High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. Previous studies suggest that polymorphisms in the E6 gene or the long control region (LCR)of HPV16 may alter the oncogenic potential of the virus.
Sun et al BMC Cancer 2013, 13:459 http://www.biomedcentral.com/1471-2407/13/459 RESEARCH ARTICLE Open Access Genetic variations of E6 and long control region of human papillomavirus type 16 from patients with cervical lesion in Liaoning, China Zhengrong Sun1, Zhitao Lu1, Jianhua Liu1,2, Guili Wang1, Weiqiang Zhou1, Lianxia Yang1, Chao Liu1, Bo Wang1 and Qiang Ruan1* Abstract Background: High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer Previous studies suggest that polymorphisms in the E6 gene or the long control region(LCR)of HPV16 may alter the oncogenic potential of the virus The aims of this study were to investigate the genetic variations of HPV16 E6 gene and LCR in isolates from Chinese population and correlation of the E6 and LCR polymorphisms with disease status of infected patients Methods: HPV16 positive endocervical specimens were collected from 304 women living in Northeast of China Sequences of E6 gene and LCR were analyzed by PCR-sequencing Results: Two lineages were found in the populations, including EUR lineage and As lineage Based on the HPV16 prototype, the most frequent variation in the E6 gene was T178A/G (48.7%), followed by mutations of G94A (12.2%) and T350G (9.9%) The rank orders of incidence of E6 variations in amino acid were as follows: D25E (46.3%), L83V (9.9%) and H78Y (4.3%) Nucleotide variations in LCR were found in all the 304 isolates from HPV16 positive cervical samples The most commonly observed LCR variations were the transition replacement G7193T, 7434CIns, G7521A and 7863ADel (100%) The As lineage was associated with HPV persistent infections and with disease status of ≥CIN2,3 The EUR lineage variants showed a negative trend of association with the severity of ≥CIN2,3 Among 41 variations found in LCR, 25 (61.0%) were located at the binding sites for transcription factors Occurrence of ≥CIN2,3 was significantly associated with the mutations of R10G/L83V in E6 and the C7294T co-variation in LCR, after adjusting for ages of infected patients Conclusions: Associations between As lineage and HPV persistent infections, and with disease status of ≥CIN2,3, and an association between the EUR lineage and negative trend of association with the severity of ≥CIN2,3 were found in this study An association between a co-variation of R10G/L83V in E6 and C7294T in LCR and an increased risk for developing CIN-2,3 was found in a HPV16 infected population of Chinese women These findings indicate that HPV16 polymorphism influences development of CIN-2,3 Keywords: HPV16, E6, LCR, Cervical lesion * Correspondence: ruanq@sj-hospital.org Present address: Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, No 36, Sanhao Street, Heping District, Shenyang 110004, China Full list of author information is available at the end of the article © 2013 Sun et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Sun et al BMC Cancer 2013, 13:459 http://www.biomedcentral.com/1471-2407/13/459 Background Persistent infections of high-risk human papillomavirus (HPV) are the necessary causes of cervical cancer However, only a minority of women infected with high-risk HPV will develop persistent infection [1] Developing risk of cervical intraepithelial neoplasia grade or (CIN2,3) is affected by viral, environmental and host factors, which are also thought to play a mediating role in cervical carcinogenesis [2] HPV has coevolved through time with humans into distinct genotypes [3,4] In a given type, the HPV genome is further classified into variants through cumulative nucleotide variations less than a 5% difference Epidemiological evidence suggests that polymorphism of HPV genome could contribute to viral pathogenicity and oncogenicity [4,5] A better understanding of the association between HPV polymorphism, persistent infection and risk for developing cervical cancer could help explain why only a subset of women with HPV infection develop cervical cancer HPV type 16 (HPV16) is the most prevalent high-risk (HR) type worldwide and is found in the majority of cervical cancer cases [6,7] The HPV16 genome is about 7,900 bp long and consists of protein-coding genes (L1, L2, E1, E2, E4, E5, E6, and E7) and non-coding regions, including the non-coding region (NCR) and the long control region (LCR) [8] Proteins encoded by E6 and E7 genes are the major oncoproteins, which are highly expressed in tumors and are involved in tumorgenesis LCR, adjacent to E6 downstream, contains the early promoter and regulatory elements involved in viral DNA replication and transcription Based principally on LCR sequences, several studies from Europe and American have suggested that HPV16 variants can influence viral persistence and development of cervical cancer [9-11] The aims of this study were to detect polymorphism of HPV16 E6 gene and LCR in isolates from women without a history of immunosuppression in northeast China, and to investigate the association of polymorphisms of HPV16 E6 gene and LCR with persistent HPV16 infection and developing risks of CIN2,3 or cervical cancer Methods Study population Three hundred and four HPV16 positive cervical cytobrush samples detected with flow-through hybridization (Hybribio Ltd, Hong Kong) were collected from women admitted to the Affiliated Shengjing hospital of China medical university According to the cytological and histological evaluations of fresh specimens, cervical disease statuses of the women were categorized as normal, cervicitis, atypical squamous cells of undetermined significance (ASCUS), cervical intraepithelial neoplasia grade 1, and (CIN1, CIN2 and CIN3) and cervical cancer (CC) Histopathological Page of diagnosis was completed by a pathologist who was unaware of the HPV detection results Informed consent was obtained from participation in the study and this study was approved by the ethics committee of the Affiliated Shengjing hospital Analysis of HPV16 E6 and LCR by PCR-sequencing To obtain HPV16 E6 and LCR sequences, a half-nested PCR was performed as that previously described [12] According to the reference strain (GenBank KO 2718), the primers used in the amplification were designed as follows: the forward primer of LCR-F (5’-ACGCAAAAAA CGTAAGCTG-3’) is located at nucleotide (nt) 7133–7151, the out backward primer of E6-R (5’-CTTCCCCATTGGT ACC TGCAG-3’) at nt 875–895 and the inner backward primer of E6-R-2 (5’-TCCATGCATGATTACAGCTGGG TT-3’) at nt 547–570 In the first-round PCR, 10–100 ng of sample DNA was used as a template; in the second-round reaction, 1μl of the first-round products were used PCR was performed in a 9700 Thermal Cycler (Perkin–Elmer GeneAmp PCR system) with the following parameters for all the amplification reactions: an initial denaturation at 94°C for min, followed by 30 cycles of 94°C for 45 sec, 50°C for 45 sec and 72°C for min, and a final elongation at 72°C for A negative control reaction without DNA template was included in all PCR tests The size of the PCR products containing HPV16 E6– LCR sequence was confirmed by gel electrophoresis The amplicons were purified using the QIAquick gel extraction kit (Qiagen Inc, Mississauga, ON) Direct double-stranded PCR sequencing was performed for the E6-LCR amplicons using the same primers as in PCR by fluorescent cyclesequencing method (BigDye terminator ready reaction kit; Perkin-Elmer) All amplicons were sequenced at least twice to exclude Taq-induced errors Sequences of the amplicons were aligned by CLUSTAL W (version 1.8) The obtained sequences were compared to the reference sequence (GenBank KO 2718) using NCBI BLAST The HPV16 sequences were classified into respective variant classes as: prototype (GenBank KO 2718), European (E), Asian (As), African (Af-1), African (Af-2) and Asian American (AA) [13-15] To assess the effects of LCR variations on binding sites for cellular transcription factors, the TFSEARCH software was used [16] Data analysis The association of HPV16 polymorphisms with persistent infection and developing risks of CIN2,3 or cervical cancer of women was investigated Persistent infection was conceptualized at least two times positive for HPV detection in a series of samples collected at more than three visits The proportion of women infected with HPV16 prototype was compared between participants with transient and persistent infection by Fisher’s exact test In Sun et al BMC Cancer 2013, 13:459 http://www.biomedcentral.com/1471-2407/13/459 this study, CIN1 or low-grade squamous intraepithelial lesion (LSIL) is not considered as a precursor lesion to cervical cancer but a morphologic consequence of HPV infection So, the controls including women without CIN or with CIN1 and LSIL are designated as