Core Promoter Mutations in Hepatitis B Virus-infected Population of North India

Short Communication

JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY

Prevalence of Genotype D and Precore/Core Promoter Mutations in Hepatitis B Virus-infected Population of North India Rajesh Kumar, Vikas Pahal, Jasbir Singh Department of Biochemistry, Kurukshetra University, Kurukshetra – 136119, Haryana, India

M

ore than 2 billion people are infected with hepatitis B virus (HBV) worldwide and 350 million are chronic carriers of the virus.1 The HBV infection is associated with a diverse clinical spectrum of liver damage ranging from asymptomatic carriers, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC).2 Hepatitis B virus is a member of the family Hepadnaviridae. It is a partially double-stranded DNA virus and contains 4 overlapping open reading frames (ORFs) encoding the surface, precore (PC)/core, polymerase, and X genes. This virus shows a remarkable genetic variability and is currently classified into 8 genotypes, designated A to H based on genomic sequence analysis.3 Different genotypes have characteristic geographical distributions. Genotype A is widely distributed in North-West Europe, North-America and Central Africa, while genotype B and C are distributed in Asia only; genotype D has been found worldwide with its highest prevalence in the Mediterranean area, the Middle East, and South-Asia, particularly in India; genotype E is found in Sub-Saharan Africa and genotype F in South and Central America; genotype G has been found in France and in the USA, while genotype H seems so far to be Keywords: Hepatitis B virus, genotypes, mutations Received: 06.06.2011; Accepted: 24.08.2011 Address for correspondence: Rajesh Kumar, Department of Biochemistry, Kurukshetra University, Kurukshetra – 136119, Haryana, India E-mail: [email protected] Abbreviations: ALT: alanine aminotransaminase; BCP: basal core promotor; BQW: best quality water; EnhII: enhancer II region; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; ORF: open reading frames; PCR: polymerase chain reaction doi: 10.1016/S0973-6883(11)60125-4 © 2011, INASL

restricted to the northern part of Latin America, including Central America and Mexico.4,5 The evidences suggest that HBV genotypes have an impact on the natural course of chronic HBV infection with differences in the severity of underlying liver disease or the treatment response.6–8 In addition to HBV genotype, mutations in the core promoter, precore have been shown to have an association with severe liver disease. The HBV has a high mutation rate compared with other DNA viruses because it lacks proofreading capacity during the replication via reverse transcription of its pregenomic RNA.9 The well known naturally occurring HBV variants include the PC stop codon mutation (G1896A), which eradicates hepatitis B e-antigen (HBeAg) production. The other common HBV variants include double mutations in the basal core promotor (BCP) region (A1762T/G1764A), which overlaps the ORF of the X gene and results in reduction of HBeAg production.10 These dual mutants have been reported in upto 50–80% of patients with HBeAg-negative chronic hepatitis B in Europe and Asia,11 and they have been implicated in HCC development.12–14 Apart from these variants, other mutations, such as T1753C/A/G in the BCP region and C1653T in the enhancer II region (EnhII) have been increasingly recognized as being associated with the outcome of chronic HBV infection, including HCC development.15–17 An attempt has been made in this communication to study different genotypes, subgenotypes and precore/core promoter mutations in HBV-infected population of Punjab (North India), as there is lack of information from this area. The sera were obtained from 21 HBsAg-positive patients from Punjab (North India) who had not taken antiviral agents such as lamivudine or interferon. All patients were

Journal of Clinical and Experimental Hepatology | September 2011 | Vol. 1 | No. 2 | 73–76

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Hepatitis B virus (HBV) isolates (21) from Punjab (North India) were studied for genotype distribution and precore/core promoter mutations. Assays of alanine aminotransaminase (ALT) and HBeAg were performed in all isolates. Genotypes were determined in all the samples by restriction fragment length polymorphism and the precore/core promoter mutations were studied by amplification and by direct sequencing of precore/core promoter region. Sixty-two percent of the isolates had higher ALT levels and 57% of the isolates were HBeAg negative. It was observed that 90% of the isolates were HBV D genotype (subgenotype D1 and D2) and 10% of the isolates were HBV A genotype (subgenotype A1). Amplification and sequencing of the precore/core promoter region showed 1762A–T and 1764G–A mutations in 29% and 19% of the isolates, respectively. 1809C/T mutation was observed in 71% of the isolates under study. Novel precore and core promoter mutations like 1690A, 1695A/T/G, 1700A/C, 1703C, 1850A and 1915A/G were observed in HBV-infected population of the state of Punjab (North India). Deletion and insertional mutations were also observed in some patients. (J CLIN EXP HEPATOL 2011;1:73–76)

