Epigenetic mechanisms of osteoarthritis risk in human skeletal development.

The epigenome, including the methylation of cytosine bases at CG dinucleotides, is intrinsically linked to transcriptional regulation. The tight regulation of gene expression during skeletal development is essential, with ~1/500 individuals born with skeletal abnormalities. Furthermore, increasing evidence is emerging to link age-associated complex genetic musculoskeletal diseases, including osteoarthritis (OA), to developmental factors including joint shape. Multiple studies have shown a functional role for DNA methylation in the genetic mechanisms of OA risk using articular cartilage samples taken from aged patients. Despite this, our knowledge of temporal changes to the methylome during human cartilage development has been limited. We quantified DNA methylation at ~700,000 individual CpGs across the epigenome of developing human articular cartilage in 72 samples ranging from 7-21 post-conception weeks, a time period that includes cavitation of the developing knee joint. We identified significant changes in 8% of all CpGs, and >9400 developmental differentially methylated regions (dDMRs). The largest hypermethylated dDMRs mapped to transcriptional regulators of early skeletal patterning including MEIS1 and IRX1. Conversely, the largest hypomethylated dDMRs mapped to genes encoding extracellular matrix proteins including SPON2 and TNXB and were enriched in chondrocyte enhancers. Significant correlations were identified between the expression of these genes and methylation within the hypomethylated dDMRs. We further identified 811 CpGs at which significant dimorphism was present between the male and female samples, with the majority (68%) being hypermethylated in female samples. Following imputation, we captured the genotype of these samples at >5 million variants and performed epigenome-wide methylation quantitative trait locus (mQTL) analysis. Colocalization analysis identified 26 loci at which genetic variants exhibited shared impacts upon methylation and OA genetic risk. This included loci which have been previously reported to harbour OA-mQTLs (including GDF5 and ALDH1A2), yet the majority (73%) were novel (including those mapping to CHST3, FGF1 and TEAD1). To our knowledge, this is the first extensive study of DNA methylation across human articular cartilage development. We identify considerable methylomic plasticity within the development of knee cartilage and report active epigenomic mediators of OA risk operating in prenatal joint tissues.

Using pre-fracture mobility to augment prediction of post-operative outcomes in hip fracture.

PURPOSE: Pre-operative scores based on patient characteristics are commonly used to predict hip fracture outcomes. Mobility, an indicator of pre-operative function, has been neglected as a potential predictor. We assessed the ability of pre-fracture mobility to predict post-operative outcomes following hip fracture. METHODS: We analysed prospectively collected data from hip fracture surgery patients at a large-volume trauma unit. Mobility was classified into four groups. Post-operative outcomes studied were mortality and residence at 30 days, medical complications within 30- or 60-days post-operatively, and prolonged length of stay (LOS, ≥ 28 days). We performed multivariate regression analyses adjusting for age and sex to assess the discriminative ability of the Nottingham Hip Fracture Score (NHFS), with and without mobility, for predicting outcomes using the area under the receiver operating characteristic curve (AUROC). RESULTS: 1919 patients were included, mean age 82.6 (SD 8.2); 1357 (70.7%) were women. Multivariate analysis demonstrated patients with worse mobility had a 1.7-5.5-fold higher 30-day mortality (p ≤ 0.001), and 1.9-3.2-fold higher likelihood of prolonged LOS (p ≤ 0.001). Worse mobility was associated with a 2.3-3.8-fold higher likelihood of living in a care home at 30-days post-operatively (p < 0.001) and a 1.3-2.0-fold higher likelihood of complications within 30 days (p ≤ 0.001). Addition of mobility improved NHFS discrimination for discharge location, AUROC NHFS 0.755 [0.733-0.777] to NHFS + mobility 0.808 [0.789-0.828], and LOS, AUROC NHFS 0.584 [0.557-0.611] to NHFS + mobility 0.616 [0.590-0.643]. CONCLUSION: Incorporating mobility assessment into risk scores may improve casemix adjustment, prognostication following hip fracture, and identify high-risk patient groups requiring enhanced post-operative care at admission.

