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Summary Objective In this review, we have highlighted the advances over the past year in genetics, genomics and epigenetics in the field of osteoarthritis (OA). Methods A literature search of PubMed was performed using the criteria: "osteoarthritis" and one of the following terms "genetic(s), genomic(s), epigenetic(s), polymorphism, noncoding RNA, microRNA, long noncoding RNA, lncRNA, circular RNA, RNA sequencing (RNA-seq), single cell sequencing, transcriptomics, or DNA methylation between April 01, 2020 and April 30, 2021. Results In total we identified 765 unique publications, which eventually reduced to 380 of relevance to the field as judged by two assessors. Many of these studies included multiple search terms. We summarised advances relating to genetics, functional genetics, genomics and epigenetics, focusing on our personal key papers during the year. Conclusions This year few studies have identified new genetic variants contributing to OA susceptibility, but a focus has been on refining risk loci or their functional validation. The use of new technologies together with investigating the cross-talk between multiple tissue types, greater sample sizes and/or better patient classification (OA subtypes) will continue to increase our knowledge of disease mechanisms and progress towards understanding and treating OA.

Background Autologous fat grafting is often a crucial aspect of reconstructive and aesthetic surgeries, yet poor graft retention is a major issue with this technique. Enriching fat grafts with adipose tissue-derived mesenchymal stem cells (AD-MSCs) improves graft survival—however, AD-MSCs represent a heterogeneous population. Selection of subpopulations of AD-MSCs would allow the targeting of specific AD-MSCs that may benefit fat graft survival more than the general AD-MSC population. Methods Human AD-MSCs were selected for the surface marker CD271 using magnetic-activated cell sorting and compared to the CD271 negative phenotype. These subpopulations were analysed for gene expression using Real-Time qPCR and RNA sequencing; surface marker characteristics using immunostaining; ability to form tubules when cultured with endothelial cells; and gene and protein expression of key angiogenic mediators when cultured with ex-vivo adipose tissue. Results Human AD-MSCs with the surface marker CD271 express angiogenic genes at higher levels, and inflammatory genes at lower levels, than the CD271− AD-MSC population. A greater proportion of CD271+ AD-MSCs also possess the typical complement of stem cell surface markers and are more likely to promote effective neoangiogenesis, compared to CD271− AD-MSCs. Conclusion Enriching grafts with the CD271+ AD-MSC subpopulation holds potential for the improvement of reconstructive and aesthetic surgeries involving adipose tissue.

ObjectivesTo collate the genes experimentally modulated in animal models of osteoarthritis (OA) and compare these data with OA transcriptomics data to identify potential therapeutic targets.MethodsPubMed searches were conducted to identify publications describing gene modulations in animal models. Analysed gene expression data were retrieved from the SkeletalVis database of analysed skeletal microarray and RNA-Seq expression data. A network diffusion approach was used to predict new genes associated with OA joint damage.ResultsA total of 459 genes were identified as having been modulated in animal models of OA, with ageing and post-traumatic (surgical) models the most prominent. Ninety-eight of the 143 genes (69%) genetically modulated more than once had a consistent effect on OA joint damage severity. Several discrepancies between different studies were identified, providing lessons on interpretation of these data. We used the data collected along with OA gene expression data to expand existing annotations and prioritise the most promising therapeutic targets, which we validated using the latest reported associations. We constructed an online database OATargets to allow researchers to explore the collated data and integrate it with existing OA and skeletal gene expression data.ConclusionsWe present a comprehensive survey and online resource for understanding gene regulation of animal model OA pathogenesis.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3′UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.

