HPV Methylation as a Biomarker of Viral Persistence and Risk of Cervical Disease

描述（由申请人提供）：美国女性人乳头瘤病毒（HPV）感染的估计流行率超过1000万，每年约有620万新感染，但大多数HPV感染是短暂的，在24个月内清除。在美国，每年大约有12000例新的宫颈癌病例被报道，只有持续的高危HPV感染的妇女才有发育不良（CIN）的风险，最终发展为宫颈癌。目前尚不清楚为什么HPV感染在大多数妇女中清除，而在其他妇女中持续存在。目前巴氏试验筛查的假阳性率（约十分之一是“异常”），导致后续阴道镜检查和活检，这在经济上和情感上都是昂贵的。因此，临床迫切需要确定能够预测少数高危HPV感染的生物标志物，这些感染将持续存在和发展，以便医疗资源可以针对真正有宫颈疾病风险的妇女。病毒DNA序列（尤其是L1和L2 ORF）的甲基化作为宿主防御机制正受到越来越多的关注。甲基化过程依赖于膳食叶酸，而去甲基化过程依赖于碱基切除修复（BER）来恢复CpG序列中的天然胞嘧啶。很少有研究前瞻性地跟踪妇女一段时间，具体目标是测量病毒的持久性。我们从一个名为卡罗莱纳妇女护理研究（CWCS）的独特队列中收集了DNA样本，该研究由国家少数民族健康和健康差异研究所（P20 MD001770）支持。这项研究的目的是确定大学年龄女性中HPV持续存在的生物标志物。从CWCS纵向收集的脱落宫颈细胞中分离的DNA将使我们能够根据以下假设发现候选生物标志物。HPV DNA甲基化的改变最终决定病毒的持久性或清除，HPV序列的表观遗传甲基化由叶酸代谢和/或DNA修复过程控制。因此，参与叶酸代谢和/或碱基切除修复的基因多态性将作为HPV持久性的优秀生物标志物，从而最终确定宫颈癌的风险。目的1将确定作为CWCS的一部分收集的脱落宫颈细胞中病毒L1和L2基因的甲基化状态，该研究对大学年龄妇女进行了长达四年的前瞻性随访，并确定了迅速清除HPV病毒感染的参与者以及其他持续HPV感染的参与者。目的2将确定CWCS队列中叶酸代谢酶和DNA修复酶多态性（snp）的流行程度，并将特定snp与高危HPV DNA甲基化水平、病毒持久性和病毒清除相关。本研究的临床和转化影响将是确定HPV甲基化、叶酸代谢酶和DNA修复酶之间的遗传相互作用，这些酶将作为HPV持久性的生物标志物，从而作为宫颈癌风险的生物标志物。

DESCRIPTION (provided by applicant): The estimated prevalence of human papillomavirus (HPV) infection for women in the U.S. is over 10 million, with approximately 6.2 million new infections annually, yet most HPV infections are transient and clear within 24 months. About 12,000 new cases of cervical cancer are reported annually in the U.S., and only women with persistent high-risk HPV infection are at risk of dysplasia (CIN) that ultimately progresses to cervical cancer. It is unknown why HPV infections clear in most women yet persist in others. The current false positive rate of pap test screening (~1 in 10 are "abnormal"), results in follow up colposcopy and biopsy that are financially and emotionally costly. Thus, there is a great clinical need to identify biomarkers that are predictive of the small number of high-risk HPV infections that will persist and progress, so that medical resources can be directed at the women truly at risk of cervical disease. Methylation of viral DNA sequences (especially in the L1 and L2 ORF) as a host defense mechanism is gaining attention. The process of methylation depends on dietary folate and the demethylation depends on base excision repair (BER) to restore native cytosine in CpG sequences. Rarely have studies prospectively followed women over time with the specific goal of measuring viral persistence. We have available for the proposed study samples of DNA collected from a unique cohort called the Carolina Women's Care Study (CWCS) supported by the National Institute on Minority Health and Health Disparities (P20 MD001770). The study was initiated to identify biomarkers of HPV persistence in college-age women. DNA isolated from exfoliated cervical cells collected longitudinally in the CWCS will allow us to uncover candidate biomarkers based on the following hypothesis. Alterations in the methylation of HPV DNA ultimately determine viral persistence or clearance, and epigenetic methylation of HPV sequences is controlled by folate metabolism and/or DNA repair processes. Accordingly, polymorphisms in genes involved in folate metabolism and/or base excision repair will serve as excellent biomarkers for HPV persistence, and thus, risk ultimately for cervical cancer. Aim 1 will determine the methylation status of the viral L1 and L2 genes in exfoliated cervical cells collected as part of the CWCS, which followed college age women prospectively for up to four years and identified participants who rapidly cleared the HPV viral infection along with others who had persistent HPV infections. Aim 2 will determine the prevalence of polymorphisms (SNPs) in folate metabolizing enzymes and DNA repair enzymes in the CWCS cohort and will correlate specific SNPs with the levels of high- risk HPV DNA methylation, viral persistence, and viral clearance. The clinical and translational impact of this study will be to identify genetic interactions between methylation of HPV, folate metabolizing enzymes and DNA repair enzymes that will serve as biomarkers for HPV persistence, and thus, risk for cervical cancer.