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SUPPLEMENTARY MATERIAL AND METHODS Patients and controls A total of 73 unrelated patients diagnosed with knee or hip OA were included in the present study. Twenty-five of these patients carried haplogroup J, 25 haplogroup U and 23 haplogroup H. The patients meeting the inclusion criteria for this study included 52 females and 21 males (mean age: 67.74 ± 8.96 years; range: 51-95), who had been diagnosed with OA according to American College of Rheumatology (ACR) criteria. Knee and hip radiographs from 148 subjects were classified according to Kellgren and Lawrence (K/L) scoring from Grade I to Grade IV. The 77 healthy controls who met the inclusion criteria for normal subjects included 39 females and 38 males (mean age: 66.01 ± 11.88; range: 42-94), not having knee or hip OA according to the ACR criteria. Twenty-five of the healthy controls carried haplogroup J, 25 haplogroup U and 27 haplogroup H. Clinical data including gender, age and smoking status were collected for all the subjects. In all cases, informed consent and the agreement of the ethical committee from the Galician Health Administration were obtained. Single Base Extension (SBE) assay SBE assay consists in the annealing of a single primer to a sequence of the mtDNA template that contains the SNP we want to interrogate, such that the 3´-end of this primer falls one base short of the SNP site present on the template. Since we are using only dideoxynucleotides (ddNTPs) in the reaction, when the complementary base is incorporated by the Taq DNA polymerase, the elongation stops and, depending on the fluorescence emitted, the SNP site will be identified. In contrast, those minor samples that did not belong to any of these 6 mtDNA haplogroups, were analyzed by PCR-RFLP. Six specific primers are designed in order to amplify the mtDNA fragments that contain each of the informative SNPs, which characterize the 6 major European mtDNA haplogroups, in one multiplex reaction. The polymorphic sites analyzed (7025, 14766, 10394, 4577, 12308 and 4216) were previously reported. Besides, another 6 specific SBE primers were also designed to interrogate each SNP site. In order to design the SBE primers, several considerations were taken into account: (i) the minimum length of each primer consisted of 20 nt, (ii) each primer had a Tm of ~60ºC and (iii) the minimum difference in length among the 6 primers was of 6nt in order to avoid overlapping. In this latest case, is possible to add unspecific nucleotides at 5´-end of the primer for increasing its length. The sequence of PCR, PCR-RFLP and SBE primers are avialabe in Rego et al 2008 [1]. Multiplex PCR mixture consisted of a final concentration of 1X Reaction Buffer (Bioline), 0.2mM of each deoxynucleotide (dNTP) (Bioline), 1.5mM MgCl 2 (Bioline), 0.025 U/µL of BioTaq DNA polymerase (Bioline) and 0.3 µM of each primer in a volume of 50 µL. Blood isolated (Invisorb Spin Blood Mini Kit from Invitek) DNA (75 ng) was added to the mixture and amplified as follows: 94ºC for 5 minutes, 35 cycles at 95ºC for 60 seconds, 55ºC for 60 seconds and 72ºC for 60 seconds, and a final extension at 70ºC for 10 minutes. After amplification, the PCR products were optionally electrophoresed in a agarose gel and uvvisualized after ethidium bromide treatment. To remove primers and unincorporated dNTPs, multiplex PCR products were treated with ExoSap-IT (Amersham) following manufacturer´s recommendations: adding 2µL of enzyme to 5 µL of PCR product (or a proportional ratio), followed by an activation of the enzyme at 37ºC for 15 minutes and a further deactivation by incubating at 80ºC for 15 minutes. Later, the samples were placed on ice. Multiplex SBE reactions were carried out following the recommendations from Applied Biosystems with some modifications. So, in a final volume of 10µL, we added 