Mitonuclear epistasis involving TP63 and haplogroup Uk: Risk of rapid progression of knee OA in patients from the OAI.

OBJECTIVE: To investigate genetic interactions between mitochondrial deoxyribonucleic acid (mtDNA) haplogroups and nuclear single nucleotide polymorphisms (nSNPs) to analyze their impact on the development of the rapid progression of knee osteoarthritis (OA). DESIGN: A total of 1095 subjects from the Osteoarthritis Initiative, with a follow-up time of at least 48-months, were included. Appropriate statistical approaches were performed, including generalized estimating equations adjusting for age, gender, body mass index, contralateral knee OA, Western Ontario and McMaster Universities Osteoarthritis Index pain, previous injury in target knee and the presence of the mtDNA variant m.16519C. Additional genomic data consisted in the genotyping of Caucasian mtDNA haplogroups and eight nSNPs previously associated with the risk of knee OA in robust genome-wide association studies. RESULTS: The simultaneous presence of the G allele of rs12107036 at TP63 and the haplogroup Uk significantly increases the risk of a rapid progression of knee OA (odds ratio = 1.670; 95% confidence interval [CI]: 1.031-2.706; adjusted p-value = 0.027). The assessment of the population attributable fraction showed that the highest proportion of rapid progressors was under the simultaneous presence of the G allele of rs12107036 and the haplogroup Uk (23.4%) (95%CI: 7.89-38.9; p-value < 0.05). The area under the curve of the cross-validation model (0.730) was very similar to the obtained for the predictive model (0.735). A nomogram was constructed to help clinicians to perform clinical trials or epidemiologic studies. CONCLUSIONS: This study demonstrates the existence of a mitonuclear epistasis in OA, providing new mechanisms by which nuclear and mitochondrial variation influence the susceptibility to develop different OA phenotypes.

Liver insulin-like growth factor-1 mediates effects of low-intensity vibration on wound healing in diabetic mice.

Chronic wounds in diabetic patients are associated with significant morbidity and mortality; however, few therapies are available to improve healing of diabetic wounds. Our group previously reported that low-intensity vibration (LIV) could improve angiogenesis and wound healing in diabetic mice. The purpose of this study was to begin to elucidate the mechanisms underlying LIV-enhanced healing. We first demonstrate that LIV-enhanced wound healing in db/db mice is associated with increased IGF1 protein levels in liver, blood, and wounds. The increase in insulin-like growth factor (IGF) 1 protein in wounds is associated with increased Igf1 mRNA expression both in liver and wounds, but the increase in protein levels preceded the increase in mRNA expression in wounds. Since our previous study demonstrated that liver was a primary source of IGF1 in skin wounds, we used inducible ablation of IGF1 in the liver of high-fat diet (HFD)-fed mice to determine whether liver IGF1 mediated the effects of LIV on wound healing. We demonstrate that knockdown of IGF1 in liver blunts LIV-induced improvements in wound healing in HFD-fed mice, particularly increased angiogenesis and granulation tissue formation, and inhibits the resolution of inflammation. This and our previous studies indicate that LIV may promote skin wound healing at least in part via crosstalk between the liver and wound. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clinical and Ecological Impact of an Educational Program to Optimize Antibiotic Treatments in Nursing Homes (PROA-SENIOR): A Cluster, Randomized, Controlled Trial and Interrupted Time-Series Analysis.

BACKGROUND: Antimicrobial stewardship programs (ASPs) are recommended in nursing homes (NHs), although data are limited. We aimed to determine the clinical and ecological impact of an ASP for NHs. METHODS: We performed a cluster, randomized, controlled trial and a before-after study with interrupted time-series analyses in 14 NHs for 30 consecutive months from July 2018 to December 2020 in Andalusia, Spain. Seven facilities implemented an ASP with a bundle of 5 educational measures (general ASP) and 7 added 1-to-1 educational interviews (experimental ASP). The primary outcome was the overall use of antimicrobials, calculated monthly as defined daily doses (DDD) per 1000 resident days (DRD). RESULTS: The total mean antimicrobial consumption decreased by 31.2% (-16.72 DRD; P = .045) with respect to the preintervention period; the overall use of quinolones and amoxicillin-clavulanic acid dropped by 52.2% (P = .001) and 42.5% (P = .006), respectively; and the overall prevalence of multidrug-resistant organisms (MDROs) decreased from 24.7% to 17.4% (P = .012). During the intervention period, 12.5 educational interviews per doctor were performed in the experimental ASP group; no differences were found in the total mean antimicrobial use between groups (-14.62 DRD; P = .25). Two unexpected coronavirus disease 2019 waves affected the centers increasing the overall mean use of antimicrobials by 40% (51.56 DRD; P < .0001). CONCLUSIONS: This study suggests that an ASP for NHs appears to be associated with a decrease in total consumption of antimicrobials and prevalence of MDROs. This trial did not find benefits associated with educational interviews, probably due to the coronavirus disease 2019 pandemic. Clinical Trials Registration. NCT03543605.

