青岛地区宫颈病变组织中HPV16 URR和E6基因突变分析

Analysis of variants of HPV16 URR and E6 among Qingdao women with cervical lesions.

目的:分析青岛地区妇女不同宫颈病变组织中人乳头瘤病毒16型(HPV16)上游调控区(URR)和E6基因序列多态性,以及序列变异与宫颈病变的相关性。方法:采用聚合酶链式反应(PCR)技术检测120例宫颈癌及254例宫颈上皮内瘤变(CIN)标本,筛选出HPV16阳性标本,进而扩增出HPV16URR和E6基因,PCR产物纯化后测序,与德国HPV标准株进行对比分析。结果:URR测序结果发现了19个突变位点,所有标本在7521位点均发生了G→A突变;63.30%(69/109)标本中检测到24,7730和7842的联合突变,为另一突变热点。E6测序结果共发现17个突变位点,突变热点为178位点(D25E),其在宫颈癌组、CINⅢ组和CINⅡ组的比例分别为61.11%(44/72)、62.00%(31/50)和50.00%(16/32),3组的差异无统计学意义(P=0.499)。仅观察到6例350位点突变(L83V)。亚洲型(AS)是最多见的突变类型,在154例HPV16阳性标本中占59.09%,其次为E-P原型(33.12%)。结论:青岛地区妇女宫颈病变组织中HPV16URR突变热点为nt7521以及nt24、nt7730和nt7842的联合突变。HPV16E6突变热点为nt178,这些突变热点可能与癌前病变的进展有密切关系。AS型和原型是青岛地区宫颈病变组织中两种主要的HPV16分支。

Objective：The study was performed to investigate genetic variants of the HPV16 URR and E6 genes derived from Qingdao women with different stages of cervical lesions,and to assess the association between the sequence variants and the cervical lesions. Methods：We use the technology of polymerase chain reaction（PCR） to detect 120 cases of cervical cancer,254 cases with cervical intraepithelial neoplasia（CIN）. Then we selected samples with HPV16 positive,and amplifies the HPV16 URR and E6 genes. The products of PCR were purified and sequenced, and then were compared with the standard strain. Results：There were 19 kinds of mutations in the URR gene. The mutation at nt 7521from G to A was most frequent. A remarkable finding was the CO-MUtations including nt 24, nt 7730 and nt 7842. The most frequent sequence variation of E6 was nt 178 ,which was 61.11% （44/72） ,62.00% （31/50）and 50.00% （16/32）in cervical cancer,CiNⅢ and CIN Ⅱ. The mutation of L83V was only found in 6 cases. In Qingdao area, two major branches of HPV16 were type AS （59.09%）, followedby prototype（33.12% ）. Conclusion：The mutational hot spots of HPV16 URR are nt7521 and a co-mutation including nt24, nt7830 and nt7842. The hot spot of E6 is nt178. These variants may be associated with the developing cervical cancer. In Qingdao area, two major branches of HPV16 are type AS and prototype.