Osteoarthritis Susceptibility Research Articles

Pravastatin ameliorated osteoarthritis susceptibility in male offspring rats induced by prenatal ethanol exposure.

Osteoarthritis (OA) is a disease associated with a disorder of cholesterol metabolism. Our previous studies showed that prenatal ethanol exposure (PEE) caused cholesterol accumulation in articular cartilage and increased the susceptibility to OA in offspring. However, we did not determine whether pravastatin, a cholesterol-lowering agent, could rescue PEE-induced susceptibility to OA. Here, fetal rats were divided into a PEE group and a control group during pregnancy. At postnatal week (PW) 8, sixteen male offspring rats from both groups were injected papain through the articular cavity. Eight of them from each group were treated with pravastatin (20mg/kg·d) by gavage for four weeks simultaneously. We found that pravastatin ameliorated papain-induced high expression of inflammatory factors [interleukin (IL)-1, IL-6], matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13], and apoptosis factors (caspase-3 and caspase-8) in the cartilage of the PEE group. Also, pravastatin significantly reduced the content of TCH in the blood and cartilage of the PEE offspring and improved cholesterol efflux pathway. Our in vitro findings further confirmed that pravastatin partially reversed cholesterol-induced inflammation and apoptosis of chondrocytes. In conclusion, pravastatin effectively reduced inflammation and matrix degradation, and thus ameliorate OA susceptibility in articular cartilage by relieving cholesterol accumulation in chondrocyte.

Discovery and functional analysis of osteoarthritis susceptibility genes using families from a large population-based cohort

Purpose: The main obstacle to the development of disease-modifying therapies for OA is poor understanding of the disease process: we do not know the cell types or molecular pathways that normally function to limit susceptibility to OA. Our goal is to discover molecular pathways that are vulnerability points for the development of OA and generate mouse models using human disease alleles. We discover these pathways by identifying rare gene mutations in coding sequences that have a strong effect on susceptibility to OA in families identified from a unique statewide population-based cohort, the Utah Population Database. From the family studies, we identify gene variants that we view as candidates for having a strong effect on OA susceptibility. To rapidly identify those candidate variants that measurably alter gene function, we quantitatively assay WT and variant gene functions in zebrafish embryos or tissue culture. We then determine whether and how the functionally altered genes confer susceptibility to OA in genetically modified mouse models. Methods: We take an approach used infrequently in the OA field. We study many unrelated families with clear-cut inherited forms of OA to identify susceptibility alleles that have strong determinate effects. To discover pathways that when modified lead to strong and unambiguous susceptibility to OA, we analyzed the exomes of >50 unrelated families from the Utah Population Database that had a significant enrichment of dominantly inherited shoulder OA, distal and proximal interphalangeal joint hand OA, erosive hand OA, and 1st metatarsophalangeal (MTP) joint OA. We use loss- and gain-of-function analyses in zebrafish to rapidly screen for gene variants that have a functional impact on protein activity. Finally, Crispr/Cas9 technology is used to introduce OA-associated disease alleles into the mouse where we analyze the how these mutations affect onset and progression of OA. Results: Our results indicate that we can use families to identify genes and pathways that confer strong susceptibility to the early stages of OA. Our approach has led to the discovery of candidate gene variants in families with different forms of OA that affect common pathways. These pathways include genes that regulate 1) the proinflammatory response, 2) mechanotransduction/mechanosensation, 3) the TGF-b and Notch signaling pathways, 3) transcription modulators, and 4) ECM components and ECM modification. We have discovered variants that are worth pursuing experimentally as they coincide with loci detected in GWAS, affect genes of related biological function, represent multiple alleles of a candidate gene, and identify genes not previously linked to OA. We have identified mutations in the NOD-RIPK2 pathway in families with interphalangeal joint, erosive hand, and 1st MTP joint OA. The NOD-RIPK2 pathway is a central regulator of the proinflammatory response. We have shown the OA-associated variants have increased proinflammatory activity relative to the wildtype protein. Based on these findings, we propose the gene variants act dominantly as a gain-of-function allele to over-stimulate the inflammatory response to naturally occurring joint damage leading to OA. To test this hypothesis we introduced a single amino acid change, encoded by the RIPK2 disease allele, into an isogenic mouse. Current results indicate mice harboring the disease allele exhibit increased severity of OA, an increased systemic inflammatory response, and altered gene expression when compared to controls. We have generated a conditional disease allele to test whether expression of the RIPK2 disease allele in innate immune cells or chondrocytes is sufficient to confer increased susceptibility to experimentally induced or spontaneous OA in the mouse. Erosive hand OA is characterized radiographically by centrally located interphalangeal joint erosions, and clinically by its rapid onset and progression. Genomic analysis of one family identified a rare coding variant in the procollagen galactosyltransferase COLGALT1. Loss- and gain-of-function analyses in the zebrafish indicate that colgalt1a/1b are necessary for cartilage formation. We are also examining if collagen structure is disrupted in articular cartilage of mice with a Colgalt1 mutation and if Colgalt1 mutant mice are more susceptible to injury induced OA. Conclusions: Our studies demonstrate our innovative approach of combining genetic analysis of families with OA and functional variant analysis in the zebrafish to identify well-supported OA-associated alleles and pathways. Our approach has led to the discovery of 1) compelling novel candidate genes, 2) multiple genes that contribute to a common pathway, 3) and identification of common pathways altered in multiple forms of OA, 4) and to the generation of new mouse models of OA. Our genetic studies of families with OA and functional analyses in the mouse support our hypothesis that variation in the inflammatory response may be a key factor in susceptibility to OA. Our work is significant in that we have taken a novel approach to identify genes that when mutated have a significant impact on the onset or progression of OA. Developing new animal models with these susceptibility genes will be useful for discovery of therapeutic drugs.

