Osteoarthritis year in review: genetics, genomics, epigenetics

  • A. Ratneswaran
    Affiliations
    Schroeder Arthritis Institute, University Health Network, Toronto, ON, Canada

    Krembil Research Institute, University Health Network, Toronto, ON, Canada
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  • M. Kapoor
    Correspondence
    Address correspondence and reprint requests to: M. Kapoor, Schroeder Arthritis Institute, Krembil Research Institute, University Health Network, 5KD-413, 60 Leonard Avenue, Toronto, ON, M5T 0S8, Canada. Tel.: 1-416-603-5800 ext. 4796.
    Affiliations
    Schroeder Arthritis Institute, University Health Network, Toronto, ON, Canada

    Krembil Research Institute, University Health Network, Toronto, ON, Canada

    Department of Surgery, Faculty of Medicine, University of Toronto, ON, Canada

    Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
    Search for articles by this author
Open AccessPublished:November 20, 2020DOI:https://doi.org/10.1016/j.joca.2020.11.003

      Summary

      Objective

      In this review, we have highlighted advances in genetics, genomics and epigenetics in the field of osteoarthritis (OA) over the past year.

      Methods

      A literature search was performed using PubMed and the criteria: “osteoarthritis” and one of the following terms “genetic(s), genomic(s), epigenetic(s), epigenomic(s), noncoding RNA, microRNA, long noncoding RNA, lncRNA, circular RNA, RNA sequencing, single cell sequencing, or DNA methylation between April 1, 2019 and April 30, 2020.

      Results

      We identified 653 unique publications, many studies spanned multiple search terms. We summarized advances relating to evolutionary genetics, pain, ethnicity specific risk factors, functional studies of gene variants, and interactions between coding and non-coding RNAs in OA pathogenesis.

      Conclusions

      Studies have identified variants contributing to OA susceptibility, candidate biomarkers for diagnosis and prognosis, as well as promising therapeutic candidates. Validation in multiple cohorts, multi-omics strategies, and machine learning aided computational analyses have all contributed to the strength of published literature. Open access data-sets, greater sample sizes to capture broader populations and understanding disease mechanisms by investigating the interactions between multiple tissue types will further aid in progress towards understanding and curing OA.

      Keywords

      Introduction

      Recent years provided the field of Osteoarthritis (OA) with some of the most ground-breaking research advances to date including the identification of several novel risk loci, functional evaluation of OA loci, profiling of microRNA (miRNA), long-non coding RNA (lncRNA) and circular RNA (circRNA) expressed in OA cartilage, as well as the first published study using single-cell RNA sequencing (scRNA-seq) to characterize human osteoarthritic cartilage (as reviewed
      • Reynard L.N.
      • Barter M.J.
      Osteoarthritis year in review 2019: genetics, genomics and epigenetics.
      ). These advances, combined with increased accessibility to OA tissue, high-throughput platforms, and online databases paved the way for studies published this year.
      The goal of this narrative review is to highlight key research studies published in OA genetics, genomics and epigenetics between April 2019 and April 2020, which comprised of 653 original research articles. Among these unique publications, themes of evolutionary genetics, ethnicity specific risk factors, genetics of pain, and the interaction of non-coding RNAs with respect to disease progression are further discussed. Emerging areas of research in genetics, genomics and epigenetics published within this timeframe are also briefly discussed. An overview of the total number of publications for OA genetics, genomics and epigenetic search terms are listed in Table I, of which the full list can be found in supplementary Table 1. The articles referenced in this review are those which the authors determined to be novel and impactful to significant advancements in the field, and fit within the themes mentioned in this review.
      Table ISearch terms and publications
      Search criteriaTotal number of articlesExcluded (Books and documents; Meta-Analysis; Review; Systematic Review)Remaining
      Osteoarthritis + genetic(s)52562463
      Osteoarthritis + genomic(s)15116135
      Osteoarthritis + epigenetic(s)491633
      Osteoarthritis + epigenomic(s)945
      Osteoarthritis + noncoding RNA17914165
      Osteoarthritis + microRNA18117164
      Osteoarthritis + circular RNA15114
      Osteoarthritis + long noncoding RNA81972
      Osteoarthritis + lncRNA961185
      Osteoarthritis + RNA sequencing49247
      Osteoarthritis + single cell sequencing13211
      Osteoarthritis + DNA methylation33825
      Total1,3811621,219
      Total (no duplicates)653
      Number of publications as indexed by PubMed search criteria within the dates April 1 2019 to April 30 2020. Some studies have overlapping search criteria and are included in more than one category. Total number, no duplicates indicates the number of unique studies overall.

       Evolution and OA heredity

      OA is a complex multifactorial disease with a strong hereditary component. To investigate whether adaptive processes from the biomechanical demands of bipedalism during human knee evolution contribute to OA risk, Richard and colleagues eloquently examined how altered chondrocyte developmental programs could lead to the development of OA
      • Richard D.
      • Liu Z.
      • Cao J.
      • Kiapour A.M.
      • Willen J.
      • Yarlagadda S.
      • et al.
      Evolutionary selection and constraint on human knee chondrocyte regulation impacts osteoarthritis risk.
      . As the development of knee features is linked to chondrocyte regulation which acts as a template for joint shape, the authors first epigenomically profiled human and mouse joint chondrocytes. After discovering knee-specific regulatory elements conserved across primates, they reported that within human knee evolution signatures there was a tendency away from modification of genetic factors that influenced knee morphology, yet towards those that would lead to OA or skeletal defects. This data led to the creation of their model proposing evolutionary mechanisms such as genetic drift and antagonistic pleiotropy (where positive selection increases a beneficial allele together with linked deleterious alleles) leads to violations to constraint of functional conserved sequences which are tolerated during knee development but can increase risk of OA development. The authors showed that individuals with OA, or at high risk for developing OA, had a significantly greater number of alternative alleles within constrained knee regulatory elements. Subsequently, they functionally evaluated GDF5 (Growth Differentiation Factor 5, a BMP family member essential to knee development across mammals but also implicated in OA risk), and identified a variant rs6060369 (overlapping with the regulatory element R4) as causative. R4 null mice formed normal joints, but by 1 year of age had morphological defects and developed OA. These effects were likely mediated through Pituitary Homeobox 1 (PITX1). This all-encompassing study showed in a series of logical experiments how the evolution in human knee anatomy resulted in the conservation of a variant that impacts morphology and OA development
      • Richard D.
      • Liu Z.
      • Cao J.
      • Kiapour A.M.
      • Willen J.
      • Yarlagadda S.
      • et al.
      Evolutionary selection and constraint on human knee chondrocyte regulation impacts osteoarthritis risk.
      .
      Further functional data demonstrated upregulation of GDF5 expression in OA cartilage, during articular cartilage repair and as a response to injured synovium in mice, which could partially explain its association with OA risk
      • Kania K.
      • Colella F.
      • Riemen A.H.K.
      • Wang H.
      • Howard K.A.
      • Aigner T.
      • et al.
      Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.
      .
      Evolutionary conservation was also implicated in an epigenetic study from Hsueh et al., one of the first studies to comprehensively profile regional patterns of protein turnover and miRNA expression in human adult lower limb cartilage during health and disease
      • Hsueh M.F.
      • Onnerfjord P.
      • Bolognesi M.P.
      • Easley M.E.
      • Kraus V.B.
      Analysis of "old" proteins unmasks dynamic gradient of cartilage turnover in human limbs.
      . This study evaluated whether human cartilage shared miRNA regulatory circuitry of the blastema, a mass of cells capable of differentiation and growth into organs or appendages, essential to limb regeneration in certain animal species. Ankle cartilage as well as surface zone cartilage, had lower ratios of deamidated proteins indicating higher tissue anabolism and greater repair capacity. Blastema miRNAS (miR-121, miR-31 and miR-181c) were enriched in ankle joint, superficial zone and OA cartilage, indicating a position dependent gradient (distal high, proximal low) of protein turnover that may have limited regenerative capacity. Therefore, strategies incorporating miRNAs to enhance cartilage anabolism and endogenous repair may be promising.
      While the above mentioned studies largely examine how evolutionarily conserved variants and circuitry may impact OA risk and development, Magnusson et al. used the world's largest twin registry to estimate sex-specific genetic contribution to knee OA surgery
      • Magnusson K.
      • Turkiewicz A.
      • Englund M.
      Nature vs nurture in knee osteoarthritis - the importance of age, sex and body mass index.
      . They found that the overall heritability of knee OA surgery was 53% but this was higher in women than men. Men had relatively low genetic contribution that varied with BMI and age. Similarly, the effects of maternal and paternal factors on hand, hip and knee OA in offspring were studied in the MUST (Musculoskeletal Pain in Ullensaker Study) and Nor-Twin (Norwegian OA Twin Study) cohorts
      • Weldingh E.
      • Johnsen M.B.
      • Hagen K.B.
      • Osteras N.
      • Risberg M.A.
      • Natvig B.
      • et al.
      The maternal and paternal effects on clinically and surgically defined osteoarthritis.
      . It was discovered maternal OA increased the risk of any OA in daughters, and sons (albeit to a lesser degree) yet having a father with OA was less likely to increase the risk of any OA in daughters or sons. These findings strongly implicate maternal genes or factors in the heredity of OA.

