Abstract：
Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.
摘要：
基因启动子的全局甲基化分析有望在高危型人乳头瘤病毒（hrHPV）阳性女性中检测高度鳞状上皮内病变或更严重的病变（HSIL）。然而,甲基化数据在单个CpG位点的诊断性能有限。我们探索了在巴布亚新几内亚自我和临床医生收集的样本中预测HSIL+的甲基化。EPB41L3（1-6个CpG位点）的甲基化，hTERT（1-10个CpG位点）和FAM19A4（1-5个CpG位点）通过焦磷酸测序从44个HPV+样品（4个癌症，19HSIL,4低度鳞状上皮内病变（LSIL），17正常）。针对FAM19A4设计了新的引物,其指向先前未探索的第一外显子区域。在临床医生收集的样本中，EPB41L3的CpG位点4和5的甲基化是癌症的最佳HSIL预测因子(AUC>0.83)和CpG位点4(0.925)。EPB41L3位点2/4加上FAM19A4位点1的组合是最佳的HSIL+标记[100%灵敏度,63.2%特异性]。FAM19A4的CpG位点5处的甲基化是自收集样品中最佳的HSIL预测因子（0.67），和FAM19A4的CpG位点1和3用于癌症(0.77)。合并,FAM19A4位点1加HPV16/18检测产生82.6%的灵敏度和61.9%的特异性。总之,EPB41L3和FAM19A4的各个CpG位点的甲基化优于全局分析，并改善了HSIL检测，保证进一步调查。
