Original Article
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World J Gastroenterol. Dec 28, 2013; 19(48): 9294-9306
Published online Dec 28, 2013. doi: 10.3748/wjg.v19.i48.9294
Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India
Deepak Gopalakrishnan, Mark Keyter, Kotacherry Trivikrama Shenoy, Kondarappassery Balakumaran Leena, Lakshmikanthan Thayumanavan, Varghese Thomas, KR Vinayakumar, Charles Panackel, Arun T Korah, Ramesh Nair, Anna Kramvis
Deepak Gopalakrishnan, Mark Keyter, Anna Kramvis, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Parktown, Johannesburg 2193, South Africa
Deepak Gopalakrishnan, KR Vinayakumar, Charles Panackel, Arun T Korah, Ramesh Nair, Department of Gastroenterology, Medical College Trivandrum 695011, India
Kotacherry Trivikrama Shenoy, Kondarappassery Balakumaran Leena, Population Health and Research Institute, Trivandrum 695011, India
Lakshmikanthan Thayumanavan, Government Rajaji Hospital, Madurai Medical College, Madurai 625002, India
Varghese Thomas, Department of Gastroenterology, Calicut Medical College, PO Calicut 673008, India
Author contributions: Gopalakrishnan D and Keyter M contributed equally to the study; Gopalakrishnan D, Shenoy KT and Kramvis A conceived the study; Gopalakrishnan D, Shenoy KT, Leena KB, Thayumanavan L, Thomas V, Vinayakumar KR, Panackel C, Korah AT and Nair R collected the clinical data; Gopalakrishnan D, Keyter M and Kramvis A conceived and designed the laboratory experiments; Kramvis A contributed reagents, materials and analysis tools; Gopalakrishnan D and Keyter M performed the experiments; Gopalakrishnan D, Keyter M and Kramvis A analyzed and interpreted the data, and wrote the paper.
Supported by The National Research Foundation of South Africa, NRF, GUN 65530 (to Kramvis A) and the Cancer Association of South Africa; postdoctoral funding from the NRF, GUN 75055 and the University of the Witwatersrand (to Gopalakrishnan D); bursaries from the University of the Witwatersrand, the Poliomyelitis Research Foundation and the Ernst and Ethel Eriksen Trust (to Keyter M)
Correspondence to: Anna Kramvis, Professor, Hepatitis Virus Diversity Research Programme, Department of Internal Medicine, School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa. anna.kramvis@wits.ac.za
Telephone: +27-11-4883100 Fax: +27-86-5296806
Received: May 16, 2013
Revised: June 20, 2013
Accepted: July 17, 2013
Published online: December 28, 2013
Abstract

AIM: To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.

METHODS: Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student’s t test. The χ2 test or Fisher’s exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05.

RESULTS: Irrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D.

CONCLUSION: Kerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.

Keywords: Hepatocellular carcinoma, Cirrhosis, Chronic hepatitis, Phylogenetic analysis, Genotype, India

Core tip: This study shows the predominance of subgenotype A1 in liver disease patients in Kerala, and its high prevalence in hepatocellular carcinoma (HCC) patients. Subgenotype A1 could be more hepatocarcinogenic and HCC could develop at an earlier age, regardless of host ethnicity. The S open reading frame of subgenotype A1 isolates from Kerala clustered separately within the ‘‘Asian’’ cluster and encoded distinct subgenotype A1 amino acids. A higher frequency of G1862T was detected compared with subgenotype A1 isolates from other geographical regions. This is the first time that preS deletion mutants have been described in Indian HCC patients.