       OA genetics: GWAS and pain

      This year, two large genome-wide association studies (GWAS) were published. The first, a GWAS of bone size derived from dual-energy X-ray absorptiometry (DXA) scans, identified eight bone area variants associated with hip or knee OA
      • Weldingh E.
      • Johnsen M.B.
      • Hagen K.B.
      • Osteras N.
      • Risberg M.A.
      • Natvig B.
      • et al.
      The maternal and paternal effects on clinically and surgically defined osteoarthritis.
      , highlighted last year
      • Reynard L.N.
      • Barter M.J.
      Osteoarthritis year in review 2019: genetics, genomics and epigenetics.
      . The second GWAS identified genetic variants associated with knee pain in 171,516 individuals from the UK Biobank cohort and verified by three independent cohorts
      • Meng W.
      • Adams M.J.
      • Palmer C.N.A.
      • andMe Research T.
      • Shi J.
      • Auton A.
      • et al.
      Genome-wide association study of knee pain identifies associations with GDF5 and COL27A1 in UK Biobank.
      . Two loci for knee pain were identified, rs143384 located in GDF5 as well as rs2808772 located near COL27A1. GDF5 is important for joint development, and variants in GDF5 contribute to increased OA risk, as emphasized above. While there are no specific studies regarding rs2808772, the adjacent COL27A1 codes for a member of the fibrillar collagen family and mutations in this gene are associated with Steel syndrome, an osteochondraldysplasia.
      This GWAS unbiasedly identified variants associated with knee pain, though the relationship between pain and OA phenotype is complex. A recent targeted study, tested 75 patients with medial compartment knee OA for pain (subjective, mechanical, heat, pressure) and identified EDNRA (Endothelin Receptor Type A), COMT (Catechol O-Methyltransferase), BDRKB1 (Bradykinin Receptor B1) and IL1B (Interleukin-1 Beta) as involved with aspects of pain
      • Schutte D.L.
      • Mukhopadhyay N.
      • Holwerda T.
      • Sluka K.
      • Rakel B.
      • Govil M.
      Genetic predictors of knee pain in persons with mild to moderate osteoarthritis.
      . Genetic variation in pain phenotype may also play a role in response to intervention
      • Govil M.
      • Mukhopadhyay N.
      • Holwerda T.
      • Sluka K.
      • Rakel B.
      • Schutte D.L.
      Effects of genotype on TENS effectiveness in controlling knee pain in persons with mild to moderate osteoarthritis.
      . Pain phenotypes such as “post-surgical pain”, “early stage OA pain” and “persistent OA” pain were supported by transcriptomic differences identifying distinct molecular pathways in the dorsal root ganglia of a mouse model of surgically induced OA
      • Miller R.E.
      • Tran P.B.
      • Ishihara S.
      • Syx D.
      • Ren D.
      • Miller R.J.
      • et al.
      Microarray analyses of the dorsal root ganglia support a role for innate neuro-immune pathways in persistent pain in experimental osteoarthritis.
      . Pathway analyses in the persistent pain phenotype suggested the innate immune system and neuroinflammation are key pathways involved in pain pathology, and genes such as CX3CL1 (Fractalkine), CCL2 (C–C Motif Chemokine Ligand 2), TLR1 (Toll-like Receptor 1), and NGF (Nerve Growth Factor) are upregulated in this phenotype. Sakurai and colleagues, also suggested an “advanced OA pain phenotype” in a rat OA model, and implicated the involvement of cyclooxygenase resistant synovial macrophages
      • Sakurai Y.
      • Fujita M.
      • Kawasaki S.
      • Sanaki T.
      • Yoshioka T.
      • Higashino K.
      • et al.
      Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors.
      . This theme is also consistent with in vitro studies of co-culture, where activation of fibroblast-like OA synoviocytes with inflammatory stimuli resulted in knee neuron sensitization
      • Chakrabarti S.
      • Hore Z.
      • Pattison L.A.
      • Lalnunhlimi S.
      • Bhebhe C.N.
      • Callejo G.
      • et al.
      Sensitization of knee-innervating sensory neurons by tumor necrosis factor-alpha-activated fibroblast-like synoviocytes: an in vitro, coculture model of inflammatory pain.
      .

       OA genetics: ethnicity based risk variants

      Recently, a number of studies identified variants within specific ethnicities, which are summarized in Table II. Many of these studies investigate polymorphisms first reported in other populations. They offer a unique opportunity to establish risk loci in sub-populations.
      Table IICharacterization of OA risk variants by ethnic populations
      Sample SizePopulationVariantsOA RiskReference
      280 KOA, 308 CxPakistanirs1862513, RETN rs3745367, RETNIncreasedNaqvi et al., 2019
      • Klein J.C.
      • Keith A.
      • Rice S.J.
      • Shepherd C.
      • Agarwal V.
      • Loughlin J.
      • et al.
      Functional testing of thousands of osteoarthritis-associated variants for regulatory activity.
      200 KOA, 200 CxIndian, Hyderabadrs2228314, SREBP2IncreasedPoornima et al., 2019
      • Lehtovirta S.
      • Makitie R.E.
      • Casula V.
      • Haapea M.
      • Niinimaki J.
      • Niinimaki T.
      • et al.
      Defective WNT signaling may protect from articular cartilage deterioration - a quantitative MRI study on subjects with a heterozygous WNT1 mutation.
      279 KOA, 287 ControlsIndian, Punjabrs1800795, IL6

      rs10499563, IL6
      IncreasedSingh et al., 2020
      • den Hollander W.
      • Boer C.G.
      • Hart D.J.
      • Yau M.S.
      • Ramos Y.F.M.
      • Metrustry S.
      • et al.
      Genome-wide association and functional studies identify a role for matrix Gla protein in osteoarthritis of the hand.
      haplotype GGGGCT