RNA Sequencing Reveals Interacting Key Determinants of Osteoarthritis Acting in Subchondral Bone and Articular Cartilage: Identification of IL11 and CHADL as Attractive Treatment Targets.

ObjectiveTo identify key determinants of the interactive pathophysiologic processes in subchondral bone and cartilage in osteoarthritis (OA).MethodsWe performed RNA sequencing on macroscopically preserved and lesional OA subchondral bone from patients in the Research Arthritis and Articular Cartilage study who underwent joint replacement surgery due to OA (n = 24 sample pairs: 6 hips and 18 knees). Unsupervised hierarchical clustering and differential expression analyses were conducted. Results were combined with data on previously identified differentially expressed genes in cartilage (partly overlapping samples) as well as data on recently identified OA risk genes.ResultsWe identified 1,569 genes that were significantly differentially expressed between lesional and preserved subchondral bone, including CNTNAP2 (fold change [FC] 2.4, false discovery rate [FDR] 3.36 × 10−5) and STMN2 (FC 9.6, FDR 2.36 × 10−3). Among these 1,569 genes, 305 were also differentially expressed, and with the same direction of effect, in cartilage, including the recently recognized OA susceptibility genes IL11 and CHADL. Upon differential expression analysis with stratification for joint site, we identified 509 genes that were exclusively differentially expressed in subchondral bone of the knee, including KLF11 and WNT4. These genes that were differentially expressed exclusively in the knee were enriched for involvement in epigenetic processes, characterized by, e.g., HIST1H3J and HIST1H3H.Conclusion IL11 and CHADL were among the most consistently differentially expressed genes OA pathophysiology–related genes in both bone and cartilage. As these genes were recently also identified as robust OA risk genes, they classify as attractive therapeutic targets acting on 2 OA‐relevant tissues.

H3K9ac of TGF\u03b2RI in human umbilical cord: a potential biomarker for evaluating cartilage differentiation and susceptibility to osteoarthritis via a two-step strategy

BackgroundEpidemiological investigation and our previous reports indicated that osteoarthritis had a fetal origin and was closely associated with intrauterine growth retardation (IUGR). Human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) could be programmable to “remember” early-life stimuli. Here, we aimed to explore an early-warning biomarker of fetal-originated adult osteoarthritis in the WJ-MSCs.MethodsFirstly, two kinds of WJ-MSCs were applied to evaluate their chondrogenic potential in vitro through inducing chondrogenic differentiation as the first step of our strategy, one from newborns with IUGR and the other from normal newborns but treated with excessive cortisol during differentiation to simulate the excessive maternal glucocorticoid in the IUGR newborns. As for the second step of the strategy, the differentiated WJ-MSCs were treated with interleukin 1β (IL-1β) to mimic the susceptibility to osteoarthritis. Then, the expression and histone acetylation levels of transforming growth factor β (TGFβ) signaling pathway and the expression of histone deacetylases (HDACs) were quantified, with or without cortisol receptor inhibitor RU486, or HDAC4 inhibitor LMK235. Secondly, the histone acetylation and expression levels of TGFβRI were further detected in rat cartilage and human umbilical cord from IUGR individuals.ResultsGlycosaminoglycan content and the expression levels of chondrogenic genes were decreased in the WJ-MSCs from IUGR, and the expression levels of chondrogenic genes were further reduced after IL-1β treatment, while the expression levels of catabolic factors were increased. Then, serum cortisol level from IUGR individuals was found increased, and similar changes were observed in normal WJ-MSCs treated with excessive cortisol. Moreover, the decreased histone 3 lysine 9 acetylation (H3K9ac) level of TGFβRI and its expression were observed in IUGR-derived WJ-MSCs and normal WJ-MSCs treated with excessive cortisol, which could be abolished by RU486 and LMK235. At last, the decreased H3K9ac level of TGFβRI and its expression were further confirmed in the cartilage of IUGR rat offspring and human umbilical cords from IUGR newborn.ConclusionsWJ-MSCs from IUGR individuals displayed a poor capacity of chondrogenic differentiation and an increased susceptibility to osteoarthritis-like phenotype, which was attributed to the decreased H3K9ac level of TGFβRI and its expression induced by high cortisol through GR/HDAC4. The H3K9ac of TGFβRI in human umbilical cord could be a potential early-warning biomarker for predicting neonatal cartilage dysplasia and osteoarthritis susceptibility.

