Molecular variants of HPV-16 associated with cervical cancer in Indian population M.R. Pillai 1 , R. Hariharan 1 , Janki Mohan Babu 1 , S. Lakshmi 2 , S.V. Chiplunkar 3,4 , M. Patkar 3,4 , H. Tongaonkar 3,4 , K. Dinshaw 3,4 , R.S. Jayshree 5 , B.K.M. Reddy 5 , M. Siddiqui 6 , Soma Roychoudury 6 , Baisakhi Saha 7 , P. Abraham 8 , M. Gnanamony 8 , A. Peedicayil 9 , J. Subhashini 10 , T.S. Ram 10 , Bindu Dey 11 , C. Sharma 12 , S.K. Jain 13 and N. Singh 12 * 1 Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India 2 Regional Cancer Centre, Thiruvananthapuram, India 3 Tata Memorial Center, Parel, Mumbai, India 4 Advanced Centre for Treatment, Research, Education in Cancer, Kharghar, Navi Mumbai, India 5 Department of Microbiology, Kidwai Memorial Institute of Oncology, Bangalore, India 6 Cancer Foundation of India, Kolkata, India 7 Bose Institute, Kolkata, India 8 Department of Clinical Virology, Christian Medical College, Vellore, India 9 Department of Obstetrics and Gynaecology, Christian Medical College, Vellore, India 10 Department of Radiation Oncology, Christian Medical College, Vellore, India 11 Department of Biotechnology, New Delhi, India 12 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India 13 Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India Human papilloma virus is a causative factor in the etiology of cer- vical cancer with HPV16 being the most prevalent genotype asso- ciated with it. Intratype variations in oncogenic E6/E7 and capsid L1 proteins of HPV 16 besides being of phylogenetic importance, are associated with risk of viral persistence and progression. The objective of this multicentric study was to identify HPV-16 E6, E7 and L1 variants prevalent in India and their possible biological effects. Squamous cell cervical cancer biopsies were collected from 6 centres in India and examined for the presence of HPV 16. Variants of HPV-16 were characterized by full length sequence analysis of L1, E6 and E7 genes in 412 samples. Similar distribu- tion of the variants was seen from the different centres/regions, with the European variant E350G being the most prevalent (58%), followed by American Asian variant (11.4%). Fifty six changes were seen in E6 region, 31 being nonsynonymous. The most frequent being L83V (72.3%), Q14H (13.1%) and H78Y (12.1%). Twenty-nine alterations were seen in E7 region, with 12 being nonsynonymous. The most frequent being F57V (9%). L1 region showed 204 changes, of which 67 were nonsynonymous. The most frequent being 448insS (100%), and 465delD (100%), H228D (94%), T292A (85%). The identified variants some new and some already reported can disrupt pentamer formation, tran- scriptional regulation of the virus, L1 protein interface interac- tion, B and T cell epitopes, p53 degradation, and thus their distri- bution is important for development of HPV diagnostics, vaccine, and for therapeutic purpose. ' 2009 UICC Key words: cervical cancer; human papilloma virus 16; variants Cervical cancer is the second most predominant cancer world- wide. 1 In India it is the most common cancer amongst women, accounting for 130,000 new cases and more than 70,000 deaths annually. Persistent infection with high risk Human papilloma virus (HPV) is the main aetiological factor in the development of cervical cancer and may depend on HPV genotypes and variants. 2 HPV-16 is the most prevalent genotype associated with cervical carcinomas globally, as well as in Indian women. 3–5 This could perhaps be because of differential HPV-16 variant frequency, with certain variants conferring greater oncogenecity. 6,7 The molecular variants or lineages differ in nucleotide sequence by no more than 2% in the coding region and 5% in the noncoding regions of the viral genome with respect to the prototype. 8 Through nucleotide sequence comparisons, it has been found that HPV-16 has evolved along 5 major phylogenetic branches i.e., European, Asian, Asian American, African-1 and African-2. 9 Intratypic variation of HPV- 16 has been shown to be an important predictor of progression to clinical relevant cervical lesion. 10 In a cohort study of young women 16 different HPV variants were found, one of which per- sisted over time, although the other variants were transiently detected. 11 Considerable intratypic diversity of HPV-16 has been reported by other studies based on DNA sequencing from cervical tumors. 12–14 The European variant is the most widely distributed worldwide except in Africa. Furthermore, variation studies per- formed on LCR, L1, L2, E6 genes of HPV-16 indicate that recom- bination between variants is rare or nonexistent. 9,14 The major L1 and minor L2 capsid proteins make up 83% and 17% of the viral coat, respectively, and thus L1 is present during the initial infection. Since L1 has the property of assembling into virus like particles (VLPs) which in turn are important in eliciting an effective immune response it has been used as an ideal target for prophylactic HPV vaccine. Few nucleotide differences found in HPV variants correspond to change in amino acids. Alterations that may interfere with the structure, functional or antigenic prop- erties of specific viral proteins is important. For instance, variant 114K of HPV-16 assembles into VLPs in a heterologous expres- sion system, whereas the reference (prototype) clone of HPV-16 does not have the same property. 15 This difference has been attrib- uted to a single amino acid change H228D of L1. Other changes in the L1/L2 region may be important for discriminating between the infectious potential of different variants and in defining epi- topes relevant to vaccine design. The oncoproteins E6 and E7 play an important role in transfor- mation of the host cell in HPV induced cervical carcinogenesis. 16 The transcriptional transactivator E6 protein targets the degrada- tion of p53, eventually accelerating cell proliferation. E7 binds to and inactivates pRb and thus triggers the cell cycle progression. It is also reported that specific variations in the HPV-16 E6 protein, interfere with the T-cell cytotoxic immune response. 17 HPV-16 sequence variations in cervical cancer have been reported from some countries. 18–20 However, there is only limited data available in the literature from India, the data is regional, sample size is small and full length sequencing is not done. Comprehensive data integrating various regions is lacking. 12,18,21,22 In this study, we present results of HPV-16 L1, E6 and E7 variants observed in Conflict of Interest: A. Peedicayil has a potential personal conflict of in- terest as he attended a meeting organized by MSD pharmaceuticals in New Delhi, India, in April 2008 on ‘‘Diseases caused by HPV.’’ *Correspondence to: Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India. Fax: 91-11-26588663. E-mail: singh_neeta@hotmail.com Received 5 November 2008; Accepted after revision 7 January 2009 DOI 10.1002/ijc.24322 Published online 3 February 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 91–103 (2009) ' 2009 UICC Publication of the International Union Against Cancer