      haplotype CGAGGC
      Increased

      Decreased
      730 HOA, 1,220 CxChinese, Hanrs187064, TLR9IncreasedYi et al., 2019
      • Shepherd C.
      • Reese A.E.
      • Reynard L.N.
      • Loughlin J.
      Expression analysis of the osteoarthritis genetic susceptibility mapping to the matrix Gla protein gene MGP.
      348 KOA, 423 CxChinese, Hanrs11564299, CDH2Increased risk, especially for females, smokers, drinkers, BMI25 Also assoc. w KL grade and CRPShang et al., 2019
      • Hindy G.
      • Akesson K.E.
      • Melander O.
      • Aragam K.G.
      • Haas M.E.
      • Nilsson P.M.
      • et al.
      Cardiometabolic polygenic risk scores and osteoarthritis outcomes: a mendelian randomization study using data from the malmo Diet and cancer study and the UK Biobank.
      237 KOA, 142 CxChinese, Hanrs1065080, SMAD3Increased risk (GG), decreased risk recessiveLu et al., 2019
      • Funck-Brentano T.
      • Nethander M.
      • Moverare-Skrtic S.
      • Richette P.
      • Ohlsson C.
      Causal factors for knee, hip, and hand osteoarthritis: a mendelian randomization study in the UK Biobank.
      866 KOA, 1,688 CxChinese, Hanrs3884606, FGF18Increased riskZhao et al., 2020
      • Zhou A.
      • Morris H.A.
      • Hypponen E.
      Health effects associated with serum calcium concentrations: evidence from MR-PheWAS analysis in UK Biobank.
      471 KOA, 532 CxChinese, Multi- siters2908004, WNT16Decreased risk females, smokers, non-drinkers, under 60 years, BMI>25Huang et al., 2019
      • Attur M.
      • Zhou H.
      • Samuels J.
      • Krasnokutsky S.
      • Yau M.
      • Scher J.U.
      • et al.
      Interleukin 1 receptor antagonist (IL1RN) gene variants predict radiographic severity of knee osteoarthritis and risk of incident disease.
      rs1799986, LRP1Decreased risk males, smokers, drinkers, under age 60, BMI>25
      393 KOA, 500 CxChinese, Hanrs1485286, rs1905786, and rs1032128, OPGIncreased riskQi et al., 2019
      • Zhao L.
      • Huang J.
      • Fan Y.
      • Li J.
      • You T.
      • He S.
      • et al.
      Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis.
      278 OA, 289 CxChinese, Hanrs2498789, AKT1 rs7646409, PIK3CAIncreased riskWang et al., 2020
      • Fu L.
      • Hu Y.
      • Song M.
      • Liu Z.
      • Zhang W.
      • Yu F.X.
      • et al.
      Up-regulation of FOXD1 by YAP alleviates senescence and osteoarthritis.
      1033 KOA, 920 CxChinese, Hanrs2234693, ESRaIncreased Risk, mild but not severe OADai et al., 2020
      • Ji Q.
      • Zheng Y.
      • Zhang G.
      • Hu Y.
      • Fan X.
      • Hou Y.
      • et al.
      Single-cell RNA-seq analysis reveals the progression of human osteoarthritis.
      423 KOA, 523 CxChinese, Hanrs4965833, PACE4Increased risk, over 55 years, BMI25He et al., 2020
      • Dicks A.
      • Wu C.L.
      • Steward N.
      • Adkar S.S.
      • Gersbach C.A.
      • Guilak F.
      Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.
      532 KOA, 927 CxChinese, Hanrs4867568, LSP1P3 rs143383, GDF5Increased riskLi et al., 2020
      • Zhang F.
      • Wei K.
      • Slowikowski K.
      • Fonseka C.Y.
      • Rao D.A.
      • Kelly S.
      • et al.
      Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.
      260 HOA, 240 KOA, 597 CxCroatianrs2275913 IL17A rs763780, rs1889570 IL17FEftedal et al., 2019
      • Kuang L.
      • Wu J.
      • Su N.
      • Qi H.
      • Chen H.
      • Zhou S.
      • et al.
      FGFR3 deficiency enhances CXCL12-dependent chemotaxis of macrophages via upregulating CXCR7 and aggravates joint destruction in mice.
      GCA haplotypeIncreased risk HOA
      ATG haplotypeIncreased risk KOA
      127 KOA, 127 CxIranianrs2275913, IL17ADecreased RiskBafrani et al., 2019
      • Bekki H.
      • Duffy T.
      • Okubo N.
      • Olmer M.
      • Alvarez-Garcia O.
      • Lamia K.
      • et al.
      Suppression of circadian clock protein cryptochrome 2 promotes osteoarthritis.
      rs763780, IL17FIncreased Risk
      Studies from April 1 2019 to April 30 2020 examining genetic variants for OA risk stratified by population are summarized above. ∗HOA = hip OA, KOA= Knee OA, Cx = Control, KL= Kellgren–Lawrence Grade CRP= C-Reactive Protein.

       OA genetics: functional evaluation of genetic risk

      Functional testing of risk variants can indicate causality, and provides insight as to how a polymorphism may impact disease pathology. Earlier this year, Klein et al. functionally tested 1,605 single nucleotide variants associated with OA
      • Klein J.C.
      • Keith A.
      • Rice S.J.
      • Shepherd C.
      • Agarwal V.
      • Loughlin J.
      • et al.
      Functional testing of thousands of osteoarthritis-associated variants for regulatory activity.
      . The most significant variant, rs473022, falls within an alternative transcriptional start site of HBP1. This variant demonstrates differential nuclear protein binding, and increased expression of an alternate isoform in multiple cell lines. As HBP1 is a transcriptional repressor of WNT pathway signalling, the authors propose by disrupting this pathway, the variant could predispose individuals to OA. WNT signalling was also explored in a functional study by Lehtovirta et al. characterizing articular cartilage composition in two Finnish families with autosomal dominant WNT1 osteoporosis. They observed less age-related cartilage degeneration in mutation-positive than mutation-negative individuals suggesting the mutation may have a cartilage protective effect
      • Lehtovirta S.
      • Makitie R.E.
      • Casula V.
      • Haapea M.
      • Niinimaki J.
      • Niinimaki T.
      • et al.
      Defective WNT signaling may protect from articular cartilage deterioration - a quantitative MRI study on subjects with a heterozygous WNT1 mutation.
      .
      In 2017, den Hollander and colleagues showed robust genome-wide significance between a DNA variant near the Matrix Gla Protein (MGP) gene and radiographic hand OA. They found the hand OA risk allele is associated with lower expression of MGP in articular cartilage and suggested the variant causes increased OA burden by decreasing levels of MGP which could lead to increased cartilage calcification
      • den Hollander W.
      • Boer C.G.
      • Hart D.J.
      • Yau M.S.
      • Ramos Y.F.M.
      • Metrustry S.
      • et al.
      Genome-wide association and functional studies identify a role for matrix Gla protein in osteoarthritis of the hand.
      . This year, Shepherd et al. functionally characterized MGP and rs4764133, confirming the risk conferring allele reduces MGP expression in cartilage, synovium and fat pad but this is opposite in peripheral blood. After allelic expression imbalance analysis they conclude that in response to OA, chondrocytes may need to make more MGP but those with low expressing alleles cannot respond adequately. This compromise may mean that in individuals carrying the risk allele, the disease is less likely to resolve
      • Shepherd C.
      • Reese A.E.
      • Reynard L.N.
      • Loughlin J.
      Expression analysis of the osteoarthritis genetic susceptibility mapping to the matrix Gla protein gene MGP.
      .
      Mendelian Randomization (MR) uses genetic variation to investigate causal relationships between risk factors and outcomes. Two studies this year used MR, the first investigated the role of cardiometabolic risk factors in hip and knee OA in individuals from the Malmö Diet and Cancer Study (MDCS), and validated their findings in a cohort from the UK Biobank. They implicated a causal role for lower Low Density Lipoprotein and greater BMI in OA
      • Hindy G.
      • Akesson K.E.
      • Melander O.
      • Aragam K.G.
      • Haas M.E.
      • Nilsson P.M.
      • et al.
      Cardiometabolic polygenic risk scores and osteoarthritis outcomes: a mendelian randomization study using data from the malmo Diet and cancer study and the UK Biobank.
      . The second study evaluated causal factors for hand, hip and knee OA using data from 384,838 individuals from the UK Biobank. MR analyses demonstrated a strong causal association of genetically determined BMI with all OA, knee OA and hip OA but not hand OA. Increased bone mineral density at the femoral neck and low systolic blood pressure were also causally associated with all OA, hip and knee OA
      • Funck-Brentano T.
      • Nethander M.
      • Moverare-Skrtic S.
      • Richette P.
      • Ohlsson C.
      Causal factors for knee, hip, and hand osteoarthritis: a mendelian randomization study in the UK Biobank.
      . MR has also been used to investigate phenome wide health effects (MR-PheWAS). Recently OA was evidenced as a downstream health outcome in a UK Biobank study using data from 337,535 individuals based on their genetic calcium score
      • Zhou A.
      • Morris H.A.
      • Hypponen E.
      Health effects associated with serum calcium concentrations: evidence from MR-PheWAS analysis in UK Biobank.
      .
      The relationship between gene variants associated with the Interleukin-1 Receptor Antagonist gene (IL1RN) with age-dependent OA severity was described in a study by Attur and colleagues study demonstrating the IL1RN TTG haplotype is associated with more severe age-dependent radiographic OA. Using the Incidence sub-cohort from the OAI, they also showed that this haplotype could predict the risk of incident knee OA (OR = 4.13 (1.75–9.72); p = 0.001). Plasma levels of IL-1Ra are lower in these patients, and chondrocytes derived from these patients secreted less IL-1Ra than those who don't carry copies of the variants
      • Attur M.
      • Zhou H.
      • Samuels J.
      • Krasnokutsky S.
      • Yau M.
      • Scher J.U.
      • et al.
      Interleukin 1 receptor antagonist (IL1RN) gene variants predict radiographic severity of knee osteoarthritis and risk of incident disease.
      .
      Functional evaluation of genetic risk has been augmented by the accessibility of efficient tools such as CRISPR/Cas9 technology. Zhao et al. used this technology to assess whether modifying key OA genes had disease mediating effects in mice
      • Zhao L.
      • Huang J.
      • Fan Y.
      • Li J.
      • You T.
      • He S.
      • et al.
      Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis.
      . Multiplex gene-editing of NGF, Il-1B, and MMP-13 relieved OA pain and mitigated structural disease progression. Gene-editing through CRISPR/Cas9 has also been used to investigate age-associated underlying OA. In a study using genetic ablation of Yes-associated protein (YAP) in human mesenchymal stem cells (hMSC), the authors find YAP knockout results in increased cellular senescence through down-regulation of forkhead box D1 (FOXD1). Gene-transfer of YAP or FOXD1 through intraarticular lentiviral injection decreases the development of OA in mice indicating that this pathway could be targeted for primary OA
      • Fu L.
      • Hu Y.
      • Song M.
      • Liu Z.
      • Zhang W.
      • Yu F.X.
      • et al.
      Up-regulation of FOXD1 by YAP alleviates senescence and osteoarthritis.
      .

