Genetics of osteoarthritis.

Osteoarthritis genetics has been transformed in the past decade through the application of large-scale genome-wide association scans. So far, over 100 polymorphic DNA variants have been associated with this common and complex disease. These genetic risk variants account for over 20% of osteoarthritis heritability and the vast majority map to non-protein coding regions of the genome where they are presumed to act by regulating the expression of target genes. Statistical fine mapping, in silico analyses of genomics data, and laboratory-based functional studies have enabled the identification of some of these targets, which encode proteins with diverse roles, including extracellular signaling molecules, intracellular enzymes, transcription factors, and cytoskeletal proteins. A large number of the risk variants correlate with epigenetic factors, in particular cartilage DNA methylation changes in cis, implying that epigenetics may be a conduit through which genetic effects on gene expression are mediated. Some of the variants also appear to have been selected as humans adapted to bipedalism, suggesting that a proportion of osteoarthritis genetic susceptibility results from antagonistic pleiotropy, with risk variants having a positive role in joint formation but a negative role in the long-term health of the joint. Although data from an osteoarthritis genetic study has not yet directly led to a novel treatment, some of the osteoarthritis associated genes code for proteins that have available therapeutics. Genetic investigations are therefore revealing fascinating fundamental insights into osteoarthritis and can expose options for translational intervention.

Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC.

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.

Discovery and analysis of methylation quantitative trait loci (mQTLs) mapping to novel osteoarthritis genetic risk signals.

OBJECTIVE: Osteoarthritis (OA) is polygenic with over 90 independent genome-wide association loci so far reported. A key next step is the identification of target genes and the molecular mechanisms through which this genetic risk operates. The majority of OA risk-conferring alleles are predicted to act by modulating gene expression. DNA methylation at CpG dinucleotides may be a functional conduit through which this occurs and is detectable by mapping methylation quantitative trait loci, or mQTLs. This approach can therefore provide functional insight into OA risk and will prioritize genes for subsequent investigation. That was our goal, with a focus on the largest set of OA loci yet to be reported. METHOD: We investigated DNA methylation, genotype and RNA sequencing data derived from the cartilage of patients who had undergone arthroplasty and combined this with in silico analyses of expression quantitative trait loci, epigenomes and chromatin interactions. RESULTS: We investigated 42 OA risk loci and in ten of these we identified 24 CpGs in which methylation correlated with genotype (false discovery rate (FDR) P-values ranging from 0.049 to 1.73x10-25). In silico analyses of these mQTLs prioritised genes and regulatory elements at the majority of the ten loci, with COLGALT2 (encoding a collagen galactosyltransferase), COL11A2 (encoding a polypeptide chain of type XI collagen) and WWP2 (encoding a ubiquitin ligase active during chondrogenesis) emerging as particularly compelling target genes. CONCLUSION: We have highlighted the pivotal role of DNA methylation as a link between genetic risk and OA and prioritized genes for further investigation.

A National Review of Neonatal Jaundice Identification.

Background Physiological neonatal hyperbilirubinemia is a normal transitional phenomenon, however bilirubin encephalopathy can develop due to exposure to very high bilirubin levels. A systematic approach to early detection of high levels can prevent this outcome. Methods We designed a questionnaire to assess local jaundice management practices in Irish maternity units. Results All 19 units responded to our clinical questionnaire. Early discharge (<48 hours) occurs in 12 units (63%). Six units universally screen all babies with a transcutaneous bilirubinometer (TCB) (32%) while 12 units only do so if clinically jaundiced (83%). 12 units follow up <5% of their babies for jaundice monitoring after discharge (67%), which is lower than expected for optimal jaundice management. Conclusion Our survey responses show a high degree of variability in jaundice identification and follow up practices around the country. As maternity units trend towards earlier discharge of mothers due to resource constraints, we need to develop national systems to stratify risk before discharge and monitor jaundice in the out-patient setting. Introduction

Age-related changes in mesenchymal stem cells identified using a multi-omics approach.

