Abstract
The glutathione S-transferase (GST) supergene family is made up of four gene families responsible for the biotransformation of drugs and other xenobiotics. Genetic variations in this supergene family influence individual detoxification levels and may contribute to the development of cancer. A hospital-based case-control study was conducted to evaluate the association between GST polymorphism among Filipino patients positive for hepatitis B virus (HBV DNA) and clinically diagnosed as either with chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma as well as normal individuals negative for HBV infection. Multiplex PCR was used to detect the presence or absence of the GSTT1 and GSTM1 polymorphisms in peripheral blood. DNA sequencing of the S gene region of the virus was used to determine the predominant genotype found among HBV-infected patients. Our results showed that the odds of having a chronic liver disease is only 0.95 (95% CI 0.58-1.57) among those with GSTT1 null genotype compared to those with GSTT1+ genotype. On the other hand, the odds of chronic liver disease is 17.85 times (95% CI 7.34-43.45) for those with GSTM1 null genotype compared to those with GSTM1+ genotype. Using the GSTT1+/GSTM1+ genotype as the reference, both GSTT1+/GSTM1- (OR 16.61; 95% CI 6.69-41.22) and GSTT1-/GSTM1- (OR 11.91; 95% CI 4.48-31.66) genotypes seem to be risk factors for chronic liver disease. From our observations, we conclude that polymorphism in GSTM1 null genotype (OR 17.85; 95% CI 7.34-43.45) seem to be associated with an increased risk of chronic liver disease among Filipinos.
Keywords: GSTT1, GSTM1, chronic liver disease, hepatitis B virus, polymorphism, Filipino
Introduction
Glutathione S-transferase (GST) enzymes are involved in the metabolism of a wide variety of carcinogenic compounds. GSTT1 and GSTM1 subfamilies exhibit homozygous deletions (null genotype) that have been considered as modifiers of individual risk of environmentally-induced cancers. These deletions are easily detected by molecular-based assays such as multiplex PCR [1].
The prevalence of the null genotype of GSTT1 and GSTM1 has been reported to vary among different ethnic populations. The GSTT1 is absent in 20% of Caucasians and 60% of Asians while GSTM1 is absent in 30% to 70% of the population [2-4]. Both genes are inherited independently from one another. The GST null genotype has been shown to be associated with various malignancies including cancers of the blood, bladder, colon, and lung [5-8].
Several studies have investigated the role of GSTT1 and GSTM1 in HBV-related liver cirrhosis (LC) and hepatocellular carcinoma (HCC) but the findings are inconsistent across studies [9-14]. It has been reported that individuals with dual null genotypes (GSTT1-/GSTM1-) were particularly susceptible to develop HCC. However, these findings were not found among Caucasians but only among Asian populations [15].
Here we aimed to determine the distribution of GSTT1 and GSTM1 null genotypes in patients with chronic liver disease and in normal individuals. Additionally, we evaluated the association between GSTT1 and GSTM1 gene polymorphisms and chronic liver disease among Filipinos and compared the frequency among various populations. To the best of our knowledge, this is the first report of the incidence of GSTT1 and GSTM1 polymorphisms among Filipino patients positive for HBV DNA and clinically diagnosed as either with chronic active hepatitis (CAH), LC, or HCC.
Materials and methods
Samples and patients
Blood samples from 107 Filipino patients infected with hepatitis B and 127 control subjects at St. Luke’s Medical Center-Quezon City were retrospectively studied. The cases include patients positive for HBV DNA and clinically diagnosed as either with CAH, LC, or HCC. A total of 52 samples positive for HBV were genotyped. The control group was composed of normal individuals negative for HBV DNA or non reactive to hepatitis B surface antigen. There were 138 (59%) males and 96 (41%) females with ages ranging from 15 to 76 years old.
Multiplex PCR amplification of GSTT1 and GSTM1 genes
Genomic DNA from normal individuals and HBV-infected patients was obtained from peripheral blood using the QIAamp DNA Blood Mini kit from Qiagen according to manufacturer’s instructions. GST gene amplification was carried with 1.0 pmol each of the primers, 1X Phusion HF Buffer (Finnzymes), 200 μM dNTPs, 0.02 U of Phusion DNA polymerase and DNA template was made up to a volume of 25 μl. Multiplex PCR step was carried out at 98°C for 1 minute, 35 cycles at 98°C for 1 minute, 63°C for 1 minute, 72°C for 1 minute and a final elongation at 72°C for 10 minutes. Multiplex PCR was done using the sense and antisense primers (Table 1). Amplicons were analyzed on 2% agarose gel followed by staining with ethidium bromide (Figure 1).
