Cindy Boer

86 | Chapter 2.1 on 10 independent OA associated loci, based on existing literature in PubMed till August 2014. Mouse gene expression and phenotype: For each investigated gene, expression in mouse bone and/or cartilage tissue during several developmental stages as well as for adult tissue was determined using data from the Jackson lab database (http://www.in- formatics.jax.org/ ). In addition mouse phenotype data was also obtained for each gene. OMIM phenotype: Using the Online Mendelian Inheritance in Man (OMIM) database we examined which genes were involved in abnormal skeletal growth syndromes when mutated (http://omim.org) . Expression quantitative trait loci: eQTL information was taken from the Blood eQTL browser (http://genenetwork.nl/bloodeqtlbrowser/) and the eQTL browser (http://www.ncbi.nlm.nih.gov/projects/gap/eqtl/index.cgi ) using the lead SNP in each locus [20]. Non-synonymous variants: Last we determined if there were any nonsynonymous variants in LD (r 2 >0.6) with the lead SNP of a locus, using HaploReg V2 and the SNP Annotation and Proxy Search (SNAP) tools[47,48]. For each gene we assigned a score based on equally weighted lines of evidence. Human cartilage gene expression We have used cartilage samples from the RAAK study to study gene expression in pre- served and affected cartilage from individuals undergoing joint replacement[19]. The ongoing Research Arthritis and Articular Cartilage (RAAK) study is aimed at the bio- banking of blood, joint materials (cartilage, bone and where available ligaments of knees and hips) and bone marrow stem cells (hip joints only) of patients and controls in the Leiden University Medical Center and collaborating outpatient clinics in the Leiden area. At the moment of collection (within 2 hours following surgery) tissue was washed extensively with phosphate buffered saline (PBS) to decrease the risk of contamination by blood, and cartilage was collected of the weight-bearing area of the joint. Cartilage was classified macroscopically and collected separately for macroscopically OA affected and preserved regions. Classification was done according to predefined features for OA related damage based on colour/whiteness of the cartilage, based on surface integrity as determined by visible fibrillation/crack formation, and based on depth and hardness of the cartilage upon sampling with a scalpel. During collection with a scalpel, care was taken to avoid contamination with bone or synovium. Collected cartilage was snap fro- zen in liquid nitrogen and stored at -80°C prior to RNA extraction. Tissues have been stored tailored to apply staining and immunohistochemistry(IHC). Furthermore, DNA and RNA have been isolated from the preserved and affected areas of the respective tissues in order to apply genetic, transcriptomic and epigenomic profiling with respect to the OA pathophysiological process. After in vitro transcription, amplification, and labeling with biotin-labeled nu- cleotides (Illumina TotalPrep RNA Amplification Kit) Illumina HumanHT-12 v3 mi-