Implementing grip strength assessment in hip fracture patients: a feasibility project.

OBJECTIVES: Risk stratification scores are used in hip fracture surgery, but none incorporate objective tests for low muscle strength. Grip strength testing is simple and cheap but not routinely assessed for patients with hip fracture. This project aimed to assess the feasibility of implementing grip strength testing into admission assessment of patients with hip fracture. METHODS: A scalable protocol and a corresponding training programme of instructional presentations and practical assessments were designed and delivered by and for physiotherapy staff. Grip strength values were collected pre-surgery on patients with hip fracture at a single centre whilst supine in bed. Implementation of the process was evaluated using narrative, quantitative and cost measures. RESULTS: 53 hip fracture patients with a mean age 80.6 (SD 10.4), of which 36 (67.9%) were female, were included. Testing was offered to 42/52 (81%) patients. Cognitive impairment prevented 14/42 (33%) of patients from completing testing; one patient declined testing. Of the 27 patients who completed testing, 14/27 (52%) had low grip strength as defined by EWGSOP2 criteria. The projected cost of testing for one year was £2.68-£2.82 per patient. Fidelity to the protocol was high using multiple criteria. CONCLUSIONS: Grip strength assessment is acceptable to physiotherapy staff and can be rapidly and cost-effectively implemented into hip fracture admission assessment.

The impact of research intercalation during medical school on post-graduate career progression.

BACKGROUND: Medical students at The University of Manchester have the option of research intercalation on the Master of Research programme. There is a paucity of evidence for the benefits of research intercalation. However, we hypothesised that research intercalation would accelerate post-graduate career progression and aimed to objectively measure the career enhancing impact, quantify the benefits and determine the alumni perception of research intercalation. METHODS: Data was collected retrospectively by electronic questionnaire (in 2018) from those commencing research intercalation between 2005 and 2012. RESULTS: Participants (n=52) returned questionnaires (68% response), demonstrating that the cohort had completed 67 postgraduate qualifications, published 304 manuscripts (median 3 publications per person (PP); range: 0-53) and made 430 presentations (median 7 PP; range: 0-37). Alumni had been awarded 49 research grants; funding disclosed on 43% totalled £823,000. Career progression of 73% of alumni had taken the minimum number of years; 27% took longer due to time spent working abroad or to gain additional experience prior to specialty training. Fifty-five publications and 71 presentations were directly related to MRes projects. CONCLUSION: Research intercalation provides graduates with an opportunity to learn valuable transferrable skills, contribute to translational research, and objectively enhances medical career progression.

Prediction of Postoperative Outcomes Following Hip Fracture Surgery: Independent Validation and Recalibration of the Nottingham Hip Fracture Score.

OBJECTIVES: Independent validation of risk scores after hip fracture is uncommon, particularly for evaluation of outcomes other than death. We aimed to assess the Nottingham Hip Fracture Score (NHFS) for prediction of mortality, physical function, length of stay, and postoperative complications. DESIGN: Analysis of routinely collected prospective data partly collected by follow-up interviews. SETTING AND PARTICIPANTS: Consecutive hip fracture patients were identified from the Northumbria hip fracture database between 2014 and 2018. Patients were excluded if they were not surgically managed or if scores for predictive variables were missing. METHODS: C statistics were calculated to test the discriminant ability of the NHFS, Abbreviated Mental Test Score (AMTS), and American Society of Anesthesiologists (ASA) grade for in-hospital, 30-day, and 120-day mortality; functional independence at discharge, 30 days, and 120 days; length of stay; and postoperative complications. RESULTS: We analyzed data from 3208 individuals, mean age 82.6 (standard deviation 8.6). 2192 (70.9%) were female. 194 (6.3%) died during the first 30 days, 1686 (54.5%) were discharged to their own home, 211 (6.8%) had no mobility at 120 days, 141 (4.6%) experienced a postoperative complication. The median length of stay was 18 days (interquartile range 8-28). For mortality, C statistics for the NHFS ranged from 0.68 to 0.69, similar to ASA and AMTS. For postoperative mobility, the C statistics for the NHFS ranged from 0.74 to 0.83, similar to AMTS (0.61-0.82) and better than the ASA grade (0.68-0.71). Length of stay was significantly correlated with each score (P < .001 by Jonckheere-Terpstra test); NHFS and AMTS showed inverted U-shaped relationships with length of stay. For postoperative complications, C statistics for NHFS (0.54-0.59) were similar to ASA grade (0.53-0.61) and AMTS (0.50-0.58). CONCLUSIONS AND IMPLICATIONS: The NHFS performed consistently well in predicting functional outcomes, moderately in predicting mortality, but less well in predicting length of stay and complications. There remains room for improvement by adding further predictors such as measures of physical performance in future analyses.

