Abstract
Background and aim
Progression of hepatitis B virus infection (HBV) might be affected by host genetic factors. The present study was undertaken to study the role of glutathione S-transferases (GST)-M1 and T1 gene polymorphisms in different stages of HBV infection: HBV inactive carrier, chronic hepatitis B and cirrhosis, and cryptogenic cirrhosis.
Methods
The study population comprised of 170 subjects; 120 cases (HBV inactive carrier, n = 30; HBV related chronic hepatitis, n = 30; HBV related cirrhosis, n = 30; cryptogenic cirrhosis, n = 30) and 50 unrelated healthy adults without liver disease as controls. Analysis of GSTM1 and GSTT1 gene polymorphisms was done by multiplex polymerase chain reaction.
Results
The GSTM1 null genotype was seen more commonly in hepatitis B cirrhosis (n = 21; 70%), chronic hepatitis B (n = 19; 63.33%) and cryptogenic cirrhosis (n = 17; 56.67%) as compared with inactive carrier (n = 9; 30%) and controls (n = 13; 26%). The GSTT1 null genotype was seen less frequently in all the groups, the observed frequencies were controls (n = 7; 14%), inactive carrier (n = 5; 16.67%), chronic hepatitis B (n = 8; 26.67%) and hepatitis B cirrhosis (n = 7; 23.33%). The difference of GSTM1 null genotype frequencies was statistically significant for hepatitis B cirrhosis vs. controls (P = 0.0002), chronic hepatitis B vs. controls (P = 0.002) and cryptogenic cirrhosis vs. controls (P = 0.01). The GSTT1 null genotype was not found to vary significantly between the groups.
Conclusion
The patients with GSTM1 null genotype are at risk of progression of liver disease as the frequency of GSTM1 null genotype was found to be significantly higher in chronic hepatitis B, hepatitis B cirrhosis and cryptogenic cirrhosis as compared with controls.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GST, glutathione S-transferase; HBV, hepatitis B virus
Keywords: hepatitis B, cryptogenic cirrhosis, GSTM1, GSTT1, null genotype
Hepatitis B is an infectious inflammatory illness of the liver caused by the hepatitis B virus (HBV). Approximately 2 billion people are exposed to HBV worldwide and more than 350 million suffer from chronic HBV infection. In India, the prevalence of HBsAg ranges from 2% to 8% in general population, which places India in the intermediate endemicity zone for HBV.1
Progression in disease is influenced by several factors, like viral genotype, demographic features, concurrent viral infections, and social factors like alcohol abuse and specific mutations. Host genetic factors may also determine the interindividual differences in disease progression from chronic hepatitis to cirrhosis. Genetic polymorphisms have been observed in enzymes that play a role in the detoxification of xenobiotics and they have been linked to various diseases especially cancers.
Glutathione S-transferases (GST) catalyze the conjugation of the reduced form of glutathione to xenobiotic substrates for the purpose of detoxification.2 α (A), μ (M), π (P), σ (S), θ (T), ω (O) and ζ (Z) are the classes of GSTs.3 The five mu class genes are situated on chromosome 1p13 in a 20 kb cluster (5′-GSTM4-GSTM2-GSTM1-GSTM5-GSTM3-3′).3, 4 GSTT1 and GSTT2, are located on chromosome 22.5 Of these GSTM1 and GSTT1 subclasses exhibit deletion polymorphisms and the null GSTM1 and GSTT1genotypes have been extensively studied for their role in susceptibility to various cancers like breast cancer,6 lung cancer,7 hepatocellular carcinoma (HCC),8 prostate cancer,9 colorectal cancer.10
GSTM1 and GSTT1 null genotypes are found in the general population and most recent studies have shown the influence of the ethnic component in the distribution of these polymorphisms.11, 12, 13 In a study done in Iran GSTM1 null genotype was found to be more prevalent in cryptogenic cirrhosis than in healthy controls.14 Later a similar study was undertaken to find the correlation if any between polymorphisms in GSTM1, GSTT1, and GSTP1 in patients with HBV-related, chronic hepatitis, liver cirrhosis and normal carriers, they observed a higher frequency of the GSTM1 null genotype in patients with cirrhosis and chronic hepatitis compared to normal carriers.15
The present study was undertaken to study the role of GSTM1 and T1 gene polymorphisms in different stages of HBV infection namely HBV inactive carrier, chronic hepatitis B and cirrhosis, and cryptogenic cirrhosis.
