Discovery of osteoblast and osteoclast bone mass effector genes using advanced genomics

骨质疏松症是一种毁灭性的骨骼疾病，影响着1000多万美国人。而细胞基础 对于骨质疏松症，包括成骨细胞骨形成失衡和破骨细胞骨吸收， 被证实与骨质疏松症直接相关的临床相关基因相对较少。有一个重要的 需要发现影响骨质疏松症发病机制的新基因。骨质疏松症新基因的发现 最终将允许骨和矿物生物学领域实现开发新的长期目标 预防和治疗这种令人衰弱的疾病的疗法。 汉肯森实验室和格兰特实验室之间现有的长期合作一直是重点 理解基因组广泛关联研究(GWAS)信号的功能意义 骨量、骨质疏松症和骨折风险。我们已经开发出使用这些信号来识别新奇事物的方法 推测与疾病发病机制有关的基因。虽然许多研究小组在GWA方面所做的努力 成功地发现了与骨密度(BMD)和骨密度相关的基因组变异 对于骨折，GWAS只报告与给定特征相关的信号，而不一定是罪魁祸首基因。 在这个方案中，我们利用了一种计算先进的多步骤过程，该过程将基因组水平的数据整合到 鉴定新的成骨细胞和破骨细胞基因。这种生物信息驱动的全基因组变异到基因 Map的努力结合了RNA-seq、atac-seq和高分辨率染色质构象捕获方法，以 牵涉到罪犯效应器基因。我们已经在成骨细胞系细胞中使用了这种方法，30%的 与骨质疏松症相关的GWAS信号被证明与这些细胞中的基因有直接的物理接触， 共86个可能的靶基因。这些目标中有几个(例如EPDR1、ING3)已有广泛的 功能性随访。然而，更多的人需要功能随访，仍有70%的骨质疏松症 仍未解决的相关GWA基因座。重要的是，我们最初的工作只专注于发现 成骨细胞相关基因，以及在破骨细胞中发挥作用的基因没有被揭示。此外， 到目前为止，我们发表的工作只集中在成骨细胞分化过程中的一个时间点， 因此，在细胞分化后期发挥作用的基因还没有被发现。 这一全面的应用将使GWAS的发现发挥作用，并在这样做的过程中，揭示涉及的新基因 在调节骨骼形成和吸收方面的作用。我们已建立的从基因发现到基因验证的管道 经过了强有力的测试，到目前为止，尽管我们的样本很小，但我们有100%的命中率 验证可能的效应基因。因此，我们的假设是，我们可以发现更多的BMD效应器 通过在破骨细胞和成骨细胞中进行高分辨率的“全基因组变异到基因图谱”来研究基因。 基因的相关性将在小鼠模型中使用体外和体内两种方法进行验证。vt.在.的基础上 完成后，我们将为骨界提供新的靶点，以寻求了解机制。

Osteoporosis is a devastating disease of bone that impacts over 10 million Americans. While the cellular basis for osteoporosis includes an imbalance in bone formation by osteoblasts and bone resorption by osteoclasts, there are relatively few validated, clinically relevant genes directly linked to osteoporosis. There is a significant need to discover new genes that influence osteoporosis pathogenesis. Discovery of new osteoporosis genes will eventually permit the field of bone and mineral biology to achieve the long-term goal of developing new therapies to both prevent and treat this debilitating disease. The existing, long-term collaboration between the Hankenson and Grant laboratories has been focused on understanding the functional significance of genome wide association study (GWAS) signals associated with bone mass, osteoporosis and fracture risk. We have developed methods to use those signals to identify novel genes putatively involved in disease pathogenesis. While GWAS efforts by numerous research groups have been successful in discovering genomic variants robustly associated with bone mineral density (BMD) and fracture, GWAS only reports signals associated with a given trait and not necessarily culprit genes. In this proposal we utilize a computationally advanced, multi-step process that integrates genome level data to identify novel osteoblast and osteoclast genes. This bioinformatically-driven `genome-wide variant to gene mapping' effort combines RNA-seq, ATAC-seq and high-resolution chromatin conformation capture methods to implicate culprit effector genes. We have already used this approach in osteoblast lineage cells and 30% of osteoporosis-associated GWAS signals were shown to have direct physical contact with genes in these cells, totaling 86 putative target genes. Several of these targets (ex. EPDR1, ING3) have already had extensive functional follow-up. However, many more need functional follow-up and there are still 70% of osteoporosis associated GWAS loci that remain unresolved. Importantly, our initial work focused only on discovering osteoblast-associated genes, and thus genes that play a role in osteoclasts were not revealed. Furthermore, our published work to date has only focused on one time-point during the osteoblast differentiation process, thus genes that play roles at later points in cell differentiation have not been discovered. This comprehensive application will functionalize GWAS findings, and in doing so, reveal novel genes involved in regulating bone formation and resorption. Our established pipeline from gene discovery to gene validation has been robustly tested and thus far, although our sampling has been small, we have had a 100% hit rate for validating putative effector genes. Thus, it is our hypothesis that we can uncover many more BMD effector genes by conducting high resolution `genome-wide variant to gene mapping' in osteoclasts and osteoblasts. The relevance of genes will be validated using both in vitro and in vivo approaches in mouse models. Upon completion, we will provide the bone community with new targets to pursue for understanding mechanism.