Activation of peroxisome proliferator activated receptor-γ (PPARG) is required for the differentiation of marrow mesenchymal stem cell into adipocytes and is associated with the development of ...age-related marrow adiposity in mice. Thiazolidinediones are agonists for PPARG and have a heterogeneous effect on bone mineral density (BMD). We postulated that genetic determinants influence the skeletal response to thiazolidinediones. We examined the effects of rosiglitazone (3 mg/kg · d for 8 wk) on BMD, body composition, and serum IGF-I in adult female mice from four inbred strains. C3H/HeJ mice showed the most significant response to treatment, exhibiting decreased femoral and vertebral BMD, reduced distal femoral bone volume fraction and a decrease in serum IGF-I. In DBA/2J, there were no changes in femoral BMD or bone volume fraction, but there was a decrease in vertebral BMD. C57BL/6J mice showed increases in marrow adiposity, without associated changes in trabecular bone volume; the skeletal effects from rosiglitazone in A/J mice were minimal. No association between trabecular bone volume and marrow adiposity was found. The effect of rosiglitazone on gene expression in the femur was then examined in the C3H/HeJ and C57BL/6J strains by microarray. Increased gene expression was observed in the PPARG signaling pathway and fatty acid metabolism in both C3H/HeJ and C57BL/6J, but a significant down-regulation of genes associated with cell cycle was noted only in the C3H/HeJ strain. The divergent skeletal responses to rosiglitazone in this study suggest the existence of a strong genetic background effect.
Treatment of four inbred strains of mice with rosiglitazone results in very distinct skeletal and metabolic responses, suggesting an important genotype by drug interaction.
This protocol describes the application of the “omnigenic” model of the genetic architecture of complex traits to identify novel “core” genes influencing a disease-associated phenotype. Core genes ...are hypothesized to directly regulate disease and may serve as therapeutic targets. This protocol leverages GWAS data, a co-expression network, and publicly available data, including the GTEx database and the International Mouse Phenotyping Consortium Database, to identify modules enriched for genes with “core-like” characteristics.
For complete details on the use and execution of this protocol, please refer to Sabik et al. (2020).
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•A protocol to identify core modules by integrating gene expression data and GWAS data•Core modules serve as the basis for identifying genes influencing complex traits•Disease gene identification by module integration with publicly available databases
This protocol describes the application of the “omnigenic” model of the genetic architecture of complex traits to identify novel “core” genes influencing a disease-associated phenotype. Core genes are hypothesized to directly regulate disease and may serve as therapeutic targets. This protocol leverages GWAS data, a co-expression network, and publicly available data, including the GTEx database and the International Mouse Phenotyping Consortium Database, to identify modules enriched for genes with “core-like” characteristics.
Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study (GWAS) in 142,487 individuals from the UK Biobank to ...identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.
Category:
Ankle; Ankle Arthritis; Basic Sciences/Biologics; Trauma
Introduction/Purpose:
Osteoarthritis (OA) of the lower limb is a debilitating, incurable, expensive and prevalent condition in ...people over 45. Although ankle posttraumatic OA (PTOA) has the highest incidence, primary OA is increasingly recognized. The difference in OA incidence between lower limb joints is partly driven by differences in cartilage architecture and chondrocyte function at each site, with ankle chondrocytes showing reduced responsiveness to inflammatory mediators and more metabolic activity than those in the knee. Synovium also contributes to the pathogenesis of OA, however its potentially differential role in knee versus ankle OA remains an unanswered question. We aim to understand the molecular contribution of synovial dysregulation in the progression of ankle and knee OA to uncover candidate pathways for the development of targeted therapeutic paradigms.
Methods:
After obtaining informed consent from patients undergoing total knee or ankle replacement, basic surgical, past medical history, and imaging data was collected. Synovium samples were harvested during surgery and processed for RNA. Bulk 150 bp, paired-end RNA seq was conducted using standard protocols using the Illumina NovaSeq6000 platform. After quality control trimming, reads were aligned to human reference genome (GRCh38) using STAR (v 2.5) using the Maximum Mappable Prefix method and HTSeq (V0.6.1) was used to index mappable reads per gene. Counts were corrected for gene length using the FPKM method. Differential expression was determined using the edgeR (v 3.16.5) package for R, after adjustment using a scaling normalization factor and P-values were corrected using the Benjamini and Hochberg method. Similarities between samples were determined using Spearman Correlation and degree of similarity was visualized considering the first three Principal Components (PC).
Results:
RNA was obtained from 6 end-stage ankle OA, 1 end-stage knee OA patients. Of the ankle samples, two were PTOA and the remaining four were primary OA. The PTOA and the primary OA samples showed high sample correlation regardless of class (correlation coefficients ranged from 0.844 to 0.941). In contrast, the total knee sample was less correlated with the ankle samples (correlation coefficients ranged from 0.719 to 0.878). By using the first three PC, we observed that there was no obvious differentiation among the ankle OA samples, but there was clear separation between ankle and total knee samples. We observed an enrichment of genes annotated to the Gene Ontology clusters 'Extracellular Structure Organization' and 'Inflammatory Response' when comparing the two types of OA.
