High microsatellite and SNP genotyping success rate established in a large number of genomic DNA samples extracted from mouth swabs and genotypes

In this article, we present the genomic DNA yield and the microsatellite and single nucleotide polymorphism (SNP) genotyping success rates of genomic DNA extracted from a large number of mouth swab samples. In total, the median yield and quality was determined in 714 individuals and the success rates in 378,480 genotypings of 915 individuals. The median yield of genomic DNA per mouth swab was 4.1 μg (range 0.1-42.2 μg) and was not reduced when mouth swabs were stored for at least 21 months prior to extraction. A maximum of 20 mouth swabs is collected per participant. Mouth swab samples showed in, respectively, 89% for 390 microsatellites and 99% for 24 SNPs a genotyping success rate higher than 75%. A very low success rate of genotyping (0%-10%) was obtained for 3.2% of the 915 mouth swab samples using microsatellite markers. Only 0.005% of the mouth swab samples showed a genotyping success rate lower than 75% (range 58%-71 %) using SNPs. Our results show that mouth swabs can be easily collected, stored by our conditions for months prior to DNA extraction and result in high yield and high-quality DNA appropriate for genotyping with high success rate including whole genome searches using microsatellites or SNPs

Family based association analyses between the Serotonin Transporter Gene Polymorphism (5-HTTLPR) and Neuroticism, Anxiety and Depression

We studied the association between the short/long promotor-based length polymorphism of the serotonin transporter gene (5-HTTLPR) and neuroticism, anxiety and depression. Subjects included twins, their siblings and parents from the Netherlands Twin Register (559 parents and 1,245 offspring). Subjects had participated between one and five times in a survey study measuring neuroticism, anxiety and depression. Offspring of these families were also approached to participate in a psychiatric interview diagnosing DSM-IV major depression. Within-family and total association between 5-HTTLPR and these traits were tested. Only three of the 36 tests showed a significant effect of 5-HTTLPR (P < 0.05). These effects were in opposite directions, i.e. both negative and positive regression coefficients were found for the s allele. No additive effect of the s allele was found for DSM-IV depression. Our results strongly suggest that there is no straightforward association between 5-HTTLPR and neuroticism, anxiety and depression

A powerful and rapid approach to human genome scanning in small quantities of genomic DNA

Dense maps of short-tandem-repeat polymorphisms (STRPs) have allowed genome-wide searches for genes involved in a great variety of diseases with genetic influences, including common complex diseases. Generally for this purpose, marker sets with a 10 cM spacing are genotyped in hundreds of individuals. We have performed power simulations to estimate the maximum possible intermarker distance that still allows for sufficient power. In this paper we further report on modifications of previously published protocols, resulting in a powerful screening set containing 229 STRPs with an average spacing of 18·3 cM. A complete genome scan using our protocol requires only 80 multiplex PCR reactions which are all carried out using one set of conditions and which do not contain overlapping marker allele sizes. The multiplex PCR reactions are grouped by sets of chromosomes, which enables on-line statistical analysis of a set of chromosomes, as sets of chromosomes are being genotyped. A genome scan following this modified protocol can be performed using a maximum amount of 2.5 μg of genomic DNA per individual, isolated from either blood or from mouth swabs.link_to_subscribed_fulltex

Genome wide DNA methylation profiling of osteoarthritic articular cartilage

Optimising joint reconstruction management in arthritis and bone tumour patient

Circulating microRNAs highly correlate to expression of cartilage genes potentially reflecting OA susceptibility: towards identification of applicable early OA biomarkers

Objective: To identify and validate circulating micro RNAs (miRNAs) that mark gene expression changes in articular cartilage early in osteoarthritis (OA) pathophysiology process. Methods: Within the ongoing RAAK study, human preserved OA cartilage and plasma (N = 22 paired samples) was collected for RNA sequencing (respectively mRNA and miRNA). Spearman correlation was determined for 114 cartilage genes consistently and significantly differentially expressed early in osteoarthritis and 384 plasma miRNAs. Subsequently, the minimal number of circulating miRNAs serving to discriminate between progressors and non-progressors was assessed by regression analysis and area under receiver operating curves (AUC) was calculated with progression data and plasma miRNA sequencing from the GARP study (N = 71). Results: We identified strong correlations (rho >= |0.7|) among expression levels of 34 unique plasma miRNAs and 21 genes, including 4 genes that correlated with multiple miRNAs. The strongest correlation was between let-7d-5p and EGFLAM (rho = -0.75, P = 6.9 x 10(-5)). Regression analysis of the 34 miRNAs resulted in a set of 7 miRNAs that, when applied to the GARP study, demonstrated clinically relevant predictive value with AUC > 0.8 for OA progression over 2 years and near-clinical value for progression over 5 years- (AUC = 0.8). Conclusions: We show that plasma miRNAs levels reflect gene expression levels in cartilage and can be exploited to represent ongoing pathophysiological processes in articular cartilage. We advocate that identified signature of 7 plasma miRNAs can contribute to direct further studies toward early biomarkers predictive for progression of osteoarthritis over 2 and 5 years.Molecular Epidemiolog

