Whole-blood samples from women exposed to hormone replacement therapy. Homo sapiens

The American Women’s Health Initiative study published in July 2002 caused considerable concern among hormone replacement therapy (HRT) users and prescribers in many countries. This study is an exploratory research comparing the genome wide expression profile in whole blood samples according to HRT use. Within the Norwegian Women and Cancer study, 100 postmenopausal women (50 HRT users and 50 non-HRT users) born between 1943 and 1949 with normal to high body mass index and no other medication use were selected. After total RNA extraction, amplification and labelling, the samples were hybridized together with a common reference (Universal human reference RNA, Stratagen) to Agilent Human 1A oligoarrays (G4110b, Agilent Technologies, Palo Alto, CA) containing 20,173 unique genes. Differentially expressed genes were used to build a classifier using the nearest shrunken centroids method (PAM). Then, we tested the significant changes in single genes by different methods like t-test, Significance Analysis of Microarrays (SAM) and Bayesian ANOVA analysis (BAM). Results did not reveal any distinct gene list which predicted accurately HRT exposure (error rate = 0.45). Classifier performance slightly improved (error rate = 0.29) including only women who were using continuous combined HRT treatment. According to the small amplitude of expression alterations observed after HRT use in whole blood, large sample sizes are needed to identify significant single genes differentially expressed. However, significant enrichments in biologic process of genes with small changes after HRT use were observed (e.g. receptor and transporter activities, immune response, frizzled signalling pathway, actin filament organization, glycogen metabolism). Keywords: gene expression profile and exposure Overall design: Indirect design with Startagen reference 47 non HRT users and 42 HRT users No technical replicates

2002年7月发表的美国妇女健康倡议（American Women’s Health Initiative）研究，引发了多国激素替代疗法（hormone replacement therapy, HRT）使用者与处方医师的广泛担忧。本研究为探索性研究，旨在根据激素替代疗法的使用情况，比较全血样本中的全基因组表达谱。
在挪威妇女与癌症研究（Norwegian Women and Cancer study）框架内，研究人员筛选出1943年至1949年间出生的100名绝经后女性，其中激素替代疗法使用者与非使用者各50名，所有受试者体重指数处于正常至偏高水平，且未服用其他药物。
完成总RNA提取、扩增与标记后，将样本与通用人类参考RNA（Universal human reference RNA, Stratagen）共同杂交至安捷伦人类1A寡核苷酸微阵列（Agilent Human 1A oligoarrays, G4110b，安捷伦科技公司，加利福尼亚州帕洛阿尔托），该微阵列包含20173个独特基因。
研究采用最近收缩质心法（PAM），以差异表达基因为基础构建分类器。随后通过t检验、微阵列显著性分析（Significance Analysis of Microarrays, SAM）以及贝叶斯方差分析（Bayesian ANOVA analysis, BAM）等方法，检验单个基因的显著表达变化。
结果显示，并未找到能够精准预测激素替代疗法暴露情况的独特基因列表（错误率=0.45）。仅纳入持续联合激素替代疗法使用者时，分类器性能略有提升（错误率=0.29）。
鉴于全血中激素替代疗法使用后基因表达改变幅度较小，需通过大样本量才能鉴定出具有显著差异表达的单个基因。不过研究观察到，激素替代疗法使用后表达小幅变化的基因在生物学过程中存在显著富集，例如受体与转运蛋白活性、免疫应答、卷曲蛋白信号通路、肌动蛋白丝组织以及糖原代谢。
关键词：基因表达谱与暴露情况
整体实验设计：采用Stratagen参考RNA作为参照的间接杂交设计；纳入非激素替代疗法使用者47名、激素替代疗法使用者42名；无技术重复。