Prioritization of Genes Relevant to Bone Fragility Through the Unbiased Integration of Aging Mouse Bone Transcriptomics and Human GWAS Analyses

Abstract

Identifying new genetic determinants of bone mineral density (BMD) and fracture promises to yield improved diagnostics and therapies for bone fragility. However, prioritizing candidate genes from genome‐wide screens can be challenging. To overcome this challenge, we prioritized mouse genes that are differentially expressed in aging mouse bone based on whether their human homolog is associated with human BMD and/or fracture. Unbiased RNA‐seq analysis of young and old male C57BL/6 mouse cortical bone identified 1499, 1685, and 5525 differentially expressed genes (DEGs) in 1, 2, and 2.5‐year‐old bone, relative to 2‐month‐old bone, respectively. Gene‐based scores for heel ultrasound bone mineral density (eBMD) and fracture were estimated using published genome‐wide association studies (GWAS) results of these traits in the UK Biobank. Enrichment analysis showed that mouse bone DEG sets for all three age groups, relative to young bone, are significantly enriched for eBMD, but only the oldest two DEG sets are enriched for fracture. Using gene‐based scores, this approach prioritizes among thousands of DEGs by a factor of 5‐ to 100‐fold, yielding 10 and 21 genes significantly associated with fracture in the two oldest groups of mouse DEGs. Though these genes were not the most differentially expressed, they included Sost, Lrp5, and others with well‐established functions in bone. Several others have, as yet, unknown roles in the skeleton. Therefore, this study accelerates identification of new genetic determinants of bone fragility by prioritizing a clinically relevant and experimentally tractable number of candidate genes for functional analysis. Finally, we provide a website (http://www.mouse2human.org) to enable other researchers to easily apply our strategy. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone-cartilage crosstalk informed by aging mouse bone transcriptomics and human osteoarthritis genome-wide association studies

“…For RNA-seq analysis, wild-type male C57BL/6 mice were allowed to age to 2 months (2mo), 11 months (1y), 23 months (2y) and 30 months (2.5y) (n = 3 animals for 2mo; n = 4 animals for others) with well controlled conditions at the Buck Institute for Research on Aging. Tissues were collected from all mice simultaneously and RNA was isolated, as described ( Kaya et al, 2022 ).…”

“…To yield RNA from aged mouse bone enriched for osteocytes, humeri were harvested, soft tissues, periosteum, and epiphyses were dissected, and bone marrow was removed by centrifugation, as described ( Halleux et al, 2012 ; Kelly et al, 2014 ). Harvested bones were immediately snap-frozen in liquid nitrogen, and RNA was isolated, as previously described ( Fowler et al, 2017 ; Mazur et al, 2019 ; Kaya et al, 2022 ). Isolated RNA samples that passed the Bioanalyzer quality control threshold of RNA Integrity Number > 7 were used for RNA library preparation using the Illumina TruSeq Stranded mRNA sample preparation kit with 500 ng of total RNA, according to manufacturer's protocol.…”

“…Using UK Biobank human fracture and bone mineral density GWAS of 426,824 individuals ( Morris et al, 2019 ) M2H prioritized <1 % of the DEGs in aged mouse bone, including SOST, the target of the latest FDA-approved therapy for osteoporosis. Mouse age-related DEGs with unknown function in bone were also prioritized, based on their association with human fracture ( Kaya et al, 2022 ), thus identifying new candidate genetic factors related to bone fracture. The goal of the current study is to prioritize analysis of clinically relevant genetic contributors to human OA identified among DEGs in aging mouse bone.…”

Pathways Controlling Formation and Maintenance of the Osteocyte Dendrite Network

“…At present, the genetic basis underlying osteocyte dendrite defects in osteoporosis remains unknown. Since the “osteocyte transcriptome” is enriched in genes linked to BMD variation [ 56 ••, 57 ••, 113 ], it is possible that specific genetic variants predispose certain individuals to accelerated deterioration of the osteocyte network over time. Future study is needed to better define how osteocyte-expressed BMD-associated genes control cellular morphology and dendrite homeostasis in the setting of skeletal disease.…”

Examining craniofacial variation among crispant and mutant zebrafish models of human skeletal diseases

“…As these associations are identified, methods such as Next Generation Sequencing and genetic modification of model organisms are used to parse out the underlying genetic mechanisms of skeletal diseases. CRISPR-modified mouse and zebrafish models have been used to understand the mechanisms for skeletal diseases such as sclerosteosis, osteoporosis, and osteogenesis imperfecta, as well as more rare variations of these disorders (Garg et al, 2022; Kaya et al, 2022; Kwon et al, 2019). Using model organisms to understand the genetic mechanism of these disorders presents opportunities to develop and test potential treatments (Kague and Karasik, 2022).…”