Human osteochondral explants: reliable biomimetic models to investigate disease mechanisms and develop personalized treatments for osteoarthritis

Introduction: Likely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment causing a large social and economic burden. In this study we set out to explore responses of aged human osteochondral explants upon different OA-related perturbing triggers (inflammation, hypertrophy and mechanical stress) for future tailored biomimetic human models.Methods: Human osteochondral explants were treated with IL-1 beta (10 ng/ml) or triiodothyronine (T3; 10 nM) or received 65% strains of mechanical stress (65% MS). Changes in chondrocyte signalling were determined by expression levels of nine genes involved in catabolism, anabolism and hypertrophy. Breakdown of cartilage was measured by sulphated glycosaminoglycans (sGAGs) release, scoring histological changes (Mankin score) and mechanical properties of cartilage.Results: All three perturbations (IL-1 beta, T3 and 65% MS) resulted in upregulation of the catabolic genes MMP13 and EPAS1. IL-1 beta abolished COL2A1 and ACAN gene expression and increased cartilage degeneration, reflected by increased Mankin scores and sGAGs released. Treatment with T3 resulted in a high and significant upregulation of the hypertrophic markers COL1A1, COL10A1 and ALPL. However, 65% MS increased sGAG release and detrimentally altered mechanical properties of cartilage.Conclusion: We present consistent and specific output on three different triggers of OA. Perturbation with the pro-inflammatory IL-1 beta mainly induced catabolic chondrocyte signalling and cartilage breakdown, while T3 initiated expression of hypertrophic and mineralization markers. Mechanical stress at a strain of 65% induced catabolic chondrocyte signalling and changed cartilage matrix integrity. The major strength of our ex vivo models was that they considered aged, preserved, human cartilage of a heterogeneous OA patient population. As a result, the explants may reflect a reliable biomimetic model prone to OA onset allowing for development of different treatment modalities.Orthopaedics, Trauma Surgery and Rehabilitatio

RNA sequencing reveals interacting key determinants of osteoarthritis acting in subchondral bone and articular cartilage: identification of IL11 and CHADL as attractive treatment targets

Objective To identify key determinants of the interactive pathophysiologic processes in subchondral bone and cartilage in osteoarthritis (OA).Methods We 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.Results We 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 x 10(-5)) and STMN2 (FC 9.6, FDR 2.36 x 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.Molecular Epidemiolog

Inhibiting thyroid activation in aged human explants prevents mechanical induced detrimental signalling by mitigating metabolic processes

Objectives To investigate whether the deiodinase inhibitor iopanoic acid (IOP) has chondroprotective properties, a mechanical stress induced model of human aged explants was used to test both repeated dosing and slow release of IOP. Methods Human osteochondral explants subjected to injurious mechanical stress (65%MS) were treated with IOP or IOP encapsulated in poly lactic-co-glycolic acid-polyethylene glycol nanoparticles (NP-IOP). Changes to cartilage integrity and signalling were determined by Mankin scoring of histology, sulphated glycosaminoglycan (sGAG) release and expression levels of catabolic, anabolic and hypertrophic markers. Subsequently, on a subgroup of samples, RNA sequencing was performed on 65%MS (n = 14) and 65%MS+IOP (n = 7) treated cartilage to identify IOP's mode of action. Results Damage from injurious mechanical stress was confirmed by increased cartilage surface damage in the Mankin score, increased sGAG release, and consistent upregulation of catabolic markers and downregulation of anabolic markers. IOP and, though less effective, NP-IOP treatment, reduced MMP13 and increased COL2A1 expression. In line with this, IOP and NP-IOP reduced cartilage surface damage induced by 65%MS, while only IOP reduced sGAG release from explants subjected to 65%MS. Lastly, differential expression analysis identified 12 genes in IOP's mode of action to be mainly involved in reducing metabolic processes (INSIG1, DHCR7, FADS1 and ACAT2) and proliferation and differentiation (CTGF, BMP5 and FOXM1). Conclusion Treatment with the deiodinase inhibitor IOP reduced detrimental changes of injurious mechanical stress. In addition, we identified that its mode of action was likely on metabolic processes, cell proliferation and differentiation.Molecular Epidemiolog

Underlying molecular mechanisms of DIO2 susceptibility in symptomatic osteoarthritis

Objectives: To investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches. Methods: Epigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid). Results: OA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (ß=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (ß=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2a/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes. Conclusions: Our findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach

Identification and characterization of two consistent osteoarthritis subtypes by transcriptome and clinical data integration

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