Table 1.
Primers used for GSTT1, GSTM1, β-globin, and HBV DNA amplification.
| Gene | Polarity | Nucleotide Sequence (5’ to 3’) | Reference |
|---|---|---|---|
| GSTT1 | Sense | TTC CTT ACT GGT CCT CAC ATC TC | [5] |
| Antisense | TCA CCG GAT CAT GGC CAG CA | ||
| GSTM1 | Sense | GAA CTC CCT GAA AAG CTA AAG C | [5] |
| Antisense | GTT GGG CTC AAA TAT ACG GTG G | ||
| β-globin | Sense | GAA GAG CCA AGG ACA GGT AC | [5] |
| Antisense | CAA CTT CAT CCA CGT TCA CC | ||
| HBV PrS2 | Outer sense | GGG ACA CCA TAT TCT TGG | [16] |
| HBV S1R | Outerantisense | TTA GGG TTT AAA TGT ATA CCC A | |
| HBV YS2 | Inner sense | GCG GGG TTT TTC TTG TTG A | [16] |
| HBV YS1 | Inner antisense | GGG ACT CAA GAT GTT GTA CAG |
Figure 1.
Ethidium bromide-stained 2% agarose gel showing the amplified products of a multiplex PCR used in the detection of GSTM1 (219-bp) and GSTT1 (480-bp) genotypes. The 268-bp fragment corresponds to the β-globin gene that was used as an internal control. Lane 1: GSTT1+/GSTM1+, Lane 2: GSTT1-/GSTM1-, Lane 3: GSTT1+/GSTM1-, Lane 4: GSTT1-/GSTM1+, Lane 5: 50-bp ladder.
Genotyping of hepatitis B virus by direct DNA sequencing
The viral DNA from HBV-infected patient plasma was extracted from peripheral blood using the QIAamp DNA and Blood Mini kit from Qiagen. The S gene amplification was carried with 10 pmol each of the primers, 1.5U HotStar Taq DNA polymerase and DNA template was made up to a volume of 50 μl. Nested PCR was carried out at 95°C for 15 minutes, 35 cycles at 94°C for 1 minute, 53°C for 45 seconds, 72°C for 1 minute and a final elongation at 74°C for 7 minutes. First round PCR was done using the outersense and outer antisense primers [16]. One μl of the first round PCR product was reamplified with internal primers for another 35 cycles under the same conditions. The 585-bp amplicon was purified prior to direct DNA sequencing (Macrogen, Korea). The sequence data were aligned with the consensus sequences of confirmed genotypes. The DNA sequences were then compared for identity with sequence from NCBI using the BLAST program.
Statistical analysis
The data were analyzed using OpenEpi 2.2. The chi-square (X2) test was used to determine significant differences in the demographic and genetic characteristics between cases and controls. The odds ratios (OR) with their corresponding 95% confidence intervals (CI) and p-values were calculated simultaneously as an estimate of the risk of having chronic liver disease given a particular variable or polymorphism.
Results
The distribution of subjects by GSTT1 and GSTM1 genotypes as well as the distribution of GSTT1 and GSTM1 genotypes in patients with chronic liver disease and normal individuals are shown in Tables 2 and 3. Out of the 107 cases, 90 (84%) were classified as CAH, 12 (11%) were LC and 5 (5%) were HCC. Most individuals were found to be GSTT1+/GSTM1- (55%). Among GSTT1+/GSTM1- individuals, 50% were CAH, 6% LC, 4% were HCC, and 40% were normal. For GSTT1-/GSTM1-, 44% were CAH, 8% were LC, and 48% were normal. Individuals who were GSTT1-/GSTM1- comprised 21% of the study subjects. Also, 21% were genotyped as GSTT1+/GSTM1+ and 3% were GSTT1-/GSTM1+. Among GSTT1+/GSTM1+ individuals, 8% had liver diseases and 92% were healthy. All GSTT1-/GSTM1+ individuals were normal.