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based treatments for many clinical diseases and injuries. Ageing is associated with various altered cellular phenotypes coupled with a variety of transcriptional, epigenetic and translational changes. Furthermore, the regeneration potential of MSCs is reduced with increasing age and is correlated with changes in cellular functions. This study used a systems biology approach to investigate the transcriptomic (RNASeq), epigenetic (miRNASeq and DNA methylation) and protein alterations in ageing MSCs in order to understand the age-related functional and biological variations, which may affect their applications to regenerative medicine. We identified no change in expression of the cellular senescence markers. Alterations were evident at both the transcriptional and post-transcriptional level in a number of transcription factors. There was enrichment in genes involved in developmental disorders at mRNA and differential methylated loci (DML) level. Alterations in energy metabolism were apparent at the DML and protein level. The microRNA miR-199b-5p, whose expression was reduced in old MSCs, had predicted gene targets involved in energy metabolism and cell survival. Additionally, enrichment of DML and proteins in cell survival was evident. Enrichment in metabolic processes was revealed at the protein level and in genes identified as undergoing alternate splicing. Overall, an altered phenotype in MSC ageing at a number of levels implicated roles for inflamm-ageing and mitochondrial ageing. Identified changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients.

Night-eating symptoms and 2-year weight change in parents enrolled in the QUALITY cohort.

BACKGROUND/OBJECTIVE: The timing of food intake may be implicated in weight gain. This study tested the hypothesis that symptoms commonly associated with night-eating syndrome are related to measures of weight gain in adults. SUBJECTS/METHODS: Parents participating in QUALITY (Québec Adipose and Lifestyle InvesTigation in Youth) completed the night eating questionnaire (NEQ) at baseline (2005-2008) and at follow-up (2008-2010). Height and weight were measured and self-report questionnaire data were available for 388 parents (59% female, mean (s.d.) age: 41.8±5.7, mean (s.d.) body mass index (BMI): 29.6±5.7). Linear regression models were used to test the associations between baseline night-eating symptoms (NEQ scores, night-eating behaviours) and percent change in each of BMI and waist circumference (WC). RESULTS: A high NEQ score predicted a small increase in percent change in BMI in nonobese parents but a decrease among those who were severely obese. Nocturnal ingestions of food predicted an increase in percent change in BMI; however, the effect size was small. Morning anorexia predicted an increase in percent change in WC. CONCLUSION: Certain night-eating symptoms may predict measures of weight gain in adults but the effects seem small and the findings need to be confirmed in more symptomatic samples.

The role of inflammation-related genes in osteoarthritis.

In this review article we examine the role of inflammation-related genes in osteoarthritis (OA) from the perspective of genetics, epigenetics and gene expression. There have been great strides in such genomic analyses of OA in recent years thanks to the study of adequately powered patient cohorts, the detailed analysis of candidate genes, and the application of genome-wide approaches. These have led to some unexpected and therefore exciting discoveries, implicating pathways that would not necessarily have been predicted to have a role in this common arthritis. Inflammatory-related genes sit firmly in the candidate camp based on prior observations that the OA disease process can have an inflammatory component. What is clear from the genetic studies published to date is that there is no compelling evidence that DNA variation in inflammatory genes is an OA risk factor. This conclusion may of course change as ever more powerful association studies are conducted. There is, however, compelling evidence that epigenetic effects involving inflammatory genes are a component of OA and that alteration in the expression of these genes is also highly relevant to the disease process. We may in fact be close to demonstrating, at the genomic level, a clear separation of OA patients into those in whom inflammation is a key driver of the disease and those in whom it is not. This has obvious implications for the design of trials of novel OA interventions and may also guide the intelligent re-purposing of anti-inflammatory therapies.

Identification and analysis of a SMAD3 cis-acting eQTL operating in primary osteoarthritis and in the aneurysms and osteoarthritis syndrome.

OBJECTIVE: The TGF-ß pathway plays a central role in joint development with polymorphism in TGF-ß pathway genes implicated in osteoarthritis susceptibility. One association is to rs12901499, within intron 1 of SMAD3. Since rs12901499 is not in linkage disequilibrium with a non-synonymous polymorphism, it is likely the association is operating by influencing expression of SMAD3. DESIGN: Using tissues from the joints of primary osteoarthritis patients who had undergone joint replacement we measured the overall expression of SMAD3 by quantitative real-time PCR. We also measured allelic expression of SMAD3 using these tissues and vascular smooth muscle cells from patients with aneurysms and osteoarthritis syndrome, a rare condition featuring early-onset osteoarthritis. We tested the functional effect of SNPs in vitro using luciferase assays and assessed association with osteoarthritis using a large osteoarthritis case-control dataset. RESULTS: We observed that genotype at rs12901499 did not correlate with overall SMAD3 expression or allelic expression. However, genotype at a 3'UTR SNP, rs8031440, did correlate with SMAD3 expression in cartilage (P = 0.005) which was supported by allelic expression data showing that the G allele correlated with decreased SMAD3 expression in joint tissues and vascular smooth muscle cells. This G allele was underrepresented in osteoarthritis cases vs controls (P = 0.027, odds ratio = 0.921). rs8031440 is in perfect linkage disequilibrium with five other SMAD3 3'UTR SNPs and our luciferase analysis identified rs3743342 and rs12595334 as being functional. CONCLUSION: SMAD3 is subject to cis-acting regulatory polymorphism in the tissues of relevance to both primary osteoarthritis and the aneurysms-osteoarthritis syndrome.