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negative for HIV and HCV infection. The ethical committee of the institute approved the study. HBeAg and alanine aminotransaminase (ALT) were determined in all the serum samples by using an auto-analyzer and commercially available kits (Span Diagnostics, Surat, India), respectively. Extraction of DNA from serum was performed using QIAmp extraction kit (Qiagen, Hilden, Germany). Finally, extracted DNA was suspended in 100 μL best quality water (BQW). Polymerase chain reaction (PCR) for the amplification of pre-S1/S2 region was done by using nested PCR. First round PCR was performed by utilizing primer 230F (5′-TCACAATACCGCAGAGTCT-3′) and 800R (5′-AACAGCGGTATAAAGGGACT-3′) primers. The PCR was performed for 40 cycles involving denaturation at 94°C for 1 min, annealing at 53°C for 50 sec and extension at 72°C for 50 sec. The initial denaturation was at 94°C for 3 min and final extension was performed at 72°C for 10 min. Second round PCR was performed using P7 (5′-GTGGTGGACTTCTCTCAATTTTC-3′) and P8 primers (5′-CGGTATAAAGGGACTCAAGAT-3′) as per the method of Lindh et al.18 The PCR was performed for 40 cycles involving denaturation at 94°C for 45 sec, annealing at 53°C for 1 min, extension at 72°C for 1 min 30 sec. Here, the initial denaturation was at 94°C for 3 min and final extension was at 72°C for 7 min. The results were analyzed on 1.5% agarose gel as shown in Figure 1. Genotyping and subgenotyping was done using restriction enzyme Hinf I and Tsp 509I as per the method of Lindh et al.18 PCR for the amplification of precore/core promoter region was done using nested PCR. First round PCR was performed utilizing primer 1622F (5′-GAACGCCCATCAGATCCTGC-3′) and 1966R (5′-GTCAGAAGGCAAAAACGAGAG-3′) primers. Then the PCR was performed for 40 cycles involving denaturation at 94°C for 30 sec, annealing at 57°C for 50 sec and extension at 72°C for 50 sec. The initial denaturation was at 94°C for 5 min and final extension was performed at 72°C for 10 min. Next, the second round PCR was performed using 1661F (5′-GACTCTTGGACTCTCAGC-3′) and 1921R primers (5′-TTTATACGGGTCAATGTC-3′) as per the method of Baptista et al.19 Again the PCR was performed for 40 cycles involving denaturation at 94°C for 30 sec, annealing at 42°C for 30 sec, extension at 72°C for 50 sec. Initial denaturation was at 94°C for 3 min, and final extension was at 72°C for 10 min. The results were analyzed on 2% agarose gel as shown in Figure 3. Nucleotide sequences of samples were compared with the wild-type sequence using BioEdit sequence comparison software (USA). The assays of ALT and HBeAg showed that 62% of the samples had higher ALT level and 57% of the HBeAg samples were negative. The banding pattern of nested PCR (using 230F and 800R primers in the first round and P7 and P8 primers in the second round, followed by restriction analysis using 74

KUMAR ET AL

Lanes

1

2

3

4

5

6

7

8

541 bp

500 bp

Figure 1 Amplification of preS1/S2 region. Lanes 2–7 show amplified products of preS1/S2 region. Lane 1 shows marker DNA and 8 shows blank. Lanes

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3

4

5

6

7

8

9

10 11 12

500 bp 300 bp 200 bp

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Figure 2 Restriction fragment length polymorphism analysis of amplified preS1/S2 products. Lane 1 show 100 bp molecular weight marker, lane 2 shows undigested 541 bp PCR product, lanes 3, 5, 7, 9, 11 (odd numbered) show PCR products incubated with restriction enzyme HinfI, lanes 4, 6, 8, 10, 12 (even numbered) show PCR products incubated with restriction enzyme Tsp 509I. Lanes

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Figure 3 Amplification of precore/core promoter region. Lanes 2–7 show amplified products of preC/CP region. Lanes 1 and 8 show marker DNA and blank, respectively.

Hinf I and Tsp 509I for genotyping) was compared with the published results of Lindh et al.18 The results of restriction analysis for the identification of genotypes/subgenotypes were shown in Figure 2 and in Table 1; the results were analyzed as per the method of Lindh et al.18 Our results showed that HBV genotype D was found in 90% (19/21) of the isolates and 10% (2/21) isolates contained genotype A. Subgenotype D2 (79%) was predominant among genotype D isolates, followed by genotype D1 (21%). Genotype A had subgenotype A1 (100%). Genotype A was found to have subgenotype A1 which appeared quite similar to the South African isolates. Our results were in accordance with Asim et al20 who observed genotype D as prevalent genotype followed by genotype A in India. Our results were also in accordance with the study © 2011, INASL

JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY

Lanes

Restriction digestion pattern (bp)