Table 2.
Distribution of subjects by GSTT1 and GSTM1 genotypes.
| Genotype | Chronic liver disease | Normal individuals | Total | p-value |
|---|---|---|---|---|
| N (%) | N (%) | N (%) | ||
| GSTT1 | NS | |||
| GSTT1- | 26 (24) | 32 (25) | 58 (25) | |
| GSTT1+ | 81 (76) | 95 (75) | 176 (75) | |
| Total | 107 (100) | 127 (100) | 234 (100) | |
| GSTM1 | <0.001 | |||
| GSTM1- | 103 (96) | 75 (59) | 178 (76) | |
| GSTM1+ | 4 (4) | 52 (41) | 56 (24) | |
| Total | 107 (100) | 127 (100) | 234 (100) | |
NS = not significant
Table 3.
Distribution of GSTT1 and GSTM1 genotypes in patients with chronic liver disease and normal individuals
| GSTT1+ | GSTT1+ | GSTT1- | GSTT1- | Total | |
|---|---|---|---|---|---|
| GSTM1+ | GSTM1- | GSTM1+ | GSTM1- | ||
| N (%) | N (%) | N (%) | N (%) | ||
| Chronic liver disease | 4 (4) | 77 (72) | 0 (0) | 26 (24) | 107 |
| Normal individuals | 44 (35) | 51 (40) | 8 (6) | 24 (19) | 127 |
| Total | 48 (21) | 128 (55) | 8 (3) | 50 (21) | 234 |
The association of age, sex, GSTT1, and GSTM1 genotypes was examined (Table 4). There is a significant difference in age distribution (p-value 0.007) but not in sex between the two groups. The odds of having a chronic liver disease is only 0.95 (95% CI 0.58-1.57) among those with GSTT1 null genotype compared to those with GSTT1+ genotype. On the other hand, the odds of chronic liver disease is 17.85 times (95% CI 7.34-43.45) for those with GSTM1 null genotype compared to those with GSTM1+ genotype. Using the GSTT1+/GSTM1+ genotype as the reference, both GSTT1+/GSTM1- (OR 16.61; 95% CI 6.69-41.22) and GSTT1-/GSTM1- (OR 11.91; 95% CI 4.48-31.66) genotypes seem to be risk factors for chronic liver disease.
Table 4.
Odds ratio and 95% confidence interval of GSTT1 and GSTM1 genotypes.
| Parameters | Chronic liver disease | Normal individuals | p-value |
|---|---|---|---|
| N=107 (%) | N=127 (%) | ||
| Age | 0.007 | ||
| ≤ 40 | 56 (52) | 49 (39) | |
| 41-64 | 46 (43) | 57 (45) | |
| ≥65 | 5 (5) | 21 (16) | |
| Sex | NS | ||
| Male | 68 (64) | 70 (55) | |
| Female | 39 (36) | 57 (45) | |
| GSTT1 | NS | ||
| Null | 26 (24) | 32 (25) | |
| Positive | 81 (76) | 95 (75) | |
| OR (95% CI) | 0.95 (0.58-1.57) | ||
| GSTM1 | <0.001 | ||
| Null | 103 (96) | 75 (59) | |
| Positive | 4 (4) | 52 (41) | |
| OR (95% CI) | 17.85 (7.34-43.45) | ||
| GSTT1+/GSTM1+* | 4 (4) | 44 (35) | <0.001 |
| GSTT1+/GSTM1- | 77 (72) | 51 (40) | |
| OR (95% CI) | 16.61 (6.69-41.22) | ||
| GSTT1-/GSTM1+ | 0 (0) | 8 (6) | |
| OR (95% CI) | 0 (0) | ||
| GSTT1-/GSTM1- | 26 (24) | 24 (19) | |
| OR (95% CI) | 11.91 (4.48-31.66) | ||
NS = not significant;
used as a reference
Out of 107 HBV positive samples, only 52 (49%) were genotyped. Sequence analysis of the S gene region showed that 24 (46%) belonged to genotype HBV-A, 14 (27%) belonged to genotype HBV-B, and 14 (27%) was of genotype HBV-C. No HBV genotypes D to H were noted.
