Distribution Of HR-HPVE6/E7 Variants In Cervical Carcinoma And Its Precursors And The Development Of SNP Microarray

Cervical cancer is the second most common cancer among women worldwide. It has been showed that cervical cancer accounted for 493,000 newly diagnosed cases, 273,000 deaths worldwide in the year 2002. Of these, more than 80% occurred in less developed countries. It has been established by molecular biological and epidemiological studies that infection with specific types of genital human papillomavirus (HPV) is an essential etiological factor for both cervical cancer and its precursor lesions( cervical intraepithelial neoplasia (CIN)).It is not enough to lead to cervical cancer with infection of HPV alone. There are many other viral or non-viral factors involved in cervical carcinogenesis. The viral aspects mainly include:HPV genotype, persistent infection of HR-HPV, encoded proteins by HPV especially of E6 /E7 and HPV variant. It has been demonstrated that HR-HPV intratypical variant had some association with persistent infection of HPV and progression of cervical neoplasias.HPV 16 and 18 are the most frequently types accounting for and responsible for nearly 80% of cervical cancers with HPV16 accounting for as much as 60%.However, accumulated studies have showed that HPV 52 and 58 are relatively more frequent among HPV-positive Chinese and other Asian women and there is 11.5-28% of prevalence across the full spectrum of cervical neoplasia in Chinese and Asian women. The incidence and mortality of cervical cancer are generally decreasing in the world in lately 60 years, which is attributed to cervical cancer screening popularization with cervical papanicolaou smear. Papanicolaou smear has been long the only preliminary screening method for its high specificity, non-invasive, and easy to operate but it has disadvantages of low sensitivity and high false negative rate, it is limited for the only preliminary screen. The fact that HPV is the independent contributor to cervical cancer has made HPV-DNA detecting as a screening method possible. To find a method of forecasting HPV persistent infection and progression of cervical neoplasias is benefits for those women with HPV positive but normal cytology and is important for screening efficiency and saving society resources.The study was designed to find the distributions of E6/E7variants of HR-HPV16/58/52 at first, then, to analyze the risk of different variants for cervical cancer and its precursors in Chinese women. According to the above findings, it is hoped to develop an earlier and more specific screening method of cervical cancer. Part?Distribution of HR-HPV E6/E7 variants in cervical carcinoma and its precursorsObjectives:To understand the distribution of Human papillomavirus (HPV) 16/58/52 E6 and E7variants in women of Zhejiang province and assess their risks for cervical cancer and its precursors.Methods:The cervical samples collected from patients who visited at cervical disease clinic in Women's Hospital, Medical school, Zhejiang University, during April 2008 and December 2009. The included patients were all HR-HPV infected and ranged in age of 21-73 years, with a mean of 38 years. A total of 2021 HPV positive cervical samples were collected included 170 samples with HPV16 positive,177 with HPV58 positive and 121 with HPV52 positive. According to histopathology,59 were classified as cervical epithelial neoplasia (CIN1),128 were cervical epithelial neoplasia (CIN2-3),58 were invasive cancer (ICC), and 205 were normal controls. Informed consent was taken from each of the subject. DNA was extracted by the standard method of proteinnase K digestion and phenol-chloroform abstraction. For the HPV genotyping, flow-through hybridization (HPV GenoArray test kit; Hybribio Ltd., Hong Kong) was used based on the principle of Reverse Dot Blot Assay which was described in previous studies. The DNA samples which positive with HPV16/58/52 were amplified by type-specific primers, and then, the amplified products were directed sequenced on automated sequencer (ABI 3100 Prism, Applied Biosystems). The sequenced product was aligned with prototype sequence on NCBI (http://blast.ncbi.nlm.nih.gov/Blast).Results:1 HPV16E6/E7 variantsThe results showed that HPV-16 Asian lineage was the most frequently (77%) and Asian variant presented a significantly higher disease risk for cervical cancer and CIN. Additionally, three novel variants at E6 (Q20P, H118Q, and Q123K) and two at E7 (D75N and T86P) were found. The substitution G368T at E6 leading to a premature stop codon was occurred in an isolate of normal control sample.2 HPV58E6/E7 variantsTotally 15 new reported variations of HPV58 were found, of which, there were 5 variations of E6 and 10 of E7. It's interesting that the presence of C632T (T20I) and G760A (G63S) variants in E7 showed a positive trend of the association with the severity of neoplasia (Ptrend<0.05,?2test for trend).3 HPV52E6/E7variantsTotally 14 new reported variations of HPV52 were found, of which, there were 3 variations of E6 and 11 variations of E7.The most frequently found variations were A379G(K93R) for E6, C751T and A801G for HPV52E7.Conclusions:1 This study reported the distribution of HPV16/58/52 E6 and E7 gene variations in women from Zhejiang province, China which is different from that showed in previous studies.2 HPV16 As-V and HPV58 E7 C632T (T20I)/G760A (G63S) had higher carcinogenic potentials. PartThe development of HPV16/18/58/52 SNP microarrayObjectives:According to the different distributions of HPV16/58/18/52E6 and E7 variants in various cervical tissues, we construted a SNP microarray consisted of these variants. It's hoped that the SNP-microarray will be used as an earlier screening tool for CIN and ICC.Methods:Selected 51 cervical samples with HPV 16/58/18/52 single infected respectively, of which, there were 35 different SNPs of E6/E7. Meanwhile, we have reviewed references about this aspect and found E6/E7variants present relatively frequent in CIN and ICC. Finally,70 SNPs were selected for positions of genoarray. Designed different probes for each SNP and made the SNP-array according to the principle of nucleotide hybridization.Results:In this experiment,1689 probes have been designed and four SNP-microarrays have been made.Obtained four hybridization results,as follows, HPV16E6 SNP-microarray hybridized with mixed samples and single sample respectively, and HPV16/58/18/52 E6 and E7 SNP-microarray hybridized with mixed samples and single sample respectively.lt was demonstrated that the detected results of HPV intratype variation microarray were consistent with that of DNA sequencing.The HPV SNP-microarray had good reproducibility and there were not cross hybridizations among different HPVs.Conclusions:1 HPVE6 and E7 SNP-microarray could detected variations precisely and detected multiple variations simutanously present in the same sample, but there were about 5% omission factor. So, it is yet to be further improved.2 The SNP-microarray had very high HPV type specifity.