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Supplementary Material toThe association of relative telomere length with symptomatic peripheral arterial disease: Results from the CAVASIC StudyJulia Raschenberger 1*, Barbara Kollerits 1*, Angelika Hammerer-Lercher 2, Barbara Rantner 1,3, Marietta Stadler4, Margot Haun 1, Peter Klein-Weigel 5, Gustav Fraedrich 3, Florian Kronenberg1*These authors contributed equally.1Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria2Central Institute of Medical and Chemical Laboratory Diagnostics, University Hospital Innsbruck, Innsbruck, Austria3Department of Vascular Surgery, Innsbruck Medical University, Innsbruck, Austria43rd Medical Department of Metabolic Diseases and Nephrology, Hietzing Hospital, Vienna, Austria5Clinic for Angiology, HELIOS Klinikum Berlin-Buch, Berlin, GermanyCorresponding Author:Florian Kronenberg, MDDivision of Genetic EpidemiologyDepartment of Medical Genetics, Molecular and Clinical Pharmacology,Innsbruck Medical UniversitySch?pfstr. 41, A-6020 Innsbruck, AUSTRIAPhone: (+43)512-9003-70560, Fax: (+43)512-9003-73560E-mail: Florian.Kronenberg@i-med.ac.atMaterials and MethodsBaseline and vascular examination and other phenotypic characterization Briefly, measurement of ankle-brachial index (ABI) was performed as follows: the systolic brachial blood pressure was measured once on both arms. On the arm with the higher value two additional measurements were done and these were averaged. On the lower extremity 3 measurements were made for each artery (dorsalis pedis artery and tibialis posterior artery, on both the left and right ankles) and the mean of the second and third for each site was taken. To calculate the ABI the mean of the lower extremity sites was divided by the averaged systolic blood pressure of the arm. The lowest ABI value from the 4 sites was used for further analysis ADDIN REFMGR.CITE <Refman><Cite><Author>Rantner</Author><Year>2008</Year><RecNum>189</RecNum><IDText>Association between the UGT1A1 TA-repeat polymorphism and bilirubin concentration in patients with intermittent claudication: results from the CAVASIC study</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>189</Ref_ID><Title_Primary>Association between the UGT1A1 TA-repeat polymorphism and bilirubin concentration in patients with intermittent claudication: results from the CAVASIC study</Title_Primary><Authors_Primary>Rantner,B.</Authors_Primary><Authors_Primary>Kollerits,B.</Authors_Primary><Authors_Primary>Anderwald-Stadler,M.</Authors_Primary><Authors_Primary>Klein-Weigel,P.</Authors_Primary><Authors_Primary>Gruber,I.</Authors_Primary><Authors_Primary>Gehringer,A.</Authors_Primary><Authors_Primary>Haak,M.</Authors_Primary><Authors_Primary>Schnapka-Kopf,M.</Authors_Primary><Authors_Primary>Fraedrich,G.</Authors_Primary><Authors_Primary>Kronenberg,F.</Authors_Primary><Date_Primary>2008/5</Date_Primary><Keywords>ASSOCIATION</Keywords><Keywords>CAVASIC,telomere</Keywords><Keywords>disease</Keywords><Keywords>Epidemiology</Keywords><Keywords>genetic</Keywords><Keywords>Male</Keywords><Reprint>Not in File</Reprint><Start_Page>851</Start_Page><End_Page>857</End_Page><Periodical>Clin Chem</Periodical><Volume>54</Volume><Issue>5</Issue><Misc_3>clinchem.2007.102046 [pii];10.1373/clinchem.2007.102046 [doi]</Misc_3><Address>Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria</Address><Web_URL>PM:18375480</Web_URL><Web_URL_Link1>file://L:\Rantner, Clin Chem, 2008 (189).pdf</Web_URL_Link1><Web_URL_Link2><u> name="System">Clin Chem</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[1].To detect symptomatic intermittent claudication the Edinburgh questionnaire was applied ADDIN REFMGR.CITE <Refman><Cite><Author>Leng</Author><Year>1992</Year><RecNum>203</RecNum><IDText>The Edinburgh Claudication Questionnaire: an improved version of the WHO/Rose Questionnaire for use in epidemiological surveys</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>203</Ref_ID><Title_Primary>The Edinburgh Claudication Questionnaire: an improved version of the WHO/Rose Questionnaire for use in epidemiological surveys</Title_Primary><Authors_Primary>Leng,G.C.