The Study Of Gene Mutation Hot Spots And Whole Genome Analysis Of Human Papillomavirus Type52Collected In Beijing

Cervical cancer (CC)is the second most common malignant disease among women worldwide, and it is the most common malignant disease among Chinese women. There are about150,000incident CC cases per year in China. The causal link between human papillomavirus (HPV) infection and cervical cancer is now established beyond doubt. Based on their association with cervical cancer and precursor lesions, HPVs can also be grouped to high-risk, possible high-risk and low-risk HPV types. High-risk HPV types include type16,18,31,33,35,39,45,51,52,56,58,59,68,73and82, associating with malignant tumors of cervix uteri, vulvae and anus. HPV16and18are two predominant prevalent types associated with CC in Euramerican countries. Data on HPV prevalence suggests that the distribution of HPV types is different in China. HPV16is the predominant type, following by HPV52,58or58,52among Chinese women. HPV52is an important HPV type associating with CC in China.The molecular mechanism underlying HPV-induced oncogenesis is unclear. At present the E6and E7oncoproteins are thought to play causative roles in the development of pre-neoplastic and malignant lesions of the uterine cervix. But the correlation between mutation of E6, E7and other ORFs with the development of pre-neoplastic and malignant lesions of the uterine cervix is still being under discussed, and will be a study hotspot. E5oncoprotein is also thought to play causative roles in the development of pre-neoplastic and malignant lesions of the uterine cervix, so the study of E5gene mutation is of gteat importance. LCR fragement contains the origin of DNA replication as well as important transcription control elements, and it is important for controlling viral gene expression. Variation in the LCR may affect replication in activities of one or more viral proteins. Presently the two prophylactic virus-like particle’ based vaccines (one bivalent vaccine against HPV16/18and a quadrivalent vaccine against HPV16/18/6/11) have demonstrated efficacy (about90-100%) against persistent infection with targeted types. But the distribution of HPV is different and the variation within the same HPV type may affect the protection of the vaccine, even under the supposition that they could prevent100%of the infection with HPV16/18, they could only be expected to reduce the cancer burden in China by a small percent. So whether the vaccines can be used in China is doubtful.Investigating the HPV infection in cervix among Chinese women, investigating the whole sequence of major HPV types and the variation within the same HPV type, and establishing standard samples of major HPV types is of important use for the CC prevention and control in China and the study of the molecular mechanism underlying HPV-induced oncogenesis.To investigate the HPV52whole sequence and to investigate HPV52E5, E6, E7and LCR sequence, hotspot mutation and their association with the development of pre-neoplastic and malignant lesions of the uterine cervix, samples were collected form outpatient women of the first hospital of Peking University, then sample DNA were extracted and checked for20HPV DNA types by DNA flow-through hybridization genotyping technique.HPV type distribution was calculated for the first1244samples. Twenty HPV types were detected, and the HPV prevalence was40.8%(507/1244), and the HPV52prevalence was5.9%, third to HPV16(14.1%) and58(10.5%).Sample DNA was extracted from79patients with single HPV52infection, of which E5, E6-E7and LCR segmenst were amplified and sequenced, and the sequence analysis result was analyzed with the clinical data. Four single-nucleotide variations and two amino acid mutation was found in E5; eight single-nucleotide variations and two amino acid mutation was found in E6; ten single-nucleotide variations and seven amino acid mutation was found in E7; and the LCR polymorphism was generated by40single-nucleotide variations, one single-nucleotide deletion site, and four fragment-deletion sites.Five HPV52single infection samples were extracted based on the result of gene mutation hot spots ananlysis, each was amplified as eight overlapped segments, and the whole sequence was acquired by combination of the eight segments with E5, E6-E7and LCR sequence known above. The whole sequence polymorphism was generated by70single-nucleotide variations, three deletion sites and one insertion sites. Four of the five HPV52single infection samples had similar sequence distribution characteristic which is different from the HPV52prototype. There may exist two HPV52isolated strains in China.Two long segments covering the whole HPV52sequence were amplified and cloned into cloning vector, mixed proportionally with C33A DNA, then the whole length HPV52standard sample for PCR were made.