       OA transcriptomics: examining phenotypes

      Last year, to the best of our knowledge, the first scRNA-seq study on human OA cartilage was published, revealing subpopulations of chondrocytes including novel phenotypes with distinct functions
      • Ji Q.
      • Zheng Y.
      • Zhang G.
      • Hu Y.
      • Fan X.
      • Hou Y.
      • et al.
      Single-cell RNA-seq analysis reveals the progression of human osteoarthritis.
      . This year, advances in single cell technologies were used to profile cell surface markers and disease associated subsets. Recently, Dicks et al. used single-cell sequencing paired with cell-surface marker screening on CRISPR/Cas9 gene-edited COL2A1-GFP reporter human induced pluripotent stem cells to identify surface cell markers that distinguish the chondroprogenitor population in order to develop more efficient and accurate methods for chondrogenic differentiation for in vitro, preclinical and tissue engineering purposes
      • Dicks A.
      • Wu C.L.
      • Steward N.
      • Adkar S.S.
      • Gersbach C.A.
      • Guilak F.
      Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.
      . Canonical correlation analysis (CCA), a machine learning method, was used to integrate data sets. Cells which were CD146+/CD166+/PDGFRβ+/CD45 exhibited high chondrogenic potential with subsequent in vitro tests demonstrating that this subset upregulates SOX9, ACAN, and COL2 expression and produces substantial extracellular matrix. The identification and purification of this cell subset will provide more efficient chondroprogenitors with less heterogeneity for downstream applications addressing a persistent challenge within this field.
      Another innovative study used scRNA-seq, bulk RNA-seq, mass and flow cytometry with CCA to identify the contribution of synovial tissue cell subsets to pathways mediating RA, OA and chronic inflammation
      • Zhang F.
      • Wei K.
      • Slowikowski K.
      • Fonseka C.Y.
      • Rao D.A.
      • Kelly S.
      • et al.
      Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.
      . They found that leukocyte poor RA tissues (low infiltration of synovial T-cells and B-cells) had cellular compositions similar to OA and synovial hyperplasia was similar between the leukocyte rich or poor RA tissues and OA. Single-cell sequencing revealed 18 clusters among five cell types, and sorted-cell bulk RNA seq indicated that genes expressed in synovial fibroblast cluster 2 (HLA-DRAhi) were more highly expressed in leukocyte rich RA than OA tissues. Genes associated with CD55+ synovial fibroblast cluster four were more highly expressed in OA samples, as were genes associated with NUPR1+ monocytes. By integrating transcriptomic and proteomic data at a single cell resolution this group was able identify unique contributions of chronic highly inflammatory pathways in arthritis. These complementary techniques and the incorporation of unique CCA analyses were necessary to extricate the unique interplay between transcriptomics and phenotype.
      The idea of targeting subsets of cells has also been explored in a study by Kuang et al., who identify Fibroblast Growth Factor Receptor 3 (FGFR3) as a key mediator of OA progression in mice
      • Kuang L.
      • Wu J.
      • Su N.
      • Qi H.
      • Chen H.
      • Zhou S.
      • et al.
      FGFR3 deficiency enhances CXCL12-dependent chemotaxis of macrophages via upregulating CXCR7 and aggravates joint destruction in mice.
      . Previous studies demonstrated macrophages accumulate in OA synovium and targeting them could be a viable strategy to attenuate disease progression. Conditional knock-out (CKO) of FGFR3 in myeloid cells demonstrate spontaneous OA after 13 months, and changes in synovitis and macrophage accumulation which precede OA development. RNA sequencing identified CXCR7 as highly differentially expressed, and inhibition of CXCR7 using a monoclonal antibody attenuated OA progression in FGFR3 CKO mice in both aging and DMM models. The RNA-sequencing employed here was crucial not only to identifying a viable therapeutic target, but also evaluating how mechanisms governing dysregulation in a subset of synovial cells could impact disease progression.
      Dysregulated circadian rhythm signaling has previously been identified in OA pathogenesis and by using RNA-seq Bekki et al., demonstrate how core clock gene cryptochrome 2 (CRY2) mediates OA progression. They show CRY2 levels are significantly depressed in OA cartilage, and that knock-out of CRY2 in mice results in greater OA severity after DMM. They identify disrupted ECM remodeling and angiogenesis as pathways involved in circadian rhythm mediation of cartilage homeostasis
      • Bekki H.
      • Duffy T.
      • Okubo N.
      • Olmer M.
      • Alvarez-Garcia O.
      • Lamia K.
      • et al.
      Suppression of circadian clock protein cryptochrome 2 promotes osteoarthritis.
      .
      An important consideration for the translatability of preclinical studies is the investigation of disease pathogenesis in multiple tissue types. In recent years, many groups have shifted away from a sole focus on articular cartilage to evaluating how other tissues within the joint affect the onset and progression of OA. Lee et al. investigated the impairment of meniscus cell homeostasis during trauma and aging by evaluating the expression and function of Forkhead Box O (FoxO), a regulator of cellular homeostasis, in human and mouse menisci. They found that the expression of FoxO 1 and 3 is reduced in aged, and degenerated meniscus
      • Lee K.I.
      • Choi S.
      • Matsuzaki T.
      • Alvarez-Garcia O.
      • Olmer M.
      • Grogan S.P.
      • et al.
      FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.
      . Deletion of FoxO using Col2Cre or AcanCreERT2 (as there are no genes exclusively expressed in meniscus that could be used as a cre driver), demonstrate spontaneous meniscal pathology, and age related damage. It also suppressed autophagy, antioxidant defense, and altered meniscus-specific genes. Both, FoxO1 and FoxO 3 protect against degeneration during aging and OA. The FOXO1 transcriptional network has also been examined in an epigenetic study using ChIP-seq on human primary chondrocytes to elucidate protective mechanisms of this transcription factor in OA
      • Duffy T.
      • Bekki H.
      • Lotz M.K.
      Genome-wide occupancy profiling reveals critical roles of FOXO1 in regulating extracellular matrix and circadian rhythm genes in human chondrocytes.
      . Pathways which are aberrantly regulated during OA pathogenesis such as homeostasis, ECM regulation, and circadian clock genes are all directly regulated by FOX01.
      In many pre-clinical studies published this year, the importance of choosing or creating models which accurately recapitulate human disease cannot be underscored enough. Studies such as the above-mentioned identification of chondroprogenitor populations using scRNA-seq play an important role in establishing models for tissue repair
      • Dicks A.
      • Wu C.L.
      • Steward N.
      • Adkar S.S.
      • Gersbach C.A.
      • Guilak F.
      Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.
      . While those delineating transcriptomic differences between healer and non-healer mouse strains
      • Duan X.
      • Cai L.
      • Schmidt E.J.
      • Shen J.
      • Tycksen E.D.
      • O'Keefe R.J.
      • et al.
      RNA-seq analysis of chondrocyte transcriptome reveals genetic heterogeneity in LG/J and SM/J murine strains.
      , and genetic differences in cartilage stem progenitor cells during age dependent OA progression in STR/Ort (spontaneous OA phenotype) and CBA (low OA susceptibility) strains
      • Zhang S.
      • An Q.
      • Hu P.
      • Wu X.
      • Pan X.
      • Peng W.
      • et al.
      Core regulatory RNA molecules identified in articular cartilage stem/progenitor cells during osteoarthritis progression.
      , aid in capturing the genetic and phenotypic diversity of OA which is integral to the creation of translational models and therapies in humans.

