogenesis via the IL-8/p-JAK2/p-STAT3 signalling pathway suggesting a pro-tumour role of ISG20 [2, 7]. Our results additionally contribute to verify the functional role of ISG20 in the pathogenesis of HCC. The risk of HCC of rs4566136 polymorphism has been assessed by comparing the frequency of genotype and alleles. Additionally, different genetic models such as dominant and recessive models and binary logistic regression adjusted for confounding factors (age and gender) have been used to determine the association of ISG20 rs4566136 polymorphism with HCC and LC. When LC group was used as a control group, the frequency of genotypes and alleles were not statistically significantly different. Our result indicated that ISG20 rs4566136 SNP had no association with an increased risk of HCC in liver cirrhosis patients. In the HCC group, the TT genotype had higher liver enzyme levels, AFP levels were increased significantly compared to genotype TC and CC. However, the differences were not statistically significant. Albumin plays an important role in liver function, when liver function was decreased, the non-branding amino acids are not synthesized thereby leading to a decrease in albumin levels in the blood. This could lead to a decrease in intravascular colloid pressure and that was the mechanism for interpreting the clinical symptoms of liver cirrhosis with HCC patients. This study has shown that individuals with TT genotypes had lower albumin concentration compared to those with TC and CC genotypes, the difference was statistically significant. We found the association between the genotype of rs4566136 polymorphism with liver cirrhosis according to Child-Pugh scores, however, we did not find any association between the genotype of rs4566136 polymorphism with disease stages according to the Barcelona classification. In conclusion, allele C and genotype CC are associated with an increased risk of HBV related HCC and liver cirrhosis. ISG20 rs4566136 SNP could be considered as a marker and need further studies to clarify the functional role in pathogenesis and prognosis of HBV- related HCC and cirrhosis. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 176 ACKNOWLEDGEMENTS We sincerely thank the patients and voluntary blood donors who participated in this study. This study is funded by the National Science and Technology Development Fund (NAFOSTED) in the subject code 108.02-2017.15. REFERENCES 1. Van Tong H, Hoan NX, Binh MT, et al. Interferon-stimulated gene 20 kDa protein serum levels and clinical outcome of hepatitis B virus-related liver diseases. Oncotarget 2018; 9(45):27858-27871. 2. Lin SL, Wu SM, Chung IH, et al. Stimulation of interferon-stimulated gene 20 by thyroid hormone enhances angiogenesis in liver cancer. Neoplasia 2018; 20(1):57-68. 3. Jemal A, Ward EM, Johnson CJ, et al. Annual report to the nation on the status of cancer. Featuring Survival. J Natl Cancer Inst, 2017; 109(9):1975-2014. 4. Leong CR, Funami K, Oshiumi H, et al. Interferon-stimulated gene of 20 kDa protein (ISG20) degrades RNA of hepatitis B virus to impede the replication of HBV in vitro and in vivo. Oncotarget, 2016; 7(42):68179-68193. 5. Schulze K, Nault JC, Villanueva A. Genetic profiling of hepatocellular carcinoma using next-generation sequencing. J Hepatol, 2016; 65(5):1031-1042. 6. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136(5):E359-386. 7. Taylor KL, Leaman DW, Grane R, et al. Identification of interferon-beta-stimulated genes that inhibit angiogenesis in vitro. J Interferon Cytokine Res 2008; 28(12):733-740.