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Osteoporosis, characterized by low bone mineral density (BMD), is the most common complex disease affecting bone and constitutes a major societal health problem. Genome-wide association studies (GWASs) have identified over 1100 associations influencing BMD. It has been shown that perturbations to long noncoding RNAs (lncRNAs) influence BMD and the activities of bone cells; however, the extent to which lncRNAs are involved in the genetic regulation of BMD is unknown. Here, we combined the analysis of allelic imbalance (AI) in human acetabular bone fragments with a transcriptome-wide association study (TWAS) and expression quantitative trait loci (eQTL) colocalization analysis using data from the Genotype-Tissue Expression (GTEx) project to identify lncRNAs potentially responsible for GWAS associations. We identified 27 lncRNAs in bone that are located in proximity to a BMD GWAS association and harbor single-nucleotide polymorphisms (SNPs) demonstrating AI. Using GTEx data we identified an additional 31 lncRNAs whose expression was associated (false discovery rate [FDR] correction 0.05) with BMD through TWAS and had a colocalizing eQTL (regional colocalization probability [RCP] 0.1). The 58 lncRNAs are located in 43 BMD associations. To further support a causal role for the identified lncRNAs, we show that 23 of the 58 lncRNAs are differentially expressed as a function of osteoblast differentiation. Our approach identifies lncRNAs that are potentially responsible for BMD GWAS associations and suggest that lncRNAs play a role in the genetics of osteoporosis. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

There are over one million cases of failed bone repair in the U.S. annually, resulting in substantial patient morbidity and societal costs. Multiple candidate genes affecting bone traits such as bone mineral density have been identified in human subjects and animal models using genome-wide association studies (GWAS). This approach for understanding the genetic factors affecting bone repair is impractical in human subjects but could be performed in a model organism if there is sufficient variability and heritability in the bone regeneration response. Diversity Outbred (DO) mice, which have significant genetic diversity and have been used to examine multiple intact bone traits, would be an excellent possibility. Thus, we sought to evaluate the phenotypic distribution of bone regeneration, sex effects and heritability of intramembranous bone regeneration on day 7 following femoral marrow ablation in 47 12-week old DO mice (23 males, 24 females). Compared to a previous study using 4 inbred mouse strains, we found similar levels of variability in the amount of regenerated bone (coefficient of variation of 86 % v. 88 %) with approximately the same degree of heritability (0.42 v. 0.49). There was a trend toward more bone regeneration in males than females. The amount of regenerated bone was either weakly or not correlated with bone mass at intact sites, suggesting that the genetic factors responsible for bone regeneration and intact bone phenotypes are at least partially independent. In conclusion, we demonstrate that DO mice exhibit variation and heritability of intramembranous bone regeneration that will be suitable for future GWAS.

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics., Osteoporosis GWAS faces two challenges, causal gene discovery and a lack of phenotypic diversity. Here, the authors use the Diversity Outbred mouse population to inform human GWAS using networks and map genetic loci for 55 bone traits, identifying new potential bone strength genes.

Osteoporosis is a condition characterized by low bone mineral density (BMD) and an increased risk of fracture. Traits contributing to osteoporotic fracture are highly heritable, indicating that a comprehensive understanding of bone requires a thorough understanding of the genetic basis of bone traits. Towards this goal, genome-wide association studies (GWASs) have identified over 500 loci associated with bone traits. However, few of the responsible genes have been identified, and little is known of how these genes work together to influence systems-level bone function. In this review, we describe how systems genetics approaches can be used to fill these knowledge gaps.

Antimalarial resistance is a major obstacle in the eradication of the human malaria parasite, Plasmodium falciparum. Genome amplifications, a type of DNA copy number variation (CNV), facilitate overexpression of drug targets and contribute to parasite survival. Long monomeric A/T tracks are found at the breakpoints of many Plasmodium resistance-conferring CNVs. We hypothesize that other proximal sequence features, such as DNA hairpins, act with A/T tracks to trigger CNV formation. By adapting a sequence analysis pipeline to investigate previously reported CNVs, we identified breakpoints in 35 parasite clones with near single base-pair resolution. Using parental genome sequence, we predicted the formation of stable hairpins within close proximity to all future breakpoint locations. Especially stable hairpins were predicted to form near five shared breakpoints, establishing that the initiating event could have occurred at these sites. Further in-depth analyses defined characteristics of these ‘trigger sites’ across the genome and detected signatures of error-prone repair pathways at the breakpoints. We propose that these two genomic signals form the initial lesion (hairpins) and facilitate microhomology-mediated repair (A/T tracks) that lead to CNV formation across this highly repetitive genome. Targeting these repair pathways in P. falciparum may be used to block adaptation to antimalarial drugs.

Bone metastasis is a complication of prostate cancer in up to 90% of men afflicted with advanced disease. Therapies that reduce androgen exposure remain at the forefront of treatment. However, most prostate cancers transition to a state whereby reducing testicular androgen action becomes ineffective. A common mechanism of this transition is intratumoral production of testosterone (T) using the adrenal androgen precursor dehydroepiandrosterone (DHEA) through enzymatic conversion by 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD and 17βHSD). Given the ability of prostate cancer to form blastic metastases in bone, we hypothesized that osteoblasts might be a source of androgen synthesis. RNA expression analyses of murine osteoblasts and human bone confirmed that at least one 3βHSD and 17βHSD enzyme isoform was expressed, suggesting that osteoblasts are capable of generating androgens from adrenal DHEA. Murine osteoblasts were treated with 100 nM and 1 μM DHEA or vehicle control. Conditioned media from these osteoblasts were assayed for intermediate and active androgens by liquid chromatography-tandem mass spectrometry. As DHEA was consumed, the androgen intermediates androstenediol and androstenedione were generated and subsequently converted to T. Conditioned media of DHEA-treated osteoblasts increased androgen receptor (AR) signaling, prostate-specific antigen (PSA) production, and cell numbers of the androgen-sensitive prostate cancer cell lines C4-2B and LNCaP. DHEA did not induce AR signaling in osteoblasts despite AR expression in this cell type. We describe an unreported function of osteoblasts as a source of T that is especially relevant during androgen-responsive metastatic prostate cancer invasion into bone. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10−12) BMD locus on Chromosome 3@52.5 Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10−5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass., Author summary Osteoporosis is a common, chronic disease characterized by low bone mineral density (BMD) that puts millions of Americans at high risk of fracture. Variation in BMD in the general population is, in large part, determined by genetic factors. To identify novel genes influencing BMD, we performed a genome-wide association study in a panel of inbred mouse strains. We identified a locus on Chromosome 3 strongly associated with BMD. Using a combination of systems genetics approaches, we connected the expression of the Lhfp gene with BMD-associated genetic variants and predicted it influenced BMD by altering the activity of bone-forming osteoblasts. Using mice deficient in Lhfp, we demonstrated that Lhfp negatively regulates bone formation and BMD. These data suggest that inhibiting Lhfp may represent a novel therapeutic strategy to increase BMD and decrease the risk of fracture.