Discussion
Polymorphism in GSTT1 and GSTM1 genes were identified through multiplex PCR and the β-globin gene was used as an internal control. It has been suggested that internal controls such as β-globin, dihydrofolate reductase (DHFR), or p53 gene must be included in order to exclude false negative reactions [9,11]. This approach was effective in accurately identifying GST null genotypes since PCR inhibition can be ruled out through a positive β-globin band when a negative result for GSTT1 and GSTM1 is obtained. Heterozygous genotypes for GSTT1 and GSTM1 cannot be distinguished using current PCR techniques including multiplex PCR [10,17]. Thus, Hardy-Weinberg equilibrium (HWE) cannot be determined in this study. As previously described, Hardy-Weinberg tests in case-control studies are not available that can be applied to dominant and recessive markers with only two alleles such as GST [18].
The frequency of the GSTT1 and GSTM1 null genotype has been reported in various ethnic backgrounds worldwide. Deng et al., and Munaka et al., have shown that GSTT1 null genotype among healthy Chinese and Japanese populations were absent in 42.7% and 47.8%, respectively. In another study, Sun et al., reported that the frequency of GSTT1 and GSTM1 null genotypes among healthy Taiwanese were 60.2%. In this study, the GSTT1 null genotype was observed in 24.2% of HBV-infected patients and 25.2% among normal individuals. On the other hand, GSTM1 null genotype was observed in 96.3% of HBV-infected patients and 59.0% among normal individuals (Table 5 and 6).
Table 5.
GSTT1 null genotype distribution in patients with chronic liver disease and normal individuals.
| Method | Case | Control | Reference | |||||
|---|---|---|---|---|---|---|---|---|
| N | Null | (%) | N | Null | (%) | |||
| Filipino | Multiplex PCR | 107 | 26 | (24.2) | 127 | 32 | (25.2) | Present study |
| Chinese | PCR | 181 | 108 | (59.7) | 360 | 154 | (42.7) | [9] |
| Italian | Multiplex PCR | 67 | 30 | (16.0) | 400 | 72 | (18.0) | [10] |
| Spanish | Multiplex PCR | 184 | 53 | (28.8) | 329 | 76 | (23.1) | [11] |
| Japanese | Multiplex PCR | 78 | 39 | (51.3) | 138 | 66 | (47.8) | [19] |
| Taiwanese | PCR | 67 | 30 | (44.8) | 128 | 77 | (60.2) | [20] |
Table 6.
GSTM1 null genotype distribution in patients with chronic liver disease and normal individuals.
| Method | Case | Control | Reference | |||||
|---|---|---|---|---|---|---|---|---|
| N | Null | (%) | N | Null | (%) | |||
| Filipino | Multiplex PCR | 107 | 103 | (96.3) | 127 | 75 | (59.0) | Present study |
| Japanese | PCR | 83 | 51 | (61.4) | 101 | 43 | (42.5) | [4] |
| Chinese | PCR | 181 | 117 | (64.6) | 360 | 172 | (47.8) | [9] |
| Italian | Multiplex PCR | 200 | 99 | (49.5) | 400 | 215 | (53.8) | [10] |
| Spanish | Multiplex PCR | 184 | 88 | (47.8) | 329 | 149 | (45.3) | [11] |
| Japanese | Multiplex PCR | 78 | 29 | (38.5) | 138 | 68 | (49.3) | [19] |
| Taiwanese | PCR | 69 | 26 | (37.7) | 128 | 77 | (60.2) | [20] |
Several associations of GSTT1 and GSTM1 polymorphisms with HBV-related LC and HCC have been reported. However, the results are conflicting among different ethnic populations and do not provide consistent evidence for an association of genetic polymorphisms on GST enzymes with the development of HCC [9-12,19-21]. One of the possible reasons for this may be explained by the selection of study participants using cohort, hospital, or community-based subjects [22]. Another possible reason is that not all study check for HWE, which can affect the result especially of case-control studies [10,17].
The present study did not look into the association between GSTT1 and GSTM1 genotypes with CAH, LC, and HCC because of the limited number of LC and HCC cases. Ideally, GSTT1 and GSTM1 association should be determined in relation to the progression of chronic liver disease to HCC as previously described [9-12,19-21]. Gene-environment interactions were also not addressed in this study due to the limited reported data. Further studies in combination with other genes and environmental factors are recommended. For instance, N-acetyltransferase-2 (NAT-2) slow acetylator genotype in combination with GSTT1 null genotype further increase the risk of chronic liver disease among cigarette smokers [10,23].