Growth differentiation factor 5 in cartilage and osteoarthritis: A possible therapeutic candidate.

Growth differentiation factor 5 (GDF‐5) is essential for cartilage development and homeostasis. The expression and function of GDF‐5 are highly associated with the pathogenesis of osteoarthritis (OA). OA, characterized by progressive degeneration of joint, particularly in cartilage, causes severe social burden. However, there is no effective approach to reverse the progression of this disease. Over the past decades, extensive studies have demonstrated the protective effects of GDF‐5 against cartilage degeneration and defects. Here, we summarize the current literature describing the role of GDF‐5 in development of cartilage and joints, and the association between the GDF‐5 gene polymorphisms and OA susceptibility. We also shed light on the protective effects of GDF‐5 against OA in terms of direct GDF‐5 supplementation and modulation of the GDF‐5‐related signalling. Finally, we discuss the current limitations in the application of GDF‐5 for the clinical treatment of OA. This review provides a comprehensive insight into the role of GDF‐5 in cartilage and emphasizes GDF‐5 as a potential therapeutic candidate in OA.

Vitamin D receptor gene variants contribute to hip and knee osteoarthritis susceptibility

Vitamin D receptor (VDR) gene polymorphisms could play a significant role in the susceptibility and pathogenesis of osteoarthritis (OA), the most common degenerative joint disorder in humans. The current study involved 94 OA patients and 100 healthy, asymptomatic controls. VDR variants FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232) and EcoRV (rs4516035) were genotyped using TaqMan-based real-time PCR. Adjusted odds ratio (OR) analysis showed that VDR TaqI and FokI polymorphisms are significantly associated with susceptibility to OA (OR=1.986, P=0.001 and OR=1.561, P=0.017, respectively). Joint-specific analysis showed that the VDR TaqI polymorphism was associated with risk of hip OA (OR=1.930, P=0.005) and knee OA (OR=1.916, P=0.028), while the VDR FokI polymorphism was associated with higher risk of knee OA (OR=2.117, P=0.012). VDR TaqI and FokI polymorphisms are associated with the occurrence of persistent pain (P=0.005 and P=0.027, respectively), while ApaI was associated with a family history of OA (p=0.004). The VDR FokI and TaqI genetic variants significantly contribute to osteoarthritis susceptibility, the occurrence of persistent pain, and potentially to joint-specific OA risk.

Multi-Tissue Epigenetic and Gene Expression Analysis Combined With Epigenome Modulation Identifies RWDD2B as a Target of Osteoarthritis Susceptibility.

Osteoarthritis (OA) is polygenic, with more than 90 risk loci currently mapped, including at the single-nucleotide polymorphism rs6516886. Previous analysis of OA cartilage DNA identified 6 CpG dinucleotides whose methylation levels correlated with the rs6516886 genotype, forming methylation quantitative trait loci (mQTLs). We undertook this study to investigate these mQTLs and to map expression quantitative trait loci (eQTLs) across joint tissues in order to prioritize a particular gene as a target of the rs6516886 association effect. Nucleic acids were extracted from the cartilage, fat pad, synovium, and peripheral blood from OA patients. Methylation of CpGs and allelic expression imbalance of potential target genes were assessed by pyrosequencing. A chondrocyte cell line expressing deactivated Cas9 (dCas9)-TET1 was used to directly alter CpG methylation levels, with effects on gene expression quantified by polymerase chain reaction. Multiple mQTLs were detected, with effects strongest in joint tissues and with methylation at CpG cg20220242 correlating most significantly with the rs6516886 genotype. CpG cg20220242 is located upstream of RWDD2B. Significant rs6516886 eQTLs were observed for this gene, with the OA risk-conferring allele of rs6516886 correlating with reduced expressionCpG methylation also correlated with allelic expression ofRWDD2B, forming methylation-expression QTLs (meQTLs). Deactivated Cas9-TET1 reduction in the methylation of cg20220242 increased expression of RWDD2B. The rs6516886 association signal is a multi-tissue meQTL involving cg20220242 and acting onRWDD2B. Modulating CpG methylation reverses the impact of the risk allele. RWDD2B codes for a protein about which little is currently known. Its further analysis as a target of OA genetic risk will provide novel insight into this complex disease.