Methods
Study Population
The study was conducted in the Department of Medicine, Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India from September 2010 to April 2012. The study included a total of 170 subjects; 120 cases (HBV inactive carrier, n = 30; HBV related chronic hepatitis, n = 30; HBV related cirrhosis, n = 30; cryptogenic cirrhosis n = 30) and 50 unrelated healthy adults without liver disease as controls. Since, it is a pilot study the samples size was not statistically verified. Informed consent was obtained from each subject included in the study and the study protocol confirms to the ethical guidelines of the 1975 Declaration of Helsinki.
Inclusion Criteria
Inactive HBsAg carrier were included when the HBsAg was positive for more than 6 months, HBeAg negative and anti-HBe positive, serum HBV DNA levels less than 2000 IU/ml and persistently normal liver enzymes levels. Chronic hepatitis B was diagnosed when the HBsAg was positive for more than 6 months, serum HBV DNA levels greater than 20,000 IU/ml (105 copies/ml) and persistent or intermittent elevation in ALT/AST levels. The diagnosis of cirrhosis was based on clinical, biochemical, and ultrasonography data.
Exclusion Criteria
Cases with viral co-infections (HCV, HAV, HEV, HIV) and alcoholics were excluded. The patients were evaluated on the basis of history, clinical examination, hematological and biochemical profile, LFT profile and serological tests for hepatitis B (HBsAg, HBeAg, Anti-HBe, IgM Anti-HBc and IgG anti-HBc) using commercially available Elisa kits.
Sample Collection
Five ml of blood was collected in ethylenediaminetetraacetic acid vials for genomic DNA extraction. Another Five ml blood was taken in plain vial for serological testing. Blood samples were stored at −70 °C until use.
Genomic DNA Isolation and Polymerase Chain Reaction (PCR)
Genomic DNA was extracted by a salting-out method.16 Analysis of GSTM1 and GSTP1 gene polymorphisms was done by multiplex PCR simultaneously in the same tube.17 Genomic DNA was amplified using the set of primers described by Arand et al.17 Briefly the PCR reaction conditions were initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min. The final extension was done at 72 °C for 7 min. GSTM1 and GSTT1 genotypes positive DNA samples yielded bands of 215 bp and 480 bp, respectively.
Statistical Analysis
The data obtained from the study was tabulated and transferred to a personal computer. Allelic frequencies were compared between different study groups and controls using Chi square test or Fisher's test as applicable using OpenEpi: Open Source Epidemiologic Statistics for Public Health, Version 2.3.1. Odds ratio (OR) with 95% confidence interval (CI) were calculated. 2-tailed P value was used to test significance. The data was considered significant when P < 0.05.
Results
The mean age of subjects in different study groups were inactive carrier 29.30 ± 13.01 years, chronic hepatitis B 31.67 ± 12.90 years, hepatitis B cirrhosis 40.17 ± 14.35 years and cryptogenic cirrhosis 39.70 ± 14.69 years.
The relative frequencies of GSTM1 and GSTT1 null genotypes are shown in (Table 1). The GSTM1 null genotype was seen more commonly in hepatitis B cirrhosis (n = 21; 70%), chronic hepatitis B (n = 19; 63.33%) and cryptogenic cirrhosis (n = 17; 56.67%) as compared with inactive carrier (n = 9; 30%) and controls (n = 13; 26%). The GSTT1 null genotype was seen less frequently in all the groups, the observed frequencies were controls (n = 7; 14%), inactive carrier (n = 5; 16.67%), chronic hepatitis B (n = 8; 26.67%), hepatitis B cirrhosis (n = 7; 23.33%) and cryptogenic cirrhosis (n = 6; 20%).
