Decoding Methylation Mediated Epigenomic Contributions to Male Osteoporosis

项目总结： 骨质疏松症是最常见的代谢性骨病，以低骨密度为主要特征。 和恶化的骨质量/强度。外周血单核细胞(PBM)可能不仅是 破骨细胞也产生对破骨细胞分化和功能重要的细胞因子，因此代表 骨代谢的主要系统细胞。DNA甲基化是基因表达的重要表观遗传调控 在人类复杂疾病的病因学中可能具有显著的和潜在的性别特异性影响。然而， 骨质疏松风险背后的全球DNA甲基化特征的重要性在很大程度上是未知的，特别是在男性中。 骨质疏松性骨折死亡率明显高于女性。 我们的假设是，PBM中DNA甲基化的变化及其相关的基因表达的变化 而破骨细胞的形成有助于男性峰值骨密度和骨质量/强度的变化。 我们的目标/期望是：i)确定与以下相关的PBM中显著的差异甲基化区域(DMR) 高加索男性的骨质疏松症风险；ii)评估潜在的性别和种族共性/特异性 以及iii)确定DNA甲基化介导的骨质疏松症的表观遗传机制，即 DMRS调节靶基因的表达和随后的破骨细胞形成。 我们将实现以下具体目标：1)识别和验证显著相关的DMRS 高加索男性的峰值骨密度和骨质量/强度(QCT和FEA)。我们将进行PBM甲基化组 双限制性内切酶还原代表性亚硫酸氢盐测序(DRRBS)分析 200名骨量高峰年龄在20-30岁的不和谐的高加索男性(“发现队列”)，其中一半患有 高峰值BMD和另一半具有低峰值BMD，并验证这两个 “发现队列”和一个由200名高加索男性组成的独立的“复制队列”不符合骨密度峰值。2) 评估性别、种族和成熟阶段--重要的DMRS/基因的共性/特异性。这个 经过验证的DMRS/目标基因将在三个独立的BMD不一致样本中进行测试，其中包括200个非洲人 美国男性，b)160名高加索女性，c)160名中国男性，以及d)1670名美国儿童的骨骼 儿童研究中的矿物质密度。3)深入研究DMRS在基因调控中的作用 表达与破骨细胞生成。我们将通过关联DNA来确定DMR调节的靶基因 400例PBM中同一组候选靶基因甲基化及基因表达水平的研究 并进行基于细胞的体外分析，以确定DNA甲基化在这些 DMR参与调控靶基因的表达，进而影响破骨细胞的形成。 结果将揭示对一般和性别/种族/发展具体情况的新的和基本的见解 骨质疏松症的表观遗传机制。所获得的知识可能最终会导致新的方法来 更好地防治骨质疏松症。

PROJECT SUMMARY: Osteoporosis is the most common metabolic bone disease mainly characterized by low bone mineral density (BMD) and deteriorated bone quality/strength. Peripheral blood monocytes (PBMs) may not only act as precursors of osteoclasts but also produce cytokines important for osteoclast differentiation and function, and thus represent major systemic cells for bone metabolism. DNA methylation as an important epigenetic regulator of gene expression may have significant and potentially sex-specific effects in the etiology of human complex diseases. However, the significance of global DNA methylation profiles underlying osteoporosis risk is largely unknown, particularly in males who suffer significantly higher mortality rate upon osteoporotic fractures than females. Our Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to variations in peak BMD and bone quality/strength in males. Our Goal/Expectation is to i) identify significant differentially methylated regions (DMRs) in PBMs associated with osteoporosis risk in Caucasian males; ii) assess the potential sex- and ethnic-generality/specificity of the significant DMRs; and iii) ascertain the DNA methylation mediated epigenetic mechanisms of osteoporosis, that is, how the DMRs regulate the expression of the target genes and subsequent osteoclastogenesis. We will accomplish the following Specific Aims: 1) Identification and validation of DMRs significantly associated with peak BMD and bone quality/strength (QCT and FEA) in Caucasian males. We will perform PBM methylome profiling analyses with double restriction-enzyme reduced representation bisulfite sequencing (dRRBS) assays in 200 discordant Caucasian males (‘Discovery cohort’) at peak bone mass ages of 20-30 years old, including half with high peak BMDs and the other half with low peak BMDs, and validate the most significant DMRs in both of the ‘Discovery cohort’ and an independent ‘Replication cohort’ of 200 Caucasian males discordant for peak BMDs. 2) Evaluation of the sex-, ethnic-, and maturation stage- generality/specificity of the significant DMRs/genes. The validated DMRs/target genes will be tested in three independent BMD-discordant samples, including a) 200 African American males, b) 160 Caucasian females, and c) 160 Chinese males, and d) in 1670 US children from the Bone Mineral Density in Childhood Study. 3) In-depth functional investigation of the roles of DMRs in regulating gene expression and osteoclastogenesis. We will identify the DMR-regulated target genes by correlating the DNA methylation and the mRNA expression levels of candidate target genes in the same sets of PBMs from the total 400 Caucasian males, and conduct in vitro cell-based assays to determine the contribution of DNA methylation at these DMRs in regulating target gene expression and subsequently influencing osteoclastogenesis. The results will reveal novel and fundamental insights into the general and sex-/ethnic-/development specific epigenetic mechanisms underlying osteoporosis. The knowledge gained may ultimately lead to novel approaches to better prevention and treatment of osteoporosis.