新疆维吾尔族妇女宫颈癌组织中HPV16型E6基因突变分析

背景与目的：高危型人乳头状瘤病毒16和18(human papillomavirus type16 and 18,HPV16,HPV18)是宫颈癌主要病因之一,尤其以HPV16最为常见,其中HPV16E6是主要癌基因之一。在一些地区,特定的E6基因突变株是宫颈癌发生的危险因素。新疆南部维吾尔族聚居区足宫颈癌高发区,我们已在前期的研究中发现该地区HPV16E6基因发生突变。本研究旨在检测该突变在新疆南部维吾尔族妇女宫颈癌组织中的分布规律,并探讨其与该地区宫颈癌高发的关系。方法：从35例中国新疆南部维吾尔族妇女宫颈癌活检标本中提取组织DNA作为模板,PCR扩增HPV16E6全长基因,PCR产物直接测序或克隆后测序,分析新疆维吾尔族妇女宫颈癌组织中HPV16E6基因的突变。结果：PCR检测结果表明宫颈癌组织中HPV16E6阳性率为82．86％(29／35);26例中E6分离片段的测序和序列分析表明,15例(57．69％)分离株E6基因与原型相同,另有11例(42．31％)E6基因突变,其中9例(34．62％)分离株发生了L83V突变,2例(7．69％)分离株发生rL83V／D63E突变。结论：中国新疆南部地区HPV16E6基囚发生变异,其原型和变异型在该地区维吾尔族宫颈癌患者巾的分布规律可能与该地区宫颈癌高发存在一定关系。     

人乳头瘤病毒16型转化基因在不同阶段宫颈上皮病变组织中的分布及基因变异特点

目的 研究人乳头瘤病毒(HPV)16 E6、E7和E5基因在湖北地区不同阶段宫颈上皮病变患者组织中的分布以及E6、E7基闪的变异特点.方法 从124例宫颈癌、17例宫颈上皮内瘤变(CIN) Ⅰ+CINⅡ级、23例CIN Ⅲ级和36例慢性宫颈炎患者活检或手术切除标本中提取组织DNA,用HPV16 E6、E7和E5特异性引物进行PCR扩增,对部分扩增的 E6 和 E7 产物片段进行测序分析.结果 在官颈炎、CIN Ⅰ+CINⅡ级、CINⅢ级和宫颈癌组织中,E6基因的阳性率分别为25.0%、29.4%、60.9%和76.6%;E7基因的阳性率分别为16.7%、41.2%、43.5%和61.3%:E5 基因的阳性率分别为5.6%、5.9%、30.4%和40.3%.E6、E7和E5基因在不同阶段宫颈上皮病变组织中的阳性率差异均有统计学意义(均P<0.01).在80例官颈癌测序组织中,有47例发生E6基因178位点的T→C突变,突变率为58.8%,相应氨基酸由天冬氨酸(Asp)改变为谷氨酸(Glu);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E6基因178位点的突变率分别为25.0%和31.8%.在30例宫颈癌测序组织中,有21例发生E7基因647位点的A-G突变,突变率为70.0%,相应氨基酸由天冬酰胺(Asn)改变为丝氨酸(Ser);而在20例宫颈炎和22例CINⅠ～Ⅲ级测序组织中,E7基因647位点的突变率分别为35.0%和40.9%.结论 HPV16 E6、E7和E5基因与宫颈癌的发生和发展有高度的相关性.但E5基因在不同阶段官颈上皮病变中可能存在不同程度的缺失.中国湖北地区流行的HPV16病毒株可能为HPV16亚洲型变异株.     

四川地区宫颈癌HPV33、52和58型E6基因突变分析

目的:调查我国四川地区妇女宫颈癌组织中HPV33、52和58型的感染率及E6基因结构特点,探讨其与宫颈癌发生的关系.方法:采用PCR技术,对四川地区2004年60例宫颈癌患者癌组织DNA进行HPV检测.结果:获得HPV52和HPV58型各4例,检出率为6.7%;HPV33型1例,检出率为1.7%.对所获得的各例HPV33、52和58 E6基因测序分析.结论:与Genbank标准株相比,四川地区发现的HPV33和HPV52E6基因有多处突变,与日本发现的HPV33,52突变株相近.     

