AbstractmicroRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280
Objective To identify methylation quantitative trait loci (mQTLs) correlating with osteoarthritis (OA) risk alleles and to undertake mechanistic characterization as a means of target gene prioritization. Methods We used genome‐wide genotyping and cartilage DNA methylation array data in a discovery screen of novel OA risk loci. This was followed by methylation, gene expression analysis, and genotyping studies in additional cartilage samples, accompanied by in silico analyses. Results We identified 4 novel OA mQTLs. The most significant mQTL contained 9 CpG sites where methylation correlated with OA risk genotype, with 5 of the CpG sites having P values <1 × 10−10. The 9 CpG sites reside in an interval of only 7.7 kb within the PLEC gene and form 2 distinct clusters. We were able to prioritize PLEC and the adjacent gene GRINA as independent targets of the OA risk. We identified PLEC and GRINA expression QTLs operating in cartilage, as well as methylation‐expression QTLs operating on the 2 genes. GRINA and PLEC also demonstrated differential expression between OA hip and non‐OA hip cartilage. Conclusion PLEC encodes plectin, a cytoskeletal protein that maintains tissue integrity by regulating intracellular signaling in response to mechanical stimuli. GRINA encodes the ionotropic glutamate receptor TMBIM3 (transmembrane BAX inhibitor 1 motif–containing protein family member 3), which regulates cell survival. Based on our results, we hypothesize that in a joint predisposed to OA, expression of these genes alters in order to combat aberrant biomechanics, and that this is epigenetically regulated. However, carriage of the OA risk–conferring allele at this locus hinders this response and contributes to disease development.
Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.
Developmental dysplasia of the hip (DDH) is the most common skeletal developmental disease. However, its genetic architecture is poorly understood. We conduct the largest DDH genome-wide association study to date and replicate our findings in independent cohorts. We find the heritable component of DDH attributable to common genetic variants to be 55% and distributed equally across the autosomal and X-chromosomes. We identify replicating evidence for association between GDF5 promoter variation and DDH (rs143384, effect allele A, odds ratio 1.44, 95% confidence interval 1.34–1.56, P = 3.55 × 10−22). Gene-based analysis implicates GDF5 (P = 9.24 × 10−12), UQCC1 (P = 1.86 × 10−10), MMP24 (P = 3.18 × 10−9), RETSAT (P = 3.70 × 10−8) and PDRG1 (P = 1.06 × 10−7) in DDH susceptibility. We find shared genetic architecture between DDH and hip osteoarthritis, but no predictive power of osteoarthritis polygenic risk score on DDH status, underscoring the complex nature of the two traits. We report a scalable, time-efficient recruitment strategy and establish for the first time to our knowledge a robust DDH genetic association locus at GDF5.
Objective Osteoarthritis (OA) is an age‐related disease characterized by articular cartilage degeneration. It is largely heritable, and genetic screening has identified single‐nucleotide polymorphisms (SNPs) marking genomic risk loci. One such locus is marked by the G>A SNP rs75621460, downstream of TGFB1. This gene encodes transforming growth factor β1, the correct expression of which is essential for cartilage maintenance. This study investigated the regulatory activity of rs75621460 to characterize its impact on TGFB1 expression in disease‐relevant patient samples (n = 319) and in Tc28a2 immortalized chondrocytes. Methods Articular cartilage samples from human patients were genotyped, and DNA methylation levels were quantified using pyrosequencing. Gene reporter and electrophoretic mobility shift assays were used to determine differential nuclear protein binding to the region. The functional impact of DNA methylation on TGFB1 expression was tested using targeted epigenome editing. Results The analyses showed that SNP rs75621460 was located within a TGFB1 enhancer region, and the OA risk allele A altered transcription factor binding, with decreased enhancer activity. Protein complexes binding to A (but not G) induced DNA methylation at flanking CG dinucleotides. Strong correlations between patient DNA methylation levels and TGFB1 expression were observed, with directly opposing effects in the cartilage and the synovium at this locus. This demonstrated biologic pleiotropy in the impact of the SNP within different tissues of the articulating joint. Conclusion The OA risk SNP rs75621460 impacts TGFB1 expression by modulating the function of a gene enhancer. We propose a mechanism by which the SNP impacts enhancer function, providing novel biologic insight into one mechanism of OA genetic risk, which may facilitate the development of future pharmacologic therapies.