Chronicling changes to the chronic disease prevention landscape in Canada's public health system 2004-2010.

The collective impact of major shifts in public health infrastructure and numerous new chronic disease prevention (CDP) capacity-building initiatives that have taken place in Canada over the last decade is unknown. The objective of this study was to determine if CDP capacity (i.e., skills and resources) and involvement in CDP programming improved in public health organizations in Canada from 2004 to 2010. Data for this repeated cross-sectional study were drawn from two waves of a national census of organizations mandated to carry out primary prevention of chronic disease and/or promotion of healthy eating, physical activity and tobacco control. Medians for continuous variables and frequencies for categorical variables were compared across time. Neither resources nor level of priority for CDP increased over time. There was little difference in the proportion of organizations with high levels of skills and involvement in core CDP practices (i.e., needs assessment, identification of relevant practices, planning, evaluation). Skills and involvement in CDP risk factor programming showed some gains, some steady states and some losses. Specifically, skill and involvement in tobacco control programming declined markedly while the proportion of organizations involved in healthy eating and physical activity programming increased. Skills to address and involvement in programming related to social determinants of health remained low over time as did involvement in programming addressing multiple risk factors concurrently. The lack of marked improvement in CDP capacity between 2004 and 2010 against a backdrop of initiatives favourable to strengthening the preventive health system in Canada suggests that efforts may have fallen short.

Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4-13). These dominant forms of MED (EDM1-3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1-3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24-p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.

Gene expression analysis reveals HBP1 as a key target for the osteoarthritis susceptibility locus that maps to chromosome 7q22.

OBJECTIVES: An osteoarthritis (OA) susceptibility locus has been mapped to chromosome 7q22, to a region of high-linkage disequilibrium encompassing six genes: PRKAR2B, HBP1, COG5, GPR22, DUS4L and BCAP29. The authors assessed whether these genes were subject to cis-acting regulatory polymorphisms that are active in joint tissues and which could contribute to the association signal. METHODS: Using joint tissues from 156 patients with OA, and control cartilage from 25 patients who had neck of the femur fractures, the authors measured the overall gene expression by quantitative PCR and the allelic expression of the genes, using an assay that can distinguish mRNA output from each allele of a transcript single nucleotide polymorphism. RESULTS: Five of the genes were expressed in joint tissues, the exception being GPR22, which the authors could not detect. In OA cartilage compared with control cartilage, significantly reduced expression levels were observed for these five genes. Carriers of the OA-associated alleles showed a significant reduction in expression of HBP1 in cartilage (p=0.0002) and synovium (p=0.02), and of DUS4L in fat pad (p=0.04). HBP1 and DUS4L also demonstrated allelic expression imbalance across a range of different joint tissues, with carriers of the associated allele showing an HBP1 allelic expression imbalance profile that was significantly different from non-carriers (p=0.008). CONCLUSION: Cis-acting regulatory polymorphisms acting on HBP1 contribute to the OA association signal at chromosome 7q22. HBP1 codes for a transcription factor and studies by the authors have enabled them to prioritise this gene for further investigation.

Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA.

