Environmental Exposure and DNA Damage

癌症发病率随着年龄的增长而增加，但原因尚不清楚。在血液DNA的小型研究中，已有与年龄相关的甲基化变化的报道，这促使我们探索年龄和DNA甲基化之间的联系。我们使用我们的姐妹研究病例-乳腺癌队列研究的数据来评估27,578个CPGS中与年龄相关的甲基化变化。参与研究的女性年龄从35岁到76岁不等，所有女性在登记时都没有患癌症。我们发现，几乎三分之一的CPG与年龄显著相关。使用三个关于血液的小型公共甲基化数据集，我们构建了749个高置信度年龄相关CPG的子集，该子集在所有数据集上显示出一致的年龄效应。使用来自癌症基因组图谱的新的公开可用的数据，我们证明了我们在血液中识别的衰老部位也广泛适用于多种人体组织。 我们注意到，与年龄相关的CPGS甲基化程度越来越高，位于许多基因的启动子区域，这些基因是细胞分化/发育程序的关键调节因素。这些基因包括CDX2(滋养外胚层)、Gata4(原始内胚层)、Sox17(内胚层)、T(中胚层)、MyoD1(肌肉)、Sox1(神经外胚层)和Hox基因(A、C和D簇，胚体计划)。发育调控基因对于癌症的表观遗传学和癌症干细胞假说是特别重要的：虽然发育基因在正常细胞和癌细胞中都被转录抑制，但它们经历了表观遗传学的转换，从不稳定的、基于组蛋白的基因抑制到永久的DNA甲基化沉默。这种永久沉默可能会为癌细胞提供进化优势，使其能够增殖但不能分化。 基于表观遗传转换假说，我们预测与年龄相关的CpG位点日益甲基化将与抑制性H3K27me3组蛋白标记相关。为了验证这一假设，我们获得了公开可用的人类胚胎干细胞(HESC)和正常分化的淋巴母细胞系(GM12878)中组蛋白修饰的ENCODE芯片序列数据。在HESC和淋巴母细胞中，约80%的年龄相关CPGS甲基化程度增加，具有抑制性的H3K27me3标记。这明显高于阵列上其他位置的40%的频率(p&lt；10-50)，并提供了证据，表明像许多发育基因一样，在胚胎干细胞和分化的细胞中，与年龄相关的甲基化位置经常与H3K27me3组蛋白标记重合。 我们接下来预测，这些日益甲基化的年龄相关部位也应该成为肿瘤组织过度甲基化的靶点。我们分析了来自癌症基因组图谱项目的正常-肿瘤对的公开甲基化数据，其中有7种癌症的数据。我们发现，在所有7种肿瘤类型中，年龄相关部位的改变方向是显著甲基化改变的有力预测因子。这些发现暗示了癌症干细胞假说的延伸：癌症发病率增加和年龄之间的关联可能部分归因于某些基因上与年龄相关的中间甲基化变化。 表观基因组研究已经确定了五个与成人吸烟相关的血液DNA甲基化降低的基因座。我们从27K CpG甲基化研究和我们的450K CpG DES研究中检查了女性吸烟史与DNA甲基化的关系。我们在F2RL3、GPR15、AHRR和2号染色体的一个基因间隔区确认了四个与吸烟相关的CPG与吸烟之间的关系。此外，我们还首次报道了共比例卟啉原氧化酶(CPOX)基因中的CpG与吸烟之间的关联。CPOX编码一种参与血红素生物合成的酶，该酶定位于线粒体膜间间隙，CPOX的突变是遗传性盆腔发育不良的原因之一。为了验证这一基因座，我们设计了CPOX第一内含子中CpG海岸位点的焦磷酸测序分析，并在Sister研究中的一组独立的476名女性身上复制了我们的吸烟结果。我们发现，与从不吸烟的人相比，既往吸烟者的甲基化水平较低(p&lt；2x10-6)，现在吸烟者的甲基化水平甚至更低(p&lt；2x10-18)。这些结果表明，与吸烟相关的氧化应激或血红素生物合成增加可能导致CPOX第一内含子甲基化持续降低。 此外，我们还完成了一项研究，研究吸烟母亲所生婴儿血液中的表观遗传学变化。孕期母亲吸烟与婴儿发病率和死亡率显著相关，并可能影响以后的疾病风险。吸烟(和其他环境因素)可能产生长期影响的一种机制是通过表观遗传修饰，如DNA甲基化。我们分析了889名婴儿出生后不久采集的血液，并使用Illumina 450K甲基化芯片检测了DNA。BWE在吸烟者的婴儿中发现了185个具有全基因组意义的甲基化改变的CPGS。这些基因对应于110个基因区域，其中7个是以前报道的，10个是根据公开的结果新确认的。其中最值得注意的是FRMD4A、ATP9A、GALNT2和MEG3，它们与尼古丁依赖、戒烟以及胎盘和胚胎发育有关。在新生儿中发现的甲基化变化可能介导了宫内母亲吸烟暴露与后来的健康结果之间的关联。

Cancer incidence increases with age, but it is not clear why. Age-related changes in methylation have been reported in small studies of blood DNA, prompting us to explore associations between age and DNA methylation. We used data from our Sister Study case-cohort study of breast cancer to assess age-related methylation change at 27,578 CpGs. The women in the study ranged in age from 35 to 76 and all women were cancer-free at the time of enrollment. We found that almost a third of the CpGs were significantly associated with age. Using three small public methylation data sets on blood, we constructed a subset of 749 high confidence age-related CpGs that showed consistent age effects across all data sets. Using new publicly-available data from The Cancer Genome Atlas, we demonstrate that the aging sites we identified in blood are also broadly applicable across multiple human tissues. We noted that increasingly methylated age-related CpGs were in the promoter regions of a number of genes that are key regulators of cell differentiation/development programs. These genes included Cdx2 (Trophectoderm), Gata4 (primitive endoderm), Sox17 (endoderm), T (mesoderm), MyoD1 (muscle), Sox1 (neuroectoderm), and Hox genes (A, C, and D clusters, embryo body plan). Developmental regulatory genes are of particular interest for the epigenetics of cancer, and in relation to the cancer stem cell hypothesis: Although developmental genes are transcriptionally repressed in both normal cells and cancer cells, they undergo epigenetic switching from less stable, histone-based, gene repression to permanent DNA-methylation silencing. This permanent silencing may provide an evolutionary advantage to a cancer cell, allowing it to proliferate but not to differentiate. Based on an epigenetic switching hypothesis we predicted that increasingly-methylated age-associated