A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis.

OBJECTIVES: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. METHODS: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. RESULTS: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. CONCLUSIONS: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

Is osteoarthritis a mitochondrial disease? What is the evidence.

PROPOSE OF REVIEW: To summarize the evidence that suggests that osteoarthritis (OA) is a mitochondrial disease. RECENT FINDINGS: Mitochondrial dysfunction together with mtDNA damage could contribute to cartilage degradation via several processes such as: (1) increased apoptosis; (2) decreased autophagy; (3) enhanced inflammatory response; (4) telomere shortening and increased senescence chondrocytes; (5) decreased mitochondrial biogenesis and mitophagy; (6) increased cartilage catabolism; (7) increased mitochondrial fusion leading to further reactive oxygen species production; and (8) impaired metabolic flexibility. SUMMARY: Mitochondria play an important role in some events involved in the pathogenesis of OA, such as energy production, the generation of reactive oxygen and nitrogen species, apoptosis, authophagy, senescence and inflammation. The regulation of these processes in the cartilage is at least partially controlled by retrograde regulation from mitochondria and mitochondrial genetic variation. Retrograde regulation through mitochondrial haplogroups exerts a signaling control over the nuclear epigenome, which leads to the modulation of nuclear genes, cellular functions and development of OA. All these data suggest that OA could be considered a mitochondrial disease as well as other complex chronic disease as cancer, cardiovascular and neurologic diseases.

Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy.

OBJECTIVES: To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. METHODS: Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. RESULTS: An altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. CONCLUSIONS: Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

Mitochondrial DNA from osteoarthritic patients drives functional impairment of mitochondrial activity: a study on transmitochondrial cybrids.

With the redefinition of osteoarthritis (OA) and the understanding that the joint behaves as an organ, OA is now considered a systemic illness with a low grade of chronic inflammation. Mitochondrial dysfunction is well documented in OA and has the capacity to alter chondrocyte and synoviocyte function. Transmitochondrial cybrids are suggested as a useful cellular model to study mitochondrial biology in vitro, as they carry different mitochondrial variants with the same nuclear background. The aim of this work was to study mitochondrial and metabolic function of cybrids with mitochondrial DNA from healthy (N) and OA donors. In this work, the authors demonstrate that cybrids from OA patients behave differently from cybrids from N donors in several mitochondrial parameters. Furthermore, OA cybrids behave similarly to OA chondrocytes. These results enhance our understanding of the role of mitochondria in the degeneration process of OA and present cybrids as a useful model to study OA pathogenesis.

Postoperative Plasma Mitochondrial DNA and Cytokine Profiles of Elderly Patients Undergoing Minimally Invasive Aortic Valve Replacement.

INTRODUCTION: Mitochondrial DNA (mtDNA) is gaining increasing interest as a marker of cellular damage and could also act as an inflammatory mediator in cardiopulmonary bypass induced postoperative inflammatory response. Although minimally invasive heart valve surgery reportedly reduces inflammation, the mtDNA and cytokine profile in this context remains unclear. MATERIALS AND METHODS: Here, we report a prospective series of 40 elderly patients with aortic stenosis who underwent bioprosthetic aortic valve replacement (AVR) through upper ministernotomy with either a sutureless (n = 20) or a conventional (n = 20) valve. Primary end points included serial plasma levels of mtDNA (T1: at baseline; T2: 4 hours after surgery; and T3: 24s hour after surgery), cytokines (interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and myocardial necrosis biomarkers (MNBs), whereas secondary end points included clinical and echocardiographic data. RESULTS: Significant increases in the postoperative plasma levels (T2) of mtDNA, cytokines, and MNBs were observed in all patients. The postoperative plasma levels of mtDNA, TNF-α, and MNBs showed no significant differences between the treatment groups, although there was a trend toward lower levels in the sutureless group. The decreases in aortic cross-clamp and cardiopulmonary bypass times seen in the sutureless group were associated with significant lower postoperative levels (T2 and T3) of IL-6. CONCLUSION: AVR through upper ministernotomy was associated with a significant increase in postoperative plasma levels of mtDNA and cytokines. There was no difference in the mtDNA levels between the sutureless and conventional valve groups, suggesting a similar level of inflammation in both groups. However, the shorter operation time observed in the sutureless valve group was associated with significantly lower postoperative levels of IL-6, indicating potential clinical benefits.