OBJECTIVE: To define for the first time the transcriptomes of normal and end-stage osteoarthritis (OA) hip cartilage. MATERIALS AND METHODS: RNA was isolated from cartilage within 2h of joint replacement surgery. Gene expression was analyzed using Agilent GeneSpring GX 11 following hybridization to Illumina Human HT-12 V3 microarrays. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to validate the expression of six genes identified by microarray as differentially expressed. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were used to investigate enriched functions or canonical pathways amongst differentially expressed genes respectively. RESULTS: In total we identified 998 differentially expressed genes (fold change ≥ ±1.5, P-value ≤ 0.01) between neck of femur fracture (NOF) (n = 10) and OA hip (n = 9) patient cartilage. These differentially expressed genes were enriched within 71 canonical pathways. A comparison between a comparable knee dataset(20) only identified 229 genes similarly differentially expressed although remarkably 34 canonical pathways overlapped between experiments. CONCLUSIONS: This study is the first to report a comprehensive gene expression analysis of human hip OA cartilage compared to control (NOF) cartilage at the whole-genome level. Our differential gene expression dataset shows excellent correlation with similar defined studies using comparable tissue but reveals discord between hip and knee OA at the individual gene status but with commonality with regards the molecular pathways involved.

Osteoarthritis year 2010 in review: genetics.

At the 2010 Osteoarthritis Research Society International (OARSI) congress in Brussels I was asked to present on "Genetics" in the "Year in Review" session. This gave me an opportunity to reflect on the talk that I gave on the same topic at the 2007 OARSI congress in Ft Lauderdale, Florida. My 2007 talk was very upbeat and ended with the hostage to fortune statement "The next few years will provide tremendous clarity in our genetic understanding of osteoarthritis".

Summary of the OA biomarkers workshop 2010 - genetics and genomics: new targets in OA.

On November fourth and fifth 2010 a group of more than 100 international investigators gathered in Atlanta for the second Osteoarthritis (OA) Biomarkers Global Initiative workshop titled "Genetics and Genomics: New Targets in OA". The first workshop took place in April 2009 and focused on in vitro (soluble) biomarkers whilst the third and final workshop will take place in 2012 and will focus on imaging biomarkers. The OA Research Society International (OARSI) has organized the workshops. In addition to OARSI, the National Institute of Arthritis, Musculoskeletal and Skin Diseases, the Arthritis Foundation, Amgen, Genzyme, the American Orthopaedic Society for Sports Medicine and Pfizer sponsored the second meeting. It was clear from this meeting that experiments in the genetics, epigenetics and genomics of OA, are yielding valuable insights into the etiology of this heterogeneous disease but that much still needs to be learnt. Combining genetic insights with conventional biomarkers and imaging modalities may provide scientists with the enhanced tools to understand this complex disease. With those tools in hand, clinicians and industry can develop protocols to ultimately improve patient care.

Deep sequencing of GDF5 reveals the absence of rare variants at this important osteoarthritis susceptibility locus.

OBJECTIVE: The common single nucleotide polymorphism (SNP) rs143383 in the 5' untranslated region (5'UTR) of growth and differentiation factor 5 (GDF5) is strongly associated with osteoarthritis (OA) and influences GDF5 allelic expression in vitro and in the joint tissues of OA patients. This effect is modulated in cis by another common SNP, also located within the 5'UTR, whilst a common SNP in the 3'UTR influences allelic expression independent of rs143383. DNA variants can be common, rare or extremely rare/unique. To therefore enhance our understanding of the allelic architecture of this very important OA susceptibility locus we sequenced the gene for potentially functional and novel rare variants. METHOD: Using the Sanger method we sequenced GDF5 in 992 OA patients and 944 controls, with DNA changes identified by sequencing software. We encompassed the protein-coding region of the two GDF5 exons, both untranslated regions and approximately 100 bp of the proximal promoter of the gene. RESULTS: We detected 13 variants. Six were extremely rare with minor allele frequencies (MAFs) of ≤ 0.0006. One is in a predicted transcription factor binding site in the GDF5 promoter whilst two substitute conserved amino acids. The remaining seven variants were common and are previously known variants, with MAFs ranging from 0.025 to 0.39. There was a complete absence of variants with frequencies in-between the extremely rare (n=6) and the common (n=7). CONCLUSIONS: This is the first report of the deep sequencing of an OA susceptibility locus. The absence of rare variants informs us that within the regions of the gene that we have sequenced GDF5 does not harbour any novel variants that are able to contribute, at a population level, to the OA association signal mediated by rs143383 nor does it harbour, at a population level, any novel variants that can influence OA susceptibility independent of rs143383.

The effect of smoking cessation counselling in pregnant women: a meta-analysis of randomised controlled trials.