Table 1.
Genotype Frequencies of Null GSTM1 and GSTT1 Polymorphism.
| Gene polymorphism | Controls (n = 50) |
Inactive carrier (n = 30) |
Chronic hepatitis B (n = 30) |
Hepatitis B cirrhosis (n = 30) |
Cryptogenic cirrhosis (n = 30) |
|||||
|---|---|---|---|---|---|---|---|---|---|---|
| n | % | n | % | n | % | n | % | N | % | |
| NullGSTM1 | 13 | 26 | 9 | 30 | 19 | 63.33 | 21 | 70 | 17 | 56.67 |
| NullGSTT1 | 7 | 14 | 5 | 16.67 | 8 | 26.67 | 7 | 23.33 | 6 | 20 |
The OR and P value comparisons for GSTM1 and GSTT1 null genotypes are shown in (Table 2). The difference of GSTM1 null genotype frequencies was statistically significant for hepatitis B cirrhosis vs. controls (P = 0.0002), chronic hepatitis B vs. controls (P = 0.002) and cryptogenic cirrhosis vs. controls (P = 0.01). The difference was not statistically significant for inactive carrier vs. controls (P = 0.89). Thus, GSTM1 null genotype was found to be more common in chronic hepatitis B and hepatitis B cirrhosis as compared to control population. There was no statistically significant difference between the genotype frequencies of GSTT1 null genotype when each of the study groups were compared against controls.
Table 2.
Odds Ratio and P Value Comparisons for GSTM1 and GSTT1 Gene Polymorphisms Against Controls.
| Investigation parameters | Inactive carrier vs. control |
Chronic hepatitis B vs. control |
Hepatitis B cirrhosis vs. control |
Cryptogenic cirrhosis vs. control |
||||
|---|---|---|---|---|---|---|---|---|
| Odds ratio (CI) | P value | Odds ratio (CI) | P value | Odds ratio (CI) | P value | Odds ratio (CI) | P value | |
| NullGSTM1 | 1.22 (0.44–3.33) | 0.89 | 4.91 (1.85–13.03) | 0.002 | 6.64 (2.43–18.13) | 0.0002 | 3.72 (1.42–9.71) | 0.01 |
| NullGSTT1 | 1.22 (0.35–4.28) | 0.98 | 2.23 (0.71–6.96) | 0.26 | 1.87 (0.58–5.98) | 0.44 | 1.53 (0.46–5.09) | 0.68 |
The difference of GSTM1 null genotype frequencies was statistically significant for chronic hepatitis B vs. inactive carrier (P = 0.01) and hepatitis B cirrhosis vs. inactive carrier (P = 0.004). There was no significant difference between hepatitis B cirrhosis vs. chronic hepatitis B. Thus GSTM1 null genotype was more common in chronic hepatitis B and hepatitis B cirrhosis compared with inactive carriers. There was no significant difference in GSTT1 null genotype in chronic hepatitis B vs. inactive carrier, hepatitis B cirrhosis vs. inactive carrier and hepatitis B cirrhosis vs. chronic hepatitis B.
Discussion
In addition to the virological factors, progression of hepatitis B virus infection might be affected by host genetic factors.18 The absence of liver detoxifying enzymes might lead to enhanced oxidative stress and cytotoxic damage in the liver. Cirrhosis is the end stage of chronic damage to the liver characterized by fibrosis and destruction of normal liver architecture. The GSTs play a key role in the protection against oxidative stress and the role of variant GST genotypes has been studied in various diseases especially cancers. Some studies have already shown the role of GSTM1 and GSTT1 null gene polymorphisms in HCC.8 GST gene polymorphisms have been studied in the past in liver diseases. These studies have shown that GSTM1 null genotype was more prevalent in cryptogenic cirrhosis as well as hepatitis B cirrhosis.14, 15 Our results also show that GSTM1 null genotype was more prevalent in chronic hepatitis B, hepatitis B cirrhosis and cryptogenic cirrhosis as compared to control population, thereby confirming previous findings. However the GSTT1 null genotypes were not found to differ significantly between any of the groups.