      OA Epigenetics: expanding understanding of disease mechanisms

      Many studies have started incorporating total RNA sequencing with other -omics technologies to expand ‘deep phenotyping’ and our understanding of disease mechanisms in diverse tissues and these interactions will be explored in the following section.

       MicroRNA

      From April 2019–April 2020, an astounding 164 articles were published on miRNA and OA; they have identified potential therapeutic targets, characterized molecular mechanisms, explored whether candidate miRNAs could be used as biomarkers, and even examined whether factors involved in miRNA biogenesis could mediate OA progression
      • Deng L.
      • Ren R.
      • Liu Z.
      • Song M.
      • Li J.
      • Wu Z.
      • et al.
      Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.
      . The utility of miRNA markers is that they are stable in circulation, and are very responsive in changes to pathology. Some potential diagnostic biomarker candidates identified this year include circulating miR-200c-3p, miR-100–5p, miR-1826, miR-29 b-3p and miR-582–5p
      • Lai Z.
      • Cao Y.
      Plasma miR-200c-3p, miR-100-5p, and miR-1826 serve as potential diagnostic biomarkers for knee osteoarthritis: randomized controlled trials.
      • Chen C.
      • Chen H.
      Clinical diagnosis value of miR-29b-3p in peripheral blood mononuclear cells and synovial fluid among osteoarthritis patients.
      • Wang P.
      • Dong R.
      • Wang B.
      • Lou Z.
      • Ying J.
      • Xia C.
      • et al.
      Genome-wide microRNA screening reveals miR-582-5p as a mesenchymal stem cell-specific microRNA in subchondral bone of the human knee joint.
      . Synovial fluid (SF) levels of miR-210 have also been implicated as a potential diagnostic biomarker due to their increase in early OA, and sustained level in late-stage radiographic disease, whereas SF levels of miR-30c-5p were found to be associated with post-operative pain outcomes
      • Xie W.
      • Su W.
      • Xia H.
      • Wang Z.
      • Su C.
      • Su B.
      Synovial fluid MicroRNA-210 as a potential biomarker for early prediction of osteoarthritis.
      ,
      • Kwak Y.H.
      • Kwak D.K.
      • Kim N.Y.
      • Kim Y.J.
      • Lim J.S.
      • Yoo J.H.
      Significant changes in synovial fluid microRNAs after high tibial osteotomy in medial compartmental knee osteoarthritis: identification of potential prognostic biomarkers.
      . The identification of these candidates is an important first step, however to have any clinical utility they must be validated in multiple cohorts, with larger sample sizes.
      Given that OA is polygenic in nature, miRNA are seen as attractive therapeutic entities as they can target multiple genes with related functions. The in vivo functions of miR-204 and miR-211 were characterized by Huang et al., who found that these homologous miRNAs are necessary to maintain joint homeostasis. Forty-two week old mice with a mesenchymal progenitor cell-specific double knockout (dKO) of miR-204/211 displayed an OA phenotype in multiple tissues. Intraarticular AAV administration of miR-204 sustained long-term protection from OA progression and pain in a surgical model of OA in mice
      • Huang J.
      • Zhao L.
      • Fan Y.
      • Liao L.
      • Ma P.X.
      • Xiao G.
      • et al.
      The microRNAs miR-204 and miR-211 maintain joint homeostasis and protect against osteoarthritis progression.
      . In contrast, Kang et al. defined miR-204 as destructive in cartilage and induced by the transcription factors NF-kB and GATA4 in response to senescence signals
      • Kang D.
      • Shin J.
      • Cho Y.
      • Kim H.S.
      • Gu Y.R.
      • Kim H.
      • et al.
      Stress-activated miR-204 governs senescent phenotypes of chondrocytes to promote osteoarthritis development.
      . When anti-miR-204 was administered to mice after DMM surgery there was decreased cartilage breakdown compared to control mice. Both studies illustrate the significant role of miR-204 in maintaining joint homeostasis.
      In women from the Os des Femmes de Lyon (OFELY) cohort, circulating miR-146a-5p and 186–5p were elevated in those with prevalent knee OA compared to healthy controls. In fact, the likelihood of prevalent knee OA increased with each quartile increase in serum miR-146a-5p
      • Rousseau J.C.
      • Millet M.
      • Croset M.
      • Sornay-Rendu E.
      • Borel O.
      • Chapurlat R.
      Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study.
      . Local levels of isoform miR-146a along with miR-140–5p were downregulated in human OA chondrocytes and associated with hypermethylation of the regulatory regions in these miRNAs
      • Papathanasiou I.
      • Trachana V.
      • Mourmoura E.
      • Tsezou A.
      DNA methylation regulates miR-140-5p and miR-146a expression in osteoarthritis.
      . Human chondrocytes transfected with miR-146a, miR-140–5p or miR-140–3p prior to stimulation with IL-1B or TNFa prevented inflammation, promoted autophagy and regulated proteins involved in oxidative stress and metabolism
      • Al-Modawi R.N.
      • Brinchmann J.E.
      • Karlsen T.A.
      Multi-pathway protective effects of MicroRNAs on human chondrocytes in an in vitro model of osteoarthritis.
      . Pre-transfection with miR-140 also prevented IL-1B induced chondrocyte senescence and intra-articular injection of miR-140 protected from surgically induced OA in rats, making it an appealing therapeutic target
      • Si H.B.
      • Yang T.M.
      • Li L.
      • Tian M.
      • Zhou L.
      • Li D.P.
      • et al.
      miR-140 attenuates the progression of early-stage osteoarthritis by retarding chondrocyte senescence.
      . The host gene of miR-140, WWP2 also exerted cartilage protective effects through mediating ADAMTS5
      • Mokuda S.
      • Nakamichi R.
      • Matsuzaki T.
      • Ito Y.
      • Sato T.
      • Miyata K.
      • et al.
      Wwp2 maintains cartilage homeostasis through regulation of Adamts5.
      .
      Autophagy is a common process to many miR examined in OA studies this year. In autophagy, damaged cytosolic components are degraded and recycled which is an important part of maintaining cellular homeostasis. In OA, the process of autophagy inhibits inflammation and reduces chondrocyte apoptosis and variants in autophagy related genes increase risk of OA
      • Xu Z.
      • Yang H.
      • Zhou X.
      • Li J.
      • Jiang L.
      • Li D.
      • et al.
      Genetic variants in mTOR-pathway-related genes contribute to osteoarthritis susceptibility.
      . In addition to miR-140–5p and miR-146a, other miR that promoted autophagy in chondrocytes include miR-34a-5p, miR-107, miR-335–5p and miR-let-7e
      • Zhao X.
      • Li H.
      • Wang L.
      MicroRNA-107 regulates autophagy and apoptosis of osteoarthritis chondrocytes by targeting TRAF3.
      • Zhong G.
      • Long H.
      • Ma S.
      • Shunhan Y.
      • Li J.
      • Yao J.
      miRNA-335-5p relieves chondrocyte inflammation by activating autophagy in osteoarthritis.
      • Tian F.
      • Wang J.
      • Zhang Z.
      • Yang J.
      LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis.
      • Feng L.
      • Feng C.
      • Wang C.X.
      • Xu D.Y.
      • Chen J.J.
      • Huang J.F.
      • et al.
      Circulating microRNA let7e is decreased in knee osteoarthritis, accompanied by elevated apoptosis and reduced autophagy.
      . MicroRNAs that decreased autophagic processes include miR-206, miR-375, miR-411, and miR-449
      • Yang F.
      • Huang R.
      • Ma H.
      • Zhao X.
      • Wang G.
      miRNA-411 regulates chondrocyte autophagy in osteoarthritis by targeting hypoxia-inducible factor 1 alpha (HIF-1 alpha).
      • Yu Q.
      • Zhao B.
      • He Q.
      • Zhang Y.
      • Peng X.B.
      microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1.
      • Li H.
      • Li Z.
      • Pi Y.
      • Chen Y.
      • Mei L.
      • Luo Y.
      • et al.
      MicroRNA-375 exacerbates knee osteoarthritis through repressing chondrocyte autophagy by targeting ATG2B.
      • Ni Z.
      • Kuang L.
      • Chen H.
      • Xie Y.
      • Zhang B.
      • Ouyang J.
      • et al.
      The exosome-like vesicles from osteoarthritic chondrocyte enhanced mature IL-1 beta production of macrophages and aggravated synovitis in osteoarthritis.
      . While autophagy is necessary to maintain cellular homeostasis in cartilage, aberrant autophagy can also have deleterious effects in OA.