BACKGROUND: Pregnant smokers are often prescribed counselling as part of multicomponent cessation interventions. However, the isolated effect of counselling in this population remains unclear, and individual randomised controlled trials (RCTs) are inconclusive. OBJECTIVE: To conduct a meta-analysis of RCTs examining counselling in pregnant smokers. SEARCH STRATEGY: We searched the CDC Tobacco Information and Prevention, Cochrane Library, EMBASE, Medline and PsycINFO databases for RCTs evaluating smoking cessation counselling. SELECTION CRITERIA: We included RCTs conducted in pregnant women in which the effect of counselling could be isolated and those that reported biochemically validated abstinence at 6 or 12 months after the target quit date. DATA COLLECTION AND ANALYSIS: Overall estimates were derived using random effects meta-analysis models. MAIN RESULTS: Our search identified eight RCTs (n = 3290 women), all of which examined abstinence at 6 months. The proportion of women that remained abstinent at the end of follow up was modest, ranging from 4 to 24% among those randomised to counselling and from 2 to 21% among control women. The absolute difference in abstinence reached a maximum of only 4%. Summary estimates are inconclusive because of wide confidence intervals, albeit with little evidence to suggest that counselling is efficacious at promoting abstinence (odds ratio 1.08, 95% confidence interval 0.84-1.40). There was no evidence to suggest that efficacy differed by counselling type. CONCLUSIONS: Available data from RCTs examining the isolated effect of smoking cessation counselling in pregnant women are limited but sufficient to rule out large treatment effects. Future RCTs should examine pharmacological therapies in this population.

Genetic indicators and susceptibility to osteoarthritis.

A large number of experiments have been performed to identify genetic loci that influence osteoarthritis (OA) susceptibility, with a particular focus on the primary form of the disease. Unfortunately, the currently reported candidate-gene, genome-wide linkage scans and genome-wide association scans have tended to highlight the heterogeneous nature of OA rather than generate statistically compelling genome signals. Nevertheless, some breakthroughs have been made. For example, genetic susceptibility within genes coding for components of the transforming growth factor ß pathway has emerged as a particularly interesting find, while completely novel loci are also being uncovered, such as the signal to a cluster of genes on chromosome 7q22. It also appears that quantitative effects on gene expression, rather than qualitative effects on protein function, are particularly important, and that we need to consider the effects of genetic susceptibility in joint formation as much as we do in joint maintenance. Nevertheless, we are still only at the beginning of our search, and much more sophisticated clinical and laboratory approaches will need to be applied before we get a clear understanding of the genetic indicators that influence OA susceptibility.

Interleukin-1 region meta-analysis with osteoarthritis phenotypes.

OBJECTIVE: Several research groups have examined osteoarthritis (OA) association with Interleukin-1 (IL-1) region markers and haplotypes. The results have been suggestive for hand OA, negative for knee OA, and conflicting for hip OA. DESIGN: Our aim was to address conflicts employing meta-analytical methods on data from 1238 European-descent cases with various OA phenotypes and 1269 European-descent controls from four study centers. We imputed some missing genotype data and reconstructed IL-1 region extended haplotypes. A previously reported 7-marker IL1A-IL1B-IL1RN extended risk haplotype was tested for association with each specific index phenotype. RESULTS: For hip OA, data from three centers showed heterogeneity of extended-risk-haplotype effect, two panels showing trend toward risk and another showing protection, with overall odds ratio (OR) 1.24 (95% Confidence interval (CI) 0.45-3.41, P 0.67). The heterogeneity fell partly along control ascertainment lines, chiefly between controls ascertained as spouses of arthroplasty patients and controls identified through population radiographic survey. For knee OA, the results showed no heterogeneity and no significant extended-risk-haplotype effect. For hand OA, the results showed little heterogeneity and a modest trend toward positive association (summary OR 1.34, 95% CI 0.83-2.17 P 0.23). Using a Bayesian partition modeling approach, the 7-marker extended haplotypes showed no significant effect on any OA phenotype examined. A 3-single-nucleotide polymorphism (SNP) IL1B-IL1RN haplotype rs1143627-rs16944-rs419598 showed a trend toward hand OA association (posterior probability of association 0.72) with the most prominent feature being protection from a specific haplotype representing a partial mirror image of the extended risk haplotype (OR estimated at 0.46). CONCLUSIONS: The meta-analysis data do not confirm but only suggest that some hand and hip OA risk could be associated with the IL-1 region, particularly centered in IL1B and possibly also IL1RN.