1.5µL of SNaPshot® Multiplex Kit, 2.5µL of purified PCR product and a final concentration of 0.2µM of the SBE primers mixture. To reach the final volume of 10µL, ddH2O was added. Thermal cycling conditions for SBE were as follows: 96ºC for 60 seconds and 25 cycles at 96ºC for 10 seconds, 60ºC for 5 seconds and 60ºC for 30 seconds. To remove unincorporated ddNTPs, the SBE reaction products were treated with Shrimp Alkaline Phosphatase (SAP) (Amserham) by adding to the mixture 1µL of enzyme, 2µL of SAP reaction buffer and 7µL ddH2O, followed by an activation of the enzyme at 37ºC for 1 hour and a further deactivation by incubating at 75ºC for 15 minutes. Subsequently, the samples were placed on ice. Finally, 9µL of Hi-DiTM Formamide (Applied Biosystems), 0.5µL of size internal standard (120 Liz Size Standard from Applied Biosystems) and 0.5µL of purified SBE product were mixed and denatured at 95ºC for 5 minutes prior to load into an ABI 3100 genetic analyzer. The configuration system is based on a 36cm length capillary filled with a Performance Optimized Polymer 4 (POP4) containing urea, from Applied Biosystems. The SBE default run module consisted in 22 seconds of injection time, 16 minutes of run time and a run voltage of 15 KVolts. Once the runs were finished, the whole data was analyzed in GeneMapper v3.5 software (Applied Biosystems), which is able to assign the different alleles (SNPs) in each locus, prior designing of a reference sequence that encompasses all the allelic variants for each locus . In order to verify the results obtained, some samples were analyzed by PCR-RFLP and direct sequencing of the PCR product. Determination of serum levels of molecular biomarkers Coll2-1, Coll2-1NO2, C2C and CPII are markers for cartilage metabolism, HA marks synovial metabolism and YKL-40 is a marker for both cartilage and synovium turnover. Determination of Coll2-1 and Coll2-1NO2 was performed at the University of Liege (Belgium), at the Bone and Cartilage Research Unit, following the below described assay. The remaining biomarkers were measured in our facilities using enzyme-linked immunosorbent assays (ELISA) and following the manufacturers’ recommendations. Serum C2C and CPII were measured using kits from Ibex Technologies (Montreal, Quebec, Canada), HA was measured using a kit from Corgenix Medical Corporation (Denver, CO, USA) and YKL-40 was measured using a kit from Quidel Corporation (San Diego, CA, USA). The determination was performed by mixing in the same run OA patients and healthy controls, regardless of their mtDNA haplogroup. Coll2-1 and Coll2-1NO2 concentrations were measured by two competitive and specific immunoassays (ELISA) [2]. The Coll2-1 immunoassay only measured the amino acid sequence 108HRGYPGLDG116 in its linear form while the Coll2-1NO2 immunoassay quantified with a high specificity and affinity the nitrated amino acids sequence. The limits of detection were 17 nM for Coll21 immunoassay and 25 pM for Coll2-1NO2 immunoassay. The intra- and interassays coefficients of variation were lower than 10 % and the dilution curves were parallel to the standard curve for both assays. The analytical recoveries were in mean 104.7 % and 121.9 % for Coll2-1 and Coll2-1NO2 assays, respectively. Briefly, microplates were coated with 200 µl of streptavidine 0.5 mg/ml during 48 hours. After washing (buffer washing: Tris 25 mM, NaCl 50 mM, Tween 20 0,2% (v/v) pH 7.3), microtiter plates were subsequently blocked with 400 µl/well of blocking buffer (KH2PO4 1.5 mM, Na2HPO4 8 mM, KCl 2 mM, NaCl 138 mM, BSA 0.5 % (v/v) pH 7.2) overnight at 4°C. Coll2-1 and Coll21NO2 were conjugated to biotin according to the method described by Rosenquist et al [3]. After washing, 100 µl of these biotinylated peptides either Coll2-1 at 2.5 ng/ml or Coll2-1NO2 at 1.25 ng/ml, were added to each well of streptavidine-coated plates and