Agu Tamm

The GDF5 rs143383 polymorphism is associated with osteoarthritis of the knee with genome-wide statistical significance

Osteoarthritis of the knee is a major cause of pain, disability and the use of healthcare resources among middle-aged and older people.1 Although osteoarthritis is multifactorial, it is known to have a significant genetic contribution and a number of studies have attempted to dissect such a contribution (see Valdes and Spector2 for review). The GDF5 gene encodes the growth differentiation factor 5, a bone morphogenetic protein involved in joint formation, expressed in different joint structures, which has been shown to ameliorate tendon, ligament and bone healing after trauma in mice.3 4 A promoter polymorphism (rs143383) in GDF5 has been found to be strongly associated with both hip and knee osteoarthritis in Asian individuals,4 and is the most widely replicated genetic association with knee osteoarthritis, although much less so for hip and hand osteoarthritis.5 This variant is functional, with the lower gene expression variant having increased genetic risk.4 A large-scale meta-analysis reported the association of the major (T) allele with knee osteoarthritis achieved OR 1.15 p=9.7×10−7 and achieved p=9×10−5 (OR 1.13, 95% CI 1.06 to 1.20) when Asian subjects were excluded.5 The genome-wide statistical significance level of p<5×10−8 is increasingly …

Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22

Objectives Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. Methods A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. Results With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. Conclusion The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, β), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.

Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Biobank samples

Abstract Background: Blood biomarkers are subject to pre-analytical variability. In many cases, the stability of important new tissue biomarkers during freeze cycles and storage has not been studied sufficiently. Methods: To test the stability of matrix metalloproteinases-7 (MMP-7) and their tissue inhibitors (TIMP-1), vascular growth factors (VEGF) and VEGF-receptor, serum samples were frozen and then thawed up to six times. The impact of storage temperature was investigated using an accelerated stability testing protocol. Stability at –20°C and –75°C was calculated using the Arrhenius equation. Results: The average concentration of TIMP-1 was stable, even after six freeze/thaw cycles. One thawing did not change the concentration of MMP-7 and VEGF-receptor. However, repeated freeze/thaw cycles increased the measured values significantly. Decreases in VEGF concentrations were dramatic, even after the first freeze/thaw cycle. According to the Arrhenius calculation, MMP-7 showed excellent stability, at least 5 years at –20°C and several 100 years at –75°C. The VEGF-receptor maintains 90% of its initial concentration at –20°C over 3 months, and decades at –75°C. TIMP-1 and VEGF showed poor stability with cryopreservation, even at –75°C. Conclusions: The stability of MMP-7, TIMP-1, VEGF or VEGF-receptor in biobanking is highly variable, and this should be taken into account in the interpretation of results. A temperature –20°C is unsuitable for prolonged storage of the biomarkers investigated, and repeated thawing of sera is not recommended. VEGF is especially unstable and should be quantitated using serum that has never been frozen.

Missense single nucleotide polymorphism of the ADAM12 gene is associated with radiographic knee osteoarthritis in middle-aged Estonian cohort

OBJECTIVE One of the recognized candidate genes of osteoarthritis (OA) is the ADAM metallopeptidase domain 12 (meltrin alpha) gene. We investigated the potential role of two single nucleotide polymorphisms (SNP) of the ADAM12 gene in susceptibility to radiographic knee OA and its progression in an Estonian cohort. METHODS The rs3740199 and rs1871054 polymorphisms were genotyped according to restriction fragment polymorphism in a population-based cohort consisting of 189 subjects selected from the age group 32-55 years. The radiological features of OA were measured in the tibio- and patellofemoral joints (PFJ). The X-ray investigation was repeated 3 years later for estimation of OA progression. RESULTS We found statistically significant association between rs3740199 polymorphism and patellofemoral OA in male patients (P=0.014), genetic risk was mostly related to CC homozygosity. The same SNP also affected the presence of advanced grade (II+III) osteophytes in the whole group (P=0.042) and the occurrence of osteophytes on the patellar margins in the PFJ (P=0.046). In OA progression the most significant association was found between joint space narrowing of the tibiofemoral joint and rs3740199 SNP in women (P=0.018). The rs1871054 polymorphism was not related to OA susceptibility or to progression traits. In our study the haplotype GC (rs3740199/rs1871054) was associated with reduced risk for development of osteophytes in the PFJ (P=0.041). CONCLUSIONS We conclude that rs3740199 polymorphism may affect occurrence of knee OA and its progression. We also hypothesize that the genetic contribution of ADAM12 to OA is remarkably gender-dependent and anatomical site-specific.