Association between growth differentiation factor 5 rs143383 genetic polymorphism and the risk of knee osteoarthritis among Caucasian but not Asian: a meta-analysis

BackgroundA few months ago, the Bioscience Reports journal showed that growth differentiation factor 5 (GDF5) rs143383 genetic polymorphism increases the susceptibility of knee osteoarthritis (KOA), but previous studies’ results have debates about available data. Considering the availability of more recent data, we focus on clarifying the relationship of KOA and GDF5 rs143383 genetic polymorphism by a meta-analysis of case-control trial data.MethodsThe eligible studies from the time of database established to Oct. 2019 were collected from PubMed, Springer, Cochrane library, Web of Science, China National Knowledge Infrastructure (CNKI), and Wan Fang library. Odds ratios (OR) and 95% confidence intervals (CI) were used to estimate the association between these polymorphisms and KOA risk. The meta-analysis was completed by STATA 18.0 software.ResultsA total of 196 studies were collected, 16 of them included in final meta-analysis (7997 cases and 12,684 controls). There was significant association between GDF5 rs143383 polymorphism and KOA in all genetic models (for Allele model (C versus T): OR = 0.84 (95% CI = 0.76–0.91); dominate model (CC+CT versus TT): OR = 0.80 (95% CI = 0.72–0.90); recessive model (CC versus CT+TT): OR = 0.79 (95% CI = 0.68–0.92); heterozygote model (CT versus CC+TT): OR = 0.89 (95% CI = 0.80–0.97); homozygous model (CC versus TT): OR = 0.71 (95% CI = 0.60–0.85)). In the subgroup analysis, we obtained the results that there is no significance among Asians.ConclusionGDF5 rs143383 genetic polymorphism increases the risk of KOA among Caucasians; CC genotype and C allele are protective factors for the susceptibility of KOA among Caucasians.

An insight into osteoarthritis susceptibility: Integration of immunological and genetic background.

Osteoarthritis (OA) is a progressive degenerative disease that affects all synovial joints, causing the disability of the main locomotor diarthrodial joints. OA pathogenesis is caused by a complex interplay between a number of genetic and environmental risk factors, involved in the early onset and progression of this chronic inflammatory joint disease. Uncovering the underlying immunological and genetic mechanisms will enable an insight into OA pathophysiology and lead to novel and integrative approaches in the treatment of OA patients, together with a reduction of the disease risk, or a delay of its onset in susceptible patients.

Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC

SummaryObjectiveIn cartilage, the osteoarthritis (OA) associated single nucleotide polymorphism (SNP) rs11780978 correlates with differential expression of PLEC, and with differential methylation of PLEC CpG dinucleotides, forming eQTLs and mQTLs respectively. This implies that methylation links chondrocyte genotype and phenotype, thus driving the functional effect of this genetic risk signal. PLEC encodes plectin, a cytoskeletal protein that enables tissues to respond to mechanical forces. We sought to assess whether these PLEC functional effects were cartilage specific.MethodCartilage, fat pad, synovium and peripheral blood were collected from patients undergoing arthroplasty. PLEC CpGs were analysed for mQTLs and allelic expression imbalance (AEI) was performed to test for eQTLs. Plectin was knocked down in a mesenchymal stem cell (MSC) line using CRISPR/Cas9 and cells phenotyped by RNA-sequencing.ResultsmQTLs were discovered in fat pad, synovium and blood. Their effects were however stronger in the joint tissues and of comparable effect between these tissues. We observed AEI in synovium in the same direction as for cartilage and correlations between methylation and PLEC expression. Knocking-down plectin impacted on pathways reported to have a role in OA, including Wnt signalling, glycosaminoglycan biosynthesis and immune regulation.ConclusionsSynovium is also a target of the rs11780978 OA association functionally operating on PLEC. In fat pad, mQTLs were identified but these did not correlate with PLEC expression, suggesting the functional effect is not joint-wide. Our study highlights interplay between genetic risk, DNA methylation and gene expression in OA, and reveals clear differences between tissues from the same diseased joint.

Ancestral contribution of the muscle-specific creatine kinase (CKM) polymorphism rs4884 in the knee osteoarthritis risk: a preliminary study.

