Irina Kerna

Genetic variation in the SMAD3 gene is associated with hip and knee osteoarthritis.

OBJECTIVE Smad3 (or, MADH3) is a key intracellular messenger in the transforming growth factor beta signaling pathway. In mice, Smad3 deficiency accelerates growth plate chondrocyte maturation and leads to an osteoarthritis (OA)-like disease. We undertook this study to investigate the role of genetic variation in SMAD3 in the risk of large-joint OA in humans. METHODS Ten tag single-nucleotide polymorphisms (SNPs) in the SMAD3 gene region were tested in a discovery set: 313 patients who had undergone total knee replacement, 214 patients who had undergone total hip replacement, and 520 controls from the UK. The SNP associated with both hip and knee OA was subsequently genotyped in 1,221 controls and 1,074 cases from 2 cohorts of patients with hip OA and 2,537 controls and 1,575 cases from 4 cohorts of patients with knee OA. RESULTS A SNP (rs12901499) mapping to intron 1 of SMAD3 was associated with both knee and hip OA (P < 0.0022 and P < 0.021, respectively) in the discovery set. In all study cohorts, the major allele (G) was increased among OA patients relative to controls. A meta-analysis for knee OA yielded an odds ratio (OR) of 1.22 (95% confidence interval [95% CI] 1.12-1.34), P < 7.5 x 10(-6). For hip OA, the OR was 1.22 (95% CI 1.09-1.36), P < 4.0 x 10(-4). No evidence for heterogeneity was found (I(2) = 0%). CONCLUSION Our data indicate that genetic variation in the SMAD3 gene is involved in the risk of both hip OA and knee OA in European populations, confirming the results from animal models on the potential importance of this molecule in the pathogenesis of OA.

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 …

LARGE SCALE META-ANALYSIS OF INTERLEUKIN-1 BETA AND INTERLEUKIN-1 RECEPTOR ANTAGONIST POLYMORPHISMS ON RISK OF RADIOGRAPHIC HIP AND KNEE OSTEOARTHRITIS AND SEVERITY OF KNEE OSTEOARTHRITIS

OBJECTIVE To clarify the role of common genetic variation in the Interleukin-1β (IL1B) and Interleukin-1R antagonist (IL1RN) genes on risk of knee and hip osteoarthritis (OA) and severity of knee OA by means of large-scale meta-analyses. METHODS We searched PubMed for articles assessing the role of IL1B and IL1RN polymorphisms/haplotypes on the risk of hip and/or knee OA. Novel data were included from eight unpublished studies. Meta-analyses were performed using fixed- and random-effects models with a total of 3595 hip OA and 5013 knee OA cases, and 6559 and 9132 controls respectively. The role of ILRN haplotypes on radiographic severity of knee OA was tested in 1918 cases with Kellgren-Lawrence (K/L) 1 or 2 compared to 199 cases with K/L 3 or 4. RESULTS The meta-analysis of six published studies retrieved from the literature search and eight unpublished studies showed no evidence of association between common genetic variation in the IL1B or IL1RN genes and risk of hip OA or knee OA (P>0.05 for rs16944, rs1143634, rs419598 and haplotype C-G-C (rs1143634, rs16944 and rs419598) previously implicated in risk of hip OA). The C-T-A haplotype formed by rs419598, rs315952 and rs9005, previously implicated in radiographic severity of knee OA, was associated with reduced severity of knee OA (odds ratio (OR)=0.71 95%CI 0.56-0.91; P=0.006, I(2)=74%), and achieved borderline statistical significance in a random-effects model (OR=0.61 95%CI 0.35-1.06 P=0.08). CONCLUSION Common genetic variation in the Interleukin-1 region is not associated with prevalence of hip or knee OA but our data suggest that IL1RN might have a role in severity of knee OA.

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.

A meta-analysis of interleukin-6 promoter polymorphisms on risk of hip and knee osteoarthritis

OBJECTIVE Interleukin-6 is a pro-inflammatory cytokine involved in the pathogenesis of osteoarthritis (OA). We investigated the role of two single nucleotide polymorphisms (SNPs) mapping to the promoter of the IL-6 gene on genetic susceptibility to hip and knee OA. METHODS The -174G/C (rs1800795) and -597G/A (rs1800797) SNPs, implicated in the literature in risk of hip and hand OA, were genotyped in 2511 controls, 1101 hip OA cases and 1904 knee OA cases from four cohorts from the UK and Estonia. Data were analysed in conjuntion with published data on rs1800797 from the Genetics of OA and Lifestyle study (UK) on 791 controls, 1034 knee and 997 hip OA cases and rs1800795 data on 75 hip OA cases and 96 controls from Italy. Cases included both radiographic OA only and radiographic and symptomatic OA. Fixed and random-effects meta-analysis models were tested. RESULTS No significant association was found with hip OA or knee OA with either SNP nor with the haplotypes formed by them. For individual SNPs the smallest P-value for hip OA was observed using a random-effects model for rs1800795 OR(Gallele)=1.066 (95% CI 0.89-1.28) P<0.49, and significant heterogeneity between cohorts (I(2)=65%, P<0.034) was detected. For knee OA the smallest P-value was seen for rs1800797 OR(Aallele)=1.055 (95%CI 0.98-1.12) P<0.18, no significant heterogeneity was observed (I(2)=0%, P<0.68). CONCLUSIONS Our data do not support a role for the -174 and -597 IL-6 promoter polymorphisms in genetic susceptibility to knee or hip OA in Caucasian populations.