</Authors_Primary><Authors_Primary>Fowkes,F.G.</Authors_Primary><Date_Primary>1992/10</Date_Primary><Keywords>Aged</Keywords><Keywords>CAVASIC,telomere,cell senescence</Keywords><Keywords>disease</Keywords><Keywords>population</Keywords><Reprint>Not in File</Reprint><Start_Page>1101</Start_Page><End_Page>1109</End_Page><Periodical>J Clin Epidemiol</Periodical><Volume>45</Volume><Issue>10</Issue><Address>Wolfson Unit for the Prevention of Peripheral Vascular Diseases, Department of Public Health Sciences, University of Edinburgh, Scotland</Address><Web_URL>PM:1474406</Web_URL><Web_URL_Link2><u> name="System">J Clin Epidemiol</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[2]. In addition, study participants underwent pulse volume recording, treadmill examination, ultrasound scanning and magnetic resonance imaging or conventional angiography. Electrocardiographic and echocardiographic evaluations were performed. In 66 patients and 25 controls echocardiographic evaluation was not available. No differences in major clinical parameters between PAD patients with and without measured ejection fraction were observed. This supports that the clinical situation of the patients was not a criterion whether patients underwent echocardiography or not. Laboratory measurements In addition, during the patients hospital stay the following parameters were measured routinely in serum: total, LDL and HDL cholesterol as well as triglycerides were measured by enzymatic colorimetric tests on a P800 analyzer (Modular, Roche Diagnostics). C-reactive protein (CRP) was determined immunturbidmetrically and glucose using an enzymatic UV test (P800 analyzer; Modular; Roche Diagnostics). Albumin was measured routinely in serum and urine by a nephelometric method (BN II; Siemens). Serum creatinine was measured using the Jaffé method (kinetic colour assay) which was corrected for unspecific protein reactions by a factor of -26.5 μmol/L. HbA1c was measured using an immunturbidimetric Latex-assay (Integra; Roche Diagnostics) and was standardized according to the DCCT/NGPS.Assessing relative telomere length (RTL)Genomic DNA was extracted from frozen EDTA-blood samples with the Invisorb? Blood Universal Kit. DNA concentrations were measured with the Tecan NanoQuant infinite M200. Samples were normalized in 96-well microtiter plates. Samples were ascertained in a singleplex, quadruplicate approach to measure the T/S-ratios. These T/S-ratios are proportional to individual relative telomere length (RTL). RTL was measured with some modifications as described below by using a quantitative real-time polymerase chain reaction (qPCR) assay, which was first described by Cawthon ADDIN REFMGR.CITE <Refman><Cite><Author>Cawthon</Author><Year>2002</Year><RecNum>4</RecNum><IDText>Telomere measurement by quantitative PCR</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>4</Ref_ID><Title_Primary>Telomere measurement by quantitative PCR</Title_Primary><Authors_Primary>Cawthon,R.M.</Authors_Primary><Date_Primary>2002/5/15</Date_Primary><Keywords>qPCR</Keywords><Keywords>Telomere</Keywords><Reprint>Not in File</Reprint><Start_Page>e47</Start_Page><Periodical>Nucleic Acids Res</Periodical><Volume>30</Volume><Issue>10</Issue><User_Def_5>PMC115301</User_Def_5><Address>Department of Human Genetics, University of Utah, 15 N 2030 E, Room 2100, Salt Lake City, UT 84112, USA. rcawthon@genetics.utah.edu</Address><Web_URL>PM:12000852</Web_URL><Web_URL_Link1>file://L:\Cawthon, Nucl Acids Res, 2002 (4).pdf</Web_URL_Link1><Web_URL_Link2><u> name="System">Nucleic Acids Res</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[3].Singleplex PCRs for telomere (T) and housekeeping gene (S) PCRs were identical composed. DNA samples were run in 15?l reactions containing 1x Quantifast TM SYBR? Green PCR mastermix (Qiagen), 10 ng of DNA, 1?M of telomere primer or 250nm of housekeeping gene 36B4 primer. The primer sequences (5’→3’) were: tel1b CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT; tel2b GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT; 36B4u CAGCAAGTGGGAAGGTGTAATCC; 36B4d CCCATTCTATCATCAACGGGTACAA ADDIN REFMGR.CITE <Refman><Cite><Author>Willeit</Author><Year>2010</Year><RecNum>3</RecNum><IDText>Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3</Ref_ID><Title_Primary>Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk</Title_Primary><Authors_Primary>Willeit,P.