Large Scale Replication Study of the Association between HLA Class II/BTNL2 Variants and Osteoarthritis of the Knee in European-Descent Populations

Osteoarthritis (OA) is the most common form of arthritis and a major cause of disability. This study evaluates the association in Caucasian populations of two single nucleotide polymorphisms (SNPs) mapping to the Human Leukocyte Antigen (HLA) region and deriving from a genome wide association scan (GWAS) of knee OA in Japanese populations. The frequencies for rs10947262 were compared in 36,408 controls and 5,749 knee OA cases from European-descent populations. rs7775228 was tested in 32,823 controls and 1,837 knee OA cases of European descent. The risk (major) allele at rs10947262 in Caucasian samples was not significantly associated with an odds ratio (OR)  = 1.07 (95%CI 0.94 -1.21; p = 0.28). For rs7775228 the meta-analysis resulted in OR = 0.94 (95%CI 0.81-1.09; p = 0.42) for the allele associated with risk in the Japanese GWAS. In Japanese individuals these two SNPs are in strong linkage disequilibrium (LD) (r2 = 0.86) with the HLA class II haplotype DRB1*1502 DQA1*0103 DQB1*0601 (frequency 8%). In Caucasian and Chinese samples, using imputed data, these SNPs appear not to be in LD with that haplotype (r2<0.07). The rs10947262 and rs7775228 variants are not associated with risk of knee OA in European descent populations and they do not appear tag the same HLA class II haplotype as they do in Japanese individuals.

The ADAM12 is upregulated in synovitis and postinflammatory fibrosis of the synovial membrane in patients with early radiographic osteoarthritis

OBJECTIVE To investigate the possible association between ADAM12 (disintegrin and metalloproteinase domain12) expression in the synovium and the histological synovitis of patients with radiographic knee osteoarthritis (rKOA). METHODS The synovial biopsy samples were harvested from 44 subjects with chronic knee complaints during arthroscopy. In all subjects, the radiographs of both knee joints were performed for rKOA assessment. Histological features of synovitis were graded 0-3 in synovial samples. Messenger RNA (mRNA) of two ADAM12 splice variants [ADAM12-S(hort) and ADAM12-L(ong)] and the identical region for both-ADAM12-B(oth) were measured by real-time reverse transcription-PCR in all synovial samples (TaqMan® gene expression assay). Immunohistochemical staining of the synovial membrane with ADAM12 antibody was performed in 42 subjects. RESULTS ADAM12 mRNA was expressed in all synovial samples, whereas the main part of overall expression consisted of its long isoform (ADAM12-L). ADAM12 protein expression was detected in 80% of the synovial samples and correlated with mRNA expression (ρ=0.30, P<0.05). The expression of ADAM12 mRNA and protein in synovium correlated with the severity of histological synovitis (ρ=0.28, P<0.05 for ADAM12-B mRNA, R2=0.20, P<0.05 for ADAM12 protein). Out of several features of synovitis the expression level of both splice variants correlated only with the grade of fibrosis in the synovium (ρ=0.30, P<0.05 for ADAM12-L and ρ=0.33, P<0.05 for ADAM12-S). CONCLUSIONS ADAM12 is upregulated in the synovial tissue during synovitis on mRNA and protein level. We suggest that ADAM12 could be implicated in the development of KOA-associated synovitis, especially in the occurrence of postinflammatory fibrosis.

Characteristics of progressive renal disease.

