Epigenomewide DNA Methylation Study for Osteoporosis Risk

描述（由申请人提供）：骨质疏松症是一种常见疾病，主要特征是骨矿物质密度（BMD）低和骨折风险增加。外周血单核细胞（PBMs）不仅可以作为破骨细胞（骨吸收细胞）的前体细胞，而且还可以产生对破骨细胞分化、活化和凋亡重要的细胞因子，因此是骨代谢的主要系统细胞。DNA甲基化改变作为基因表达的重要表观遗传调节因子，在肿瘤的病因学中具有重要意义 复杂的人类疾病。体外研究表明，DNA甲基化参与破骨细胞生成;然而，在人类潜在的骨质疏松症风险中，整体DNA甲基化谱（甲基化组）的体内意义尚不清楚。 我们的假设是，PBMs中DNA甲基化谱的改变以及相关的基因表达和破骨细胞生成的变化有助于人类峰值BMD的变化。 我们的目标/期望是：i）在具有极高峰BMD的绝经前妇女和具有极低峰值BMD的绝经前妇女之间，在整个甲基化组水平上鉴定PBM中的差异甲基化区域（DMR）; ii）研究骨质疏松症的潜在表观遗传机制，即鉴定的DMR如何通过影响相关基因的表达和随后的破骨细胞生成来影响峰值BMD变化。 研究方法：1）PBM及其DNA和总RNA将从160名年龄为25-40岁的绝经前高加索女性中提取，包括80名具有极高峰值高峰BMD和80名具有极低峰值BMD（但在其他方面匹配）。2)DMR将通过使用尖端技术MeDIP-seq（甲基化DNA免疫沉淀试验，然后进行下一代测序）在80例受试者（包括40例高BMD受试者和40例低BMD受试者）的发现样本中进行最先进的甲基化组分析研究来确定。3)确定的DMR将在独立复制样本（包括40例高BMD和40例低BMD）中通过亚硫酸氢盐测序进行确认，并通过将DNA甲基化数据与总计160例受试者PBM中潜在候选靶基因的mRNA表达水平相关联来确定其靶基因。4)将通过基于细胞的体外试验进一步研究已鉴定的最重要DMR相关靶基因对破骨细胞生成的作用。 这个高度新颖的R 01项目具有很大的获奖希望，在骨质疏松症研究领域取得突破。这些结果可能会导致一个重大的范式转变，通过扩大目前的遗传流行病学研究骨质疏松症，从经典的DNA变异的DNA修饰的新的表观遗传学/表观基因组学机制。因此，这些结果对于了解骨质疏松症的分子机制，从而帮助预防和治疗骨质疏松症具有重要意义。 公共卫生相关性：骨质疏松症是一个严重的公共卫生问题，导致严重的骨丢失和老年受试者，特别是妇女骨折的风险增加。这项研究将确定具有不一致BMD表型的受试者之间的差异甲基化区域（DMR），并揭示BMD变异和骨质疏松风险的一些表观遗传机制。这些发现将有助于更好和更全面地了解骨质疏松症的分子机制，从而有助于预防和治疗。

DESCRIPTION (provided by applicant): Osteoporosis is a common disease mainly characterized by low bone mineral density (BMD) and increased risk of fractures. Peripheral blood monocytes (PBMs) may not only act as precursors of osteoclasts, the bone resorption cells, but also produce cytokines important for osteoclast differentiation, activation, and apoptosis, and thus represent major systemic cells for bone metabolism. Alterations in DNA methylation as an important epigenetic regulator of gene expression, is significant in the etiology of human complex diseases. In vitro studies have shown that DNA methylation is involved in osteoclastogenesis; however, the in vivo significance of global DNA methylation profiles (methylome) in humans underlying osteoporosis risk is unknown. Our Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to peak BMD variation in humans. Our Goal/Expectation is to i) identify differentially methylated regions (DMRs) in PBMs at the whole methylome level between premenopausal women with extremely high peak BMD and those with extremely low peak BMD; ii) study potential epigenetic mechanisms of osteoporosis, namely, how the DMRs identified may influence the peak BMD variation through affecting the expression of the relevant genes and subsequent osteoclastogenesis. Methods: 1) PBMs and their DNAs and total RNAs will be extracted from 160 premenopausal Caucasian females aged 25-40 years, including 80 with extremely high peak BMD and 80 with extremely low peak BMD (but otherwise matched). 2) DMRs will be identified by performing state-of-the-art methylome profiling studies with the cutting-edge technology MeDIP-seq (methylated DNA immunoprecipitation assays followed by next-generation sequencing) in a discovery sample of 80 subjects (including 40 with high and 40 with low BMD). 3) The identified DMRs will be subject to confirmation by bisulfite sequencing in an independent replication sample (including 40 with high and 40 with low BMD), and their target genes will be identified by correlating the DNA methylation data with the mRNA expression levels of the potential candidate target genes in PBMs of the total 160 subjects. 4) The roles of the identified most significant DMR-affiliated target genes on osteoclastogenesis will be further investigated by cell based in vitro assays. This highly novel R01 project holds great promise of award to generate breakthroughs in the osteoporosis research field. The results may lead to a major paradigm shift by expanding current genetic epidemiology studies of osteoporosis, from classical DNA variants to novel epigenetics/epigenomics mechanisms of DNA modification. Therefore, the results will be highly important for understanding the underlying molecular mechanisms, and thus help prevention and treatment, of osteoporosis. PUBLIC HEALTH RELEVANCE: Osteoporosis is a serious public health problem leading to severe bone loss and increased risk of fractures in elderly subjects, especially women. The proposed study will identify differentially methylated regions (DMRs) between subjects with discordant BMD phenotypes and reveal some epigenetic mechanisms of BMD variation and osteoporosis risk. The findings will contribute to a better and more comprehensive understanding of molecular mechanisms, and thus help prevention and treatment, of osteoporosis.