Articular cartilage and periarticular muscle tissues are strongly affected during knee osteoarthritis (OA). Creatine kinase (CK) is an enzyme expressed in several tissues, but the isoform CK-MM is specific of skeletal muscle, and its serum concentration is used as a biomarker of muscle damage. Genetic variants of the CKM gene have been associated with various pathologies, but to date, there are no reports of association with OA. Due to the rs4884 polymorphism being well represented in the Mexican population, it is used as an ancestry informative marker; thus, the goal of this preliminary report was to evaluate the association of this polymorphism in primary knee OA Mexican patients. Eighty-seven patients with primary knee OA were compared with 107 healthy controls. Serum CK-MM was determined using the dot blot system, and genotyping was performed using the OpenArray system. Logistic regression models were used to assess the association between the rs4884 polymorphism and OA susceptibility adjusting by gender, age, and body mass index. There were no significant differences in serum CK-MM values between patients and controls. The GG genotype and the G allele had a higher frequency in the control group compared with the OA group (24.3% vs. 12.6%, OR = 0.34, 95% CI = 0.14-0.84, P = 0.019; and 40.2% vs. 28.2%, OR = 0.51, 95% CI = 0.32-0.82, P = 0.005, respectively). Our results suggest a protection role of the rs4884 polymorphism against knee OA development; further studies are required to confirm it. Key Points • CK-MM enzyme catalyzes the conversion of creatine and ATP to create phosphocreatine and ADP; this reaction is reversible. • In tissues that consume ATP rapidly, such as skeletal muscle, the phosphocreatine serves as an important energy reservoir. • During knee OA, peripheral muscle tissues of the joint may be affected. • The rs4884 polymorphism of the CKM gene may participate as a protective factor in the development of OA.

Common variants in IL17F gene contributed to the risk of hip osteoarthritis susceptibility in Han Chinese population.

The prevalence of hip and knee osteoarthritis (OA) varies by ethnicity, suggesting genetic heterogeneity in populations and predilection sites. Given the unknown mechanism of IL17F gene in the etiology of OA, it is necessary to examine the potential shared susceptibility of IL17F gene between knee OA and hip OA (HOA). This study aimed to evaluate the association of the IL17F gene and susceptibility to HOA in a Han Chinese population. A total of 2650 study subjects, comprising 796 HOA patients and 1854 controls, were recruited into the present study. Seven tag single nucleotide polymorphisms (SNPs) were selected for genotyping. Single marker-based genetic association analyses were conducted at both the genotypic and allelic levels. χ2 statistics were calculated for statistical testing, and odds ratios were obtained to estimate the effects of genotypes and alleles for each SNP. The SNP rs763780 was identified to be significantly associated with the risk of HOA at both genotypic (χ2 =12.45, P = .002) and allelic levels (χ2 =11.83, P = .0006). A linkage disequilibrium (LD) block comprised of 3 SNPs (rs12201582-rs12203736-rs722323) was also significantly associated with the risk of HOA. In addition, rs2294835 was identified to be associated with HOA severity (χ2 =12.10, P = .02). Our results showed that IL17F gene contributed to the risk of HOA in a Han Chinese population, which would help to elucidate the pathogenesis of OA and facilitate the development of novel medicines and treatments for OA.

Association between the MMP-1-1607 1G/2G Polymorphism and Osteoarthritis Risk: A Systematic Review and Meta-Analysis.

Background Osteoarthritis (OA) is a common disease characterized by articular cartilage degeneration and secondary hyperosteogenesis. Genetic factors are associated with the occurrence of OA. While several studies have shown that the matrix metalloproteinase-1- (MMP-1-) 1607 1G/2G (rs1799750) polymorphism may be related to the occurrence and development of OA, there is inconsistency in the literature. To better estimate the relationship between the MMP-1 gene polymorphism and OA, a comprehensive meta-analysis of relevant literature was carried out. Results In total, seven studies comprising 1245 OA patients and 1230 controls were included in this meta-analysis. The combined results revealed no significant association between the MMP-1-1607 1G/2G polymorphism and risk of OA in the five genetic models. However, after Bonferroni correction, the results of subgroup analysis revealed a significant correlation between the MMP-1-1607 1G/2G polymorphism and OA susceptibility in the temporomandibular joint (TMJ) OA subgroup (allelic: 2G vs. 1G: OR = 1.575, 95%CI = 1.259–1.972, P < 0.01; recessive: 2G2G vs. 1G1G+1G2G: OR = 2.411, 95%CI = 1.658–3.504, P < 0.01; and homozygote: 2G2G vs. 1G1G: OR = 2.313, 95%CI = 1.341, 3.991, P = 0.003), the younger subgroup (aged less than 60 years; allelic: 2G vs. 1G: OR = 1.635, 95%CI = 1.354, 1.974, P < 0.01; dominant: 2G1G+2G2G vs. 1G1G: OR = 1.622, 95%CI = 1.158, 2.271, P = 0.005; recessive: 2G2G vs. 1G1G+1G2G: OR = 2.209, 95%CI = 1.718, 2.840, P < 0.01; and homozygote: 2G2G vs. 1G1G: OR = 2.578, 95%CI = 1.798, 3.696, P < 0.01), the larger subgroup (N > 300), and the hospital-based case-control study (HCC) subgroup. The sensitivity analysis suggested that the results of the meta-analysis were stable and reliable. Begg's funnel plot and Egger's test indicated that there was no publication bias in this study. Conclusion Our meta-analysis indicated that although the MMP-1-1607 1G/2G polymorphism was not significantly associated with OA susceptibility among the whole sample, it played a key role in the etiology and development of TMJ OA and OA in people aged less than 60 years.