Conclusion:
These initial pilot results of an ongoing study support our hypothesis that synovial transcriptome differs between the knee and ankle in end-stage osteoarthritis and that anatomic site is a larger driver of synovial expression differences than OA type. This is supported by previous differences reported between ankle and knee chondrocytes in end-stage OA. These findings emphasize the importance of investigating synovial tissue to better understand the microenvironment of the ankle joint and pathogenesis of all etiologies of OA. The ultimate goal is the development of clinical tests and therapeutics aimed at slowing or discontinuing the degenerative process in ankle arthritis.
Category:
Ankle Arthritis; Arthroscopy; Basic Sciences/Biologics; Trauma
Introduction/Purpose:
Post-traumatic Osteoarthritis (OA) of the ankle is a debilitating condition that impacts millions of ...people. However, little is known about the genetic and epigenetic risk factors for progression to OA following injury. In order to better understand these factors, a thorough knowledge of the molecular signature in ankle OA relative to non-arthritic ankles is critical. The objective of this study was to perform RNA-sequencing of ankle joint synovial samples from OA and non-OA ankles to better characterize the synovial transcriptome. We compared synovial gene expression patterns in patients with end-stage OA with ankle synovium from non-OA ankles to investigate the unique biological pathways altered in ankle OA.
Methods:
Patients undergoing total ankle arthroplasty or ankle arthrodesis for end stage OA and patients undergoing ankle arthroscopic surgery for non-arthritic conditions (negative control), were consented for participation. Patients with immune disorders or inflammatory arthropathies were excluded. Patient demographics and medical history were collected. Synovial samples were harvested during surgery and RNA was isolated. Bulk 150 bp, paired-end RNA seq was performed using the Illumina NovaSeq6000 platform. Differential expression was determined using the edgeR (v3.16.5) package for R, after adjustment using a scaling normalization factor and P-values were corrected using the Benjamini Hochberg method. Similarities between samples were determined using Spearman Correlation and degree of similarity was visualized considering the first three Principal Components. Gene Ontology terms were identified to classify gene properties.
Results:
A total of 8 synovial samples from total ankle OA and 24 synovial samples from ankle non-OA were available for analysis. We found 808 genes uniquely expressed in OA synovium and another 786 different genes expressed in synovium from the control group. GO enrichment term analysis revealed an upregulation in endomembrane system organization, ubiquitin system/proteasomal degradation, and membrane transport processes including cytoskeletal, endosomal, and vacuolar in ankle OA samples compared to controls (Figure 1). These signatures differ from those previously identified in knee OA. Reactome analysis revealed pathways to those previously identified in OA of other joints, including TGF-beta signaling modulation. However, several signatures unique to ankle OA included extracellular matrix organization, integrin signaling, and AKT signaling modulation, among others. In addition, unlike in knee OA, reactome analyses of ankle OA synovium did not present with an inflammatory component associated with M1 macrophage infiltration.
Conclusion:
Ankle synovium in patients with end-stage ankle OA exhibit a transcriptomic signature distinct from ankle synovium collected from non-OA patients. More intriguing, we find that the ankle OA synovial transcriptome signature is distinct from previously published datasets studying synovium from patients with OA in other joints, particularly the knee. GO term and Reactome analyses identified signatures in bulk membrane transport, proteasome, matrix organization, integrin signaling, and AKT signaling, which are different from phenotypes previously reported in the knee. We conclude that development of effective disease modifying treatments for OA require attention to joint-specific biology and disease progression.
Western diets are becoming increasingly common around the world. Western diets have high omega 6 (ω-6) and omega 3 (ω-3) fatty acids and are linked to bone loss in humans and animals. Dietary fats ...are not created equal; therefore, it is vital to understand the effects of specific dietary fats on bone. We aimed to determine how altering the endogenous ratios of ω-6:ω-3 fatty acids impacts bone accrual, strength, and fracture toughness. To accomplish this, we used the
transgenic mice, which carry a gene responsible for encoding a ω-3 fatty acid desaturase that converts ω-6 to ω-3 fatty acids. Male and female
positive mice (
) and
negative littermates (WT) were given either a high-fat diet (HFD) or low-fat diet (LFD) at 4 wk of age for 16 wk. The
transgene reduced fracture toughness in males. Additionally, male BMD, measured from DXA, decreased over the diet duration for HFD mice. In males, neither HFD feeding nor the presence of the
transgene impacted cortical geometry, trabecular architecture, or whole-bone flexural properties, as detected by main group effects. In females,
-LFD mice experienced increases in BMD compared to WT-LFD mice; however, cortical area, distal femur trabecular thickness, and cortical stiffness were reduced in
mice compared to pooled WT controls. However, reductions in stiffness were caused by a decrease in bone size and were not driven by changes in material properties. Together, these results demonstrate that the endogenous ω-6:ω-3 fatty acid ratio influences bone material properties in a sex-dependent manner. In addition,
mediated fatty acid conversion was not able to mitigate the adverse effects of HFD on bone strength and accrual.