       Circular RNA

      Circular RNAs (circRNA) are covalently-closed loop single stranded RNA that can affect biological functions by “sponging” and acting as inhibitors of miRNA and protein inhibitors
      • Kristensen L.S.
      • Andersen M.S.
      • Stagsted L.V.W.
      • Ebbesen K.K.
      • Hansen T.B.
      • Kjems J.
      The biogenesis, biology and characterization of circular RNAs.
      . Due to their stable and often cell-specific or tissue-specific expression they are promising biomarkers and therapeutic targets
      • Li H.Z.
      • Lin Z.
      • Xu X.H.
      • Lin N.
      • Lu H.D.
      The potential roles of circRNAs in osteoarthritis: a coming journey to find a treasure.
      . A few more studies have characterized the circRNA transcriptome of cartilage and synovium using unbiased transcriptomics
      • Li H.
      • Yang H.H.
      • Sun Z.G.
      • Tang H.B.
      • Min J.K.
      Whole-transcriptome sequencing of knee joint cartilage from osteoarthritis patients.
      • Xiao K.
      • Xia Z.
      • Feng B.
      • Bian Y.
      • Fan Y.
      • Li Z.
      • et al.
      Circular RNA expression profile of knee condyle in osteoarthritis by illumina HiSeq platform.
      • Xiang S.
      • Li Z.
      • Bian Y.
      • Weng X.
      RNA sequencing reveals the circular RNA expression profiles of osteoarthritic synovium.
      , finding 42, 197 and 122 differentially expressed circRNA between OA and healthy individuals. Other studies have largely focused on the interactions between circRNA and miRNA in chondrocytes. Both circ_0136474 and circ_CDR1as have been reported to act on MMP-13, a catabolic protease in articular cartilage. Circ_0136474 competitively binds to miR-127–5p thus inhibiting its expression and prevents it from suppressing the expression of its direct target, MMP-13
      • Li Z.
      • Yuan B.
      • Pei Z.
      • Zhang K.
      • Ding Z.
      • Zhu S.
      • et al.
      Circ_0136474 and MMP-13 suppressed cell proliferation by competitive binding to miR-127-5p in osteoarthritis.
      . Whereas circ-CDR1as sponges miR-641 to affect FGF2 which then increases expression of MMP-13 and decreases expression of COL2 in chondrocytes via the MEK/ERK signalling pathway
      • Zhang W.
      • Zhang C.
      • Hu C.
      • Luo C.
      • Zhong B.
      • Yu X.
      Circular RNA-CDR1as acts as the sponge of microRNA-641 to promote osteoarthritis progression.
      . MicroRNA-7 had previously been reported as a direct target of circ-CDR1as, also known as ciRS-7
      • Li P.
      • Yang X.
      • Yuan W.
      • Yang C.
      • Zhang X.
      • Han J.
      • et al.
      CircRNA-Cdr1as exerts anti-oncogenic functions in bladder cancer by sponging MicroRNA-135a.
      . Circ-CDR1as can act on miR-7 to attenuate its stimulatory effect on IL-1B in chondrocytes thereby mediating inflammation, apoptosis, and proliferation
      • Zhou X.
      • Jiang L.
      • Fan G.
      • Yang H.
      • Wu L.
      • Huang Y.
      • et al.
      Role of the ciRS-7/miR-7 axis in the regulation of proliferation, apoptosis and inflammation of chondrocytes induced by IL-1 beta.
      .

