PROJECT SUMMARY Osteoporosis is the most prevalent metabolic bone disease during aging, characterized by low bone mineral density (BMD); deteriorated bone quality/strength/microarchitecture (measured by finite element analyses [FEA] and trabecular bone score [TBS]); and increased risk of osteoporotic fractures (OFs). While studies of osteoporosis have focused on BMD, the mechanisms of the disease, especially in vivo in humans and for OFs in particular, remain unclear. Omics studies of OFs are rare but necessary given that BMD and OFs share only ~50% of genetic determination. Joint omics studies of OF and bone risk factors (BMD/FEA/TBS) are imperative to identify mechanisms that are both relevant to OFs and amenable for intervention from the aspect of bone metabolism. Bone metabolism is mainly influenced by cells in osteoblastogenic (e.g., bone marrow mesenchymal stem cells [BMSCs], osteoblasts, osteocytes), and osteoclastogenic (e.g., monocytes, osteoclasts) lineages. These classical cell types can be further classified into subtypes with differential biological functions. To date, functional omics studies of osteoporosis—on bone biopsies, peripheral blood mononuclear cells (PBMCs) and monocytes (PBMs – potential) (precursors of osteoclasts)—have been largely performed without distinguishing between cell (sub)types and without considering cell composition (CC), hence, may yield false-positive/negative results. In Project 2, our Goal is to conduct an innovative and comprehensive single cell RNA-sequencing (scRNA-seq)- based trans-omics study of osteoporosis in vivo in humans on both osteoblastogenic and osteroclastogenic lineages. Our Hypothesis is that traditional bone lineage cells, composed of functionally distinct cell subtypes, have differential genes and gene regulatory networks (GRNs) that, together with cell–cell interactions (CCIs) and individual CCs, are significant for osteoporosis. This will be tested through the following Specific Aims: 1) Identify/discover functionally distinct cell subtypes, their corresponding marker genes and GRNs and explore their developmental relationships/trajectories using scRNA-seq on freshly isolated bone, bone marrow, and PBMs obtained from 150 subjects with OFs and 150 matched (including matching for age) controls (age ≥ 55; recruited through Clinical Core [Core B]). Significant differences in transcriptomic profiles, GRNs, CCs and CCIs will be determined between OF cases and controls. The effects of age, sex, ethnicity, and other covariates will be assessed/adjusted throughout. 2) With the sc-seq data collected in Aim 1 as reference, use innovative multi-omics approaches to impute sc- transcriptomic profiles in >5,300 subjects that have been characterized both phenotypically and by various omics through our ongoing U19 and Louisiana Osteoporosis Study; PBM methylome data from these subjects will also be used as reference to impute epigenomic data in the 300 participants recruited through Core B. 3) Ident...