子宫颈脱落细胞中DAPK1、RAR—β和MGMT基因启动子的甲基化水平及其临床意义

Promoter methylation of DAPK1, RAR-I5 and MGMT in exfoliated cervical cytology and its clinical application

目的检测宫颈脱落细胞中DAPK1、RAR-β、MGMT基因启动子的甲基化水平，探讨其筛查高级别宫颈病变[包括宫颈上皮内瘤变（CIN）Ⅱ、Ⅲ]及宫颈鳞癌的价值。方法采用简单随机法选取2010年3-10月间北京协和医院临床使用后剩余的宫颈脱落细胞标本共103份，其中，细胞学诊断包括正常宫颈细胞12份、不典型鳞状上皮细胞（ASC）17份、低度鳞状上皮内病变（LSIL）30份、高度鳞状上皮内病变（HSIL）30份、宫颈鳞癌14份；组织学诊断包括正常宫颈组织27份、CINI19份、CINⅡ17份、CINⅢ25份、宫颈鳞癌15份。采用甲基化特异性高分辨率熔解曲线分析（MS—HRM）法检测宫颈脱落细胞中DAPKl、RAR—B、MGMT基因启动子的甲基化率，并评价MS．HRM法检测的敏感性；采用第二代杂交捕获技术（HCⅡ）检测宫颈脱落细胞中高危型HPV载量（以HPV-HCⅡ值表示）；评价DAPK1、RAR—β、MGMT基因启动子的甲基化和HCⅡ单独或联合检测筛查高级别宫颈病变及宫颈鳞癌的价值。结果MS—HRM法可以检测出标准样本中的1％DNA甲基化率。在不同细胞学病变中，DAPK1、RAR—β、MGMT基因启动子的甲基化率分别比较，差异均有统计学意义（P值分别为0．000、0．011、0．002）；在不同组织学病变中，DAPK1和RAR-β基因启动子的甲基化率分别比较，差异均有统计学意义（P值分别为0．000、0．021）。高级别宫颈病变＋宫颈鳞癌与正常宫颈＋CINI组织中DAPK1基因启动子的甲基化率分别为1．47％和20．98％，两者比较，差异有统计学意义（P=0．000）。DAPK1基因启动子的甲基化检测筛查高级别宫颈病变及宫颈鳞癌的灵敏度为0．571、特异度为0．791，曲线下面积（AUC）为0．709；DAPK1基因启动子的甲基化联合HCⅡ检测的灵敏度为0．825、特异度为0．565，AUC为0．695。结论MS—HRM法检测宫颈脱落细胞的甲基化水平是可行的。DAPK1、RAR—β基因启动子甲基化可以作为筛查宫颈病变的分子标记。DAPK1基因启动子的甲基化联合HCⅡ检测有助于筛查高级别宫颈病变及宫颈鳞癌。

Objective To assess the correlation of promoter methylation of DAPK1, RAR-β and MGMT with cervical lesions from cytology to histology, and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia （ CIN）. Methods A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology, Peking Union Medical Collage Hospital from March 2010 to October 2010. Informed consent was obtained from each woman before the initiation of the study. The methylation seusitive-high resolution melt （MS-HRM） assay was used to evaluate promoter methylation of three genes （DAPK1, RAR-β and MGMT） in 103 biopsy-confirmed liquid-based cervical cytology samples. Methylation levels and high-risk HPV DNA loading （ HC Ⅱ values ） were analyzed in relation to both cytological and histological diagnosis. Results The methylation level of all three genes showed significant difference among the different cytological groups （P = 0.000, 0.011 and 0.002, respectively）. The methylation level of DAPK1 and RAR-~ showed significant difference among the different histological groups （ P = 0. 000 and 0. 021 ）, while there was no significant difference for MGMT. DAPK1 methylation levels was 1.47% in the CIN lI/high-grade precancerous lesions group, and 20. 98% in the normal/CIN ｜ groups （ P = 0. 000） , but there was no significant difference between CIN II/high-grade precancerous lesions and normal/CIN I groups for RAR-~ and MGMT. The combination of DAPK1/HR- HPV loading showed a sensitivity of 0. 825 and an area under the receiver operating characteristic curve （ROC） curve （AUC） of 0. 695 as diagnostic methods for detecting CIN U/high-grade precancerous lesions. Conclusions DNA methylation such as DAPK1 and RAR-13, in combination with HR-HPV detection, may serve as biomarkers to detect CIN ]I/high-grade precancerous lesions. Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.