Characterization of medical relevant anaerobic microorganisms by isothermal microcalorimetry.

Detection of anaerobe bacteria by culture methods requires appropriate media, special growth conditions, additional detection techniques and it typically takes several days. Therefore, anaerobes are often missed in patient specimens under routine culture conditions. Microcalorimetry may provide a simple and accurate real-time method for faster and better detection of anaerobes. An isothermal calorimeter which detect minimal changes of temperature over time was used for the calorimetric experiments. In order to find optimal growth conditions, seven reference or clinical strains of medical relevant anaerobe bacteria were tested under different circumstances. First, the strains were tested with different growth media. After determining the optimal medium for each strain, the gas phase was modified by adding 3 mL or 4 mL medium, to evaluate growth under conditions with less oxygen. Cooked Meat Medium was best supporting growth of the tested strains, including Cutibacterium acnes, Fusobacterium nucleatum, Finegoldia magna, Parvimonas micra, Bacteroides fragilis and Actinomyces odontolyticus, followed by thioglycolate. The best medium to detect Clostridioides difficile was H-Medium. All tested strains showed better growth in 4 mL medium than in 3 mL. The detection time ranged between 10 and 72 h. Our results demonstrated that the sensitivity and the detection time of anaerobe bacteria can be improved by isothermal calorimetry with optimization of growth conditions. Therefore, calorimetric detection, a practical, quick and easy-to-do method, has the potential to replace current microbiological methods.

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast-derived ADAMTS-7 and -12.

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS-7 and -12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK-Runx2 axis and Wnt/ß-catenin signalling in ADAMTS-12 and ADAMTS-7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL-1ß or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS-12 expression in OA-SF, also reducing Fn-fs-induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS-7 and COMP degradation in OA-SF as well. In addition, Wnt7B expression was induced by IL-1ß and by itself, also increasing ADAMTS-7. Our results could contribute to the development of disease-modifying OA drugs targeting ADAMTS-7 and -12 for the prevention of extracellular matrix components degradation like COMP.

In vitro antimicrobial activity against Abiotrophia defectiva and Granulicatella elegans biofilms.

OBJECTIVES: To determine the efficacy of different antibiotics (alone or in combination) against Abiotrophia defectiva and Granulicatella elegans biofilms and to investigate the anti-biofilm activity of gentamicin alone versus blood culture isolates from both species. METHODS: The activity of benzylpenicillin, clindamycin, daptomycin, fosfomycin, gentamicin, levofloxacin and rifampicin against 24-hour-old biofilms of A. defectiva and G. elegans was investigated in vitro by conventional microbiological methods and isothermal microcalorimetry. RESULTS: For planktonic bacteria, the MIC values of tested antibiotics ranged from 0.016 to 64 mg/L, as determined by microcalorimetry. Higher antibiotic concentrations, ranging from 1 to >1024 mg/L, were needed to produce an effect on biofilm bacteria. Gentamicin was an exception as it was active at 1 mg/L against both planktonic and biofilm G. elegans. A synergistic effect was observed when daptomycin was combined with benzylpenicillin, gentamicin or rifampicin against A. defectiva biofilms and when gentamicin was combined with rifampicin or levofloxacin against G. elegans biofilms. A. defectiva clinical isolates displayed greater variability in gentamicin susceptibility as compared with G. elegans strains. CONCLUSIONS: Antimicrobial susceptibility profiles vary widely between Abiotrophia and Granulicatella biofilms, and synergistic effects of the tested antibiotics were heterogeneous. The clinical relevance of these in vitro observations needs to be confirmed in experimental in vivo conditions and human trials, before guidelines for the treatment of A. defectiva and G. elegans infections are established. This study suggests the benefit of further clinical exploration of antibiotic combinations with anti-biofilm effect.

Real-Time Antimicrobial Susceptibility Assay of Planktonic and Biofilm Bacteria by Isothermal Microcalorimetry.

