Project 3: Analysis of Methylome for Osteoporosis Risk in Males

项目总结 骨质疏松症是最常见的与衰老有关的代谢性骨病，主要特征是骨矿物质含量低 骨密度(BMD)和骨质质量/强度恶化。外周血单核细胞(PBM)不仅可以作为 破骨细胞的前体也产生对破骨细胞分化和功能重要的细胞因子，因此 代表骨骼代谢的主要系统细胞。DNA甲基化是基因的重要表观遗传调控 在复杂疾病的病因学中，表达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。 骨质疏松性骨折的发病率和死亡率明显高于女性。 我们的一般假设是，PBM中DNA甲基化特征的改变和相关基因的变化 在男性中，骨密度峰值和骨质量/强度的变化与表达和破骨细胞生成有关。 我们的目标/期望是1)在表观基因组水平上识别和表征差异甲基化 外周血单核细胞中与高加索男性骨质疏松风险相关的区域(DMR)；2)确定DNA甲基化 介导的骨质疏松症的表观遗传机制，即DMRS如何调节编码/非编码基因的表达 编码目标基因和随后的破骨细胞生成。 我们将实现以下具体目标：1)识别/验证与以下方面密切相关的DMR 高加索男性的峰值骨密度和骨质量/强度(QCT和FEA)。我们将进行系统的表观基因组学- 100例高加索人群外周血多核细胞DNA甲基化的广泛性和区域性比较研究 男性(发现队列)的骨量在20-30岁之间达到顶峰，其中一半的人骨密度极高，另一半人的骨密度极高 一半的人具有极低的BMD，并验证“发现队列”和 由100名高加索男性组成的独立“复制队列”不符合骨密度峰值。2)性别的确定- 以及有意义的DMR的种族共性/特异性。复制的DMRS将在三个独立的 样本，包括a)160名高加索女性(通过对女性骨质疏松症的DNA甲基化研究， R01AR059781)，b)100名非裔美国人男性，c)160名中国男性。3)深入开展分子生物学研究 验证的DMRS在调节编码/非编码基因表达和破骨细胞生成中的功能作用。 我们将通过对DNA甲基化的综合分析来确定潜在的DMR顺式调控的编码/非编码基因 来自相同100的同一组PBM中的数据、转录数据(Proj 2)和DNA序列数据(Proj 1 发现队列中的高加索男性，并进行基于细胞的体外功能分析，以评估DNA如何 这些DMRS上的甲基化调控靶基因的表达和破骨细胞的形成。 这一新颖的项目有望在骨质疏松症研究领域取得突破。这个 这一结果将对骨质疏松症的表观遗传机制提供新的见解。所获得的知识可能 最终导致更好地预防和治疗骨质疏松症的新方法。

PROJECT SUMMARY Osteoporosis is the most common aging-related metabolic bone disease mainly characterized by low bone mineral density (BMD) and deteriorated bone quality/strength. Peripheral blood monocytes (PBMs) can 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 potential sex-specific effects in the etiology of complex diseases. However, the significance of global DNA methylation profiles underlying osteoporosis risk is largely unknown, particularly in males who suffer significantly higher morbidity and mortality rates upon osteoporotic fractures than females. Our General Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to peak BMD and bone quality/strength variation in males. Our Goals/Expectations are to 1) identify and characterize, at the epigenome-wide level, differentially methylated regions (DMRs) in PBMs associated with osteoporosis risk in Caucasian males; 2) ascertain the DNA methylation mediated epigenetic mechanisms of osteoporosis, that is, how the DMRs regulate the expression of the coding/non- coding target genes and subsequent osteoclastogenesis. We will accomplish the following Specific Aims: 1) Identification/validation of DMRs significantly associated with peak BMD and bone quality/strength (QCT and FEA) in Caucasian males. We will perform systematic epigenome- wide and regional focused comparative DNA methylation profiling studies in PBMs of 100 discordant Caucasian males (‘Discovery cohort’) at peak bone mass aged 20-30, including half with extremely high BMDs and the other half with extremely low BMDs, and validate the top most significant DMRs in both of the ‘Discovery cohort’ and an independent ‘Replication cohort’ of 100 Caucasian males discordant for peak BMDs. 2) Determination of the sex- and ethnic-generality/specificity of the significant DMRs. The replicated DMRs will be tested in three independent samples, including a) 160 Caucasian females (through a DNA methylation study for female osteoporosis, R01AR059781), b) 100 African American males, and c) 160 Chinese males. 3) In-depth molecular investigation of the functional roles of the validated DMRs in regulating coding/non-coding gene expression and osteoclastogenesis. We will identify potential DMR cis-regulated coding/non-coding genes by integrative analyses of DNA methylation data, transcriptomic data (Proj 2), and DNA sequence data (Proj 1) in the same set of PBMs from the same 100 Caucasian males in the Discovery Cohort, and conduct in vitro cell-based functional assays to assess how DNA methylation at these DMRs regulates target gene expression and osteoclastogenesis. This novel project holds a great promise of award to generate breakthroughs in the osteoporosis research field. The results will give new insights into the epigenetic mechanisms underlying osteoporosis. The knowledge gained may ultimately lead to novel approaches to better prevention and treatment of osteoporosis.