Summary Objective In 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. Method Cartilage, 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. Results mQTLs 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. Conclusions Synovium 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.
Osteoarthritis (OA) is a common joint disease characterised by the focal loss of the protective cartilage layer at the ends of the bones. It is painful, disabling, multifactorial and polygenic. The growth differentiation factor 5 gene GDF5 was one of the first reported OA susceptibility signals that showed consistent association to OA, with the transcript single nucleotide polymorphism (SNP) rs143383 demonstrating association in Asians and Europeans. The functional effect of the signal is reduced expression of the gene. The GDF5 protein is an extracellular matrix signalling molecule that is active during chondrogenesis and in mature chondrocytes. Due to the functional impact of the susceptibility, we previously assessed the effect of supplementing chondrocytes from OA patients with exogenous GDF5. Their response was highly discordant, precluding the application of GDF5 as a simple means of attenuating the genetic deficit. Since GDF5 is also active during development, we have now assessed the effect of exogenous GDF5 on bone marrow derived mesenchymal stem cells (MSCs) that are undergoing chondrogenesis during cartilage disc formation. MSCs from healthy donors and OA patients were studied and the effect of GDF5 was assessed by measuring the wet mass of the discs, by histological staining, and by monitoring the change in expression of anabolic, catabolic and hypertrophic protein-coding genes. The MSCs expressed the three principal GDF5 receptor genes and responded in a significantly anabolic manner (increase in wet mass, p = 0.0022; Bonferroni corrected p = 0.018) to a variant form of GDF5 that targets the most abundantly expressed receptor, BMPR-IA. GDF5 elicited significant (p < 0.05) changes in the expression of anabolic, catabolic and hypertrophic genes with several consistent effects in healthy donors and in OA patients. Our data implies that, unlike OA chondrocytes, OA MSCs do respond in a predictable, anabolic manner to GDF5, which could therefore provide a route to modulate the genetic deficit mediated by the rs143383 association signal.
Running headline: Epigenetic analysis of the PLEC OA risk locusObjective 1 Osteoarthritis (OA) associated single nucleotide polymorphism (SNP) rs11780978 correlates 2 with differential expression of PLEC, and methylation quantitative trait loci (mQTLs) at PLEC 3 CpGs in cartilage. This implies that methylation links chondrocyte genotype and phenotype, 4 thus driving the functional effect. PLEC encodes plectin, a cytoskeletal protein that enables 5 tissues to respond to mechanical forces. We sought to assess whether PLEC functional effects 6 were cartilage specific. 7 8 Method 9Cartilage, fat pad, synovium and peripheral blood were collected from patients undergoing 10 arthroplasty. PLEC CpGs were analysed for mQTLs and allelic expression imbalance (AEI) 11 was performed. We focussed on previously reported mQTL clusters neighbouring cg19405177 12 and cg14598846. Plectin was knocked down in a mesenchymal stem cell (MSC) line using 13 CRISPR/Cas9 and cells phenotyped by RNA-sequencing. 14 15Results 16 Novel mQTLs were discovered in fat pad, synovium and peripheral blood at both clusters. The 17 genotype-methylation effect of rs11780978 was stronger in cg14598846 than in cg19405177 18 and stronger in joint tissues than in peripheral blood. We observed AEI in synovium in the 19 same direction as for cartilage. Knocking-down plectin impacted on pathways reported to have 20 a role in OA, including Wnt signalling, glycosaminoglycan biosynthesis and immune 21 regulation. 22 23 Conclusions 24Synovium is also a target of the rs11780978 OA association functionally operating on PLEC. 25In fat pad, mQTLs were identified but these did not correlate with PLEC expression, suggesting 26 the functional effect is not joint-wide. Our study highlights interplay between genetic risk, 27 DNA methylation and gene expression in OA, and reveals clear differences between tissues 28 from the same diseased joint. 29 30
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