Environmental Exposure and DNA Damage

乳腺癌是女性最常见的癌症之一，但即使发病率稳定，死亡率下降，今年美国仍将有超过18万名女性被诊断出乳腺癌，超过4万名女性将死于乳腺癌。 一项荟萃分析表明，至少有一个一级亲属被诊断患有乳腺癌的女性的风险增加了近2倍，并且一级亲属预后不良的女性乳腺癌生存率降低。 BRCA 1和BRCA 2突变与家族性乳腺癌密切相关，但这些突变仅在一小部分乳腺癌患者中发现。 最近的全基因组关联研究已经确定了几个与乳腺癌相关的多态性，尽管这些多态性的风险是适度的。 我们的研究小组正在测试某些DNA修饰与乳腺癌风险相关的假设。 这种改变可能是由环境和遗传因素决定的，预计会在妇女的一生中发生变化。 甲基化：我们的实验室已经建立了焦磷酸测序，以量化4个基因（BRCA 1，RASSF 1A，RAR β和GSTP 1）启动子区域中多个CpG位点的甲基化，这些基因在乳腺癌中经常发生异常甲基化，并测量分散在基因组中的LINE-1重复元件的整体甲基化。 我们最近完成了使用姐妹研究样本的病例队列研究的实验室阶段，现在正在完成数据分析。 此外，我们最近使用Illumina 27 K甲基化芯片完成了对这些相同样品的分析。 最近开始对这些数据进行分析。 端粒：我们的实验室优化了实时定量PCR技术，用于估计相对端粒长度。 使用该技术，端粒引发事件的数量以及端粒扩增产物的数量与端粒长度成比例。 该方法已被验证对传统的终端限制性片段（TRF）的端粒长度基于Southern分析，需要少得多的DNA，并可以有效地进行流行病学研究。 我们是第一批采用改良方法的研究小组之一，该方法在单拷贝基因引物上使用GC夹，以便将产物的熔点温移到89 ℃。 这使我们能够在同一管中测量端粒和参考单拷贝基因产物，从而提高估计的精度并减少测定所需的DNA量。 我们已经将该方法从96孔格式调整为384孔格式，以便于流行病学研究所需的高通量分析。 我们最近使用病例队列设计完成了对姐妹研究样本的分析，并正在分析这些数据。 miRNA：改变的miRNA表达是癌症的中心特征，并且已经显示miRNA表达特征与诊断、分期、预后和对治疗的反应相关。 癌症的表达模式显示出高组织特异性，使其成为癌症筛查的潜在标志物。 乳腺癌特异性miRNAs已被证明与分期、血管浸润、增殖指数和ER/PR状态相关。 最近，已经在人血浆和血清中发现了足够水平的miRNA以允许谱分析，具有足够的能力来区分患有转移性前列腺癌的男性与没有癌症的男性和患有卵巢癌的女性。 我们的实验室一直在研究优化从血清样品中提取miRNA的方法，并在与AffyphidomiRNA阵列杂交时最大化信号强度和重现性。 一旦优化，我们计划使用Sister Study样本来研究乳腺癌女性与非乳腺癌女性的miRNA表达。

Breast cancer is one of the most common cancers in women, yet even with a stabilizing incidence rate and decreased mortality, more than 180,000 women will be diagnosed and more than 40,000 women will die in the U.S. from the disease this year. A meta-analysis demonstrated an almost 2-fold increased risk for women with at least one first-degree relative diagnosed with breast cancer and there is decreased survival for breast cancer among women with a first-degree relative who also had a poor prognosis. BRCA1 and BRCA2 mutations are strongly associated with familial breast cancer however these mutations are found in only a small percentage of women with breast cancer. Recent genome-wide association studies have identified several polymorphisms associated with breast cancer although risks from these polymorphisms are modest. Our group is testing the hypothesis that certain DNA modifications are associated with breast cancer risk. Such modifications may be determined by both environmental and genetic factors, and would be expected to change over a woman's lifetime. Methylation: Our laboratory has established pyrosequencing to quantify methylation at multiple CpG sites in the promoter regions of 4 genes (BRCA1, RASSF1A, RAR Beta, and GSTP1) shown to have frequent aberrant methylation in breast cancer and, to measure global methylation, in LINE-1 repetitive elements scattered across the genome. We have recently completed the laboratory phase of a case-cohort study using Sister Study samples and are now completing data analysis. In addition we have recently completed analysis of these same samples using the Illumina 27K methylation chip. Analysis of these data has recently begun. Telomeres: Our laboratory has optimized a real-time quantitative PCR technique for estimating relative telomere length. Using this technique the number of telomere priming events, and thus the telomere amplification product, is proportional to telomere length. The method has been validated against traditional terminal restriction fragment (TRF) Southern-based analysis of telomere length, requires much less DNA, and can be efficiently done in epidemiologic studies. We are one of the first groups to employ a modified method that uses a GC clamp on the single copy gene primers in order to temperature-shift the melting point of the product to 89C. This allows us to measure both telomere and the reference single copy gene product in the same tube, thus improving the precision of the estimate and decreasing the amount of DNA required for the assay. We have adapted the method from 96- to 384-well format in order to facilitate the high throughput analysis required for epidemiology studies. We have recently completed analysis of Sister Study samples using a case-cohort design and are in the process of analyzing these data. miRNA: Altered miRNA expression is a central feature of cancer and miRNA expression signatures have been shown to be associated with diagnosis, stage, prognosis, and response to treatment. Expression patterns for cancer show high tissue specificity making them potential markers for cancer screening. Breast cancer specific miRNAs have been shown to correlate with stage, vascular invasion, proliferative index, and ER/PR status. Recently, sufficient levels of miRNAs have been found in human plasma and serum to permit profiling, with sufficient power to distinguish men with metastatic prostate cancer from men without cancer and women with ovarian cancer. Our laboratory has been working out methods to optimize miRNA extraction from serum samples and to maximize signal intensities and reproducibility in hybridizing to Affymetrix miRNA arrays. Once optimized, we plan to use Sister Study samples to study miRNA expression in women who develop breast cancer vs non-cases.