Sobel et al., reported that approximately seven million Filipinos are chronically infected with HBV. Considering the importance of determining the HBV genotypes, several methods have been developed for genotyping of HBV strains such as restriction fragment length polymorphism, line probe assay, and quantitative real-time PCR [16,25-30]. Here we report the prevalent genotype found among HBV-infected Filipino patients using DNA sequencing. Results showed that HBV-A (46%) is the predominant genotype found among 52 samples analyzed in this study. Our data corroborates the findings that HBV-A and HBV-C are the common genotypes found among patients with chronic HBV infection in Cebu City, Philippines [31]. HBV genotyping have major implications for molecular epidemiologic studies, as well as investigating clinical outcomes particularly among patients infected with HBV-A, HBV-B, or HBV-C. It has been reported that patients infected with genotype HBV-C have been found to have more severe liver disease than genotype HBV-B and a lower response rate to treatment in comparison with those individuals infected with genotypes HBV-A and HBV-B [31-33]. However, the present study did not look into the disease progression and treatment response. Thus, we cannot confirm such findings. We are also aware of the limitation in sample size and other data in this study which prevents the analysis of confounding factors and detection of significant effects in some statistical tests.
From our observations, the GSTT1 did not show significant association with chronic liver disease in the available data, but the genetic polymorphism in GSTM1 null genotype seem to be associated with an increased risk of chronic liver disease among Filipinos. The limitation of this conclusion is due to the fact that spurious association cannot be easily ruled out with the available data at hand. However, our findings may have prognostic value to identify high risk groups and possible genetic factors in future studies on chronic liver disease.
Acknowledgement
This work was supported by a research grant to the St. Luke’s Liver Diseases Study Group (Project No: 05-022) through the Research and Biotechnology Division, St. Luke’s Medical Center-Quezon City, Philippines.
References
- 1.Abdel-Rahman SZ, El-Zein RA, Anwar WA, Au WW. A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies. Cancer Letters. 1996;107:229–233. doi: 10.1016/0304-3835(96)04832-x. [DOI] [PubMed] [Google Scholar]
- 2.Landi S. Mammalian class theta GST and differential susceptibility to carcinogens: a review. Mutat Res. 2000;463:247–283. doi: 10.1016/s1383-5742(00)00050-8. [DOI] [PubMed] [Google Scholar]
- 3.Rebbeck TR. Molecular epidemiology of the human glutathione S transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol Biomarkers Prev. 1997;6:733–743. [PubMed] [Google Scholar]
- 4.Katoh T, Inatomi H, Nagaoka A, Sugita A. Cytochrome P4501A1 gene polymorphism and homozygous deletion of the glutathione S transferase M1 gene in urothelial cancer patients. Carcinogenesis. 1995;16:655–657. doi: 10.1093/carcin/16.3.655. [DOI] [PubMed] [Google Scholar]
- 5.Chen CL, Liu Q, Pui CH, Rivera GK, Sandlund JT, Ribeiro R, Evans WE, Relling MV. Higher frequency of glutathione S transferase deletions in black children with acute lymphoblastic leukemia. Blood. 1997;89:1701–1707. [PubMed] [Google Scholar]
- 6.Bell DA, Taylor JA, Paulson DF, Robertson CN, Mohler JL, Lucier GW. Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J Natl Cancer Inst. 1993;85:1159–1164. doi: 10.1093/jnci/85.14.1159. [DOI] [PubMed] [Google Scholar]