Virtually all renal diseases progress to terminal renal failure relatively independently of the initial disease. Arresting the rate of the deterioration of kidney failure has a great impact on reducing the number of patients reaching the stage of expensive renal replacement therapy. Understanding the mechanisms of the progression of kidney disease has greatly been improved during recent years. The nature of the progressive renal damage with various etiologies includes various well-known factors where hemodynamics, renin-angiotensin system (RAS) and progressive proteinuria play the central roles. Proteinuria has to be shown as an independent risk factor for renal disease progression. Also, disturbances in lipid metabolism as well as the later structural lesions contribute to the progression. Various modalities have been used for the prevention of progressive renal disease, e.g. low-protein diet, antihypertensive therapy, antifibrotic therapy. Many recent experimental and clinical studies have shown that besides the systemic blood pressure lowering effect, RAS blocking agents provide renal protective effects via direct, hemodynamic, and indirect, non-hemodynamic, pathways: (1) lowering intraglomerular capillary hydraulic pressure, and increasing the glomerular ultrafiltration coefficient; (2) lowering proteinuria; (3) lowering hyperlipidemia; (4) diminishing kidney growth; (5) diminishing infiltration of macrophages; (6) downregulation of proinflammatory cytokines. Therefore, RAS blocking agents are widely prescribed not only for antihypertensive but also for renoprotective purposes in diabetic and non-diabetic nephropathies.

Two Single-Nucleotide Polymorphisms in ADAM12 Gene Are Associated with Early and Late Radiographic Knee Osteoarthritis in Estonian Population

Objectives. To investigate associations of selected single-nucleotide polymorphisms (SNPs) in ADAM12 gene with radiographic knee osteoarthritis (rKOA) in Estonian population. Methods. The rs3740199, rs1871054, rs1278279, and rs1044122 SNPs in ADAM12 gene were genotyped in 438 subjects (303 women) from population-based cohort, aged 32 to 57 (mean 45.4). The rKOA features were evaluated in the tibiofemoral joint (TFJ) and patellofemoral joint. Results. The early rKOA was found in 51.4% of investigated subjects (72% women) and 12.3% of participants (63% women) had advanced stage of diseases. The A allele of synonymous SNP rs1044122 was associated with early rKOA in TFJ, predominantly with the presence of osteophytes in females (OR 1.57; 95% CI 1.08–2.29, P = 0.018). The C allele of intron polymorphism rs1871054 carried risk for advanced rKOA, mostly to osteophyte formation in TFJ in males (OR 3.03; 95% CI 1.11–7.53, P = 0.018). Also the CCAA haplotype of ADAM12 was associated with osteophytosis, again mostly in TFJ in males (P = 0.014). For rs3740199 and rs1278279, no statistically significant associations were observed. Conclusion.   ADAM12 gene variants are related to rKOA risk during the early and late stages of diseases. The genetic risk seems to be predominantly associated with the appearance of osteophytes—a marker of bone remodelling and neochondrogenesis.

Urinary osteocalcin and other markers of bone metabolism: the effect of risedronate therapy

Objective. Serum osteocalcin (S‐OC) is widely used as an index of bone formation. However, there is evidence that some urinary fragments of OC reflect resorption and might be useful in monitoring antiresorptive therapy. Here, we report 6‐month changes in urinary midfragments of osteocalcin (U‐MidOC) and other bone turnover markers in response to risedronate treatment. Material and methods. The study group comprised 19 patients with postmenopausal osteoporosis, aged 49–66 years, and receiving risedronate therapy. Fifty‐four premenopausal women served as controls. Osteoporosis was diagnosed by lumbal bone mineral density (BMD). Urinary osteocalcin was measured by the U‐MidOC assay for midfragments. Bone formation was assessed by S‐PINP and S‐OC, and resorption by S‐CTx‐I. Results. At baseline, U‐MidOC was significantly correlated only with S‐OC. After the 1st month of therapy, a similar decrease was observed in the values of U‐MidOC and S‐CTx‐I, but in formation markers S‐P1NP and S‐OC only after three months. The rapid decrease in U‐MidOC, analogous to S‐CTX‐I, and the different kinetics for urinary and serum OC suggest that urinary OC midfragments are more associated with resorption than S‐OC. An association was also observed between the 1‐month change in U‐MidOC and 12‐month gain in lumbar BMD. The response in U‐MidOC after only the 1st month of therapy makes it a potential marker for monitoring the effect of risedronate, presumably reflecting different aspects of bone resorption than S‐CTx‐I does.

308 KNEE INJURY AND OSTEOARTHRITIS OUTCOME SCORE (KOOS) DURING ONE YEAR AFTER ARTHROSCOPIC INTERVENTION

311 – Table 1 Predictors ipsi arthro open No ipsi proc Significance (n=1,910) (n=9,607) test**