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.

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.

Synonymous SNP influences Adam12 mRNA expression level in synovial tissue

Several studies have established that in addition to age, sex, body mass index and trauma, genetic background also contributes to the risk of osteoarthritis (OA). ADAM12, one of the main proteolytic enzymes that regulates extracellular matrix turnover in OA joint tissues, is likely to be one of such genes, because genetic association studies have indicated a link between ADAM12 genetic variants and OA susceptibility and progression traits (Valdes et al, 2004). However, the functional impact of ADAM12 polymorphisms in osteoarthritic joint tissues has not yet been studied. Previously, the allele-specific expression of several OA-associated genes (GDF5, DIO2) has been reported in cartilage and other tissue of the synovial joints, emphasizing the need to consider the OA as involving the entire joint (van Meurs and Uitterlinden, 2012). One of the key factors in the OA pathophysiology is inflammation of the synovial membrane, which is associated with risk of progressive cartilage degradation and signs and symptoms of diseases (Sellam and Berenbaum, 2010). We aimed to evaluate the influence of the ADAM12 SNP on mRNA expression in the synovial tissue of patients with early knee OA (KOA). The synovial tissue samples were harvested in 44 middle-aged subjects (aged 32–60, mean 46.7 years, 24 women) who had undergone arthroscopy due to chronic knee complaints. X-rays of the knee joints were performed on all participants for the estimation of radiographic KOA using the Nagaosa et Doherty grading system (Nagaosa et al, 2000). The expression of ADAM12 mRNA, assessed by 2-ΔΔCT method, was measured in synovial samples by TaqMan® Gene Expression Assay (Hs01106104; Applied Biosystems, Foster City, CA). The synovial tissue harvested from macroscopically intact synovia was used as a control sample. Four SNPs in the ADAM12 gene (rs3740199, rs1871054, rs1044122, rs1278279) were genotyped in all subjects using TaqMan® SNP Genotyping Assays. The expression of ADAM12 mRNA in synovia was compared with genotypes of investigated polymorphisms in the ADAM12 gene, as well as with detailed phenotypical features of OA (presence of osteophytes, joint space narrowing). The Wilcox exact test (WET) was applied for evaluation of the association between expression level (assessed by 2-ΔΔCT) and polymorphisms of the ADAM12 gene. We found that the synonymous polymorphism rs1278279 (p. c.1515G > A, p.N505N) in exon 14 of the ADAM12 gene influences the overall expression of ADAM12 mRNA in synovial tissue. The rs1278279 genotypes in the study groups are distributed as follows: 36 GG homozygotes and 8 GA heterozygotes. In our study group, GG homozygotes had a lower relative expression level of ADAM12 mRNA in synovia compared to subjects with AG genotype (Wilcox exact test; p = 0.03, Figure 1). Separate analyses in men and women did not reveal statistically significant differences in mRNA expression, probably due to the smaller number of subjects in the groups; however, a trend for higher expression in AG heterozygotes was noticed in males (p= 0.06, WET). For other SNPs, no difference was observed in ADAM12 mRNA expression between distinct genotypes. Additionally, there was no association between severity of radiographic KOA and expression level of ADAM12 mRNA. Figure 1. Relative expression of ADAM12 mRNA in synovial tissue in different genotypes of rs1278279 SNP. GG homozygotes had significant lower ADAM12 expression level compared to AG genotype. The rs1278279 is a synonymous polymorphism in exon 14 of the ADAM12 gene, which results in asparagine-coding triplet substitution (AAC→AAT). As our previous data showed rs1278279 and rs1044122 demonstrate strong linkage disequilibrium (LD) across (D`>80%) (Kerna et al, 2013). The same study revealed that rs1044122 carried the higher risk for early knee OA, whereas no statistically significant associations were found for rs1278279. The possible reason for that could be insufficient power of the study regarding detection of associations for rs1278279 with low minor allele frequency (MAF 16%); however, reported associations of several polymorphisms of ADAM12 with OA could suggest the putative relation of this gene region to specific pathophysiological pathways of OA. The mechanism, which could be responsible for regulation of mRNA expression level by rs1278279, is presently unknown. Most frequently, the spectrum of the action by which cis-acting polymorphisms could influence gene expression includes transcriptional control, relative isoform expression, and mRNA stability (Pastinen et al, 2006). Synonymous mutations do not alter the encoded protein, but they can influence gene expression via changes in secondary structures of mRNA and thereby alter the length of pause cycles during translation, the overall rate of translation, or protein folding (Kimchi-Sarfaty et al, 2007; Bartoszewski et al, 2010). Indeed, the ubiquitous long pauses can lead to translational frame shifting and to protein misfolding (Wen et al, 2008). The assumption that triplet AAT (AG heterozygote in our study) may result in shorter pause time during translation could putatively explain higher expression levels of ADAM12 mRNA. Briefly, our results suggest that synonymous variant rs1278279 in the ADAM12 gene could influence ADAM12 mRNA expression in the synovial membrane of the knee joint. The mechanism of regulation for ADAM12 mRNA and protein expression is currently unclear and needs to be clarified by further investigation. A better understanding of molecular mechanisms of ADAM12 mRNA/protein expression regulation in OA joint tissue could potentially help in the development of new therapeutic approaches in the field of OA.