- 7.Welfare M, Adeokun AM, Bassendine MF, Daly AK. Polymorphisms in GSTP1 GSTM1 and GSTT1 and susceptibility to colorectal cancer. Cancer Epidemiol Biomarkers Prev. 1999;8:289–292. [PubMed] [Google Scholar]
- 8.Dialyna IA, Miyakis S, Georgatou N, Spandidos DA. Genetic polymorphisms of CYP1A1 GSTM1 and GSTT1 genes and lung cancer risk. Oncology Reports. 2003;10:1829–1835. [PubMed] [Google Scholar]
- 9.Deng ZL, Wei YP, Ma Y. Polymorphism of glutathione S transferase mu1 and theta 1 genes and hepatocellular carcinoma in Southern Guangxi China. World J Gastroenterol. 2005;11:272–274. doi: 10.3748/wjg.v11.i2.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gelatti U, Covolo L, Talamini R, Tagger A, Barbone F, Martelli C, Cremaschini F, Franceschi S, Ribero ML, Garte S, Nardi G, Donadon V, Donato F. N-acetyltansferase-2 glutathione S transferase M1 and T1 genetic polymorphisms cigarette smoking and hepatocellular carcinoma: a case-control study. Int J Cancer. 2005;115:301–306. doi: 10.1002/ijc.20895. [DOI] [PubMed] [Google Scholar]
- 11.Ladero JM, Martíez C, Garcí-Martí E, Ropero P, Briceñ O, Villegas A, Díz-Rubio M, Agúdez JA. Glutathione S transferase M1 and T1 genetic polymorphisms are not releated to the risk of hepatocellular carcinoma: a study in the Spanish population. Eur J Cancer. 2006;42:73–77. doi: 10.1016/j.ejca.2005.08.033. [DOI] [PubMed] [Google Scholar]
- 12.Mohammadzadeh GS, Yaghmaei B, Allameh A, Hassani P, Noorinayer B, Zali MR. Polymorphisms of glutathione S transferase M1 T1 and P1 in patients with HBV related liver cirrhosis chronic hepatitis and normal carriers. Clin Biochem. 2006;39:46–49. doi: 10.1016/j.clinbiochem.2005.10.004. [DOI] [PubMed] [Google Scholar]
- 13.Kandemir O, Tamer L, Tasdelen B. Effects of GSTT1 GSTM1 and GSTP1 gene polymorphism on the course of hepatitis B virus infection. Hepatogastroenterology. 2008;55:1729–1733. [PubMed] [Google Scholar]
- 14.Yu L, Wang CY, Xi B, Sun L, Wang RQ, Yan YK, Zhu LY. GST polymorphisms are associated with hepatocellular carcinoma risk in Chinese population. World J Gastroenterol. 2011;17:3248–3256. doi: 10.3748/wjg.v17.i27.3248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Wang B, Huang G, Wang D, Li A, Xu Z, Dong R, Zhang D, Zhou W. Null genotypes of GSTM1 and GSTT1 contribute to hepatocellular carcinoma risk: evidence from an updated meta-analysis. J Hepatol. 2010;53:508–518. doi: 10.1016/j.jhep.2010.03.026. [DOI] [PubMed] [Google Scholar]
- 16.Zeng GB, Wen SJ, Wang ZH, Yan L, Sun J, Hou JL. A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons. World J Gastroenterol. 2004;10:3132–3136. doi: 10.3748/wjg.v10.i21.3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Rimando MG, Chua MN, Yuson E, de Castro-Bernas G, Okamoto T. Prevalence of GSTT1 GSTM1 and NQ01 (609C>T) in Filipino children with acute lymphoblastic leukemia. Biosci Rep. 2008;28:117–124. doi: 10.1042/BSR20070010. [DOI] [PubMed] [Google Scholar]
- 18.Esser C, Tomiukw J. Reporting Hardy Weinberg tests in case control studies: reasons for caution but not for panic reactions. J Invest Dermatol. 2005;124:1082–1083. doi: 10.1111/j.0022-202X.2005.23709.x. [DOI] [PubMed] [Google Scholar]
- 19.Munaka M, Munaka M, Kohshi K, Kawamoto T, Takasawa S, Nagata N, Itoh H, Oda S, Kato T. Genetic polymorphisms of tobacco and alcohol related metabolizing enzymes and the risk of hepatocellular carcinoma. J Cancer Res Clin Oncol. 2003;129:355–360. doi: 10.1007/s00432-003-0439-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Sun CA, Wang LY, Chen CJ, Lu SN, You SL, Wang LW, Wang Q, Wu DR, Santella RM. Genetic polymorphisms of glutathione S transferases M1 and T1 associated with susceptibility to aflatoxin-related hepatocarcinogenesis among chronic hepatitis B carriers: a nested case-control study in Taiwan. Carcinogenesis. 2001;22:1289–1294. doi: 10.1093/carcin/22.8.1289. [DOI] [PubMed] [Google Scholar]
