Epigenomewide DNA Methylation Study for Osteoporosis Risk

描述(申请人提供)：骨质疏松症是一种常见疾病，主要特征是骨密度(BMD)低和骨折风险增加。外周血单核细胞不仅可以作为破骨细胞的前体，也是骨吸收细胞，还可以产生对破骨细胞分化、激活和凋亡至关重要的细胞因子，从而代表骨代谢的主要系统细胞。DNA甲基化的改变作为基因表达的重要表观遗传调节，在病因学上具有重要意义 人类复杂的疾病。体外研究表明，DNA甲基化参与了破骨细胞的形成；然而，全球DNA甲基化特征(甲基组)在人类骨质疏松风险中的体内意义尚不清楚。我们的假设是，PBM中DNA甲基化特征的改变以及与之相关的基因表达和破骨细胞形成的变化导致了人类骨密度的峰值变化。我们的目标/期望是：i)在绝经前峰值骨密度极高的妇女和峰值骨密度极低的妇女之间，在整个甲基组水平上识别外周血中的差异甲基化区域；ii)研究骨质疏松的潜在表观遗传学机制，即所识别的甲基化区域如何通过影响相关基因的表达和随后的破骨细胞形成来影响峰值骨密度的变化。方法：1)提取160例25~40岁绝经前高加索女性PBM及其DNA和总RNA，其中80例为极高峰值骨密度组，80例为极低峰值骨密度组(其他年龄相匹配)。2)将通过使用尖端技术MeDIP-seq(甲基化DNA免疫沉淀分析随后的下一代测序)对80名发现样本(包括40名高BMD和40名低BMD)进行最先进的甲基化DNA图谱研究来识别DMRS。3)鉴定的DMRS将在一个独立的复制样本(包括40个高BMD和40个低BMD)中进行亚硫酸氢盐测序，并通过将DNA甲基化数据与潜在候选靶基因在总共160名受试者的PBM中的mRNA表达水平相关联来识别其靶基因。4)已确定的最重要的DMR相关靶基因在破骨细胞形成中的作用将通过基于细胞的体外实验进一步研究。这一极具新颖性的R01项目有望在骨质疏松研究领域取得突破。这一结果可能会导致一次重大的范式转变，扩大目前骨质疏松症的遗传流行病学研究，从经典的DNA变异到DNA修饰的新的表观遗传学/表观基因组学机制。因此，这一结果对于了解骨质疏松症的潜在分子机制，从而有助于预防和治疗具有重要意义。 公共卫生相关性：骨质疏松症是一个严重的公共健康问题，会导致老年人，特别是妇女严重的骨质流失和骨折风险增加。这项拟议的研究将确定骨密度表型不一致的受试者之间的差异甲基化区域(DMR)，并揭示骨密度变异和骨质疏松风险的一些表观遗传学机制。这些发现将有助于更好和更全面地了解骨质疏松的分子机制，从而有助于预防和治疗骨质疏松症。

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.