</Authors_Primary><Authors_Primary>Willeit,J.</Authors_Primary><Authors_Primary>Brandst&#xE4;tter,A.</Authors_Primary><Authors_Primary>Ehrlenbach,S.</Authors_Primary><Authors_Primary>Mayr,A.</Authors_Primary><Authors_Primary>Gasperi,A.</Authors_Primary><Authors_Primary>Weger,S.</Authors_Primary><Authors_Primary>Oberhollenzer,F.</Authors_Primary><Authors_Primary>Reindl,M.</Authors_Primary><Authors_Primary>Kronenberg,F.</Authors_Primary><Authors_Primary>Kiechl,S.</Authors_Primary><Date_Primary>2010/8</Date_Primary><Reprint>Not in File</Reprint><Start_Page>1649</Start_Page><End_Page>1656</End_Page><Periodical>Arterioscler Thromb Vasc Biol</Periodical><Volume>30</Volume><Issue>8</Issue><Misc_3>ATVBAHA.110.205492 [pii];10.1161/ATVBAHA.110.205492 [doi]</Misc_3><Address>Department of Neurology, Innsbruck Medical University, Innsbruck, Austria</Address><Web_URL>PM:20508208</Web_URL><Web_URL_Link1>file://C:\Papers\Willeit, arterioscler Thromb Vasc Biol, 2010 (3).pdf</Web_URL_Link1><Web_URL_Link2><u> name="System">Arterioscler Thromb Vasc Biol</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[4].Each qPCR was carried out in 384-well format which was vertically segmented in two parts: one for the telomeres (T) and one for the housekeeping gene 36B4 (S). Each 384-well plate contained the standard DNA, a quality control (commercially available DNA-Human Genomic DNA, Roche) and a non template control (NTC) in quadruplicate. All sample transfers and dilution steps were performed with a Tecan robotic workstation. Relative qPCR was carried out on an Applied Biosystems Taqman Fast Real-Time PCR 7900HT System. The thermal cycling began with the initial polymerase activation step (10 min at 95°C) and was followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. A melting curve analysis to verify the specificity and identity of the products was performed. The relative quantities were determined by the efficiency correction method ADDIN REFMGR.CITE <Refman><Cite><Author>Pfaffl</Author><Year>2001</Year><RecNum>17</RecNum><IDText>A new mathematical model for relative quantification in real-time RT-PCR</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>17</Ref_ID><Title_Primary>A new mathematical model for relative quantification in real-time RT-PCR</Title_Primary><Authors_Primary>Pfaffl,M.W.</Authors_Primary><Date_Primary>2001/5/1</Date_Primary><Keywords>efficiency correction</Keywords><Keywords>qPCR</Keywords><Keywords>real time</Keywords><Keywords>Telomere</Keywords><Reprint>Not in File</Reprint><Start_Page>e45</Start_Page><Periodical>Nucleic Acids Res</Periodical><Volume>29</Volume><Issue>9</Issue><User_Def_5>PMC55695</User_Def_5><Address>Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Germany. pfaffl@weihenstephan.de</Address><Web_URL>PM:11328886</Web_URL><Web_URL_Link1>file://C:\Papers\Pfaffl, Nucl Acids Res, 2001 (17).pdf</Web_URL_Link1><Web_URL_Link2><u> name="System">Nucleic Acids Res</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[5], which does not need calibration curves and includes the individual real-time PCR efficiencies. This mathematical model calculates the ratio of a target gene (telomere) from the efficiencies and Ct-values of an experimental sample versus a reference gene (housekeeping gene) referring to a standard. To calculate PCR efficiencies of both the reference gene and the target gene PCR raw data were imported into the program LinRegPCR (version 12.5.0) ADDIN REFMGR.CITE <Refman><Cite><Author>Ruijter</Author><Year>2009</Year><RecNum>18</RecNum><IDText>Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>18</Ref_ID><Title_Primary>Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data</Title_Primary><Authors_Primary>Ruijter,J.M.