In this study the difference of GSTM1 null genotype frequencies was statistically significant for hepatitis B cirrhosis vs. controls (P = 0.0002), chronic hepatitis B vs. controls (P = 0.002) and cryptogenic cirrhosis vs. controls (P = 0.01). The difference was not statistically significant for inactive carrier vs. controls (P = 0.89). Thus, GSTM1 null genotype was found to be more common in chronic hepatitis B, hepatitis B cirrhosis and cryptogenic cirrhosis as compared to control population. Hence, we can assume that a decrease in GSTM1 activity may contribute to the progression of liver disease. Since the frequency of GSTM1 null genotype was increased in both hepatitis B cirrhosis and cryptogenic cirrhosis we can say that GSTM1 null genotype is associated with progression of liver disease in general and not restricted to the hepatitis B related liver disease group.
In this study the difference in genotype frequencies of GSTT1 null genotype was not found to be statistically significant for any of the study groups. These results are similar to previous reports.14, 15 Thus our findings lead us to believe that absence of GSTT1 gene probably does not influence the progression of liver disease.
A study by Kiran et al.,19 to evaluate the association of GSTT1 and GSTM1 null genotype polymorphisms with hepatitis B and C virus related HCC risk in an Indian population, reported that GSTT1 null genotype was associated with 2.23-fold (P < 0.05) increased risk for HCC development as compared to the control group. However, GSTM1 null genotype was found to have a protective effect when hepatitis patients were considered. In our study it was observed that GSTM1 null genotype was found to be associated with a higher risk for hepatitis B cirrhosis when compared to control (OR = 6.19), chronic hepatitis B when compared to controls (OR = 4.62) and cryptogenic cirrhosis compared to controls (OR = 3.52).
From our study it can be inferred that patients with GSTM1 null genotype are at risk of progression to chronic hepatitis B and HBV related cirrhosis as the frequency of GSTM1 null genotype was found to be increased in these groups. The GSTM1 null genotype increases the risk of progression to cirrhosis as this genotype was found to be associated with hepatitis B cirrhosis as well as cryptogenic cirrhosis. The GSTT1 null genotype does not show any evidence of risk for progression of liver disease. Thus GSTM1 null genotype may be used as a potential molecular marker for assessing the risk of progression of chronic hepatitis B to cirrhosis. However, larger population based studies are required to validate these findings.
Conflicts of Interest
The authors have none to declare.
References
- 1.Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat. 2004;11:97–107. doi: 10.1046/j.1365-2893.2003.00487.x. [DOI] [PubMed] [Google Scholar]
- 2.Strange R.C., Jones P.W., Fryer A.A. Glutathione S-transferase: genetics and role in toxicology. Toxicol Lett. 2000;112-113:357–363. doi: 10.1016/s0378-4274(99)00230-1. [DOI] [PubMed] [Google Scholar]
- 3.Hayes J.D., Flanagan J.U., Jowsey I.R. Glutathione transferases. Annu Rev Pharmacol Toxicol. 2005;45:51–88. doi: 10.1146/annurev.pharmtox.45.120403.095857. [DOI] [PubMed] [Google Scholar]
- 4.Xu S.J., Wang Y.P. Characterization of the human class mu glutathione S-transferase gene cluster and the GSTM1 deletion. J Biol Chem. 1998;273:3517–3527. doi: 10.1074/jbc.273.6.3517. [DOI] [PubMed] [Google Scholar]
