Epigenetic Differences in Human Cartilage Between Mild and Severe OA Florentine C. Moazedi-Fuerst, 1 Manuela Hofner, 2 Gerald Gruber, 3 Andreas Weinhaeusel, 2 Martin H. Stradner, 1 Hannes Angerer, 1 Daniela Peischler, 1 Birgit Lohberger, 3 Mathias Glehr, 3 Andreas Leithner, 3 Markus Sonntagbauer, 2 Winfried B. Graninger 1 1 Department of Rheumatology and Immunology, Medical University Graz, Austria, 2 Department for Health and Environment, Austrian Institute of Technology, Vienna, Austria, 3 University Clinic of Orthopaedic Surgery, Medical University Graz, Austria Received 13 March 2014; accepted 24 July 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22722 ABSTRACT: The development of osteoarthritis (OA) depends on genetic and environmental factors, which influence the biology of the chondrocyte via epigenetic regulation. Changes within the epigenome might lead the way to discovery of new pathogenetic pathways. We performed a genome-wide methylation screening to identify potential differences between paired mild and severe osteoarthritic human cartilage. Sixteen female patients suffering from OA underwent total knee joint replacement. Cartilage specimens collected from corresponding macroscopically undamaged and from damaged areas were processed for DNA extraction and histology to evaluate the histological grading of the disease. Paired specimens were analysed for the methylation status of the whole genome using human promoter microarrays (Agilent, Santa Clara, CA). Selected target genes were then validated via methylation-specific qPCR. One thousand two hundred and fourteen genetic targets were identified differentially methylated between mild and severe OA. One thousand and seventy of these targets were found hypermethylated and 144 hypomethylated. The descriptive analysis of these genes by Gene Ontology (GO), KEGG pathway and protein domain analyses points to pathways of development and differentiation. We identified a list of genes which are differently methylated in mild and severe OA cartilage. Within the pathways of growth and development new therapeutic targets might arise by improving our understanding of pathogenetic mechanisms in OA. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Keywords: osteoarthritis; methylation; whole-genome array While osteoarthritis (OA) is a very common condition, impaired function of the weight bearing joints and the associated pain have a major impact on the patient’s quality of life and the socioeconomic system. 1 Degrada- tion of human articular cartilage is triggered by obvious pathogenetic factors like mechanical load and trauma; in addition, potential heritability is suggested by twin studies. Within the chondrocytes, signalling along autocrine pathways involves several cytokines and growth factors and leads to metabolic changes and ultimately to the activation of matrix degrading enzymes and net reduction of proteoglycan and colla- gen synthesis followed by chondrocyte death. 2 Among the molecular pathways involved in chondrocyte neme- sis are the TGF-beta pathway, WNT signalling, - and integrin mediated cell–cell adhesion pathways. 3–5 TGF- beta functions as a growth factor, enhancing proteogly- can synthesis, but depending on concentration, culture conditions and modification of signalling, TGF-beta can also be responsible for cartilage breakdown in vivo. 6,7 Epigenetic processes like DNA methylation and chromatin/histone modifications are important players in gene regulation, controlling quantity, location and timing of gene expression. 8 Classical concepts correlate DNA hypermethylation and histone deacetylation with gene silencing, while DNA hypomethylation and his- tone acetylation are promoting gene expression. Pro- moter hypomethylation events have been associated with increased expression of several matrix metallopro- teinases involved in cartilage degradation. 9–11 Whole genome methylation microarrays allow the investiga- tion of the methylation status of most human promoters with CpG islands. We performed a genome wide screen for differences in promoter methylation in chondrocytes, comparing the macroscopically damaged appearing car- tilage (severe) and the macroscopically undamaged cartilage (mild) within the OA joint. We identified a set of differentially methylated genes and performed a variety of functional and pathway analyses. MATERIALS AND METHODS Patient Samples We collected cartilage samples from 15 female patients undergoing unilateral total knee arthroplasty due to severe OA affecting the medial or lateral femorotibial and femoropa- tellar compartment of the knee. Each patient agreed to donate two cartilage cylinders, removed from her knee during surgery. Two experienced orthopaedic surgeons (GG and GM) collected the specimen. One specimen was taken from the macroscopically healthy appearing area between the femoral condyles, an area which is not affected from mechanical overloading in general (mild OA). The other sample was taken from an area of the same femur, which was macroscopically damaged but there was still enough cartilage present to collect a specimen (severe OA). Informed consent was given by patients and the protocol was approved [Correction added on 22 September 2014, after first online publication 12 September 2014: The authorship of the article was updated to include the following names: Hannes Angerer and Daniela Peischler.] Moazedi-Fuerst F, Study design, data acquisition, manuscript preparation; Hofner M, Data acquisition, statistical analysis, manuscript preparation; Gruber G, Data acquisition, sample collection; Stradner MH, Histological analysis, manuscript prep- aration; Weinhaeusel A, Statistical analysis; Angerer H, Data acquisition; Lohberger B, Data acquisition; Glehr M, Data acquisition; Sonntagbauer M, Data acquisition; Graninger WB, Manuscript preparation, supervision. Conflict of interest: None. Grant sponsor: Medical University Graz, Department of Rheu- matology and Immunology. Correspondence to: Gerald Gruber (T: 004331638587434; F: 004331638517778; E-mail: gerald.gruber@klinikum-graz.at) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2014 1