       Long non-coding RNA

      Long non-coding RNAs (lncRNA) are non-coding RNA transcripts greater than 200 nucleotides in length. They are involved in many pathologies due to their ability to modulate mRNA stability, translation and post-translational modification in the cytoplasm
      • Yao R.W.
      • Wang Y.
      • Chen L.L.
      Cellular functions of long noncoding RNAs.
      . Studies this year identified differentially expressed lncRNA in OA synovium
      • Shui X.
      • Xie Q.
      • Chen S.
      • Zhou C.
      • Kong J.
      • Wang Y.
      Identification and functional analysis of long non-coding RNAs in the synovial membrane of osteoarthritis patients.
      , and long-intergenic non-coding RNA in hip OA cartilage
      • Ajekigbe B.
      • Cheung K.
      • Xu Y.
      • Skelton A.J.
      • Panagiotopoulos A.
      • Soul J.
      • et al.
      Identification of long non-coding RNAs expressed in knee and hip osteoarthritic cartilage.
      .
      OA patient plasma samples express high levels of the lncRNA HOTAIR, and low levels of lncRNA PACER relative to healthy individuals. These lncRNA are inversely correlated, and in chondrocytes PACER can decrease cell apoptosis by mediating levels of HOTAIR
      • Jiang M.
      • Liu J.
      • Luo T.
      • Chen Q.
      • Lu M.
      • Meng D.
      LncRNA PACER is down-regulated in osteoarthritis and regulates chondrocyte apoptosis and lncRNA HOTAIR expression.
      . In chondrocytes, HOTAIR is demonstrated to act through inhibition of miR-130a-5p to increase apoptosis, and decrease autophagy with increases in Bax as well as decreases in Bcl-2 and survivin
      • He B.
      • Jiang D.
      HOTAIR-induced apoptosis is mediated by sponging miR-130a-3p to repress chondrocyte autophagy in knee osteoarthritis.
      . In a mouse DMM model, HOTAIR is upregulated in OA cartilage, and silencing HOTAIR decreases cartilage damage and increases expression of anabolic genes. These effects are mediated by HOTAIR inhibition of mir-20 b
      • Chen Y.
      • Zhang L.
      • Li E.
      • Zhang G.
      • Hou Y.
      • Yuan W.
      • et al.
      Long-chain non-coding RNA HOTAIR promotes the progression of osteoarthritis via sponging miR-20b/PTEN axis.
      . Conversely silencing HOTAIR in the synovium in a rat model of OA has the opposite effect on apoptosis, though it decreased synovitis and proliferation of synoviocytes
      • Mao T.
      • He C.
      • Wu H.
      • Yang B.
      • Li X.
      Silencing lncRNA HOTAIR declines synovial inflammation and synoviocyte proliferation and promotes synoviocyte apoptosis in osteoarthritis rats by inhibiting Wnt/beta-catenin signaling pathway.
      .
      MALAT1 is a lncRNA differentially expressed in synovium from obese patients with OA compared to non-obese OA patients and non-OA patients. Knockdown of MALAT1 in OA synovial fibroblasts inhibited proliferation, increased the protein expression of CXCL8 (interleukin-8) and increased expression of genes which have roles in cell growth, proliferation and the inflammatory response
      • Nanus D.E.
      • Wijesinghe S.N.
      • Pearson M.J.
      • Hadjicharalambous M.R.
      • Rosser A.
      • Davis E.T.
      • et al.
      Regulation of the inflammatory synovial fibroblast phenotype by metastasis-associated lung adenocarcinoma transcript 1 long noncoding RNA in obese patients with osteoarthritis.
      . Similar anti-apoptotic and anti-inflammatory effects were seen in chondrocytes in vitro with pcDNA transfection of MALAT1
      • Gao G.C.
      • Cheng X.G.
      • Wei Q.Q.
      • Chen W.C.
      • Huang W.Z.
      Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1 beta-induced inflammation in articular chondrocytes via the JNK signaling pathway.
      , as well as in LPS treated chondrocytes where MALAT1 mediated genes regulating ECM catabolism, inflammation and apoptosis through inhibition of miR-146a
      • Li H.
      • Xie S.
      • Li H.
      • Zhang R.
      • Zhang H.
      LncRNA MALAT1 mediates proliferation of LPS treated-articular chondrocytes by targeting the miR-146a-PI3K/Akt/mTOR axis.
      . Using Resveratrol supressed activation of MALAT1 and decreased pro-inflammatory characteristics by modulating miR-9
      • Zhang G.
      • Zhang H.
      • You W.
      • Tang X.
      • Li X.
      • Gong Z.
      Therapeutic effect of Resveratrol in the treatment of osteoarthritis via the MALAT1/miR-9/NF-kappaB signaling pathway.
      . Many recent studies on non-coding RNA studies were cartilage focused, and interactions between circRNA, lncRNA, with miRNA and target genes are illustrated in Fig. 1.
      Fig. 1
      Fig. 1Molecular cross-talk between circRNA, lncRNA, and miRNA indicate the complex polygenic roles of non-coding RNAs. Common mechanisms examined in cartilage focused studies from April 1 2019 to April 30 2020 characterizing the roles of lncRNA and circRNA are displayed above, manually created from articles referenced in this manuscript and visualized using Whimsical (https://whimsical.com/). Processes are illustrated in purple hexagons, circRNA are red circles, lncRNA are orange rhombuses, and gene and protein expression changes are denoted in blue rectangles. Dotted grey arrows indicate decreases/negative associations, while solid purple arrows indicate increases/positive associations.

       DNA methylation

      DNA methylation is an epigenetic modification where a methyl group is added to DNA at a CPG-site and is generally correlated with gene silencing
      • Miranda-Duarte A.
      DNA methylation in osteoarthritis: current status and therapeutic implications.
      . In recent years, the DNA methyltransferase enzyme, Dnmt3b was identified as a promising target by Shen and colleagues. They demonstrated that Dnmt3b is highly expressed in human and murine cartilage and that its deletion from cartilage led to OA through increased mitochondrial respiration
      • Shen J.
      • Wang C.
      • Li D.
      • Xu T.
      • Myers J.
      • Ashton J.M.
      • et al.
      DNA methyltransferase 3b regulates articular cartilage homeostasis by altering metabolism.
      . This year they identified a downstream target of Dnmt3b, 4-aminobutyrate aminotransferase (Abat), as a mediator of mitochondrial respiration and catabolic gene changes in chondrocytes. Lentivirus-based-knockdown of Abat protects against articular cartilage degeneration and subchondral bone sclerosis in a surgically induced mouse model of OA. Subsequently, the authors used an FDA-approved small-molecule inhibitor of Abat, vigabatrin, to determine whether pharmacological blocking of Abat could modify disease activity in vivo. Vigatrabin successfully prevented articular cartilage degeneration and subchondral bone sclerosis in mice after surgical induction of OA, indicating that it could serve as a feasible target for OA therapy
      • Shen J.
      • Wang C.
      • Ying J.
      • Xu T.
      • McAlinden A.
      • O'Keefe R.J.
      Inhibition of 4-aminobutyrate aminotransferase protects against injury-induced osteoarthritis in mice.
      . DNA methyl transferases 1 and 3a (Dnmt1, 3a) are implicated in a study by Zhu et al. investigating epigenetic suppression of PPARγ in OA development. They discovered that mouse and human OA cartilage expressed decreased levels of PPARγ and this could be attributed to hypermethylation of PPARγ promoters induced by aberrant levels of Dnmt1 and 3. Inhibiting these enzymes in vivo was able to mediate levels of PPARγ and protect from cartilage degeneration in a DMM model of OA in mice. This protection was also conferred by inhibiting Dnmt1 and 3a in PPARγ KO mice indicating that targeting epigenetic modifications of PPARγ could be a productive avenue for therapeutic development
      • Zhu X.
      • Chen F.
      • Lu K.
      • Wei A.
      • Jiang Q.
      • Cao W.
      PPARgamma preservation via promoter demethylation alleviates osteoarthritis in mice.
      .
      DNA hydroxymethylation is an epigenetic modification where Ten Eleven Translocation (TET) enzymes catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), also known as “DNA demethylation”
      • Santiago M.
      • Antunes C.
      • Guedes M.
      • Sousa N.
      • Marques C.J.
      TET enzymes and DNA hydroxymethylation in neural development and function - how critical are they?.
      . The authors previously described that 5hmC accumulates at OA associated genes
      • Taylor S.E.
      • Li Y.H.
      • Wong W.H.
      • Bhutani N.
      Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes.
      ,
      • Taylor S.E.
      • Smeriglio P.
      • Dhulipala L.
      • Rath M.
      • Bhutani N.
      A global increase in 5-hydroxymethylcytosine levels marks osteoarthritic chondrocytes.
      . In this study, they show differential hydroxymethylation of approximately 40,000 sites precedes structural disease progression in mice and that this is mediated by TET1. Tet 1 knockout mice are protected from OA development post-DMM surgery and this effect is recapitulated using 2-hydroxyglutarate (2-HG, a small molecule inhibitor of TET1), indicating another appealing target for OA therapy
      • Smeriglio P.
      • Grandi F.C.
      • Davala S.
      • Masarapu V.
      • Indelli P.F.
      • Goodman S.B.
      • et al.
      Inhibition of TET1 prevents the development of osteoarthritis and reveals the 5hmC landscape that orchestrates pathogenesis.
      .
      Methylation quantitative trait loci (MQTL) are the genetic variants at specific loci that could affect DNA methylation patterns. Rice et al. combined DNA methylation, genotyping, and RNA sequencing data with in-silico analyses to investigate 42 OA risk loci. In ten loci, they identified 24 CpGs where methylation and genotype were correlated. Of these, COLGALT2 (collagen beta (1-O) galactosyl transferase 2), COL11A2 (collagen 11 type A 2), and WWP2 (WW domain containing E3 ubiquitin protein ligase 2) emerged as key targets
      • Rice S.J.
      • Cheung K.
      • Reynard L.N.
      • Loughlin J.
      Discovery and analysis of methylation quantitative trait loci (eqtls) mapping to novel osteoarthritis genetic risk signals.
      . PLEC (plectin) and GRINA (Glutamate Ionotropic Receptor NMDA Type Subunit Associated) were also identified in a subsequent study prioritizing target genes by correlating mQTLs with OA risk and mechanistic evaluation
      • Rice S.J.
      • Tselepi M.
      • Sorial A.K.
      • Aubourg G.
      • Shepherd C.
      • Almarza D.
      • et al.
      Prioritization of PLEC and GRINA as osteoarthritis risk genes through the identification and characterization of novel methylation quantitative trait loci.
      . Interestingly, mitochondrial DNA variation was discovered to differentially associate with methylation status of articular cartilage by acting on key pathological processes such as apoptosis, and dysregulated metabolism and development
      • Cortes-Pereira E.
      • Fernandez-Tajes J.
      • Fernandez-Moreno M.
      • Vazquez-Mosquera M.E.
      • Relano S.
      • Ramos-Louro P.
      • et al.
      Differential association of mitochondrial DNA haplogroups J and H with the methylation status of articular cartilage: potential role in apoptosis and metabolic and developmental processes.
      .