Genetic correlations between cartilage regeneration and degeneration reveal an inverse relationship

The etiology of osteoarthritis (OA) is unknown, however, there appears to be a significant contribution from genetics. We have identified recombinant inbred strains of mice derived from LG/J (large) and SM/J (small) strains that vary significantly in their ability to repair articular cartilage and susceptibility to post-traumatic OA due to their genetic composition. Here, we report cartilage repair phenotypes in the same strains of mice in which OA susceptibility was analyzed previously, and determine the genetic correlations between phenotypes. We used 12 recombinant inbred strains, including the parental strains, to test three phenotypes: ear-wound healing (n=263), knee articular cartilage repair (n=131), and post-traumatic OA (n=53) induced by the surgical destabilization of the medial meniscus (DMM). Genetic correlations between various traits were calculated as Pearson's correlation coefficients of strain means. We found a significant positive correlation between ear-wound healing and articular cartilage regeneration (r=0.71; P=0.005). We observed a strong inverse correlation between articular cartilage regeneration and susceptibility to OA based on maximum (r=-0.54; P=0.036) and summed Osteoarthritis Research Society International (OARSI) scores (r=-0.56; P=0.028). Synovitis was not significantly correlated with articular cartilage regeneration but was significantly positively correlated with maximum (r=0.63; P=0.014) and summed (r=0.70; P=0.005) OARSI scores. Ectopic calcification was significantly positively correlated with articular cartilage regeneration (r=0.59; P=0.021). Using recombinant inbred strains, our study allows, for the first time, the measurement of genetic correlations of regeneration phenotypes with degeneration phenotypes, characteristic of OA (cartilage degeneration, synovitis). We demonstrate that OA is positively correlated with synovitis and inversely correlated with the ability to repair cartilage. These results suggest an addition to the risk paradigm for OA from a focus on degeneration to regeneration.

Genetic influence of candidate genes on the progression of knee osteoarthritis

Purpose: Osteoarthritis (OA) is a common disease worldwide leading to significant morbidity. The underlying disease process is multifactorial however there is increasing focus on molecular mechanisms. We examined the role of 11 single-nucleotide polymorphisms (SNPs) from 5 candidate genes in OA susceptibility and progression.

The molecular genetics and epigenetics of COLGALT2, a risk locus for osteoarthritis

Purpose: Genome wide association studies (GWAS) have identified over 90 association signals for osteoarthritis (OA). The majority of these mediate OA susceptibility by affecting the expression of their target genes, and are known as expression quantitative trait loci (eQTLs). Epigenetic modifications, such as DNA methylation (DNAm), can also influence gene expression and act as mediators between genotype at a single nucleotide polymorphism (SNP) and gene expression. Correlation between SNP genotype and DNAm at a CG dinucleotide (CpG) is known as a methylation quantitative trait locus (mQTL).

Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC

Purpose: We previously demonstrated that genotype at the OA associated SNP rs11780978, which resides within an intron of PLEC, correlates with differential expression of this gene and with differential methylation of PLEC CG dinucleotides (CpGs). These genetic, transcriptomic and epigenetic analyses were undertaken on cartilage DNA and RNA, and implied that DNA methylation links chondrocyte genotype and phenotype, with this epigenetic mechanism driving the functional impact of the rs11780978 risk signal. PLEC encodes Plectin, a cytoskeletal protein that regulates signalling from the extracellular space to the nucleus. In so doing, Plectin enables cells to respond to external mechanical stimuli and maintain tissue integrity. PLEC is expressed widely and in this study, we set out to test whether the PLEC functional effects that we had discovered were cartilage specific or whether they were active in other joint or non-joint tissues. Methods: RNA and genomic DNA were extracted from cartilage (n=35), infrapatellar fat-pad (n=68), synovium (n=79) and peripheral whole blood (n=54) tissue samples from patients who had undergone hip or knee arthroplasty for OA. Allelic expression imbalance (AEI) at PLEC was assessed by pyrosequencing in individuals heterozygous for the PLEC transcript SNP rs11783799 (pairwise r2=0.93 relative to rs11780978). The AEI was performed on DNA (providing the 1:1 ratio of allelic balance) and cDNA synthesised from the tissue RNA. DNA ratios were compared to cDNA ratios and P-values were calculated using a Mann-Whitney 2-tailed exact test. Methylation analysis of PLEC CpGs was performed by bisulphite conversion of tissue DNA followed by pyrosequencing. Patients were stratified by rs11780978 genotype and methylation levels compared, with P-values calculated using the Kruskal-Wallis test. Results: We observed AEI in synovium (P=0.005) in the same direction as for cartilage, with decreased expression of PLEC correlating with the OA risk-conferring A allele of rs11780978. We did not observe AEI in fat pad (P>0.05) and were unable to test for it in blood due to low expression level of the gene as confirmed by qPCR. The CpG analysis focussed on our two previously reported PLEC CpG clusters; cg19405177 and 7 nearby CpGs (in a 112bp interval), and cg14598846 and 3 adjacent CpGs (in a 23bp interval). In cartilage DNA, we replicated the correlation between rs11780978 genotype and methylation at the cg19405177 cluster (risk-conferring A allele of rs11780978 correlating with increased methylation; P<0.01) and at the cg14598846 cluster (risk-conferring A allele of rs11780978 correlating with decreased methylation; P<0.0001). We observed the same effects and in the same directions for synovium (cg19405177 cluster, P<0.001; cg14598846 cluster, P<0.0001), fat pad (cg19405177 cluster, P<0.05; cg14598846 cluster, P<0.0001) and blood (cg19405177 cluster, P<0.001; cg14598846 cluster, P<0.0001) DNAs. We next created a heat-map displaying the percentage effect of rs11780978 genotype on CpG methylation at the two clusters (Figure 1). This confirmed the effect of genotype on methylation across the four tissue types but highlighted that the effects are strongest in the joint tissues. Conclusions: Our study has revealed that as for cartilage, synovium is a target of the OA association that is functionally operating on PLEC expression and methylation; we observed AEI and a correlation between rs11780978 genotype and methylation for this tissue. In fat pad, although we observed methylation correlations, there was no AEI. This suggests that the functional effect of the association signal is not joint-wide and that there is a difference in sensitivity to epigenetic regulation of PLEC between cartilage/synovium and fat pad. These distinctions point toward regulatory variability in PLEC expression mediated by the same association signal depending on tissue type. This was exemplified further by our analysis of blood, with very low levels of PLEC expression and with a much weaker effect of rs11780978 genotype on methylation. Our study highlights the interplay between OA genetic risk, DNA methylation and gene expression, but reveals clear differences in these effects even for tissues from the same diseased joint.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Discovery and functional analysis of osteoarthritis susceptibility genes using pedigrees from a statewide population-based cohort