incubated for two hours at room temperature. After washing, 50 µl of calibrators (synthetic peptide) or unknown samples, diluted in incubation buffer (10 mM phosphate buffer saline (PBS), 138 mM NaCl, 7 % (w/v) BSA, 0.1 % (v/v) Tween 20 pH 7.0 for the Coll2-1 immunoassay and in 50 mM Tris, 138 mM NaCl, 7 % (w/v) BSA, 0.1% (v/v) Tween 20 pH 8.0 for the Coll2-1NO2 immunoassay), were added to the wells, followed by either 100 µl of D3 antibody (for Coll2-1), diluted 1/40,000 or 100 µl of D37 antibody (for Coll2-1NO2), diluted 1/500,000, and incubated one hour at room temperature. The dilutions of the antisera and of the secondary antibody were done in buffer dilution [10 mM PBS, 138 mM NaCl, 0.2% (w/v) BSA, 0.1% (v/v) Tween 20 pH 7.0 for the Coll2-1 immunoassay and in 50 mM Tris, 138 mM NaCl, 0.2% (v/v) BSA, 0.1% (v/v) Tween 20 pH 8.0 for the Coll 2-1NO2 immunoassay]. After washing, 100 µl of peroxidase-conjugated goat antibodies to rabbit IgG (Biosource, Belgium), diluted 1/5,000 in incubation buffer, were incubated one hour at room temperature. After washing, 100 µl of freshly prepared enzyme substrate (TMB, Biosource, Belgium) were added into each well. After 15 minutes, the reaction was stopped with 100 µl of 4M H 3PO4. The coloration was read with a microplate reader (Labsystem, Finland) at 450 nm, corrected for absorbance at 650 nm. Statistical analysis The Coll2-1NO2/Coll2-1 ratio was utilized as an additional measure of oxidative stress-mediated cartilage degradation, and the C2C/CPII ratio as a further index of cartilage breakdown. As a first approach, a non-parametric study was performed. The MannWhitney U-test was used to compare serum molecular marker concentrations between OA patients and healthy controls, and the Kruskal-Wallis test was used to compare the molecular marker concentrations among the three different haplogroups (J, U and H), regardless of diagnosis. As a second approach, a multiple regression analysis (MRA) was performed to evaluate the influence of gender, age, smoking status, haplogroup, diagnosis and possible interactions between haplogroup (J, U or H) and diagnosis (OA patient or healthy control) on serum levels of the molecular markers (dependent variable). A distribution analysis using the Kolmogorov-Smirnov test showed that most of the molecular markers were not normally distributed; therefore, concentrations of markers were log, square root or inverse transformed to obtain a normal distribution for this approach. When appropiate, the Bonferroni post hoc multiple comparisons test was also performed. The influence of radiographic grade to the serum levels of the molecular markers was tested using both the non-parametric Jonckheere-Terpstra test for ordered groups and MRA. For this approach we obtained, as indicated above, the radiological grade of 148 subjects out of the 150 that are part of the study. Using K/L scores, subjects were divided into three radiographic groups: group A included 77 healthy controls (K/L grade 0 and grade I), group B consisted of 47 OA patients with K/L grades II and III, and group C included 24 OA patients with K/L grade IV. References (not cited in the main manuscript) 1. Rego I, Fernández-Moreno M, Relaño S, et al. The use of single base extension (SBE) for the assessment of European mitochondrial DNA haplogroups in Galician population. The open cell development & biology journal. 2008;1:10-16 2. Deberg M, Labasse A, Christgau S, et al. New serum biochemical markers (Coll 2-1 and Coll 2-1 NO2) for studying oxidative-related type II collagen network degradation in patients with osteoarthritis and rheumatoid arthritis. Osteoarthritis Cartilage 2005; 13: 258-265. 3. Rosenquist C, Fledelius C, Christgau S, et al. Serum CrossLaps One Step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from Cterminal telopeptides of type I collagen. Clin Chem 1998; 44: 22812289.