- 21.Yu MW, Yang SY, Pan IJ, Lin CL, Liu CJ, Liaw YF, Lin SM, Chen PJ, Lee SD, Chen CJ. Polymorphisms in XRCC1 and glutathione S transferase genes and hepatitis B related hepatocellular carcinoma. J National Cancer Institute. 2003;95:1485–1488. doi: 10.1093/jnci/djg051. [DOI] [PubMed] [Google Scholar]
- 22.White DL, Li D, Nurgalieva Z, El-Serag HB. Genetic variants of glutathione S-transferase as possible risk factors for hepatocellular carcinoma: a HuGE systematic review and meta-analysis. Am J Epidemiol. 2008;167:377–389. doi: 10.1093/aje/kwm315. [DOI] [PubMed] [Google Scholar]
- 23.Moore LE, Baris DR, Figueroa JD, Garcia-Closas M, Karagas MR, Schwenn MR, Johnson AT, Lubin JH, Hein DW, Dagnall CL, Colt JS, Kida M, Jones MA, Schned AR, Cherala SS, Chanock SJ, Cantor KP, Silverman DT, Rothman N. GSTM1 null and NAT2 slow acetylation genotypes smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis. Carcinogenesis. 2011;32:182–189. doi: 10.1093/carcin/bgq223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sobel HL, Mantaring JB 3rd, Cuevas F, Ducusin JV, Thorley M, Hennessey KA, Nyunt-U S. Implementing a national policy for hepatitis B birth dose vaccination in Philippines: lessons for improved delivery. Vaccine. 2011;29:941–945. doi: 10.1016/j.vaccine.2010.11.047. [DOI] [PubMed] [Google Scholar]
- 25.Grandjacques C, Pradat P, Stuyver L, Chevallier M, Chevallier P, Pichoud C, Maisonnas M, Tréo C, Zoulim F. Rapid detection of genotypes and mutations in the pre core promoter and the pre core region of hepatitis B virus genome: correlation with viral persistence and disease severity. J Hepatol. 2000;3:430–439. doi: 10.1016/s0168-8278(00)80279-2. [DOI] [PubMed] [Google Scholar]
- 26.Zhao Y, Zhang XY, Guo JJ, Zeng AZ, Hu JL, Huang WX, Shan YL, Huang AL. Simultaneous genotyping and quantification of hepatitis B virus for genotypes B and C by real time PCR assay. J Clin Microbiol. 2010;48:3690–3697. doi: 10.1128/JCM.00741-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Malmstrom S, Berglin-Enquist I, Lindh M. Novel method for genotyping hepatitis B virus on the basis of TaqMan real time PCR. J Clin Microbiol. 2010;48:1105–1111. doi: 10.1128/JCM.01442-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Ali MM, Hasan F, Ahmad S, Al-Nakib W. Comparative evaluation of INNO-LiPA HBV assay direct DNA sequencing and subtractive PCR RFLP for genotyping of clinical HBV isolates. Virol J. 2010;7:111. doi: 10.1186/1743-422X-7-111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ma Y, Ding Y, Juan F, Dou XG. Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang China. Virol J. 2011;8:315. doi: 10.1186/1743-422X-8-315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Malmströ S, Berglin-Enquist I, Lindh M. Novel method for genotyping hepatitis B virus on the basis of TaqMan real time PCR. J Clin Microbiol. 2010;48:1105–1111. doi: 10.1128/JCM.01442-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Batoctoy KS, Tseng TC, Kao JH, Quiza FE, Garcia LH Sr, Lao-Tan J. HBV/A and HBV/C genotype predominance among patients with chronic hepatitis B virus infection in Cebu City Philippines. Hepatol Int. 2011;5:774–781. doi: 10.1007/s12072-011-9263-1. [DOI] [PubMed] [Google Scholar]
- 32.Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology. 2000;118:554–559. doi: 10.1016/s0016-5085(00)70261-7. [DOI] [PubMed] [Google Scholar]
- 33.Valsamakis A. Molecular testing in the diagnosis and management of chronic hepatitis B. Clin Microbiol Rev. 2007;20:426–439. doi: 10.1128/CMR.00009-07. [DOI] [PMC free article] [PubMed] [Google Scholar]