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

描述(申请人提供)：据估计，美国女性中人乳头瘤病毒(HPV)感染的流行率超过1000万，每年约有620万新感染，但大多数HPV感染是短暂的，并在24个月内消失。在美国，每年约有12000例新的宫颈癌病例报告，只有持续高危HPV感染的女性才有患上异型增生(CIN)的风险，最终发展为宫颈癌。目前尚不清楚为什么HPV感染在大多数女性中都很明显，但在其他女性中却持续存在。目前纸片检测筛查假阳性率(约1/10是“异常”)，导致随访阴道镜和活检在经济和情感上都是昂贵的。因此，有很大的临床需要确定预测少数高危HPV感染的生物标志物，这些感染将持续并进展，以便医疗资源能够针对真正有宫颈疾病风险的妇女。病毒DNA序列的甲基化(特别是在L1和L2ORF中)作为一种宿主防御机制正受到人们的关注。甲基化的过程依赖于饮食中的叶酸，去甲基化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女性一段时间，以衡量病毒持续时间的具体目标。对于拟议的研究，我们有从一个独特的队列中收集的DNA样本，该队列名为卡罗莱纳妇女护理研究(CWCS)，由国家少数民族健康和健康差距研究所(P20 MD001770)支持。这项研究是为了确定大学年龄女性中HPV持续存在的生物标记物。从CWCS纵向收集的宫颈脱落细胞中分离出DNA将使我们能够基于以下假设发现候选生物标记物。HPV DNA甲基化的改变最终决定病毒的存留或清除，而HPV序列的表观遗传甲基化受叶酸代谢和/或DNA修复过程的控制。因此，参与叶酸代谢和/或碱基切除修复的基因的多态将成为HPV持续存在的极好的生物标志物，从而最终导致宫颈癌的风险。目的1将确定作为CWCS的一部分收集的宫颈脱落细胞中病毒L1和L2基因的甲基化状态，CWCS对大学年龄女性进行了长达四年的前瞻性跟踪，并确定了迅速清除HPV病毒感染的参与者和其他持续HPV感染的参与者。目的2将确定CWCS队列中叶酸代谢酶和DNA修复酶的多态(SNPs)的发生率，并将特定的SNPs与高危HPV DNA甲基化水平、病毒持久性和病毒清除相关。这项研究的临床和翻译影响将是确定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，从而成为宫颈癌的风险。

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.