宫颈癌组织中人乳头瘤病毒16E6基因突变分析

背景与目的：高危型人乳头瘤病毒（humanpa pillomavirus,HPV）以16型最为常见,HPV16E6是宫颈癌主要的致癌基因之一,特定的E6突变是宫颈癌发生的主要因素之一。本研究主要观察兰州地区宫颈癌组织中是否存在E6突变,并探讨了突变与宫颈癌发生的关系。方法：以23例宫颈癌手术切除标本及5例正常宫颈组织的DNA作为模板,PCR扩增HPV-16E6基因201-523位,PCR产物直接测序．分析其HPV16E6基因的突变规律。结果：PCR扩增结果表明5例正常宫颈组织中HPV16E6阳性率为0％（0／5）,宫颈癌组织中HPV16E6阳性率为82．61％（19／23）,对18例样本PCR产物的测序和序列分析结果表明,6例（33．33％）E6基因与原型相同,12例（66．67％）E6基因发生突变,其中11例（61．11％）发生了350G突变。同时,在1例样本（5．56％）发现了249G突变。结论：兰州地区宫颈癌组织中存在着非常高的HPV感染率,且多数HP116E6发生了突变。     

四川地区宫颈癌HPV33、52和58型E6基因突变分析

目的:调查我国四川地区妇女宫颈癌组织中HPV33、52和58型的感染率及E6基因结构特点,探讨其与宫颈癌发生的关系。方法:采用PCR技术,对四川地区2004年60例宫颈癌患者癌组织DNA进行HPV检测。结果:获得HPV52和HPV58型各4例,检出率为6．7％;HPV33型1例,检出率为1．7％。对所获得的各例 HPV33、52和58 E6基因测序分析。结论:与Genbank标准株相比,四川地区发现的HPV33和HPV52 E6基因有多处突变,与日本发现的HPV33,52突变株相近。     

HPV16病毒中国山西襄垣流行株基因 HPV16Z 的序列分析

目的: 〖HT5"SS〗分析中国山西襄垣地区人乳头瘤病毒16型HPV16Z基因的结构特点。〖HT5W〗方法: 〖HT5"SS〗对HPV16Z进行双向测序,并将其基因全序列与德国标准株进行对比分析。〖HT5W〗结果: 〖HT5"SS〗DNA序列分析表明,HPV16Z与已发表的德国标准株基因长度相等,其中LCR和L1、E6部位的核酸片段存在变异。LCR的变异部位分别位于7431～7432 nt、7433 nt、7495 nt、7852 nt,变异方式分别为碱基插入、碱基替代及碱基缺失突变;L1的变异部位分别位于6169～6171 nt、6901～6902 nt、6949～6951 nt,其变异方式为碱基替代、碱基插入及碱基缺失突变;E6的变异部位位于350 nt,其变异方式为碱基替代突变。〖HT5W〗结论:　〖HT5"SS〗 中国山西襄垣地区妇女宫颈癌患者组织中HPV16型病毒HPV16Z基因结构与德国标准株HPV16基因之间存在一定的差异。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的：探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法：采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型，从舍有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6，经DNA序列测定法检测其基因变异，进而分析其同源性。结果：50例宫颈癌组织HPV-DNA的检出率为78％．其中HPV16和HPV18型混合感染18例，单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E 625例。其中178位核苷酸变异较大，变异率为72％，其相应氨基酸均由天冬氨酸变为谷氨酸。结论：HPV16E6 DNA序列发生碱基替换的区域主要在氨基端94～241位，羧基端相对保守，未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

