Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and skeletal system morphogenesis as major pathways involved in osteoarthritis

Abstract

Background: Evidence suggests that epigenetics plays a role in osteoarthrits (OA). The aim of the study was to describe the genome wide DNA methylation changes in hip and knee OA and identify novel genes and pathways involved in OA by comparing the DNA methylome of the hip and knee osteoarthritic cartilage tissues with those of OA-free individuals.

Methods: Cartilage samples were collected from hip or knee joint replacement patients either due to primary OA or hip fractures as controls. DNA was extracted from the collected cartilage and assayed by Illumina Infinium HumanMethylation450 BeadChip array, which allows for the analysis of >480,000 CpG sites. Student T-test was conducted for each CpG site and those sites with at least 10 % methylation difference and a p value <0.0005 were defined as differentially methylated regions (DMRs) for OA. A sub-analysis was also done for hip and knee OA separately. DAVID v6.7 was used for the functional annotation clustering of the DMR genes. Clustering analysis was done using multiple dimensional scaling and hierarchical clustering methods.

Results: The study included 5 patients with hip OA, 6 patients with knee OA and 7 hip cartilage samples from OA-free individuals. The comparisons of hip, knee and combined hip/knee OA patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the overlaps was 239, among which 151 sites were annotated to 145 genes. One-fifth of these genes were reported in previous studies. The functional annotation clustering of the identified genes revealed clusters significantly enriched in skeletal system morphogenesis and development. The analysis revealed significant difference among OA and OA-free cartilage, but less different between hip OA and knee OA.

Conclusions: We found that a number of CpG sites and genes across the genome were differentially methylated in OA patients, a remarkable portion of which seem to be involved in potential etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic organ morphogenesis may be a potential area for further OA studies.

Fig. 1

Multiple dimensions scaling of hip OA, knee OA, and OA-free hip cartilage. *Similarities between hip OA, knee OA, and OA-free cartilage, drawn from log-spectral decompositions for each subject as represented in the two-dimensional space by multiple dimensional scaling (MDS). Each dot represents one sample. Colors represent the type of involvement and the site samples obtained. X- and Y- axes represent the first and the second dimension reductions

Fig. 2

Hierarchical clustering and heat map of hip OA, knee OA, and OA-free controls*. *Top: Cluster dendogram was created using the genome wide information; Bottom: Heat map shows the top 800 CpG sites with the most variation across hip OA, knee OA, and OA-free hip cartilage samples. Rows represent CpG sites. Columns represent samples. Dark blue indicates hypermethylation and light blue/white indicates hypomethylation