The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

中文翻译：

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健康个体肿瘤抑制启动子甲基化的评估。

生殖系突变与癌症风险相关的肿瘤抑制基因的数量正在稳步增加。然而，虽然最近的报告将 BRCA1 和 MLH1 的正常组织启动子甲基化与卵巢癌和结肠癌风险联系起来，但表观遗传改变作为癌症风险因素的作用仍然很大程度上未知，这是未来研究的一个重要领域。目前，我们缺乏快速、灵敏的方法来评估已知肿瘤抑制基因的启动子甲基化状态。在本文中，我们提出了一种新的基于 NGS 的方法，用于评估一大组已定义的肿瘤抑制基因的启动子甲基化状态，以达到碱基对分辨率。该方法省略了与常用数组方法相关的限制。我们的面板包括 565 个目标区域，涵盖 283 个定义的肿瘤抑制因子的启动子，这些区域按照预先指定的标准进行选择，并应用于快速靶向甲基化特异性 NGS。该方法的可行性是通过分析来自 34 名健康绝经后妇女的正常组织 DNA（白细胞，WBC）样本并通过对这些个体中肿瘤抑制因子的甲基化情况进行初步评估来评估的。平均目标覆盖率为 189.6 倍，灵敏度为 0.53%，足以评估 BRCA1 等低水平甲基化基因的启动子甲基化。在这个有限的测试集中，我们检测到位于 149 个基因的启动子中的 206 个区域以 > 99% 的置信水平差异甲基化（超或低）。基因启动子中的七个目标区域（CIITA、RASSF1、CHN1、PDCD1LG2、GSTP1、XPA 和 ZNF668) 被发现在少数个体中被高甲基化，个体之间在整个区域的平均甲基化差异 > 20%。在探索性层次聚类分析中，我们发现所分析的个体可以根据其在 283 个肿瘤抑制基因启动子中的 WBC 甲基化谱分为两个主要组。我们的肿瘤抑制基因组的甲基化特异性 NGS 对健康个体的差异甲基化进行了详细评估，为识别癌症的新型表观遗传风险因素提供了一种可行的方法。我们发现，根据 283 个肿瘤抑制基因启动子的 WBC 甲基化谱，分析的个体可能分为两个主要组。我们的肿瘤抑制基因组的甲基化特异性 NGS 对健康个体的差异甲基化进行了详细评估，为识别癌症的新型表观遗传风险因素提供了一种可行的方法。我们发现，根据 283 个肿瘤抑制基因启动子的 WBC 甲基化谱，分析的个体可能分为两个主要组。我们的肿瘤抑制基因组的甲基化特异性 NGS 对健康个体的差异甲基化进行了详细评估，为识别癌症的新型表观遗传风险因素提供了一种可行的方法。