Oxidative stress is proposed as an important factor in osteoarthritis (OA).
To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA.
SOD expression was ...determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin-Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression.
All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin-Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression.
This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.
We have developed a mouse in which the
Cre recombinase gene has been targeted to exon 1 of the matrilin-1 gene (
Matn1) to investigate the origins of articular chondrocytes and the development of the ...knee joint. Analysis of joints from offspring of
Matn1-Cre/R26R crosses demonstrated that articular chondrocytes are derived from cells that have never expressed matrilin-1 whereas the remainder of the chondrocytes in the cartilage anlagen expresses matrilin-1. A band of chondrocytes adjacent to the developing interzone in the E13.5 day knee joint became apparent because these chondrocytes did not turn on expression of matrilin-1 in contrast to the other chondrocytes of the anlagen. The chondrocytes of the presumptive articular surface therefore appear to arise directly from a subpopulation of early chondrocytes that do not activate matrilin-1 expression rather than by redifferentiation from the flattened cells of the interzone. In addition, lineage tracing using both
Matn1-Cre/R26R and
Col2a1-Cre/R26R lines indicated that non-cartilaginous structures in the knee such as cruciate ligament, synovium and some blood vessels are formed by cells derived from the early chondrocytes of the anlagen.
Obesity as measured by body mass index (BMI) is one of the major risk factors for osteoarthritis. In addition, genetic overlap has been reported between osteoarthritis and normal adult height ...variation. We investigated whether this relationship is due to a shared genetic aetiology on a genome-wide scale.
We compared genetic association summary statistics (effect size, p value) for BMI and height from the GIANT consortium genome-wide association study (GWAS) with genetic association summary statistics from the arcOGEN consortium osteoarthritis GWAS. Significance was evaluated by permutation. Replication of osteoarthritis association of the highlighted signals was investigated in an independent dataset. Phenotypic information of height and BMI was accounted for in a separate analysis using osteoarthritis-free controls.
We found significant overlap between osteoarthritis and height (p=3.3×10(-5) for signals with p≤0.05) when the GIANT and arcOGEN GWAS were compared. For signals with p≤0.001 we found 17 shared signals between osteoarthritis and height and four between osteoarthritis and BMI. However, only one of the height or BMI signals that had shown evidence of association with osteoarthritis in the arcOGEN GWAS was also associated with osteoarthritis in the independent dataset: rs12149832, within the FTO gene (combined p=2.3×10(-5)). As expected, this signal was attenuated when we adjusted for BMI.
We found a significant excess of shared signals between both osteoarthritis and height and osteoarthritis and BMI, suggestive of a common genetic aetiology. However, only one signal showed association with osteoarthritis when followed up in a new dataset.
Objective
To test whether previously reported hip morphology or osteoarthritis (OA) susceptibility loci are associated with proximal femur shape as represented by statistical shape model (SSM) modes ...and as univariate or multivariate quantitative traits.
Methods
We used pelvic radiographs and genotype data from 929 subjects with unilateral hip OA who had been recruited previously for the Arthritis Research UK Osteoarthritis Genetics Consortium genome‐wide association study. We built 3 SSMs capturing the shape variation of the OA‐unaffected proximal femur in the entire mixed‐sex cohort and for male/female‐stratified cohorts. We selected 41 candidate single‐nucleotide polymorphisms (SNPs) previously reported as being associated with hip morphology (for replication analysis) or OA (for discovery analysis) and for which genotype data were available. We performed 2 types of analysis for genotype–phenotype associations between these SNPs and the modes of the SSMs: 1) a univariate analysis using individual SSM modes and 2) a multivariate analysis using combinations of SSM modes.
Results
The univariate analysis identified association between rs4836732 (within the ASTN2 gene) and mode 5 of the female SSM (P = 0.0016) and between rs6976 (within the GLT8D1 gene) and mode 7 of the mixed‐sex SSM (P = 0.0003). The multivariate analysis identified association between rs5009270 (near the IFRD1 gene) and a combination of modes 3, 4, and 9 of the mixed‐sex SSM (P = 0.0004). Evidence of associations remained significant following adjustment for multiple testing. All 3 SNPs had previously been associated with hip OA.
Conclusion
These de novo findings suggest that rs4836732, rs6976, and rs5009270 may contribute to hip OA susceptibility by altering proximal femur shape.
Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton. Both GDF5 and ERG are proposed as regulators of chondrocyte differentiation ...during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Erg expression was identified in mouse knee joints at E13.5. Expression analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence and absence of GDF5 and ERG. At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant GDF5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by GDF5. Erg regulation by GDF5 in induced mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2 and/or Ube2b. Combined, these data suggest a possible pathway linking GDF5, ERG and downstream factors in the processes of chondrocyte differentiation during articular joint formation.
The knee joint consists of multiple interacting tissues that are prone to injury‐ and disease‐related degeneration. Although much is known about the structure and function of the knee's constituent ...tissues, relatively little is known about their cellular origin and the mechanisms governing their segregation. To investigate the origin and segregation of knee tissues in vivo we performed lineage tracing using a Col2a1‐Cre/R26R mouse model system and compared the data obtained with actual Col2a1 expression. These studies demonstrated that at E13.5 the interzone at the presumptive joint site forms when cells within the Col2a1‐expressing anlagen cease expression of Col2a1 and not through cellular invasion into the anlagen. Later in development these interzone cells form the cruciate ligament and inner medial meniscus of the knee. At E14.5, after interzone formation, cells that had never expressed Col2a1 appeared in the joint and formed the lateral meniscus. Furthermore, cells with a Col2a1‐positive expression history combined with the negative cells to form the medial meniscus. The invading cells started to express Col2a1 1 week after birth, resulting in all cells within the meniscus synthesizing collagen II. These findings support a model of knee development in which cells present in the original anlagen combine with invading cells in the formation of this complex joint.
Background. Despite a decreasing incidence of listeriosis in the United States, molecular subtyping has increased the number of recognized outbreaks. In September 2000, the New York City Department ...of Health identified a cluster of infections caused by Listeria monocytogenes isolates with identical molecular subtypes by pulsed-field gel electrophoresis (PFGE) and ribotyping. Methods. To determine the magnitude of the outbreak and identify risk factors for infection, we notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with Listeria monocytogenes whose isolate yielded the outbreak PFGE pattern. Controls were patients infected with Listeria monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. Results. Between May and December 2000, there were 30 clinical isolates of Listeria monocytogenes with identical PFGE patterns identified in 11 US states. Cases of infection caused by these isolates were associated with 4 deaths and 3 miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95% confidence interval, 1.2–43.3). A traceback investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade previously. Conclusions. Prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.
HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we ...investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.
Osteoarthritis (OA) has a complex aetiology with a strong genetic component. Genome-wide association studies implicate several nuclear genes in the aetiology, but a major component of the ...heritability has yet to be defined at the molecular level. Initial studies implicate maternally inherited variants of mitochondrial DNA (mtDNA) in subgroups of patients with OA based on gender and specific joint involvement, but these findings have not been replicated.
The authors studied 138 maternally inherited mtDNA variants genotyped in a two cohort genetic association study across a total of 7393 OA cases from the arcOGEN consortium and 5122 controls genotyped in the Wellcome Trust Case Control consortium 2 study.
Following data quality control we examined 48 mtDNA variants that were common in cohort 1 and cohort 2, and found no association with OA. None of the phenotypic subgroups previously associated with mtDNA haplogroups were associated in this study.
We were not able to replicate previously published findings in the largest mtDNA association study to date. The evidence linking OA to mtDNA is not compelling at present.
Imputation is an extremely valuable tool in conducting and synthesising genome-wide association studies (GWASs). Directly typed SNP quality control (QC) is thought to affect imputation quality. It ...is, therefore, common practise to use quality-controlled (QCed) data as an input for imputing genotypes. This study aims to determine the effect of commonly applied QC steps on imputation outcomes. We performed several iterations of imputing SNPs across chromosome 22 in a dataset consisting of 3177 samples with Illumina 610 k (Illumina, San Diego, CA, USA) GWAS data, applying different QC steps each time. The imputed genotypes were compared with the directly typed genotypes. In addition, we investigated the correlation between alternatively QCed data. We also applied a series of post-imputation QC steps balancing elimination of poorly imputed SNPs and information loss. We found that the difference between the unQCed data and the fully QCed data on imputation outcome was minimal. Our study shows that imputation of common variants is generally very accurate and robust to GWAS QC, which is not a major factor affecting imputation outcome. A minority of common-frequency SNPs with particular properties cannot be accurately imputed regardless of QC stringency. These findings may not generalise to the imputation of low frequency and rare variants.
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