Genotype/ subgenotype

3&9 4 & 10

HinfI → 526 Tsp 509I → 173, 164 & 109

D/D1

5&7 6&8

HinfI → 274 & 252 Tsp 509I → 173, 164 & 109

D/D2

11 12

HinfI → 274 & 252 Tsp 509I → 207, 126 & 109

A/A1

of Sharma et al,21 who observed genotype D and genotype A in North Indian patients, and in accordance with the study of Madan et al,22 who observed genotype D and A but we did not observe genotype C as the study of Madan et al,22 and in accordance with the study of Singh et al,23 who observed genotype D and A in North Indian population. But, we did not observe genotype E in our population as the study of Singh et al,23 in accordance with the study of Kar et al.24 Chattopadhyay et al25 reported predominance of genotype D followed by genotype A among patients with chronic liver disease from New Delhi. Amplification and sequencing of the precore/core promoter region shows 1762A–T and 1764G–A mutations. A 1762A–T mutation was observed in 29% of the samples; 50% had elevated ALT levels and 67% of the samples were negative for HBeAg. A 1764G–A mutation was observed in 19% of the samples, and 75% had elevated ALT and 50% of the samples were negative for HBeAg. 1762A–T and 1764G–A mutations were higher in HBeAg negative patients and found more in genotype D than in genotype A, but with high ALT in our study as observed by Chauhan et al.26 1762A–T 1764G–A double mutation is commonly detected in HBV genotypes A and C but less frequently in genotypes B and D.27,28 However, in our study, it was observed more in genotype D than genotype A. 1762A–T and 1764G–A mutations suppress HBeAg synthesis and may contribute to hepatocarcinogenesis.19,29 1809C/T mutation was observed in 71.4% of the patients. 1812T mutation was observed in 61.9% of the patients in our study. 1809C/T1812T mutations were found more in HBeAg positive patients and 86% of the patients were having high ALT level. 1809C/T1812T is a missense mutation, which is found in 80% of African blacks19 and represents the wild-type. 1888A mutation was found in 66.7% of the patients in our study. Ninety-two percent of the patients were having high ALT level and this mutation was found more in HBeAg positive patients. 1888A mutation in the precore region, are found exclusively in subgenotype A1 isolates.30,31 However, we observe this mutation in genotype D. So, our observation is not in accordance with the observation of Kramvis et al30 and Kimbi et al.31 1888A mutation has a stabilizing effect on encapsidation signal30 and it also possibly affects reverse

transcription, and hence this mutation affects the translation of the core protein.32 Therefore, this communication reports the prevalence of genotype D as the predominant genotype circulating in Punjab (Northern India) followed by genotype A and some novel precore and core promoter mutations like 1690A, 1695A/T/G, 1700C/A, 1703C, 1850A and 1915A/G in addition to 1719G, 1727G, 1757G, 1762A–T 1764G–A, 1773T, 1809C/T 1812T, 1858C, 1862T/C, 1888A, 1896A/C, and deletion and insertional mutations in HBV-infected population of state of Punjab (India).

ACKNOWLEDGMENT Rajesh Kumar is thankful to the CSIR (Council of Scientific and Industrial Research), New Delhi (CSIR award no. F. No. 10-2(5)2003(I)-EU.II) and DST (Department of Science and Technology), New Delhi for providing financial assistance to carry out this work.

CONFLICTS OF INTEREST All authors have none to declare. REFERENCES 1. Lee WM. Hepatitis B virus infection. N Engl J Med 1997;337: 1733–45. 2. Ganem D, Prince AM. Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med 2004;350:1118–29. 3. Kramvis A, Kew M, Francois G. Hepatitis B virus genotypes. Vaccine 2005;23:2409–23. 4. Miyakawa Y, Mizokami M. Classifying hepatitis B virus genotypes. Intervirology 2003;46:329–38. 5. Echevarrìa JM, Avellòn A, Magnius LO. Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing. J Med Virol 2005;76:176–84. 6. Sumi H, Yokosuka O, Seki N, et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology 2003;37:19–26. 7. Chan HLY, Tsang SWC, Liew CT, et al. Viral genotype and hepatitis B virus DNA levels are correlated with histological liver damage in HBeAg-negative chronic hepatitis B virus infection. Am J Gastroenterol 2002;97:406–12. 8. Liu CJ, Kao JH, Chen DS. Therapeutic implications of hepatitis B virus genotypes. Liver Int 2005;25:1097–107. 9. Kay A, Zoulim F. Hepatitis B virus genetic variability and evolution. Virus Res 2007;127:164–76. 10. Wai CT, Fontana RJ. Clinical significance of hepatitis B virus genotypes, variants, and mutants. Clin Liver Dis 2004;8:321–52. 11. Funk ML, Rosenberg DM, Lok AS. Worldwide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants. J Viral Hepat 2002;9:52–61. 12. Chen CH, Changchien CS, Lee CM, et al. Combined mutations in pre-s/surface and core promoter/precore regions of hepatitis B virus increase the risk of hepatocellular carcinoma: a case-control study. J Infect Dis 2008;198:1634–42. 13. Kao JH, Chen PJ, Lai MY, Chen DS. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 2003;124: 327–34.

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Table 1 Digestion pattern of amplified preS1/S2 products with restriction enzymes (Hinf I and Tsp 509I).

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