Genetic analyses with mouse congenic strains for distal Chr1 have identified three closely linked QTLs regulating femoral vBMD, mid‐diaphyseal cortical thickness, and trabecular microstructure in a ...sex‐dependent fashion. The homologous relationship between distal mouse Chr 1 and human 1q21–24 offers the possibility of finding common regulatory genes for cortical and trabecular bone.
Introduction: The distal third of mouse chromosome 1 (Chr 1) has been shown to carry a major quantitative trait locus (QTL) for BMD from several inbred mouse strain crosses. Genetic and functional analyses are essential to identify genes and cellular mechanisms for acquisition of peak bone mass.
Materials and Methods: Nested congenic sublines of mice were developed with a C57BL/6J (B6) background carrying <1‐ to 9‐Mbp‐sized segments donated from C3H/HeJ (C3H). Isolated femurs from 16‐wk‐old female and male mice were measured by pQCT and μCT40 for volumetric (v)BMD, mid‐diaphyseal cortical thickness, and distal trabecular phenotypes. Static and dynamic histomorphologic data were obtained on selected females and males at 16 wk.
Results and Conclusions: We found that the original BMD QTL, Bmd5, mapped to distal Chr 1 consists of three QTLs with different effects on vBMD and trabecular bone in both sexes. Compared with B6 controls, femoral vBMD, BMD, and cortical thickness (p < 0.0001) were significantly increased in congenic subline females, but not in males, carrying C3H alleles at QTL‐1. Both females and males carrying C3H alleles at QTL‐1 showed marked increases in BV/TV by μCT compared with B6 mice (p < 0.0001). Females increased BV/TV by increasing trabecular thickness, whereas males increased trabecular number. In addition, the μCT40 data showed two unique QTLs for male trabecular bone, QTL‐2 and QTL‐3, which may interact to regulate trabecular thickness and number. These QTLs are closely linked with and proximal to QTL‐1. The histomorphometric data revealed sex‐specific differences in cellular and bone formation parameters. Mice and humans share genetic homology between distal mouse Chr 1 and human Chr 1q20–24 that is associated with adult human skeletal regulation. Sex‐ and compartment‐specific regulatory QTLs in the mouse suggest the need to partition human data by sex to improve accuracy of mapping and genetic loci identification.
The etiology of skeletal disease is driven by genetic and environmental factors. Genome-wide association studies (GWAS) of osteoporotic phenotypes have identified novel candidate genes, but have only ...uncovered a small proportion of the trait variance explained. This “missing heritability” is caused by several factors, including the failure to consider gene-by-environmental (G*E) interactions. Some G*E interactions have been investigated, but new approaches to integrate environmental data into genomic studies are needed. Advances in genotyping and meta-analysis techniques now allow combining genotype data from multiple studies, but the measurement of key environmental factors in large human cohorts still lags behind, as do the statistical tools needed to incorporate these measures in genome-wide association meta-studies. This review focuses on discussing ways to enhance G*E interaction studies in humans and how the use of rodent models can inform genetic studies. Understanding G*E interactions will provide opportunities to effectively target intervention strategies for individualized therapy.
Abstract Hepatic osteodystrophy (HOD) denotes the alterations in bone morphology and metabolism frequently observed in patients with chronic liver diseases, in particular in case of cholestatic ...conditions. The molecular mechanisms underlying HOD are only partially understood. In the present study, we characterized the bone phenotypes of the ATP-binding cassette transporter B4 knockout mouse ( Abcb4 − / − ), a well-established mouse model of chronic cholestatic liver disease, with the aim of identifying and characterizing a mouse model for HOD. Furthermore, we investigated the influence of vitamin D on bone quality in this model. The bone morphology analyses revealed reduced bone mineral contents as well as changes in trabecular bone architecture and decreased cortical bone densities in Abcb4 − / − mice with severe liver fibrosis. We observed dysregulation of genes involved in bone remodeling (osteoprotegerin, osteocalcin, osteopontin) and vitamin D metabolism (7-dehydrocholesterol reductase, Gc-globulin, Cyp2r1 , Cyp27a1 ) as well as alterations in calcium and vitamin D homeostasis. In addition, serum RANKL and TGF-β levels were increased in Abcb4 − / − mice. Vitamin D dietary intervention did not restore the bone phenotypes of Abcb4 − / − animals. We conclude that the Abcb4 − / − mouse provides an experimental framework and a preclinical model to gain further insights into the molecular pathobiology of HOD and to study the systemic effects of therapeutic interventions.
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