Statins, bone biology and revision arthroplasty: review of clinical and experimental evidence.

Osteoarthritis is a painful, disabling condition which is increasing in prevalence as a result of an ageing population. With no recognized disease-limiting therapeutics, arthroplasty of the hip and knee is the most common and effective treatment for lower limb osteoarthritis, however lower limb arthroplasty has a finite life-span and a proportion of patients will require revision arthroplasty. With increasing life expectancy and an increasing proportion of younger (<65 years) patients undergoing arthroplasty, the demand for revision arthroplasty after implant failure is also set to increase. Statins are cholesterol-modulating drugs widely used for cardiovascular risk reduction which have been noted to have pleiotropic effects including potentially influencing arthroplasty survival. In vitro studies have demonstrated pleiotropic effects in human bone cells, including enhancement of osteoblastogenesis following simvastatin exposure, and in vivo studies have demonstrated that intraperitoneal simvastatin can increase peri-implant bone growth in rats following titanium tibial implant insertion. There is evidence that statins may also influence osseointegration, enhancing bone growth at the bone-implant interface, subsequently improving the functional survival of implants. Data from the Danish Hip Arthroplasty Registry and the Clinical Practice Research Datalink in the UK suggest a reduction in the risk of lower limb revision arthroplasty in statin ever-users versus never-users, and a time-dependent effect of statins in reducing the risk of revision. In this article we review the clinical and experimental evidence linking statins and risk of revision arthroplasty.

Effect of Timing and Duration of Statin Exposure on Risk of Hip or Knee Revision Arthroplasty: A Population-based Cohort Study.

OBJECTIVES: To determine whether the timing and duration of statin exposure following total hip/knee arthroplasty (THA/TKA) influence the risk of revision arthroplasty. METHODS: Subjects from the Clinical Practice Research Datalink, a large population-based clinical database, who had THA/TKA from 1988 to 2016, were included. Propensity score adjusted Cox regression models were used to determine the association between statin exposure and the risk of revision THA/TKA, (1) at any time, and (2) if first exposed 0-1, 1-5, or > 5 years following THA/TKA. We also investigated the effect of duration of statin exposure (< 1, 1-2, 2-3, 3-4, 4-5, > 5 yrs). RESULTS: The study included 151,305 participants. There were 65,032 (43%) exposed to statins during followup and 3500 (2.3%) had revision arthroplasty. In a propensity score adjusted model, exposure to statins was associated with a reduced risk of revision arthroplasty (HR 0.82, 95% CI 0.75-0.90). Participants first exposed within 1 year and between 1 and 5 years following THA/TKA (vs unexposed) had a reduced risk of revision arthroplasty (HR 0.82, 95% CI 0.74-0.91 and HR 0.76, 95% CI 0.65-0.90, respectively). In relation to duration of statin therapy, participants exposed for more than 5 years in total (vs < 1 yr) had a reduced risk of revision (HR 0.74, 95% CI 0.62-0.88). CONCLUSION: Statin therapy initiated up to 5 years following THA/TKA may reduce the risk of revision arthroplasty.

Prioritization of PLEC and GRINA as Osteoarthritis Risk Genes Through the Identification and Characterization of Novel Methylation Quantitative Trait Loci.

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.

Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk.

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.