Project 3: Analysis of Methylome for Osteoporosis Risk in Males

项目摘要 骨质疏松症是最常见的与年龄相关的代谢性骨病，主要表现为骨矿含量降低 骨密度（BMD）和骨质/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，而且还产生对破骨细胞分化和功能重要的细胞因子， 代表了骨代谢的主要系统细胞。DNA甲基化是基因的重要表观遗传调节因子 表达可能在复杂疾病的病因学中具有显著和潜在的性别特异性作用。但 总体DNA甲基化谱潜在骨质疏松风险的意义在很大程度上是未知的，特别是在男性中 女性的发病率和死亡率明显高于女性。 我们的一般假设是PBMs中DNA甲基化谱的改变和基因表达的相关变化， 表达和破骨细胞生成有助于男性的峰值BMD和骨质量/强度变化。 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的 在白种男性中，PBM中与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DMR如何调节编码/非编码基因的表达。 编码靶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/验证与以下方面显著相关的DMR 高加索男性峰值BMD和骨质量/强度（QCT和FEA）。我们将进行系统的表观基因组- 在100名不一致的高加索人PBM中进行的广泛和区域集中的比较DNA甲基化谱研究 20-30岁峰值骨量的男性（“发现队列”），其中一半具有极高的BMD，另一半具有极高的BMD。 其中一半的BMD极低，并验证了“发现队列”和 100名峰值BMD不一致的高加索男性的独立“复制队列”。2)性别的确定- 和重要DMR的种族一般性/特异性。复制的DMR将在三个独立的 样本，包括a）160名高加索女性（通过女性骨质疏松症的DNA甲基化研究， R 01 AR 059781），B）100例非洲裔美国男性，和c）160例中国男性。3)深入的分子研究 经验证的DMR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DNA甲基化的综合分析来识别潜在的DMR顺式调节编码/非编码基因。 来自同一100个PBM的同一组PBM中的数据、转录组数据（Proj 2）和DNA序列数据（Proj 1） 发现队列中的高加索男性，并进行体外细胞功能测定，以评估DNA 这些DMR的甲基化调节靶基因表达和破骨细胞生成。 这一新颖的项目具有很大的获奖希望，以产生骨质疏松症研究领域的突破。的 结果将为骨质疏松症的表观遗传机制提供新的见解。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.