      In silico and beyond: next steps for progress in genetics, genomics and epigenomics

      OA research has made tremendous progress in recent years and the number of publications within Genetics, Genomics and Epigenomics continues to rise concurrently with increased accessibility to high-throughput technology and knowledge transfer between research organizations. Within this timeframe, the content of manuscripts has largely evolved from studies that may use a singular technique to discover a genetic target to using multiple approaches connecting the “what” and “how” of genetic regulation. In particular, the identification of genetic risk variants integrated with the elucidation epigenetic mediators and their mechanisms provides new possibilities for therapeutic development in OA
      • Rice S.J.
      • Beier F.
      • Young D.A.
      • Loughlin J.
      Interplay between genetics and epigenetics in osteoarthritis.
      . The integration of multiple discovery techniques, or comparison of whole tissue or specific cell populations have been made possible through extensive in-silico techniques. In-silico methods have been used to discover gene targets, identify pathways, predict relationships, characterize mechanisms, and validate findings in other cohorts
      • van den Bosch M.H.J.
      • Ramos Y.F.M.
      • den Hollander W.
      • Bomer N.
      • Nelissen R.
      • Bovee J.
      • et al.
      Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.
      • Porings A.S.
      • Lowin T.
      • Dufner B.
      • Grifka J.
      • Straub R.H.
      A thyroid hormone network exists in synovial fibroblasts of rheumatoid arthritis and osteoarthritis patients.
      • Kim S.
      • Han S.
      • Kim Y.
      • Kim H.S.
      • Gu Y.R.
      • Kang D.
      • et al.
      Tankyrase inhibition preserves osteoarthritic cartilage by coordinating cartilage matrix anabolism via effects on SOX9 PARylation.
      • Li P.
      • Ning Y.
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      • Ma M.
      • et al.
      Integrating transcriptome-wide study and mRNA expression profiles yields novel insights into the biological mechanism of chondropathies.
      • Zhou Y.
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      • Chen X.
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      • et al.
      Identification of differentially expressed miRNAs and mRNAs in synovial of osteoarthritis via RNA-sequencing.
      . Techniques such as CCA, enable the amalgamation of multi-omics experiments while neutralizing batch effects to distill crucial information. Computational modelling has even been used to build a biologist-friendly visually-based modeling tool that can be used to investigate dynamic networks in cartilage development and OA
      • Schivo S.
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      • Scholma J.
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      • Zhong L.
      • et al.
      ECHO, the executable CHOndrocyte: a computational model to study articular chondrocytes in health and disease.
      . While genomic, transcriptomic and epigenomic techniques are becoming more affordable and more accessible it is important that we continue to build research capacity through open access to datasets, and transparent, reproducible computational analyses. Although high-throughput sequencing has provided us with a multitude of targets, these must be validated across populations and across disease stages. Combining data-sets could also be used to identify patterns across populations, and to evaluate potential targets among pre-clinical models
      • Soul J.
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      • Young D.A.
      OATargets: a knowledge base of genes associated with osteoarthritis joint damage in animals.
      . These are necessary next steps for the field to aggregate and compare data in order to draw meaningful and applicable conclusions needed for building diagnostic tools, as well as disease intervention. Much of the work in OA genomics is still chondro-centric, and using multiple tissues may elucidate the reciprocal contributions of joint tissues to disease pathogenesis
      • Chou C.H.
      • Jain V.
      • Gibson J.
      • Attarian D.E.
      • Haraden C.A.
      • Yohn C.B.
      • et al.
      Synovial cell cross-talk with cartilage plays a major role in the pathogenesis of osteoarthritis.
      ,
      • Zhao G.H.
      • Yang L.
      • Lammi M.J.
      • Guo X.
      A preliminary analysis of microRNA profiles in the subchondral bone between Kashin-Beck disease and primary knee osteoarthritis.
      , this could also delineate which avenues for drug delivery may be most appropriate. Though recent studies have clarified roles for a variety of risk factors and their interplay with genetics, future efforts are required in defining the genetic basis of OA and devising appropriate interventions. The development of the OAI cohort among others facilitate the connection of genotype and phenotype data along with the potential to study complex interactions among risk factors, and the path forward will hopefully include the integration of multiple levels of data across systems and disease states.

      Author contributions

      AR and MK were involved in conceptualization, design, literature review, writing, editing and reviewing the final version of the manuscript.

      Conflict of interest

      Authors declare no conflict of interest related to this review article.

      Funding sources

      This work is supported by grants to MK by the Canadian Institute of Health Research (CIHR; #156299 ). AR is a recipient of the Postdoctoral award from CIHR. MK is a recipient of Tier 1 Canada Research Chair (CRC) Award.

      Acknowledgement

      Authors would like to thank Sarah Gabrial for her assistance with the literature search.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:
      • Supplementary Table 1

        List of curated studies for OA Year in Review. Total list of publications alphabetically indexed by search term (category) and including PubMed ID (PMID), and pertinent article information for OA studies published between April 1 2019 and April 30 2020.

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