- 5.Coggan M., Whitbread L., Whittington A., Board P. Structure and organization of the human theta-class glutathione-S-transferase and d-dopachromet automerase gene complex. Biochem J. 1998;334:617–623. doi: 10.1042/bj3340617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Millikan R., Pittman G., Tse C.K., Savitz D.A., Newman B., Bell D. Glutathione S-transferases M1, T1, and P1 and breast cancer. Cancer Epidemiol Biomarkers Prev. 2000;9:567–573. [PubMed] [Google Scholar]
- 7.Hosgood H.D., Berndt S.I., Lan Q. GST genotypes and lung cancer susceptibility in Asian populations with indoor air pollution exposures: a meta-analysis. Mutat Res. 2007;636:134–143. doi: 10.1016/j.mrrev.2007.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.White D.L., Li D., Nurgalieva Z., El-Serag H.B. Genetic variants of glutathione S-transferase as possible risk factors for hepatocellular carcinoma: a HuGE systematic review and meta-analysis. Am J Epidemiol. 2008;167(4):377–389. doi: 10.1093/aje/kwm315. [DOI] [PubMed] [Google Scholar]
- 9.Mo Z., Gao Y., Cao Y. An updating meta-analysis of the GSTM1, GSTT1, and GSTP1 polymorphisms and prostate cancer: a HuGE review. Prostate. 2009;69:662–688. doi: 10.1002/pros.20907. [DOI] [PubMed] [Google Scholar]
- 10.Liao C., Cao Y., Wu L., Huang J., Gao F. An updating meta-analysis of the glutathione S-transferase T1 polymorphisms and colorectal cancer risk: a HuGE review. Int J Colorectal Dis. 2010;25(1):25–37. doi: 10.1007/s00384-009-0805-0. [DOI] [PubMed] [Google Scholar]
- 11.Bolt H.M., Parra F.C., Amado R.C. Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology. Curr Drug Metab. 2006;7:613–628. doi: 10.2174/138920006778017786. [DOI] [PubMed] [Google Scholar]
- 12.Mishra D.K., Kumar A., Srivastava D.S. Allelic variation of GSTT1, GSTM1 and GSTP1 genes in North Indian population. Asian Pac J Cancer Prev. 2004;5:362–365. [PubMed] [Google Scholar]
- 13.Vettriselvi V., Vijayalakshmi K., Paul S.F., Venkatachalam P. Genetic variation of GSTM1, GSTT1 and GSTP1 genes in a South Indian population. Asian Pac J Cancer Prev. 2006;7:325–328. [PubMed] [Google Scholar]
- 14.Mohammadzadeh G.S., Yaghmaei B., Bakayev V. GSTP1, GSTM1, and GSTT1 genetic polymorphisms in patients with cryptogenic liver cirrhosis. J Gastrointest Surg. 2004;8(4):423–427. doi: 10.1016/j.gassur.2004.02.005. [DOI] [PubMed] [Google Scholar]
- 15.Mohammadzadeh G.S., Yaghmaei B., Allameh A., Hassani P., Noorinayer B., Zali M.R. Polymorphisms of glutathione S-transferase M1, T1, and P1 in patients with HBV-related liver cirrhosis, chronic hepatitis, and normal carriers. Clin Biochem. 2006;39(1):46–49. doi: 10.1016/j.clinbiochem.2005.10.004. [DOI] [PubMed] [Google Scholar]
- 16.Miller S.A., Dykes D.D., Polesky H.F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Arand M., Muhlbauer R., Hengstler J. A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S transferase GSTM1 and GSTT1 polymorphisms. Anal Biochem. 1996;236(1):184–186. doi: 10.1006/abio.1996.0153. [DOI] [PubMed] [Google Scholar]
- 18.Wang F.S. Current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J Gastroenterol. 2003;9:641–644. doi: 10.3748/wjg.v9.i4.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kiran M., Chawla Y.K., Kaur J. Glutathione-S-transferase and microsomal epoxide hydrolase polymorphism and viral-related hepatocellular carcinoma risk in India. DNA Cell Biol. 2008;27(12):687–694. doi: 10.1089/dna.2008.0805. [DOI] [PubMed] [Google Scholar]