CpG sites would be associated with the repressive H3K27me3 histone mark. To test this hypothesis we obtained publicly available ENCODE CHIP-seq data for histone modifications in a human embryonic stem cell (HESC), and in a normal differentiated lymphoblastoid cell line (GM12878) . In both HESC and lymphoblastic cells about 80% of increasingly-methylated age-related CpGs had the repressive H3K27me3 mark. This was significantly higher (p< 10-50) than the 40% frequency at other sites on the array, and provides evidence that, like many developmental genes, the age-related methylation sites often coincide with the H3K27me3 histone mark in both embryonic stem and differentiated cells. We next predicted that these increasingly-methylated age-related sites should also be targets for overmethylation in tumor tissues. We analyzed publicly available methylation data for normal-tumor pairs from The Cancer Genome Atlas project that had data for 7 cancers. We found that the direction of change at age-related sites was a powerful predictor of significant methylation change in all 7 tumor types. These finding suggest an extension to the cancer-stem cell hypothesis: The association between increasing cancer incidence and age may in part be attributable to intermediate age-related methylation change at certain genes. Epigenome-wide studies have identified five loci where there is decreased blood DNA methylation associated with adult smoking. We examined smoking history in relation to DNA methylation in women from our 27K CpG methylation study and in women from our 450K CpG study of DES. We confirm smoking associations for four previously identified smoking-related CpGs in F2RL3, GPR15, AHRR and an intergenic region of chromosome 2. In addition we provide the first report of an association between a CpG in the coproporphyrinogen oxidase (CPOX) gene. CPOX codes for an enzyme involved in heme biosynthesis that is localized to the intermembrane space of the mitochondria, and mutations in CPOX are a cause of hereditary coproporphyria. In order to validate this locus we designed a pyrosequencing assay for the CpG shore site in the first intron of CPOX, and replicated our smoking result in an independent set of 476 women from the Sister Study. We found that relative to the levels in never smokers, there was lower methylation in former smokers (p < 2x10-6) and even lower methylation levels among current smokers (p < 2x10-18). These results suggest that the oxidative stress or the increased heme biosynthesis associated with smoking might lead to persistent decreased methylation in the first intron of CPOX. In addition we have completed a study epigenetic changes in blood of babies born to mothers who smoke. Maternal smoking during pregnancy is associated with significant infant morbidity and mortality, and may influence later disease risk. One mechanism by which smoking (and other environmental factors) might have long-lasting effects is through epigenetic modifications such as DNA methylation. We analyzed blood collected from 889 infants shortly after delivery and examined the DNA using the Illumina 450K methylation chip. BWe identified 185 CpGs with altered methylation in infants of smokers at genome-wide significance. These correspond to 110 gene regions, of which 7 have been previously reported and 10 are newly confirmed using publically-available results. Among these 10 the most noteworthy are FRMD4A, ATP9A, GALNT2, and MEG3, implicated in processes related to nicotine dependence, smoking cessation, and placental and embryonic development. The methylation changes identified in newborns may mediate the association between in utero maternal smoking exposure and later health outcomes.