应用TDI-FP技术分析宫颈癌组织HPV16 E7基因A647G点突变

模板指导的末端碱基掺入反应结合荧光偏振检测技术(template direct dye-terminator incorporation with fluorescence- polarization，TDI-FP) 是SNP检测新技术. 应用TDI-FP方法分析中国陕西HPV16阳性宫颈组织HPV16 E7基因第647位核苷酸A→G热点突变(即A647G)，首先在HPV16阳性的91例宫颈癌及49例正常/宫颈炎妇女宫颈DNA标本中，PCR扩增含647位点在内的HPV16 E7部分基因, 然后将紧邻647位点5′端的寡核苷酸探针与PCR产物内的模板杂交，并延伸一个与647位点碱基互补的荧光标记碱基：TAMRA-ddTTP或R110-ddCTP. 用荧光偏振仪读取荧光偏振 (FP) 值，根据升高的相应FP值判断647位点碱基. 结果表明，宫颈组织HPV16 E7 A647G的总体检出率为35.71% (50/140). 宫颈癌组的A→G突变率为42.86% (39/91)，显著高于正常/宫颈炎组22.45% (11/49) 的突变率 (x2 = 5.778, P = 0.016)，两组间的OR值为2.59 (95% CI = 1.17~5.71). 提示TDI-FP 可用于HPV有意义点突变的分析；我国陕西地区妇女HPV 16 A647G突变率及其对宫颈癌的警示性与其他地区相比有明显差异，该地区携带此突变病毒株的妇女患宫颈癌的风险可能较高

Application of TDI-FP for Analysis of A647G Variation in HPV 16 E7 Gene From Cervical Cancer Patients

Template direct dye-terminator incorporation with fluorescence-polarization (TDI-FP assay) is a technology for genotyping single nucleotide polymorphisms (SNPs). To apply this method in analyses of A647G variation in human papillomavirus (HPV) 16 E7 gene from HPV 16-positive cervical tissues, a total of 91 and 49 HPV 16-positive DNA samples obtained from women with cervical cancer and normal/inflamed cervices living in Shaanxi in northwest China were subjected to the partial E7 gene PCR with nucleotide (nt) 647 in the products. Then, the oligonucleotide probe designed to anneal immediately to nt 647 was hybridized to the template within the PCR amplicons, and extended specifically by TAMRA-ddTTP or R110-ddCTP directed by the base at nt 647. The increasing FP values were read and the base at nt 647 was identified. The prevalence of nt 647 A→G was 35.71% (50/140). The variation 647G detected in 42.86% (39/91) of women with cervical cancer was significantly higher than 22.45% (11/49) detected in those with normal/inflamed cervices (x2 = 5.778, P = 0.016). The odds ratio (OR) between these two groups was 2.59 (95% confidence interval=l.17～5.71). The results demonstrate that TDI-FP method can be potentially applied in analysis of interest point mutations in HPVs. The incidence and risk implication of HPV 16 A647G variant infection in Shaanxi, China, displays significant geographic difference from other areas. The HPV 16 with E7 gene A647G point mutation appears to have a higher risk for invasive cervical cancer in women living in Shaanxi.