</Authors_Primary><Authors_Primary>Ramakers,C.</Authors_Primary><Authors_Primary>Hoogaars,W.M.</Authors_Primary><Authors_Primary>Karlen,Y.</Authors_Primary><Authors_Primary>Bakker,O.</Authors_Primary><Authors_Primary>van den Hoff,M.J.</Authors_Primary><Authors_Primary>Moorman,A.F.</Authors_Primary><Date_Primary>2009/4</Date_Primary><Keywords>efficiency</Keywords><Keywords>LinReg PCR</Keywords><Keywords>qPCR</Keywords><Keywords>Telomere</Keywords><Reprint>Not in File</Reprint><Start_Page>e45</Start_Page><Periodical>Nucleic Acids Res</Periodical><Volume>37</Volume><Issue>6</Issue><User_Def_5>PMC2665230</User_Def_5><Misc_3>gkp045 [pii];10.1093/nar/gkp045 [doi]</Misc_3><Address>Heart Failure Research Center, Academic Medical Center, University of Amsterdam, The Netherlands. j.m.ruijter@amc.uva.nl</Address><Web_URL>PM:19237396</Web_URL><Web_URL_Link1>file://C:\Papers\Ruijter, Nucl Acids Res, 2009 (18).pdf</Web_URL_Link1><Web_URL_Link2><u> name="System">Nucleic Acids Res</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>[6]. Efficiencies were computed for all replicates of each sample. To check PCR data for outliers, the coefficients of variation (intra-assay CVs) were assessed for the Ct-values and the efficiency-values of quadruplicates in each gene. All single outlying values (CV>5%) were removed from further analyses. In the case of the housekeeping gene 36B4, less than 0.8% and in case of telomere, about 1.8% were removed. For further mathematical analysis, the mean value of all efficiency-values of each gene on each plate and the mean Ct-value of the four replicates for each gene and each sample were used. Relative T/S-ratios reflect relative telomere length differences. QUOTE RelativeTS-Ratio= eff tel, sampleCt tel, sample eff ref, sampleCt ref, sample÷ eff tel, standardCt tel, standard eff ref, standardCt ref, standard RelativeTS-Ratio= eff tel, sampleCt tel, sample eff ref, sampleCt ref, sample÷ eff tel, standardCt tel, standard eff ref, standardCt ref, standardTo test the reproducibility of RTL measurement, the inter-assay CV of T/S-ratios was calculated according to the following formula:2i=1nxi –yi2 2nX+Y÷2. 20% of all samples were analyzed in duplicate. Duplicate samples were never positioned on the same plate or at the same plate position. They were taken from different original DNA plates. Inter-assay CV of T/S-ratios was 7.9%. As second RTL measurement quality control, we analyzed the relative telomere length of a commercially available DNA, which was positioned on each 384-well plate (a total of 16 plates). Inter-assay CV of T/S-ratios of this 16 times analyzed sample was 7.6%. The personnel were blinded about the case-control status of study participants when analyzing the batches of blood samples. Moreover, patients and controls were placed in random order on each plate. ADDIN REFMGR.REFLIST Reference List[1] Rantner B, Kollerits B, Anderwald-Stadler M, Klein-Weigel P, Gruber I, Gehringer A et al. Association between the UGT1A1 TA-repeat polymorphism and bilirubin concentration in patients with intermittent claudication: results from the CAVASIC study. Clin Chem 2008; 54(5): 851-7.[2] Leng GC, Fowkes FG. The Edinburgh Claudication Questionnaire: an improved version of the WHO/Rose Questionnaire for use in epidemiological surveys. J Clin Epidemiol 1992; 45(10): 1101-9.[3] Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res 2002; 30(10): e47.[4] Willeit P, Willeit J, Brandst?tter A, Ehrlenbach S, Mayr A, Gasperi A et al. Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk. Arterioscler Thromb Vasc Biol 2010; 30(8): 1649-56.[5] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29(9): e45.[6] Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, Moorman AF. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 2009; 37(6): e45. ................
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