Most antimicrobials currently used in the clinical practice are tested as growth inhibitors against free-floating microorganisms in a liquid suspension, rather than against sessile cells constituting biofilms. Hence, reliable, fast, and reproducible methods for assessing biofilm susceptibility to antimicrobials are strongly needed. Isothermal microcalorimetry (IMC) is a nondestructive sensitive technique that allows for the real-time monitoring of microbial viability in the presence or absence of antimicrobial compounds. Therefore, the efficacy of specific antimicrobials, alone or in combination, may be promptly validated supporting the development of new drugs and avoiding the administration of ineffective therapies. Furthermore, the susceptibility of both planktonic and biofilm cells to antimicrobials can be conveniently assessed without the need for elaborated staining procedures and under nontoxic working conditions. Quantitative data regarding the antimicrobial effect against different strains might be collected by monitoring the microbial cell replication, and, more importantly, a dose-dependent activity can be efficiently detected by measuring the delay and decrease in the heat flow peak of the treated samples. A limitation of IMC for anti-biofilm susceptibility test is the inability to directly quantify the non-replicating cells in the biofilm or the total biomass. However, as IMC is a nondestructive method, the samples can be also analyzed by using different techniques, acquiring more information complementary to calorimetric data. IMC finds application also for the investigation of antibiotic eluting kinetics from different biomaterials, as well as for studying bacteriophages activity against planktonic and biofilm bacteria. Thus, the wide applicability of this ultra-sensitive and automated technique provides a further advance in the field of clinical microbiology and biomedical sciences.

Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis.

OBJECTIVES: To determine the antimicrobial activity against streptococcal biofilm in species mostly isolated from implant-associated infections and examine the effect of enzyme treatment of biofilm on the antimicrobial activity of different antibiotics. METHODS: The activities of fosfomycin, rifampicin, benzylpenicillin, daptomycin, gentamicin, levofloxacin, proteinase K and their combinations on planktonic and/or biofilm-embedded standard laboratory strains of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis were investigated in vitro by standard methods and isothermal microcalorimetry. RESULTS: MIC values obtained for the tested antimicrobials against planktonic bacteria ranged from 0.016 to 128 mg/L for the three species tested. Higher antibiotic concentrations were usually required to reduce biofilm in comparison with planktonic bacteria, with the exception of gentamicin, for which similar concentrations (4-16 mg/L) exerted an effect on both planktonic and biofilm cells. A synergistic effect against the streptococcal biofilm of the three species was observed when gentamicin was combined with benzylpenicillin or with rifampicin. Moreover, antibiotic concentrations comparable to the MIC observed against planktonic cells induced a strong reduction of viable bacteria in proteinase K pre-treated biofilm. CONCLUSIONS: This study shows that the combination of gentamicin with either benzylpenicillin or rifampicin exerts a synergistic effect against biofilms produced by the tested streptococci strains in vitro. Our results also suggest that coupling a dispersal agent with conventional antibiotics may facilitate their access to the bacteria within the biofilm. In vivo and clinical studies are needed in order to confirm whether such a strategy may be effective in the treatment of implant-associated infections caused by streptococci.

Mitochondrial DNA haplogroups influence the risk of incident knee osteoarthritis in OAI and CHECK cohorts. A meta-analysis and functional study.

OBJECTIVE: To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and therapeutic targets. METHODS: Two prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 2579 subjects of the incidence subcohort, and the cohort hip and cohort knee (CHECK) included 635, both with 8-year follow-up. The analysis included the association of mtDNA haplogroups with the rate of incident knee OA in subjects from both cohorts followed by a subsequent meta-analysis. Transmitochondrial cybrids harbouring haplogroup J or H were constructed to detect differences between them in relation to physiological features including specific mitochondrial metabolic parameters, reactive oxygen species production, oxidative stress and apoptosis. RESULTS: Compared with H, the haplogroup J associates with decreased risk of incident knee OA in subjects from OAI (HR=0.680; 95% CI 0.470 to 0.968; p<0.05) and CHECK (HR=0.728; 95% CI 0.469 to 0.998; p<0.05). The subsequent meta-analysis including 3214 cases showed that the haplogroup J associates with a lower risk of incident knee OA (HR=0.702; 95% CI 0.541 to 0.912; p=0.008). J cybrids show a lower free radical production, higher cell survival under oxidative stress conditions, lower grade of apoptosis as well as lower expression of the mitochondrially related pro-apoptotic gene BCL2 binding component 3 (BBC3). In addition, J cybrids also show a lower mitochondrial respiration and glycolysis leading to decreased ATP production. CONCLUSIONS: The physiological effects of the haplogroup J are beneficial to have a lower rate of incident knee OA over time. Potential drugs to treat OA could focus on emulating the mitochondrial behaviour of this haplogroup.

A replication study and meta-analysis of mitochondrial DNA variants in the radiographic progression of knee osteoarthritis.