宫颈癌组织中HPV16E6序列多态性及同源性分析

目的探讨广东地区宫颈癌组织中HPV16肿瘤相关性抗原E6基因序列的多态性及同源性。方法采用通用引物PCR直接测序法对宫颈癌标本中的HPV分型,从含有HPV16型的标本中采用自行设计的多重引物通过巢式PCR扩增出HPV16E6,经DNA序列测定法检测其基因变异,进而分析其同源性。结果50例宫颈癌组织HPV-DNA的检出率为78%,其中HPV16和HPV18型混合感染18例,单纯HPV16型感染15例。含有HPV16型的标本34例中扩增出HPV16E625例。其中178位核苷酸变异较大,变异率为72%,其相应氨基酸均由天冬氨酸变为谷氨酸。结论HPV16E6DNA序列发生碱基替换的区域主要在氨基端94~241位,羧基端相对保守,未见变异。广东地区宫颈癌组织中HPV16E6的热点突变为Nt178。     

人乳头瘤病毒16型(湖北株)E_7蛋白免疫学性质的初步研究

人乳头瘤病毒(HPV)16型与人类宫颈癌等多种恶性肿瘤的关系十分密切.HPV_(16)E_7基因及其蛋白产物的作用最为重要,这是因为E_7基因是HPV最主要的转化基因.1994年本室从宫颈癌组织中分离到HPV_(16)E_7基因的变异株(称为HB-E_7),DNA测序发现HB-E_7基因与标准株相比有两处突变.第43位密码子由CAA突变为终密码子TAA,产生了无义突变,另一突变发生在第76位密码子,由CGT突变为TGT.其中前者使E_7蛋白由标准株的98个氨基酸残基     

荧光偏振法检测高危HPV16型E6基因350位(T→G)突变

目的:检测宫颈癌人类乳头瘤病毒(HPV)16 E6基因350位(T→G)点突变情况.方法:应用TDI -FP方法检测宫颈癌患者HPV16F6基因350位(T→G)点突变情况.TDI - FP技术是一种在聚合酶链反应的基础上具备液相探针杂交和碱基掺入三重特异反应的检测新方法,具有极高的特异性和敏感性.结果:应用TDI - FP法检测宫颈癌组织中HPV16型E6基因350位(T→G)突变率为6.06％,HPV16 E6基因350位突变与对照组相比无统计学差异(P=0.603.P＞0.05).结论:本地区宫颈癌患者中HPV16型E6 350位(T→G)很少发生突变,有别于欧洲的HPVI6型E6 350位(T→G)高突变.     

Human papillomavirus 16E6 oncogene mutation in cervical cancer

Objective: Cervical cancer (CC) is the second most common type of cancer in women worldwide, after breast cancer. High-risk human papillomaviruses (HR-HPVs) are considered to be the major causes of cervical cancer. HPV16 is the most common type of HR-HPVs and HPV16 E6 gene is one of the major oncogenes. Specific mutations are considered as dangerous factors causing CC. This study was designed to find mutations of HPV16 E6 and the relationship between the mutations and the happening of CC.Methods: The tissue DNA was extracted from 15 biopsies of CC. Part of HPV16 E6 gene (nucleotide 201-523) was amplified by polymerase chain reaction (PCR) from the CC tissue DNA. The PCR fragments were sequenced and analyzed.Results: The result of PCR showed that the positive rate of HPV16 E6 was 93.33% (14/15). After sequencing and analyzing, in the 13 out of 14 PCR fragments, 4 maintained prototype (30.77%), 8 had a same 350G mutation (61.54%), and 1 had a 249G mutation (7.69%).Conclusion: This study suggest that there is a high infection rate of HPV in cervical cancer and most of the HPV16 E6 gene has mutations. Those mutations may have an association with the development of cervical cancer.     

