Search

Background In the last years the study of the mitochondria in the context of osteoarthritis (OA) attracted much attention. In the present work we aim to analyze the role of the mtDNA haplogroups in the development of incident knee OA in well-characterized study cohorts Objectives To analyze the influence of the mtDNA haplogroups in the rate of incident knee OA in the prospective cohorts of CHECK and OAI and to perform functional analysis with transmitochondrial cybrids to explore the consequences of the mitochondrial background in the pathogenesis of Osteoarthritis Methods The influence of the haplogroups in the development of incident knee OA at eight years in 2579 subjects from the Osteoarthritis Initiative (OAI) and 635 subjects from the Cohort hip and Cohort knee (CHECK) was assessed. A subsequent meta-analysis was conducted to strengthen the association of these individual studies. Transmitochondrial cybrids carrying the haplogroups J and H were constructed to study the influence of the mitochondrial background in different OA-related features. Results The haplogroup J significantly associates with a decreased risk of incident knee OA in subjects from the OAI (HR=0.680;95%CI=0.470–0.968;p Compared with the haplogroup H, haplogroup J shows a lower free radical production, specially peroxide/peroxynitrite (52.51±11.34 vs 41.26±7.48;p Conclusions The haplogroup J reduces the risk of incident knee OA. This mitochondrial variant constitute a protective phenotype against the development of OA, emerging as a powerful OA biomarker and even leading to the consideration of the mitochondrion as a potential therapeutic target in OA Acknowledgement We9d like to acknowledge to the participants of CHECK and OAI cohorts Disclosure of Interest None declared

Background Significant influence of the mtDNA haplogroups on radiographic progression of knee OA (KOA) has been reported; however, further validation in other well-defined prospective cohorts is necessary Objectives To validate, in patients from the CHECK cohort, the results obtained in patients from the OAI by which the mtDNA haplogroups influence the radiographic progression of knee Osteoarthritis. The CHECK cohort is a running 10-year prospective multi-center cohort of subjects, with pain and/or stiffness of one and/or both knees Methods We assigned the mtDNA haplogroups to the entire CHECK cohort (n=1002 subjects). In accordance with the early-stage OA in CHECK subjects (van Spil et al 2012), we selected subjects that did show radiographic KOA at baseline (K&L grade=1 for the left and/or right knee), reaching a total of n=417 subjects. Progression in these subjects was defined as an increase of ≥1 K&L grade for the left and/or right knee during 5 years follow-up period.Appropriate statistical analyses, including Receiver Operating Curves (ROC) with their respective Area Under the Curve (AUC), were carried out using SPSS software (v.19) Results The 417 subjects that met the eligibility criteria to study radiographic progression included 81.1% females and 18.9% males, they were older than 45 years (mean age: 56.24±5.07 years; range: 45-66) and had a mean BMI of 26.80 kg/m2 (range: 15.43-48.85). Chi-square analysis revealed that the mtDNA haplogroup T significantly associated with lower risk of radiographic progression (OR=0.467; CI=0.250-0.874; p=0.015); analysing by mtDNA clusters, the cluster TJ significantly associated with lower risk of progression too (OR=0.586; CI=0.366-0.939; p=0.025). By comparing each of the mtDNA haplogroups with the most common haplogroup H in the regression model, we detected that carriers of the mtDNA haplogroup T were at lower risk for KL CI=0.204-0.844; p=0.015); in terms of mtDNA clusters, subjects in cluster TJ were at lower risk of progression than subjects in the cluster HV (OR=0.551; CI=0.317-0.955; p=0.034). Additionally, either including mtDNA haplogroups or mtDNA clusters in the regression model, both BMI (OR=1.064; CI=1.010-1.120; p=0.020) and bilateral KL=1 at baseline (OR=3.595; CI=2.345-5.510; p Conclusions As noted in KOA patients of the OAI cohort, the early identification and classification of patients with haplogroup T and the most common haplogroup H would permit to identify those patients more prone to a rapid radiographic progression of the disease References Van Spil WE, et al. Cros-sectional and predictive associations between plasma adipokines and radiographic signs of early-stage knee osteoarthritis: data from CHECK. Osteoarthritis Cartilage 2012;20:1278-1285 Acknowledgements We would like to thank the participants, principal investigators, coinvestigators and staff of the CHECK study. Disclosure of Interest None declared