OBJECTIVE: To conduct a replication study and meta-analysis involving the study of mtDNA variants in the radiographic progression of OA in different cohorts worldwide, including Cohort Hip and Cohort Knee (CHECK), the OA Initiative and a cohort from Spain. METHODS: The influence of the haplogroups in the rate of radiographic progression at 96 months in 431 subjects from CHECK was assessed in terms of Kellgren and Lawrence (KL) grade. Progression was defined as a change from KL ⩾ 1 at baseline to any higher grade during the follow-up. Extended Cox proportional hazard models were used to analyse the influence of mtDNA variants in the rate of radiographic knee OA progression. A subsequent meta-analysis of 1603 subjects following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was conducted to combine the data of individual studies. A sensitivity analysis was performed to validate the stability of the results. RESULTS: CHECK subjects carrying the haplogroup T showed the lowest rate of radiographic knee OA progression [hazard ratio (HR) 0.645 (95% CI 0.419, 0.978); P < 0.05]. When pooled, subjects within the superhaplogroup JT showed the same trend [HR 0.707 (95% CI 0.501, 0.965); P < 0.05]. BMI [HR 1.046 (95% CI 1.018, 1.073); P < 0.05] and bilateral OA [HR 2.266 (95% CI 1.733, 2.954); P < 0.05] at baseline are risk factors for radiographic knee OA progression as well. In the meta-analysis there was a reduced rate of radiographic progression in subjects with haplogroup T [HR 0.612 (95% CI 0.454, 0.824); P = 0.001] or in the superhaplogroup JT [HR 0.765 (95% CI 0.624, 0.938); P = 0.009]. Sensitivity analysis revealed that the results were robust. CONCLUSION: The mtDNA variants in the superhaplogroup JT associate with a reduced rate of radiographic OA progression. The mtDNA polymorphisms in the superhaplogroup JT emerge as potential complementary genetic biomarkers for disease progression.

Mitochondrial DNA haplogroups modulate the radiographic progression of Spanish patients with osteoarthritis.

Not all patients with osteoarthritis (OA) show the same disease progression, as some of them remain relatively stable over time, while others progress to severe structural deterioration of the joint. In this sense, the main goal of both genetic and protein biomarkers in OA is to predict not only the risk of OA at an earlier stage of the disease but also which OA patients are more likely to progress to severe disease. Taking into account the incidence of the mitochondria and the mtDNA haplogroups in the pathogenesis of OA, the main objective of this work was to evaluate the incidence of the mtDNA haplogroups in the radiographic progression of the OA disease in a well-characterized follow-up cohort of Spanish patients. DNA from 281 OA patients from Hospital Universitario A Coruña was isolated to determine the European mtDNA haplogroups. Knee or hip radiographs from all affected joints were obtained at two time points with at least 36 months apart. Radiographs were evaluated using the Kellgren/Lawrence (K/L) scale; radiographic OA progression was defined as any radiographic worsening of the K/L joint score. Statistical analyses included Kaplan-Meier survival curves and Cox regression models. Patients belonging to the cluster TJ showed a slower radiographic OA progression than patients in the cluster KU (p = 0.036). Moreover, patients carrying the most common mtDNA haplogroup H are more apt to require total joint replacement surgery than non-H patients (p = 0.049). The inherited mitochondrial variants influence the radiographic progression of OA and could be considered among the genetic variants taken into account when the radiographic progression of OA is analyzed.

Genome-wide DNA methylation analysis of articular chondrocytes reveals a cluster of osteoarthritic patients.

OBJECTIVE: Alterations in DNA methylation patterns have been found to correlate with several diseases including osteoarthritis (OA). The aim of this study was to identify, for the first time, the genome-wide DNA methylation profiles of human articular chondrocytes from OA cartilage and healthy control cartilage samples. METHODS: DNA methylation profiling was performed using Illumina Infinium HumanMethylation27 in 25 patients with OA and 20 healthy controls. Subsequent validation was performed by genome-wide expression analysis using the Affymetrix Human Gene 1.1 ST array in an independent cohort of 24 patients with OA. Finally, the most consistent genes in both assays were amplified by quantitative reverse transcriptase PCR in a validation cohort of 48 patients using microfluidic real-time quantitative PCR. Appropriate bioinformatics analyses were carried out using R bioconductor software packages and qBase plus software from Biogazelle. RESULTS: We found 91 differentially methylated (DM) probes, which permitted us to separate patients with OA from healthy controls. Among the patients with OA, we detected 1357 DM probes that identified a tight cluster of seven patients who were different from the rest. This cluster was also identified by genome-wide expression in which 450 genes were differentially expressed. Further validation of the most consistent genes in an independent cohort of patients with OA permitted us to identify this cluster, which was characterised by increased inflammatory processes. CONCLUSIONS: We were able to identify a tight subgroup of patients with OA, characterised by an increased inflammatory response that could be regulated by epigenetics. The identification and isolation of this subgroup may be critical for the development of effective treatment and disease prevention.