Uneven distribution of HPV 16 E6 prototype and variant (L83V) oncoprotein in cervical neoplastic lesions

A previous Swedish study revealed that both prototype and variant HPV16 E6 oncoprotein, occur in about equal numbers in high-grade cervical intraepithelial neoplasia (HCIN), whereas variant HPV16 predominates in invasive cervical squamous carcinoma. Most of the malignant HPV16 variants contain a common mutation, L83V, in the E6 oncoprotein. In the present investigation, 28 HPV16 positive, invasive cervical adenocarcinomas were collected from a total number of 131 adenocarcinomas. These HPV16-positive cases were evaluated with analysis of the E6 gene, using a recently described PCR-SSCP method for identification of the specific mutation (L83V) in the E6 gene. The results obtained were correlated to findings in 103 preinvasive, HCIN, and 31 invasive cervical squamous carcinomas also infected with HPV16. The HPV16 E6 variant L83V was present in 40% of the HCIN lesions, in 54% of the invasive adenocarcinomas, in comparison to 81% of the invasive squamous carcinomas. The difference between HCIN and squamous carcinomas was statistically significant, P < 0.001, whereas the difference between HCIN and invasive adenocarcinomas was not statistically significant, P = 0.604. Prototype HPV16 and its E6 variant L83V are both prevalent in preinvasive and invasive cervical lesions in Swedish women. However, the obvious predominance of HPV16 variant in squamous carcinomas was not seen in adenocarcinomas. A single amino-acid shift in the HPV16 E6 gene appears to result in a different transforming potential in squamous and glandular cervical lesions.     

山东青岛地区宫颈癌组织中ＨＰＶ１６型Ｅ６和Ｅ２基因突变分析

目的　通过分析山东青岛地区宫颈组织中ＨＰＶ１６型Ｅ６和Ｅ２基因突变情况,探讨其与该地区宫颈癌的关系。方法　从１０４例青岛地区宫颈疾病组织中提取ＤＮＡ作为模板,采用聚合酶链式反应（ＰＣＲ）技术筛选出高危型ＨＰＶ及ＨＰＶ１６阳性标本,扩增出ＨＰＶ１６型Ｅ６、Ｅ２全长基因,ＰＣＲ产物纯化后测序,与德国ＨＰＶ标准株进行比对分析。结果　宫颈组织中高危型ＨＰＶ阳性率为９３.２７％（９７／１０４）,ＨＰＶ１６阳性率为６９.２３％（７２／１０４）。ＨＰＶ１６阳性标本中扩增出Ｅ６基因３７例,有５例与标准株序列相同,３２例存在突变,其中２５例突变型别为Ｔ１７８Ｇ或Ｔ１７８Ａ（Ｄ２５Ｅ）。在Ｅ２基因全序列测序中,２３例均存在Ｃ３６８４Ａ（Ｔ-Ｋ）,１４例同时存在Ｔ３５２４Ｃ、Ｃ３６８４Ａ（Ｔ-Ｋ）和Ｃ３７８７Ａ（Ｄ-Ｅ）,９例同时存在Ａ２９２６Ｇ、Ｃ３１５９Ａ（Ｔ-Ｋ）、Ｇ３２４９Ａ（Ｒ-Ｑ）、Ｔ３３８４Ｃ（Ｉ-T）、Ｃ３４１０Ｔ（Ｐ-Ｓ）和Ｃ３６８４Ａ（Ｔ-Ｋ）。结论　山东青岛地区宫颈癌患者ＨＰＶ１６型Ｅ６、Ｅ２基因与德国标准株比较存在多处变异,Ｅ６与Ｅ２基因突变可能存在相关性。     

Uniform distribution of HPV 16 E6 and E7 variants in patients with normal histology, cervical intra-epithelial neoplasia and cervical cancer.

