Serum Cartilage Oligomeric Matrix Protein (COMP) in Knee Osteoarthritis: A Novel Diagnostic and Prognostic Biomarker Priyanka Verma, Krishna Dalal Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India Received 4 October 2012; accepted 23 January 2013 Published online 19 February 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22324
ABSTRACT: A case–control study was conducted to estimate the association of cartilage oligomeric matrix protein (COMP) with knee osteoarthritis (OA) and to examine the potential utility of COMP as a diagnostic and prognostic biomarker in early knee OA. The COMP levels were estimated in the blood sera of 150 subjects belonging to study group (n ¼ 100) and control one (n ¼ 50). Patients with confirmed clinical isolated knee OA diagnosed through American College of Rheumatology criteria were included and were without any other cause of knee pain. ELISA was used to determine the levels of COMP, interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a). The median (range) serum COMP levels were observed to be 1117.21 ng/ml (125.03–4209.75 ng/ml) in OA patients and 338.62 ng/ml (118–589 ng/ml) in control subjects with p < 0.001. The COMP levels of study group were negatively correlated (correlation factor 0.88) with disease duration and positively correlated with age, BMI, pain score and IL-1b with correlation factors 0.86, 0.63, 0.76, and 0.79, respectively with p < 0.001. Gender differentiation was found in study group with 52% higher COMP level in males as compared to that of females. There was no significant correlation of COMP levels with radiological grading, erythrocyte sedimentation rate (ESR), hemoglobin (Hb), and TNF-a. The serum COMP levels may be used as a diagnostic OA marker along with prognostic value in determining the patients at risk of rapidly progressing this debilitating joint disease. The serum COMP level remains significantly high in first 3 years of disease duration. # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:999–1006, 2013. Keywords: COMP; prognosis; diagnosis; knee osteoarthritis; biomarker
Osteoarthritis (OA) is currently defined by the American College of Rheumatology1 as a “heterogeneous group of conditions that leads to joint symptoms and signs which are associated with defective integrity of articular cartilage, in addition to related changes in the underlying bone at the joint margins.” The aetiology of OA is based on various factors such as inflammation, mechanical and physical injury, and other metabolic causes. A number of environmental risk factors like obesity, occupation, and trauma, can also initiate various pathological pathways which may lead to OA. OA is mainly characterized by the degeneration of articular cartilage. Articular cartilage is made up of extracellular matrix and collagen fibrils. The extracellular matrix provides this tissue with its great strength in order to dissipate the load and handle the forces generated within the joint.2 It also provides the resistance against deformation. The colla-
Abbreviations: COMP, cartilage oligomeric matrix protein; OA, osteoarthritis; BMI, body mass index; ESR, erythrocyte sedimentation rate; ECM, extra cellular matrix; Hb, hemoglobin; JSN, joint space narrowing; MMP, matrix metalloproteinases; VAS, visual analog scale Authors’ contribution: K.D. and P.V. designed the study. P.V. conducted the study. P.V. performed data collection. K.D. and P. V. analyzed the data. K.D. performed data interpretation. P.V. drafted the manuscript. K.D. and P.V. revised the manuscript content: K.D. and P.V. K.D. approved the final version of manuscript. P.V. take the responsibility for the integrity of the data analyses. All authors state that they have no conflicts of interest. Grant sponsor: Council of Scientific and Industrial Research. Correspondence to: Priyanka Verma (T: 91-11-26593215 ext. 919818795588; F: 91-11-26588641 ext. 91-11-26588663; E-mail:
[email protected]) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
gen fibrillar network provides tensile strength, and the aggregate of water-laden proteoglycan aggrecan contributes to its compressive stiffness.3 As there is no vascularity in articular cartilage, it is not able to recruit the chondroprogenitor stem cells which can do the effective repair once growth ceases. Therefore, in knee OA, the amount of cartilage matrix synthesis in relation to its degradation may prove of great importance in determining the disease progression.4 The degenerative process are found to be more apparent with aging, and in a majority of the population over age 60 years, this process may result in OA.4 Since the earliest pathological changes in OA take place periarticularly, they are not captured well by radiographs but are evident only through the costly MRI. Therefore, the field of OA study is in desperate need of biomarkers which can change the process of OA prediction, OA management, and the efficacy of drug therapies and our understanding of disease pathogenesis. The possibility to objectively determine the OA status through serum would significantly increase the possibilities of diagnosing the disease with greater ease and with much cost-effective method than radiographic methods. It has been studied that in the course of the disease, when the erosion of the cartilage is taking place, it is possible that immune reactions to other cartilage proteins are initiated and contribute to the disease course. The pro-inflammatory cytokines like interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a) are known to play important roles in the development and progression of OA.5–7 It has been documented that they stimulate the production of MMPs8 by regulating the collagenases and aggrecanase production, and are known to suppress chondrocyte synthesis of aggrecan and type II collagen which is required to restore the JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2013