Purpose: Lack of understanding of the biological processes conferring susceptibility to OA and the paucity of animal models are key limitations to the development of effective therapies. Our goal is to discover molecular pathways that are vulnerability points for the development of OA and generate mouse models using human disease alleles. We discover these pathways by identifying rare gene mutations in coding sequences that have a strong effect on susceptibility to OA in families identified from a unique statewide population-based cohort, the Utah Population Database. From the family studies, we identify gene variants that we view as candidates for having a strong effect on OA susceptibility. To rapidly identify those candidate variants that measurably alter gene function, we quantitatively assay WT and variant gene functions in zebrafish embryos. We then determine whether and how the functionally altered genes confer susceptibility to OA in genetically modified mouse models. Methods: To discover pathways that when modified lead to strong and unambiguous susceptibility to OA, we analyzed the exomes of 50 unrelated families from the Utah Population Database that had a significant enrichment of shoulder OA, distal and proximal interphalangeal joint hand OA, erosive hand OA, and 1st metatarsophalangeal (MTP) joint foot OA. We use loss- and gain-of-function analyses in zebrafish to rapidly screen for gene variants that have a functional impact on protein activity. Finally, Crispr/Cas9 technology is used to introduce OA-associated disease alleles into the mouse where we analyze the how these mutations affect onset and progression of OA. Results: Our results indicate that we can use families to identify genes and pathways that confer strong susceptibility to the early stages of OA. Our approach has led to the discovery of candidate gene variants in families with different forms of OA that affect common pathways. These pathways include genes that regulate i) the proinflammatory response, ii) mechanotransduction/mechanosensation, iii) the TGF-b and Notch signaling pathways, and iv) collagen modification. We have identified mutations in the NOD-RIPK2 pathway in families with interphalangeal joint, erosive hand, and 1st MTP joint OA. The NOD-RIPK2 pathway is a central regulator of the proinflammatory response. We have shown the OA-associated variants have increased proinflammatory activity relative to the wildtype protein. Based on these findings, we propose the gene variants act dominantly as a gain-of-function allele to over-stimulate the inflammatory response to naturally occurring joint damage leading to OA. To test this hypothesis we introduced a single amino acid change, encoded by the RIPK2 disease allele, into an isogenic mouse. Current results indicate mice harboring the disease allele exhibit increased severity of OA when compared to controls. Erosive hand OA is characterized radiographically by centrally located interphalangeal joint erosions, and clinically by its rapid onset and progression. Genomic analysis of one family identified a rare coding variant in the procollagen galactosyltransferase COLGALT1. We are currently analyzing the functional impact of the COLGLAT1 gene variant in zebrafish and examining if collagen structure is disrupted in articular cartilage of mice with a Colgalt1 mutation. Conclusions: Our studies indicate that we can use families to identify genes and pathways that confer strong susceptibility to the early stages of OA. Our approach has led to the discovery of i) compelling novel candidate genes, ii) multiple genes that contribute to a common pathway, iii) and identification of common pathways altered in multiple forms of OA, iv) and to the generation of new mouse models of OA. Our genetic studies of families with OA and functional analyses in the mouse support our hypothesis that variation in the inflammatory response may be a key factor in susceptibility to OA. Our work is significant in that we have taken a novel approach to identify genes that when mutated have a significant impact on the onset or progression of OA. Developing new animal models with these susceptibility genes will be useful for discovery of therapeutic drugs.

GR/HDAC2/TGFβR1 pathway contributes to prenatal caffeine induced-osteoarthritis susceptibility in male adult offspring rats

Prenatal caffeine exposure (PCE) induces developmental toxicity of multi-organ and susceptibility to multi-disease in offspring. However, the effects of PCE on osteoarthritis susceptibility in adult offspring and its intrauterine programming mechanism remain to be further investigated. Here, we found that PCE induced susceptibility to osteoarthritis in male adult offspring rats, which was related to the inhibited function of cartilage matrix synthesis from fetuses to adults. Meanwhile, PCE consistently downregulated the H3K9ac and expression levels of transforming growth factor β receptor 1 (TGFβR1), and then blocked TGFβ signaling pathway, which contributed to the suppressed cartilage matrix synthesis. Moreover, the high level of corticosterone caused by PCE reduced the H3K9ac level on TGFβR1 promoter region through acting on glucocorticoids receptor (GR) and recruiting histone deacetylase 2 (HDAC2) into the nucleus of fetal chondrocytes. Taken together, PCE induced osteoarthritis susceptibility in male adult offspring rats, which was attributed to the low-functional programming of TGFβR1 induced by corticosterone via GR/HDAC2 signaling.

Association between IL-17A and IL-17F gene polymorphism and susceptibility in inflammatory arthritis: A meta-analysis

ObjectivesThe association between Interleukin (IL)-17A and IL-17F gene polymorphism with inflammatory arthritis were inconsistent among previous studies. This meta-analysis aimed to determine the association between IL-17A and IL-17F gene polymorphism with ankylosing spondylitis (AS), osteoarthritis (OA) and rheumatoid arthritis (RA). MethodsWe searched Medline up to August 2019. The summary Odds Ratio (OR) with corresponding 95% confidence intervals (CIs) was calculated to evaluate the relationship between IL-17A and IL-17F gene polymorphism with genetic susceptibility of AS, OA and RA. ResultsA total of 19 studies with 5298 cases and 5675 healthy controls were included. There were significant associations between rs2275913 G allele with OA, RA susceptibility (P < .05) but not AS. Subgroup analysis by ethnicity indicated that rs763780 C allele was closely related to AS and OA in Caucasian populations (P < .001) but not Mongolians. A significant association between rs763780 and RA susceptibility was detected in Caucasian populations (P < .05). ConclusionIL-17F gene rs763780 C allele confers increased risk of inflammatory arthritis in Caucasians; IL-17A gene rs2275913 G allele are protective for OA susceptibility in Mongolians. More well-designed studies with larger sample size are needed to elucidate the role of IL-17A gene rs2275913 G allele in inflammatory arthritis, especially AS.