High-risk human papillomaviruses (HPV), particularly HPV 16, are associated with invasive cervical cancer (ICC), and persistent high-risk HPV infection is considered to be a marker for progressive cervical intra-epithelial neoplasia (CIN). However, most high-risk, HPV-infected, pre-cancerous lesions will not progress to invasion. Several reports suggest that specific HPV 16 E6 and/or E7 sequence variations may be associated with a high risk for progression. No data from German patients have so far been reported. Therefore, we analyzed intra-type variations of these oncogenes in women with normal histology or CIN 1 (< or = CIN 1), CIN 2/3 or ICC. Cervical scrapes from 75 patients with normal histology or CIN and biopsies from 37 ICC patients all positive for HPV 16 were analyzed. The open reading frames of oncogenes HPV 16 E6 and E7 were amplified by nested PCR followed by primer cycle sequencing. From each cervical scrape, 2 independent PCR amplicons were generated and sequenced from both orientations. The prototype sequence of HPV 16 E6 and E7 was identified in 33% and 87% of < or = CIN 1, in 62% and 69% of CIN 2/3 and in 43% and 86% of ICC, respectively (not significant). Of all variants identified, the E6 variant 350G (L83V) and the E7 variant 822G were most frequently detected irrespective of histology and showed prevalence rates of 27% to 43% and 7% to 20%, respectively. No statistically significant differences in the prevalence of the E6 or E7 prototype sequences, any variants or multivariants in German women with < or = CIN 1, CIN 2/3 or ICC were found.     

Human papillomavirus genotype in cervical intraepithelial neoplasia grades 2 and 3 of Taiwanese women

We aimed to assess the distribution of human papillomavirus (HPV) genotypes in high‐grade cervical lesions in Taiwan. The study included 1,086 paraffin‐embedded, formaldehyde‐fixed cervical intraepithelial neoplasia (CIN) 2/3 specimens. HPV genotyping was performed using polymerase chain reaction (PCR)‐based methods. Multiple HPV types were validated by E6 type‐specific PCR, direct sequencing and/or real‐time PCR. HPV DNA was detected in 995 (91.6%) specimens, and multiple HPV types were identified in 192 (19.3%) samples. The leading HPV types were HPV16 (24%), HPV52 (20%), HPV58 (20%), HPV33 (13%), HPV31 (8%) and HPV18 (4.6%). Although the leading six types consisted of 87.6%, HPV16 or 18 comprised only 30.9%. The prevalence of different HPV types showed a significant association with age. In women older than 50 yr, HPV16 and 18 comprised 21.3% (83/389), while HPV52, 58 and 33 represented 55.5% (216/389). In women aged less than 50 yr, HPV16 and 18 comprised 32.1% (224/697, p < 0.0001), while HPV 52, 58 and 33 represented 47.9% (334/697, p = 0.02). The distribution of HPV genotypes was compared with previously reported findings for Taiwanese women with cervical cancer (CC). The overall HPV16 positivity rate was significantly higher in CC than in CIN 2/3 (odds ratio: 2.14, 95% CI: 1.91–2.40). In addition, HPV18, 39 and 45 were significantly overrepresented in CC, whereas HPV52, 58, 33, 31, 35, 51 and 53 were underrepresented. We concluded that an effective vaccine against the most common HPV types could prevent a significant proportion of cervical cancer cases that occur in Taiwan.     

DETECTION OF HUMAN PAPILLOMAVIRUS L1 -16 AND -18 DNA AND EPSTEIN-BARR VIRUS DNA IN LARYNGEAL CARCINOMA

Objective: To look for the further evidence for HPV LI HPVI6 E6, HPV 18 E6 and EBV as carcinogenic factors in laryngeal carcinoma. Method: we examined representative numbers of specimens from laryngeal cancer with highly sensitive PCR technique for the presence ofHPV LI and high-risk types HPV16 E6, HPV18 E6 and EBV LMPI. Results: Using PCRdetection, 7.3% samples were HPV LI positive, 52.03% were HPV16 E6 positive, 30.89% were HPV18 E6 positive and 9.13% were EBV LMPI positive. The low incidence of HPV L1 and high incidence of HPV-16 E6 and HPVI8 E6 genes suggest that HPV might be integrated into tumor cells. Our results support a role of HPV-16 and HPV-18 infection in the pathogenesis of laryngeal carcinoma in China. Conclusion: Integration of E6 into host genome and stable expression of these genes may be associated with the carcinogenesis of laryngeal carcinoma. HPV-16 and HPV-18 may synergistically function on the pathogenesis of laryngeal carcinoma. Our results suggest an association of laryngeal carcinogenesis and infection with the high-risk HPV types 16, HPV 18 and EBV.