Objective To identify OA subtypes based on cartilage transcriptomic data in cartilage tissue and characterize their underlying pathophysiological processes and/or clinically relevant characteristics. Methods This study includes n = 66 primary OA patients (41 knees and 25 hips), who underwent a joint replacement surgery, from which macroscopically unaffected (preserved, n = 56) and lesioned (n = 45) OA articular cartilage were collected [Research Arthritis and Articular Cartilage (RAAK) study]. Unsupervised hierarchical clustering analysis on preserved cartilage transcriptome followed by clinical data integration was performed. Protein–protein interaction (PPI) followed by pathway enrichment analysis were done for genes significant differentially expressed between subgroups with interactions in the PPI network. Results Analysis of preserved samples (n = 56) resulted in two OA subtypes with n = 41 (cluster A) and n = 15 (cluster B) patients. The transcriptomic profile of cluster B cartilage, relative to cluster A (DE-AB genes) showed among others a pronounced upregulation of multiple genes involved in chemokine pathways. Nevertheless, upon investigating the OA pathophysiology in cluster B patients as reflected by differentially expressed genes between preserved and lesioned OA cartilage (DE-OA-B genes), the chemokine genes were significantly downregulated with OA pathophysiology. Upon integrating radiographic OA data, we showed that the OA phenotype among cluster B patients, relative to cluster A, may be characterized by higher joint space narrowing (JSN) scores and low osteophyte (OP) scores. Conclusion Based on whole-transcriptome profiling, we identified two robust OA subtypes characterized by unique OA, pathophysiological processes in cartilage as well as a clinical phenotype. We advocate that further characterization, confirmation and clinical data integration is a prerequisite to allow for development of treatments towards personalized care with concurrently more effective treatment response.

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptg knockout (Gnptgko) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgko chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptab knock-in (Gnptabki) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint. This article has an associated First Person interview with the first author of the paper., Summary: Analyses of Gnptg knockout mice indicate that functional joint abnormalities observed in mucolipidosis type III gamma patients are caused by imbalanced metabolism, leading to structural extracellular matrix changes in cartilage and tendons.

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology, however the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5’ and 3’ end, with >99% having one of two seed sequences (5 ‘ bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3 ‘UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published datasets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

Breast cancer is a major disease posing many therapeutic and societal challenges. The major problem remains the diversity of the disease. Breast cancers are divided in several distinct subtypes, but the specific mechanisms involved in each subtype are not understood. We apply advanced network medicine techniques to the analysis of transcriptomics data of several breast cancer cell lines. We show that different subtypes are characterized by highly heterogeneous pathway activity. Using the overlap between differentially expressed genes and drug-induced expression changes, we present candidate drugs for repurposing. Furthermore, we find different active subnetworks across the subtypes, regulated by distinct transcription factors. All these results are made available via a user-friendly portal at http://phenome.manchester.ac.uk/breastcancer/

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific ‘disease signature’ of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation., Author summary A significant proportion of genetic skeletal diseases result from misfolding of structural proteins within the endoplasmic reticulum (ER) of cartilage cells (chondrocytes). Interestingly, these diseases often share transcriptomic changes with non-skeletal conditions such as Alzheimer’s disease and diabetes and include changes in the unfolded protein response (UPR). UPR signals are conveyed through three effector molecules, IRE1, ATF6 and PERK, understanding of which is of utmost importance for the development of novel therapeutic approaches. IRE1 is the most conserved UPR pathway, with XBP1 as its main effector. Interestingly, in our study we show that chondrocyte response to ER stress is highly modulated by the differentiation state of the cell. We have deleted the IRE1/XBP1 pathway activity in the chondrocytes of a mouse model of multiple epiphyseal dysplasia (MED), expressing misfolding matrilin-3 predominantly in proliferating chondrocytes, and in metaphyseal chondrodysplasia type Schmid (MCDS), resulting from mutations in type X collagen, expressed by the hypertrophic chondrocytes. Here, using deep phenotyping and extensive transcriptomic analysis we demonstrate that whilst the IRE1/XBP1 pathway is redundant in hypertrophic chondrocyte pathology, it is in fact essential for UPR responses in proliferating cells, making this highly conserved pathway an attractive therapeutic target for a broad range of UPR related conditions.

Motivation Skeletal diseases are prevalent in society, but improved molecular understanding is required to formulate new therapeutic strategies. Large and increasing quantities of available skeletal transcriptomics experiments give the potential for mechanistic insight of both fundamental skeletal biology and skeletal disease. However, no current repository provides access to processed, readily interpretable analysis of this data. To address this, we have developed SkeletalVis, an exploration portal for skeletal gene expression experiments. Results The SkeletalVis data portal provides an exploration and comparison platform for analysed skeletal transcriptomics data. It currently hosts 287 analysed experiments with 739 perturbation responses with comprehensive downstream analysis. We demonstrate its utility in identifying both known and novel relationships between skeletal expression signatures. SkeletalVis provides users with a platform to explore the wealth of available expression data, develop consensus signatures and the ability to compare gene signatures from new experiments to the analysed data to facilitate meta-analysis. Availability and implementation The SkeletalVis data portal is freely accessible at http://phenome.manchester.ac.uk. Supplementary information Supplementary data are available at Bioinformatics online.