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ECM.7,9–11 It thus results in the degradation of cartilage matrix. However, the development and progression of OA is affected by multiple active mediators but still it is essential to elucidate the effect of these two cytokines in progression of OA and to find if there is any correlation between them and studied biomarker level. It has also been observed that during turnover of cartilage matrix in normal and diseased joints, fragments of extracellular matrix molecules, and other degradation products of cartilage metabolism are released into the synovial fluid12–14 and thereafter into the blood serum.15 One such biomarker which was most investigated till date to predict knee OA progression is cartilage oligomeric matrix protein (COMP). COMP is an important degradation product of articular cartilage16 and it may prove to be a promising diagnostic and prognostic marker in serum for diagnosis of knee OA. COMP is a pentameric non-collagenous glycoprotein belonging to the heterogeneous family of thrombospondin which can bind type to collagen type I, II, and IX.17 COMP pentamer bound up to five collagen molecules thereby retaining them in close proximity. By this process, COMP facilitates the collagen–collagen interactions and microfibril formation. Several studies conducted in past suggested that COMP is mainly produced by articular chondrocytes18,19 and reached the consensus that COMP levels in synovial fluid and serum might be related to cartilage damage.20–22 It was also reported that COMP level elevates in the knee joint synovial fluids of patients with reactive rheumatoid arthritis and in serum of patients with juvenile chronic arthritis compared to healthy children.23 Despite these results, the role of COMP as a marker of a disease process remained to be determined. The use of serum COMP to disclose disease status is not yet thoroughly studied in knee OA as the elevated serum COMP levels correlated with a more rapid disease course in RA.24,25 There is also evidence that an increase in serum COMP may serve as an indicator of radiographic OA progression.26,27 Therefore, this study has been devoted to evaluate the COMP in serum for assessing the knee OA disease progression, and to find out its association with duration of disease, patient’s gender, BMI, radiological grading, visual analog scale (VAS) score, erythrocyte sedimentation rate (ESR), hemoglobin (Hb), and levels of cytokines.
MATERIALS AND METHODS Participants This case control study was carried out in a tertiary care medical institute located in New Delhi, India with the permission of the institute ethics committee and was conducted among the populations of the plain lands of India. The study was comprised of two groups: control group (n ¼ 50) and study group (n ¼ 100). The data was collected in person with the submission of the signed individual’s informed consent form of one’s participation prior to the JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2013
initiation of the study. The details of both the group subjects are stated in the sub-sections to follow. Study Group This group consisted of patients with primary OA of the knee joint with effusions, screened in the outpatients Department of Medicine and other medical specialty services of the study institute for their confirmed knee OA as per the definition of American College of Rheumatology criteria for diagnosis of primary knee OA.1 The medical visits of the patients were prompted by the acute onset of pain, swelling, and stiffness in the affected region. Patients with primary knee OA unilateral or bilateral and with chronic knee pain of more than 3 months and radiological evidence of early OA were included in the study group. The grading of knee severity on knee X-ray was done according to Kellgren/Lawrence scale.28 The study group was consisted of 100 patients with the age group 40–80 years and disease duration of 4 months to 12 years. The patients were excluded if they presented with secondary OA, previous knee injury, or intra-articular fracture, steroid injection into the affected knee joint within 3 months of recruitment for the study, OA in joints other than the knee joint, osteoporosis, and other rheumatic diseases. Also, the patients with inflammed joints other than knee joint were excluded. The process of OA evaluation also included VAS scores for pain. Control Group Fifty individuals without any history of knee pain were chosen from the Casualty Department of the study institute and from patient’s allies to serve as a control group. The control group did not report any evidence of fracture or meniscal injury in past. They were matched for genders and age with the OA group but there was no subject with age more than 60 years who could be defined as controls as per the inclusion criteria. Outcome Variables The potential determinants of the outcome measures explored included the following: demographic data together with the BMI, radiological grading, VAS score, and serum levels of COMP, ESR, and Hb. Every patient prior to blood sampling procedure was asked to take a rest of 30 min. In a subject 5 ml of blood sample was withdrawn for estimating serum COMP and for carrying out other relevant investigations. Each blood sample was dispensed in 1.5 ml micro centrifuge tube (MCT). Blood was allowed to clot for 1–2 h at room temperature (25–26˚C). The clotted blood was rimmed (so as to detach fibrin from the walls of the tube) and centrifuged at 2,000–3,000 rpm for 10 min. Serum was separated using a micropipette and aliquoted in triplicates to a fresh set of MCTs. The tubes were labeled and stored at 20˚C until use for investigative purposes. Biochemical Analysis The quantitative measurement of overall COMP level was performed on the serum samples using an immunoassay ELISA. The kit was manufactured by BioVendor Laboratory Medicine, Inc., BioVendor GmbH, Heidelberg, Germany; Cat. no. RD 194080200R (Cat. No.: RD194080200R). The detection limit of the assay was 0.4 ng/ml, and the intra-assay and inter-assay coefficients of variation were 4% and 3.1%, respectively. In case of TNF-a, the quantitative measurement was performed on the serum samples using an immunoassay by RayBio Human TNF-alpha ELISA Kit
SERUM COMP IN KNEE OA
(U.S.; Cat#: ELH-TNF-a-001) having detection limit of 0.03 ng/ml, and the intra-assay and inter-assay coefficients of variation were