Background Previous studies have showed the mtDNA haplogroup J are associated with incidence and progression of OA. Transmitochondrial cybrids are optimal cellular models to study mitochondrial biology and function since they carry different mitochondrial variants with the same nuclear background, excluding those variations from the nuclear genome Objectives The aim of this work is to test the real role of mtDNA haplogroups in cellular activity, using cybrids with mtDNA haplogroup H and J Methods Cybrids were developed using 143B.TK- Rho-0 cell line and platelets from healthy (without OA) and OA donors with mtDNA haplogroups H and J. The metabolic status was evaluated by lactic acid production and glucose consumption. OXPHOS function was evaluated by O2 consumption (Oroboros®). The mitochondrial ROS production and percentage of apoptotic cells were measured by Flow Cytometry using DHR 123 and Annexin-V respectively. The expression levels of OA-related genes (Mn-SOD and IL-6), were evaluated by qRT-PCR. Appropriate statistical analyses were performed with GraphPad Prism v5 and qBase software Results J cybrids had higher lactic acid production than H (51.42 mg/ml and 64.22 mg/ml p

Background Oxidative stress and reactive oxygen species (ROS) production are key factors in the development of OA. Some reports have shown that those mtDNA haplogroups related with the mild uncoupling of the mitochondrial oxidative phosphorylation system (OXPHOS) are less prone to produce ROS. Objectives Taking into account that the mtDNA haplogroup J is a protective factor against OA in Spanish populations, the aim of this work is to compare the frequency distribution of the mtDNA haplogroups in OA patients and healthy controls between United Kingdom (UK) and Spain. Methods We used the single base extension (SBE) assay combined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to obtain the European mtDNA haplogroups in 1471 OA patients and 406 healthy controls from Spain, and 453 OA patients and 280 healthy controls from UK. Some of the differential single nucleotide polymorphisms (SNPs) haplogroup J-related between both populations were also analyzed using the SBE assay. The resulting frequencies were analyzed with SPSS software (v.18) following appropriate statistical analyses. Results The mtDNA haplogroup J appeared significantly underrepresented in OA patients from the north of Spain when compared with healthy controls (OR=0.614; 95% CI: 0.426–0.884; p=0.008). Individuals from UK carrying the mtDNA haplogroup T showed a significantly decreased risk of OA (OR=0.581; 95% CI: 0.365–0.926; p=0.021). The mtDNA haplogroup J bordered the statistical significance towards a decreased presence in healthy controls from UK (OR=1.686; 95% CI: 0.929–3.061; p=0.083). The analysis of the differentially expressed haplogroup J-related SNPs in Spain and UK, m.14798t>c, m.15257g>a and m.3394t>c, showed that the SNP m.3394t>c appeared significantly underrepresented in the UK cohort (p=0.029). Conclusions The mitochondrial uncoupling mechanism, derived from the mtDNA haplogroups, is a protective factor against OA in Spain and UK populations. This association is probably due to a reduced reactive oxygen species production as a consequence of the climate adaptation of these mtDNA haplogroups. Disclosure of Interest None Declared

Background Many genes, many of them still unknown, are involved in the etiology and development of OA. Today, different tools are available to try to identify some of the key genes related to the OA process. Objectives To perform a genome-wide expression assay in order to identify different expression profiles in the OA disease Methods Total RNA from OA cartilage samples was isolated with RNeasy Kit (Qiagen, Madrin, Spain) following manufacturer’s instructions. RNA was checked for integrity and purity with the Agilent Bioanalyzer (Agilent Technologies) and NanoDrop spectrophotometer (Thermo Scientific). 150 nanograms of total RNA were used for cDNA synthesis using the Ambion WT Expression kit (Ambion). The fragmented cDNA was hybridized against the Human Gene 1.1 ST array strip (Affymetrix) and scanned using the GeneTitan system (Affymetrix). Quality controls, normalization, pre-processing, differential gene expression and functional analyses were carried out with Bioconductor packages using R software. Results Human Gene 1.1 ST Array, which interrogates more than 28,000 well-annotated genes of 33,297 probes, was used for studying the genome wide expression profile of 23 OA-patients tissue samples. A non-specific filtering was previously applied for removing those probes with non-annotation information and with low intra-array variation. An unsupervised machine learning approach revealed a group of samples highly different (Figure 1). The differential expression analysis between this cluster, comprising a total of five OA-patients, and the rest of the samples allowed for the identification of 176 differentially expressed probes with an adjusted p value below 0.0001. The 20 differentially expressed probes with higher log Fold Change are presented in Table 1. The analysis of the biological processes related to these differentially expressed genes showed that inflammation and immune processes were the main pathways found to be altered when a gene set enrichment analysis was applied. Conclusions The genome-wide expression analysis shows a clearly distinct profile for a group of OA patients. Both inflammation and immune response processes appeared to be altered and therefore are revealed as key factors in the development of the OA disease. Disclosure of Interest None Declared