Osteoarthritis (OA) is a heterogeneous and complex disease. We have used a network biology approach based on genome-wide analysis of gene expression in OA knee cartilage to seek evidence for pathogenic mechanisms that may distinguish different patient subgroups.Results from RNA-Sequencing (RNA-Seq) were collected from intact knee cartilage at total knee replacement from 44 patients with OA, from 16 additional patients with OA and 10 control patients with non-OA. Results were analysed to identify patient subsets and compare major active pathways.The RNA-Seq results showed 2692 differentially expressed genes between OA and non-OA. Analysis by unsupervised clustering identified two distinct OA groups: Group A with 24 patients (55%) and Group B with 18 patients (41%). A 10 gene subgroup classifier was validated by RT-qPCR in 16 further patients with OA. Pathway analysis showed increased protein expression in both groups. PhenomeExpress analysis revealed group differences in complement activation, innate immune responses and altered Wnt and TGFβ signalling, but no activation of inflammatory cytokine expression. Both groups showed suppressed circadian regulators and whereas matrix changes in Group A were chondrogenic, in Group B they were non-chondrogenic with changes in mechanoreceptors, calcium signalling, ion channels and in cytoskeletal organisers. The gene expression changes predicted 478 potential biomarkers for detection in synovial fluid to distinguish patients from the two groups.Two subgroups of knee OA were identified by network analysis of RNA-Seq data with evidence for the presence of two major pathogenic pathways. This has potential importance as a new basis for the stratification of patients with OA for drug trials and for the development of new targeted treatments.

Summary: PhenomeScape is a Cytoscape app which provides easy access to the PhenomeExpress algorithm to interpret gene expression data. PhenomeExpress integrates protein interaction networks with known phenotype to gene associations to find active sub-networks enriched in differentially expressed genes. It also incorporates cross-species phenotypes and associations to include results from animal models of disease. With expression data imported into PhenomeScape, the user can quickly generate and visualise interactive sub-networks. PhenomeScape thus enables researchers to use prior knowledge of a disease to identify differentially regulated sub-networks and to generate an overview of altered biologically processes specific to that disease. Availability and Implementation: Freely available for download at https://github.com/soulj/PhenomeScape Contact: jamie.soul@postgrad.manchester.ac.uk or jean-marc.schwartz@manchester.ac.uk

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.

Introduction Osteoarthritis (OA) is a heterogeneous and complex disease. We have used a network biology approach based on genome-wide analysis of gene expression in OA knee cartilage to seek evidence for pathogenic mechanisms that may distinguish different patient subgroups. Methods Results from RNA-Sequencing (RNA-Seq) were collected from intact knee cartilage at total knee replacement from 44 patients with OA, from 16 additional patients with OA and 10 control patients with non-OA. Results were analysed to identify patient subsets and compare major active pathways. Results The RNA-Seq results showed 2692 differentially expressed genes between OA and non-OA. Analysis by unsupervised clustering identified two distinct OA groups: Group A with 24 patients (55%) and Group B with 18 patients (41%). A 10 gene subgroup classifier was validated by RT-qPCR in 16 further patients with OA. Pathway analysis showed increased protein expression in both groups. PhenomeExpress analysis revealed group differences in complement activation, innate immune responses and altered Wnt and TGFβ signalling, but no activation of inflammatory cytokine expression. Both groups showed suppressed circadian regulators and whereas matrix changes in Group A were chondrogenic, in Group B they were non-chondrogenic with changes in mechanoreceptors, calcium signalling, ion channels and in cytoskeletal organisers. The gene expression changes predicted 478 potential biomarkers for detection in synovial fluid to distinguish patients from the two groups. Conclusions Two subgroups of knee OA were identified by network analysis of RNA-Seq data with evidence for the presence of two major pathogenic pathways. This has potential importance as a new basis for the stratification of patients with OA for drug trials and for the development of new targeted treatments.